releases during and after natural disasters: a review: Sci. Total Environ.322:3-20. **Part 2**

## **New Approaches to the TB Diagnosis**

236 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Young, S., L. Balluz, and J. Malilay. 2004. Natural and technologic hazardous material

**11** 

*tuberculosis*

*Perth, Western Australia,* 

*Australia* 

**New Diagnostics for** *Mycobacterium* 

Michaela Lucas1,2, Andrew Lucas1 and Silvana Gaudieri1,3 *1Institute for Immunology and Infectious Diseases, Murdoch University,* 

*3School of Anatomy and Human Biology and Centre for Forensic Science,* 

**1.1 Tuberculosis as a global health issue and the need for reliable diagnostics in** 

Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of morbidity and mortality worldwide with an estimated one third of the world's population infected by Mtb resulting in about two million deaths per year (Lonnroth and Raviglione, 2008; Wallis et al., 2010). Developing countries are burdened with the highest levels of Mtb infections and conversely have the lowest financial resources available to improve this situation (Figures 1 and 2). High rates of infection are associated with poverty, low levels of public hygiene and often with a high prevalence of HIV+ individuals who are at particular risk of infection, all factors that contribute to an uncontrolled spread of Mtb infection (Corbett et al., 2006;

Mtb can be spread from person to person via droplet nuclei that contain Mtb organisms. Droplet nuclei are primarily produced when people with pulmonary Mtb cough or sneeze and these particles can remain in the air for long periods of time. If inhaled, these droplet nuclei can reach the alveoli within the lungs where Mtb replicates. Individuals with Mtb infection can exhibit a wide range of clinical features that challenge current diagnostic approaches including acute active pulmonary infection with infective sputum, latent disease with risk of reactivation (especially in the immune-compromised host), sputum-negative and extrapulmonary Mtb infection and childhood tuberculosis (Wallis et al., 2010). In the majority of cases an infection with Mtb cannot be cleared and is contained by an effective immune response and the infection becomes latent and asymptomatic. About 10% of latently infected individuals progress to active reactivated disease during their lifetime. Thus individuals with latent Mtb infection act as infective foci of recurrent active disease and newly infect people in close contact. This large pool of undetected and untreated

**1. Introduction** 

**primary care settings** 

Wright et al., 2009).

disease hampers eradication programs.

*2Department of Health, Perth, Western Australia,* 

*University of Western Australia, Perth, Western Australia,* 

## **New Diagnostics for** *Mycobacterium tuberculosis*

Michaela Lucas1,2, Andrew Lucas1 and Silvana Gaudieri1,3

*1Institute for Immunology and Infectious Diseases, Murdoch University, Perth, Western Australia, 2Department of Health, Perth, Western Australia, 3School of Anatomy and Human Biology and Centre for Forensic Science, University of Western Australia, Perth, Western Australia, Australia* 

#### **1. Introduction**

#### **1.1 Tuberculosis as a global health issue and the need for reliable diagnostics in primary care settings**

Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of morbidity and mortality worldwide with an estimated one third of the world's population infected by Mtb resulting in about two million deaths per year (Lonnroth and Raviglione, 2008; Wallis et al., 2010). Developing countries are burdened with the highest levels of Mtb infections and conversely have the lowest financial resources available to improve this situation (Figures 1 and 2). High rates of infection are associated with poverty, low levels of public hygiene and often with a high prevalence of HIV+ individuals who are at particular risk of infection, all factors that contribute to an uncontrolled spread of Mtb infection (Corbett et al., 2006; Wright et al., 2009).

Mtb can be spread from person to person via droplet nuclei that contain Mtb organisms. Droplet nuclei are primarily produced when people with pulmonary Mtb cough or sneeze and these particles can remain in the air for long periods of time. If inhaled, these droplet nuclei can reach the alveoli within the lungs where Mtb replicates. Individuals with Mtb infection can exhibit a wide range of clinical features that challenge current diagnostic approaches including acute active pulmonary infection with infective sputum, latent disease with risk of reactivation (especially in the immune-compromised host), sputum-negative and extrapulmonary Mtb infection and childhood tuberculosis (Wallis et al., 2010). In the majority of cases an infection with Mtb cannot be cleared and is contained by an effective immune response and the infection becomes latent and asymptomatic. About 10% of latently infected individuals progress to active reactivated disease during their lifetime. Thus individuals with latent Mtb infection act as infective foci of recurrent active disease and newly infect people in close contact. This large pool of undetected and untreated disease hampers eradication programs.

New Diagnostics for *Mycobacterium tuberculosis* 241

Pulmonary Sputum difficult

reduced sensitivity in HIV+ individuals

turnaround time due to slow growth of bacteria, requires biosafety level 3 facilities

Pulmonary Long

Pleuritis, pericarditis, peritonitis

Pulmonary and extrapulmonary

Pulmonary and extrapulmonary

Active and latent Mtb disease

Variable sensitivity especially in smear -ve

and

disease

Variable sensitivity

Inconsistent estimates of sensitivity and specificity

extrapulmonary

to obtain from children, proportion of individuals smear negative,

**Disadvantages Advantages** 

ADA levels in

FM 10% more sensitive than conventional

no need for darkroom

microscopy, LED FM associated with low cost, durability and

Liquid culture more sensitive than solid cultures and higher turnover rate

pleural, pericardial

High specificity and positive predictive

Quick and relatively

Fast turnaround and can be used for children

easy assay to perform

and ascitic fluid has high specificity and sensitivity for extrapulmonary Mtb infection

value

**site** 

**Diagnosis of active tuberculosis**

Conventional, FM, LED

Liquid

deaminase

**Assays Description Disease/** 

Microscopic observation of stained acid-fast bacilli

Monitor changes in media resources to detect bacterial growth

Detection of host enzyme released in response to intracellular pathogen

Detection of Mtb genetic material

Detection of circulating Mtb antigens

of host humoral response to pathogen

**Platform/ Target** 

Sputum-Smear Microscopy

Culture Solid,

Biochemical Adenosine

Pathogen Nucleic

acid amplification tests

Mtb antigens

Serological Detection

Fig. 1. Global Mtb burden of disease (2009). Data extracted from Global TB report 2010.

Global efforts to control Mtb center around improving both the rate of detection of cases and the treatment of infective subjects as reflected in the "The Stop TB strategy" published by the World Health Organisation (WHO) (WHO, 2006). The strategy aims to reverse the trend of rising incidence, halve the 1990 prevalence and mortality due to Mtb infection by 2015 and eliminate Mtb as a public health problem by 2050 (Maher et al., 2007). Successful implementation of the Stop TB strategy relies on accurate diagnostics for Mtb infection. Such an accurate diagnostic for Mtb infection should include the ability to identify adults and children with active infection, predict durable treatment success, and indicate and forecast reactivation of latent disease (Wallis et al., 2010). The ideal diagnostic tool would also need to remain trustworthy even in the setting of malnutrition and immunodeficiency and be performed within the primary care setting (Lucas et al., 2010). Given the enormity of the problem and the high prevalence in developing countries, the test(s) should also be simple and cheap. Despite major research efforts, a diagnostic assay or set of assays for Mtb infection that exhibit these properties is currently not available.

#### **2. Current diagnostic tools for active infection: Focus on pathogen detection and early immune activation**

Sputum-smear microscopy and chest radiography are still the primary tools to identify active Mtb infection in the typically resource-poor countries with a high-burden of Mtb disease (Figure 2). These tools can sometimes perform poorly and sputum is difficult to obtain from children. However, there are efforts to improve diagnostic assays available to developing countries including the development of assays that directly assay the pathogen. A summary of the main features of current diagnostics for active Mtb infection is given in Table 1.


#### **Diagnosis of active tuberculosis**

240 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Fig. 1. Global Mtb burden of disease (2009). Data extracted from Global TB report 2010.

infection that exhibit these properties is currently not available.

**and early immune activation** 

Table 1.

Global efforts to control Mtb center around improving both the rate of detection of cases and the treatment of infective subjects as reflected in the "The Stop TB strategy" published by the World Health Organisation (WHO) (WHO, 2006). The strategy aims to reverse the trend of rising incidence, halve the 1990 prevalence and mortality due to Mtb infection by 2015 and eliminate Mtb as a public health problem by 2050 (Maher et al., 2007). Successful implementation of the Stop TB strategy relies on accurate diagnostics for Mtb infection. Such an accurate diagnostic for Mtb infection should include the ability to identify adults and children with active infection, predict durable treatment success, and indicate and forecast reactivation of latent disease (Wallis et al., 2010). The ideal diagnostic tool would also need to remain trustworthy even in the setting of malnutrition and immunodeficiency and be performed within the primary care setting (Lucas et al., 2010). Given the enormity of the problem and the high prevalence in developing countries, the test(s) should also be simple and cheap. Despite major research efforts, a diagnostic assay or set of assays for Mtb

**2. Current diagnostic tools for active infection: Focus on pathogen detection** 

Sputum-smear microscopy and chest radiography are still the primary tools to identify active Mtb infection in the typically resource-poor countries with a high-burden of Mtb disease (Figure 2). These tools can sometimes perform poorly and sputum is difficult to obtain from children. However, there are efforts to improve diagnostic assays available to developing countries including the development of assays that directly assay the pathogen. A summary of the main features of current diagnostics for active Mtb infection is given in

New Diagnostics for *Mycobacterium tuberculosis* 243

Ziehl-Neelsen sputum-smear microscopy with a conventional light microscope is commonly used to identify acid-fast bacilli in sputum to diagnose active Mtb infection in low and middle-income countries with a high prevalence of Mtb disease. This assay identifies the most infectious patients and is a quick and relatively easy procedure that is widely applicable but also requires multiple sample collections over several days (usually 3 days) and is dependent on the quality and bacterial load of the sputum specimen. The use of acidfast fluorochrome dyes with fluorescence-based microscopy (FM) is a standard assay performed in high-income countries to detect Mtb. FM has greater sensitivity than conventional microscopy and can be performed in less time but the need for a dark room and considerable outlay costs make it less amenable for resource poor countries. The recent development of light-emitting diode (LED) fluorescence microscopy may overcome some of the difficulties associated with the widespread implementation of FM (Cuevas et al., 2011). In the setting of laboratories, which contribute to a well-functioning external quality assurance system, revised WHO guidelines for the diagnosis of pulmonary Mtb infection include the reduction in the minimum number of samples that need to be tested (from three to two), given that the inclusion of a third sample only increases sensitivity by 2-5% (Pai et al., 2008). This move could significantly reduce the local collection and testing costs and increase the successful collection rate of samples. Furthermore, the addition of simple sputum processing methods (including the use of household bleach and centrifugation) can improve sensitivity of sputum-smear microscopy. However, it is important to remember that a positive acid-fast staining result may represent the presence of non-tuberculosis

Clinical specimens suspected of containing Mtb can be inoculated onto a culture media. Culture of Mtb on solid media (typically egg or agar-based) is more sensitive than sputumsmear microscopy for the diagnosis of active Mtb infection and can differentiate between species of mycobacteria but can take weeks to perform due to the slow growth of Mtb and related organisms. The sensitivity of cultures is generally between 80-85% with a specificity of about 98% (Prevention, 2000). The use of liquid cultures can reduce bacterial growth times (1-3 weeks compared to 3-8 weeks), can be automated and have sensitivity and specificity levels close to 100%. However, the use of such cultures requires a biosafety level 3 environment and equipment and consumables which are relatively expensive, although

The genus Mycobacterium consists of over 80 species and many appear similar on acid-fast staining; a limitation of sputum-smear microscopy. Although cultures offer some differentiation between species, these assays have a slow turnaround time that results in a delay in diagnosing Mtb infection. Amplification and detection of Mtb DNA directly from specimens can be an efficient and sensitive method to detect Mtb infection and may also allow for the detection of mutations in the Mtb genome associated with drug resistance.

cheaper products may become available for developing countries (WHO, 2006).

**2.1.3 Direct detection of pathogen nucleic acid** 

**2.1 Pathogen detection** 

**2.1.1 Microscopy** 

mycobacteria.

**2.1.2 Microbiological culture** 


**Diagnosis of active tuberculosis**

References include (Wallis et al., 2010), (Daley et al., 2007; Greco et al., 2003; Mase et al., 2007), (Dinnes et al., 2007), (Jiang et al., 2007), (Flores et al., 2005; Greco et al., 2006; Ling et al., 2008), (Pai et al., 2003; Pai et al., 2004), (Sarmiento et al., 2003; Steingart et al., 2007a; Steingart et al., 2007b; Steingart et al., 2006a; Steingart et al., 2006b), (Pai et al., 2010), (Pai et al., 2008), (Goto et al., 2003; Kalantri et al., 2005), (Liang et al., 2008; Riquelme et al., 2006; Tuon et al., 2006).

Table 1. Review of current diagnostics for Mtb infection.

Fig. 2. Average gross national product (\$USD) for 2009 for selected nations. Data extracted from Global TB report 2010 and The World Bank.

#### **2.1 Pathogen detection**

#### **2.1.1 Microscopy**

242 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

**Disadvantages Advantages** 

Quick and relatively easy assay to perform

High specificity and unaffected by previous BCG vaccination

BCG vaccinated subjects more likely to be positive

Cannot distinguish between latent and active Mtb infection, sensitivity may be lower in HIV+ subjects

**site** 

References include (Wallis et al., 2010), (Daley et al., 2007; Greco et al., 2003; Mase et al., 2007), (Dinnes et al., 2007), (Jiang et al., 2007), (Flores et al., 2005; Greco et al., 2006; Ling et al., 2008), (Pai et al., 2003; Pai et al., 2004), (Sarmiento et al., 2003; Steingart et al., 2007a; Steingart et al., 2007b; Steingart et al., 2006a; Steingart et al., 2006b), (Pai et al., 2010), (Pai et al., 2008), (Goto et al., 2003; Kalantri et al., 2005),

Fig. 2. Average gross national product (\$USD) for 2009 for selected nations. Data extracted

**Diagnosis of active tuberculosis**

**Assays Description Disease/** 

of induration as a result of exposure to intradermal tuberculin

of interferon gamma released from

lymphocytes when stimulated with Mtb antigens

TST Measurement

IGRAs Measurement

(Liang et al., 2008; Riquelme et al., 2006; Tuon et al., 2006).

from Global TB report 2010 and The World Bank.

Table 1. Review of current diagnostics for Mtb infection.

**Platform/ Target** 

Immunological markers

Ziehl-Neelsen sputum-smear microscopy with a conventional light microscope is commonly used to identify acid-fast bacilli in sputum to diagnose active Mtb infection in low and middle-income countries with a high prevalence of Mtb disease. This assay identifies the most infectious patients and is a quick and relatively easy procedure that is widely applicable but also requires multiple sample collections over several days (usually 3 days) and is dependent on the quality and bacterial load of the sputum specimen. The use of acidfast fluorochrome dyes with fluorescence-based microscopy (FM) is a standard assay performed in high-income countries to detect Mtb. FM has greater sensitivity than conventional microscopy and can be performed in less time but the need for a dark room and considerable outlay costs make it less amenable for resource poor countries. The recent development of light-emitting diode (LED) fluorescence microscopy may overcome some of the difficulties associated with the widespread implementation of FM (Cuevas et al., 2011). In the setting of laboratories, which contribute to a well-functioning external quality assurance system, revised WHO guidelines for the diagnosis of pulmonary Mtb infection include the reduction in the minimum number of samples that need to be tested (from three to two), given that the inclusion of a third sample only increases sensitivity by 2-5% (Pai et al., 2008). This move could significantly reduce the local collection and testing costs and increase the successful collection rate of samples. Furthermore, the addition of simple sputum processing methods (including the use of household bleach and centrifugation) can improve sensitivity of sputum-smear microscopy. However, it is important to remember that a positive acid-fast staining result may represent the presence of non-tuberculosis mycobacteria.

#### **2.1.2 Microbiological culture**

Clinical specimens suspected of containing Mtb can be inoculated onto a culture media. Culture of Mtb on solid media (typically egg or agar-based) is more sensitive than sputumsmear microscopy for the diagnosis of active Mtb infection and can differentiate between species of mycobacteria but can take weeks to perform due to the slow growth of Mtb and related organisms. The sensitivity of cultures is generally between 80-85% with a specificity of about 98% (Prevention, 2000). The use of liquid cultures can reduce bacterial growth times (1-3 weeks compared to 3-8 weeks), can be automated and have sensitivity and specificity levels close to 100%. However, the use of such cultures requires a biosafety level 3 environment and equipment and consumables which are relatively expensive, although cheaper products may become available for developing countries (WHO, 2006).

#### **2.1.3 Direct detection of pathogen nucleic acid**

The genus Mycobacterium consists of over 80 species and many appear similar on acid-fast staining; a limitation of sputum-smear microscopy. Although cultures offer some differentiation between species, these assays have a slow turnaround time that results in a delay in diagnosing Mtb infection. Amplification and detection of Mtb DNA directly from specimens can be an efficient and sensitive method to detect Mtb infection and may also allow for the detection of mutations in the Mtb genome associated with drug resistance.

New Diagnostics for *Mycobacterium tuberculosis* 245

growth of Mtb. As an alternative, the enzyme adenosine deaminase (ADA), which is widely distributed in pleural, meningeal and pericardial fluids, is used in the diagnosis of Mtb pleuritis, pericarditis and peritonitis. The host enzyme is released from lymphocytes in response to infection with intracellular pathogens (Pai et al., 2010). The biochemical assay used to quantity ADA is quick and non-invasive. A recent systematic review of 10 studies involving 1364 participants showed the ADA assay exhibited a mean sensitivity of 79% and mean specificity of 91% for Mtb meningitis (Xu et al., 2010). However, the study stresses that a negative ADA assay does not rule out Mtb meningitis and should not be used alone to

Initial pathogen recognition of Mtb by macrophages occurs via toll-like receptors (TLRs), resulting in the induction of transcription of pro-inflammatory cytokine genes essential to direct the subsequent immune response (Berrington and Hawn, 2007). Mtb ligands for TLRs include CpG DNA, triggering the intracellular TLR-9, and LAM and mannosylated phosphotidylinositol acting mainly via TLR-2 (in association with TLR-1, -4 and -6) (Constantoulakis et al., 2010). Once uptake into macrophages has occurred, Mtb has unique mechanisms to survive within the phagocytes, for example by blocking biogenesis of the phagolysosome. A recent study assessed mRNA expression of a combination of these innate markers, namely TLR-2, Coronin 1, a protein which arrests the maturation of the phagolysosome within macrophages, and Sp110, a protein complex important for monocyte differentiation and apoptosis in Mtb infection. This study revealed significantly elevated levels of mRNA of all three proteins in subjects with active and latent Mtb disease as compared to healthy uninfected subjects (Chen et al., 2010). However, larger case-controlled studies are needed to confirm these findings and evaluate these factors as future diagnostic

Another potential immune marker of Mtb infection is Neopterin. Neopterin is secreted when macrophages are activated through exposure to interferon gamma (IFN-γ) and its concentration in serum is increased in the early stages of infection whilst its levels decrease following successful treatment and increasing again upon relapse. As such it can be used as a non-specific pro-inflammatory marker for Mtb as its detection is also associated with other chronic infections that commonly coexist with Mtb, such as HIV, malaria, Hepatitis B and C

Other examples of innate markers that have been shown to be increased in Mtb infection are the soluble intercellular adhesion molecules (Walzl et al., 2008), the acute phase reactant proteins (Djoba Siawaya et al., 2008), soluble urokinase plasminogen activator receptor (Eugen-Olsen et al., 2002), CXCL10/IP10 and Pentraxin 3 (Azzurri et al., 2005), and procalcitonin (Baylan et al., 2006; Kandemir et al., 2003; Nyamande and Lalloo, 2006; Prat et

Recently, an elegant study by Anne O'Garra and colleagues identified a 393-whole blood transcript signature for active Mtb, which correlated with clinical and radiological disease as well as treatment response. One of the surprising findings of this study was the dominant induction of genes of the IFN-γ and type I interferon pathways in neutrophils

(Fuchs et al., 1984; Immanuel et al., 2001; Turgut et al., 2006; Wallis et al., 1996).

**2.2.2 The potential role of early immune markers in the diagnosis of acute** 

make clinical decisions regarding treatment of a patient.

**Mtb infection** 

biomarkers for Mtb infection.

al., 2006; Schleicher et al., 2005).

In theory, nucleic acid amplification tests (NAATs) could identify a single mycobacterium at the species level but first generation NAATs were not sufficiently reliable to replace conventional diagnostic methods for Mtb infection (Greco et al., 2006). False negative results using these assays may have been due to sampling issues (given small volumes needed for test) and possible presence in specimens of inhibitors of the amplification process. False positive results may have been due to contaminations given the inherent increased risk in these assays due to the common amplification step. Subsequent improvements using internal controls (to identify assay inhibitors) and automated systems using a single sealed tube (reduce contamination) have improved the sensitivity and specificity of these assays.

A review of several commercial NAATs showed a mean sensitivity of 96% and specificity of 85% for smear-positive cases and 66% sensitivity and 98% specificity for smear negative cases (Greco et al., 2006). Importantly, these NAATs could exclude Mtb in patients with smear positive microscopy in which environmental mycobacteria is suspected.

The fully automated Xpert Mtb device, which was developed by a consortium that included both commercial and publicly funded organisations (Cepheid Inc.; Foundation for Innovative New Diagnostics) has been endorsed by WHO for use in Mtb endemic countries (http://www.who.int/mediacentre/news/releases/2010/tb\_test\_20101208/en/index.html). The device does not require extensive staff training and produces results from the assay on the same day (WHO, 2006). The first assessment of the new system suggests it is highly sensitive for both smear positive and smear negative samples (Wallis et al., 2010) and the negotiation that the price per test would be reduced by 75% in countries most effected by TB should make this broadly accessible as an effective point-of-care diagnostic tool.

#### **2.1.4 Mtb antigen detection**

Assays that directly assess the presence of circulating Mtb antigens in serum, sputum, urine, cerebrospinal and pleural fluid to diagnose active Mtb disease are widely used. However, a recent review of Mtb antigen assays showed that of 47 studies examining pulmonary Mtb, the sensitivity of the assays varied from 2-100% and specificity varied from 33-100% (Flores et al., 2011). Furthermore, 21 studies examining extrapulmonary Mtb using Mtb antigen assays showed sensitivity levels varying from 0-100% and specificity from 62-100%. Most assays utilised the Mtb cell wall protein lipoarabinomannan (LAM) but some assays used multiple Mtb antigens. Interestingly, the detection of LAM in urine samples tended to have higher sensitivity in HIV+ patients than in HIV negative patients for Mtb diagnosis. However, much research is needed to improve the performance of these assays given the relative ease of translation into the primary care setting and is likely to be centred on identifying Mtb antigens that are abundantly expressed, specific to Mtb and resistant to the host's immune response.

#### **2.2 Early immune activation markers**

#### **2.2.1 Biochemical**

The diagnosis of Mtb meningitis is difficult due to the low sensitivity of identifying acid-fast bacilli in cerebrospinal fluid with microscopy and the length of time required for the culture

In theory, nucleic acid amplification tests (NAATs) could identify a single mycobacterium at the species level but first generation NAATs were not sufficiently reliable to replace conventional diagnostic methods for Mtb infection (Greco et al., 2006). False negative results using these assays may have been due to sampling issues (given small volumes needed for test) and possible presence in specimens of inhibitors of the amplification process. False positive results may have been due to contaminations given the inherent increased risk in these assays due to the common amplification step. Subsequent improvements using internal controls (to identify assay inhibitors) and automated systems using a single sealed tube (reduce contamination) have improved the sensitivity

A review of several commercial NAATs showed a mean sensitivity of 96% and specificity of 85% for smear-positive cases and 66% sensitivity and 98% specificity for smear negative cases (Greco et al., 2006). Importantly, these NAATs could exclude Mtb in patients with

The fully automated Xpert Mtb device, which was developed by a consortium that included both commercial and publicly funded organisations (Cepheid Inc.; Foundation for Innovative New Diagnostics) has been endorsed by WHO for use in Mtb endemic countries (http://www.who.int/mediacentre/news/releases/2010/tb\_test\_20101208/en/index.html). The device does not require extensive staff training and produces results from the assay on the same day (WHO, 2006). The first assessment of the new system suggests it is highly sensitive for both smear positive and smear negative samples (Wallis et al., 2010) and the negotiation that the price per test would be reduced by 75% in countries most effected by TB

Assays that directly assess the presence of circulating Mtb antigens in serum, sputum, urine, cerebrospinal and pleural fluid to diagnose active Mtb disease are widely used. However, a recent review of Mtb antigen assays showed that of 47 studies examining pulmonary Mtb, the sensitivity of the assays varied from 2-100% and specificity varied from 33-100% (Flores et al., 2011). Furthermore, 21 studies examining extrapulmonary Mtb using Mtb antigen assays showed sensitivity levels varying from 0-100% and specificity from 62-100%. Most assays utilised the Mtb cell wall protein lipoarabinomannan (LAM) but some assays used multiple Mtb antigens. Interestingly, the detection of LAM in urine samples tended to have higher sensitivity in HIV+ patients than in HIV negative patients for Mtb diagnosis. However, much research is needed to improve the performance of these assays given the relative ease of translation into the primary care setting and is likely to be centred on identifying Mtb antigens that are abundantly expressed, specific to Mtb and resistant to the

The diagnosis of Mtb meningitis is difficult due to the low sensitivity of identifying acid-fast bacilli in cerebrospinal fluid with microscopy and the length of time required for the culture

smear positive microscopy in which environmental mycobacteria is suspected.

should make this broadly accessible as an effective point-of-care diagnostic tool.

and specificity of these assays.

**2.1.4 Mtb antigen detection** 

host's immune response.

**2.2.1 Biochemical** 

**2.2 Early immune activation markers** 

growth of Mtb. As an alternative, the enzyme adenosine deaminase (ADA), which is widely distributed in pleural, meningeal and pericardial fluids, is used in the diagnosis of Mtb pleuritis, pericarditis and peritonitis. The host enzyme is released from lymphocytes in response to infection with intracellular pathogens (Pai et al., 2010). The biochemical assay used to quantity ADA is quick and non-invasive. A recent systematic review of 10 studies involving 1364 participants showed the ADA assay exhibited a mean sensitivity of 79% and mean specificity of 91% for Mtb meningitis (Xu et al., 2010). However, the study stresses that a negative ADA assay does not rule out Mtb meningitis and should not be used alone to make clinical decisions regarding treatment of a patient.

#### **2.2.2 The potential role of early immune markers in the diagnosis of acute Mtb infection**

Initial pathogen recognition of Mtb by macrophages occurs via toll-like receptors (TLRs), resulting in the induction of transcription of pro-inflammatory cytokine genes essential to direct the subsequent immune response (Berrington and Hawn, 2007). Mtb ligands for TLRs include CpG DNA, triggering the intracellular TLR-9, and LAM and mannosylated phosphotidylinositol acting mainly via TLR-2 (in association with TLR-1, -4 and -6) (Constantoulakis et al., 2010). Once uptake into macrophages has occurred, Mtb has unique mechanisms to survive within the phagocytes, for example by blocking biogenesis of the phagolysosome. A recent study assessed mRNA expression of a combination of these innate markers, namely TLR-2, Coronin 1, a protein which arrests the maturation of the phagolysosome within macrophages, and Sp110, a protein complex important for monocyte differentiation and apoptosis in Mtb infection. This study revealed significantly elevated levels of mRNA of all three proteins in subjects with active and latent Mtb disease as compared to healthy uninfected subjects (Chen et al., 2010). However, larger case-controlled studies are needed to confirm these findings and evaluate these factors as future diagnostic biomarkers for Mtb infection.

Another potential immune marker of Mtb infection is Neopterin. Neopterin is secreted when macrophages are activated through exposure to interferon gamma (IFN-γ) and its concentration in serum is increased in the early stages of infection whilst its levels decrease following successful treatment and increasing again upon relapse. As such it can be used as a non-specific pro-inflammatory marker for Mtb as its detection is also associated with other chronic infections that commonly coexist with Mtb, such as HIV, malaria, Hepatitis B and C (Fuchs et al., 1984; Immanuel et al., 2001; Turgut et al., 2006; Wallis et al., 1996).

Other examples of innate markers that have been shown to be increased in Mtb infection are the soluble intercellular adhesion molecules (Walzl et al., 2008), the acute phase reactant proteins (Djoba Siawaya et al., 2008), soluble urokinase plasminogen activator receptor (Eugen-Olsen et al., 2002), CXCL10/IP10 and Pentraxin 3 (Azzurri et al., 2005), and procalcitonin (Baylan et al., 2006; Kandemir et al., 2003; Nyamande and Lalloo, 2006; Prat et al., 2006; Schleicher et al., 2005).

Recently, an elegant study by Anne O'Garra and colleagues identified a 393-whole blood transcript signature for active Mtb, which correlated with clinical and radiological disease as well as treatment response. One of the surprising findings of this study was the dominant induction of genes of the IFN-γ and type I interferon pathways in neutrophils

New Diagnostics for *Mycobacterium tuberculosis* 247

was not possible in 37 (11%) children due to non-attendance at clinic appointments or absence of the child at scheduled home visits (Lucas et al., 2010). The interpretation of results and determination of cut-offs also varies and is infuenced by age, previous BCG vaccination and Mtb infection risk. In addition, this method is now recognised to have other limitations, such as poor specificity due to cross reactivity with both non-tuberculous

More recently, measurement of IFN-γ -producing memory T cells specific for Mtb has been introduced into the clinical practice of many countries. Two blood-based IFN-γ release assays (IGRAs) are available for diagnostic use, the Quantiferon-TB gold in-tube (QFT-GIT; Cellestis, Carnegie, Australia) and the T-SPOT.TB (Oxford Immunotech,Oxford, UK). Both assays have high specificity for adult Mtb infection including in BCG-vaccinated populations (Lucas et al., 2010). Both assays measure T cell IFN-γ production in response to antigens encoded by the RD1 gene which is present in all strains of Mtb but is not present in the Mycobacterium bovis genome from which BCG is derived. This eliminates BCG crossreactivity, however cross-reactivity to a limited number of non-Mtb mycobacteria (M. kansasii, M. szulgai, M. marinum) remains. T-SPOT.TB is an enzyme-linked immunospot (ELISpot) assay that measures the response to two antigens, early-secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10). The QFT-GIT assay is a whole blood IGRA assay that includes an additional Mtb-specific antigen (TB7.7). In the few head-to head comparisons of the latest generation tests, T-SPOT.TB and QFT-GIT both perform satisfactorily. Some studies however suggest that the use of the T-SPOT.TB assay may be the preferred option in subjects with primary and secondary (HIV, iatrogenic)

To date, there has been limited focus on high incidence paediatric populations, which are at greater risk of reactivation and extrapulmonary manifestations of Mtb, including TB meningitis. We recently published a prospective comparative study of IGRAs and TST for the diagnosis of LTBI in 524 refugee children from countries with a high prevalence of MTb that resettled to Australia (Lucas et al., 2010). This study included 182 children <5 years of age. In our study, the T-SPOT.TB and QFT-GIT had similar rates of positivity (8% and 10%, respectively) and showed good concordance when both tests gave definitive results (kappa= 0.78; p<0.0001). Surprisingly, both IGRAs had significant failure rates: 15% of QFT-GIT gave indeterminate results due to failed mitogen response and 14% of T-SPOT.TB results were inconclusive because of insufficient mononuclear leukocyte yields. Failure of the QFT-GIT mitogen response was associated with African ethnicity and comorbid infections, particularly with helminths. Overall, the TST results showed low concordance (about 50%) with both IGRAs. This study highlights the influence of age, ethnicity and clinical status on IGRA results and the limitations of using these T cell based

In general, IGRAs are likely to be too complex and expensive for point-of-care testing in the Developing World, but their use in developed countries where the Mtb burden is much smaller, is currently justified. In addition, they have a role in the identification of subjects with latent Mtb prior to immunosuppressive therapy, including treatment with novel

biologic drugs such as TNF- antagonists (Bellofiore et al., 2009).

mycobacteria and BCG (National Health Service, 2005) (Table 1).

immunodeficiencies (Lalvani, 2007; Pai et al., 2008b).

tests in refugee children.

(Berry et al., 2010). This indicates the potential power of using innate responses as surrogate markers of Mtb infection and the strength of using a combination of markers for multiplex analyses.

Despite the lack of specificity to Mtb disease associated with these innate immune markers, the measurement of these surrogate markers is technically straightforward and the assays are often readily available. It may be that by combining multiple markers distinct pathogen associated patterns will be described. Certainly the development of multiplexed assays for soluble proteins and the definition of gene expression profiles for Mtb disease by gene expression analysis (Jacobsen et al., 2007; Maertzdorf et al., 2010), such as those offered for the Luminex platform (Luminex Corp.), will continue to make this process more feasible.

#### **3. Current diagnostic tools for latent infection: Focus on immune memory and cytokine based diagnostics**

Overall, human T cell responses to Mtb involve CD4+, CD8+ and gamma-delta T cells. Akin to chronic viral infections, a broad T cell repertoire able to recognise many different types of bacterial epitopes (proteins as well as lipids) enhances the efficiency of the immune response against Mtb (Boom et al., 2003). The balance between different T helper subsets, especially Th1, T (Fox P3+) regulatory cells and Th17 helper cells may also be a key factor (Korn et al., 2007; Liang et al., 2006; Marin et al., 2010; Torrado and Cooper, 2010) and could potentially be explored for diagnostic purposes.

For some time, assays assessing the presence of Mtb specific memory CD8 and CD4 T cells have been used to identify those who previously have been infected with Mtb. Mtb-specific T cells are detectable within 2 – 3 weeks of acute infection in the peripheral blood and mark the end of the phase of rapid bacterial replication and bacterial containment. Interestingly, studies in mice suggest a late adaptive T cell response, which is linked to a delay in activation of T helper cells (Wolf et al., 2008). The measurement of Mtb specific T cells is therefore diagnostically insensitive in acute infection. This apparent problem may be overcome by the detection of Mtb-specific T cells directly at the site of infection, such as in fluid from broncho-alveolar lavage and cerebrospinal fluid, during early stages of disease in selected cohorts (Jafari et al., 2009; Thomas et al., 2008). On the other hand, the long-term maintenance of memory T cell responses to pathogens in peripheral blood (Semmo et al., 2006) make the measurements of Mtb specific T cells useful as a diagnostic cross-sectional assay to test for prior exposure to Mtb.

#### **3.1 Tuberculin skin test and Interferon- release asssays (IGRA)**

One of the current well established diagnostic approaches, which is based on a T cell mediated delayed hypersensitivity reaction, is the tuberculin skin test (TST). The TST is generally performed by an intradermal injection of 5 tuberculin units using purified protein derivative (PPD) following the Mantoux method. The transverse diameter of the skin induration occuring after 48-72 hours is typically measured. Therefore at least two clinic visits are required for a valid test which may prove a problem. In our recent study of Western Australian refugee children, the TST was initiated in 341 children; however reading

(Berry et al., 2010). This indicates the potential power of using innate responses as surrogate markers of Mtb infection and the strength of using a combination of markers for

Despite the lack of specificity to Mtb disease associated with these innate immune markers, the measurement of these surrogate markers is technically straightforward and the assays are often readily available. It may be that by combining multiple markers distinct pathogen associated patterns will be described. Certainly the development of multiplexed assays for soluble proteins and the definition of gene expression profiles for Mtb disease by gene expression analysis (Jacobsen et al., 2007; Maertzdorf et al., 2010), such as those offered for the Luminex platform (Luminex Corp.), will continue to make

**3. Current diagnostic tools for latent infection: Focus on immune memory** 

Overall, human T cell responses to Mtb involve CD4+, CD8+ and gamma-delta T cells. Akin to chronic viral infections, a broad T cell repertoire able to recognise many different types of bacterial epitopes (proteins as well as lipids) enhances the efficiency of the immune response against Mtb (Boom et al., 2003). The balance between different T helper subsets, especially Th1, T (Fox P3+) regulatory cells and Th17 helper cells may also be a key factor (Korn et al., 2007; Liang et al., 2006; Marin et al., 2010; Torrado and Cooper, 2010) and could potentially

For some time, assays assessing the presence of Mtb specific memory CD8 and CD4 T cells have been used to identify those who previously have been infected with Mtb. Mtb-specific T cells are detectable within 2 – 3 weeks of acute infection in the peripheral blood and mark the end of the phase of rapid bacterial replication and bacterial containment. Interestingly, studies in mice suggest a late adaptive T cell response, which is linked to a delay in activation of T helper cells (Wolf et al., 2008). The measurement of Mtb specific T cells is therefore diagnostically insensitive in acute infection. This apparent problem may be overcome by the detection of Mtb-specific T cells directly at the site of infection, such as in fluid from broncho-alveolar lavage and cerebrospinal fluid, during early stages of disease in selected cohorts (Jafari et al., 2009; Thomas et al., 2008). On the other hand, the long-term maintenance of memory T cell responses to pathogens in peripheral blood (Semmo et al., 2006) make the measurements of Mtb specific T cells useful as a diagnostic cross-sectional

One of the current well established diagnostic approaches, which is based on a T cell mediated delayed hypersensitivity reaction, is the tuberculin skin test (TST). The TST is generally performed by an intradermal injection of 5 tuberculin units using purified protein derivative (PPD) following the Mantoux method. The transverse diameter of the skin induration occuring after 48-72 hours is typically measured. Therefore at least two clinic visits are required for a valid test which may prove a problem. In our recent study of Western Australian refugee children, the TST was initiated in 341 children; however reading

multiplex analyses.

this process more feasible.

**and cytokine based diagnostics** 

be explored for diagnostic purposes.

assay to test for prior exposure to Mtb.

**3.1 Tuberculin skin test and Interferon- release asssays (IGRA)** 

was not possible in 37 (11%) children due to non-attendance at clinic appointments or absence of the child at scheduled home visits (Lucas et al., 2010). The interpretation of results and determination of cut-offs also varies and is infuenced by age, previous BCG vaccination and Mtb infection risk. In addition, this method is now recognised to have other limitations, such as poor specificity due to cross reactivity with both non-tuberculous mycobacteria and BCG (National Health Service, 2005) (Table 1).

More recently, measurement of IFN-γ -producing memory T cells specific for Mtb has been introduced into the clinical practice of many countries. Two blood-based IFN-γ release assays (IGRAs) are available for diagnostic use, the Quantiferon-TB gold in-tube (QFT-GIT; Cellestis, Carnegie, Australia) and the T-SPOT.TB (Oxford Immunotech,Oxford, UK). Both assays have high specificity for adult Mtb infection including in BCG-vaccinated populations (Lucas et al., 2010). Both assays measure T cell IFN-γ production in response to antigens encoded by the RD1 gene which is present in all strains of Mtb but is not present in the Mycobacterium bovis genome from which BCG is derived. This eliminates BCG crossreactivity, however cross-reactivity to a limited number of non-Mtb mycobacteria (M. kansasii, M. szulgai, M. marinum) remains. T-SPOT.TB is an enzyme-linked immunospot (ELISpot) assay that measures the response to two antigens, early-secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10). The QFT-GIT assay is a whole blood IGRA assay that includes an additional Mtb-specific antigen (TB7.7). In the few head-to head comparisons of the latest generation tests, T-SPOT.TB and QFT-GIT both perform satisfactorily. Some studies however suggest that the use of the T-SPOT.TB assay may be the preferred option in subjects with primary and secondary (HIV, iatrogenic) immunodeficiencies (Lalvani, 2007; Pai et al., 2008b).

To date, there has been limited focus on high incidence paediatric populations, which are at greater risk of reactivation and extrapulmonary manifestations of Mtb, including TB meningitis. We recently published a prospective comparative study of IGRAs and TST for the diagnosis of LTBI in 524 refugee children from countries with a high prevalence of MTb that resettled to Australia (Lucas et al., 2010). This study included 182 children <5 years of age. In our study, the T-SPOT.TB and QFT-GIT had similar rates of positivity (8% and 10%, respectively) and showed good concordance when both tests gave definitive results (kappa= 0.78; p<0.0001). Surprisingly, both IGRAs had significant failure rates: 15% of QFT-GIT gave indeterminate results due to failed mitogen response and 14% of T-SPOT.TB results were inconclusive because of insufficient mononuclear leukocyte yields. Failure of the QFT-GIT mitogen response was associated with African ethnicity and comorbid infections, particularly with helminths. Overall, the TST results showed low concordance (about 50%) with both IGRAs. This study highlights the influence of age, ethnicity and clinical status on IGRA results and the limitations of using these T cell based tests in refugee children.

In general, IGRAs are likely to be too complex and expensive for point-of-care testing in the Developing World, but their use in developed countries where the Mtb burden is much smaller, is currently justified. In addition, they have a role in the identification of subjects with latent Mtb prior to immunosuppressive therapy, including treatment with novel biologic drugs such as TNF- antagonists (Bellofiore et al., 2009).

New Diagnostics for *Mycobacterium tuberculosis* 249

currently available commercial serological tests exhibit low specificity and sensitivity leading to misdiagnosis, mistreatment and potential harm to public health (http://www.who.int/mediacentre/news/releases/2011/tb\_20110720n/index.html).

The role of a protective antibody response in Mtb has been less investigated. Approximately 90% of patients produce antibodies to Mtb proteins with antibody profiles showing great inter-individual variation. So far, a clear correlation between antibody profiles and disease status has not been clearly established, making its use for routine diagnostics problematic (Lyashchenko et al., 1998; Wu et al., 2010). Recently, a major contribution to the field has been published by Kunnath-Velayudhan and colleagues (Kunnath-Velayudhan et al., 2010). They screened 500 sera from suspected Mtb infected subjects against the entire Mtb proteome using high-throughput microarray technology and identified signatures of antibody responses in subjects with active Mtb, with some variation across subjects, thus providing novel insights into the biology of the humoral response against Mtb and

providing further steps to develop effective humoral immunodiagnostics.

**4. Diagnosis of reactivation disease or low level pathogen detection** 

Upon inhalation of Mtb, mycobacteria are phagocytosed by alveolar macrophages that recruit mononuclear cells to the site of infection. This leads to the characteristic granuloma formation consisting of macrophages, monocytes and neutrophils. At later stages, the granuloma becomes more organised and infiltrated by lymphocytes (Russell et al., 2010). For the majority of cases Mtb is contained by the immune system. How the transition between the detection and control of acute infection to the establishment of latent Mtb infection is mediated, remains unknown. A critical factor, however, is the pathogen's ability to evade its complete elimination by the immune system. Studies have shown that even asymptomatic infected hosts harbour virulent bacteria in their tuberculous granulomas (Bouley et al., 2001; Tufariello et al., 2003). It is even possible that the granulomas paradoxically offer a niche for long-term survival of the bacteria. The barrier of activated macrophages and giant multinucleated cells that surround Mtb infected cells within granulomas are relatively impermeable to T cell infiltration although T cells remain closely associated at the site of infection (Tufariello et al., 2003). Thus paradoxically the combined efforts of the innate and adaptive immune response often contain and control the infection but fail to eliminate it. In fact the presence of Mtb antigens released into the host are thought to maintain the presence of an Mtb specific effector-memory population, which is absent in cases who have successfully eliminated the pathogen and only show evidence of a Mtb-specific central memory T cells (Millington et al., 2010). Ongoing adaptive immunity is essential in preventing reactivation of infection at a later stage (Boom et al., 2003). Reactivation disease occurs when latent bacteria from old, scarred granulomatous lesions are reactivated into an active, virulent state. A increased risk of reactivation arises when the host's immune system is compromised, which may be secondarily due to immune suppressive drugs, especially those that modify Mtb-specific immunity such as TNF antagonists, due to cancer, malnutrition or chronic viral infections, such as HIV. HIV+ individuals with advanced disease and low CD4+ T cell count face an approximate 10% risk per year of Mtb reactivation and co-infection of Mtb with HIV is now well documented (Shen et al., 2004). In the developing world therefore, Mtb has become one of the leading causes of death (Tufariello et al., 2003). This also highlights the problem that those with the greatest risk of reactivation are often cared for by those that are most vulnerable for Mtb infection and re-

#### **3.2 Assessment of the role of other cytokines in Mtb infection**

As the cooperation between macrophages and T lymphocytes is critical for acute and longterm control of Mtb, cytokines produced during their interaction, such as IL-1, IL-2, IFN-γ, TNF-, IL-12 and IL-23 are thought to play a critical role, all of which have potential to be used as surrogate markers of anti-Mtb immunity (Doherty et al., 2009). In addition, there is increasing evidence that during acute Mtb infection many of these pro-inflammatory cytokines are counteracted by induction of immune suppressive regulators (such as IL-10, TGF- RII, IL1-R and IDO) in addition to upregulation of intracellular molecules, e.g. IRAK-M and suppressor of cytokine signalling (SOCS) (Almeida et al., 2009). Mtb-activated CD4+ T cells also release TNF- which can trigger cell lysis in infected macrophages and may kill intracellular Mtb (Canaday et al., 2001) and Mtb specific TNF- secreting CD4+ T cells have been shown to be more frequent during active than latent disease (Harari et al., 2011). In addition, IL- 18 from macrophages and DCs has recently been explored for its protective immunity against tuberculosis (Schneider et al., 2010). Newer diagnostic approaches that allow the measurement of mulitple cytokine simultaneously may aid in the diagnosis of latent infection, but also are promising tools for the identification of acute infection/reactivation/infection of Mtb.

#### **3.3 Antibody responses to Mtb**

Serological blood tests detect the host's humoral response (antibodies) to a pathogen that can remain circulating in the blood for several years. However, as with any pathogen specific immune response (antibody or T cell) this response develops after initial infection, and therefore its use at early infection timepoints is limited. Overall, these tests can be quick and inexpensive to perform using either ELISA or immunochromatographic formats. In the case of Mtb infection, serological assays would also be more practical for children, for whom sputum samples are difficult to obtain.

It is estimated that more than a million Mtb serological tests are performed each year, predominantly in high disease burden countries (Steingart et al., 2011). However, many of these assays vary in the antigens used, source and type of antigen and the class of immunoglobulin investigated. First generation antibody assays used crude mixtures of Mtb that tended to give low specificity results; most likely due to shared antigens between mycobacteria species. A review to assess the efficacy of "in-house" serological assays showed that assays with a combination of antigens gave higher sensitivity and specificity than earlier assays (Steingart et al., 2009). Furthermore, assays detecting IgG and IgA anti-Mtb antibodies gave higher sensitivity values for the detection of pulmonary Mtb than IgMbased assays. IgM-based assays may be better suited to the detection of acute Mtb infection as IgM is typically expressed early in the infection but then dissipates over time.

A recent systematic review commissioned by the WHO clearly showed that commercial serological assays for active Mtb infection exhibited substantial variation in sensitivity and specificity for pulmonary Mtb infection ((Steingart et al., 2011); Table 1). The review was based on 67 studies with 5,147 participants including 48% from low-middle income countries. Accordingly, on the 20th July 2011, WHO released a press statement warning against the use of serological tests for the diagnosis of active Mtb infection, stating that the

As the cooperation between macrophages and T lymphocytes is critical for acute and longterm control of Mtb, cytokines produced during their interaction, such as IL-1, IL-2, IFN-γ, TNF-, IL-12 and IL-23 are thought to play a critical role, all of which have potential to be used as surrogate markers of anti-Mtb immunity (Doherty et al., 2009). In addition, there is increasing evidence that during acute Mtb infection many of these pro-inflammatory cytokines are counteracted by induction of immune suppressive regulators (such as IL-10, TGF- RII, IL1-R and IDO) in addition to upregulation of intracellular molecules, e.g. IRAK-M and suppressor of cytokine signalling (SOCS) (Almeida et al., 2009). Mtb-activated CD4+ T cells also release TNF- which can trigger cell lysis in infected macrophages and may kill intracellular Mtb (Canaday et al., 2001) and Mtb specific TNF- secreting CD4+ T cells have been shown to be more frequent during active than latent disease (Harari et al., 2011). In addition, IL- 18 from macrophages and DCs has recently been explored for its protective immunity against tuberculosis (Schneider et al., 2010). Newer diagnostic approaches that allow the measurement of mulitple cytokine simultaneously may aid in the diagnosis of latent infection, but also are promising tools for the identification of acute infection/re-

Serological blood tests detect the host's humoral response (antibodies) to a pathogen that can remain circulating in the blood for several years. However, as with any pathogen specific immune response (antibody or T cell) this response develops after initial infection, and therefore its use at early infection timepoints is limited. Overall, these tests can be quick and inexpensive to perform using either ELISA or immunochromatographic formats. In the case of Mtb infection, serological assays would also be more practical for children, for whom

It is estimated that more than a million Mtb serological tests are performed each year, predominantly in high disease burden countries (Steingart et al., 2011). However, many of these assays vary in the antigens used, source and type of antigen and the class of immunoglobulin investigated. First generation antibody assays used crude mixtures of Mtb that tended to give low specificity results; most likely due to shared antigens between mycobacteria species. A review to assess the efficacy of "in-house" serological assays showed that assays with a combination of antigens gave higher sensitivity and specificity than earlier assays (Steingart et al., 2009). Furthermore, assays detecting IgG and IgA anti-Mtb antibodies gave higher sensitivity values for the detection of pulmonary Mtb than IgMbased assays. IgM-based assays may be better suited to the detection of acute Mtb infection

A recent systematic review commissioned by the WHO clearly showed that commercial serological assays for active Mtb infection exhibited substantial variation in sensitivity and specificity for pulmonary Mtb infection ((Steingart et al., 2011); Table 1). The review was based on 67 studies with 5,147 participants including 48% from low-middle income countries. Accordingly, on the 20th July 2011, WHO released a press statement warning against the use of serological tests for the diagnosis of active Mtb infection, stating that the

as IgM is typically expressed early in the infection but then dissipates over time.

**3.2 Assessment of the role of other cytokines in Mtb infection** 

activation/infection of Mtb.

**3.3 Antibody responses to Mtb** 

sputum samples are difficult to obtain.

currently available commercial serological tests exhibit low specificity and sensitivity leading to misdiagnosis, mistreatment and potential harm to public health (http://www.who.int/mediacentre/news/releases/2011/tb\_20110720n/index.html).

The role of a protective antibody response in Mtb has been less investigated. Approximately 90% of patients produce antibodies to Mtb proteins with antibody profiles showing great inter-individual variation. So far, a clear correlation between antibody profiles and disease status has not been clearly established, making its use for routine diagnostics problematic (Lyashchenko et al., 1998; Wu et al., 2010). Recently, a major contribution to the field has been published by Kunnath-Velayudhan and colleagues (Kunnath-Velayudhan et al., 2010). They screened 500 sera from suspected Mtb infected subjects against the entire Mtb proteome using high-throughput microarray technology and identified signatures of antibody responses in subjects with active Mtb, with some variation across subjects, thus providing novel insights into the biology of the humoral response against Mtb and providing further steps to develop effective humoral immunodiagnostics.

#### **4. Diagnosis of reactivation disease or low level pathogen detection**

Upon inhalation of Mtb, mycobacteria are phagocytosed by alveolar macrophages that recruit mononuclear cells to the site of infection. This leads to the characteristic granuloma formation consisting of macrophages, monocytes and neutrophils. At later stages, the granuloma becomes more organised and infiltrated by lymphocytes (Russell et al., 2010). For the majority of cases Mtb is contained by the immune system. How the transition between the detection and control of acute infection to the establishment of latent Mtb infection is mediated, remains unknown. A critical factor, however, is the pathogen's ability to evade its complete elimination by the immune system. Studies have shown that even asymptomatic infected hosts harbour virulent bacteria in their tuberculous granulomas (Bouley et al., 2001; Tufariello et al., 2003). It is even possible that the granulomas paradoxically offer a niche for long-term survival of the bacteria. The barrier of activated macrophages and giant multinucleated cells that surround Mtb infected cells within granulomas are relatively impermeable to T cell infiltration although T cells remain closely associated at the site of infection (Tufariello et al., 2003). Thus paradoxically the combined efforts of the innate and adaptive immune response often contain and control the infection but fail to eliminate it. In fact the presence of Mtb antigens released into the host are thought to maintain the presence of an Mtb specific effector-memory population, which is absent in cases who have successfully eliminated the pathogen and only show evidence of a Mtb-specific central memory T cells (Millington et al., 2010). Ongoing adaptive immunity is essential in preventing reactivation of infection at a later stage (Boom et al., 2003). Reactivation disease occurs when latent bacteria from old, scarred granulomatous lesions are reactivated into an active, virulent state. A increased risk of reactivation arises when the host's immune system is compromised, which may be secondarily due to immune suppressive drugs, especially those that modify Mtb-specific immunity such as TNF antagonists, due to cancer, malnutrition or chronic viral infections, such as HIV. HIV+ individuals with advanced disease and low CD4+ T cell count face an approximate 10% risk per year of Mtb reactivation and co-infection of Mtb with HIV is now well documented (Shen et al., 2004). In the developing world therefore, Mtb has become one of the leading causes of death (Tufariello et al., 2003). This also highlights the problem that those with the greatest risk of reactivation are often cared for by those that are most vulnerable for Mtb infection and re-

New Diagnostics for *Mycobacterium tuberculosis* 251

cytokines in lung epithelia cells ((Liu et al., 2009); reviewed in (Vannberg et al., 2011)). Although the role of this gene in Mtb susceptibility has not been clarified, it certainly

But it is important to remember that Mtb is also a variable pathogen and it is likely that variation in the genetics of the host and pathogen are likely to be relevant in determining

Microbiological detections systems are likely to remain the gold standard for detection and identification of extracellular pathogens aided by the increasing complementary usage of PCR to shorten the time and improve the accuracy of identification. Future developments may also include screening of other less invasive sample types including volatile samples from the breath, for the presence of characteristic pathogen associated molecules or metabolic bi-products (Phillips et al., 2007). Sensitive physical detection technologies like those based on mass spectrophotometry (Metzger et al., 2010) may also make their way into

Newly described cytokine networks such as those associated with inflammasome complexes (IL-1β, IL-18, IL-33) (Church et al., 2008), the IL-23/IL-17 inflammatory pathway (Kikly et al., 2006) are being studied at a basic research level and are likely to contribute additional insights into anti-pathogen immunity. The continued development of better assays for established biomarkers (e.g. monokine assay; Chakera et al., 2011) to improve sensitivity and specificity and the speed in which results are obtained or give additional insights into

The complexities that underlie effective pathogen specific immunity is still incompletely understood and will continue to be informed by data generated using molecular approaches that measure changes in pathways that influence the response to infection. Advances in the CHIP technology (Weinmann et al., 2002), which involves the selection of specific DNA binding proteins by antibodies, will add additional dimension to the data being obtained and show which genetic levers are being pulled during a response to a given pathogen. Furthermore, this technology is complemented by Next-generation sequencing that will allow the detection of low frequency viral and host variants as well as transcripts. It is hoped that insights generated by such approaches, along with further iterations of these technologies will result in more sophisticated approaches and tools to be developed,

Almeida, A. S., P. M. Lago, et al. (2009). "Tuberculosis is associated with a down-modulatory

Azzurri, A., O. Y. Sow, et al. (2005). "IFN-gamma-inducible protein 10 and pentraxin 3

Baylan, O., A. Balkan, et al. (2006). "The predictive value of serum procalcitonin levels in adult patients with active pulmonary tuberculosis." Jpn J Infect Dis 59(3): 164-7.

Mycobacterium tuberculosis infection." Microbes Infect 7(1): 1-8.

lung immune response that impairs Th1-type immunity." J Immunol 183(1): 718-31.

plasma levels are tools for monitoring inflammation and disease activity in

remains a gene of interest.

larger clinical laboratories to assist in sample analysis.

the immune responses to the pathogen will be central to these advances.

affordable to the countries burdened with the highest levels of Mtb.

infection outcome.

**6. Conclusion** 

**7. References** 

infection. Not surprisingly, this vicious cycle of reactivation/infection in developing countries has led to a flourishing Mtb spread and extensive resistance to most anti-Mtb drugs.

To-date no reliable routine diagnostics that allow pathogen recognition at low levels or immune markers that predict reactivation are available. In general, the principles underlying the diagnosis of reactivation disease will be similar to those used for the detection of early acute infection; namely pathogen based assays and assays which rely on immune markers associated with the innate immune system (see above). In addition, there are reports which suggest that a change in T cell based immunity against Mtb exists in acute and latent infection.

Casey et al., for example, expands the current technology of the T-SPOT.TB assay by measuring IFN-γ and IL-2 responses by Mtb specific T cells after stimulation with ESAT 6 and CFP 10 antigens, thus aiming to test T cells for polyfunctionality in active, treated and latent Mtb (Casey et al., 2010). The authors use a dual fluorescent ELISpot to measure two cytokines simultaneously, which could be adapted to routine –albeit expensive- clinical practice. They demonstrated that active untreated Mtb, compared to latent Mtb infection, was dominated by IFN-γ-only producing effector T cells. In addition, sequential testing of successfully treated patients revealed a shift from IFN-γ only producing T cells to a higher number of effector-memory cells secreting IL-2 and IFN-γ which confirms previous data, using a flowcytometric approach, by Caccamo et al (Caccamo et al., 2010).

#### **5. Use of genetic studies to identify novel genes involved in Mtb pathogenesis**

Recent technological advances has allowed the large-scale sampling of the human genome in a cost-effective manner and now allows researchers to examine, without a priori knowledge, genetic variations that may influence Mtb infection outcome. Furthermore, the deposit of whole genome sequences from individuals from different ethnic backgrounds into public databases allows a more comprehensive view of human genetic variation that better reflects the individuals at most risk of Mtb infection.

Genome-wide linkage studies to identify the transfer of chromosomal regions containing susceptibility genes, typically using families of affected individuals, have been performed on Mtb (reviewed in (Moller et al., 2009)). However, of the studies to date, none of the linkage peaks reach genome-wide significance. The majority of these studies targeted different populations and not surprisingly there is no obvious overlap between highlighted chromosome regions for each study.

The only reported genome-wide association study to date for Mtb involves the African TB Genetics consortium and the Wellcome Trust Case Control Consortium (Thye et al., 2010). In this study, individuals with tuberculosis and unaffected controls from the West African nations Ghana and The Gambia were genotyped for single nucleotide polymorphisms (SNPs) covering the entire genome. The initial study identified 17 loci associated with disease with a p<10-5. However, subsequent replication studies found that the SNP rs4331426 had the highest association signal (OR 1.19; total number of individuals 11,425). Interestingly, this SNP is located within a gene-poor region of chromosome 18 (18q11.2) but does contain the gene GATA6, which encodes a transcription factor known to regulate arachidonate 15-lipoxygenase (ALOX15); a molecule involved in regulating the release of cytokines in lung epithelia cells ((Liu et al., 2009); reviewed in (Vannberg et al., 2011)). Although the role of this gene in Mtb susceptibility has not been clarified, it certainly remains a gene of interest.

But it is important to remember that Mtb is also a variable pathogen and it is likely that variation in the genetics of the host and pathogen are likely to be relevant in determining infection outcome.

#### **6. Conclusion**

250 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

infection. Not surprisingly, this vicious cycle of reactivation/infection in developing countries

To-date no reliable routine diagnostics that allow pathogen recognition at low levels or immune markers that predict reactivation are available. In general, the principles underlying the diagnosis of reactivation disease will be similar to those used for the detection of early acute infection; namely pathogen based assays and assays which rely on immune markers associated with the innate immune system (see above). In addition, there are reports which suggest that a change in T cell based immunity against Mtb exists in acute

Casey et al., for example, expands the current technology of the T-SPOT.TB assay by measuring IFN-γ and IL-2 responses by Mtb specific T cells after stimulation with ESAT 6 and CFP 10 antigens, thus aiming to test T cells for polyfunctionality in active, treated and latent Mtb (Casey et al., 2010). The authors use a dual fluorescent ELISpot to measure two cytokines simultaneously, which could be adapted to routine –albeit expensive- clinical practice. They demonstrated that active untreated Mtb, compared to latent Mtb infection, was dominated by IFN-γ-only producing effector T cells. In addition, sequential testing of successfully treated patients revealed a shift from IFN-γ only producing T cells to a higher number of effector-memory cells secreting IL-2 and IFN-γ which confirms previous data,

Recent technological advances has allowed the large-scale sampling of the human genome in a cost-effective manner and now allows researchers to examine, without a priori knowledge, genetic variations that may influence Mtb infection outcome. Furthermore, the deposit of whole genome sequences from individuals from different ethnic backgrounds into public databases allows a more comprehensive view of human genetic variation that

Genome-wide linkage studies to identify the transfer of chromosomal regions containing susceptibility genes, typically using families of affected individuals, have been performed on Mtb (reviewed in (Moller et al., 2009)). However, of the studies to date, none of the linkage peaks reach genome-wide significance. The majority of these studies targeted different populations and not surprisingly there is no obvious overlap between highlighted

The only reported genome-wide association study to date for Mtb involves the African TB Genetics consortium and the Wellcome Trust Case Control Consortium (Thye et al., 2010). In this study, individuals with tuberculosis and unaffected controls from the West African nations Ghana and The Gambia were genotyped for single nucleotide polymorphisms (SNPs) covering the entire genome. The initial study identified 17 loci associated with disease with a p<10-5. However, subsequent replication studies found that the SNP rs4331426 had the highest association signal (OR 1.19; total number of individuals 11,425). Interestingly, this SNP is located within a gene-poor region of chromosome 18 (18q11.2) but does contain the gene GATA6, which encodes a transcription factor known to regulate arachidonate 15-lipoxygenase (ALOX15); a molecule involved in regulating the release of

has led to a flourishing Mtb spread and extensive resistance to most anti-Mtb drugs.

using a flowcytometric approach, by Caccamo et al (Caccamo et al., 2010).

**5. Use of genetic studies to identify novel genes involved in** 

better reflects the individuals at most risk of Mtb infection.

and latent infection.

**Mtb pathogenesis** 

chromosome regions for each study.

Microbiological detections systems are likely to remain the gold standard for detection and identification of extracellular pathogens aided by the increasing complementary usage of PCR to shorten the time and improve the accuracy of identification. Future developments may also include screening of other less invasive sample types including volatile samples from the breath, for the presence of characteristic pathogen associated molecules or metabolic bi-products (Phillips et al., 2007). Sensitive physical detection technologies like those based on mass spectrophotometry (Metzger et al., 2010) may also make their way into larger clinical laboratories to assist in sample analysis.

Newly described cytokine networks such as those associated with inflammasome complexes (IL-1β, IL-18, IL-33) (Church et al., 2008), the IL-23/IL-17 inflammatory pathway (Kikly et al., 2006) are being studied at a basic research level and are likely to contribute additional insights into anti-pathogen immunity. The continued development of better assays for established biomarkers (e.g. monokine assay; Chakera et al., 2011) to improve sensitivity and specificity and the speed in which results are obtained or give additional insights into the immune responses to the pathogen will be central to these advances.

The complexities that underlie effective pathogen specific immunity is still incompletely understood and will continue to be informed by data generated using molecular approaches that measure changes in pathways that influence the response to infection. Advances in the CHIP technology (Weinmann et al., 2002), which involves the selection of specific DNA binding proteins by antibodies, will add additional dimension to the data being obtained and show which genetic levers are being pulled during a response to a given pathogen. Furthermore, this technology is complemented by Next-generation sequencing that will allow the detection of low frequency viral and host variants as well as transcripts. It is hoped that insights generated by such approaches, along with further iterations of these technologies will result in more sophisticated approaches and tools to be developed, affordable to the countries burdened with the highest levels of Mtb.

#### **7. References**


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**12** 

*Portugal* 

*Universidade Nova de Lisboa* 

**Nanodiagnostics for Tuberculosis** 

Bruno Veigas, Gonçalo Doria and Pedro V. Baptista\*

*CIGMH, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia,* 

Tuberculosis (TB) remains one of the most serious infectious diseases in the world requiring new and more effective diagnostics and treatments (World Health Organization [WHO], 2010). Several approaches have been developed to improve TB diagnostics, reducing the time from weeks to a few days that still require demanding expertise technical personal for labor intensive and expensive methods, which hamper application in resource-poor countries where the main TB epidemic is observed. Nanotechnology has triggered the development of new and cheaper approaches for biomolecular recognition that may circumvent the current limitations of conventional molecular diagnostic methods used in the global fight against TB. This new era of molecular nanodiagnostics may provide a rapid and sensitive detection of the main TB etiologic agent in humans, i.e. *Mycobacterium* 

Nanodiagnostics can be defined as the use of nano-sized materials, devices or systems for diagnostics purposes. Biological tests measuring the presence or activity of selected analytes become quicker, more sensitive and more flexible when nanoscale particles are put to work as tags or labels, with numerous advantages over more traditional procedures, for example fluorescence and chemiluminescence technology. Here we will provide a closer look into nanodiagnostics systems developed for TB diagnostics and/or *M. tuberculosis* detection and characterization, such as nanoparticle-based systems (e.g. gold, silver, silica and quantum dots) and nanocantilevers. These techniques are already showing to be more sensitive and specific than conventional commercial molecular diagnostics methodologies although many aspects of nanodiagnostics for TB still need further evaluation and validation. Current advances in nanofabrication may enable the construction of cheap and full-automated devices, extending the limits of current molecular diagnostics and enable point- of-care

Tuberculosis is caused by *M. tuberculosis*, a member of the *Mycobacterium tuberculosis* complex (MTBC) and, according to the most current statistics of the World Health

**1. Introduction** 

*tuberculosis.*

diagnostics.

**2. Tuberculosis** 

\* Corresponding Author


## **Nanodiagnostics for Tuberculosis**

Bruno Veigas, Gonçalo Doria and Pedro V. Baptista\*

*CIGMH, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa Portugal* 

#### **1. Introduction**

256 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

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susceptibility locus for tuberculosis on chromosome 18q11.2." Nat Genet 42(9): 739-

Tuberculosis (TB) remains one of the most serious infectious diseases in the world requiring new and more effective diagnostics and treatments (World Health Organization [WHO], 2010). Several approaches have been developed to improve TB diagnostics, reducing the time from weeks to a few days that still require demanding expertise technical personal for labor intensive and expensive methods, which hamper application in resource-poor countries where the main TB epidemic is observed. Nanotechnology has triggered the development of new and cheaper approaches for biomolecular recognition that may circumvent the current limitations of conventional molecular diagnostic methods used in the global fight against TB. This new era of molecular nanodiagnostics may provide a rapid and sensitive detection of the main TB etiologic agent in humans, i.e. *Mycobacterium tuberculosis.*

Nanodiagnostics can be defined as the use of nano-sized materials, devices or systems for diagnostics purposes. Biological tests measuring the presence or activity of selected analytes become quicker, more sensitive and more flexible when nanoscale particles are put to work as tags or labels, with numerous advantages over more traditional procedures, for example fluorescence and chemiluminescence technology. Here we will provide a closer look into nanodiagnostics systems developed for TB diagnostics and/or *M. tuberculosis* detection and characterization, such as nanoparticle-based systems (e.g. gold, silver, silica and quantum dots) and nanocantilevers. These techniques are already showing to be more sensitive and specific than conventional commercial molecular diagnostics methodologies although many aspects of nanodiagnostics for TB still need further evaluation and validation. Current advances in nanofabrication may enable the construction of cheap and full-automated devices, extending the limits of current molecular diagnostics and enable point- of-care diagnostics.

#### **2. Tuberculosis**

Tuberculosis is caused by *M. tuberculosis*, a member of the *Mycobacterium tuberculosis* complex (MTBC) and, according to the most current statistics of the World Health

<sup>\*</sup> Corresponding Author

Nanodiagnostics for Tuberculosis 259

Several new technologies are under development, which will enable the presumptive diagnosis of MDR-TB in just one to two days, compared with two or more months when using conventional culture and drug susceptibility tests. Rapid diagnosis of MDR-TB will have several benefits: earlier treatment of patients, reduction of time spent on inappropriate and ineffective treatment (thereby promoting the development of further drug resistance),

Nanotechnology introduced new paradigms for molecular diagnostics – nanodiagnostics, where the increased sensitivity, specificity, speed and reduced cost constitutes an appealing alternative to conventional techniques (Azzazy et al., 2006). Because we are dealing with nanometer sized objects and/or nanometer scale, bulk matter physics does not apply and amazing and extraordinary new properties arise. This interesting field relies on the knowledge and technological developments emerging from transdisciplinary research efforts that bring together a plethora of expertise from areas as diverse as Materials Science, Physics, Biology and Biotechnology, Chemistry and Medicine. From the intersection of these complementarities new and radical approaches can be explored towards application in platforms for biomolecular recognition (e.g. nucleic acids, antibodies, proteins, etc.) that can be miniaturized for point of care utilization and/or for enhanced portability for small laboratory settings in remote areas without the standard access to conventional laboratory equipment and apparatus. Nanodiagnostics have redefined the standards for molecular diagnostics, triggering the development of new approaches in biomolecular recognition and analytical systems, where the most promising approaches include nanoparticles (NPs), nanotubes, nanopores and nanocantilever technologies (Baptista et al., 2008; Branton et al., 2008; Jain, 2007; Rosi & Mirkin, 2005; Wang et al., 2009). Their potential arises from recognition events occurring at one-to-one interactions between analytes and signalgenerating nanostructures, allowing for an increased sensitivity and specificity at lower

Different nanodiagnostics systems have been developed for the molecular diagnostics of TB. Despite the wide range of nanoscale systems being used for biomolecular assays in general (e.g. electromechanical, electrochemical) (Azzazy et al., 2006; Das et al., 2010; Jain, 2007), nanoparticle based systems, such as gold, silver, silica and quantum dots (QDs), have been the most widely used for TB diagnostics due to their unique physicochemical properties, that offer greater sensitivity than conventional reporter molecules and can be easily tuned and functionalized by simple chemistry modulation and derivatization (Azzazy, 2009). Table 1 summarizes existing nanotechnology based systems applied to TB diagnostics.

Nanoparticles are typically in the size range of 1–100 nm and can have different shapes and compositions (Liu, 2006). They are structurally robust and have very specific size dependent properties that differ considerably from those observed on microparticles or bulk materials. Depending on their size and composition they exhibit peculiar properties, such as quantum confinement in semiconductor nanocrystals, surface plasmon resonance (SPR) in some metal NPs and superparamagnetism in magnetic materials (Vollath, 2008). They also provide large

and reduction of MDR-TB spreading in congregate settings.

**3. Nanodiagnostics for TB** 

costs.

**3.1 Nanoparticles** 

Organization, remains one of the most serious infectious diseases in the world, being responsible for 1.7 million deaths and 9.4 million new cases in 2009 alone (WHO, 2010). The emergence of multidrug-resistant TB also represents a serious threat to the TB control and an increasing public health problem (Deun et al., 2010), leading to a global need for rapid drug susceptibility testing. Single nucleotide sequence variations (mutations and/or polymorphisms) within *M. tuberculosis* genome have been associated with antibiotic resistance for all first-line drugs (isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin), and for several second-line and newer drugs (ethionamide, fluoroquinolones, macrolides, and nitroimidazopyrans), making these sequences ideal targets for the development of molecular drug susceptibility testing (Abebe et al., 2011; Miller et al., 1994; Musser, 1995; Soini & Musser, 2001; Telenti et al., 1993).

The mainstay for TB diagnostics in endemic developing countries is sputum smear microscopy (Perkins, 2009). However, the sensitivity of this technology is low as it can only detect roughly half of all active cases of tuberculosis when properly used – in people with co-infections and in children the sensitivity is even lower. Moreover, though routinely described as a simple technology, microscopy is actually complex, and highly dependent on the training and diligence of the technician, requiring multiple examinations which may take weeks to complete, with the consequence that many patients drop out during the diagnostic process. Several diagnostic approaches have brought incremental improvements for the direct detection, species identification and drug susceptibility testing of mycobacteria that are capable of reducing the laboratorial time from weeks to a few days (Barnard et al., 2008; D'Amato et al., 1995; De Beenhouwer et al., 1995; Griffith et al., 2007; Hillemann et al., 2005; Hirano et al., 1999; Moore et al., 2006; Park et al., 2002; Rossau et al., 1997; Sharma et al., 2003; Traore et al., 2000). Most of these approaches for direct detection of TB and drug susceptibility from clinical specimens, including several commercial tests, rely on complex and expensive DNA amplification based procedures (e.g. PCR), whereas the need is for affordable, simple and high-throughput systems with the possibility to use small amounts of sample (Cheng et al., 2005; Das et al., 2010; Shamputa et al., 2004; Watterson et al., 1998). These molecular recognition assays still need to make the way to widespread utilization in some technological advanced countries, which will definitely delay the required validation and setup for simplified platforms for general use at more remote and less equipped areas.

Some of the diagnostic tools expected to be introduced into control programs will be incremental improvements on existing technologies while others will be radically new. The speed and extent of adoption of new technologies will depend on the balance between the benefits they bring and the degree of disruption their implementation causes. For instance, a simplified microscopy method may see greater adoption than a novel alternative that necessitates changes in the way testing or case notification are carried out. On the other hand, a new method that rapidly identifies all smear-positive and many smear-negative cases might, if suitably robust and specific, see widespread use and could substantially replace microscopy. Point-of-care diagnosis is instrumental to TB control because, despite having the necessary treatment, strategies in some regions are rather ineffective (see South Africa as an example). Identifying new cases very quickly and getting patients immediately on to treatment are crucial in addressing this pandemic. New diagnostic tools for drug resistant TB (TB that is resistant to drugs – multi-drug resistant TB, MDR-TB) are urgently needed for reducing diagnostic time from months to days.

Organization, remains one of the most serious infectious diseases in the world, being responsible for 1.7 million deaths and 9.4 million new cases in 2009 alone (WHO, 2010). The emergence of multidrug-resistant TB also represents a serious threat to the TB control and an increasing public health problem (Deun et al., 2010), leading to a global need for rapid drug susceptibility testing. Single nucleotide sequence variations (mutations and/or polymorphisms) within *M. tuberculosis* genome have been associated with antibiotic resistance for all first-line drugs (isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin), and for several second-line and newer drugs (ethionamide, fluoroquinolones, macrolides, and nitroimidazopyrans), making these sequences ideal targets for the development of molecular drug susceptibility testing (Abebe et al., 2011; Miller et al., 1994;

The mainstay for TB diagnostics in endemic developing countries is sputum smear microscopy (Perkins, 2009). However, the sensitivity of this technology is low as it can only detect roughly half of all active cases of tuberculosis when properly used – in people with co-infections and in children the sensitivity is even lower. Moreover, though routinely described as a simple technology, microscopy is actually complex, and highly dependent on the training and diligence of the technician, requiring multiple examinations which may take weeks to complete, with the consequence that many patients drop out during the diagnostic process. Several diagnostic approaches have brought incremental improvements for the direct detection, species identification and drug susceptibility testing of mycobacteria that are capable of reducing the laboratorial time from weeks to a few days (Barnard et al., 2008; D'Amato et al., 1995; De Beenhouwer et al., 1995; Griffith et al., 2007; Hillemann et al., 2005; Hirano et al., 1999; Moore et al., 2006; Park et al., 2002; Rossau et al., 1997; Sharma et al., 2003; Traore et al., 2000). Most of these approaches for direct detection of TB and drug susceptibility from clinical specimens, including several commercial tests, rely on complex and expensive DNA amplification based procedures (e.g. PCR), whereas the need is for affordable, simple and high-throughput systems with the possibility to use small amounts of sample (Cheng et al., 2005; Das et al., 2010; Shamputa et al., 2004; Watterson et al., 1998). These molecular recognition assays still need to make the way to widespread utilization in some technological advanced countries, which will definitely delay the required validation and setup for simplified platforms for general use at more remote and less equipped areas. Some of the diagnostic tools expected to be introduced into control programs will be incremental improvements on existing technologies while others will be radically new. The speed and extent of adoption of new technologies will depend on the balance between the benefits they bring and the degree of disruption their implementation causes. For instance, a simplified microscopy method may see greater adoption than a novel alternative that necessitates changes in the way testing or case notification are carried out. On the other hand, a new method that rapidly identifies all smear-positive and many smear-negative cases might, if suitably robust and specific, see widespread use and could substantially replace microscopy. Point-of-care diagnosis is instrumental to TB control because, despite having the necessary treatment, strategies in some regions are rather ineffective (see South Africa as an example). Identifying new cases very quickly and getting patients immediately on to treatment are crucial in addressing this pandemic. New diagnostic tools for drug resistant TB (TB that is resistant to drugs – multi-drug resistant TB, MDR-TB) are urgently

Musser, 1995; Soini & Musser, 2001; Telenti et al., 1993).

needed for reducing diagnostic time from months to days.

Several new technologies are under development, which will enable the presumptive diagnosis of MDR-TB in just one to two days, compared with two or more months when using conventional culture and drug susceptibility tests. Rapid diagnosis of MDR-TB will have several benefits: earlier treatment of patients, reduction of time spent on inappropriate and ineffective treatment (thereby promoting the development of further drug resistance), and reduction of MDR-TB spreading in congregate settings.

### **3. Nanodiagnostics for TB**

Nanotechnology introduced new paradigms for molecular diagnostics – nanodiagnostics, where the increased sensitivity, specificity, speed and reduced cost constitutes an appealing alternative to conventional techniques (Azzazy et al., 2006). Because we are dealing with nanometer sized objects and/or nanometer scale, bulk matter physics does not apply and amazing and extraordinary new properties arise. This interesting field relies on the knowledge and technological developments emerging from transdisciplinary research efforts that bring together a plethora of expertise from areas as diverse as Materials Science, Physics, Biology and Biotechnology, Chemistry and Medicine. From the intersection of these complementarities new and radical approaches can be explored towards application in platforms for biomolecular recognition (e.g. nucleic acids, antibodies, proteins, etc.) that can be miniaturized for point of care utilization and/or for enhanced portability for small laboratory settings in remote areas without the standard access to conventional laboratory equipment and apparatus. Nanodiagnostics have redefined the standards for molecular diagnostics, triggering the development of new approaches in biomolecular recognition and analytical systems, where the most promising approaches include nanoparticles (NPs), nanotubes, nanopores and nanocantilever technologies (Baptista et al., 2008; Branton et al., 2008; Jain, 2007; Rosi & Mirkin, 2005; Wang et al., 2009). Their potential arises from recognition events occurring at one-to-one interactions between analytes and signalgenerating nanostructures, allowing for an increased sensitivity and specificity at lower costs.

Different nanodiagnostics systems have been developed for the molecular diagnostics of TB. Despite the wide range of nanoscale systems being used for biomolecular assays in general (e.g. electromechanical, electrochemical) (Azzazy et al., 2006; Das et al., 2010; Jain, 2007), nanoparticle based systems, such as gold, silver, silica and quantum dots (QDs), have been the most widely used for TB diagnostics due to their unique physicochemical properties, that offer greater sensitivity than conventional reporter molecules and can be easily tuned and functionalized by simple chemistry modulation and derivatization (Azzazy, 2009). Table 1 summarizes existing nanotechnology based systems applied to TB diagnostics.

#### **3.1 Nanoparticles**

Nanoparticles are typically in the size range of 1–100 nm and can have different shapes and compositions (Liu, 2006). They are structurally robust and have very specific size dependent properties that differ considerably from those observed on microparticles or bulk materials. Depending on their size and composition they exhibit peculiar properties, such as quantum confinement in semiconductor nanocrystals, surface plasmon resonance (SPR) in some metal NPs and superparamagnetism in magnetic materials (Vollath, 2008). They also provide large


Nanodiagnostics for Tuberculosis 261

> Specific detection of *M. tuberculosis* by nanostructured zinc oxide (nsZnO) films.

biosensor integrated into

Das et al., 2010; Lee et al., 2010; Wang et al., 1997; Prabhakar et al., 2008;

specific identification of *M. tuberculosis* complex

> Amorphous/ nanocrystalline

an optoelectronic platform for the

members.

surface to volume ratio with the same size range of biomolecules and cellular organelles, allowing a nearly one-on-one interaction between the NP and the biomolecule of interest (Azzazy et al., 2006, 2007; Jain, 2005) and making them of high potential for use in *in vitro* diagnostics. The most promising NPs already applied to TB diagnostics are gold, magnetic and silica NPs, and QDs. Size-dependent properties of the NPs also enable modification of the surface for conjugation with various biomolecules allowing for a wide range of bioassay

Nobel metal NPs have attracted considerable attention in molecular diagnostic applications due to their simplicity and versatility, becoming a critical component in the development of nanotechnology-based detection of pathogens (Liu, 2006). Gold NPs (AuNPs), in particular, have been extensively used due to their unique optical properties with their typical bright red color in colloidal solutions associated with a well-defined SPR band in the visible region of the spectrum (Halfpenny & Wright, 2010). This SPR is originated from the collective oscillation of conduction band electrons at the NPs' surface induced by the interacting electromagnetic radiation of light. The SPR band is weakly dependent on size of the NP and refractive index of the surrounding media, but changes considerably with the composition, shape and inter-particle distance (Johnson et al., 2007). In the latter case, the aggregation of AuNPs leads to a pronounced color transition from red to blue due to plasmon coupling between NPs (Jain, 2007). Another remarkable property of AuNPs is the easiness of chemical functionalization via the use of thiol-ligands (e.g. thiol-modified oligonucleotides, antibodies or other biomolecules) that form quasi-covalent bonds with the NP's gold surface, rendering gold nanoprobes for specific target recognition (Daniel & Astruc, 2004). Most AuNPs based methods rely on the colorimetric changes of the colloidal solution upon aggregation either mediated by a change to the dielectric medium or by recognition of a specific target. The design of these systems is centered in the ability of complementary targets to balance and control inter-particle attractive and repulsive forces, which determine

**Technology Description Application(s) Reference(s)** 

Electrochemical

Table 1. Nanotechnology systems for TB diagnostics

applications (Salata, 2004).

**3.1.1 Noble metal nanoparticles** 

nanofabricated sensors. Portable microfluidic nuclear magnetic resonance biosensor for rapid, quantitative, and multiplexed detection of biological targets. Reduced cost of the automated sensitive detection. Ideal for point-of-care applications.

**Electrochemical devices** 


> Specific detection of *M tuberculosis* complex, *M. avium* complex, *M. avium subsp. paratuberculosis*, *M. bovis* and

Baptista et al., 2006; Costa et al., 2010; Liandris et al., 2009; Silva

Veigas et al., 2010

Kaittanis et al., 2007; Lee et al., 2009

Rotem et al., 2006; Gazouli et al., 2010

Qin

et al., 2007, 2008

et al., 2008, 2010; Soo et al., 2009;

> Detection of *rpo*B mutations

> High sensitive detection of *bacillus Calmette-Guérin*.

*M. tuberculosis*.

resistance.

associated with drug

> Conjugation of

streptavidin-coated QDs with specific bacteriophage. > Integration with magnetic NPs for the detection of *M. tuberculosis* and *M. avium subsp. paratuberculosis*.

> Detection of *M. tuberculosis* by combining luminescent

> Improved two-color flowcytometry assay by a combination of the bioconjugated fluorescent silica NPs and SYBR Green I to avoid false positives.

NPs and indirect immunofluorescence

microscopy.

**Technology Description Application(s) Reference(s)** 

Detection relies on the evaluation of SPR change upon aggregation and the concomitant colorimetric changes that can be assessed by the naked

eye.

Detection by measurement of the spin-spin relaxation time. Minimal sample preparation needed, without the need for sample amplification.

Fluorescence detection of inorganic

dependent

and narrow fluorescence. Highly sensitive. Optimal for multiplex assays.

fluorophores with size-

optical properties, bright

Fluorescence detection of NPs with large quantities of fluorophore molecules inside a polymer or silica matrix. Easy conjugation with several biomolecules and fluorophore making. Ideal for multiplex assays.

**Noble metal** 

**Magnetic NPs** 

**Quantum Dots** 

**Silica NPs** 

**NPs** 


Table 1. Nanotechnology systems for TB diagnostics

surface to volume ratio with the same size range of biomolecules and cellular organelles, allowing a nearly one-on-one interaction between the NP and the biomolecule of interest (Azzazy et al., 2006, 2007; Jain, 2005) and making them of high potential for use in *in vitro* diagnostics. The most promising NPs already applied to TB diagnostics are gold, magnetic and silica NPs, and QDs. Size-dependent properties of the NPs also enable modification of the surface for conjugation with various biomolecules allowing for a wide range of bioassay applications (Salata, 2004).

#### **3.1.1 Noble metal nanoparticles**

Nobel metal NPs have attracted considerable attention in molecular diagnostic applications due to their simplicity and versatility, becoming a critical component in the development of nanotechnology-based detection of pathogens (Liu, 2006). Gold NPs (AuNPs), in particular, have been extensively used due to their unique optical properties with their typical bright red color in colloidal solutions associated with a well-defined SPR band in the visible region of the spectrum (Halfpenny & Wright, 2010). This SPR is originated from the collective oscillation of conduction band electrons at the NPs' surface induced by the interacting electromagnetic radiation of light. The SPR band is weakly dependent on size of the NP and refractive index of the surrounding media, but changes considerably with the composition, shape and inter-particle distance (Johnson et al., 2007). In the latter case, the aggregation of AuNPs leads to a pronounced color transition from red to blue due to plasmon coupling between NPs (Jain, 2007). Another remarkable property of AuNPs is the easiness of chemical functionalization via the use of thiol-ligands (e.g. thiol-modified oligonucleotides, antibodies or other biomolecules) that form quasi-covalent bonds with the NP's gold surface, rendering gold nanoprobes for specific target recognition (Daniel & Astruc, 2004).

Most AuNPs based methods rely on the colorimetric changes of the colloidal solution upon aggregation either mediated by a change to the dielectric medium or by recognition of a specific target. The design of these systems is centered in the ability of complementary targets to balance and control inter-particle attractive and repulsive forces, which determine

Nanodiagnostics for Tuberculosis 263

marker for isoniazid resistance allowing to predict, with a high degree of confidence,

Fig. 1. Non-cross-linking detection of MTBC members. A DNA sample is extracted from a patient and amplified by a first round PCR. The resulting PCR product is characterized using gold nanoprobes and following a non-cross-linking approach that consists of a visual comparison between solutions before and after salt induced nanoprobe aggregation: 'Blank', nanoprobe alone; 'MycoNEG', nanoprobe in the presence of a non-complementary DNA sequence; and 'MycoPOS', nanoprobe in the presence of a complementary DNA sequence. Optimization of the above strategy allowed detection and identification of members of the MTBC at the species level. Three different nanoprobes based on the *gyr*B *locus*, allowed the specific identification of MTBC, *M. bovis* and *M. tuberculosis* (Costa et al., 2010). Based on the conserved *gyr*B gene sequence between species from the MTBC, a set of primers was used to PCR amplify a specific 1020 bp fragment of the gene from MTBC species only. *In silico* alignment of the *gyr*B gene sequences showed three regions that allowed discrimination between MTBC members. As proof-of-concept, one probe was designed to identify this genomic region shared by all the members of the MTBC, and two probes were designed to specifically identify *M. tuberculosis* and *M. bovis*, respectively. The MTBC probe positively identified the members of the MTBC used in the assay, while clearly discriminating the nonmembers. The *M. tuberculosis* and *M. bovis* probes unequivocally identified the respective species. Also, a blind assay using mycobacteria strains isolated from fifteen different clinical

whether the strain is indeed a multidrug-resistant TB (Hillemann et al., 2005).

whether AuNPs are stabilized or aggregated and, consequently, the SPR band and color of the solution remains unaltered or changes, respectively. For example, a specific complementary target can hybridize to the gold nanoprobes and promote an inter-particle cross-linking aggregation (e.g. when using two nanoprobes with contiguous target recognition) or stabilize the nanoprobes against the changes of the dielectric medium, which otherwise would induce a non-cross-linking aggregation of the nanoprobes in the absence of a complementary target (e.g. exploring the differential salt induced non-cross-linking aggregation of the nanoprobes) (Baptista et al., 2005).

The first application of AuNPs for the molecular diagnostics of *M. tuberculosis* was introduced by Baptista et al. (Baptista et al., 2006). The method consists in differential stabilization of gold nanoprobes in the presence of different DNA targets. The presence of a complementary target prevents nanoprobe aggregation and the solution remains red, while non-complementary/mismatched targets or their absence do not prevent gold nanoprobe aggregation, resulting in a visible change of color from red to blue. The gold nanoprobes were functionalized with thiol-modified oligonucleotides harboring a sequence derived from the *M. tuberculosis* RNA polymerase β-subunit gene sequence suitable for mycobacteria identification. The methodology was tested in clinical samples demonstrating high efficiency when combined with an initial round of PCR for target amplification (Baptista et al., 2006) – see Figure 1.

The attained results have shown a 100% concordance with the available commercial molecular TB diagnostics test INNO-LiPA Rif.TB. Following optimization towards detection of single base mismatches (Doria et al., 2010), this strategy was applied to the rapid detection of MTBC strains and simultaneous characterization of the presence of mutations associated with rifampicin resistance (Veigas et al., 2010). This low-complexity assay enabled the detection of mutations D516V and S531L from MTBC clinical specimens with remarkable sensitivity in just a few hours. Based on the molecular signatures of MTBC members and the most common mutations associated with RIF resistance in *M. tuberculosis*, a two-step approach based on the PCR amplification of a fragment of *rpo*B gene and subsequent hybridization with specific nanoprobes, namely a probe for the rpoB locus shared by all the members of the MTBC and a probe specific to MTBC members, was developed. Three additional sets of probes specific for the most common point mutations associated with RIF resistance (D516V; H526D; S531L) were also designed and synthesized. Each set composed of two probes: one complementary to the wild-type sequence and the other complementary to the mutation. A limit of detection could be set at 75 nM, however, for robust single base mismatch determination, 117 nM of DNA target were used per assay. This non-cross-linking approach correctly detected the presence of DNA from members of the MTBC in 83.3% of all samples, when compared to the INNO-LiPA Rif.TB assay. By means of a set of two probes for each mutation associated to RIF resistance to be screened (mutations in codons 516, 526 and 531 of the rpoB gene), it was possible to correctly score the presence of at least one of the mutations in 81% of all samples also screened via the INNO-LiPA Rif.TB assay. Following PCR amplification, the method takes only 90 min to yield a colorimetric result which, through the use of a suitable photodetector (e.g. UV/visible spectrophotometer, microplate reader, etc.), may be used in medium throughput analysis at a peripheral laboratory or point-of-care. Fast and reliable identification of MTBC members and mutations within the *rpo*B gene is of great advantage as it is a secondary

whether AuNPs are stabilized or aggregated and, consequently, the SPR band and color of the solution remains unaltered or changes, respectively. For example, a specific complementary target can hybridize to the gold nanoprobes and promote an inter-particle cross-linking aggregation (e.g. when using two nanoprobes with contiguous target recognition) or stabilize the nanoprobes against the changes of the dielectric medium, which otherwise would induce a non-cross-linking aggregation of the nanoprobes in the absence of a complementary target (e.g. exploring the differential salt induced non-cross-linking

The first application of AuNPs for the molecular diagnostics of *M. tuberculosis* was introduced by Baptista et al. (Baptista et al., 2006). The method consists in differential stabilization of gold nanoprobes in the presence of different DNA targets. The presence of a complementary target prevents nanoprobe aggregation and the solution remains red, while non-complementary/mismatched targets or their absence do not prevent gold nanoprobe aggregation, resulting in a visible change of color from red to blue. The gold nanoprobes were functionalized with thiol-modified oligonucleotides harboring a sequence derived from the *M. tuberculosis* RNA polymerase β-subunit gene sequence suitable for mycobacteria identification. The methodology was tested in clinical samples demonstrating high efficiency when combined with an initial round of PCR for target amplification (Baptista et

The attained results have shown a 100% concordance with the available commercial molecular TB diagnostics test INNO-LiPA Rif.TB. Following optimization towards detection of single base mismatches (Doria et al., 2010), this strategy was applied to the rapid detection of MTBC strains and simultaneous characterization of the presence of mutations associated with rifampicin resistance (Veigas et al., 2010). This low-complexity assay enabled the detection of mutations D516V and S531L from MTBC clinical specimens with remarkable sensitivity in just a few hours. Based on the molecular signatures of MTBC members and the most common mutations associated with RIF resistance in *M. tuberculosis*, a two-step approach based on the PCR amplification of a fragment of *rpo*B gene and subsequent hybridization with specific nanoprobes, namely a probe for the rpoB locus shared by all the members of the MTBC and a probe specific to MTBC members, was developed. Three additional sets of probes specific for the most common point mutations associated with RIF resistance (D516V; H526D; S531L) were also designed and synthesized. Each set composed of two probes: one complementary to the wild-type sequence and the other complementary to the mutation. A limit of detection could be set at 75 nM, however, for robust single base mismatch determination, 117 nM of DNA target were used per assay. This non-cross-linking approach correctly detected the presence of DNA from members of the MTBC in 83.3% of all samples, when compared to the INNO-LiPA Rif.TB assay. By means of a set of two probes for each mutation associated to RIF resistance to be screened (mutations in codons 516, 526 and 531 of the rpoB gene), it was possible to correctly score the presence of at least one of the mutations in 81% of all samples also screened via the INNO-LiPA Rif.TB assay. Following PCR amplification, the method takes only 90 min to yield a colorimetric result which, through the use of a suitable photodetector (e.g. UV/visible spectrophotometer, microplate reader, etc.), may be used in medium throughput analysis at a peripheral laboratory or point-of-care. Fast and reliable identification of MTBC members and mutations within the *rpo*B gene is of great advantage as it is a secondary

aggregation of the nanoprobes) (Baptista et al., 2005).

al., 2006) – see Figure 1.

marker for isoniazid resistance allowing to predict, with a high degree of confidence, whether the strain is indeed a multidrug-resistant TB (Hillemann et al., 2005).

Fig. 1. Non-cross-linking detection of MTBC members. A DNA sample is extracted from a patient and amplified by a first round PCR. The resulting PCR product is characterized using gold nanoprobes and following a non-cross-linking approach that consists of a visual comparison between solutions before and after salt induced nanoprobe aggregation: 'Blank', nanoprobe alone; 'MycoNEG', nanoprobe in the presence of a non-complementary DNA sequence; and 'MycoPOS', nanoprobe in the presence of a complementary DNA sequence.

Optimization of the above strategy allowed detection and identification of members of the MTBC at the species level. Three different nanoprobes based on the *gyr*B *locus*, allowed the specific identification of MTBC, *M. bovis* and *M. tuberculosis* (Costa et al., 2010). Based on the conserved *gyr*B gene sequence between species from the MTBC, a set of primers was used to PCR amplify a specific 1020 bp fragment of the gene from MTBC species only. *In silico* alignment of the *gyr*B gene sequences showed three regions that allowed discrimination between MTBC members. As proof-of-concept, one probe was designed to identify this genomic region shared by all the members of the MTBC, and two probes were designed to specifically identify *M. tuberculosis* and *M. bovis*, respectively. The MTBC probe positively identified the members of the MTBC used in the assay, while clearly discriminating the nonmembers. The *M. tuberculosis* and *M. bovis* probes unequivocally identified the respective species. Also, a blind assay using mycobacteria strains isolated from fifteen different clinical

Nanodiagnostics for Tuberculosis 265

with the complementary target resulted in the formation of a polymeric cross-linked network, bringing the AuNPs close enough to induce a color change from red to blue (Beermann et al., 2007; Li et al., 2006; Liandris et al., 2009; Storhoff et al., 2005). The efficacy of such cross-linking assay was evaluated by analyzing sputum specimens. Results were compared with traditional culture and biochemical identification methods together with patients clinical assessments. The detection limit of this assay was measured using IS6110 DNA amplified from *M. tuberculosis* H37Rv chromosome. This methodology was able to detect as low as 0.5 pmol of DNA target within two hours. The assay comprises two main steps, namely, the target DNA amplification by single or nested PCR, followed by nanoprobe detection. The gold nanoprobes are added to the heat denatured PCR products, and incubated at 55ºC for DNA hybridization with increased stringency. In the presence of complementary DNA the nanoprobes aggregated upon hybridization to the target, resulting in decrease in absorbance of the solution at 525 nm. On the other hand, the color and absorbance pattern did not change when specific complementary target DNAs were absent in the solution. The methodology was evaluated by directly and simultaneously detecting MTBC and *M. tuberculosis* from 600 clinical strains and comparing the results with those from conventional culture methods and biochemical identification in combination with clinical assessment. The assay presented 96.6% sensitivity and 98.9% specificity towards detection of MTBC, and 94.7% sensitivity and 99.6% specificity for detection of *M.* 

Magnetic properties are largely dependent on the composition and molecular structure of the NPs (Lu et al., 2007). Different materials can exhibit diamagnetic, paramagnetic or ferromagnetic behavior (Sato et al., 2003). In most cases, the particles range from 1 to 100 nm in size and may display supermagnetism when the thermal energy is enough to change the direction of magnetization of the NPs (Neubergera et al., 2005). Superparamagnetic NPs made of magnetic materials (e.g. iron, nickel, cobalt, or alloys of magnetic metals) are preferred for biomedical applications, due to the fact that they behave non-magnetically when they are not under the influence of an external magnetic field, thus preventing undesired self-magnetic agglomeration. In the presence of an external magnetic field gradient, the large magnetic moments of all the atoms align with the field and the superparamagnetic NPs can be manipulated to interact with different biomolecules (Jain, 2007). Removing the external magnetic field causes the NPs to lose their alignment with the field and relax into random directions of magnetization. To make the superparamagnetic NPs biocompatible, they are coated with a material such as silica or polyethylene glycol, and then functionalized with the relevant targeting biomolecule for the desired application, such as antibodies, proteins or oligonucleotides (Jain, 2005). As an example, superparamagnetic NPs have been used in the development of a magnetic immunoassay. The presence of a target analyte allows the superparamagnetic NPs to bind to a magnetic sensor in a sandwich conformation, which creates a local magnetic field that is detected by the sensor once an applied external field is used to induce a magnetic moment in the superparamagnetic NPs,

Kaitanis and coworkers designed superparamagnetic iron oxide nanoprobes coupled with a magnetic relaxation methodology to detect *Mycobacterium avium* spp. *paratuberculosis*, in

*tuberculosis*.

(Sato et al., 2003).

**3.1.2 Magnetic nanoparticles** 

samples showed 100% concordance with the results attained by the *gyr*B-PCR-RFLP method.

Towards a point-of-care application, Baptista and co-workers further integrated the noncross-linking nanoprobe-based method in an innovative optoelectronic platform that allows an analytical measurement of the colorimetric changes, hence to detect a target without the need of experienced personnel. The device integrates an amorphous/nanocrystalline biosensor and a light emission source with the non-cross-linking method for specific DNA detection. This low cost, fast and simple optoelectronic platform was optimized for the specific identification of MTBC members and the consequent improvement of the laboratorial diagnostics algorithms of TB (Bernacka-Wojcik, et al., 2010; Silva et al., 2008, 2010). The integration of these technologies together with the possibility of miniaturization are of utmost importance for the development of an integrated biosensor suitable for peripheral laboratories and/or point-of-care diagnostics, providing a new tool in the fight against TB.

Recently, Liandris et al. have developed a non-cross-linking approach to the detection of TB without the need of target amplification (Liandris et al., 2009). The method relied on the same non-cross-linking hybridization approach of Baptista and co-workers, whereas the aggregation of the gold nanoprobes was induced by an increasing acid concentration instead of salt. The detection is based on the fact that double and single-stranded oligonucleotides have different electrostatic properties. After hybridization, single-stranded DNA becomes double-stranded DNA. As a result, the double-stranded DNA cannot uncoil sufficiently like the single-stranded DNA to expose its bases toward the gold nanoprobe. Therefore, the nanoprobe undergoes aggregation in an acidic environment. Liandris and coworkers designed an array of gold nanoprobes to collectively detect the main mycobacterial pathogens in clinical samples, namely MTBC, *M. avium* complex and *M. avium* subsp. *paratuberculosis*. A nanoprobe harboring 20 nucleotides was designed to harbor a conserved genus region sequence of 16s–23s ITS DNA of the most common mycobacterial pathogens. In order to obtain an indication of the method's performance on clinical samples, the assay was tested for the detection of *M. avium* subsp. *paratuberculosis* DNA in feces. For this purpose, 12 fecal samples were collected from an equal number of goats from a herd with a well-established record of *M. avium* subsp. *paratuberculosis* and the results were compared to those obtained by a real-time PCR assay. The quantification was performed using *M. avium* subsp. *paratuberculosis* DNA of known concentration, and the standard curve as obtained by real-time PCR. The evaluation of the specificity and repeatability of this noncross-linking approach indicated a reliable and highly specific detection of a broad spectrum of mycobacteria without cross reactions with related bacteria (the concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%). Moreover, the methodology demonstrated to be highly sensitive, where even the lowest concentration of the targeted sequence was easily detected by simple visual observation of the test and the control tubes (Liandris et al., 2009).

Following a cross-linking approach, Soo et al. designed a set of gold nanoprobes to specifically hybridize with target DNAs of MTBC and *M. tuberculosis* strains (Soo et al., 2009). The nanoprobes were oriented in a tail-to-tail arrangement, one probe functionalized via a thiol moiety located at the 5'end of the sequence and other at 3' end, with both sequences being contiguous to each other. This way the hybridization of the nanoprobes

samples showed 100% concordance with the results attained by the *gyr*B-PCR-RFLP

Towards a point-of-care application, Baptista and co-workers further integrated the noncross-linking nanoprobe-based method in an innovative optoelectronic platform that allows an analytical measurement of the colorimetric changes, hence to detect a target without the need of experienced personnel. The device integrates an amorphous/nanocrystalline biosensor and a light emission source with the non-cross-linking method for specific DNA detection. This low cost, fast and simple optoelectronic platform was optimized for the specific identification of MTBC members and the consequent improvement of the laboratorial diagnostics algorithms of TB (Bernacka-Wojcik, et al., 2010; Silva et al., 2008, 2010). The integration of these technologies together with the possibility of miniaturization are of utmost importance for the development of an integrated biosensor suitable for peripheral laboratories and/or point-of-care diagnostics, providing a new tool in the fight

Recently, Liandris et al. have developed a non-cross-linking approach to the detection of TB without the need of target amplification (Liandris et al., 2009). The method relied on the same non-cross-linking hybridization approach of Baptista and co-workers, whereas the aggregation of the gold nanoprobes was induced by an increasing acid concentration instead of salt. The detection is based on the fact that double and single-stranded oligonucleotides have different electrostatic properties. After hybridization, single-stranded DNA becomes double-stranded DNA. As a result, the double-stranded DNA cannot uncoil sufficiently like the single-stranded DNA to expose its bases toward the gold nanoprobe. Therefore, the nanoprobe undergoes aggregation in an acidic environment. Liandris and coworkers designed an array of gold nanoprobes to collectively detect the main mycobacterial pathogens in clinical samples, namely MTBC, *M. avium* complex and *M. avium* subsp. *paratuberculosis*. A nanoprobe harboring 20 nucleotides was designed to harbor a conserved genus region sequence of 16s–23s ITS DNA of the most common mycobacterial pathogens. In order to obtain an indication of the method's performance on clinical samples, the assay was tested for the detection of *M. avium* subsp. *paratuberculosis* DNA in feces. For this purpose, 12 fecal samples were collected from an equal number of goats from a herd with a well-established record of *M. avium* subsp. *paratuberculosis* and the results were compared to those obtained by a real-time PCR assay. The quantification was performed using *M. avium* subsp. *paratuberculosis* DNA of known concentration, and the standard curve as obtained by real-time PCR. The evaluation of the specificity and repeatability of this noncross-linking approach indicated a reliable and highly specific detection of a broad spectrum of mycobacteria without cross reactions with related bacteria (the concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%). Moreover, the methodology demonstrated to be highly sensitive, where even the lowest concentration of the targeted sequence was easily detected

by simple visual observation of the test and the control tubes (Liandris et al., 2009).

Following a cross-linking approach, Soo et al. designed a set of gold nanoprobes to specifically hybridize with target DNAs of MTBC and *M. tuberculosis* strains (Soo et al., 2009). The nanoprobes were oriented in a tail-to-tail arrangement, one probe functionalized via a thiol moiety located at the 5'end of the sequence and other at 3' end, with both sequences being contiguous to each other. This way the hybridization of the nanoprobes

method.

against TB.

with the complementary target resulted in the formation of a polymeric cross-linked network, bringing the AuNPs close enough to induce a color change from red to blue (Beermann et al., 2007; Li et al., 2006; Liandris et al., 2009; Storhoff et al., 2005). The efficacy of such cross-linking assay was evaluated by analyzing sputum specimens. Results were compared with traditional culture and biochemical identification methods together with patients clinical assessments. The detection limit of this assay was measured using IS6110 DNA amplified from *M. tuberculosis* H37Rv chromosome. This methodology was able to detect as low as 0.5 pmol of DNA target within two hours. The assay comprises two main steps, namely, the target DNA amplification by single or nested PCR, followed by nanoprobe detection. The gold nanoprobes are added to the heat denatured PCR products, and incubated at 55ºC for DNA hybridization with increased stringency. In the presence of complementary DNA the nanoprobes aggregated upon hybridization to the target, resulting in decrease in absorbance of the solution at 525 nm. On the other hand, the color and absorbance pattern did not change when specific complementary target DNAs were absent in the solution. The methodology was evaluated by directly and simultaneously detecting MTBC and *M. tuberculosis* from 600 clinical strains and comparing the results with those from conventional culture methods and biochemical identification in combination with clinical assessment. The assay presented 96.6% sensitivity and 98.9% specificity towards detection of MTBC, and 94.7% sensitivity and 99.6% specificity for detection of *M. tuberculosis*.

#### **3.1.2 Magnetic nanoparticles**

Magnetic properties are largely dependent on the composition and molecular structure of the NPs (Lu et al., 2007). Different materials can exhibit diamagnetic, paramagnetic or ferromagnetic behavior (Sato et al., 2003). In most cases, the particles range from 1 to 100 nm in size and may display supermagnetism when the thermal energy is enough to change the direction of magnetization of the NPs (Neubergera et al., 2005). Superparamagnetic NPs made of magnetic materials (e.g. iron, nickel, cobalt, or alloys of magnetic metals) are preferred for biomedical applications, due to the fact that they behave non-magnetically when they are not under the influence of an external magnetic field, thus preventing undesired self-magnetic agglomeration. In the presence of an external magnetic field gradient, the large magnetic moments of all the atoms align with the field and the superparamagnetic NPs can be manipulated to interact with different biomolecules (Jain, 2007). Removing the external magnetic field causes the NPs to lose their alignment with the field and relax into random directions of magnetization. To make the superparamagnetic NPs biocompatible, they are coated with a material such as silica or polyethylene glycol, and then functionalized with the relevant targeting biomolecule for the desired application, such as antibodies, proteins or oligonucleotides (Jain, 2005). As an example, superparamagnetic NPs have been used in the development of a magnetic immunoassay. The presence of a target analyte allows the superparamagnetic NPs to bind to a magnetic sensor in a sandwich conformation, which creates a local magnetic field that is detected by the sensor once an applied external field is used to induce a magnetic moment in the superparamagnetic NPs, (Sato et al., 2003).

Kaitanis and coworkers designed superparamagnetic iron oxide nanoprobes coupled with a magnetic relaxation methodology to detect *Mycobacterium avium* spp. *paratuberculosis*, in

Nanodiagnostics for Tuberculosis 267

Although the proof of concept only used a single phage–host system, the method may be expanded for the detection of multiple bacterial strains by their specific phages. This concept could be applied to any slow growing pathogen, such as *M. tuberculosis* for TB

Only recently QDs have been used for the detection and imaging of respiratory pathogens and, in particular, for TB diagnostics. Gazouli and coworkers developed and evaluated a detection assay for specific DNA sequences combining fluorescent semiconductor QDs with magnetic beads allowing for a fast identification of two members of the *Mycobacterium* genus (*M. tuberculosis* and *M. avium* subsp. *paratuberculosis*) without the need for DNA amplification (Gazouli et al., 2010). The assay involves two biotinylated oligonucleotide probes to recognize and detect specific complementary mycobacterial target DNA through a sandwich like hybridization. Five 30-bp-long genus-specific probes were designed for the detection of *Mycobacterium* based on the 23S rRNA gene, which is highly conserved among the mycobacterial species. For the detection of *M. tuberculosis* and *M. avium* subsp. *paratuberculosis*, 2 sets of five 30-bp-long probes were designed based on IS6110 and IS900, respectively. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a fluorescent signal, while the magnetic beads conjugated with streptavidin and genus-specific probes were used to isolate and concentrate the DNA targets. The minimum detection limit of the assay was defined to be 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance with clinical samples, the system was compared with conventional diagnostics methodologies, namely Ziehl-Neelsen staining and real-time PCR. Additionally, to assess the performance of the assay with clinical material, DNA isolated from bronchoalveolar lavage samples, formalin-fixed paraffin-embedded tissues or feces was used for the detection of *M. tuberculosis* and *M. avium* subsp. *paratuberculosis.* With regard to *M. tuberculosis*, the assessment relied on DNA isolated from bronchoalveolar lavage samples from 48 patients with clinical tuberculosis and 12 bronchoalveolar lavage samples from healthy individuals, both confirmed by culture and real-time PCR, with the exception of a bronchoalveolar lavage sample that reacted negatively by the latter method. The overall concordance of this assay was 84.61% and 100% with regard to positive and negative results, respectively. This approach of capturing and detection in two steps by different building blocks minimizes false-positives associated with low specificity. Given that the capture and detection probes of the QD assay are complementary to different genes of the mycobacterial genome, the chances of false-negative results due to DNA fragmentation are inevitably increased. Nonetheless, this weakness can be circumvented by the use of a different set of DNA probes that anneal closer to each other, allowing an assessment that minimizes falsepositive results associated with low specificity (Gazouli et al., 2010). Additionally, the method avoids the drawback of PCR-based diagnostic assays that are prone to falsenegative results generated by inhibitors commonly found in clinical samples such as feces.

Fluorescence-based detection techniques have been extensively used in both biological research and clinical diagnostics, due to the extremely high sensitivity. Dye-doped silica NPs contain large quantities of dye (fluorophore) molecules inside a polymer or silica matrix, amplifying the fluorescence of each interaction event. This signal enhancement

diagnostics.

**3.1.4 Silica nanoparticles** 

milk and blood (Kaittanis et al., 2007). The methodology could quickly quantify the bacterial target with high sensitivity, and was not susceptible to interferences caused by other bacteria. The principle underlying the detection by these nanosensors is their ability to change between disperse and clustered (or assembled) state upon target interaction, with a concomitant change in the spin-spin relaxation time of the solution's water protons. This approach, apart from sensitive and fast, is independent of the sample's optical properties and requires minimum sample preparation. More recently, Lee et al. developed a very similar methodology where bacteria were targeted by highly magnetic NPs with a large Fe core and a thin ferrite shell NPs, concentrated into a microfluidic chamber, and detected by nuclear magnetic resonance (Lee et al., 2009). The clinical utility of this diagnostic platform was evaluated by detecting TB using the *bacillus* Calmette-Guérin (BCG) as a surrogate for *M. tuberculosis*. Following liquefaction, the samples were subjected to standard TB diagnostic tests, namely culture and acid-fast bacilli smear microscopy, to be compared with the magnetic NPs-based nuclear magnetic resonance measurements. This methodology shown similar results to those attained with the standard culture-based methods (detection limit of ~20 colony forming units) with the advantage to be less prone to human error and less labor-intensive. The nuclear magnetic resonance-based detection was much faster (< 30 min) and performed on a single microfluidic chip, markedly contrasting with the culturebased test that was time-consuming (> 2 weeks) and facility-dependent (e.g. incubators).

Both these methodologies are in their first stages of development and present great advantages over current techniques such as speed, easiness of procedure and minimum sample preparation.

#### **3.1.3 Quantum dots**

Quantum dots are inorganic fluorophores with size-dependent optical properties, exhibiting strong light absorbance, bright and narrow symmetric emission bands and high photo stability, due to three-dimensional quantum confinement effects (Chan et al., 2002; Yezhelyev et al., 2006). The size and composition of QDs determine their emission wavelength and color (Coto-García et al., 2011; Michalet et al., 2005; Sukhanova & Nabiev, 2008). Moreover, the QDs can maintain these properties upon conjugation to biomolecules (Alivisatos et al., 2005; Fortina et al., 2005; Salata, 2004) making them preferred fluorescent probes for imaging applications (Halfpenny & Wright, 2010). The fact that QDs do not depend on the presence of a variety of different fluorescence dyes also allows their application for multiplexing analysis (Azzazy & Mansour, 2009). Although QDs are typically insoluble in water, they can be made biocompatible by several strategies including silanization and coating with a polymer shell, thus enabling their utilization in biological systems. Target specificity is achieved by conjugating them to a variety of biomolecules, such as antibodies, streptavidin and oligonucleotides, enabling their application in conventional molecular biological methodologies, such as fluorescent *in situ* hybridization (FISH), immunological assays or northern/southern/western blots (Michalet et al., 2005). In fact, QDs have already been used in a number of biological applications including studies of protein trafficking, DNA detection and dynamic studies of cell mobility (Zrazhevskiy et al., 2010). Rotem and coworkers reported the use of QDs for the detection of pathogenic bacteria combining *in vivo* biotinylation of engineered host-specific bacteriophage with the conjugation of the phage to streptavidin-coated fluorescent QDs (Rotem et al., 2006).

milk and blood (Kaittanis et al., 2007). The methodology could quickly quantify the bacterial target with high sensitivity, and was not susceptible to interferences caused by other bacteria. The principle underlying the detection by these nanosensors is their ability to change between disperse and clustered (or assembled) state upon target interaction, with a concomitant change in the spin-spin relaxation time of the solution's water protons. This approach, apart from sensitive and fast, is independent of the sample's optical properties and requires minimum sample preparation. More recently, Lee et al. developed a very similar methodology where bacteria were targeted by highly magnetic NPs with a large Fe core and a thin ferrite shell NPs, concentrated into a microfluidic chamber, and detected by nuclear magnetic resonance (Lee et al., 2009). The clinical utility of this diagnostic platform was evaluated by detecting TB using the *bacillus* Calmette-Guérin (BCG) as a surrogate for *M. tuberculosis*. Following liquefaction, the samples were subjected to standard TB diagnostic tests, namely culture and acid-fast bacilli smear microscopy, to be compared with the magnetic NPs-based nuclear magnetic resonance measurements. This methodology shown similar results to those attained with the standard culture-based methods (detection limit of ~20 colony forming units) with the advantage to be less prone to human error and less labor-intensive. The nuclear magnetic resonance-based detection was much faster (< 30 min) and performed on a single microfluidic chip, markedly contrasting with the culturebased test that was time-consuming (> 2 weeks) and facility-dependent (e.g. incubators).

Both these methodologies are in their first stages of development and present great advantages over current techniques such as speed, easiness of procedure and minimum

Quantum dots are inorganic fluorophores with size-dependent optical properties, exhibiting strong light absorbance, bright and narrow symmetric emission bands and high photo stability, due to three-dimensional quantum confinement effects (Chan et al., 2002; Yezhelyev et al., 2006). The size and composition of QDs determine their emission wavelength and color (Coto-García et al., 2011; Michalet et al., 2005; Sukhanova & Nabiev, 2008). Moreover, the QDs can maintain these properties upon conjugation to biomolecules (Alivisatos et al., 2005; Fortina et al., 2005; Salata, 2004) making them preferred fluorescent probes for imaging applications (Halfpenny & Wright, 2010). The fact that QDs do not depend on the presence of a variety of different fluorescence dyes also allows their application for multiplexing analysis (Azzazy & Mansour, 2009). Although QDs are typically insoluble in water, they can be made biocompatible by several strategies including silanization and coating with a polymer shell, thus enabling their utilization in biological systems. Target specificity is achieved by conjugating them to a variety of biomolecules, such as antibodies, streptavidin and oligonucleotides, enabling their application in conventional molecular biological methodologies, such as fluorescent *in situ* hybridization (FISH), immunological assays or northern/southern/western blots (Michalet et al., 2005). In fact, QDs have already been used in a number of biological applications including studies of protein trafficking, DNA detection and dynamic studies of cell mobility (Zrazhevskiy et al., 2010). Rotem and coworkers reported the use of QDs for the detection of pathogenic bacteria combining *in vivo* biotinylation of engineered host-specific bacteriophage with the conjugation of the phage to streptavidin-coated fluorescent QDs (Rotem et al., 2006).

sample preparation.

**3.1.3 Quantum dots** 

Although the proof of concept only used a single phage–host system, the method may be expanded for the detection of multiple bacterial strains by their specific phages. This concept could be applied to any slow growing pathogen, such as *M. tuberculosis* for TB diagnostics.

Only recently QDs have been used for the detection and imaging of respiratory pathogens and, in particular, for TB diagnostics. Gazouli and coworkers developed and evaluated a detection assay for specific DNA sequences combining fluorescent semiconductor QDs with magnetic beads allowing for a fast identification of two members of the *Mycobacterium* genus (*M. tuberculosis* and *M. avium* subsp. *paratuberculosis*) without the need for DNA amplification (Gazouli et al., 2010). The assay involves two biotinylated oligonucleotide probes to recognize and detect specific complementary mycobacterial target DNA through a sandwich like hybridization. Five 30-bp-long genus-specific probes were designed for the detection of *Mycobacterium* based on the 23S rRNA gene, which is highly conserved among the mycobacterial species. For the detection of *M. tuberculosis* and *M. avium* subsp. *paratuberculosis*, 2 sets of five 30-bp-long probes were designed based on IS6110 and IS900, respectively. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a fluorescent signal, while the magnetic beads conjugated with streptavidin and genus-specific probes were used to isolate and concentrate the DNA targets. The minimum detection limit of the assay was defined to be 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance with clinical samples, the system was compared with conventional diagnostics methodologies, namely Ziehl-Neelsen staining and real-time PCR. Additionally, to assess the performance of the assay with clinical material, DNA isolated from bronchoalveolar lavage samples, formalin-fixed paraffin-embedded tissues or feces was used for the detection of *M. tuberculosis* and *M. avium* subsp. *paratuberculosis.* With regard to *M. tuberculosis*, the assessment relied on DNA isolated from bronchoalveolar lavage samples from 48 patients with clinical tuberculosis and 12 bronchoalveolar lavage samples from healthy individuals, both confirmed by culture and real-time PCR, with the exception of a bronchoalveolar lavage sample that reacted negatively by the latter method. The overall concordance of this assay was 84.61% and 100% with regard to positive and negative results, respectively. This approach of capturing and detection in two steps by different building blocks minimizes false-positives associated with low specificity. Given that the capture and detection probes of the QD assay are complementary to different genes of the mycobacterial genome, the chances of false-negative results due to DNA fragmentation are inevitably increased. Nonetheless, this weakness can be circumvented by the use of a different set of DNA probes that anneal closer to each other, allowing an assessment that minimizes falsepositive results associated with low specificity (Gazouli et al., 2010). Additionally, the method avoids the drawback of PCR-based diagnostic assays that are prone to falsenegative results generated by inhibitors commonly found in clinical samples such as feces.

#### **3.1.4 Silica nanoparticles**

Fluorescence-based detection techniques have been extensively used in both biological research and clinical diagnostics, due to the extremely high sensitivity. Dye-doped silica NPs contain large quantities of dye (fluorophore) molecules inside a polymer or silica matrix, amplifying the fluorescence of each interaction event. This signal enhancement

Nanodiagnostics for Tuberculosis 269

surface area for DNA molecules loading and detect genomic target DNA up to 100 pM,

These nanofabricated sensors have the capability of reducing the costs of the automated sensitive detection, making them ideal for point-of-care applications. Moreover, these platforms demonstrate the most promising trends in bioanalytical and biochemical methods,

Nanocantilevers are one of the most promising nanotechnologies for identification of biomolecules capable of providing label-free detection with high sensitivity and specificity (Craighead, 2007). These systems can operate either statically, by measuring absolute cantilever deflection, or dynamically, by measuring resonance frequency shifts. In the *static mode*, the main parameter measured is the differential surface stress produced when molecular adsorption is produced on one side of the cantilever (Fritz et al., 2000). In *dynamic mode*, sensing relies on the observation of the dynamical properties of a resonant cantilever (e.g. vibration amplitude, resonance frequency), which has been increasingly applied for mass sensing (Craighead, 2007; Waggoner & Craighead, 2007). Sensors based on static operation have demonstrated their potential for selective detection of DNA and proteins in liquid. Mass sensors based on dynamic mode can potentially achieve sub-femtomolar sensitivity (Llic et al., 2005). The ultrahigh mass sensitivity is counterbalanced with a very low selectivity due to the device contamination with non-sought molecules and salt debris (Varshney et al., 2008). Thus, in practice small bio-molecules, such as proteins or oligonucleotides, can be detected at low concentrations. Recent developments merged this nanotechnology with AuNPs as sensitizing agents (Wittenberg & Haynes, 2009). These NPs act as mass enhancers, allowing detection of biomolecules at the femtomolar level and beyond, while maintaining the possibility of performing parallel analyses and working with minute sample volumes. Despite these advantages, to our knowledge, nano-

which enables the direct detection of pathogens in clinical samples at point of care.

the fusion of different approaches, methods and technologies into a single platform.

electromechanical systems have not yet been applied to the diagnostics of TB.

In the last decades we witnessed the development of nanotechnology in isolated fields of research, introducing new and revolutionary approaches for molecular detection. Today the most promising advances are made at the interface, merging two or more technological architectures for new hybrid approaches, circumventing current limitations of each existing techniques for biomolecule analysis protocols. Tremendous advancements in the development and performance of new technological approaches for the rapid diagnostics of TB and prediction of drug resistance have been made in the last few years. The obvious advantages of nanodiagnostics based schemes are their ability to provide results within hours, with increased sensitivities and specificities at a fraction of a cost when compared to conventional microbiological and molecular biology methodologies, such as sputum smear microscopy and nucleic acid amplification based techniques. Nevertheless, thus far, only a very small number of these new nanodiagnostics platforms have been translated to the clinical setting for TB molecular diagnostics. The great efforts put into the development of proof-of-concept approaches most of the time lack the connection and the robustness to

**4. Conclusions** 

**3.3 Nano-electromechanical devices – nanocantilevers** 

facilitates ultrasensitive analyte determination otherwise undetectable with conventional fluorescence labeling techniques. Furthermore, the polymer and silica matrix serves as a protective shell, reducing photo-bleaching (Zhao et al., 2007) and allows a high versatility towards different surface modification protocols (Tan et al., 2004). These silica NPs are also more hydrophilic, biocompatible and not subject to microbial degradation, swelling or porosity changes with varying pH (Jain et al., 1998). Several systems based in silica NPs have been widely applied in biological imaging and ultrasensitive bioassays, including cell staining (Santra et al., 2001a), DNA detection (He et al., 2006; Zhao et al., 2003), cell surface receptor targeting (Her et al., 2006; Santra et al., 2001a, 2001b, 2004), and ultrasensitive detection of pathogens (Zhao et al., 2004). Moreover, these NPs can also be conjugated with QDs in a core shell like structure, taking advantage of the capabilities of QDs and taking advantage of silica chemistries versatility, while reducing toxicity (Qin et al., 2007).

The first application of dye-doped silica NPs for TB molecular diagnostics was published by Qin and co-workers, where a rapid immunological method combining highly luminescent RuBpy-doped silica NPs with indirect immunofluorescence microscopy allowed for detection of *M. tuberculosis* in both mixed bacterial and sputum samples (Qin et al., 2007). Later on, this approach was improved by using two-color flow-cytometry and adding SYBR Green I to avoid false positives (Qin et al., 2008). Briefly, *M. tuberculosis* is first recognized by the antibody-conjugated RuBpy-doped silica NPs, and then stained with a nucleic acid dye SYBR Green I to discriminate bacterial cells from background particles, followed by multiparameter determination with flow-cytometry. This way, the population of *M. tuberculosis* dual stained with antibody-conjugated RuBpy-doped silica NPs and SYBR-I could be discerned as a distinct population and the false positives caused by aggregates of NP-bioconjugates and nonspecific binding of NP-bioconjugates to background debris could be decreased dramatically, when compared with the initial one-color approach. Moreover, the decrease of false positives also allowed achieving a higher sensitivity for detection of *M. tuberculosis*. This later dual-color approach allowed for detection of TB in buffer and spiked urine, with higher sensitivities than the conventional flow cytometry techniques, maintaining the same simplicity, speed and usability.

#### **3.2 Nano-fabricated devices**

Several biosensors for the determination of short sequences from the *M. tuberculosis* DNA have been described (Buijtels et al., 2008; Csako, 2006; McGlennen, 2001). Wang and coworkers developed a sensor that relies on the modification of the carbon-paste transducer oligonucleotide probe and their hybridization to complementary strands from the *M. tuberculosis* DNA (Wang et al., 1997). Chronopotentiometry was employed as transducer and the sensor allowed detection down to nanograms per milliliter of *M. tuberculosis* DNA. Prabhakar et al. used cysteine modified NH2-end peptide nucleic acid probes and 5′-thiol end labeled DNA probes immobilized onto BK-7 gold coated glass plates for specific detection of *M. tuberculosis* using SPR (Prabhakar et al., 2008). More recently, Das and coworkers developed nanostructured zinc oxide films on conducting indium–tin–oxide coated glass plate to immobilize a DNA probe and specifically detect *M. tuberculosis* based on the strong electrostatic interactions between ZnO and the complementary target (Das et al., 2010). The presence of nanostructured ZnO films allowed to increase the electro-active

facilitates ultrasensitive analyte determination otherwise undetectable with conventional fluorescence labeling techniques. Furthermore, the polymer and silica matrix serves as a protective shell, reducing photo-bleaching (Zhao et al., 2007) and allows a high versatility towards different surface modification protocols (Tan et al., 2004). These silica NPs are also more hydrophilic, biocompatible and not subject to microbial degradation, swelling or porosity changes with varying pH (Jain et al., 1998). Several systems based in silica NPs have been widely applied in biological imaging and ultrasensitive bioassays, including cell staining (Santra et al., 2001a), DNA detection (He et al., 2006; Zhao et al., 2003), cell surface receptor targeting (Her et al., 2006; Santra et al., 2001a, 2001b, 2004), and ultrasensitive detection of pathogens (Zhao et al., 2004). Moreover, these NPs can also be conjugated with QDs in a core shell like structure, taking advantage of the capabilities of QDs and taking

advantage of silica chemistries versatility, while reducing toxicity (Qin et al., 2007).

maintaining the same simplicity, speed and usability.

**3.2 Nano-fabricated devices** 

The first application of dye-doped silica NPs for TB molecular diagnostics was published by Qin and co-workers, where a rapid immunological method combining highly luminescent RuBpy-doped silica NPs with indirect immunofluorescence microscopy allowed for detection of *M. tuberculosis* in both mixed bacterial and sputum samples (Qin et al., 2007). Later on, this approach was improved by using two-color flow-cytometry and adding SYBR Green I to avoid false positives (Qin et al., 2008). Briefly, *M. tuberculosis* is first recognized by the antibody-conjugated RuBpy-doped silica NPs, and then stained with a nucleic acid dye SYBR Green I to discriminate bacterial cells from background particles, followed by multiparameter determination with flow-cytometry. This way, the population of *M. tuberculosis* dual stained with antibody-conjugated RuBpy-doped silica NPs and SYBR-I could be discerned as a distinct population and the false positives caused by aggregates of NP-bioconjugates and nonspecific binding of NP-bioconjugates to background debris could be decreased dramatically, when compared with the initial one-color approach. Moreover, the decrease of false positives also allowed achieving a higher sensitivity for detection of *M. tuberculosis*. This later dual-color approach allowed for detection of TB in buffer and spiked urine, with higher sensitivities than the conventional flow cytometry techniques,

Several biosensors for the determination of short sequences from the *M. tuberculosis* DNA have been described (Buijtels et al., 2008; Csako, 2006; McGlennen, 2001). Wang and coworkers developed a sensor that relies on the modification of the carbon-paste transducer oligonucleotide probe and their hybridization to complementary strands from the *M. tuberculosis* DNA (Wang et al., 1997). Chronopotentiometry was employed as transducer and the sensor allowed detection down to nanograms per milliliter of *M. tuberculosis* DNA. Prabhakar et al. used cysteine modified NH2-end peptide nucleic acid probes and 5′-thiol end labeled DNA probes immobilized onto BK-7 gold coated glass plates for specific detection of *M. tuberculosis* using SPR (Prabhakar et al., 2008). More recently, Das and coworkers developed nanostructured zinc oxide films on conducting indium–tin–oxide coated glass plate to immobilize a DNA probe and specifically detect *M. tuberculosis* based on the strong electrostatic interactions between ZnO and the complementary target (Das et al., 2010). The presence of nanostructured ZnO films allowed to increase the electro-active surface area for DNA molecules loading and detect genomic target DNA up to 100 pM, which enables the direct detection of pathogens in clinical samples at point of care.

These nanofabricated sensors have the capability of reducing the costs of the automated sensitive detection, making them ideal for point-of-care applications. Moreover, these platforms demonstrate the most promising trends in bioanalytical and biochemical methods, the fusion of different approaches, methods and technologies into a single platform.

#### **3.3 Nano-electromechanical devices – nanocantilevers**

Nanocantilevers are one of the most promising nanotechnologies for identification of biomolecules capable of providing label-free detection with high sensitivity and specificity (Craighead, 2007). These systems can operate either statically, by measuring absolute cantilever deflection, or dynamically, by measuring resonance frequency shifts. In the *static mode*, the main parameter measured is the differential surface stress produced when molecular adsorption is produced on one side of the cantilever (Fritz et al., 2000). In *dynamic mode*, sensing relies on the observation of the dynamical properties of a resonant cantilever (e.g. vibration amplitude, resonance frequency), which has been increasingly applied for mass sensing (Craighead, 2007; Waggoner & Craighead, 2007). Sensors based on static operation have demonstrated their potential for selective detection of DNA and proteins in liquid. Mass sensors based on dynamic mode can potentially achieve sub-femtomolar sensitivity (Llic et al., 2005). The ultrahigh mass sensitivity is counterbalanced with a very low selectivity due to the device contamination with non-sought molecules and salt debris (Varshney et al., 2008). Thus, in practice small bio-molecules, such as proteins or oligonucleotides, can be detected at low concentrations. Recent developments merged this nanotechnology with AuNPs as sensitizing agents (Wittenberg & Haynes, 2009). These NPs act as mass enhancers, allowing detection of biomolecules at the femtomolar level and beyond, while maintaining the possibility of performing parallel analyses and working with minute sample volumes. Despite these advantages, to our knowledge, nanoelectromechanical systems have not yet been applied to the diagnostics of TB.

#### **4. Conclusions**

In the last decades we witnessed the development of nanotechnology in isolated fields of research, introducing new and revolutionary approaches for molecular detection. Today the most promising advances are made at the interface, merging two or more technological architectures for new hybrid approaches, circumventing current limitations of each existing techniques for biomolecule analysis protocols. Tremendous advancements in the development and performance of new technological approaches for the rapid diagnostics of TB and prediction of drug resistance have been made in the last few years. The obvious advantages of nanodiagnostics based schemes are their ability to provide results within hours, with increased sensitivities and specificities at a fraction of a cost when compared to conventional microbiological and molecular biology methodologies, such as sputum smear microscopy and nucleic acid amplification based techniques. Nevertheless, thus far, only a very small number of these new nanodiagnostics platforms have been translated to the clinical setting for TB molecular diagnostics. The great efforts put into the development of proof-of-concept approaches most of the time lack the connection and the robustness to

Nanodiagnostics for Tuberculosis 271

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make an impact in the analytical laboratory and very few techniques are available for direct application in respiratory specimens (Lee et al., 2009, 2010). It is expected that in the next few years, some of the strategies depicted throughout this short chapter can take their rightful place at the front line of fighting TB.

Future trends in nanodiagnostics will continue through miniaturization of biochip technology to the nanoscale range for point-of-care diagnostics with a sample-in answer-out approach that hampers user-error, thus enabling their use by non-specialized personnel.

#### **5. Acknowledgment**

The authors thank CIGMH – FCT/MCTES for financial support.

#### **6. References**


make an impact in the analytical laboratory and very few techniques are available for direct application in respiratory specimens (Lee et al., 2009, 2010). It is expected that in the next few years, some of the strategies depicted throughout this short chapter can take their

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**13** 

*Brazil* 

**Sputum Smear Microscopy for Tuberculosis:** 

*Centre for Electronic and Information Technology/Federal University of Amazonas* 

Since 1997 the World Health Organization has published an annual report on global control of tuberculosis (TB) with the purpose of providing a comprehensive and up-to-date assessment of the TB epidemic. According to the Global TB control report of 2010 (World Health Organization [WHO], 2010), the global burden of disease caused by TB in 2009 is as follows: 9.4 million incident cases, 14 million prevalent cases, 1.3 million deaths among non

The absolute number of cases continues to increase from year to year. The slow reduction in incident rates per capita is outweighed by increases in population. The greatest number of cases are in Asia (55%) and Africa (30%). Other regions have lower numbers of cases: Eastern Mediterranean Region (7%), European Region (4%) and American Region (3%). The main effort of WHO today concerning TB is to attain the targets included in the Millennium

Adopted by world leaders in 2000, the MDGs are a blueprint that guides the efforts of the United Nations Development Program and various and various aid agencies, providing concrete, numerical benchmarks for tackling extreme poverty in its many dimensions to be achieved by 2015. The MDGs define 8 goals (United Nations [UN], 2010) with 21 targets that are measured by 60 indicators. TB falls under the 6th goal related to fighting disease epidemics, aiming to: "Combat HIV/AIDS, Malaria and other diseases". Within this goal the following target refers to TB: "Halt and begin to reverse the incidence of malaria and other major diseases". Related to this target, the following indicator refers to TB: Halt and begin to reverse TB incidence by 2015; Reduce prevalence and deaths due to

To achieve these indicators the WHO adopted a Partnership Global Plan to Stop TB (WHO, 2011). Launched in January 2006, it includes sputum smear microscopy as the main diagnostic tool. Indeed, one of the targets of this plan is stated as follows: "A treatment success rate among sputum smear positive case of 90%". The main reason for sputum smear

HIV-positive people and 0.38 million deaths among HIV positive people.

**1. Introduction** 

Development Goals (MDGs).

TB by 50% compared with a baseline of 1990.

**Evaluation of Autofocus Functions** 

**and Automatic Identification of** 

**Tuberculosis Mycobacterium** 

Cicero F. F. Costa Filho and Marly G. F. Costa


## **Sputum Smear Microscopy for Tuberculosis: Evaluation of Autofocus Functions and Automatic Identification of Tuberculosis Mycobacterium**

Cicero F. F. Costa Filho and Marly G. F. Costa *Centre for Electronic and Information Technology/Federal University of Amazonas Brazil* 

#### **1. Introduction**

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Since 1997 the World Health Organization has published an annual report on global control of tuberculosis (TB) with the purpose of providing a comprehensive and up-to-date assessment of the TB epidemic. According to the Global TB control report of 2010 (World Health Organization [WHO], 2010), the global burden of disease caused by TB in 2009 is as follows: 9.4 million incident cases, 14 million prevalent cases, 1.3 million deaths among non HIV-positive people and 0.38 million deaths among HIV positive people.

The absolute number of cases continues to increase from year to year. The slow reduction in incident rates per capita is outweighed by increases in population. The greatest number of cases are in Asia (55%) and Africa (30%). Other regions have lower numbers of cases: Eastern Mediterranean Region (7%), European Region (4%) and American Region (3%). The main effort of WHO today concerning TB is to attain the targets included in the Millennium Development Goals (MDGs).

Adopted by world leaders in 2000, the MDGs are a blueprint that guides the efforts of the United Nations Development Program and various and various aid agencies, providing concrete, numerical benchmarks for tackling extreme poverty in its many dimensions to be achieved by 2015. The MDGs define 8 goals (United Nations [UN], 2010) with 21 targets that are measured by 60 indicators. TB falls under the 6th goal related to fighting disease epidemics, aiming to: "Combat HIV/AIDS, Malaria and other diseases". Within this goal the following target refers to TB: "Halt and begin to reverse the incidence of malaria and other major diseases". Related to this target, the following indicator refers to TB: Halt and begin to reverse TB incidence by 2015; Reduce prevalence and deaths due to TB by 50% compared with a baseline of 1990.

To achieve these indicators the WHO adopted a Partnership Global Plan to Stop TB (WHO, 2011). Launched in January 2006, it includes sputum smear microscopy as the main diagnostic tool. Indeed, one of the targets of this plan is stated as follows: "A treatment success rate among sputum smear positive case of 90%". The main reason for sputum smear

Sputum Smear Microscopy for Tuberculosis: Evaluation of Autofocus

Larsen, 1966);

skill (Toman, 2004b).

(Sotaquirá, 2009).

methods.

methods could not be done.

microscopy (Steingart et. al., 2006).

range from 0.94 to 1 (Steingart et. al., 2006).

2009; Makkapati, et. al., 2009; Khutlang et. al., 2010).

Functions and Automatic Identification of Tuberculosis Mycobacterium 279

1. Fluorescence microscopy has the following main advantages over conventional microscopy: 1) Fluorescence microscopy uses a lower power objective lens (typically 25x), while conventional microscopy uses a higher power objective lens (typically 100x). As a consequence fluorescence microscopy allows the same area of a smear to be scanned in a much shorter time than with conventional microscopy (Bennedesen &

2. Fluorescence microscopy is on average 10% more sensitive than conventional

The main shortcomings of fluorescence microscopy are: 1) The relatively high costs of the microscopy unit and its maintenance when compared with the conventional microscopy unit; 2) The handling and maintenance of the optical equipment require advanced technical

The sensitivity of tuberculosis diagnostic through sputum smear analysis reported in the literature varies greatly. While reported sensitivities of conventional microscopy range from 0.32 to 0.94, reported sensitivities of fluorescence microscopy range from 0.52 to 0.97. On average the specificity of fluorescence microscopy is similar to conventional microscopy and

In addition to the huge variability in sensitivity, the manual screening for bacillus identification is a labor-intensive task that consumes between 40 minutes and 3 hours, depending on patient's level of infection and it is needed to analyse 40-100 images

Automatic methods for bacilli screening were first developed for fluorescence microscopy images (Veropoulos et. al., 1998; Forero et. al., 2003). The first methods for automatic bacilli screening in conventional microscopy were published only in 2008 (Costa et. al., 2008; Sadaphal et. al., 2008; Raof et. al., 2008). Some other methods for automatic bacilli screening were published in recent years (Forero, 2004, 2006; Lenseigne et. al., 2007; Sotaquira et. al.,

Some authors (Forero et. al., 2006; Sotaquira, 2009; Khutlang, 2010) claimed that the main advantages of an automatic bacilli screening over a manual one are better reproducible values for sensitivity and specificity and a faster screening process. Table 2 shows reported values for sensitivity, specificity and time waste for one image analysis using automatic

The sensitivity and specificity values previously cited for manual screening methods refer to tuberculosis diagnosis. The sensitivity and specificity values for automatic methods shown in Table 2 refer to object classification as bacillus or not bacillus. Therefore, a rigorous comparison of sensitivities and specificities between manual and automatic screening

Only one paper of Table 2 cited time wasted for one image analysis, 1.87s. To compute the time consumed with a TB automatic diagnosis it is necessary to take into account the number of images needed to achieve a correct diagnosis. As previously cited, in order to achieve a correct diagnosis, it is necessary to analyze between 20 and 100 fields of one slide. With an automatic procedure, it is also necessary to take into account the time spent with focusing computations, image acquisition and microscopy displacement. According to

microscopy to be included is that it is the main non-invasive technique employed for TB diagnosis. Other non-invasive techniques include culture and chest radiography.

Sputum smear microscopy has several operational advantages over culture as a diagnostic tool (Luelmo, 2004): "The results are available soon, correlate with infectiousness, and identify both patients at high risk of death from tuberculosis if untreated and patients who require more drugs in the initial treatment regimen because of greater bacterial load". In addition sputum smear microscopy has an important role in follow up of TB treatment. Only when the smears are negative can the intensive phase of the treatment be suspended.

Despite the historical importance of chest radiography in TB diagnosis, it is not used today as a diagnostic tool alone. The following reasons justify this practice: 1) Some other diseases of the lung show a similar appearance in radiographic picture. Consequently radiographic exam is not specific to TB; 2) Lesions of pulmonary tuberculosis can take almost any form in a radiographic image (American Thoracic Society [ATS], 2000) .

Two main facts enable the use of sputum smear microscopy for TB diagnosis. The first one is that special dyes allow to differentiating the bacillus from the background. The second one is that there is a positive correlation between the number of bacillus in the smear and the probability of their being identified by microscopy.

To support the last statement, Table 1 (David, 1976, as cited in Toman, 2004a) shows the positive correlation that exists between the number of bacillus present in a sputum specimen, the number of bacillus in a smear and the probability of finding theses bacillus by microscopy. For this study 0.01 ml of sputum was placed on a slide and spread over an area of 200mm2. The magnification of the microscope used allowed for observing 10.000 fields on this slide.


Table 1. Number of observed bacilli, concentration of bacilli in sputum specimen (culture results) and probability of a positive result

Two techniques are used for TB diagnostic with sputum smear microscopy: Fluorescence microscopy and conventional microscopy. Fluorescence microscopy uses an acid-fast fluorochrome dye (eg, auramine O or auramine-rhodamine), while conventional microscopy uses the carbolfuchsin Ziehl-Neelsen - ZN or Kinyoun acid-fast stains. While the first one uses an intense light source, such as a halogen or high-pressure mercury vapor lamp, the second one uses a conventional artificial light source.

microscopy to be included is that it is the main non-invasive technique employed for TB

Sputum smear microscopy has several operational advantages over culture as a diagnostic tool (Luelmo, 2004): "The results are available soon, correlate with infectiousness, and identify both patients at high risk of death from tuberculosis if untreated and patients who require more drugs in the initial treatment regimen because of greater bacterial load". In addition sputum smear microscopy has an important role in follow up of TB treatment. Only when the smears are negative can the intensive phase of the treatment be

Despite the historical importance of chest radiography in TB diagnosis, it is not used today as a diagnostic tool alone. The following reasons justify this practice: 1) Some other diseases of the lung show a similar appearance in radiographic picture. Consequently radiographic exam is not specific to TB; 2) Lesions of pulmonary tuberculosis can take almost any form in

Two main facts enable the use of sputum smear microscopy for TB diagnosis. The first one is that special dyes allow to differentiating the bacillus from the background. The second one is that there is a positive correlation between the number of bacillus in the smear and the

To support the last statement, Table 1 (David, 1976, as cited in Toman, 2004a) shows the positive correlation that exists between the number of bacillus present in a sputum specimen, the number of bacillus in a smear and the probability of finding theses bacillus by microscopy. For this study 0.01 ml of sputum was placed on a slide and spread over an area of 200mm2. The magnification of the microscope used allowed for observing 10.000 fields on

> **Estimated concentration of bacilli per ml of especimen**

0 in 100 or more field <1000 <10% 1-2 in 300 fields 5000-10000 50% 1-9 in 100 fields about 30 000 80% 1-9 in 10 fields about 50 000 90% 1-9 per field about 100 000 96.2% 10 or more per field about 500 000 99.95%

Table 1. Number of observed bacilli, concentration of bacilli in sputum specimen

Two techniques are used for TB diagnostic with sputum smear microscopy: Fluorescence microscopy and conventional microscopy. Fluorescence microscopy uses an acid-fast fluorochrome dye (eg, auramine O or auramine-rhodamine), while conventional microscopy uses the carbolfuchsin Ziehl-Neelsen - ZN or Kinyoun acid-fast stains. While the first one uses an intense light source, such as a halogen or high-pressure mercury vapor lamp, the

**Probability of a positive result**

diagnosis. Other non-invasive techniques include culture and chest radiography.

a radiographic image (American Thoracic Society [ATS], 2000) .

probability of their being identified by microscopy.

(culture results) and probability of a positive result

second one uses a conventional artificial light source.

**No. of bacilli observed**

suspended.

this slide.


The main shortcomings of fluorescence microscopy are: 1) The relatively high costs of the microscopy unit and its maintenance when compared with the conventional microscopy unit; 2) The handling and maintenance of the optical equipment require advanced technical skill (Toman, 2004b).

The sensitivity of tuberculosis diagnostic through sputum smear analysis reported in the literature varies greatly. While reported sensitivities of conventional microscopy range from 0.32 to 0.94, reported sensitivities of fluorescence microscopy range from 0.52 to 0.97. On average the specificity of fluorescence microscopy is similar to conventional microscopy and range from 0.94 to 1 (Steingart et. al., 2006).

In addition to the huge variability in sensitivity, the manual screening for bacillus identification is a labor-intensive task that consumes between 40 minutes and 3 hours, depending on patient's level of infection and it is needed to analyse 40-100 images (Sotaquirá, 2009).

Automatic methods for bacilli screening were first developed for fluorescence microscopy images (Veropoulos et. al., 1998; Forero et. al., 2003). The first methods for automatic bacilli screening in conventional microscopy were published only in 2008 (Costa et. al., 2008; Sadaphal et. al., 2008; Raof et. al., 2008). Some other methods for automatic bacilli screening were published in recent years (Forero, 2004, 2006; Lenseigne et. al., 2007; Sotaquira et. al., 2009; Makkapati, et. al., 2009; Khutlang et. al., 2010).

Some authors (Forero et. al., 2006; Sotaquira, 2009; Khutlang, 2010) claimed that the main advantages of an automatic bacilli screening over a manual one are better reproducible values for sensitivity and specificity and a faster screening process. Table 2 shows reported values for sensitivity, specificity and time waste for one image analysis using automatic methods.

The sensitivity and specificity values previously cited for manual screening methods refer to tuberculosis diagnosis. The sensitivity and specificity values for automatic methods shown in Table 2 refer to object classification as bacillus or not bacillus. Therefore, a rigorous comparison of sensitivities and specificities between manual and automatic screening methods could not be done.

Only one paper of Table 2 cited time wasted for one image analysis, 1.87s. To compute the time consumed with a TB automatic diagnosis it is necessary to take into account the number of images needed to achieve a correct diagnosis. As previously cited, in order to achieve a correct diagnosis, it is necessary to analyze between 20 and 100 fields of one slide. With an automatic procedure, it is also necessary to take into account the time spent with focusing computations, image acquisition and microscopy displacement. According to

$$T\_{ad} = 100 \text{x} (1.87 + 1.8 + 0.7) = 437 \text{s} \cong 7 \text{ minutes} \tag{1}$$


$$F\_{th\\_grad} = \Sigma\_M \Sigma\_N |g(i, j+1) - g(i, j)|\tag{2}$$

$$\text{while } |g(i, j+1) - g(i, j)| \ge \theta^i$$

$$F\_{sq\\_grad} = \Sigma\_M \Sigma\_N |g(l, j+1) - g(l, f)|^2 \tag{3}$$

$$while \ |g(l, j+1) - g(l, f)| \ge \theta$$

$$F\_{teman} = \Sigma\_M \Sigma\_N \, T[g(i, j)] \tag{4}$$

$$\mathcal{F}[g(i, j)] = G\_\mathcal{X}^2(i, j) + G\_\mathcal{Y}^2(i, j)$$

$$F\_{brenner} = \Sigma\_{\mathcal{M}} \Sigma\_{\mathcal{N}} |g(i, j + \mathcal{Z}) - g(i, j)|^2 \tag{5}$$
 
$$while \ |g(i, j + 1) - g(i, j)| \ge \theta$$

$$F\_{\text{Laplace}} = \Sigma\_{\text{M}} \Sigma\_{\text{N}} \left( g(l, j+1) + g(l, j-1) + g(l+1, j) + g(l-1, j) - 4g(l, j) \right)^2 \tag{6}$$

$$\begin{aligned} \boldsymbol{F}\_{\text{Gaussian}} &= \frac{1}{NM} \sum\_{\mathbf{N}} \sum\_{\mathbf{N}} \Big\{ \mathbf{g}(\mathbf{l}, \mathbf{j}) \, \* \, \mathbf{G}\_{\mathbf{x}}(\mathbf{x}, \mathbf{y}, \sigma) \Big\}^2 + \Big\{ \mathbf{g}(\mathbf{l}, \mathbf{j}) \, \* \, \mathbf{G}\_{\mathbf{y}}(\mathbf{x}, \mathbf{y}, \sigma) \Big\}^2 \\ \boldsymbol{G}\_{\mathbf{x}}(\mathbf{x}, \mathbf{y}, \sigma) \text{ and } \boldsymbol{G}\_{\mathbf{y}}(\mathbf{x}, \mathbf{y}, \sigma) \text{ are the first order Gaussian derivatives in the x and y directions.} \end{aligned}$$

$$F\_{var} = \frac{1}{m} \Sigma\_M \Sigma\_N |g(l, f) - \vec{g}|\tag{7}$$

$$F\_{var} = \frac{1}{m\mathcal{N}\mathcal{g}} \Sigma\_{\mathcal{M}} \Sigma\_{\mathcal{N}} |g(i, f) - \mathcal{g}| \tag{8}$$

$$F\_{entr} = -\Sigma\_l p\_l \log p\_l \tag{9}$$

$$F\_{var\\_log} = \Sigma\_l \{ l - E\_{log} \{ l \} \} \log p\_l \tag{10}$$

$$\Sigma\_g \{ l \} = \sum \log p\_l \text{ is the expected value of } \log \text{histogram}$$

$$F\_{autocorr} = \Sigma\_{\mathcal{M}} \Sigma\_{\mathcal{N}} g(i+1, f) g(i, f) \ - \Sigma\_{\mathcal{M}} \Sigma\_{\mathcal{N}} g(i+2, f) g(i, f) \tag{11}$$

$$F\_{autocorr} = \Sigma\_M \Sigma\_N g(l+1, f) g(l, f) \ -MN\bar{g}^2 \tag{12}$$


Fig. 4. Typical shape of a focus function

The horizontal coordinate of the graph of Figure 4 corresponds to the z position of the microscope vertical axis. To plot the focus function it is necessary to vary this z position and obtain a stack of points described in equation (13). The z position movement for obtaining the image stack is illustrated in Figure 5.

$$\text{Stack} = \{ (\text{FM}\_{\text{l}} \text{ z}\_{1}), (\text{FM}\_{\text{l}} \text{z}\_{2}), \dots, \dots (\text{FM}\_{\text{n}/2} \text{z}\_{\text{n}/2}), \dots (\text{FM}\_{\text{n}-1} \text{z}\_{\text{n}-1}) \text{ (FM}\_{\text{n}} \text{z}\_{\text{n}}) \} \tag{13}$$

.

Sputum Smear Microscopy for Tuberculosis: Evaluation of Autofocus

focus function departs from the correct focal position;

from an ideal behavior the following algorithm is used:

maximum;

narrowness of the peak.

position of maximum focus.

the series are obtained.

(Osibote et. al., 2010) can be used:

obtained.

standard deviation equal to unity.

curve and according to four features (Firestone at. al., 1991), defined as:

Number of false maxima: number of spurious focus function maxima;

Santos et al. (1997) introduced a 5th feature, Execution Time, as follows:

Functions and Automatic Identification of Tuberculosis Mycobacterium 285

to gray scale. The performance of a focus measure is frequently evaluated using the focus

1. Accuracy: expressed here as the number of steps by which the maximum of a particular

Range: the number of steps between the two neighboring local minima around the global

Width: computed at 50% of the height focus curve. This criterion describes the sharpness or

Execution Time: the time taken for an algorithm to compute the focus plot and locate the

According to Santos (Santos et. al., 1997) a quantitative evaluation may compare a focus curve to an ideal function with respect to each of these features. The authors define an ideal focus function as having a value of 0 for execution time, accuracy, width and number of false maxima and a range determined by multiplying the number of images in the stack used to plot the focus function and the step size between each position in the stack (adjustment step of the microscopy). To obtain a measure of how a focus measure departs

1. A series of focus measure curves is obtained (these series should contain images with different background content). The mean and the standard deviation of each feature in

2. The five feature values of each image series are normalized by subtracting the corresponding mean and dividing by the standard deviation. This produces values for the different features that can be compared as they all now have mean zero and

3. For each feature the distance from the ideal function is computed. First the differences between the feature value in the function and in the ideal function are obtained. Then

4. Finally, to produce a final figure of this function, the mean value of the five distances is

When doing a TB diagnosis with sputum smear microscopy, a bacilli count on a number of fields of one slide is necessary. A time-consuming autofocus procedure determines the optimal focus through the acquisition of the focus function for each field. To reduce lens motion and achieve faster autofocus times the following procedure proposed by Osibote

1. Obtaining the focus position for the first field of the slide through the acquisition of a full image stack of the focus measure, ensuring a perfect evaluation of this field to avoid

2. Adopt a simplified procedure to determine the optimal focus position in subsequent fields, using the optimal focus position of the previous field as a reference. For this purpose the procedure proposed by Yanzdafar (Yanzdafar et. al., 2008) can be used.

locating the optimal focus in a false minimum position;

the square root of the addition of the squares of these results is computed.

Where: FMi = Focus measure at position zi

Fig. 5. Z position movement for obtaining the image stack

The in-focus image normally is the central image of the stack. Varying the z position changes the image sharpness and hence the degree of focus. Each image in a stack, therefore, is at a different focus level. For focus measure computation, images are converted from RGB

The horizontal coordinate of the graph of Figure 4 corresponds to the z position of the microscope vertical axis. To plot the focus function it is necessary to vary this z position and obtain a stack of points described in equation (13). The z position movement for obtaining

Stack = {(FM1, z1), (FM2,z2),……..(FMn/2zn/2)….(FMn-1zn-1) (FMn,zn)} (13)

The in-focus image normally is the central image of the stack. Varying the z position changes the image sharpness and hence the degree of focus. Each image in a stack, therefore, is at a different focus level. For focus measure computation, images are converted from RGB

.

The shape of a focus function typically resembles a Gaussian curve as shown if Figure 4.

Fig. 4. Typical shape of a focus function

the image stack is illustrated in Figure 5.

Where: FMi = Focus measure at position zi

Fig. 5. Z position movement for obtaining the image stack

to gray scale. The performance of a focus measure is frequently evaluated using the focus curve and according to four features (Firestone at. al., 1991), defined as:


Number of false maxima: number of spurious focus function maxima;

Width: computed at 50% of the height focus curve. This criterion describes the sharpness or narrowness of the peak.

Santos et al. (1997) introduced a 5th feature, Execution Time, as follows:

Execution Time: the time taken for an algorithm to compute the focus plot and locate the position of maximum focus.

According to Santos (Santos et. al., 1997) a quantitative evaluation may compare a focus curve to an ideal function with respect to each of these features. The authors define an ideal focus function as having a value of 0 for execution time, accuracy, width and number of false maxima and a range determined by multiplying the number of images in the stack used to plot the focus function and the step size between each position in the stack (adjustment step of the microscopy). To obtain a measure of how a focus measure departs from an ideal behavior the following algorithm is used:


When doing a TB diagnosis with sputum smear microscopy, a bacilli count on a number of fields of one slide is necessary. A time-consuming autofocus procedure determines the optimal focus through the acquisition of the focus function for each field. To reduce lens motion and achieve faster autofocus times the following procedure proposed by Osibote (Osibote et. al., 2010) can be used:


Sputum Smear Microscopy for Tuberculosis: Evaluation of Autofocus

Edge detection techniques: Canny

Edge detection techniques (Canny operator) + Adaptive color thresholding (RGB

color space)

Edge detection techniques (Canny operator) + Adaptive color thresholding

**Color space techniques:**  Adaptive global threshold; **Color space:** RGB

Bayesian segmentation; **Color space:** RGB

**Color space techniques:**  First derivative of histogram;

**Color space:** YCbCr, Lab

**Color space techniques:**  Pixel classifiers (Baye's, Linear regression, quadratic discriminant); **Color space:** RGB

Table 4. Published papers involving Automated Sputum Smear Microscopy

Conventional **Color space techniques:**  Thresholding; **Color space:** RGB

operator

applied to the image on the left side of Figure 7.

vary: RGB, YCbCr and Lab

et. al, 1998 Fluorescence

Fluorescence

Fluorescence

Conventional

al., 2008 Conventional **Color space techniques:** 

Veropoulos

Forero et. al, 2004

Forero et. al, 2006

Costa et. al., 2008

Sadaphal et.

Raof et. al., 2008

Sotaquirá et.

Khutlang et.

al. , 2009 Conventional

al. (2010) Coventional

Functions and Automatic Identification of Tuberculosis Mycobacterium 287

operators, such as canny operators (Veropoulos et. al., 1998; Forero et. al., 2004). Intermediate steps for edge linking and boundary tracing are also employed. Figure 8 shows the results of the segmentation procedure used by Forero (Forero et. al., 2004) when

In conventional microscopy images, the bacilli are not easily separated from the background with a threshold operation. In this case, for bacilli segmentation, colour space techniques are used. As shown in Table 4, the techniques found in the literature vary: histogram based techniques, Bayesian pixel classifiers, KNN pixel classifiers, etc. The colour spaces used also

**Author Microscopy Bacilli segmentation Bacilli Classification Results**

**Shape Descriptors:** 15 Fourier descriptors; **Classifier:** Back-propagation (BP), RBF networks, KNN, Kernel Regression (KR)

**Shape Descriptors:**  compactness, eccentricity and Hu's moments descriptors; **Classifier:** Classification tree

**Shape Descriptors:** Hu's moments descriptors; **Classifier:** Gaussian mixture

**Shape Descriptors:** Axis ratio, eccentricity;

**Classifier:** Classification tree

**Shape Descriptors:** Fourier features, color moments, eccentricity, compactness; **Classifier:** Probabilistic neural network, kNN, SVM

Size filters **Sensitivity:** 76.65%

12%

models

**Accuracy:**  BP - 97.57% RBF - 88.06% KNN - 91.80 KR - 95.24%

**Specificity, Sensitivity:**  99.74%, 73.33% 94.96%, 86.66%

**Specificity, Sensitivity:**  97.89%, 94.67% 98.10%, 92.9%

**False Positive Rate:** 

No information

No information

**False detection:** 9.78%

**Accuracy:** 98.55% **Sensitivity:** 97.77% **Specificity:** 99.13%

**Accuracy:**  96.3%

#### **3. Automated sputum smear microscopy**

According to Forero (Forero et. al., 2006) bacilli are structures that have a length between 1 and 10μm and a width between 0.2 and 0.6μm presenting a straight, curve or bent shape, as shown if Figure 6.

Fig. 6. Different shapes of bacilli

Depending on the staining procedures used, the bacilli assume different appearances. When the sputum smear is stained with an acid-fast fluorochrome dye, as is the case when fluorescence microscopy is used, the bacilli fluoresce in the range between green and yellow up to white, while the background is dark. Otherwise, when the sputum smear is stained with carbolfuchsin Ziehl-Neelsen - ZN or Kinyoun acid-fast stains, as is the case when conventional microscopy is used, the bacilli may have different colours, varying from light fuchsia to dark purple. In Figure 7 we show images of both microscopy types.

Fig. 7. Fluorescence microscopy (after Forero et al., 2004) and conventional microscopy sputum smear image

The block diagram of Figure 2 shows the main steps involved in automated bacilli recognition. Table 4 shows the main methods used in the literature for each step of this block diagram.

As shown in Figure 2, after image capture, bacilli segmentation is performed. The segmentation procedures adopted in both types of images shown in Figure 7 are completely different from each other.

In fluorescence microscopy images, the bacilli are easily separated from the background with a threshold operation. Afterwards, the segmentation is performed using edge detection

According to Forero (Forero et. al., 2006) bacilli are structures that have a length between 1 and 10μm and a width between 0.2 and 0.6μm presenting a straight, curve or bent shape,

Depending on the staining procedures used, the bacilli assume different appearances. When the sputum smear is stained with an acid-fast fluorochrome dye, as is the case when fluorescence microscopy is used, the bacilli fluoresce in the range between green and yellow up to white, while the background is dark. Otherwise, when the sputum smear is stained with carbolfuchsin Ziehl-Neelsen - ZN or Kinyoun acid-fast stains, as is the case when conventional microscopy is used, the bacilli may have different colours, varying from light

The block diagram of Figure 2 shows the main steps involved in automated bacilli recognition. Table 4 shows the main methods used in the literature for each step of this

As shown in Figure 2, after image capture, bacilli segmentation is performed. The segmentation procedures adopted in both types of images shown in Figure 7 are completely

In fluorescence microscopy images, the bacilli are easily separated from the background with a threshold operation. Afterwards, the segmentation is performed using edge detection

Fig. 7. Fluorescence microscopy (after Forero et al., 2004) and conventional microscopy

fuchsia to dark purple. In Figure 7 we show images of both microscopy types.

**3. Automated sputum smear microscopy** 

as shown if Figure 6.

Fig. 6. Different shapes of bacilli

sputum smear image

different from each other.

block diagram.

operators, such as canny operators (Veropoulos et. al., 1998; Forero et. al., 2004). Intermediate steps for edge linking and boundary tracing are also employed. Figure 8 shows the results of the segmentation procedure used by Forero (Forero et. al., 2004) when applied to the image on the left side of Figure 7.

In conventional microscopy images, the bacilli are not easily separated from the background with a threshold operation. In this case, for bacilli segmentation, colour space techniques are used. As shown in Table 4, the techniques found in the literature vary: histogram based techniques, Bayesian pixel classifiers, KNN pixel classifiers, etc. The colour spaces used also vary: RGB, YCbCr and Lab


Table 4. Published papers involving Automated Sputum Smear Microscopy

After the segmentation step is finished, not only bacilli are segmented. Some structures fluoresce the same way as bacilli in fluorescence microscopy images. Similarly some structures have the same colour properties as bacilli in conventional microscopy images. confused with bacilli. These structures, also called noise, could be debris or cells present in the background. To illustrate this point, near the lower left corner of Figure 7, a circular structure can be seen that fluoresces the same way as a bacillus, but because of its circular shape could not be classified as one. Nevertheless, this structure is segmented the same way as a bacillus, as shown in Figure 8.

Sputum Smear Microscopy for Tuberculosis: Evaluation of Autofocus

Functions and Automatic Identification of Tuberculosis Mycobacterium 289

Fig. 9. Conventional microscopy image showing some examples of touching bacilli

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**4. References** 

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Fig. 8. Objects resulting from segmentation procedures applied in the left image of Figure 7.

To separate noise from bacilli in the segmented images an additional step, called object classification in the block diagram of Figure 2 is normally employed. For this purpose classifiers using shape descriptors are used. As the bacilli may have different sizes, positions and orientations, the shape descriptors used must be rotation, translation and scale invariant. As shown in Table 4, the most used descriptors used are: compactness, eccentricity, Hu's moments and Fourier Descriptors. Varied classifiers such as classification trees, Support Vector Machines and Neural Networks were employed by some authors in order to recognize the bacilli.

The results presented in Table 4 show that, in bacilli detection, results for sensitivity and specificity as good as 97.77% and 99.13% are cited. It is noteworthy, however that the authors who cited these values, do not consider touching bacilli. In some cases, as the one shown in Figure 9, these bacilli are present in large quantities. Disregarding these bacilli implies a different count of what is done by manual means. Because of this, we believe that other ways of removing noise than those that use shape descriptors must be investigated.

Fig. 9. Conventional microscopy image showing some examples of touching bacilli

#### **4. References**

288 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

After the segmentation step is finished, not only bacilli are segmented. Some structures fluoresce the same way as bacilli in fluorescence microscopy images. Similarly some structures have the same colour properties as bacilli in conventional microscopy images. confused with bacilli. These structures, also called noise, could be debris or cells present in the background. To illustrate this point, near the lower left corner of Figure 7, a circular structure can be seen that fluoresces the same way as a bacillus, but because of its circular shape could not be classified as one. Nevertheless, this structure is segmented the same way

Fig. 8. Objects resulting from segmentation procedures applied in the left image of Figure 7. To separate noise from bacilli in the segmented images an additional step, called object classification in the block diagram of Figure 2 is normally employed. For this purpose classifiers using shape descriptors are used. As the bacilli may have different sizes, positions and orientations, the shape descriptors used must be rotation, translation and scale invariant. As shown in Table 4, the most used descriptors used are: compactness, eccentricity, Hu's moments and Fourier Descriptors. Varied classifiers such as classification trees, Support Vector Machines and Neural Networks were employed by some authors in

The results presented in Table 4 show that, in bacilli detection, results for sensitivity and specificity as good as 97.77% and 99.13% are cited. It is noteworthy, however that the authors who cited these values, do not consider touching bacilli. In some cases, as the one shown in Figure 9, these bacilli are present in large quantities. Disregarding these bacilli implies a different count of what is done by manual means. Because of this, we believe that other ways of removing noise than those that use shape descriptors must be

as a bacillus, as shown in Figure 8.

order to recognize the bacilli.

investigated.


Sputum Smear Microscopy for Tuberculosis: Evaluation of Autofocus

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Bennedsen, J. & Larsen, S. O. (1966), Examination for tubercle bacilli by fluorescence, *Scandinavian Journal of Respiratory Disease*, Vol. 47, pp.114–20, ISSN 0036-5572 Costa, M. G. F., Costa Filho, C. F. F., Sena, J. F., Salen, J. & Lima, M. O. (2008), Automatic

Firestone, L., Cook, K., Culp, K., Talsania, N. & Preston Jr, K. (1991), Comparison of autofocus methods for automated microscopy, *Cytometry*, Vol. 12, pp.195-206 Forero, M. G. & Cristóbal, G. (2003), Automatic identification techniques of tuberculosis

Forero, M.G., Sroubek, F. & Cristóbal, G. (2004). Identification of tuberculosis bacteria based on shape and color, *Real Time Imaging*, Vol. 10, pp. 251–262, ISSN 1077-2014 Forero, M.G., Cristóbal, G. & Desco, M. (2006), Automatic identification of Mycobacterium

Geusebrock, J., Cornelissen, F., Smeulders, A.W.M. & Geerts, G. (2000), Robust auto focusing

Kautsky, J., Flusser, .J, Zitova, B. S. & Imberova, S., (2002), A new waveletbased measure of

Khutlang, R., Krishnan, S., Dendere, R., Whitelaw, A., Veropoulos, K., Learmonth, G. &

Kimura Junior, A., Costa, M., Costa Filho, C. F. F., Fujimoto, L. B.M. & Salem, J. (2010),

tuberculosis, *32th Annual International IEEE EMBS Conference*, pp. 3041-3044 Krotkov, E. (1987), Focusing, *International Journal of Computer Vision*, Vol. 1, pp. 223-227,

Lenseigne, B., Brodin, P., Christophe, T. & Genovesio, A. (2007), Support vector machines

Luelmo, F. (2004), What is the role of sputum microscopy in patients attending health

Makkapati, V., Agrawal, R. & Acharya, R. (2009), Segmentation and Classification of

Osibote, O.A., Dendere, R., Krishnan, S. & Douglas, T.S. (2010), Automated focusing in

tuberculosis by Gaussian mixture models, *Journal of Microscopy*, Vol. 223, pp. 120–

Douglas, T. S. (2010), Classification of Mycobacterium tuberculosis in Images of ZN-Stained Sputum Smears, *IEEE Transactions on Information Technology in* 

Evaluation of autofocus functions of conventional sputum smear microscopy for

for automatic detection of tuberculosis bacteria in confocal microscopy images, *Proceedings of 4th IEEE International Symposium on Biomedical Imaging*, pp. 85-88,

facilities?, In: *Toman's Tuberculosis Case detection, treatment, and monitoring –questions and answers*, T. Frieden, pp. 7-10, World Health Organization, ISBN 9241546034,

Tuberculosis Bacilli from ZN-stained Sputum Smear Images, *Proceddings of 5th Annual IEEE Conference on Automation Science and Engineering*, pp. 217-220, ISBN

bright-field microscopy for tuberculosis detection, *Journal of Microscopy*, Vol. 240, 2,

385,Vancouver, British Columbia, Canada

132, ISSN 0022-2720

ISSN 0920-5691

Hong Kong, China

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ISBN 0-7803-7585-8, Arlington, VA, USA

978-1-60566-750-8 , Bangalore, India

pp.155-163, ISSN 0022-2720

image focus. *Pattern Recognition Letters*, Vol. 23, pp. 1785–1794.

identification of mycobacterium tuberculosis with conventional light microscopy, *Proceedings of the 30th Annual International Conference of the IEEE EMBS*, pp. 382-


Yazdanfar, S., Kenny, K.B., Tasimi, K., Corwin, A.D., Dixon, E.L. & Filkins R.J. (2008), Simple and robust image-based auto focusing for digital microscopy. *Optics Express*. Vol. 16, pp. 8670–8677.

**14** 

**The Use of Phage for Detection, Antibiotic** 

Bacteriophage are bacterial viruses which may attack and destroy bacterial cells. While many have a narrow host range and are used in sub-typing techniques, some infect many members of a genus or species and these have been used to develop rapid detection methods for a variety of bacterial pathogens (Rees and Loessner, 2008). The use of bacteriophage (or phage) in assays for detecting bacteria was first reported over half a century ago when an assay to detect *Salmonella* using phage Felix 01 was described by Cherry et al*.* (1954). Since then other bacteriophage-based detection methods have been developed that take advantage of the specificity of the host-phage interaction and its ability, once inside the host, to replicate rapidly. This is particularly useful when studying slowgrowing organisms such as *Mycobacterium tuberculosis* and other slow growing mycobacteria such as *Mycobacterium avium* subsp. *paratuberculosis* (see Stanley et al., 2007; Grant & Rees, 2009, Botsaris et al., 2010). This chapter will provide an introduction to phage biology and will then describe the different phage-based detection methods that have been described for *M. tuberculosis* – including one that has been developed into a commercial product. In addition adaptations of the phage test are described that allow the antibiotic sensitivity of isolates to be rapidly determined, and also how a further modification can be used to allow rapid estimation of viable cell number. However, all these indirect methods have their

Mycobacteriophage, which are phage that infect any members of the *Mycobacterium* genus, were first isolated and characterized by Gardner and Weiser in 1947 and further investigation was prompted in the 1950s by their utility in typing of clinical isolates. Phage typing is a method used to sub-type members of a bacterial species based the sensitivity of a particular host strain to a panel of bacteriophage that have been shown to have a limited host range within the group (see Rees and Loessner, 2008, for a description of this method). So far over 200 different mycobacteriophage have been described, infecting a broad variety of mycobacterial hosts and these have been isolated from a variety of environmental sources, such as soil or surface water (Froman et al*.,* 1954; Caroli & Avio, 1975), and stool

limitations and these too will be discussed in each case.

**2. Mycobacteriophage** 

**1. Introduction** 

**Sensitivity Testing and Enumeration** 

Catherine Rees1 and George Botsaris2

*1University of Nottingham and 2Cyprus University of Technology* 

*1United Kingdom* 

*2Cyprus* 

## **The Use of Phage for Detection, Antibiotic Sensitivity Testing and Enumeration**

Catherine Rees1 and George Botsaris2 *1University of Nottingham and 2Cyprus University of Technology 1United Kingdom 2Cyprus* 

#### **1. Introduction**

292 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Yazdanfar, S., Kenny, K.B., Tasimi, K., Corwin, A.D., Dixon, E.L. & Filkins R.J. (2008), Simple

16, pp. 8670–8677.

and robust image-based auto focusing for digital microscopy. *Optics Express*. Vol.

Bacteriophage are bacterial viruses which may attack and destroy bacterial cells. While many have a narrow host range and are used in sub-typing techniques, some infect many members of a genus or species and these have been used to develop rapid detection methods for a variety of bacterial pathogens (Rees and Loessner, 2008). The use of bacteriophage (or phage) in assays for detecting bacteria was first reported over half a century ago when an assay to detect *Salmonella* using phage Felix 01 was described by Cherry et al*.* (1954). Since then other bacteriophage-based detection methods have been developed that take advantage of the specificity of the host-phage interaction and its ability, once inside the host, to replicate rapidly. This is particularly useful when studying slowgrowing organisms such as *Mycobacterium tuberculosis* and other slow growing mycobacteria such as *Mycobacterium avium* subsp. *paratuberculosis* (see Stanley et al., 2007; Grant & Rees, 2009, Botsaris et al., 2010). This chapter will provide an introduction to phage biology and will then describe the different phage-based detection methods that have been described for *M. tuberculosis* – including one that has been developed into a commercial product. In addition adaptations of the phage test are described that allow the antibiotic sensitivity of isolates to be rapidly determined, and also how a further modification can be used to allow rapid estimation of viable cell number. However, all these indirect methods have their limitations and these too will be discussed in each case.

#### **2. Mycobacteriophage**

Mycobacteriophage, which are phage that infect any members of the *Mycobacterium* genus, were first isolated and characterized by Gardner and Weiser in 1947 and further investigation was prompted in the 1950s by their utility in typing of clinical isolates. Phage typing is a method used to sub-type members of a bacterial species based the sensitivity of a particular host strain to a panel of bacteriophage that have been shown to have a limited host range within the group (see Rees and Loessner, 2008, for a description of this method). So far over 200 different mycobacteriophage have been described, infecting a broad variety of mycobacterial hosts and these have been isolated from a variety of environmental sources, such as soil or surface water (Froman et al*.,* 1954; Caroli & Avio, 1975), and stool

The Use of Phage for Detection, Antibiotic Sensitivity Testing and Enumeration 295

Schematic representation of the Lytic and the Lysogenic Cycles. Lytic Cycle: Infection is

bacteriophage inserts the genomic DNA located inside the virus capsid into the cell (1 & 2)

bacteriophage nucleic acids and proteins (3 & 4 and 5). Finally the phage produces enzymes (lysins) that breaks open the cell and the mature bacteriophage particles are released (6). Lysogenic Cycle: adsorption occurs in the same way (1) but the bacteriophage DNA is not replicated and instead integrates into the host cell's genome (2b). It is then replicated along with the host cell DNA at cell division and the lytic genes are not expressed (3b and 4b). When the cell divides a copy of the bacteriophage DNA is transferred along with the host chromosome (5b). Following induction into the lytic phase, the integrated phage DNA is excised from the host cell genome and the lytic genes are then expressed leading to phage

Mycobacteriophage have been used to detect slow fastidious mycobacteria, such as members of the TB complex. The main two techniques developed for detection of

mediated via receptors found in the phage tail structures. During infection the

and then it takes over the cell replication machinery and directs the synthesis of

Fig. 2. The Bacteriophage Replication Cycle

**3. Use of phage in detection assays** 

replication.

and resection specimens of patients with tuberculosis or sarcoidosis (Mankiewicz, 1961; Mankiewicz & Liivak, 1967). The collection of Mycobacteriophage is continually being expanded due to an education programme developed by researchers at the University of Pittsburgh, supported by the Howard Hughes Medical Institute Science Education Alliance. This has recently resulted in the publication of a multi-author paper describing the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations by freshmen attending Universities across within the United States (Pope et al., 2011).

Like all viruses, phage consist of a nucleic acid core and a protein coat, and considerable variation in structure has been reported. However all mycobacteriophage that have been described to date are double-stranded DNA viruses consisting of icosahedral heads with a tail which may be either short or long and either flexible (Siphoviridae) or contractile (Myoviridae) (Hatfull et al., 2008; see Figure 1). These tail structures play an essential role in host cell recognition and penetration of the bacterial cell wall structure. The comparative genomic analysis of mycobacteriophage reveals that they have relatively large genomes (average length approx. 70 kbp), contain large numbers of previously unidentified genes, and are highly diverse at both the nucleotide and amino acid sequence levels (Pedulla et al., 2003; Hatfull et al., 2010; Pope et al., 2011). Once inside the host cell the phage take over the host cell biosynthetic machinery and use this to replicate themselves, usually producing hundreds of progeny phage per infected cell (Figure 2). At the end of the replication cycle many phage produce enzymes (lysins) which degrade the cell wall and result in the lysis of the host cell and release of the progeny phage. However some phage do not always enter this lytic cycle. Instead, after entering the cell, they can enter a dormant state resulting in a latent infection. This state is known as lysogeny, but these lysogenic (or temperate) phage can be induced back into the lytic cycle, often in response to environmental conditions that either damage cellular structures or induce a stress response in the host cell (Figure 2). Both lytic and lysogenic Mycobacteriophage have been identified.

and resection specimens of patients with tuberculosis or sarcoidosis (Mankiewicz, 1961; Mankiewicz & Liivak, 1967). The collection of Mycobacteriophage is continually being expanded due to an education programme developed by researchers at the University of Pittsburgh, supported by the Howard Hughes Medical Institute Science Education Alliance. This has recently resulted in the publication of a multi-author paper describing the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations by freshmen attending Universities across within the

Like all viruses, phage consist of a nucleic acid core and a protein coat, and considerable variation in structure has been reported. However all mycobacteriophage that have been described to date are double-stranded DNA viruses consisting of icosahedral heads with a tail which may be either short or long and either flexible (Siphoviridae) or contractile (Myoviridae) (Hatfull et al., 2008; see Figure 1). These tail structures play an essential role in host cell recognition and penetration of the bacterial cell wall structure. The comparative genomic analysis of mycobacteriophage reveals that they have relatively large genomes (average length approx. 70 kbp), contain large numbers of previously unidentified genes, and are highly diverse at both the nucleotide and amino acid sequence levels (Pedulla et al., 2003; Hatfull et al., 2010; Pope et al., 2011). Once inside the host cell the phage take over the host cell biosynthetic machinery and use this to replicate themselves, usually producing hundreds of progeny phage per infected cell (Figure 2). At the end of the replication cycle many phage produce enzymes (lysins) which degrade the cell wall and result in the lysis of the host cell and release of the progeny phage. However some phage do not always enter this lytic cycle. Instead, after entering the cell, they can enter a dormant state resulting in a latent infection. This state is known as lysogeny, but these lysogenic (or temperate) phage can be induced back into the lytic cycle, often in response to environmental conditions that either damage cellular structures or induce a stress response in the host cell (Figure 2). Both

Myoviridae (panel A) typically have short, rigid contractile tails which often have associated

tail fibres. Siphoviridae (panle B) have longer, flexible, non-contractile tails which termimates in a base-plate structure and tail fibres are absent. Both types of phage have double stranded DNA genomes. The majority of Mycobacteriophage are Siphoviridae.

United States (Pope et al., 2011).

lytic and lysogenic Mycobacteriophage have been identified.

Fig. 1. Common Morphotypes of Mycobacteriophage

Schematic representation of the Lytic and the Lysogenic Cycles. Lytic Cycle: Infection is mediated via receptors found in the phage tail structures. During infection the bacteriophage inserts the genomic DNA located inside the virus capsid into the cell (1 & 2) and then it takes over the cell replication machinery and directs the synthesis of bacteriophage nucleic acids and proteins (3 & 4 and 5). Finally the phage produces enzymes (lysins) that breaks open the cell and the mature bacteriophage particles are released (6). Lysogenic Cycle: adsorption occurs in the same way (1) but the bacteriophage DNA is not replicated and instead integrates into the host cell's genome (2b). It is then replicated along with the host cell DNA at cell division and the lytic genes are not expressed (3b and 4b). When the cell divides a copy of the bacteriophage DNA is transferred along with the host chromosome (5b). Following induction into the lytic phase, the integrated phage DNA is excised from the host cell genome and the lytic genes are then expressed leading to phage replication.

#### **3. Use of phage in detection assays**

Mycobacteriophage have been used to detect slow fastidious mycobacteria, such as members of the TB complex. The main two techniques developed for detection of

The Use of Phage for Detection, Antibiotic Sensitivity Testing and Enumeration 297

compared. If light is produced from both samples then the organism detected is not M. tuberculosis. However if light is suppressed in the sample containing NAP, this indicates that *M. tuberculosis* cells have been detected (Riska et al. 1997). Using this combination of tests, Banaiee et al. (2001) reported that 94% of strains tested could be correctly identified.

Schematic representation for Reporter Phage Assays. Following the insertion of the reporter gene into the phage genome the phage infects the targeted host cell. The reporter gene is then expressed from the phage genome and as the reporter protein accumulates the signal is

Although these initial reports were promising there have been very few recent reports of the use of reporter phage as a diagnostic tool for TB. Recently, Dusthackeer et al. (2008) reported the construction of a new *Fflux* Reporter Phage using the temperate phage, Che12 – again arguing that integration of these phage would produce more sustained light levels – and also using promoters to direct the expression of the *Fflux* that were predicted to be more highly expressed in dormant bacilli (isocitrate lyase (*icl*) and alpha crystallin protein (*acr*)). They also re-engineered the promoter sequences gene in the defective TM4-based phage phAE129 (Carriere et al., 1997) to determine if light output could be improved. Interestingly, by comparing the results obtained for the two phage they showed that while these promoters did increase light output when the phage infected dormant cells, the Che12 Reporter Phage only poorly infected dormant bacilli. This is probably because it lacks a peptidoglycan hydrolase TM3 motif found in the TM4 tape- measure protein and this is required to allow phage to efficiently infect *M. smegmatis* in stationary phase when it has a

Two new Reporter Phage constructs have also been described which have been engineered to contain genes encoding the fluorescent proteins GFP or ZsYellow (Piuri et al., 2009). These were introduced into the conditionally-replicating TM4 derivative phAE87 under the control of the constitutive *M. bovis* BCG Hsp60 promoter to create the fluorophage phAE87::*hsp60*-*EGFP* and phAE87::*hsp60*-*ZsYellow.* However these were evaluated for use as a rapid, semi-automoated method for determining antibiotic sensitivity of *M. tuberculosis*

Fig. 3. General Schematic for Reporter Phage Assays

thicker or more highly cross-linked peptidoglycan layer.

detected.

mycobacteria using phage are recombinant Reporter Phage and the Phage Amplification Assay (PAA), also known as Phage Amplified Biologically (PhaB) assay. These two methods differ in how the host cell is detected at the end of the assay and both are described in more detail in the following sections.

#### **3.1 Reporter Phage assays for mycobacteria**

The Reporter Phage detection method uses genetic engineering to introduce a reporter gene into a phage genome. Since genes are not expressed inside the virion particle, no signal is produced from the reporter gene until it enters a host cell during infection. At this point the reporter gene is expressed along with the phage replication genes and this can be detected to indicate that an infection event has occurred (see Figure 3). The limitation here is the packaging constraint of the phage being used; this is the natural limit on the size of phage genome that can be packaged into a phage head. If the size of the gene being introduced into the phage exceeds the packaging constraint, the recombinant phage particles are defective. However the firefly luciferase reporter gene (*Fflux* or *luc*) has been successfully introduced into a number of mycobacteria phage (Jacobs et al., 1993; Sarkis et a., 1995; Pearson et al., 1996; Riska et al., 1997). This enzyme requires a source of ATP to produce light (bioluminescence), and this is provided by the infected cell. When the luciferin substrate for the enzyme is added (exogenously) the light can be sensitively detected by a luminometer.

The first Mycobacterial Reporter Phage described were based on the lytic phage TM4 since it was argued that a lytic phage would not be able to enter the dormant lysogenic state and therefore would produce high levels of reporter signal. However but it was found that these lysed the *Mycobacterium* cells too rapidly so that only low levels of luciferase were produced. This reduced the limit of detection when using this phage to approximately 104 mycobacterial cells (Jacobs et al., 1993). The same group also constructed a reporter phage using the temperate phage L5 and found that when the phage integrated into the chromosome there was prolonged expression of the reporter gene and this then reduced the limit of detection to approximately 102 cells after a 40 h incubation period, or 103 cells after 20 h (Sarkis et al., 1995). Although this demonstrated for the first time that reporter phage could be used to rapidly and sensitively detect Mycobacteria, this phage had a limited host range and therefore could not be developed as a practical test for *M. tuberculosis.* Instead the TM4-based reporter phage was improved by changing the site of insertion of the *Fflux* gene in the phage genome and isolating mutants of the phage to improve host range. This lead to the isolation of a TM4-based reporter phage that could detect as few as 120 *Mycobacterium bovis* BCG after 12 h of incubation (Carriere et al., 1997) which is significantly faster than any culture-based method can achieve and when tested on clinical samples it was shown that smear-positive sputum samples could be detected within 24-48 h (Riska et al., 1997). One limitation of the Reporter Phage assay for direct identification of *M. tuberculosis* in clinical samples is that these phage are able to infect a number of species of *Mycobacterium*, for instance TM4 also infects *M. bovis* and members of the *M. avium* complex (Timme and Brennan, 1984). This results in reduced specificity of the assay and hence produces falsepositive test results. To overcome this, the reporter phage have been used in combination with p-nitro-a-acetylamino-b-hydroxy propiophenone (NAP) which specifically inhibits the growth of *M. tuberculosis* complex bacteria (Eidus et al. 1960). Parallel samples, with and without the addition of NAP, are used and the results of the Reporter Phage assay

mycobacteria using phage are recombinant Reporter Phage and the Phage Amplification Assay (PAA), also known as Phage Amplified Biologically (PhaB) assay. These two methods differ in how the host cell is detected at the end of the assay and both are described in more

The Reporter Phage detection method uses genetic engineering to introduce a reporter gene into a phage genome. Since genes are not expressed inside the virion particle, no signal is produced from the reporter gene until it enters a host cell during infection. At this point the reporter gene is expressed along with the phage replication genes and this can be detected to indicate that an infection event has occurred (see Figure 3). The limitation here is the packaging constraint of the phage being used; this is the natural limit on the size of phage genome that can be packaged into a phage head. If the size of the gene being introduced into the phage exceeds the packaging constraint, the recombinant phage particles are defective. However the firefly luciferase reporter gene (*Fflux* or *luc*) has been successfully introduced into a number of mycobacteria phage (Jacobs et al., 1993; Sarkis et a., 1995; Pearson et al., 1996; Riska et al., 1997). This enzyme requires a source of ATP to produce light (bioluminescence), and this is provided by the infected cell. When the luciferin substrate for the enzyme is added (exogenously) the light can be sensitively detected by a luminometer. The first Mycobacterial Reporter Phage described were based on the lytic phage TM4 since it was argued that a lytic phage would not be able to enter the dormant lysogenic state and therefore would produce high levels of reporter signal. However but it was found that these lysed the *Mycobacterium* cells too rapidly so that only low levels of luciferase were produced. This reduced the limit of detection when using this phage to approximately 104 mycobacterial cells (Jacobs et al., 1993). The same group also constructed a reporter phage using the temperate phage L5 and found that when the phage integrated into the chromosome there was prolonged expression of the reporter gene and this then reduced the limit of detection to approximately 102 cells after a 40 h incubation period, or 103 cells after 20 h (Sarkis et al., 1995). Although this demonstrated for the first time that reporter phage could be used to rapidly and sensitively detect Mycobacteria, this phage had a limited host range and therefore could not be developed as a practical test for *M. tuberculosis.* Instead the TM4-based reporter phage was improved by changing the site of insertion of the *Fflux* gene in the phage genome and isolating mutants of the phage to improve host range. This lead to the isolation of a TM4-based reporter phage that could detect as few as 120 *Mycobacterium bovis* BCG after 12 h of incubation (Carriere et al., 1997) which is significantly faster than any culture-based method can achieve and when tested on clinical samples it was shown that smear-positive sputum samples could be detected within 24-48 h (Riska et al., 1997). One limitation of the Reporter Phage assay for direct identification of *M. tuberculosis* in clinical samples is that these phage are able to infect a number of species of *Mycobacterium*, for instance TM4 also infects *M. bovis* and members of the *M. avium* complex (Timme and Brennan, 1984). This results in reduced specificity of the assay and hence produces falsepositive test results. To overcome this, the reporter phage have been used in combination with p-nitro-a-acetylamino-b-hydroxy propiophenone (NAP) which specifically inhibits the growth of *M. tuberculosis* complex bacteria (Eidus et al. 1960). Parallel samples, with and without the addition of NAP, are used and the results of the Reporter Phage assay

detail in the following sections.

**3.1 Reporter Phage assays for mycobacteria** 

compared. If light is produced from both samples then the organism detected is not M. tuberculosis. However if light is suppressed in the sample containing NAP, this indicates that *M. tuberculosis* cells have been detected (Riska et al. 1997). Using this combination of tests, Banaiee et al. (2001) reported that 94% of strains tested could be correctly identified.

Fig. 3. General Schematic for Reporter Phage Assays

Schematic representation for Reporter Phage Assays. Following the insertion of the reporter gene into the phage genome the phage infects the targeted host cell. The reporter gene is then expressed from the phage genome and as the reporter protein accumulates the signal is detected.

Although these initial reports were promising there have been very few recent reports of the use of reporter phage as a diagnostic tool for TB. Recently, Dusthackeer et al. (2008) reported the construction of a new *Fflux* Reporter Phage using the temperate phage, Che12 – again arguing that integration of these phage would produce more sustained light levels – and also using promoters to direct the expression of the *Fflux* that were predicted to be more highly expressed in dormant bacilli (isocitrate lyase (*icl*) and alpha crystallin protein (*acr*)). They also re-engineered the promoter sequences gene in the defective TM4-based phage phAE129 (Carriere et al., 1997) to determine if light output could be improved. Interestingly, by comparing the results obtained for the two phage they showed that while these promoters did increase light output when the phage infected dormant cells, the Che12 Reporter Phage only poorly infected dormant bacilli. This is probably because it lacks a peptidoglycan hydrolase TM3 motif found in the TM4 tape- measure protein and this is required to allow phage to efficiently infect *M. smegmatis* in stationary phase when it has a thicker or more highly cross-linked peptidoglycan layer.

Two new Reporter Phage constructs have also been described which have been engineered to contain genes encoding the fluorescent proteins GFP or ZsYellow (Piuri et al., 2009). These were introduced into the conditionally-replicating TM4 derivative phAE87 under the control of the constitutive *M. bovis* BCG Hsp60 promoter to create the fluorophage phAE87::*hsp60*-*EGFP* and phAE87::*hsp60*-*ZsYellow.* However these were evaluated for use as a rapid, semi-automoated method for determining antibiotic sensitivity of *M. tuberculosis*

The Use of Phage for Detection, Antibiotic Sensitivity Testing and Enumeration 299

Grapic representation of the phage amplification assay. On the top the processing steps of the assay are presented and on the bottom the scientific details of the assay are shown. Following the appropriate sampling preparation the assay is carried out following the

transferred to a dedicated microbiology laboratory. This was perceived to be a disadvantage over conventional sputum smear testing when diagnosis was being performed in clinics in remote areas where reliable, temperature controlled transportation of samples is difficult

The issue of specificity of the phage test arises from the fact that the test utilizes the broad host range of phage D29 to allow both slow growing and fast growing Mycobacteria to be infected. As discussed in relation to the Reporter Phage assays, additional tests are therefore required to determine if the cell detected is TB or a non-tuberculosis *Mycobacterium* spp. For the commercial phage assays a cut-off of 20 plaques is applied, which is the expected number of plaques that will result from the presence of non-pathogenic Mycobacteria that have been fortuitously introduced into sputum, so that samples with fewer than 20 plaques are scored as negative. Patients with active disease generally produced samples with much higher plaque numbers due to growth of the bacteria. However Stanley et al*.* (2007) have demonstrated that PCR can also be used to increase the specificity of the D29 phage

In this report the assay was being used for the detection of MAP in raw milk samples, but the presence of other pathogenic or non-pathogenic Mycobacteria in these samples could not be ruled out. To overcome this problem, DNA was extracted from the center of the plaques formed at the end of the assay and used for PCR amplification of signature sequences so that the identity of the cell detected by the phage could be determined (see Figure 4). Since *M. tuberculosis* and *M. bovis* were both likely to be present in raw milk samples a multiplex plaque PCR assay was developed to allow simultaneous discrimination between these three organisms. The signature sequences chosen were all multicopy *IS* elements (MAP, IS*900*; TB complex IS*6110* and IS*1081*) with the size of the PCR product

Fig. 4. Diagram of Phage Amplification Assay

procedure described and presented in this figure.

(Mbulo et al., 2004; Prakash et al., 2009).

amplification assay.

isolates rather than as a detection method (see section 4) and this seems to be now accepted as the most useful application of the Reporter Phage in TB diagnostics.

However there may still be examples where the Reporter Phage may provide a useful tool for the detection of other types of pathogenic Mycobacteria. For instance Sasahara et al*.* (2004) described the use of Reporter Phage for the detection of the cattle pathogen *Mycobacterium avium* subspecies *paratuberculosis* (MAP). Using phage phAE85 >1000 cells /mL were detectable within 24-48 h. When applied to milk, the authors reported that MAP was detectable at 100 cells/ml in skim milk and 1,000 cells/ml in whole milk. While this is a useful demonstration of the rapid detection of pathogenic Mycobacteria in a food matrix, it is unlikely that this will be adopted as a rapid testing method by the food industry since these Reporter Phage are considered to be GMOs and any laboratory using them is required to work according to the local GM regulations, and the cost of implementing these is not generally compatible with food microbiology testing.

#### **3.2 Phage amplification assays**

Unlike the Reporter Phage assays, the PAA or PhaB assays do not use recombinant phage. In these assays a positive indication of the presence of mycobacteria is the formation of plaques at the end of the assay. There have been several variations of these types of assay published but the general principle is a phage protection assay (see Rees and Loessner, 2008). To initiate the assays the sample containing the target cell is first mixed with a high titre of the bacteriophage. The samples are then incubated to allow time for cell infection to occur and for the phage to enter the eclipse phase. At this point any exogenous phage that have not entered an appropriate host cell are destroyed by the addition of a virucide. Various chemicals have been described that can be used as the virucide, but the essential feature of the chemical chosen is fast inactivation of phage particles while having no effect on the viability of the host cells (Stewart et al., 1998). These internalized phage now must be detected, and in its simplest form the phage released at the end of the lytic cycle are detected by the formation of plaques (areas of no growth) in a bacterial lawn. Many of the Mycobacteria phage, such as TM4 and D29, have a broad host range and can also infect the fast growing, non-pathogen *M. smegmatis.* Hence this is often used as a rapidly growing host to produce lawns of bacterial to detect the newly released phage (see Figure 4).

This assay has been produced as commercial kits (the *FAST*plaqueTBTM or Phage Tek MB assays; www.biotec.com) and also "in house" versions have been described (McNerney et al., 2004) and its performance has been extensively reviewed (see Kalantri et al., 2005; Palomino, 2005; Dinnes et al., 2007). The assessment of the performance of these tests have been variable, with some groups reporting that that they perform very well (Albert et al., 2002; Muzaffar et al., 2002; Shenai et al., 2002; Kiraz et al., 2007) while others have reported problems with sample contamination leading to loss of results (Mbulo et al. 2004; Bonnet et al., 2009), and hence decreased sensitivity of the test. This problem has been addressed by the manufacturers by the introduction of an antibiotic supplement containing nystatin, oxacillin and aztreonam (NOA) which suppresses the growth of a wide range of Grampositive and Gram-negative bacteria and yet does not lead to significant reduction in assay sensitivity while increasing the proportion of interpretable results obtained (Albert et al., 2007; Mole et al., 2007). Although the phage assay is simple to perform, inexpensive and does not require any sophisticated or dedicated equipment, it does require the samples to be

Fig. 4. Diagram of Phage Amplification Assay

298 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

isolates rather than as a detection method (see section 4) and this seems to be now accepted

However there may still be examples where the Reporter Phage may provide a useful tool for the detection of other types of pathogenic Mycobacteria. For instance Sasahara et al*.* (2004) described the use of Reporter Phage for the detection of the cattle pathogen *Mycobacterium avium* subspecies *paratuberculosis* (MAP). Using phage phAE85 >1000 cells /mL were detectable within 24-48 h. When applied to milk, the authors reported that MAP was detectable at 100 cells/ml in skim milk and 1,000 cells/ml in whole milk. While this is a useful demonstration of the rapid detection of pathogenic Mycobacteria in a food matrix, it is unlikely that this will be adopted as a rapid testing method by the food industry since these Reporter Phage are considered to be GMOs and any laboratory using them is required to work according to the local GM regulations, and the cost of implementing these is not

Unlike the Reporter Phage assays, the PAA or PhaB assays do not use recombinant phage. In these assays a positive indication of the presence of mycobacteria is the formation of plaques at the end of the assay. There have been several variations of these types of assay published but the general principle is a phage protection assay (see Rees and Loessner, 2008). To initiate the assays the sample containing the target cell is first mixed with a high titre of the bacteriophage. The samples are then incubated to allow time for cell infection to occur and for the phage to enter the eclipse phase. At this point any exogenous phage that have not entered an appropriate host cell are destroyed by the addition of a virucide. Various chemicals have been described that can be used as the virucide, but the essential feature of the chemical chosen is fast inactivation of phage particles while having no effect on the viability of the host cells (Stewart et al., 1998). These internalized phage now must be detected, and in its simplest form the phage released at the end of the lytic cycle are detected by the formation of plaques (areas of no growth) in a bacterial lawn. Many of the Mycobacteria phage, such as TM4 and D29, have a broad host range and can also infect the fast growing, non-pathogen *M. smegmatis.* Hence this is often used as a rapidly growing host

to produce lawns of bacterial to detect the newly released phage (see Figure 4).

This assay has been produced as commercial kits (the *FAST*plaqueTBTM or Phage Tek MB assays; www.biotec.com) and also "in house" versions have been described (McNerney et al., 2004) and its performance has been extensively reviewed (see Kalantri et al., 2005; Palomino, 2005; Dinnes et al., 2007). The assessment of the performance of these tests have been variable, with some groups reporting that that they perform very well (Albert et al., 2002; Muzaffar et al., 2002; Shenai et al., 2002; Kiraz et al., 2007) while others have reported problems with sample contamination leading to loss of results (Mbulo et al. 2004; Bonnet et al., 2009), and hence decreased sensitivity of the test. This problem has been addressed by the manufacturers by the introduction of an antibiotic supplement containing nystatin, oxacillin and aztreonam (NOA) which suppresses the growth of a wide range of Grampositive and Gram-negative bacteria and yet does not lead to significant reduction in assay sensitivity while increasing the proportion of interpretable results obtained (Albert et al., 2007; Mole et al., 2007). Although the phage assay is simple to perform, inexpensive and does not require any sophisticated or dedicated equipment, it does require the samples to be

as the most useful application of the Reporter Phage in TB diagnostics.

generally compatible with food microbiology testing.

**3.2 Phage amplification assays** 

Grapic representation of the phage amplification assay. On the top the processing steps of the assay are presented and on the bottom the scientific details of the assay are shown. Following the appropriate sampling preparation the assay is carried out following the procedure described and presented in this figure.

transferred to a dedicated microbiology laboratory. This was perceived to be a disadvantage over conventional sputum smear testing when diagnosis was being performed in clinics in remote areas where reliable, temperature controlled transportation of samples is difficult (Mbulo et al., 2004; Prakash et al., 2009).

The issue of specificity of the phage test arises from the fact that the test utilizes the broad host range of phage D29 to allow both slow growing and fast growing Mycobacteria to be infected. As discussed in relation to the Reporter Phage assays, additional tests are therefore required to determine if the cell detected is TB or a non-tuberculosis *Mycobacterium* spp. For the commercial phage assays a cut-off of 20 plaques is applied, which is the expected number of plaques that will result from the presence of non-pathogenic Mycobacteria that have been fortuitously introduced into sputum, so that samples with fewer than 20 plaques are scored as negative. Patients with active disease generally produced samples with much higher plaque numbers due to growth of the bacteria. However Stanley et al*.* (2007) have demonstrated that PCR can also be used to increase the specificity of the D29 phage amplification assay.

In this report the assay was being used for the detection of MAP in raw milk samples, but the presence of other pathogenic or non-pathogenic Mycobacteria in these samples could not be ruled out. To overcome this problem, DNA was extracted from the center of the plaques formed at the end of the assay and used for PCR amplification of signature sequences so that the identity of the cell detected by the phage could be determined (see Figure 4). Since *M. tuberculosis* and *M. bovis* were both likely to be present in raw milk samples a multiplex plaque PCR assay was developed to allow simultaneous discrimination between these three organisms. The signature sequences chosen were all multicopy *IS* elements (MAP, IS*900*; TB complex IS*6110* and IS*1081*) with the size of the PCR product

The Use of Phage for Detection, Antibiotic Sensitivity Testing and Enumeration 301

Reporter Phage assays had the highest accuracy (sensitivity = 99.3%, specificity = 98.6%), with in-house phage amplification assays also performing well (sensitivity = 98.5%, specificity = 97.9%). Estimates from studies evaluating the commercial *FAST*plaque kits were slightly lower (sensitivity = 95.5%, specificity = 95.0%); however, this difference was not statistically significant (based on overlapping confidence intervals) from the LRP and inhouse assays (Minion and Pai, 2010). However this report did highlight the fact that the phage-based methods currently provide the fastest phenotypic assay for antibiotic susceptibility testing and if levels of technical failure can be reduced these assays could be a

Perhaps the most overlooked potential of these phage based assays is as a research tool for the rapid evaluation of viable count of laboratory cultures. Simple modifications of the Phage Amplification assay allow cell number to be rapidly determined, with results being available within 24 h. This method was first reported by Stanley et al. (2007) in a paper describing the development of a phage amplification assay for MAP. In this case 10-fold serial dilutions of MAP cultures were prepared and a sample of each dilution tested using the Phage Amplification assay and it was found that the number of plaques detected correlated well with the number of MAP cells in the test sample (Stanley, 2005). Rather than diluting the sample and testing each one, a modification of the *FAST*plaque assay method was devised that allowed the viable count of a sample containing an unknown number of cells. In this case the sample is simply diluted immediately prior to plating so that the plaque number can be accurately counted and then the number of cells present in the original sample determined according to the dilution factor (Botsaris et al., 2009; Figure 5).

useful clinical tool to improve patient outcomes.

Fig. 5. Extraction of Plaques and Molecular Identification

PCR tube for DNA extraction.

Schematic representation of the plaque extraction method. The plaques containing the genome of the initial targeted cell are extracted using a sterile loop. The loop is used to cut gently all 4 sides of the plaque area and then the plaque is gently lifted and placed into a

It is clear that there is not always a 1:1 relationship between the cfu and pfu counts obtained, however results for a given isolate grown under the same conditions seem to be relatively

**5. Enumeration assays** 

indicating which element had been amplified. Combining the phage assay with PCR provides a significant advantage over direct PCR detection methods since the phage test provides live/dead discrimination while the PCR assay achieves definitive molecular molecular identification of the cell detected.

By reducing sample loss due to contamination by the introduction of antibiotic supplements and increasing the specificity of the phage-based test by combining with PCR, it should be possible that a rapid and robust assay format can be achieved. However the need to perform PCR increases the complexity, cost and time required to complete the assay and the usefulness of this combination assay has yet to be evaluated for clinical cases of TB.

#### **4. Antibiotic sensitivity testing**

While the usefulness of phage detection tests for direct identification of *M. tuberculosis* in clinical samples remains questionable, the use of both Reporter Phage and PAA/PhaB assays for determining antibiotic susceptibility of clinical isolates has proven to be more a valuable tool. The principle of these tests was first outlined by Jacobs et al*.* (1993) using Reporter Phage when they demonstrated that phage growth and gene expression was inhibited if rifampicin was added to antibiotic-sensitive cells whereas if the cells were resistant to the antibiotic, signal generation was unaffected. They showed that the reporter gene signal could be detected within minutes of infection of *M. tuberculosis* with a Fflux Reporter Phage, and by comparing the results of Reporter Phage infection in the presence and absence of the antibiotic, the sensitivity of *M. tuberculosis* isolates could be determined within days. As before, this type of assay is not specific to *M. tuberculosis* and Williams et al*.* (1999) described the use of *Fflux* Reporter phage to rapidly determination of drug susceptibilities of MAP giving faster results and, in this case, also being used to determine the minimum inhibitory concentration (MIC) of the antibiotics tested more rapidly and accurately.

Similar assays have been developed for the non-recombinant phage assays by showing that phage growth is inhibited, but in this case it is the formation of plaques that is inhibited rather than the signal from a reporter gene (Wilson et al., 1997 and reviewed in Minion and Pai, 2010), and again these have been produced as a commercial test (*FAST*plaque-Response™; www.biotec.com) which can be used on direct patient specimens as well as indirect isolates. Other assay formats using microtitre plates and alternative methods of detecting phage growth have also been described (Gali et al., 2003; McNerney et al., 2007;, which have been designed to shorten the time required for testing. Most recently Bainee et al., (2008) have described a microtitre format Reporter Phage assay that can be semiautomated. Isolates were inoculated in to the wells of the microtitre dish, treated with either rifampicin (RIF), isoniazid (INH) or no antibiotic and then incubated for 40 h before samples were infected with the TM4-based Reporter Phage phAE142. After 3 h of infection the light levels in each well are determined using a microplate luminometer and the sensitivity of the strains determined by comparing the light levels of the treated and control samples. This method was shown to be able to determine both the RIF and INH resistance of clinical isolates of M. tuberculosis with a median test result time of 2 days.

Minion and Pai (2010) carried out a meta-analysis of 31 studies describing the use of the different phage assays used to determine rifampicin resistance and concluded that the Reporter Phage assays had the highest accuracy (sensitivity = 99.3%, specificity = 98.6%), with in-house phage amplification assays also performing well (sensitivity = 98.5%, specificity = 97.9%). Estimates from studies evaluating the commercial *FAST*plaque kits were slightly lower (sensitivity = 95.5%, specificity = 95.0%); however, this difference was not statistically significant (based on overlapping confidence intervals) from the LRP and inhouse assays (Minion and Pai, 2010). However this report did highlight the fact that the phage-based methods currently provide the fastest phenotypic assay for antibiotic susceptibility testing and if levels of technical failure can be reduced these assays could be a useful clinical tool to improve patient outcomes.

#### **5. Enumeration assays**

300 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

indicating which element had been amplified. Combining the phage assay with PCR provides a significant advantage over direct PCR detection methods since the phage test provides live/dead discrimination while the PCR assay achieves definitive molecular

By reducing sample loss due to contamination by the introduction of antibiotic supplements and increasing the specificity of the phage-based test by combining with PCR, it should be possible that a rapid and robust assay format can be achieved. However the need to perform PCR increases the complexity, cost and time required to complete the assay and the

While the usefulness of phage detection tests for direct identification of *M. tuberculosis* in clinical samples remains questionable, the use of both Reporter Phage and PAA/PhaB assays for determining antibiotic susceptibility of clinical isolates has proven to be more a valuable tool. The principle of these tests was first outlined by Jacobs et al*.* (1993) using Reporter Phage when they demonstrated that phage growth and gene expression was inhibited if rifampicin was added to antibiotic-sensitive cells whereas if the cells were resistant to the antibiotic, signal generation was unaffected. They showed that the reporter gene signal could be detected within minutes of infection of *M. tuberculosis* with a Fflux Reporter Phage, and by comparing the results of Reporter Phage infection in the presence and absence of the antibiotic, the sensitivity of *M. tuberculosis* isolates could be determined within days. As before, this type of assay is not specific to *M. tuberculosis* and Williams et al*.* (1999) described the use of *Fflux* Reporter phage to rapidly determination of drug susceptibilities of MAP giving faster results and, in this case, also being used to determine the minimum inhibitory concentration (MIC) of the antibiotics tested more rapidly and

Similar assays have been developed for the non-recombinant phage assays by showing that phage growth is inhibited, but in this case it is the formation of plaques that is inhibited rather than the signal from a reporter gene (Wilson et al., 1997 and reviewed in Minion and Pai, 2010), and again these have been produced as a commercial test (*FAST*plaque-Response™; www.biotec.com) which can be used on direct patient specimens as well as indirect isolates. Other assay formats using microtitre plates and alternative methods of detecting phage growth have also been described (Gali et al., 2003; McNerney et al., 2007;, which have been designed to shorten the time required for testing. Most recently Bainee et al., (2008) have described a microtitre format Reporter Phage assay that can be semiautomated. Isolates were inoculated in to the wells of the microtitre dish, treated with either rifampicin (RIF), isoniazid (INH) or no antibiotic and then incubated for 40 h before samples were infected with the TM4-based Reporter Phage phAE142. After 3 h of infection the light levels in each well are determined using a microplate luminometer and the sensitivity of the strains determined by comparing the light levels of the treated and control samples. This method was shown to be able to determine both the RIF and INH resistance of clinical

Minion and Pai (2010) carried out a meta-analysis of 31 studies describing the use of the different phage assays used to determine rifampicin resistance and concluded that the

isolates of M. tuberculosis with a median test result time of 2 days.

usefulness of this combination assay has yet to be evaluated for clinical cases of TB.

molecular identification of the cell detected.

**4. Antibiotic sensitivity testing** 

accurately.

Perhaps the most overlooked potential of these phage based assays is as a research tool for the rapid evaluation of viable count of laboratory cultures. Simple modifications of the Phage Amplification assay allow cell number to be rapidly determined, with results being available within 24 h. This method was first reported by Stanley et al. (2007) in a paper describing the development of a phage amplification assay for MAP. In this case 10-fold serial dilutions of MAP cultures were prepared and a sample of each dilution tested using the Phage Amplification assay and it was found that the number of plaques detected correlated well with the number of MAP cells in the test sample (Stanley, 2005). Rather than diluting the sample and testing each one, a modification of the *FAST*plaque assay method was devised that allowed the viable count of a sample containing an unknown number of cells. In this case the sample is simply diluted immediately prior to plating so that the plaque number can be accurately counted and then the number of cells present in the original sample determined according to the dilution factor (Botsaris et al., 2009; Figure 5).

Fig. 5. Extraction of Plaques and Molecular Identification

Schematic representation of the plaque extraction method. The plaques containing the genome of the initial targeted cell are extracted using a sterile loop. The loop is used to cut gently all 4 sides of the plaque area and then the plaque is gently lifted and placed into a PCR tube for DNA extraction.

It is clear that there is not always a 1:1 relationship between the cfu and pfu counts obtained, however results for a given isolate grown under the same conditions seem to be relatively

The Use of Phage for Detection, Antibiotic Sensitivity Testing and Enumeration 303

development of experimental design to initiate new areas of research. Once the normal pfu:cfu ratio has been established for a particular strain grown under a particular condition, the phage assay can be used to rapidly determine the number of viable cells present in a

The use of bacteriophage to type bacterial cells has been accepted as a standard microbiological method for many decades. First reports of the use of this technique for Mycobacteria appear in the 1960's and it continued to be used as a standard method for investigations of epidemiological investigations until the 1980's (see Snider et al., 1984). However the advent of molecular methods of identification and subtyping has made such phage-based methods obsolete. The difficulty of culturing with these slow growing and fastidious bacteria may have driven the development of alternative methods for detection and identification. To this end the rapid growth of phage within these cells has been exploited to produce a range of different assay methods and assay formats, all with the aim of producing rapid, simple, economic tests that can be adopted in areas where more expensive molecular diagnostic tests cannot be supported. However to date none of these has gained widespread acceptance in the clinical setting. Perhaps this is because all of these methods still require a degree of staff expertise to be able to perform the tests and therefore they do not yet fulfil the criteria required to be of use in less developed countries. However there is potential for these assays to be used effectively as an adjunct to standard culture methods to further our understanding of Mycobacteria, and perhaps as our understanding of the phage-host interaction increases we may be able to solve some of the limitations of the current phage based assays and allow them to eventually realise their potential in

Albert, H., Heydenrych, A., Brookes, R., Mole, R.J., Harley, B., et al. (2002) Performance of a

Altic L.C., Rowe M.T., and Grant I.R. (2007) UV Light inactivation of *Mycobacterium avium*

Banaiee, N., Bobadilla-del-Valle, M., Bardarov, S.Jr, Riska, P.F., Small, P.M., et al. (2001)

Banaiee, N., V. January, C. Barthus, M. Lambrick, D. RoDiti, M.A. Behr, W.R. Jacobs Jr., &

sputum specimens in South Africa. *Int J Tuberc Lung Dis.* 6: 529–537. Albert, H., A.P. Trollip, K. Linley, C. Abrahams, T. Seaman and R.J. Mole. (2007)

culture. *Applied and Environmental Microbiology*. 73: 3728–3733

rapid phage-based test, *FASTPlaque*TB, to diagnose pulmonary tuberculosis from

Development of an antimicrobial formulation for control of specimen-related contamination in phage-based diagnostic testing for tuberculosis. *Journal of Applied* 

subsp. *paratuberculosis* in milk as assessed by *FASTPlaque*TB phage assay and

Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of *Mycobacterium tuberculosis* in Mexico. *Journal of Clinical* 

L.M. Steyn (2008) Evaluation of a semi-automated reporter phage assay for susceptibility testing of *Mycobacterium tuberculosis* isolates in South Africa.

sample without the need for extended periods of incubation.

**6. Conclusion** 

combating human disease.

*Microbiology*. 103: 892–899

*Microbiology*. 39: 3883–3888

*Tuberculosis*. 88: 64–68

**7. References** 

constant (Stanley, 2005). Foddai et al*.* (2009) described the optimisation of the MAP Phage Amplification assay to accurately detect and enumerate MAP cells. They investigated the optimal buffer conditions for D29 phage infection of MAP cells, determined the minimal time for D29 incubation with MAP to produce extracellular phage (burst time) and the incubation time required before virucide treatment and assessed the impact of changing these parameters on the correlation between plaque count and corresponding colony count. To achieve a correlation closer to 1:1 between pfu/ml and cfu/ml value when detecting MAP, the authors suggested modifications to the standard protocol including supplementing the medium with 2 mM calcium chloride to enhance phage infection, incubating cells at 37 °C overnight before infection with D29 and extending the time allowed for phage infection to 2 h so that phage successfully infect a higher percentage of cells.

Fig. 6. Modification of the PAA for Enumeration of cells

Enumeration of cells using the PAA. 1 ml of the sample is infected with phage. Following adsorption exogenous phage are destroyed by the addition of a virucide which is then neutralised. Serial dilutions are made and plated on a lawn of M. smegmatis cells. Plaques (representing the target cells) are enumerated after overnight incubation.

A limited number of reports of the use of phage for enumeration have appeared in the literature – all for MAP rather than for *M. tuberculosis* – but these do demonstrate the usefulness of the method, especially when following inactivation kinetics. Altic et al. (2007) used the FPTB assay in UV inactivation studies and the authors observed that the colony counts were consistently 1 to 2 log10 higher than the plaque counts. However the rate of inactivation measured by both culture and Phage Amplification Assay were identical, so that even though a proportion of the population was detected, these cells were inactivated in the same rate as the rest of the culture. Similarly Donaghy et al. (2009) used the Phage Amplification assay to monitor UV inactivation of MAP using a novel pilot-scale UV treatment for milk. Again there was a discrepancy between the cfu and pfu values obtained, and some evidence of large differences in the infectivity of different strains of MAP, but despite this the inactivation curves obtained were identical and the pfu data was available within 24 h whereas the culture results required up to 18 weeks for growth of the colonies. This demonstrates the power of the phage-based assays to provide rapid data to allow development of experimental design to initiate new areas of research. Once the normal pfu:cfu ratio has been established for a particular strain grown under a particular condition, the phage assay can be used to rapidly determine the number of viable cells present in a sample without the need for extended periods of incubation.

#### **6. Conclusion**

302 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

constant (Stanley, 2005). Foddai et al*.* (2009) described the optimisation of the MAP Phage Amplification assay to accurately detect and enumerate MAP cells. They investigated the optimal buffer conditions for D29 phage infection of MAP cells, determined the minimal time for D29 incubation with MAP to produce extracellular phage (burst time) and the incubation time required before virucide treatment and assessed the impact of changing these parameters on the correlation between plaque count and corresponding colony count. To achieve a correlation closer to 1:1 between pfu/ml and cfu/ml value when detecting MAP, the authors suggested modifications to the standard protocol including supplementing the medium with 2 mM calcium chloride to enhance phage infection, incubating cells at 37 °C overnight before infection with D29 and extending the time allowed for phage infection to 2 h so that phage successfully infect a higher percentage of cells.

Fig. 6. Modification of the PAA for Enumeration of cells

Enumeration of cells using the PAA. 1 ml of the sample is infected with phage. Following adsorption exogenous phage are destroyed by the addition of a virucide which is then neutralised. Serial dilutions are made and plated on a lawn of M. smegmatis cells. Plaques

A limited number of reports of the use of phage for enumeration have appeared in the literature – all for MAP rather than for *M. tuberculosis* – but these do demonstrate the usefulness of the method, especially when following inactivation kinetics. Altic et al. (2007) used the FPTB assay in UV inactivation studies and the authors observed that the colony counts were consistently 1 to 2 log10 higher than the plaque counts. However the rate of inactivation measured by both culture and Phage Amplification Assay were identical, so that even though a proportion of the population was detected, these cells were inactivated in the same rate as the rest of the culture. Similarly Donaghy et al. (2009) used the Phage Amplification assay to monitor UV inactivation of MAP using a novel pilot-scale UV treatment for milk. Again there was a discrepancy between the cfu and pfu values obtained, and some evidence of large differences in the infectivity of different strains of MAP, but despite this the inactivation curves obtained were identical and the pfu data was available within 24 h whereas the culture results required up to 18 weeks for growth of the colonies. This demonstrates the power of the phage-based assays to provide rapid data to allow

(representing the target cells) are enumerated after overnight incubation.

The use of bacteriophage to type bacterial cells has been accepted as a standard microbiological method for many decades. First reports of the use of this technique for Mycobacteria appear in the 1960's and it continued to be used as a standard method for investigations of epidemiological investigations until the 1980's (see Snider et al., 1984). However the advent of molecular methods of identification and subtyping has made such phage-based methods obsolete. The difficulty of culturing with these slow growing and fastidious bacteria may have driven the development of alternative methods for detection and identification. To this end the rapid growth of phage within these cells has been exploited to produce a range of different assay methods and assay formats, all with the aim of producing rapid, simple, economic tests that can be adopted in areas where more expensive molecular diagnostic tests cannot be supported. However to date none of these has gained widespread acceptance in the clinical setting. Perhaps this is because all of these methods still require a degree of staff expertise to be able to perform the tests and therefore they do not yet fulfil the criteria required to be of use in less developed countries. However there is potential for these assays to be used effectively as an adjunct to standard culture methods to further our understanding of Mycobacteria, and perhaps as our understanding of the phage-host interaction increases we may be able to solve some of the limitations of the current phage based assays and allow them to eventually realise their potential in combating human disease.

#### **7. References**


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*Mycobacterium tuberculosis*. *J. Clin. Microbiol.* 35:3232-3239

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*of Laboratory and Clinical Medicine*. 44: 51–55

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Inactivation of *Mycobacterium avium* ssp. *paratuberculosis* in milk by UV treatment.

Hassan S., Nagamaiaha, S., Chan, J., Paranji Rama N. (2008) Construction and evaluation of luciferase reporter phages for the detection of active and non-

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Keenan, L., Bardarov, S., Kriakov, J., Lawrence, J.G., Jacobs, W.R. Jr., Hendrix, R.W. and Hatfull, G.F. (2003) Origins of highly mosaic mycobacteriophage genomes. *Cell*. 113: 171–182

**15** 

**Molecular Imaging in TB:** 

*1Imperial College London, 2University of Auckland,* 

> *1United Kingdom 2New Zealand*

**From the Bench to the Clinic** 

Nuria Andreu1, Paul T. Elkington1 and Siouxsie Wiles1,2

Despite all efforts, tuberculosis (TB) still constitutes a serious global health threat with 9.4 million new cases and 1.7 million deaths worldwide in 2009 (World Health Organisation, 2010). Furthermore, an estimated one third of the worlds' population is infected with the bacterium responsible, *Mycobacterium tuberculosis*. The main handicaps in fighting TB include a vaccine which works poorly in the most affected populations, and an arduous treatment regimen, involving a combination of several drugs taken over many months. This is further complicated by the emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) *M. tuberculosis* strains, which require even longer treatment times with less well-tolerated drugs. Eradication of TB will require the development of new drugs and vaccines, alongside improved methods for diagnosis and monitoring treatment efficacy. With the vast burden of disease falling in resource poor settings, the challenge will also be to develop methodologies that can be deployed with minimal investment in infrastructure,

Recent decades have seen the emergence of the new discipline of molecular imaging. In essence, molecular imaging enables the non-invasive visualisation, characterisation, and quantification of biological processes taking place within intact living subjects, be it a mouse or man (Filippi & Rocca, 2011; Horky & Treves, 2011; Pysz et al., 2010; Sandhu et al., 2010). Imaging has long been applied to managing TB; simple chest x-rays have allowed clinicians to visualise TB in people for over a century (Singh & Nath, 1994). However, the new molecular imaging techniques are revolutionising medical research, with the potential to translate into significant changes in clinical practice. In this chapter we describe the new generation of imaging modalities and how these are being applied to eradicating TB, from

Molecular imaging is broadly defined as the visualisation, characterisation and quantification of biological processes, at the cellular and subcellular level, within living subjects. Importantly, the non-invasive nature of the techniques enables the study of disease

**1. Introduction** 

maintenance and staff expertise.

the laboratory bench and in to the clinic.

**2. Molecular imaging modalities** 


## **Molecular Imaging in TB: From the Bench to the Clinic**

Nuria Andreu1, Paul T. Elkington1 and Siouxsie Wiles1,2 *1Imperial College London, 2University of Auckland, 1United Kingdom 2New Zealand* 

#### **1. Introduction**

306 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Keenan, L., Bardarov, S., Kriakov, J., Lawrence, J.G., Jacobs, W.R. Jr., Hendrix, R.W. and

Piuri, M., Jacobs, W. R., Jr & Hatfull, G. F. (2009). Fluoromycobacteriophages for rapid,

Pope, W.,H., Jacobs-Sera, D., Russell, D. A., Peebles, C. L., Al-Atrache, Z., et al. (2011)

Prakash, S., Katiyar, S. K., Purwar, S., Singh, J. P. (2009) Clinical evaluation of the

Sarkis, G.J., Jacobs, W.R., and Hatfull, G.F. (1995) L5 Luciferase reporter

Sasahara, K.C., Gray, M.J., Shin, S.J. and Boor, K.J. (2004) Detection of viable *Mycobacterium* 

Shenai, S., Rodrigues, C., and Mehta, A.P. (2002) Evaluation of a new phage amplification

Snider, D. E. Jr., Jones, W. D., Good, R. C. (1984) The usefulness of phage typing *Mycobacterium tuberculosis* isolates. *Am Rev Respir Dis*. 130(6):1095-9. Stanley E. (2005) The development of a rapid detection method for *Mycobacterium avium* subspecies *paratuberculosis* in milk PhD Thesis, University of Nottingham. Stanley, E.C., Mole, R.J., Smith, R.J., Glenn, S.M., Barer, M.R., McGowan, M. and Rees,

respiratory specimens. *Indian Journal of Medical Microbiology*. 27 (2): 134-8 Rees C.E.D and M.J. Loessner. (2008) Phage Identification of Bacteria, In: *Practical Handbook of Microbiology*".2nd edition E. Goldman & L. Green CRC Press, pp85-99. Riska, P.F., Jacobs, W.R.,Jr, Bloom, B.R., McKitrick, J. and Chan, J. (1997) Specific

Architecture and Evolution. *PLoS ONE* 6(1): e16329.

*of Clinical Microbiology*. 35: 3225–3231

*and Disease.* 1: 258–266

*Microbiology*. 20: 194-199

Mycobacteria. *Molec. Microbiol*., 15:1055-1067

amplification. *J Appl Microbiol.* 84(5):777-83.

serocomplex. *J Gen Microbiol*. 130, 2059–2066.

113: 171–182

*PLoS One.* 4, e4870

Hatfull, G.F. (2003) Origins of highly mosaic mycobacteriophage genomes. *Cell*.

specific, and sensitive antibiotic susceptibility testing of *Mycobacterium tuberculosis*.

Expanding the Diversity of Mycobacteriophages: Insights into Genome

mycobacteriophage-based assay in rapid detection of *Mycobacterium tuberculosis* in

identification of *Mycobacterium tuberculosis* with the luciferase reporter mycobacteriophage: use of p-nitro--acetylamino-ß-hydroxy propiophenone. *Journal* 

mycobacteriophages - A sensitive tool for the detection and assay of live

*avium* subsp. *paratuberculosis* using luciferase reporter systems. *Foodborne Pathogens* 

technology for rapid diagnosis of tuberculosis. *Indian Journal of Medical* 

C.E.D. (2007) Development of a new, combined rapid method using phage and PCR for detection and identification of viable *Mycobacterium paratuberculosis* bacteria within 48 hours, *Applied and Environmental Microbiology*. 73: 1851-1857 Stewart, G.S., Jassim, S.A., Denyer, S.P., Newby, P., Linley, K., Dhir, V.K. (1998) The specific

and sensitive detection of bacterial pathogens within 4 h using bacteriophage

*Mycobacterium avium*, *Mycobacterium intracellulare*, *Mycobacterium scrofulaceum*

based assay for determining antimicrobial susceptibility of *Mycobacterium avium*

Evaluation of a new rapid bacteriophage-based method for the drug susceptibility

Timme, T.L. and Brennan, P.J. (1984) Induction of bacteriophages from members of the

Williams, S.L., Harris, N.B. and Barletta, R.G. (1999) Development of a firefly luciferase-

Wilson, S. M., Al-Suwaidi, Z., McNerney, R., Porter, J., and Drobniewski, F. (1997)

subsp. *paratuberculosis*. *Journal of Clinical Microbiology.* 37: 304-309

testing of *Mycobacterium tuberculosis*. *Nat Med*. 3: 465– 468.

Despite all efforts, tuberculosis (TB) still constitutes a serious global health threat with 9.4 million new cases and 1.7 million deaths worldwide in 2009 (World Health Organisation, 2010). Furthermore, an estimated one third of the worlds' population is infected with the bacterium responsible, *Mycobacterium tuberculosis*. The main handicaps in fighting TB include a vaccine which works poorly in the most affected populations, and an arduous treatment regimen, involving a combination of several drugs taken over many months. This is further complicated by the emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) *M. tuberculosis* strains, which require even longer treatment times with less well-tolerated drugs. Eradication of TB will require the development of new drugs and vaccines, alongside improved methods for diagnosis and monitoring treatment efficacy. With the vast burden of disease falling in resource poor settings, the challenge will also be to develop methodologies that can be deployed with minimal investment in infrastructure, maintenance and staff expertise.

Recent decades have seen the emergence of the new discipline of molecular imaging. In essence, molecular imaging enables the non-invasive visualisation, characterisation, and quantification of biological processes taking place within intact living subjects, be it a mouse or man (Filippi & Rocca, 2011; Horky & Treves, 2011; Pysz et al., 2010; Sandhu et al., 2010). Imaging has long been applied to managing TB; simple chest x-rays have allowed clinicians to visualise TB in people for over a century (Singh & Nath, 1994). However, the new molecular imaging techniques are revolutionising medical research, with the potential to translate into significant changes in clinical practice. In this chapter we describe the new generation of imaging modalities and how these are being applied to eradicating TB, from the laboratory bench and in to the clinic.

#### **2. Molecular imaging modalities**

Molecular imaging is broadly defined as the visualisation, characterisation and quantification of biological processes, at the cellular and subcellular level, within living subjects. Importantly, the non-invasive nature of the techniques enables the study of disease

Molecular Imaging in TB: From the Bench to the Clinic 309

Radiation exposure Poor soft tissue contrast Moderately expensive

Limited sensitivity

Radiation exposure

Low spatial resolution

Radiation exposure

Low spatial resolution

Limited depth penetration Whole body imaging of humans

Long acquisition times (minutes to

Long acquisition times (minutes to

PET cyclotron or generator needed

Long acquisition times (minutes to

Expensive

Expensive

hours)

hours)

not possible

hours)

Modality Advantages Disadvantages

Whole body imaging of animals and

Whole body imaging of animals and

Whole body imaging of animals and

Whole body imaging of animals and

radionuclides, so multiple processes can be imaged simultaneously

Highly sensitive and quantitative Whole body imaging of animals Can be combined with CT for anatomical imaging of animals

Table 2. Advantages and disadvantages of imaging modalities (Adapted from Massoud &

The spatial resolution of CT is primarily limited by scanning times, the size of the x-ray source, and the sensitivity of the detection system. In addition, CT has relatively poor soft tissue contrast; generally, iodinated molecules are applied as contrast agents, owing to the high x-ray absorption coefficient of iodine (McClennan, 1994). Current iodine-based contrast agents have several limitations, including adverse reactions, renal toxicity, vascular permeation and rapid renal clearance resulting in limited imaging times. As a result, alternative contrast agents have been suggested, such as polymer-coated Bi2S3 (Rabin et al., 2006) or gold nanoparticles (Hainfield et al., 2006). Indeed, gadolinium chelate-coated gold nanoparticles have been reported as dual imaging probes for CT and magnetic resonance

Short aquisition times (minutes)

Unlimited depth penetration High spatial resolution

Good soft tissue contrast Non-ionising radiation Anatomical imaging

Unlimited depth penetration

Can be combined with CT for

Unlimited depth penetration

Can be combined with CT for

anatomical imaging

anatomical imaging Can distinguish between

Short aquisition times

Inexpensive

Anatomical imaging

Unlimited depth penetration High spatial resolution

humans

humans

humans

humans

CT

MRI

PET

SPECT

Optical

Gambhir, 2003)

processes longitudinally within the same subjects, a powerful tool indeed for elucidating host-pathogen interactions and treatment efficacy. A number of imaging modalities have emerged, which vary in their methods of image generation, spatial resolution, depth penetration and detection thresholds (Table 1). As a result each modality has different advantages and disadvantages (Table 2), suggesting the techniques should be used to complement each other to answer specific research questions.


Table 1. Features of currently employed imaging modalities (Adapted from Massoud & Gambhir, 2003)

#### **2.1 Computed Tomography (CT)**

CT imaging combines low-dose x-rays and computing to produce reconstructions of the internal organs and tissues. This is possible because diverse tissue types differentially absorb x-rays as they pass through the body. CT is not a molecular imaging tool per se, but can provide important information on anatomical changes which arise as a result of disease processes. Widely used in clinical settings, there are now a number of miniaturised machines suitable for scanning of small animals (often referred to as micro-CT). To collect data, the subject is placed on a motorised table, which then moves into the lead-encased CT machine. Inside, an x-ray source and a set of x-ray detectors rotate 360 degrees around the subject in synchrony. At every angle, the detectors record the x-rays passing through the subject to provide a digital projection which is collected and sent to a computer. The x-ray source produces a narrow, fan-shaped beam, with widths ranging from 1 to 20 mm. In axial CT, which is commonly used for head scans, the table is stationary during a rotation, after which it is moved along for the next slice. In helical CT, which is commonly used for body scans, the table moves continuously as the x-ray source and detectors rotate, producing a spiral or helical scan. Clinical machines typically have multiple rows of detectors operating side by side, so that many slices (currently up to 64) can be imaged simultaneously, reducing the overall scanning time. As an alternative to the fan-shaped x-ray beam, small animal scanners may instead use a cone-shaped beam, where the scanned subject is captured completely in one rotation, speeding up the imaging process. The data are processed by computer to produce a series of image slices representing two-dimensional (2D) or three-dimensional (3D) views of the target organ or body region.

processes longitudinally within the same subjects, a powerful tool indeed for elucidating host-pathogen interactions and treatment efficacy. A number of imaging modalities have emerged, which vary in their methods of image generation, spatial resolution, depth penetration and detection thresholds (Table 1). As a result each modality has different advantages and disadvantages (Table 2), suggesting the techniques should be used to

Modality Image generation Spatial resolution Depth penetration

1-2 mm No limit

1-2 mm No limit

(CT) x-rays 50-200 μm No limit

imaging (MRI) Radiowaves 25-100 μm No limit

Optical Visible light 2-5 mm 1-2 cm

CT imaging combines low-dose x-rays and computing to produce reconstructions of the internal organs and tissues. This is possible because diverse tissue types differentially absorb x-rays as they pass through the body. CT is not a molecular imaging tool per se, but can provide important information on anatomical changes which arise as a result of disease processes. Widely used in clinical settings, there are now a number of miniaturised machines suitable for scanning of small animals (often referred to as micro-CT). To collect data, the subject is placed on a motorised table, which then moves into the lead-encased CT machine. Inside, an x-ray source and a set of x-ray detectors rotate 360 degrees around the subject in synchrony. At every angle, the detectors record the x-rays passing through the subject to provide a digital projection which is collected and sent to a computer. The x-ray source produces a narrow, fan-shaped beam, with widths ranging from 1 to 20 mm. In axial CT, which is commonly used for head scans, the table is stationary during a rotation, after which it is moved along for the next slice. In helical CT, which is commonly used for body scans, the table moves continuously as the x-ray source and detectors rotate, producing a spiral or helical scan. Clinical machines typically have multiple rows of detectors operating side by side, so that many slices (currently up to 64) can be imaged simultaneously, reducing the overall scanning time. As an alternative to the fan-shaped x-ray beam, small animal scanners may instead use a cone-shaped beam, where the scanned subject is captured completely in one rotation, speeding up the imaging process. The data are processed by computer to produce a series of image slices representing two-dimensional

Table 1. Features of currently employed imaging modalities (Adapted from Massoud &

High energy γ-rays

Lower energy γ-rays

(2D) or three-dimensional (3D) views of the target organ or body region.

complement each other to answer specific research questions.

Computed tomography

Magnetic resonance

Positron emission tomography (PET)

Single photon emission (SPE) CT

**2.1 Computed Tomography (CT)** 

Gambhir, 2003)


Table 2. Advantages and disadvantages of imaging modalities (Adapted from Massoud & Gambhir, 2003)

The spatial resolution of CT is primarily limited by scanning times, the size of the x-ray source, and the sensitivity of the detection system. In addition, CT has relatively poor soft tissue contrast; generally, iodinated molecules are applied as contrast agents, owing to the high x-ray absorption coefficient of iodine (McClennan, 1994). Current iodine-based contrast agents have several limitations, including adverse reactions, renal toxicity, vascular permeation and rapid renal clearance resulting in limited imaging times. As a result, alternative contrast agents have been suggested, such as polymer-coated Bi2S3 (Rabin et al., 2006) or gold nanoparticles (Hainfield et al., 2006). Indeed, gadolinium chelate-coated gold nanoparticles have been reported as dual imaging probes for CT and magnetic resonance

Molecular Imaging in TB: From the Bench to the Clinic 311

PET imaging is based on the fact that the incorporated radionucleotide undergoes positive β decay and emits a positron. The positron travels a few mm before it annihilates with an electron to emit a pair of photons moving in approximately opposite directions. These photons are then detected by the scanning device. As the photons are travelling at approximately 180º to each other, it is possible to localise their source along a straight line of coincidence known as a line of response (LOR). The distribution pattern of the LORs is then used to reconstruct an image of the radioactivity distribution within the subject. One minor limitation of utilising photons is that they are differentially attenuated as they traverse different thicknesses of tissue. This attenuation results in the reconstruction of structures deep within the body as having falsely low uptake of the radiotracer. However this

Despite the great promise of PET imaging, there are a number of significant disadvantages. One is the use of ionising radiation, although this is minimised by the use of radioisotopes with short half-lives. However, these short half-lives require both costly cyclotron generators and chemical synthesis apparatus within close proximity to the scanning facility for the production of the radiotracers. This certainly limits the use of the technology within resource poor settings. Furthermore, scanning times are typically long, from minutes to

Like PET, SPECT imaging is based on the distribution and uptake of a radiolabelled tracer after injection into a subject. Unlike PET tracers, SPECT radionucleotides undergo radioactive decay and emit γ-rays of a particular energy, which are then captured by an external detector. A number of 2D projections are captured from multiple angles which, when combined, form a 3D image. Radiotracers based upon radioactive metals, such as 111In, 188Re, 131I, and 133Xe, are often used. For a summary of available SPECT radiotracers see Pysz et al., 2010. PET and SPECT imaging share many of the same advantages and disadvantages. However, while PET is more sensitive, SPECT imaging is much cheaper largely thanks to the availability of different radiotracers which are longer lived and easier to obtain. Moreover, different SPECT radiotracers have different energies enabling multiple

The electromagnetic radiation we refer to as light undergoes a range of interactions when propagating through tissue. Importantly, these interactions depend on the structural arrangement and physical properties of the micro-environment. Such interactions have led to the development of the field of optical imaging which encompasses a wide variety of methods and approaches (Table 3), from visualising tissue anatomy on the microscopic scale (Zonios et al., 2001) to the 3D localisation of a photonic signal in whole animals using

In this chapter we will focus on biophotonic imaging (BPI), a preclinical imaging technique based on the ability of light to travel through flesh. This principle is easily demonstrated by placing a torch underneath ones hand and observing the light emerging through the fingers. BPI involves the detection of visible light which arises from either the excitation of a

attenuation can be corrected for by combining PET with CT imaging.

hours, and the technique provides low spatial resolution.

tracers to be used to image different processes.

fluorescence molecular tomography (Ntziachristos, 2006).

**2.5 Optical Imaging** 

**2.4 Single Photon Emission Computed Tomography (SPECT)** 

imaging (MRI) (Alric et al, 2008). Tissue contrast can also be improved by using a dualenergy x-ray method in which the projection data are acquired using two different x-ray spectra (Taschereau et al., 2010). However, one of the major limitations of CT is radiation exposure, and while the doses are low, they are not negligible and this can limit repeated imaging of the subject.

#### **2.2 Magnetic Resonance Imaging (MRI)**

MRI is based on the interactions of atoms and molecules in a tissue of interest, upon exposure to a magnetic field. In addition to providing detailed structural images, MRI can obtain physiological information through the use of specific contrast agents. While the proton 1H is most widely used in MRI, due to the abundance of water within soft tissues, other paramagnetic atoms such as 13C, 17O, 19F, 23Na and 31P are also useful. Within an MRI scanner, a strong 'coiled' magnet produces a magnetic field with a gradient in the X, Y and Z directions, which causes nuclei to align themselves. The device also contains a radiofrequency (RF) coil which is used to produce a temporary RF pulse, resulting in a change in nuclei alignment. Following the pulse, the protons return to their baseline orientation (known as relaxation) which is detected as a change in electromagnetic flux.

The behaviour of the energy inserted into the system is described by two relaxation constants: the longitudinal relaxation time (T1) or the transverse relaxation time (T2). Different tissues have different relaxation times and this can be used to produce endogenous contrast between different tissues. Addition of exogenous contrast agents can further enhance tissue contrast by selectively shortening either T1 or T2 in a tissue of interest. According to their magnetic properties, contrast agents can be classified as paramagnetic (for example, gadolinium based agents) or superparamagnetic (for example, iron oxide nanoparticles) (reviewed in Geraldes & Laurent, 2009). Depending on their biodistribution patterns, different contrast agents can also be utilised to image specific anatomical regions. MRI is becoming widely used in both clinical and preclinical settings, with dedicated MRI machines available for humans and rodents. An advantage of MRI is that it does not involve ionising radiation, has unlimited depth penetration and good soft tissue contrast. However, it is expensive and scanning times are typically long, from minutes to hours.

#### **2.3 Positron Emission Tomography (PET)**

PET imaging involves the visualisation of a radiotracer, a biomarker labelled with a positron emitter. The positron emitters typically used are isotopes with short half-lives (several hours to a few minutes), such as 11C, 13N, 15On and 18F. Radiotracers are typically made to reflect compounds normally used by the body, such as glucose or ammonia, or molecules that bind to specific receptors. Once the radiotracer is injected into a subject, it therefore distributes based on its similarity to the original biomarker compound. The most commonly used radiotracer is an analogue of glucose labelled with 18F, [18F]-2-fluoro-deoxy-D-glucose, ([18F]- FDG). A major advantage of PET imaging is that it can be used to trace the fate of any compound, provided it can be radiolabeled with a PET isotope. As a result, the processes that can be probed using PET imaging are virtually limitless, and radiotracers for new target molecules and processes continue to be developed. For a summary of available PET radiotracers see Pysz et al., 2010. Dedicated clinical and small animal PET scanners are now available.

imaging (MRI) (Alric et al, 2008). Tissue contrast can also be improved by using a dualenergy x-ray method in which the projection data are acquired using two different x-ray spectra (Taschereau et al., 2010). However, one of the major limitations of CT is radiation exposure, and while the doses are low, they are not negligible and this can limit repeated

MRI is based on the interactions of atoms and molecules in a tissue of interest, upon exposure to a magnetic field. In addition to providing detailed structural images, MRI can obtain physiological information through the use of specific contrast agents. While the proton 1H is most widely used in MRI, due to the abundance of water within soft tissues, other paramagnetic atoms such as 13C, 17O, 19F, 23Na and 31P are also useful. Within an MRI scanner, a strong 'coiled' magnet produces a magnetic field with a gradient in the X, Y and Z directions, which causes nuclei to align themselves. The device also contains a radiofrequency (RF) coil which is used to produce a temporary RF pulse, resulting in a change in nuclei alignment. Following the pulse, the protons return to their baseline orientation (known as relaxation) which is detected as a change in electromagnetic flux.

The behaviour of the energy inserted into the system is described by two relaxation constants: the longitudinal relaxation time (T1) or the transverse relaxation time (T2). Different tissues have different relaxation times and this can be used to produce endogenous contrast between different tissues. Addition of exogenous contrast agents can further enhance tissue contrast by selectively shortening either T1 or T2 in a tissue of interest. According to their magnetic properties, contrast agents can be classified as paramagnetic (for example, gadolinium based agents) or superparamagnetic (for example, iron oxide nanoparticles) (reviewed in Geraldes & Laurent, 2009). Depending on their biodistribution patterns, different contrast agents can also be utilised to image specific anatomical regions. MRI is becoming widely used in both clinical and preclinical settings, with dedicated MRI machines available for humans and rodents. An advantage of MRI is that it does not involve ionising radiation, has unlimited depth penetration and good soft tissue contrast. However,

PET imaging involves the visualisation of a radiotracer, a biomarker labelled with a positron emitter. The positron emitters typically used are isotopes with short half-lives (several hours to a few minutes), such as 11C, 13N, 15On and 18F. Radiotracers are typically made to reflect compounds normally used by the body, such as glucose or ammonia, or molecules that bind to specific receptors. Once the radiotracer is injected into a subject, it therefore distributes based on its similarity to the original biomarker compound. The most commonly used radiotracer is an analogue of glucose labelled with 18F, [18F]-2-fluoro-deoxy-D-glucose, ([18F]- FDG). A major advantage of PET imaging is that it can be used to trace the fate of any compound, provided it can be radiolabeled with a PET isotope. As a result, the processes that can be probed using PET imaging are virtually limitless, and radiotracers for new target molecules and processes continue to be developed. For a summary of available PET radiotracers see Pysz et al., 2010. Dedicated clinical and small animal PET scanners are now

it is expensive and scanning times are typically long, from minutes to hours.

imaging of the subject.

**2.2 Magnetic Resonance Imaging (MRI)** 

**2.3 Positron Emission Tomography (PET)** 

available.

PET imaging is based on the fact that the incorporated radionucleotide undergoes positive β decay and emits a positron. The positron travels a few mm before it annihilates with an electron to emit a pair of photons moving in approximately opposite directions. These photons are then detected by the scanning device. As the photons are travelling at approximately 180º to each other, it is possible to localise their source along a straight line of coincidence known as a line of response (LOR). The distribution pattern of the LORs is then used to reconstruct an image of the radioactivity distribution within the subject. One minor limitation of utilising photons is that they are differentially attenuated as they traverse different thicknesses of tissue. This attenuation results in the reconstruction of structures deep within the body as having falsely low uptake of the radiotracer. However this attenuation can be corrected for by combining PET with CT imaging.

Despite the great promise of PET imaging, there are a number of significant disadvantages. One is the use of ionising radiation, although this is minimised by the use of radioisotopes with short half-lives. However, these short half-lives require both costly cyclotron generators and chemical synthesis apparatus within close proximity to the scanning facility for the production of the radiotracers. This certainly limits the use of the technology within resource poor settings. Furthermore, scanning times are typically long, from minutes to hours, and the technique provides low spatial resolution.

#### **2.4 Single Photon Emission Computed Tomography (SPECT)**

Like PET, SPECT imaging is based on the distribution and uptake of a radiolabelled tracer after injection into a subject. Unlike PET tracers, SPECT radionucleotides undergo radioactive decay and emit γ-rays of a particular energy, which are then captured by an external detector. A number of 2D projections are captured from multiple angles which, when combined, form a 3D image. Radiotracers based upon radioactive metals, such as 111In, 188Re, 131I, and 133Xe, are often used. For a summary of available SPECT radiotracers see Pysz et al., 2010. PET and SPECT imaging share many of the same advantages and disadvantages. However, while PET is more sensitive, SPECT imaging is much cheaper largely thanks to the availability of different radiotracers which are longer lived and easier to obtain. Moreover, different SPECT radiotracers have different energies enabling multiple tracers to be used to image different processes.

#### **2.5 Optical Imaging**

The electromagnetic radiation we refer to as light undergoes a range of interactions when propagating through tissue. Importantly, these interactions depend on the structural arrangement and physical properties of the micro-environment. Such interactions have led to the development of the field of optical imaging which encompasses a wide variety of methods and approaches (Table 3), from visualising tissue anatomy on the microscopic scale (Zonios et al., 2001) to the 3D localisation of a photonic signal in whole animals using fluorescence molecular tomography (Ntziachristos, 2006).

In this chapter we will focus on biophotonic imaging (BPI), a preclinical imaging technique based on the ability of light to travel through flesh. This principle is easily demonstrated by placing a torch underneath ones hand and observing the light emerging through the fingers. BPI involves the detection of visible light which arises from either the excitation of a

Molecular Imaging in TB: From the Bench to the Clinic 313

to image colonic pathology (Hsiung et al., 2008). The advantages of BPI are that it is

*M. tuberculosis*, the infectious agent of TB, can infect many animals in addition to its natural human host. Although the study of TB in patients is extremely useful, a detailed analysis of the pathogenesis and the interactions of *M. tuberculosis* with the host requires the use of well-defined models that can be infected in a controlled manner. Furthermore, animal models can be easily manipulated, can be used in statistically significant numbers, and the results are obtained in a relatively short time frame. In addition, they are particularly useful in drug and vaccine efficacy testing before moving the most promising candidates to clinical

For both practical and economical reasons, laboratory mice remain the most extensively used animal model of TB: they are easy to manipulate and house, there is a wide range of mutant and genetically modified strains, and there are many immunological reagents available. However, latent infection is difficult to achieve in the mouse model, and the pathology, with poorly organised granulomas, differs considerably to that observed in humans. By contrast, guinea pigs and particularly rabbits display a spectrum of pathology that better represents the human disease. Moreover, guinea pigs are extremely susceptible to *M. tuberculosis* infection and relatively inexpensive compared to other larger animal models, which makes this model very useful for vaccine efficacy studies. Even so, studies with guinea pigs and rabbits are limited by the narrow range of immunological reagents available. This is not the case in non-human primates, which are the closest model to humans in terms of pathology and disease development and therefore constitute the most relevant model to predict treatment and vaccine efficacy. Nevertheless, work with nonhuman primates presents many limitations regarding space requirements, animal availability, and costs. In summary, each animal model presents both advantages and disadvantages which must be carefully considered when designing a new study. A more detailed description of these animal models of TB can be found elsewhere (Dharmadhikari

The use of animals in research is accompanied by ethical responsibilities and most countries promote the three Rs: replacement, reduction and refinement. Replacement refers to methods that avoid the use of animals, for example, *in silico* computer modelling, or using established human and animal cell lines and non-mammalian models such as the nematode *Caenorhabditis elegans* or the embryo of the zebrafish, *Danio rerio*. Reduction refers to methods which minimise the use of animals and enable researchers to obtain comparable levels of information from fewer animals or to obtain more information from the same number of animals, thereby reducing the future use of animals. Refinement refers to improvements to scientific procedures and husbandry which minimise actual or potential pain, suffering, distress or lasting harm and/or improve animal welfare. Molecular imaging is a very powerful tool for implementation of two of the 3Rs, refinement and reduction. Using traditional disease models, infected animals are sacrificed at defined time points and tissues excised for determination of pathogen numbers and localisation. In contrast, the nondestructive nature of molecular imaging allows the course of an infection to be monitored simply by repeated imaging of the same group of animals. Importantly, this allows disease

inexpensive, sensitive and requires short imaging times.

& Nardell, 2008; Flynn, 2006; Gupta & Katoch, 2005).

studies.

**3. Use of molecular imaging in animal models of TB** 


fluorescent protein (FP), or molecule, or from an enzyme-catalysed oxidation reaction (a phenomenon known as bioluminescence).

Key: A, Absorption; Fl, fluorescence; S, Scattering; E, Emission.

Table 3. Optical imaging techniques (taken from N. Andreu et al., 2011).

Bioluminescence arises from the oxidation of a substrate (a luciferin) by an enzyme (a luciferase), which usually requires energy (in the form of FMNH2 and ATP) and oxygen. Luciferin and luciferase are generic terms as none of the major classes share sequence homology. Most widely studied are the systems belonging to luminous beetles in the family Lampyridae (such as the firefly *Photinus pyralis*), the sea pansy *Renilla reniformans*, the marine copepod *Gaussia princeps* and numerous luminous bacteria (such as *Vibrio* sp. and *Photorhabdus luminescens*). In contrast, fluorescence arises when a fluorescent compound is irradiated with light of a suitable wavelength. This leads to the transition of an electron in the molecule to a higher energy state, a process known as excitation. This process is almost instantaneous, taking around 10-15 seconds. Upon return of the electron to a lower energy level (around 10 ns), light of lower energy is emitted, giving the fluorescent signal.

Although the emitted light may be dim, it can be detected externally using sensitive photon detectors such as those based on cooled, or intensified, charge coupled device (CCD) cameras, mounted within light-tight specimen chambers. As light passes through a range of tissue types (including skin, muscle and bone) it is possible to observe and quantify the spatial and temporal distribution of light production from within living subjects (N. Andreu et al, 2011). In general, imaging of luminescence is much more sensitive than imaging fluorescence as a result of better signal-to-noise ratios. This is mainly due to the high levels of background fluorescence *in vivo* compared to luminescence, due to endogenously produced fluorophores such as keratin, porphyrins, NAD(P)H, collagen and elastin (Troy et al., 2004). A major limitation of BPI is the limited depth penetration through tissue. Hence BPI is currently only applied to imaging small animals, although visualisation of bioluminescence from within infant monkeys (the long-tailed macaque, *Macaca fascicularis*) has been reported (Tarantal et al., 2006). Alternatively, the light could potentially be detected internally using an endoscopic device, such as reported by Hsiung and colleagues

fluorescent protein (FP), or molecule, or from an enzyme-catalysed oxidation reaction (a

Resolution Technique Contrast Depth

Bioluminescence arises from the oxidation of a substrate (a luciferin) by an enzyme (a luciferase), which usually requires energy (in the form of FMNH2 and ATP) and oxygen. Luciferin and luciferase are generic terms as none of the major classes share sequence homology. Most widely studied are the systems belonging to luminous beetles in the family Lampyridae (such as the firefly *Photinus pyralis*), the sea pansy *Renilla reniformans*, the marine copepod *Gaussia princeps* and numerous luminous bacteria (such as *Vibrio* sp. and *Photorhabdus luminescens*). In contrast, fluorescence arises when a fluorescent compound is irradiated with light of a suitable wavelength. This leads to the transition of an electron in the molecule to a higher energy state, a process known as excitation. This process is almost instantaneous, taking around 10-15 seconds. Upon return of the electron to a lower energy

level (around 10 ns), light of lower energy is emitted, giving the fluorescent signal.

Although the emitted light may be dim, it can be detected externally using sensitive photon detectors such as those based on cooled, or intensified, charge coupled device (CCD) cameras, mounted within light-tight specimen chambers. As light passes through a range of tissue types (including skin, muscle and bone) it is possible to observe and quantify the spatial and temporal distribution of light production from within living subjects (N. Andreu et al, 2011). In general, imaging of luminescence is much more sensitive than imaging fluorescence as a result of better signal-to-noise ratios. This is mainly due to the high levels of background fluorescence *in vivo* compared to luminescence, due to endogenously produced fluorophores such as keratin, porphyrins, NAD(P)H, collagen and elastin (Troy et al., 2004). A major limitation of BPI is the limited depth penetration through tissue. Hence BPI is currently only applied to imaging small animals, although visualisation of bioluminescence from within infant monkeys (the long-tailed macaque, *Macaca fascicularis*) has been reported (Tarantal et al., 2006). Alternatively, the light could potentially be detected internally using an endoscopic device, such as reported by Hsiung and colleagues

Epi microscopy A, Fl 20 µm Confocal microscopy Fl 500 µm Multi-photon microscopy Fl 800 µm

Optical projection tomography A, Fl 15 mm Optical coherence tomography S 2 mm Laser speckle imaging S 1 mm

Hyperspectral imaging A, S, Fl <5 mm Endoscopy A, S, Fl <5 mm Fluorescence reflectance imaging (FRI) A, Fl <7 mm Diffuse optical tomography (DOT) A, Fl <20 cm Fluorescence resonance imaging (FRI) A, Fl <7 mm Fluorescence molecular tomography (FMT) Fl <20 cm Biophotonic Imaging (BPI) Fl, E < 3cm

phenomenon known as bioluminescence).

Key: A, Absorption; Fl, fluorescence; S, Scattering; E, Emission.

Table 3. Optical imaging techniques (taken from N. Andreu et al., 2011).

Microscopic

Mesoscopic

Macroscopic

to image colonic pathology (Hsiung et al., 2008). The advantages of BPI are that it is inexpensive, sensitive and requires short imaging times.

#### **3. Use of molecular imaging in animal models of TB**

*M. tuberculosis*, the infectious agent of TB, can infect many animals in addition to its natural human host. Although the study of TB in patients is extremely useful, a detailed analysis of the pathogenesis and the interactions of *M. tuberculosis* with the host requires the use of well-defined models that can be infected in a controlled manner. Furthermore, animal models can be easily manipulated, can be used in statistically significant numbers, and the results are obtained in a relatively short time frame. In addition, they are particularly useful in drug and vaccine efficacy testing before moving the most promising candidates to clinical studies.

For both practical and economical reasons, laboratory mice remain the most extensively used animal model of TB: they are easy to manipulate and house, there is a wide range of mutant and genetically modified strains, and there are many immunological reagents available. However, latent infection is difficult to achieve in the mouse model, and the pathology, with poorly organised granulomas, differs considerably to that observed in humans. By contrast, guinea pigs and particularly rabbits display a spectrum of pathology that better represents the human disease. Moreover, guinea pigs are extremely susceptible to *M. tuberculosis* infection and relatively inexpensive compared to other larger animal models, which makes this model very useful for vaccine efficacy studies. Even so, studies with guinea pigs and rabbits are limited by the narrow range of immunological reagents available. This is not the case in non-human primates, which are the closest model to humans in terms of pathology and disease development and therefore constitute the most relevant model to predict treatment and vaccine efficacy. Nevertheless, work with nonhuman primates presents many limitations regarding space requirements, animal availability, and costs. In summary, each animal model presents both advantages and disadvantages which must be carefully considered when designing a new study. A more detailed description of these animal models of TB can be found elsewhere (Dharmadhikari & Nardell, 2008; Flynn, 2006; Gupta & Katoch, 2005).

The use of animals in research is accompanied by ethical responsibilities and most countries promote the three Rs: replacement, reduction and refinement. Replacement refers to methods that avoid the use of animals, for example, *in silico* computer modelling, or using established human and animal cell lines and non-mammalian models such as the nematode *Caenorhabditis elegans* or the embryo of the zebrafish, *Danio rerio*. Reduction refers to methods which minimise the use of animals and enable researchers to obtain comparable levels of information from fewer animals or to obtain more information from the same number of animals, thereby reducing the future use of animals. Refinement refers to improvements to scientific procedures and husbandry which minimise actual or potential pain, suffering, distress or lasting harm and/or improve animal welfare. Molecular imaging is a very powerful tool for implementation of two of the 3Rs, refinement and reduction. Using traditional disease models, infected animals are sacrificed at defined time points and tissues excised for determination of pathogen numbers and localisation. In contrast, the nondestructive nature of molecular imaging allows the course of an infection to be monitored simply by repeated imaging of the same group of animals. Importantly, this allows disease

Molecular Imaging in TB: From the Bench to the Clinic 315

To our knowledge, MRI was the first molecular imaging method reported for an animal model of TB, when Kraft and colleagues used the technique to assess lesion distribution and lesion numbers as an indication of disease burden in BCG-vaccinated and unvaccinated guinea pigs infected with *M. tuberculosis* by the aerosol route (Kraft et al., 2004). 3D lung images were reconstructed from images taken of 2 mm slices of formalin-fixed and agaroseembedded lungs, and lung volumes, lymph node volumes and total nodular burden were quantified. Small nodules were observed 15 days post-infection, which developed into granulomatous lesions 20 days later. Lesions were uniformly distributed in the lungs, which suggested that aerosol delivery of *M. tuberculosis* results in a homogenous infection. Additionally, lesions numbers supported the hypothesis that a single bacillus establishes a single lesion. In terms of vaccine efficacy, the authors found the same number of lesions in vaccinated and unvaccinated animals but the lesions were smaller in the vaccinated group, thus suggesting that BCG has an effect on disease development rather than on the initial establishment of the infection. All in all, they found that MRI was a useful method to assess disease burden in terms of lesion distribution, size and number. The main limitation was a

More recently, the same laboratory used MRI to assess treatment efficacy in guinea pigs infected with *M. tuberculosis* (Ordway et al., 2010). The treatment had a dramatic effect on bacterial load with a 4-6 log decrease in viable counts (as determined by colony forming units [CFUs]) both in the lungs and lymph nodes in just 25 days. However, the effect on lesion burden, as quantified by MRI, was slower and could only be detected in the lungs after 50 days of therapy (Figure 1). In addition, the lesions in the lymph nodes of the treated group were smaller, although the differences with the untreated group were only obvious at later time points. These results were corroborated by histological analysis, although the number of lesions in the lungs of the treated animals was already lower than in the control

MRI has also been applied to studies in non-human primates (Sharpe et al., 2009, 2010). Disease burden in this animal model has been traditionally assessed by a range of ante- and post-mortem methods such as clinical signs (behaviour, weight, and body temperature), laboratory markers (haemoglobin levels, erythrocyte sedimentation rate, and immunology),

Day 29 Day 50 Day 78 Day 105

Fig. 1. MRI showing lesions resolving/disappearing during treatment of *M. tuberculosis*infected guinea pigs with a cocktail of anti-TB drugs (given as days post-treatment).

**3.2 Magnetic Resonance Imaging (MRI)** 

group by day 25.

low sensitivity when dealing with very small (< 1 mm) lesions.

progression to be followed with extreme accuracy, while allowing each animal to act as its own control. Furthermore, we have demonstrated that BPI can provide real time information on the effectiveness of the inoculation method (Wiles et al., 2007). As a result, errors in administration can be detected immediately (N. Andreu et al., 2011) and animals eliminated from further study – thus minimising any potential pain, suffering and distress for the animal and reducing variation by removing flawed scientific data.

One major drawback to working with *M. tuberculosis* is the slow growth of the organism. This lengthens the time required to carry out *in vivo* experiments extraordinarily, and delays the quantification of bacterial burdens by about four weeks, which is the time required for *M. tuberculosis* to form visible colonies on agar. Therefore, the use of molecular imaging to track infection dynamics in real time would be a major advantage as it would enable researchers to make on-the-spot decisions, shortening the length of the experiment if clear differences (for example, between control and vaccinated groups) were observed. There is, therefore, an increasing interest in the development of molecular imaging techniques in animal models of TB. Moreover, the developments and knowledge acquired through the use of these techniques in animal models may eventually translate into the clinic.

#### **3.1 Computed Tomography (CT)**

CT imaging has mostly been used as a complementary technique to PET and SPECT imaging (see sections 3.3 and 3.4), as it gives high-resolution anatomical information for a better localisation of the radionuclide signal. However, CT has also been evaluated as an imaging method on its own to assess disease burden in macaques (Lewinsohn et al., 2006). To this end, four animals were infected by bronchoscopy instillation of *M. tuberculosis*, and disease progression was monitored every four weeks clinically (weight, body temperature, complete blood count and erythrocyte sedimentation rate), immunologically (ELISPOT), bacteriologically (quantitative *M. tuberculosis* culture from bronchoalveolar lavage), and by CT imaging. In addition, a necropsy was performed at the end of the experiment (12 weeks post-infection) which included histopathology and bacterial burden quantification from selected organs. Clinical indicators failed to provide information about disease progression, as most of them were fairly constant through the whole experiment. Most bacterial cultures from bronchoalveolar lavage were positive, although some cultures were negative even though CT imaging and post-mortem analysis showed infection. Even bacterial cultures from post-mortem lung samples were not consistently positive, which was attributed to a non-uniform infection of the lungs and therefore biased tissue sampling. In contrast, CT imaging provided a reliable readout of disease progression in the whole lung and also allowed monitoring of other organs, such as the liver and spleen. Moreover, different types of lesions were observed, and progression of the lesions from small nodules to cavitation and necrosis was evident. CT findings were corroborated by post-mortem histopathology and, together with immunological monitoring, provided a non-invasive, accurate, and rapid assessment of TB in this animal model. It is important to note that even though a CT scanner was not available in the animal biosafety level 3 (BSL3) containment facility, the authors were able to image infected animals in a scanner localised within a non-containment facility, by transporting and imaging the anesthetised macaques in a box fitted with HEPA filters. This is a solution that has also been adopted for other imaging techniques like PET/CT, SPECT/CT and BPI (see below).

#### **3.2 Magnetic Resonance Imaging (MRI)**

314 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

progression to be followed with extreme accuracy, while allowing each animal to act as its own control. Furthermore, we have demonstrated that BPI can provide real time information on the effectiveness of the inoculation method (Wiles et al., 2007). As a result, errors in administration can be detected immediately (N. Andreu et al., 2011) and animals eliminated from further study – thus minimising any potential pain, suffering and distress

One major drawback to working with *M. tuberculosis* is the slow growth of the organism. This lengthens the time required to carry out *in vivo* experiments extraordinarily, and delays the quantification of bacterial burdens by about four weeks, which is the time required for *M. tuberculosis* to form visible colonies on agar. Therefore, the use of molecular imaging to track infection dynamics in real time would be a major advantage as it would enable researchers to make on-the-spot decisions, shortening the length of the experiment if clear differences (for example, between control and vaccinated groups) were observed. There is, therefore, an increasing interest in the development of molecular imaging techniques in animal models of TB. Moreover, the developments and knowledge acquired through the use

CT imaging has mostly been used as a complementary technique to PET and SPECT imaging (see sections 3.3 and 3.4), as it gives high-resolution anatomical information for a better localisation of the radionuclide signal. However, CT has also been evaluated as an imaging method on its own to assess disease burden in macaques (Lewinsohn et al., 2006). To this end, four animals were infected by bronchoscopy instillation of *M. tuberculosis*, and disease progression was monitored every four weeks clinically (weight, body temperature, complete blood count and erythrocyte sedimentation rate), immunologically (ELISPOT), bacteriologically (quantitative *M. tuberculosis* culture from bronchoalveolar lavage), and by CT imaging. In addition, a necropsy was performed at the end of the experiment (12 weeks post-infection) which included histopathology and bacterial burden quantification from selected organs. Clinical indicators failed to provide information about disease progression, as most of them were fairly constant through the whole experiment. Most bacterial cultures from bronchoalveolar lavage were positive, although some cultures were negative even though CT imaging and post-mortem analysis showed infection. Even bacterial cultures from post-mortem lung samples were not consistently positive, which was attributed to a non-uniform infection of the lungs and therefore biased tissue sampling. In contrast, CT imaging provided a reliable readout of disease progression in the whole lung and also allowed monitoring of other organs, such as the liver and spleen. Moreover, different types of lesions were observed, and progression of the lesions from small nodules to cavitation and necrosis was evident. CT findings were corroborated by post-mortem histopathology and, together with immunological monitoring, provided a non-invasive, accurate, and rapid assessment of TB in this animal model. It is important to note that even though a CT scanner was not available in the animal biosafety level 3 (BSL3) containment facility, the authors were able to image infected animals in a scanner localised within a non-containment facility, by transporting and imaging the anesthetised macaques in a box fitted with HEPA filters. This is a solution that has also been adopted for other imaging techniques like PET/CT,

for the animal and reducing variation by removing flawed scientific data.

of these techniques in animal models may eventually translate into the clinic.

**3.1 Computed Tomography (CT)** 

SPECT/CT and BPI (see below).

To our knowledge, MRI was the first molecular imaging method reported for an animal model of TB, when Kraft and colleagues used the technique to assess lesion distribution and lesion numbers as an indication of disease burden in BCG-vaccinated and unvaccinated guinea pigs infected with *M. tuberculosis* by the aerosol route (Kraft et al., 2004). 3D lung images were reconstructed from images taken of 2 mm slices of formalin-fixed and agaroseembedded lungs, and lung volumes, lymph node volumes and total nodular burden were quantified. Small nodules were observed 15 days post-infection, which developed into granulomatous lesions 20 days later. Lesions were uniformly distributed in the lungs, which suggested that aerosol delivery of *M. tuberculosis* results in a homogenous infection. Additionally, lesions numbers supported the hypothesis that a single bacillus establishes a single lesion. In terms of vaccine efficacy, the authors found the same number of lesions in vaccinated and unvaccinated animals but the lesions were smaller in the vaccinated group, thus suggesting that BCG has an effect on disease development rather than on the initial establishment of the infection. All in all, they found that MRI was a useful method to assess disease burden in terms of lesion distribution, size and number. The main limitation was a low sensitivity when dealing with very small (< 1 mm) lesions.

More recently, the same laboratory used MRI to assess treatment efficacy in guinea pigs infected with *M. tuberculosis* (Ordway et al., 2010). The treatment had a dramatic effect on bacterial load with a 4-6 log decrease in viable counts (as determined by colony forming units [CFUs]) both in the lungs and lymph nodes in just 25 days. However, the effect on lesion burden, as quantified by MRI, was slower and could only be detected in the lungs after 50 days of therapy (Figure 1). In addition, the lesions in the lymph nodes of the treated group were smaller, although the differences with the untreated group were only obvious at later time points. These results were corroborated by histological analysis, although the number of lesions in the lungs of the treated animals was already lower than in the control group by day 25.

MRI has also been applied to studies in non-human primates (Sharpe et al., 2009, 2010). Disease burden in this animal model has been traditionally assessed by a range of ante- and post-mortem methods such as clinical signs (behaviour, weight, and body temperature), laboratory markers (haemoglobin levels, erythrocyte sedimentation rate, and immunology),

Fig. 1. MRI showing lesions resolving/disappearing during treatment of *M. tuberculosis*infected guinea pigs with a cocktail of anti-TB drugs (given as days post-treatment).

Molecular Imaging in TB: From the Bench to the Clinic 317

defined necrotic granulomas), and evaluated three different treatments: (i) first line tuberculosis regimen (rifampin + pyrazinamide + isoniazid), (ii) a more bactericidal regimen (rifampin + pyrazinamide + moxifloxacin), and (iii) a bacteriostatic regimen (ethambutol). The animals were imaged and CFUs obtained at different time points during the 12 weeks of treatment. Furthermore, one group of BALB/c mice was followed for 22 weeks after completion of the bactericidal treatments to assess relapse of the infection. For the imaging, anesthetised mice were contained in a sealed device with holes for passage of gases fitted with 0.22 m filters. In both mouse models, CFU counts perfectly reflected the efficiency of the three treatments being evaluated, with a faster decrease in bacterial numbers when moxifloxacin was used instead of isoniazid, and stabilization of bacterial burden when mice

Fig. 2. 3D co-registered PET and CT images from a live C3HeB/FeJ mouse infected with a low-dose aerosol of *M. tuberculosis.* The brightness of the lesions represents FDG activity, with brighter lesions being more active. The heart also takes up FDG and can therefore be seen on the left. The bony structure (rib cage and scapula), shown in grey, were extracted

The use of PET/CT imaging allowed differentiation between the bacteriostatic and bactericidal treatments, as the [18F]-FDG activity was higher in the mice treated with ethambutol. However, unexpectedly, [18F]-FDG activity was higher in mice treated with moxifloxacin than in those treated with isoniazid during the first four weeks of treatment. The authors suggested that this could be due to the limited statistical power of the study since only three mice per group were used, or that it could be an inherent limitation of using [18F]-FDG, whereby an increased killing of *M. tuberculosis* would cause an increased Tumour Necrosis Factor (TNF)-mediated inflammation and therefore increased [18F]-FDG activity even though bacterial numbers were decreasing. Relapse was detected in both groups of mice by PET imaging and by CFU counts. In summary, PET/CT allowed the non-invasive monitoring of disease progression in real-time. Moreover, individual lesions could be observed in the C3HeB/FeJ mouse model; as treatment response has been suggested to be lesion-dependent, the possibility of monitoring individual lesions would be very useful. However, it is important to take into account that this method does not specifically image infection but only measures inflammation which, as illustrated by the treatment results of this work, does not always correlate with bacterial burden. Nevertheless, this method has some advantages over using, for example, CFU counts: it uses a reduced number of animals, and the same animals can be repeatedly imaged which allows a more easy detection of

from the CT (Davis, S.L. & Jain, S.K.; unpublished data).

were treated with ethambutol only.

untimed events such as relapse.

chest x-ray, gross pathology, and histology. However, most of these methods are qualitative and subjective. Moreover, the most common alternative method, the quantitative estimation of total lesion numbers in the lung by manual counting, is laborious and particularly difficult in animals with more severe disease, as individual lesions become difficult to distinguish. Sharpe and colleagues used MRI and stereology (a statistical method that extracts quantitative information of a 3D structure from measurements made on planar sections of the material) to quantify lesion volume relative to lung volume in macaques infected with a range of doses of *M. tuberculosis* by the aerosol route (Sharpe et al., 2009). Similarly to what was previously seen in guinea pigs, the authors observed a uniform distribution of lesions in the lungs. In addition, the lesion-to-lung volume ratio increased with the infectious dose, and this ratio revealed subtle differences in the level of pulmonary disease and correlated well with other measures of disease burden. By contrast, methods such as gross pathology and chest x-ray were less sensitive and did not differentiate between the levels of disease in the animals exposed to the highest infectious doses. In conclusion, MRI together with stereology makes up a sensitive, quantitative, systematic and consistent method to assess disease burden in the macaque model of tuberculosis. Moreover, when MRI was compared with more traditional methods to measure vaccine efficacy, it was found that MRI combined with stereology was the only readout that distinguished between the unvaccinated and the vaccinated groups, and it was even able to show differences between survivor and non-survivor animals within the vaccinated groups, thus highlighting the sensitivity of the method (Sharpe et al., 2010).

In summary, the use of MRI appears to be a reliable method to assess disease burden in the lungs of *M. tuberculosis*-infected animals. However, it should be noted that the studies described here were performed on fixed lungs where the bacteria had been inactivated, as the use of MRI under BSL3 containment was not available. Initially, the whole lung was fixed and used for imaging to reduce sample error. As a result the tissue could not be used for other procedures, such as determination of bacterial load. However, the results discussed above illustrate that aerosol delivery of *M. tuberculosis* results in an even distribution of the lesions in the lungs and, therefore, samples can be taken and used for other techniques without compromising its reliability. Similarly to what has been done to image live animals by CT scanning, MRI of live animals could be done by using a sealed box with filters to transport the animals to the MRI facility and contain them during imaging. When available, MRI of live animals will allow longitudinal monitoring of disease progression, and real-time observation of vaccine and drug efficacy. The *ex vivo* results discussed here, together with the excellent soft tissue contrast of MRI and the development of faster MRI devices that reduce the artefacts induced by respiratory motion, suggest that *in vivo* MRI may become a very useful technique for the study of TB in animal models.

#### **3.3 Positron Emission Tomography (PET)**

Another technique which is gaining popularity in TB research is PET combined with CT imaging (PET/CT). The PET radiotracer [18F]-FDG is used to image inflammation at the infection site, as it accumulates in inflammatory cells such as neutrophils and activated macrophages. This technology has been used to image TB infection (Figure 2) and to assess drug treatment efficacy in mice (Davis et al., 2009b). The authors infected two strains of mice, BALB/c (which develop diffused granulomas) and C3HeB/FeJ (which develop well-

chest x-ray, gross pathology, and histology. However, most of these methods are qualitative and subjective. Moreover, the most common alternative method, the quantitative estimation of total lesion numbers in the lung by manual counting, is laborious and particularly difficult in animals with more severe disease, as individual lesions become difficult to distinguish. Sharpe and colleagues used MRI and stereology (a statistical method that extracts quantitative information of a 3D structure from measurements made on planar sections of the material) to quantify lesion volume relative to lung volume in macaques infected with a range of doses of *M. tuberculosis* by the aerosol route (Sharpe et al., 2009). Similarly to what was previously seen in guinea pigs, the authors observed a uniform distribution of lesions in the lungs. In addition, the lesion-to-lung volume ratio increased with the infectious dose, and this ratio revealed subtle differences in the level of pulmonary disease and correlated well with other measures of disease burden. By contrast, methods such as gross pathology and chest x-ray were less sensitive and did not differentiate between the levels of disease in the animals exposed to the highest infectious doses. In conclusion, MRI together with stereology makes up a sensitive, quantitative, systematic and consistent method to assess disease burden in the macaque model of tuberculosis. Moreover, when MRI was compared with more traditional methods to measure vaccine efficacy, it was found that MRI combined with stereology was the only readout that distinguished between the unvaccinated and the vaccinated groups, and it was even able to show differences between survivor and non-survivor animals within the vaccinated groups,

In summary, the use of MRI appears to be a reliable method to assess disease burden in the lungs of *M. tuberculosis*-infected animals. However, it should be noted that the studies described here were performed on fixed lungs where the bacteria had been inactivated, as the use of MRI under BSL3 containment was not available. Initially, the whole lung was fixed and used for imaging to reduce sample error. As a result the tissue could not be used for other procedures, such as determination of bacterial load. However, the results discussed above illustrate that aerosol delivery of *M. tuberculosis* results in an even distribution of the lesions in the lungs and, therefore, samples can be taken and used for other techniques without compromising its reliability. Similarly to what has been done to image live animals by CT scanning, MRI of live animals could be done by using a sealed box with filters to transport the animals to the MRI facility and contain them during imaging. When available, MRI of live animals will allow longitudinal monitoring of disease progression, and real-time observation of vaccine and drug efficacy. The *ex vivo* results discussed here, together with the excellent soft tissue contrast of MRI and the development of faster MRI devices that reduce the artefacts induced by respiratory motion, suggest that *in* 

*vivo* MRI may become a very useful technique for the study of TB in animal models.

Another technique which is gaining popularity in TB research is PET combined with CT imaging (PET/CT). The PET radiotracer [18F]-FDG is used to image inflammation at the infection site, as it accumulates in inflammatory cells such as neutrophils and activated macrophages. This technology has been used to image TB infection (Figure 2) and to assess drug treatment efficacy in mice (Davis et al., 2009b). The authors infected two strains of mice, BALB/c (which develop diffused granulomas) and C3HeB/FeJ (which develop well-

**3.3 Positron Emission Tomography (PET)** 

thus highlighting the sensitivity of the method (Sharpe et al., 2010).

defined necrotic granulomas), and evaluated three different treatments: (i) first line tuberculosis regimen (rifampin + pyrazinamide + isoniazid), (ii) a more bactericidal regimen (rifampin + pyrazinamide + moxifloxacin), and (iii) a bacteriostatic regimen (ethambutol). The animals were imaged and CFUs obtained at different time points during the 12 weeks of treatment. Furthermore, one group of BALB/c mice was followed for 22 weeks after completion of the bactericidal treatments to assess relapse of the infection. For the imaging, anesthetised mice were contained in a sealed device with holes for passage of gases fitted with 0.22 m filters. In both mouse models, CFU counts perfectly reflected the efficiency of the three treatments being evaluated, with a faster decrease in bacterial numbers when moxifloxacin was used instead of isoniazid, and stabilization of bacterial burden when mice were treated with ethambutol only.

Fig. 2. 3D co-registered PET and CT images from a live C3HeB/FeJ mouse infected with a low-dose aerosol of *M. tuberculosis.* The brightness of the lesions represents FDG activity, with brighter lesions being more active. The heart also takes up FDG and can therefore be seen on the left. The bony structure (rib cage and scapula), shown in grey, were extracted from the CT (Davis, S.L. & Jain, S.K.; unpublished data).

The use of PET/CT imaging allowed differentiation between the bacteriostatic and bactericidal treatments, as the [18F]-FDG activity was higher in the mice treated with ethambutol. However, unexpectedly, [18F]-FDG activity was higher in mice treated with moxifloxacin than in those treated with isoniazid during the first four weeks of treatment. The authors suggested that this could be due to the limited statistical power of the study since only three mice per group were used, or that it could be an inherent limitation of using [18F]-FDG, whereby an increased killing of *M. tuberculosis* would cause an increased Tumour Necrosis Factor (TNF)-mediated inflammation and therefore increased [18F]-FDG activity even though bacterial numbers were decreasing. Relapse was detected in both groups of mice by PET imaging and by CFU counts. In summary, PET/CT allowed the non-invasive monitoring of disease progression in real-time. Moreover, individual lesions could be observed in the C3HeB/FeJ mouse model; as treatment response has been suggested to be lesion-dependent, the possibility of monitoring individual lesions would be very useful. However, it is important to take into account that this method does not specifically image infection but only measures inflammation which, as illustrated by the treatment results of this work, does not always correlate with bacterial burden. Nevertheless, this method has some advantages over using, for example, CFU counts: it uses a reduced number of animals, and the same animals can be repeatedly imaged which allows a more easy detection of untimed events such as relapse.

Molecular Imaging in TB: From the Bench to the Clinic 319

sensitive (and expensive) radionuclides such as 123I or 124I. Other limitations of the technique include: the limited blood supply at the centre of the granulomas could limit accessibility to imaging substrates; TK requires ATP, which could be restricted in latent bacteria; and the presence of non-specific signal in tissues such as liver, gall bladder, or stomach, that either

Bioluminescence imaging is one of the most widely used imaging techniques in the study of infectious diseases (N. Andreu et al., 2011). Luciferases have been used in mycobacterial research for more than 20 years; the two most widely used are the firefly luciferase (FFluc) and the luciferase of the bacterium *Vibrio harveyi* (LuxAB). Both luciferases produce light in the presence of a combination of a substrate and a cofactor, namely D-luciferin and ATP (for FFluc) and n-decanal and FMNH2 (for LuxAB). As the co-factors are only found in live cells, the production of light by the luciferases provides a sensitive indicator of cell viability. The bacterial luciferase system has a major advantage when compared with the FFluc: the genes for the synthesis of the substrate are known and can be co-expressed with the *luxAB* genes as a convenient gene set (*luxCABDE*) that renders the bacteria autoluminescent, that is, no external addition of substrate is needed for light production. Light-emitting mycobacteria have been used as an easier and faster approach than commonly used methods to assess bacterial numbers *in vitro* and in macrophages, for example, in drug screening assays (Arain et al., 1996). The first approach to use luminescent mycobacteria in animal models consisted of measuring luminescence *ex vivo* in organ homogenates (Hickey et al., 1996). This method generated results in a much quicker time frame than using CFU counts and has been

applied to drug and vaccine efficacy testing (Hickey et al., 1996; Snewin et al., 1999).

More recently, a recombinant *M. bovis* BCG strain expressing the bacterial luciferase enzyme LuxAB has been used to monitor mycobacterial infection *in vivo* (Heuts et al., 2009). In this work, only the luciferase genes were expressed and, therefore, the n-decanal substrate had to be injected before imaging. Although n-decanal is very toxic, the authors were able to deliver it dissolved in a mixture of olive oil and ethanol by injection into the mouse peritoneum. To assess the usefulness of the system, immunodeficient RAG2−/−/cR−/− mice were intravenously infected with the luminescent BCG strain, and bioluminescence imaging was performed at different time points for 11 weeks. A signal coming from the spleen was detected four weeks post-infection, when the bacterial load was around 5x107 CFUs. The signal increased over time and extended to the abdomen of the animal but no signal was observed in the lungs, even though CFU counting showed a bacterial burden in this organ of 107 CFUs at eight weeks post-infection. However, luminescence was detected in the excised lungs, suggesting that tissue attenuation was responsible for the failure to detect the signal in the whole animal. The same luminescent BCG strain was also used to assess drug efficacy and the host immune response. A reduction in light emission, which paralleled the reduction in bacterial numbers, was observed in treated mice compared to the untreated mice, as well as in inmunocompetent BALB/c and T-cell reconstituted RAG2−/−/cR−/− mice compared to immunosupressed RAG2−/−/cR−/− mice. Therefore, bioluminescence imaging allows monitoring of mycobacterial infection in mice. However, the system was not useful for imaging infection in the lungs, and a toxic substrate had to be administered to the mice

metabolize or excrete FIAU or its iodinated derivatives.

**3.5 Biophotonic Imaging (BPI)** 

before imaging.

PET/CT imaging is also being used in non-human primates, although the results have only been presented in meetings and no peer-reviewed article has been published to date. For example, PET/CT has been used to monitor disease progression and drug efficacy in macaques (Lin et al., 2009). Using CT imaging, lesions as small as 1 mm were detected in the lungs and lymph nodes of infected animals. Moreover, lesion progression could be followed over time. Interestingly, co-registered [18F]-FDG–PET images revealed that individual granulomas differed in their [18F]-FDG affinity: whereas some granulomas exhibited high uptake values, others seemed devoid of [18F]-FDG. The imaging results were complemented with post-mortem histology and bacterial burden analysis of individual lesions. The authors found a complex, lesion-specific response to drug treatment that included changes in [18F]- FDG avidity. These remarkable results show that even though PET and CT are two complementary techniques, images should be first analysed separately, and that caution should be taken when interpreting the results of PET activity in terms of [18F]-FDG accumulation.

#### **3.4 Single Photon Emission Computed Tomography (SPECT)**

SPECT/CT has also been used for imaging of TB infection in mice (Davis et al., 2009a). The authors used the radiotracer 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analogue, together with an engineered *M. tuberculosis* strain that stably expressed the enzyme thymidine kinase (TK) which phosphorylates [125I]-FIAU leading to its accumulation within the bacteria. In contrast to [18F]-FDG–PET imaging, this technique specifically images the bacteria instead of the inflammatory response, as [125I]- FIAU is a poor substrate for mammalian TK. Using this technique, the authors were able to image individual necrotic granulomas in the lungs of C3HeB/FeJ infected mice (Figure 3). The presence of the lesions was subsequently corroborated by histopathology. However, the limit of detection was found to be 5x106 to 1x107 CFUs, a rather high bacterial burden for mice infected with *M. tuberculosis*. The authors suggested that the sensitivity of the method could be improved by increasing the expression of TK in the bacilli or by using more

Fig. 3. Co-registered SPECT and CT images from a live C3HeB/FeJ mouse infected with a low-dose aerosol of an *M. tuberculosis* strain expressing bacterial thymidine kinase (TK) under the control of a strong mycobacterial promoter. TB lesions were imaged 8-weeks after this infection, using [125I]-FIAU, a nucleoside analog substrate for bacterial TK*.* The FIAU-SPECT signal localizes to the TB lesion (crosshairs) in the lungs, indicating uptake of FIAU by the bacteria (Davis, S.L. & Jain, S.K.; unpublished data).

sensitive (and expensive) radionuclides such as 123I or 124I. Other limitations of the technique include: the limited blood supply at the centre of the granulomas could limit accessibility to imaging substrates; TK requires ATP, which could be restricted in latent bacteria; and the presence of non-specific signal in tissues such as liver, gall bladder, or stomach, that either metabolize or excrete FIAU or its iodinated derivatives.

#### **3.5 Biophotonic Imaging (BPI)**

318 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

PET/CT imaging is also being used in non-human primates, although the results have only been presented in meetings and no peer-reviewed article has been published to date. For example, PET/CT has been used to monitor disease progression and drug efficacy in macaques (Lin et al., 2009). Using CT imaging, lesions as small as 1 mm were detected in the lungs and lymph nodes of infected animals. Moreover, lesion progression could be followed over time. Interestingly, co-registered [18F]-FDG–PET images revealed that individual granulomas differed in their [18F]-FDG affinity: whereas some granulomas exhibited high uptake values, others seemed devoid of [18F]-FDG. The imaging results were complemented with post-mortem histology and bacterial burden analysis of individual lesions. The authors found a complex, lesion-specific response to drug treatment that included changes in [18F]- FDG avidity. These remarkable results show that even though PET and CT are two complementary techniques, images should be first analysed separately, and that caution should be taken when interpreting the results of PET activity in terms of [18F]-FDG

SPECT/CT has also been used for imaging of TB infection in mice (Davis et al., 2009a). The authors used the radiotracer 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analogue, together with an engineered *M. tuberculosis* strain that stably expressed the enzyme thymidine kinase (TK) which phosphorylates [125I]-FIAU leading to its accumulation within the bacteria. In contrast to [18F]-FDG–PET imaging, this technique specifically images the bacteria instead of the inflammatory response, as [125I]- FIAU is a poor substrate for mammalian TK. Using this technique, the authors were able to image individual necrotic granulomas in the lungs of C3HeB/FeJ infected mice (Figure 3). The presence of the lesions was subsequently corroborated by histopathology. However, the limit of detection was found to be 5x106 to 1x107 CFUs, a rather high bacterial burden for mice infected with *M. tuberculosis*. The authors suggested that the sensitivity of the method could be improved by increasing the expression of TK in the bacilli or by using more

Fig. 3. Co-registered SPECT and CT images from a live C3HeB/FeJ mouse infected with a low-dose aerosol of an *M. tuberculosis* strain expressing bacterial thymidine kinase (TK) under the control of a strong mycobacterial promoter. TB lesions were imaged 8-weeks after this infection, using [125I]-FIAU, a nucleoside analog substrate for bacterial TK*.* The FIAU-SPECT signal localizes to the TB lesion (crosshairs) in the lungs, indicating uptake of FIAU

by the bacteria (Davis, S.L. & Jain, S.K.; unpublished data).

**3.4 Single Photon Emission Computed Tomography (SPECT)** 

accumulation.

Bioluminescence imaging is one of the most widely used imaging techniques in the study of infectious diseases (N. Andreu et al., 2011). Luciferases have been used in mycobacterial research for more than 20 years; the two most widely used are the firefly luciferase (FFluc) and the luciferase of the bacterium *Vibrio harveyi* (LuxAB). Both luciferases produce light in the presence of a combination of a substrate and a cofactor, namely D-luciferin and ATP (for FFluc) and n-decanal and FMNH2 (for LuxAB). As the co-factors are only found in live cells, the production of light by the luciferases provides a sensitive indicator of cell viability. The bacterial luciferase system has a major advantage when compared with the FFluc: the genes for the synthesis of the substrate are known and can be co-expressed with the *luxAB* genes as a convenient gene set (*luxCABDE*) that renders the bacteria autoluminescent, that is, no external addition of substrate is needed for light production. Light-emitting mycobacteria have been used as an easier and faster approach than commonly used methods to assess bacterial numbers *in vitro* and in macrophages, for example, in drug screening assays (Arain et al., 1996). The first approach to use luminescent mycobacteria in animal models consisted of measuring luminescence *ex vivo* in organ homogenates (Hickey et al., 1996). This method generated results in a much quicker time frame than using CFU counts and has been applied to drug and vaccine efficacy testing (Hickey et al., 1996; Snewin et al., 1999).

More recently, a recombinant *M. bovis* BCG strain expressing the bacterial luciferase enzyme LuxAB has been used to monitor mycobacterial infection *in vivo* (Heuts et al., 2009). In this work, only the luciferase genes were expressed and, therefore, the n-decanal substrate had to be injected before imaging. Although n-decanal is very toxic, the authors were able to deliver it dissolved in a mixture of olive oil and ethanol by injection into the mouse peritoneum. To assess the usefulness of the system, immunodeficient RAG2−/−/cR−/− mice were intravenously infected with the luminescent BCG strain, and bioluminescence imaging was performed at different time points for 11 weeks. A signal coming from the spleen was detected four weeks post-infection, when the bacterial load was around 5x107 CFUs. The signal increased over time and extended to the abdomen of the animal but no signal was observed in the lungs, even though CFU counting showed a bacterial burden in this organ of 107 CFUs at eight weeks post-infection. However, luminescence was detected in the excised lungs, suggesting that tissue attenuation was responsible for the failure to detect the signal in the whole animal. The same luminescent BCG strain was also used to assess drug efficacy and the host immune response. A reduction in light emission, which paralleled the reduction in bacterial numbers, was observed in treated mice compared to the untreated mice, as well as in inmunocompetent BALB/c and T-cell reconstituted RAG2−/−/cR−/− mice compared to immunosupressed RAG2−/−/cR−/− mice. Therefore, bioluminescence imaging allows monitoring of mycobacterial infection in mice. However, the system was not useful for imaging infection in the lungs, and a toxic substrate had to be administered to the mice before imaging.

Molecular Imaging in TB: From the Bench to the Clinic 321

when travelling through tissues. This is exemplified by the finding that as few as 105 CFUs of a BCG strain that expressed tdTomato (excitation 554 nm, emission 581 nm) can be detected after being subcutaneously injected into mice, in comparison to 107 CFUs of BCG expressing enhanced GFP (EGFP) (excitation 484 nm, emission 510 nm) (Kong et al., 2009). Therefore, far-red reporters show a lot of promise for fluorescence *in vivo* imaging of *M. tuberculosis* infection in animal models, and the expression of several red FPs in

The strategies presented above use recombinant bacteria that express an exogenous FP. A much more versatile strategy consists of using an activatable fluorescent agent that is nonfluorescent in its native (quenched) state but produces fluorescence through enzymemediated release of its fluorochrome. This strategy has been widely used in cancer and inflammation research but, until recently, not in infectious diseases. Using a near-infrared fluorogenic substrate for -lactamase, an enzyme that is endogenously expressed by *M. tuberculosis* but not by eukaryotic cells, it is possible to detect 106 CFUs of *M. tuberculosis* subcutaneously injected in mice (Kong et al., 2010). The maximal signal was produced 48 h after substrate injection, and no signal was detected 48 h later, which suggests that repetitive imaging of the same animals can be done every 96 h. Surprisingly, the limit of the detection in the lungs of live animals was 104 CFUs, which is far lower than the limit of detection subcutaneously, even though the lungs are localised much deeper in the body. The signal was localised laterally, close to the armpit of the animal, and 3D fluorescence molecular tomography (FMT) and imaging of the excised lungs proved that the lungs were the source of the signal. The amount of fluorescence correlated with bacterial numbers when the imaging was performed 24 h post-substrate administration, but at later time-points substrate accumulation lead to a similar level of fluorescence independently of bacterial numbers. In addition, the technique was used to assess drug efficacy by imaging treated and untreated mice, showing that the signal increased in the untreated group while it decreased in the treated group. Although much work needs to be done to assess the usefulness of the technique, one can imagine many potential applications not only in *in vivo* imaging but also *in vitro* using fluorescence microscropy or FACS, as well as for TB diagnosis (e.g. detection of bacilli in sputum or imaging tuberculosis in patients). However, a limitation that needs to be considered is the fact that other bacteria, such as *Pseudomonas aeruginosa* or *Staphylococcus aureus*, also express -lactamase and therefore may give a false signal. Thus, alternative fluorogenic substrates that are activated by other endogenous enzymes are currently under study; for example, certain trehalose analogues that are substrates for the *M. tuberculosis*

mycobacteria has recently been optimised (Carroll et al., 2010).

mycolyltransesterases Ag85A, Ag85B and Ag85C (Backus et al., 2011).

The plain radiograph was first described in 1895 by Röntgen, at the same time that the TB pandemic was peaking in wealthy Western countries such as Victorian England. Consequently, there is a long experience and literature of plain radiographic imaging of TB, which has been reviewed previously (McAdams et al., 1995; J. Andreu et al., 2004; Curvo-Semedo et al., 2005). In summary, TB can cause a wide array of chest x-ray appearances, but classically causes consolidation and cavitation in the apices of the upper lobes. TB can also cause disease at the apices of the lower lobes, which appears in the mid-zone on chest

**4. Imaging TB in human disease** 

**4.1 Radiography** 

To overcome these difficulties, our group has recently optimised the expression of FFluc and the complete bacterial luciferase system in *M. tuberculosis* (N. Andreu et al., 2010). The resulting mycobacterial strains express either the optimised gene encoding FFluc (which is the brightest luciferase and uses a non-toxic substrate) or the optimised *luxCABDE* gene set from *Photorhabdus luminescens* (which results in autoluminescent strains that do not need the exogenous addition of substrate to produce light). Both *M. tuberculosis* strains were imaged *in vivo* in the lungs and spleens of infected mice (Figure 4), with limits of detection of around 105-106 CFU per lung and 105 CFU per spleen, whereas as few as 104 CFU can be imaged in the dissected organs (our unpublished results). Further work will assess the usefulness of these luminescent mycobacteria in drug efficacy testing and in other small animal models such as guinea pigs.

Fig. 4. Visualisation of bioluminescent bacteria within living mice infected with *FFluc*expressing *M. tuberculosis* after administration of luciferin*.* The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image, where red represents the most intense light emission while blue correspond to the weakest signal (Andreu, N. & Wiles, S; unpublished data).

Fluorescence imaging has had a more limited use in the study of infectious diseases, although it has been widely applied to other research fields such as cancer research (N. Andreu et al., 2011). In a first attempt to develop fluorescence imaging of *M. tuberculosis* infection, a GFP-expressing *M. tuberculosis* strain was used to infect mice and guinea pigs, and five weeks post-infection the lungs were imaged using a photon imager (Sugawara et al., 2006). Granulomas as small as 1 mm of diameter were detected, and the results were corroborated by histopathology examination. The same fluorescence technique was used to visualize granulomas in a latent model of TB in guinea pigs (Sugawara et al., 2009). In this case, the animals were subcutaneously infected with *M. tuberculosis* and the infection was followed for 10 months. No clinical signs of infection were evident in any of the animals for the length of the experiment, although the bacteriological analysis of lungs and spleens 180 and 300 days post-infection showed the presence of a few bacteria. Similarly, even though no granulomas were detected by gross pathology examination, microgranulomas were observed in the histological analysis. According to the authors, these small lesions corresponded to the fluorescent spots detected by photon imaging of sliced lungs and spleens. More work is still needed to validate these results and to be able to use this technology *in vivo*.

FPs that emit light in the far-red region of the spectrum are more appropriate for *in vivo* imaging than for example GFP, as red light is less affected by absorption and scattering

To overcome these difficulties, our group has recently optimised the expression of FFluc and the complete bacterial luciferase system in *M. tuberculosis* (N. Andreu et al., 2010). The resulting mycobacterial strains express either the optimised gene encoding FFluc (which is the brightest luciferase and uses a non-toxic substrate) or the optimised *luxCABDE* gene set from *Photorhabdus luminescens* (which results in autoluminescent strains that do not need the exogenous addition of substrate to produce light). Both *M. tuberculosis* strains were imaged *in vivo* in the lungs and spleens of infected mice (Figure 4), with limits of detection of around 105-106 CFU per lung and 105 CFU per spleen, whereas as few as 104 CFU can be imaged in the dissected organs (our unpublished results). Further work will assess the usefulness of these luminescent mycobacteria in drug efficacy testing and in other small animal models

Fig. 4. Visualisation of bioluminescent bacteria within living mice infected with *FFluc*expressing *M. tuberculosis* after administration of luciferin*.* The image was obtained using an IVIS Spectrum and is displayed as a pseudocolour image, where red represents the most intense light emission while blue correspond to the weakest signal (Andreu, N. & Wiles, S;

Fluorescence imaging has had a more limited use in the study of infectious diseases, although it has been widely applied to other research fields such as cancer research (N. Andreu et al., 2011). In a first attempt to develop fluorescence imaging of *M. tuberculosis* infection, a GFP-expressing *M. tuberculosis* strain was used to infect mice and guinea pigs, and five weeks post-infection the lungs were imaged using a photon imager (Sugawara et al., 2006). Granulomas as small as 1 mm of diameter were detected, and the results were corroborated by histopathology examination. The same fluorescence technique was used to visualize granulomas in a latent model of TB in guinea pigs (Sugawara et al., 2009). In this case, the animals were subcutaneously infected with *M. tuberculosis* and the infection was followed for 10 months. No clinical signs of infection were evident in any of the animals for the length of the experiment, although the bacteriological analysis of lungs and spleens 180 and 300 days post-infection showed the presence of a few bacteria. Similarly, even though no granulomas were detected by gross pathology examination, microgranulomas were observed in the histological analysis. According to the authors, these small lesions corresponded to the fluorescent spots detected by photon imaging of sliced lungs and spleens. More work is still needed to validate these results and to be able to use this

FPs that emit light in the far-red region of the spectrum are more appropriate for *in vivo* imaging than for example GFP, as red light is less affected by absorption and scattering

such as guinea pigs.

unpublished data).

technology *in vivo*.

when travelling through tissues. This is exemplified by the finding that as few as 105 CFUs of a BCG strain that expressed tdTomato (excitation 554 nm, emission 581 nm) can be detected after being subcutaneously injected into mice, in comparison to 107 CFUs of BCG expressing enhanced GFP (EGFP) (excitation 484 nm, emission 510 nm) (Kong et al., 2009). Therefore, far-red reporters show a lot of promise for fluorescence *in vivo* imaging of *M. tuberculosis* infection in animal models, and the expression of several red FPs in mycobacteria has recently been optimised (Carroll et al., 2010).

The strategies presented above use recombinant bacteria that express an exogenous FP. A much more versatile strategy consists of using an activatable fluorescent agent that is nonfluorescent in its native (quenched) state but produces fluorescence through enzymemediated release of its fluorochrome. This strategy has been widely used in cancer and inflammation research but, until recently, not in infectious diseases. Using a near-infrared fluorogenic substrate for -lactamase, an enzyme that is endogenously expressed by *M. tuberculosis* but not by eukaryotic cells, it is possible to detect 106 CFUs of *M. tuberculosis* subcutaneously injected in mice (Kong et al., 2010). The maximal signal was produced 48 h after substrate injection, and no signal was detected 48 h later, which suggests that repetitive imaging of the same animals can be done every 96 h. Surprisingly, the limit of the detection in the lungs of live animals was 104 CFUs, which is far lower than the limit of detection subcutaneously, even though the lungs are localised much deeper in the body. The signal was localised laterally, close to the armpit of the animal, and 3D fluorescence molecular tomography (FMT) and imaging of the excised lungs proved that the lungs were the source of the signal. The amount of fluorescence correlated with bacterial numbers when the imaging was performed 24 h post-substrate administration, but at later time-points substrate accumulation lead to a similar level of fluorescence independently of bacterial numbers. In addition, the technique was used to assess drug efficacy by imaging treated and untreated mice, showing that the signal increased in the untreated group while it decreased in the treated group. Although much work needs to be done to assess the usefulness of the technique, one can imagine many potential applications not only in *in vivo* imaging but also *in vitro* using fluorescence microscropy or FACS, as well as for TB diagnosis (e.g. detection of bacilli in sputum or imaging tuberculosis in patients). However, a limitation that needs to be considered is the fact that other bacteria, such as *Pseudomonas aeruginosa* or *Staphylococcus aureus*, also express -lactamase and therefore may give a false signal. Thus, alternative fluorogenic substrates that are activated by other endogenous enzymes are currently under study; for example, certain trehalose analogues that are substrates for the *M. tuberculosis* mycolyltransesterases Ag85A, Ag85B and Ag85C (Backus et al., 2011).

#### **4. Imaging TB in human disease**

#### **4.1 Radiography**

The plain radiograph was first described in 1895 by Röntgen, at the same time that the TB pandemic was peaking in wealthy Western countries such as Victorian England. Consequently, there is a long experience and literature of plain radiographic imaging of TB, which has been reviewed previously (McAdams et al., 1995; J. Andreu et al., 2004; Curvo-Semedo et al., 2005). In summary, TB can cause a wide array of chest x-ray appearances, but classically causes consolidation and cavitation in the apices of the upper lobes. TB can also cause disease at the apices of the lower lobes, which appears in the mid-zone on chest

Molecular Imaging in TB: From the Bench to the Clinic 323

radiography, but does not give information about the molecular events occurring at the site

Fig. 6. CT imaging reveals a small left mid zone cavity (indicated by arrow) that was not visible on plain chest x-ray (left panel) and a tree-in-bud bronchial filling in a 33 year old man with pulmonary TB (right panel), which appears as branched opacities in the lung field

MRI provides the best imaging of the meninges and spinal cord, and so is useful in the diagnosis of cerebral TB, TB meningitis and paraspinal TB abscesses. MRI is a rapidly developing area, and so may emerge as a modality which can provide insight into molecular events in TB. The advantage of MRI is that it involves no ionizing radiation and provides excellent anatomical resolution. New MRI modalities to investigate inflammatory diseases are under development (Pirko et al., 2004), but these have not yet entered the clinical arena

PET imaging is a widely used nuclear medicine technique which has the potential to study pathological events at a molecular level before extensive anatomical changes are observed on plain radiography. PET imaging is commonly combined with CT scanning in patients to provide both functional and anatomical information. [18F]-FDG accumulates in metabolically active cells after phosphorylation, and so is taken up by metabolically active macrophages within the TB granuloma and other inflammatory foci. A primary limitation of PET imaging for TB is the high cost and low availability in developing world. Increased PET uptake is well described in both pulmonary and extrapulmonary TB lesions (Matsuura et al., 2000; Bakheet et al., 1998; C.M. Yang et al., 2003), and can cause diagnostic uncertainty with

PET scanning is clinically useful in certain patients with TB. For example, when patients have normal radiology but symptoms highly suggestive of active TB, PET scans may identify occult foci of infection which can then be sampled to confirm the diagnosis and for culture (Figure 7, arrow). Furthermore, PET imaging has been proposed for monitoring the resolution of TB disease (Hofmeyr et al., 2007), although the benefit must be weighed against the increased radiation exposure. Current research questions which need to be

of infection.

adjacent to arrowhead.

for investigation in TB.

**4.3 Magnetic Resonance Imaging (MRI)** 

**4.4 Positron Emission Tomography (PET)** 

malignancy and other infections (Chen et al., 2004; Li et al., 2008).

radiograph (Figure 5). However, TB can result in a wide range of other features, such as miliary disease with small millet seed-sized nodules throughout the lungs, pleural effusions, mediastinal lymphadenopathy and extrapulmonary disease. In the era of HIV infection, where the host immune system is compromised, the appearances of pulmonary TB are often atypical (Kwan & Ernst, 2011), ranging from classical cavitation to areas of pneumonia to a normal chest x-ray even in the presence of a high mycobacterial load. This illustrates the importance of the adaptive immune response in driving lung inflammation, resulting in consolidation and tissue destruction.

Fig. 5. Radiograph illustrating right mid zone cavitation on a 17 year old patient with pulmonary TB.

As TB is treated, areas of consolidation tend to gradually resolve, leaving an area of fibrosis or scar tissue which persists for life. Cavities remain even after cure, because the lung cannot reconstruct the intricate extracellular matrix after it has been destroyed. However, it is well recognised that radiographic appearances may often worsen before they improve (Leung, 1999), and similarly some lesions may increase in size and density while others appear to resolve. Even in HIV negative patients with drug-sensitive disease, such "paradoxical" reactions may occur (Cheng, 2002). This demonstrates the different behaviour of inflammatory lesions even in the same patient, and one challenge for modern imaging techniques is to define the molecular mechanisms underlying this immune response to improve our understanding of what constitutes an effective as opposed to deleterious immune response to TB.

#### **4.2 Computed Tomography (CT)**

CT involves cross-sectional imaging of patients and so permits a much greater degree of resolution of anatomical structures, although it results in a higher radiation dose and higher cost than plain radiography. CT can demonstrate cavity formation with much greater sensitivity and will demonstrate subtle changes which may be missed on plain chest x-rays (Figure 6 [left panel]). For example, filling of small airways with inflammatory debris may result in a "tree-in-bud" pattern (Figure 6 [right panel], arrow), which should immediately alert the physician to the possibility of mycobacterial infection. Therefore, CT scanning provides information of changes at a much more precise anatomical level than plain

radiograph (Figure 5). However, TB can result in a wide range of other features, such as miliary disease with small millet seed-sized nodules throughout the lungs, pleural effusions, mediastinal lymphadenopathy and extrapulmonary disease. In the era of HIV infection, where the host immune system is compromised, the appearances of pulmonary TB are often atypical (Kwan & Ernst, 2011), ranging from classical cavitation to areas of pneumonia to a normal chest x-ray even in the presence of a high mycobacterial load. This illustrates the importance of the adaptive immune response in driving lung inflammation, resulting in

Fig. 5. Radiograph illustrating right mid zone cavitation on a 17 year old patient with

As TB is treated, areas of consolidation tend to gradually resolve, leaving an area of fibrosis or scar tissue which persists for life. Cavities remain even after cure, because the lung cannot reconstruct the intricate extracellular matrix after it has been destroyed. However, it is well recognised that radiographic appearances may often worsen before they improve (Leung, 1999), and similarly some lesions may increase in size and density while others appear to resolve. Even in HIV negative patients with drug-sensitive disease, such "paradoxical" reactions may occur (Cheng, 2002). This demonstrates the different behaviour of inflammatory lesions even in the same patient, and one challenge for modern imaging techniques is to define the molecular mechanisms underlying this immune response to improve our understanding of what constitutes an effective as opposed to deleterious

CT involves cross-sectional imaging of patients and so permits a much greater degree of resolution of anatomical structures, although it results in a higher radiation dose and higher cost than plain radiography. CT can demonstrate cavity formation with much greater sensitivity and will demonstrate subtle changes which may be missed on plain chest x-rays (Figure 6 [left panel]). For example, filling of small airways with inflammatory debris may result in a "tree-in-bud" pattern (Figure 6 [right panel], arrow), which should immediately alert the physician to the possibility of mycobacterial infection. Therefore, CT scanning provides information of changes at a much more precise anatomical level than plain

consolidation and tissue destruction.

pulmonary TB.

immune response to TB.

**4.2 Computed Tomography (CT)** 

radiography, but does not give information about the molecular events occurring at the site of infection.

Fig. 6. CT imaging reveals a small left mid zone cavity (indicated by arrow) that was not visible on plain chest x-ray (left panel) and a tree-in-bud bronchial filling in a 33 year old man with pulmonary TB (right panel), which appears as branched opacities in the lung field adjacent to arrowhead.

#### **4.3 Magnetic Resonance Imaging (MRI)**

MRI provides the best imaging of the meninges and spinal cord, and so is useful in the diagnosis of cerebral TB, TB meningitis and paraspinal TB abscesses. MRI is a rapidly developing area, and so may emerge as a modality which can provide insight into molecular events in TB. The advantage of MRI is that it involves no ionizing radiation and provides excellent anatomical resolution. New MRI modalities to investigate inflammatory diseases are under development (Pirko et al., 2004), but these have not yet entered the clinical arena for investigation in TB.

#### **4.4 Positron Emission Tomography (PET)**

PET imaging is a widely used nuclear medicine technique which has the potential to study pathological events at a molecular level before extensive anatomical changes are observed on plain radiography. PET imaging is commonly combined with CT scanning in patients to provide both functional and anatomical information. [18F]-FDG accumulates in metabolically active cells after phosphorylation, and so is taken up by metabolically active macrophages within the TB granuloma and other inflammatory foci. A primary limitation of PET imaging for TB is the high cost and low availability in developing world. Increased PET uptake is well described in both pulmonary and extrapulmonary TB lesions (Matsuura et al., 2000; Bakheet et al., 1998; C.M. Yang et al., 2003), and can cause diagnostic uncertainty with malignancy and other infections (Chen et al., 2004; Li et al., 2008).

PET scanning is clinically useful in certain patients with TB. For example, when patients have normal radiology but symptoms highly suggestive of active TB, PET scans may identify occult foci of infection which can then be sampled to confirm the diagnosis and for culture (Figure 7, arrow). Furthermore, PET imaging has been proposed for monitoring the resolution of TB disease (Hofmeyr et al., 2007), although the benefit must be weighed against the increased radiation exposure. Current research questions which need to be

Molecular Imaging in TB: From the Bench to the Clinic 325

al., 1992; Kao et al., 1994; A. Yang et al, 2007), which significantly decreases the value of this radiotracer in the differential diagnosis of pulmonary TB from other lung pathologies.

A fundamental challenge of TB research is to develop applications which are useful in resource poor settings and can be deployed with minimal investment in infrastructure, maintenance and staff expertise. Furthermore, any application needs to be equally applicable in urban and rural settings. Most modern imaging techniques are useful in the developed world, but are not available to the vast majority of patients with TB who live in

However, the detailed study of a small number of patients may identify pathophysiological markers of TB which can then be simplified to develop new diagnostic and therapeutic approaches applicable in resource poor settings. Ironically, when one considers molecular imaging of TB in this light, developing an imaging technique based on plain chest radiography is currently the only widely deliverable approach in the near future. For example, if a highly radio-dense specific TB ligand was developed, a diagnostic test might involve taking an initial chest x-ray, injecting the labelled ligand, and then taking a second x-ray to identify high uptake in the region of TB. This might be useful in the common clinical scenario when a patient presents with upper zone fibrosis, which may be caused by either old self-healed TB or new active TB. If the patient is sputum smear negative, an expensive and invasive bronchoalveolar lavage is required, so a non-invasive test to

Another frequent clinical scenario is a patient with immunological evidence of infection, but with a normal chest x-ray and a cryptic location of disease. An investigation whereby one could locate the site of disease for aspiration and culture analysis would be clinically useful. This assay might either rely on antimycobacterial ligands, potentially using the "dock and lock" strategy (Goldenberg et al., 2007), whereby a primary antibody is first injected which docks on the mycobacterial target, and then 24 hours later a second radiolabelled antibody is injected which locks onto the primary antibody, or alternatively might focus on the host immune response, such as looking for increased metalloproteinase activity at the site of

In additional to diagnosis, a secondary role of imaging is to determine the prognostic and therapeutic correlates of host immunity. Currently, standard treatment lasts for six months. A recent trial comparing short-course therapy for four months in patients with low risk features was stopped because of increased recurrence in the short course treatment group (Daley, 2010). We need better markers to identify patients who will respond rapidly to treatment and imaging modalities to define mycobacterial load, the effectiveness of the host

It is clear that molecular imaging technologies will play an important role in improving our understanding of the host-pathogen interactions that occur in animal models of TB, and should speed up preclinical testing of novel vaccine candidates and therapeutic regimes. In

**5. Future prospects** 

resource-poor settings.

determine disease activity would be useful.

disease (Elkington et al., 2011).

immune response and TB cure.

**6. Conclusions** 

addressed are whether PET imaging can be useful to define cure, especially in the context of drug-resistant TB where treatment regimes may exceed 18 months, and also investigate whether active foci can be identified in patients with clinically "latent" disease.

Fig. 7. PET imaging reveals increased uptake of [18F]-FDG in a right hilar lymph node, appearing as bright white (as indicated by arrow).

In addition to [18F]-FDG, a wide array of radiopharmaceuticals have been developed at the preclinical level which might be applied to TB (Signore & Glaudemans, 2011). However, the potential of these in man have not yet been confirmed. For example, radiolabelling the antibiotic ciprofloxacin looked promising initially to investigate cryptic foci of infection (Britton et al., 2002), but has not entered clinical practice widely. The ability to detect a wide range of pathophysiological markers suggests that PET imaging may emerge as a powerful modality to investigate the biology of TB in man, but currently most prospective candidates require further study in model systems before clinical studies in man can be considered.

#### **4.5 Single Photon Emission Computed Tomography (SPECT)**

A number of SPECT radiotracers have been applied to the management of TB including 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) (Ahmadihosseini, 2008), 67Ga (Liu et al., 2007) and 111In-octreotide (Vanhagen et al., 1994). 99mTc-MIBI is a widely used myocardial perfusion agent, which can accumulate in tumours and inflammatory lesions (Aktolun et al., 1991; Caner et al., 1992; Kao et al., 1994; A. Yang et al, 2007). Ahmadihosseini and colleagues studied 36 patients with either proven active or inactive treated pulmonary TB and found that 99mTc-MIBI uptake was increased in 23 out of 24 patients (95.8%) with active pulmonary TB but none of those with inactive TB (Ahmadihosseini et al., 2008). *M. tuberculosis* has been demonstrated to have significantly higher 99mTc-MIBI uptake compared with fibroblasts and myocytes cultures (Stefanescu et al., 2007), suggesting the bacilli themselves contribute to the signal detected on 99mTc-MIBI SPECT images.

A number of studies have found positive SPECT images in sputum smear negative patients subsequently found to have a positive sputum culture for *M. tuberculosis* (Ahmadihosseini et al., 2008; Önsel et al., 1996; Stefanescu et al., 2006), suggesting that SPECT imaging may be very useful while awaiting culture results. However, as previously stated, many benign and malignant etiologies can also demonstrate 99mTc-MIBI uptake (Aktolun et al., 1991; Caner et al., 1992; Kao et al., 1994; A. Yang et al, 2007), which significantly decreases the value of this radiotracer in the differential diagnosis of pulmonary TB from other lung pathologies.

### **5. Future prospects**

324 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

addressed are whether PET imaging can be useful to define cure, especially in the context of drug-resistant TB where treatment regimes may exceed 18 months, and also investigate

whether active foci can be identified in patients with clinically "latent" disease.

Fig. 7. PET imaging reveals increased uptake of [18F]-FDG in a right hilar lymph node,

In addition to [18F]-FDG, a wide array of radiopharmaceuticals have been developed at the preclinical level which might be applied to TB (Signore & Glaudemans, 2011). However, the potential of these in man have not yet been confirmed. For example, radiolabelling the antibiotic ciprofloxacin looked promising initially to investigate cryptic foci of infection (Britton et al., 2002), but has not entered clinical practice widely. The ability to detect a wide range of pathophysiological markers suggests that PET imaging may emerge as a powerful modality to investigate the biology of TB in man, but currently most prospective candidates require further study in model systems before clinical

A number of SPECT radiotracers have been applied to the management of TB including 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) (Ahmadihosseini, 2008), 67Ga (Liu et al., 2007) and 111In-octreotide (Vanhagen et al., 1994). 99mTc-MIBI is a widely used myocardial perfusion agent, which can accumulate in tumours and inflammatory lesions (Aktolun et al., 1991; Caner et al., 1992; Kao et al., 1994; A. Yang et al, 2007). Ahmadihosseini and colleagues studied 36 patients with either proven active or inactive treated pulmonary TB and found that 99mTc-MIBI uptake was increased in 23 out of 24 patients (95.8%) with active pulmonary TB but none of those with inactive TB (Ahmadihosseini et al., 2008). *M. tuberculosis* has been demonstrated to have significantly higher 99mTc-MIBI uptake compared with fibroblasts and myocytes cultures (Stefanescu et al., 2007), suggesting the bacilli themselves contribute to

A number of studies have found positive SPECT images in sputum smear negative patients subsequently found to have a positive sputum culture for *M. tuberculosis* (Ahmadihosseini et al., 2008; Önsel et al., 1996; Stefanescu et al., 2006), suggesting that SPECT imaging may be very useful while awaiting culture results. However, as previously stated, many benign and malignant etiologies can also demonstrate 99mTc-MIBI uptake (Aktolun et al., 1991; Caner et

appearing as bright white (as indicated by arrow).

the signal detected on 99mTc-MIBI SPECT images.

**4.5 Single Photon Emission Computed Tomography (SPECT)** 

studies in man can be considered.

A fundamental challenge of TB research is to develop applications which are useful in resource poor settings and can be deployed with minimal investment in infrastructure, maintenance and staff expertise. Furthermore, any application needs to be equally applicable in urban and rural settings. Most modern imaging techniques are useful in the developed world, but are not available to the vast majority of patients with TB who live in resource-poor settings.

However, the detailed study of a small number of patients may identify pathophysiological markers of TB which can then be simplified to develop new diagnostic and therapeutic approaches applicable in resource poor settings. Ironically, when one considers molecular imaging of TB in this light, developing an imaging technique based on plain chest radiography is currently the only widely deliverable approach in the near future. For example, if a highly radio-dense specific TB ligand was developed, a diagnostic test might involve taking an initial chest x-ray, injecting the labelled ligand, and then taking a second x-ray to identify high uptake in the region of TB. This might be useful in the common clinical scenario when a patient presents with upper zone fibrosis, which may be caused by either old self-healed TB or new active TB. If the patient is sputum smear negative, an expensive and invasive bronchoalveolar lavage is required, so a non-invasive test to determine disease activity would be useful.

Another frequent clinical scenario is a patient with immunological evidence of infection, but with a normal chest x-ray and a cryptic location of disease. An investigation whereby one could locate the site of disease for aspiration and culture analysis would be clinically useful. This assay might either rely on antimycobacterial ligands, potentially using the "dock and lock" strategy (Goldenberg et al., 2007), whereby a primary antibody is first injected which docks on the mycobacterial target, and then 24 hours later a second radiolabelled antibody is injected which locks onto the primary antibody, or alternatively might focus on the host immune response, such as looking for increased metalloproteinase activity at the site of disease (Elkington et al., 2011).

In additional to diagnosis, a secondary role of imaging is to determine the prognostic and therapeutic correlates of host immunity. Currently, standard treatment lasts for six months. A recent trial comparing short-course therapy for four months in patients with low risk features was stopped because of increased recurrence in the short course treatment group (Daley, 2010). We need better markers to identify patients who will respond rapidly to treatment and imaging modalities to define mycobacterial load, the effectiveness of the host immune response and TB cure.

#### **6. Conclusions**

It is clear that molecular imaging technologies will play an important role in improving our understanding of the host-pathogen interactions that occur in animal models of TB, and should speed up preclinical testing of novel vaccine candidates and therapeutic regimes. In

Molecular Imaging in TB: From the Bench to the Clinic 327

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#### **7. Acknowledgements**

This work has been supported in part by a grant to the Imaging TB consortium from the Bill and Melinda Gates Foundation TB Drug Accelerator Program. PE is funded by the UK National Institute for Health Research (NIHR) and is grateful for support from the NIHR Biomedical Research Centre (BRC) scheme at Imperial College London. SW is funded by a Sir Charles Hercus Fellowship from the Health Research Council of New Zealand. The authors would like to thank Prof. Ian Orme (Colorado State University) and Dr. Sanjay Jain MD (Johns Hopkins) for the provision of figures.

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Zonios, G., Bykowski, J. & Kollias, N. (2001). Skin melanin, hemoglobin, and light scattering properties can be quantitatively assessed in vivo using diffuse reflectance spectroscopy. *Journal of Investigative Dermatology*, Vol. 117, No. 6, December 2001, pp. 1452-1457, ISSN 0022-202X

**16** 

*India* 

**Data Mining Techniques in the** 

**Diagnosis of Tuberculosis** 

 *Bangalore Institute of Technology,* 

 *PES Institute of Technology,* 

T. Asha1, S. Natarajan2 and K. N. B. Murthy3 *1Department of Information Science & Engineering,* 

*2,3Department of Information Science and Engineering,* 

**Data mining** is the knowledge discovery process which helps in extracting interesting patterns from large amount of data. With the amount of data doubling every three years, data mining is becoming an increasingly important tool to transform these data into information. It is commonly used in a wide range of profiling practices, such as marketing, surveillance, fraud detection, medical and scientific discovery (J.Han & M.Kamber,2006).

Humans have been manually extracting patterns from data for centuries, but the increasing volume of data in modern times has called for more automated approaches. As data sets have grown in size and complexity, direct hands-on data analysis has increasingly been augmented with indirect, automatic data processing. This has been aided by other discoveries in computer science, such as neural networks, clustering, genetic algorithms (1950s), decision trees (1960s) and support vector machines (1980s). Data mining (DM) is the process of applying these methods to data with the intention of uncovering hidden patterns.

Generally KDD is an iterative and interactive process involving several steps. This KDD process was chosen (Figure 1) according to UNESCO definition because of its simplicity and

The first step is to understand the application domain and to formulate the problem. This step is clearly a prerequisite for extracting useful knowledge and for choosing appropriate data mining methods in the third step according to the application target and the nature of data.

The second step is to collect and pre-process the data. Today's real-world databases are susceptible to noisy, missing, and inconsistent data due to their typically huge size (often

**1. Introduction** 

**1.1 Data mining process** 

**1.1.1 Problem identification and definition** 

**1.1.2 Obtaining and preprocessing data** 

comprehensiveness.

## **Data Mining Techniques in the Diagnosis of Tuberculosis**

T. Asha1, S. Natarajan2 and K. N. B. Murthy3 *1Department of Information Science & Engineering, Bangalore Institute of Technology, 2,3Department of Information Science and Engineering, PES Institute of Technology, India* 

#### **1. Introduction**

332 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Zonios, G., Bykowski, J. & Kollias, N. (2001). Skin melanin, hemoglobin, and light scattering

pp. 1452-1457, ISSN 0022-202X

properties can be quantitatively assessed in vivo using diffuse reflectance spectroscopy. *Journal of Investigative Dermatology*, Vol. 117, No. 6, December 2001,

> **Data mining** is the knowledge discovery process which helps in extracting interesting patterns from large amount of data. With the amount of data doubling every three years, data mining is becoming an increasingly important tool to transform these data into information. It is commonly used in a wide range of profiling practices, such as marketing, surveillance, fraud detection, medical and scientific discovery (J.Han & M.Kamber,2006).

> Humans have been manually extracting patterns from data for centuries, but the increasing volume of data in modern times has called for more automated approaches. As data sets have grown in size and complexity, direct hands-on data analysis has increasingly been augmented with indirect, automatic data processing. This has been aided by other discoveries in computer science, such as neural networks, clustering, genetic algorithms (1950s), decision trees (1960s) and support vector machines (1980s). Data mining (DM) is the process of applying these methods to data with the intention of uncovering hidden patterns.

#### **1.1 Data mining process**

Generally KDD is an iterative and interactive process involving several steps. This KDD process was chosen (Figure 1) according to UNESCO definition because of its simplicity and comprehensiveness.

#### **1.1.1 Problem identification and definition**

The first step is to understand the application domain and to formulate the problem. This step is clearly a prerequisite for extracting useful knowledge and for choosing appropriate data mining methods in the third step according to the application target and the nature of data.

#### **1.1.2 Obtaining and preprocessing data**

The second step is to collect and pre-process the data. Today's real-world databases are susceptible to noisy, missing, and inconsistent data due to their typically huge size (often

Data Mining Techniques in the Diagnosis of Tuberculosis 335

The final step is to put the discovered knowledge in practical use. Putting the results in practical use is certainly the ultimate goal of the knowledge discovery. The information achieved can be used later to explain current or historical phenomenon, predict the future,

Data Mining functionalities are specifically of two categories: descriptive data mining and predictive data mining. Descriptive methods find human-interpretable patterns that describe the data. Predictive methods perform inference on the current data in order to

 Classification - Arranges the data into predefined groups. For example an email program might attempt to classify an email as legitimate or spam. Common algorithms include Decision Tree Learning, Nearest neighbor, Naive Bayesian classification and

 Association rule learning - Searches for relationships between variables. For example a supermarket might gather data on customer purchasing habits. Using association rule learning, the supermarket can determine which products are frequently bought together and use this information for marketing purposes. This is sometimes referred to

Clustering - Is like classification but the groups are not predefined, so the algorithm will

Data mining finds its applications in various fields. **Web mining -** is the application of data mining techniques to discover patterns from the Web. According to analysis targets, web mining can be divided into three different types, which are Web Usage Mining (WUM), Web Content Mining (WCM) and Web Structure Mining (WSM). It is called **Spatial Data mining** if we apply data mining techniques to spatial data. **Multimedia Data mining**- is the application of data mining techniques to multimedia data such as audio, video, image, graphics etc. **Text mining**- applying data mining techniques on unstructured or semistructured text data such as news group, email, documents. Bioinformatics and Bio-data

Data mining draws ideas from many fields such as Machine learning/Artificial Intelligence, Pattern Recognition, Statistics, and Database Systems. In recent years, data mining has been widely used in the area of genetics, medicine, bioinformatics with its applications applied to biomedical data as facilitated by domain ontologies and mining clinical trial data which is

Different types of medical data are now available on the web, where DM algorithms and applications can be applied, helping in easy diagnosis. Efficient and scalable algorithms can

Regression -Attempts to find a function which models the data with the least error.

and help decision-makers make policy from the existed facts (ho, nd).

**1.1.5 Using discovered knowledge** 

**1.2 Data mining tasks and functionalities** 

make predictions (J.Han & M.Kamber, 2006).

The predictive tasks of data mining are:

The descriptive tasks of data mining are:

as "market basket analysis".

analysis on biological data.

also called medical data mining.

try to group similar items together.

Neural Network.

several gigabytes or more), and their likely origin from multiple, heterogeneous sources. Low quality data will lead to low quality mining results. Data pre-processing is an essential step for knowledge and data mining. Data pre-processing include the data integration, removal of noise or outliers, the treatment of missing data, data transformation and reduction of data etc. This step usually takes the most time needed for the whole KDD process.

Fig. 1. Structure of Data Mining (KDD) Process

#### **1.1.3 Selection of data mining / knowledge discovery in database**

The third step is data mining that extracts patterns and models hidden in data. This is an essential process where intelligent methods are applied in order to extract data patterns. In this step we have to first select data mining tasks and then data mining method. The major classes of data mining methods are predictive modeling such as classification and regression; segmentation (clustering) and association rules which are explained in detail in the next section.

#### **1.1.4 Interpretation and evaluation of results**

The fourth step is to interpret (post-process) discovered knowledge, especially the interpretation in terms of description and prediction which is the two primary goals of discovery system in practice. Experiments show that discovered patterns or models from data are not always of interest or direct use, and the KDD process is necessarily iterative with judgement of discovered knowledge. One standard way to evaluate induced rules is to divide the data into two sets, training on the first set and testing on the second. One can repeat this process a number of times with different splits, and then average the results to estimate the rules performance.

#### **1.1.5 Using discovered knowledge**

334 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

several gigabytes or more), and their likely origin from multiple, heterogeneous sources. Low quality data will lead to low quality mining results. Data pre-processing is an essential step for knowledge and data mining. Data pre-processing include the data integration, removal of noise or outliers, the treatment of missing data, data transformation and reduction of data etc. This step usually takes the most time needed for the whole KDD

process.

Fig. 1. Structure of Data Mining (KDD) Process

**1.1.4 Interpretation and evaluation of results** 

estimate the rules performance.

the next section.

**1.1.3 Selection of data mining / knowledge discovery in database** 

The third step is data mining that extracts patterns and models hidden in data. This is an essential process where intelligent methods are applied in order to extract data patterns. In this step we have to first select data mining tasks and then data mining method. The major classes of data mining methods are predictive modeling such as classification and regression; segmentation (clustering) and association rules which are explained in detail in

The fourth step is to interpret (post-process) discovered knowledge, especially the interpretation in terms of description and prediction which is the two primary goals of discovery system in practice. Experiments show that discovered patterns or models from data are not always of interest or direct use, and the KDD process is necessarily iterative with judgement of discovered knowledge. One standard way to evaluate induced rules is to divide the data into two sets, training on the first set and testing on the second. One can repeat this process a number of times with different splits, and then average the results to The final step is to put the discovered knowledge in practical use. Putting the results in practical use is certainly the ultimate goal of the knowledge discovery. The information achieved can be used later to explain current or historical phenomenon, predict the future, and help decision-makers make policy from the existed facts (ho, nd).

#### **1.2 Data mining tasks and functionalities**

Data Mining functionalities are specifically of two categories: descriptive data mining and predictive data mining. Descriptive methods find human-interpretable patterns that describe the data. Predictive methods perform inference on the current data in order to make predictions (J.Han & M.Kamber, 2006).

The predictive tasks of data mining are:


The descriptive tasks of data mining are:


Data mining finds its applications in various fields. **Web mining -** is the application of data mining techniques to discover patterns from the Web. According to analysis targets, web mining can be divided into three different types, which are Web Usage Mining (WUM), Web Content Mining (WCM) and Web Structure Mining (WSM). It is called **Spatial Data mining** if we apply data mining techniques to spatial data. **Multimedia Data mining**- is the application of data mining techniques to multimedia data such as audio, video, image, graphics etc. **Text mining**- applying data mining techniques on unstructured or semistructured text data such as news group, email, documents. Bioinformatics and Bio-data analysis on biological data.

Data mining draws ideas from many fields such as Machine learning/Artificial Intelligence, Pattern Recognition, Statistics, and Database Systems. In recent years, data mining has been widely used in the area of genetics, medicine, bioinformatics with its applications applied to biomedical data as facilitated by domain ontologies and mining clinical trial data which is also called medical data mining.

Different types of medical data are now available on the web, where DM algorithms and applications can be applied, helping in easy diagnosis. Efficient and scalable algorithms can

Data Mining Techniques in the Diagnosis of Tuberculosis 337

**Tuberculosis (TB)** is a common and often deadly infectious disease caused by mycobacterium; in humans it is mainly *Mycobacterium tuberculosis*. It usually spreads through the air and attacks low immune bodies such as patients with Human Immunodeficiency Virus (HIV). It is a disease which can affect virtually all organs, not sparing even the relatively inaccessible sites. The microorganisms usually enter the body by inhalation through the lungs. They spread from the initial location in the lungs to other parts of the body via the blood stream. They present a diagnostic dilemma even for physicians with a great deal of experience in this disease. Hence Tuberculosis (TB) is a contagious bacterial disease caused by mycobacterium which affects usually lungs and is

It is a great problem for most developing countries because of the low diagnosis and treatment opportunities. Tuberculosis has the highest mortality level among the diseases caused by a single type of microorganism. Thus, tuberculosis is a great health concern all

Symptoms of TB depend on where in the body the TB bacteria are growing. TB bacteria usually grow in the lungs. TB in the lungs may cause symptoms such as a bad cough that lasts 3 weeks or longer pain in the chest coughing up blood or sputum. Other symptoms of active TB disease are: weakness or fatigue, weight loss, no appetite, chills, fever and

Although common and deadly in the third world, Tuberculosis was almost non-existent in the developed world, but has been making a recent resurgence. Certain drug-resistant strains are emerging and people with immune suppression such as AIDS or poor health are

The medical dataset we are using includes 700 real records of patients suffering from TB obtained from a city hospital. The entire dataset is put in one file having many records. Each record corresponds to most relevant information of one patient. Initial queries by doctor as symptoms and some required test details of patients have been considered as main attributes. Totally there are 12 attributes (symptoms) and last attribute is considered as class in case of Associative Classification. The symptoms of each patient such as age, chronic cough(weeks), loss of weight, intermittent fever(days), night sweats, Sputum, Bloodcough, chestpain, HIV, radiographic findings, wheezing and TBtype are considered as attributes. Table 1 shows names of 12 attributes considered along with their Data Types (DT). Type N-

Association Rule Mining (ARM) is an important problem in the rapidly growing field called data mining and knowledge discovery in databases (KDD). The task of association rule mining is to mine a set of highly correlated attributes/features shared among a large number of records in a given database. For example, consider the sales database of a bookstore, where the

**2. Tuberculosis** 

sweating at night.

becoming carriers.

**2.1 Data set description** 

indicates numerical and C is categorical.

**3. Association Rule Mining** 

often co-infected with HIV/AIDS.

over the world, and in India as well (wikipedia.org).

be implemented both in sequential and parallel mode thus improving the performance. Such type of mining is called medical data mining.

#### **1.3 Medical data mining**

In recent years, data mining has been widely used in the area of genetics and medicine, called medical data mining. In the past two decades we have witnessed revolutionary changes in biomedical research and bio-technology. There is an explosive growth of biomedical data, ranging from those collected in pharmaceutical studies and cancer therapy investigations to those identified in genomics and proteomics research. The rapid progress of biotechnology and bio-data analysis methods has led to the emergence and fast growth of a promising new field: Bioinformatics. On the other hand, recent progress in data mining research has led to the developments of numerous efficient and scalable methods for mining interesting patterns and knowledge in large databases, ranging from efficient classification methods to clustering, outlier analysis, frequent, sequential and structured pattern analysis methods, and visualization and spatial/temporal data analysis tools. The question becomes how to bridge the two fields, Data Mining and Bioinformatics, for successful data mining in biomedical data. Especially, we should analyze how data mining may help efficient and effective bio-medical data analysis and outline some research problems that may motivate the further developments of powerful data mining tools for bio data or medical data analysis.

Data mining is a process that involves aggregating raw data stored in a database and analyzing them to identify trends, patterns and anomalies. Medical data mining is an active research area under data mining since medical databases have accumulated large quantities of information about patients and their clinical conditions. Relationships and patterns hidden in this data can provide new medical knowledge as has been proved in a number of medical data mining applications. A Doctor quickly swung into action after a renowned pharmaceutical company in the USA announced in 2001 that it was withdrawing a cholesterol-lowering drug following the deaths of more than 30 people. Using his medical records database, his staff was able to identify all patients taking the cholesterol-lowering drug and notify them within 24 hours of the announcement. What the doctor did is technically known as Data Mining. Very few doctors, however, were able to act on the situation, because they did not have accessible raw data in the electronic format.

Not only does disciplined storage of medical data helps the physicians and healthcare institutions, but it also helps pharmaceutical companies to mine the data to see the trends in diseases. It also helps prioritize product development and clinical trials based on the accurate demands visible from the data that is mined.

Various data mining tasks can be applied on different diseases data set. This helps even the doctor to identify hidden associations between various symptoms. Research has been carried out on gene data, proteonomic data and attributes related to diseases covering even risk factors. Prediction of diseases has also been done on scanned images leading to medical imaging, which is the fastest growing area. Lot of Research has been carried out leading to breast cancer, liver diseases and other types of cancer and also diseases related to heart. There are very few articles related to Tuberculosis.

#### **2. Tuberculosis**

336 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

be implemented both in sequential and parallel mode thus improving the performance.

In recent years, data mining has been widely used in the area of genetics and medicine, called medical data mining. In the past two decades we have witnessed revolutionary changes in biomedical research and bio-technology. There is an explosive growth of biomedical data, ranging from those collected in pharmaceutical studies and cancer therapy investigations to those identified in genomics and proteomics research. The rapid progress of biotechnology and bio-data analysis methods has led to the emergence and fast growth of a promising new field: Bioinformatics. On the other hand, recent progress in data mining research has led to the developments of numerous efficient and scalable methods for mining interesting patterns and knowledge in large databases, ranging from efficient classification methods to clustering, outlier analysis, frequent, sequential and structured pattern analysis methods, and visualization and spatial/temporal data analysis tools. The question becomes how to bridge the two fields, Data Mining and Bioinformatics, for successful data mining in biomedical data. Especially, we should analyze how data mining may help efficient and effective bio-medical data analysis and outline some research problems that may motivate the further developments of powerful data mining tools for bio data or medical data

Data mining is a process that involves aggregating raw data stored in a database and analyzing them to identify trends, patterns and anomalies. Medical data mining is an active research area under data mining since medical databases have accumulated large quantities of information about patients and their clinical conditions. Relationships and patterns hidden in this data can provide new medical knowledge as has been proved in a number of medical data mining applications. A Doctor quickly swung into action after a renowned pharmaceutical company in the USA announced in 2001 that it was withdrawing a cholesterol-lowering drug following the deaths of more than 30 people. Using his medical records database, his staff was able to identify all patients taking the cholesterol-lowering drug and notify them within 24 hours of the announcement. What the doctor did is technically known as Data Mining. Very few doctors, however, were able to act on the

situation, because they did not have accessible raw data in the electronic format.

accurate demands visible from the data that is mined.

There are very few articles related to Tuberculosis.

Not only does disciplined storage of medical data helps the physicians and healthcare institutions, but it also helps pharmaceutical companies to mine the data to see the trends in diseases. It also helps prioritize product development and clinical trials based on the

Various data mining tasks can be applied on different diseases data set. This helps even the doctor to identify hidden associations between various symptoms. Research has been carried out on gene data, proteonomic data and attributes related to diseases covering even risk factors. Prediction of diseases has also been done on scanned images leading to medical imaging, which is the fastest growing area. Lot of Research has been carried out leading to breast cancer, liver diseases and other types of cancer and also diseases related to heart.

Such type of mining is called medical data mining.

**1.3 Medical data mining** 

analysis.

**Tuberculosis (TB)** is a common and often deadly infectious disease caused by mycobacterium; in humans it is mainly *Mycobacterium tuberculosis*. It usually spreads through the air and attacks low immune bodies such as patients with Human Immunodeficiency Virus (HIV). It is a disease which can affect virtually all organs, not sparing even the relatively inaccessible sites. The microorganisms usually enter the body by inhalation through the lungs. They spread from the initial location in the lungs to other parts of the body via the blood stream. They present a diagnostic dilemma even for physicians with a great deal of experience in this disease. Hence Tuberculosis (TB) is a contagious bacterial disease caused by mycobacterium which affects usually lungs and is often co-infected with HIV/AIDS.

It is a great problem for most developing countries because of the low diagnosis and treatment opportunities. Tuberculosis has the highest mortality level among the diseases caused by a single type of microorganism. Thus, tuberculosis is a great health concern all over the world, and in India as well (wikipedia.org).

Symptoms of TB depend on where in the body the TB bacteria are growing. TB bacteria usually grow in the lungs. TB in the lungs may cause symptoms such as a bad cough that lasts 3 weeks or longer pain in the chest coughing up blood or sputum. Other symptoms of active TB disease are: weakness or fatigue, weight loss, no appetite, chills, fever and sweating at night.

Although common and deadly in the third world, Tuberculosis was almost non-existent in the developed world, but has been making a recent resurgence. Certain drug-resistant strains are emerging and people with immune suppression such as AIDS or poor health are becoming carriers.

#### **2.1 Data set description**

The medical dataset we are using includes 700 real records of patients suffering from TB obtained from a city hospital. The entire dataset is put in one file having many records. Each record corresponds to most relevant information of one patient. Initial queries by doctor as symptoms and some required test details of patients have been considered as main attributes. Totally there are 12 attributes (symptoms) and last attribute is considered as class in case of Associative Classification. The symptoms of each patient such as age, chronic cough(weeks), loss of weight, intermittent fever(days), night sweats, Sputum, Bloodcough, chestpain, HIV, radiographic findings, wheezing and TBtype are considered as attributes.

Table 1 shows names of 12 attributes considered along with their Data Types (DT). Type Nindicates numerical and C is categorical.

#### **3. Association Rule Mining**

Association Rule Mining (ARM) is an important problem in the rapidly growing field called data mining and knowledge discovery in databases (KDD). The task of association rule mining is to mine a set of highly correlated attributes/features shared among a large number of records in a given database. For example, consider the sales database of a bookstore, where the

Data Mining Techniques in the Diagnosis of Tuberculosis 339

Apriori algorithm employs two actions join step and prune step as explained in the


 Generate length (k+1) candidate itemsets from length k frequent itemsets [join step] Prune candidate itemsets containing subsets of length k that are infrequent [prune

Eliminate candidates that are infrequent, leaving only those that are frequent

Once the frequent itemsets from transactions in a database D have been found, it is straightforward to generate strong association rules from them where strong association rules satisfy both minimum support and minimum confidence. This is calculated from the

Confidence(A->B) = support\_count(AỤB) */* support\_count(A)

 For every nonempty subset s of l, output the rule " s -> (l-s)" if support\_count(l) */*  support\_count(s) is greater than or equal to min\_conf, where min\_conf is the

Since the processing of the Apriori algorithm requires plenty of time, its computational efficiency is a very important issue. In order to improve the efficiency of Apriori, many

researchers have proposed modified association rule-related algorithms.


following algorithm to find frequent itemsets.

*XY X Y sX sY* , :( ) ( ) ( )




minimum confidence threshold.

Reduce passes of transaction database scans

Facilitate support counting of candidates

Tedious workload of support counting for candidates

Multiple scans of transaction database

Huge number of candidates

**Improving Apriori: general ideas** 

Shrink number of candidates

Count the support of each candidate by scanning the DB

Based on the above equation association rules can be generated as follows: For each frequent itemset l, generate all non empty subsets of l.

be frequent

**Apriori algorithm** 

step]

**Rule Generation** 

following equation

**Challenges of Apriori** 


records represent customers and the attributes represent books. The mined patterns are the set of books most frequently bought together by the customer. An example could be that, 60% of the people who buy Design and Analysis of Algorithms also buy Data Structure. The store can use this knowledge for promotions, self-placement etc. There are many application areas for association rule mining techniques, which include catalog design, store layout, customer segmentation, telecommunication alarm diagnosis and so on.


Table 1. List of Attributes and their Datatypes

#### **3.1 Definition of association rule**

Here we give the classical definition of association rules. Let { t1, t2,…..tn} be a set of transactions and let I be a set of items, I={ I1,I2,….Im }. An association rule is an implication of the form XY, where X, Y are disjoint subsets of item I and X∩Y=ф. X is called the *antecedent* and Y is called the *consequent* of the rule. In general, a set of items such as the antecedent or consequent of a rule is called an *Itemset*. Each *itemset* has an associated measure of statistical significance called *support*. *support(x)=s* is the fraction of the transactions in the database containing X. The rule has a measure of strength called *confidence* defined as the ratio *support(X Ụ Y) / support(X)* (J.Han & M.Kamber, 2006).

Given a set of transactions T, the goal of association rule mining is to find all rules having support ≥ *minsup* threshold and confidence ≥ *minconf* threshold.

Mining Association rule is a Two-step approach:

	- Generate all itemsets whose support minsup.
	- Generate high confidence rules from each frequent itemset, where each rule is a binary partitioning of a frequent itemset.

#### **Frequent Itemset Generation**

The two important algorithms for frequent itemset generation are Apriori algorithm (first proposed by Agrawal, Imielinski and swami VLDB 1994) and Frequent pattern tree growth (FP-Tree) (FPgrowth—Han, Pei & Yin @SIGMOD'00).

Apriori algorithm employs two actions join step and prune step as explained in the following algorithm to find frequent itemsets.

	- Support of an itemset never exceeds the support of its subsets
		- *XY X Y sX sY* , :( ) ( ) ( )

#### **Apriori algorithm**


338 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

records represent customers and the attributes represent books. The mined patterns are the set of books most frequently bought together by the customer. An example could be that, 60% of the people who buy Design and Analysis of Algorithms also buy Data Structure. The store can use this knowledge for promotions, self-placement etc. There are many application areas for association rule mining techniques, which include catalog design, store layout, customer

> No **Name DT**  1 Age N 2 chroniccough(weeks) N 3 weightloss C 4 intermittentfever(days) N 5 nightsweats C 6 Bloodcough C 7 chestpain C 8 HIV C 9 Radiographicfindings C 10 Sputum C 11 wheezing C 12 TBType C

Here we give the classical definition of association rules. Let { t1, t2,…..tn} be a set of transactions and let I be a set of items, I={ I1,I2,….Im }. An association rule is an implication of the form XY, where X, Y are disjoint subsets of item I and X∩Y=ф. X is called the *antecedent* and Y is called the *consequent* of the rule. In general, a set of items such as the antecedent or consequent of a rule is called an *Itemset*. Each *itemset* has an associated measure of statistical significance called *support*. *support(x)=s* is the fraction of the transactions in the database containing X. The rule has a measure of strength called

Given a set of transactions T, the goal of association rule mining is to find all rules having

Generate high confidence rules from each frequent itemset, where each rule is a

The two important algorithms for frequent itemset generation are Apriori algorithm (first proposed by Agrawal, Imielinski and swami VLDB 1994) and Frequent pattern tree growth

*confidence* defined as the ratio *support(X Ụ Y) / support(X)* (J.Han & M.Kamber, 2006).

support ≥ *minsup* threshold and confidence ≥ *minconf* threshold.

Generate all itemsets whose support minsup.

binary partitioning of a frequent itemset.

(FP-Tree) (FPgrowth—Han, Pei & Yin @SIGMOD'00).

Mining Association rule is a Two-step approach:


**Frequent Itemset Generation** 


segmentation, telecommunication alarm diagnosis and so on.

Table 1. List of Attributes and their Datatypes

**3.1 Definition of association rule** 

	- Generate length (k+1) candidate itemsets from length k frequent itemsets [join step]
	- Prune candidate itemsets containing subsets of length k that are infrequent [prune step]
	- Count the support of each candidate by scanning the DB
	- Eliminate candidates that are infrequent, leaving only those that are frequent

#### **Rule Generation**

Once the frequent itemsets from transactions in a database D have been found, it is straightforward to generate strong association rules from them where strong association rules satisfy both minimum support and minimum confidence. This is calculated from the following equation

Confidence(A->B) = support\_count(AỤB) */* support\_count(A)

Based on the above equation association rules can be generated as follows:


#### **Challenges of Apriori**


#### **Improving Apriori: general ideas**


Since the processing of the Apriori algorithm requires plenty of time, its computational efficiency is a very important issue. In order to improve the efficiency of Apriori, many researchers have proposed modified association rule-related algorithms.

Data Mining Techniques in the Diagnosis of Tuberculosis 341

**4 <= Chronic cough (weeks) < 8**

**Null HIV = Positive HIV = Negative Sputum = Yes Sputum = Null**  7 8 9 10 11

In the above tables, note that Age is a numerical attribute and its cut off point is <25 & >=25. Similarly HIV is a categorical attribute where positive value is assigned one number and negative another. The value Null for categorical attribute weightloss is equivalent to No and is assigned a unique number. By using the schema table above we map each tuple in the original data of table 2 to a resulting normalized table shown in table 4. Resulting table has the same number of columns as the original table but filled with unique integer values.

Age **Chronic cough(weeks) Weight loss HIV Sputum** 

a. **Frequent Itemsets from TB data:** The following figure 2 and 3 shows some of the frequent itemsets generated with 70% support, 90% confidence and 60% support,

[7] {chroniccough(weeks)<39.0 weightloss=null intermittentfever(days)<91.25} = 526

[17] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 chestpain=null} = 539

1 2 3 4 5 6

**Chronic cough (weeks) = Null**

**Weight loss = Yes** 

**Age < 25** 

**Age >= 25** 

**Weight loss =** 

Table 3. Schema Table

Table 4. Normalized Table

**3.2.2 TB rules generation** 

[2] {weightloss=null} = 550

[8] {nightsweats=null} = 496 [9] {Bloodcough=null} = 671

[14] {chestpain=null} = 570

[1] {chroniccough(weeks)<39.0} = 692

[4] {intermittentfever(days)<91.25} = 669

**Chronic cough (weeks)< 4**

[3] {chroniccough(weeks)<39.0 weightloss=null} = 544

[6] {weightloss=null intermittentfever(days)<91.25} = 530

[10] {chroniccough(weeks)<39.0 Bloodcough=null} = 663

[13] {intermittentfever(days)<91.25 Bloodcough=null} = 645

[15 {chroniccough(weeks)<39.0 chestpain=null} = 564 [16] {intermittentfever(days)<91.25 chestpain=null} = 542

[11] {weightloss=null Bloodcough=null} = 534

[18] {Bloodcough=null chestpain=null} = 546

[5] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25} = 664

[12] {chroniccough(weeks)<39.0 weightloss=null Bloodcough=null} = 528

Fig. 2. Frequent Itemsets with 70%support and 90% confidence

#### **Advantages of frequent itemset generation and rule generation**

	- What products were often purchased together?— Beer and diapers?!
	- What are the subsequent purchases after buying a PC?
	- What kinds of DNA are sensitive to this new drug?
	- Can we automatically classify web documents?
	- Basket data analysis, cross-marketing, catalog design, sale campaign analysis, Web log (click stream) analysis, and DNA sequence analysis.

#### **3.2 Tuberculosis association rules**

**T**uberculosis association rules can be generated by applying data mining ARM technique with the following steps:


#### **3.2.1 Pre-processing**

Incomplete, noisy, and inconsistent data are common among real world databases. Hence it is necessary to preprocess such data before using it. The most common topics under data preprocessing are Data cleaning, Data integration, Data Transformation, Data reduction, Data discretization and automatic generation of concept hierarchies.

Discretization and Normalization are the two data transformation procedures that help in representing the data and their relationships precisely in a tabular format that makes the database easy to understand and operationally efficient. This also reduces data redundancy and enhances performance.

The above TB attributes are normalized and discretized to a suitable binary format. A categorical data field has a value selected from an available list of values. Such data items can be normalized by allocating a unique column number to each possible value. Numerical data fields are discretized by taking values that are within some range defined by minimum and maximum limits. In such cases we can divide the given range into a number of subranges and allocate a unique column number to each sub-range respectively.

Here we give a small example of five patients medical records with five attributes. Table 2 shows original data. Table 3 contains schema of how the attributes are mapped to individual column numbers. Table 4 is the final translated or normalized data.


Table 2. Original (raw) Data


Table 3. Schema Table

340 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Basket data analysis, cross-marketing, catalog design, sale campaign analysis, Web

**T**uberculosis association rules can be generated by applying data mining ARM technique

Incomplete, noisy, and inconsistent data are common among real world databases. Hence it is necessary to preprocess such data before using it. The most common topics under data preprocessing are Data cleaning, Data integration, Data Transformation, Data reduction,

Discretization and Normalization are the two data transformation procedures that help in representing the data and their relationships precisely in a tabular format that makes the database easy to understand and operationally efficient. This also reduces data redundancy

The above TB attributes are normalized and discretized to a suitable binary format. A categorical data field has a value selected from an available list of values. Such data items can be normalized by allocating a unique column number to each possible value. Numerical data fields are discretized by taking values that are within some range defined by minimum and maximum limits. In such cases we can divide the given range into a number of sub-

Here we give a small example of five patients medical records with five attributes. Table 2 shows original data. Table 3 contains schema of how the attributes are mapped to individual

**Weight** 

Yes Yes Null Yes Yes

**loss HIV Sputum** 

Yes Yes Yes Null Yes

Negative Negative Negative Positive Positive

What products were often purchased together?— Beer and diapers?!

**Advantages of frequent itemset generation and rule generation** 

 What are the subsequent purchases after buying a PC? What kinds of DNA are sensitive to this new drug? Can we automatically classify web documents?

log (click stream) analysis, and DNA sequence analysis.

 Pre-processing the dataset by discretizing and normalizing Generating rules by applying apriori on preprocessed range data

Data discretization and automatic generation of concept hierarchies.

ranges and allocate a unique column number to each sub-range respectively.

column numbers. Table 4 is the final translated or normalized data.

**cough(weeks)** 

3 6 6 Null Null

**Age Chronic** 

Table 2. Original (raw) Data

Finding inherent regularities in data

**3.2 Tuberculosis association rules** 

with the following steps:

**3.2.1 Pre-processing** 

and enhances performance.

Applications

In the above tables, note that Age is a numerical attribute and its cut off point is <25 & >=25. Similarly HIV is a categorical attribute where positive value is assigned one number and negative another. The value Null for categorical attribute weightloss is equivalent to No and is assigned a unique number. By using the schema table above we map each tuple in the original data of table 2 to a resulting normalized table shown in table 4. Resulting table has the same number of columns as the original table but filled with unique integer values.


Table 4. Normalized Table

#### **3.2.2 TB rules generation**

a. **Frequent Itemsets from TB data:** The following figure 2 and 3 shows some of the frequent itemsets generated with 70% support, 90% confidence and 60% support,

```
Fig. 2. Frequent Itemsets with 70%support and 90% confidence 
 [1] {chroniccough(weeks)<39.0} = 692
 [2] {weightloss=null} = 550 
 [3] {chroniccough(weeks)<39.0 weightloss=null} = 544 
 [4] {intermittentfever(days)<91.25} = 669 
 [5] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25} = 664 
 [6] {weightloss=null intermittentfever(days)<91.25} = 530 
 [7] {chroniccough(weeks)<39.0 weightloss=null intermittentfever(days)<91.25} = 526 
 [8] {nightsweats=null} = 496 
 [9] {Bloodcough=null} = 671 
 [10] {chroniccough(weeks)<39.0 Bloodcough=null} = 663 
 [11] {weightloss=null Bloodcough=null} = 534 
 [12] {chroniccough(weeks)<39.0 weightloss=null Bloodcough=null} = 528 
 [13] {intermittentfever(days)<91.25 Bloodcough=null} = 645 
 [14] {chestpain=null} = 570 
 [15 {chroniccough(weeks)<39.0 chestpain=null} = 564 
 [16] {intermittentfever(days)<91.25 chestpain=null} = 542 
 [17] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 chestpain=null} = 539 
 [18] {Bloodcough=null chestpain=null} = 546
```
Data Mining Techniques in the Diagnosis of Tuberculosis 343

(1) {intermittentfever(days)<91.25 chestpain=null} -> {chroniccough(weeks)<39.0}

(7) {weightloss=null intermittentfever(days)<91.25} -> {Bloodcough=null} 97.54 (8) {chroniccough(weeks)<39.0 weightloss=null intermittentfever(days)<91.25} ->

(2) {intermittentfever(days)<91.25 Bloodcough=null chestpain=null} ->

(9) {chroniccough(weeks)<39.0 weightloss=null Bloodcough=null} ->

(11) {Bloodcough=null chestpain=null} -> {chroniccough(weeks)<39.0

(2) {chroniccough(weeks)<39.0 HIV=Negative} -> {TBtype=PTB} 100.0

(6) {HIV=Negative TBtype=PTB} -> {chroniccough(weeks)<39.0} 98.7 (7) {Bloodcough=null TBtype=PTB} -> {chroniccough(weeks)<39.0} 98.69 (8) {chroniccough(weeks)<39.0 nightsweats=null Bloodcough=null} ->

(12) {Bloodcough=null TBtype=PTB} -> {chroniccough(weeks)<39.0

(4) {Bloodcough=null HIV=Negative} -> {TBtype=PTB} 100.0

(3) {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 HIV=Negative} ->

(5) {intermittentfever(days)<91.25 Bloodcough=null HIV=Negative} -> {TBtype=PTB}

(9) {chroniccough(weeks)<39.0 TBtype=PTB} -> {intermittentfever(days)<91.25} 98.06 (10) {TBtype=PTB} -> {chroniccough(weeks)<39.0 intermittentfever(days)<91.25}

(11) {weightloss=null Bloodcough=null} -> {intermittentfever(days)<91.25} 96.81

(13) {chroniccough(weeks)<39.0 weightloss=null} -> {chestpain=null} 82.53

(12) {chestpain=null} -> {chroniccough(weeks)<39.0 Bloodcough=null} 94.73 (13) {chroniccough(weeks)<39.0 chestpain=null} -> {intermittentfever(days)<91.25

(14) {chestpain=null} -> {intermittentfever(days)<91.25 Bloodcough=null} 91.57 (15) {chestpain=null} -> {chroniccough(weeks)<39.0 intermittentfever(days)<91.25

(3) {nightsweats=null} -> {chroniccough(weeks)<39.0} 98.99 (4) {chestpain=null} -> {chroniccough(weeks)<39.0} 98.94 (5) {weightloss=null} -> {chroniccough(weeks)<39.0} 98.9 (6) {Bloodcough=null} -> {chroniccough(weeks)<39.0} 98.8

Fig. 4. Rule generation with 70%support and 90% confidence

(1) {HIV=Negative} -> {TBtype=PTB} 100.0

{intermittentfever(days)<91.25} 98.1

(10) {weightloss=null} -> {Bloodcough=null} 97.09

99.44

{chroniccough(weeks)<39.0} 99.42

{intermittentfever(days)<91.25} 97.15

intermittentfever(days)<91.25} 95.05

{Bloodcough=null} 97.52

Bloodcough=null} 92.02

Bloodcough=null} 91.05

{TBtype=PTB} 100.0

100.0

96.82

Fig. 5. Rule generation with 60% support and 80% confidence

intermittentfever(days)<91.25 HIV=Negative} 95.2

80%confidence. The Format of the rule is: [N] {I} = S, where N is a sequential number, I is the item set converted from normalized numerical value to schema text (symptoms) and S the support.

```
[1] {chroniccough(weeks)<39.0} = 692 
[2] {weightloss=null} = 550 
[3] {chroniccough(weeks)<39.0 weightloss=null} = 544 
[4] {intermittentfever(days)<91.25} = 669 
[5] {Bloodcough=null} = 671 
[6] {chroniccough(weeks)<39.0 Bloodcough=null} = 663 
[7] {weightloss=null Bloodcough=null} = 534 
[8] {chestpain=null} = 570 
[9] {chroniccough(weeks)<39.0 chestpain=null} = 564 
[10] {weightloss=null chestpain=null} = 454 
[11] {HIV=Negative} = 465 
[12] {chroniccough(weeks)<39.0 HIV=Negative} = 459 
[13] {intermittentfever(days)<91.25 HIV=Negative} = 455 
[14] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 HIV=Negative} = 450 
[15] {Bloodcough=null HIV=Negative} = 452 
[16] {Sputum=yes} = 422 
[17] {TBtype=PTB} = 472 
[18] {chroniccough(weeks)<39.0 TBtype=PTB} = 466 
[19] {intermittentfever(days)<91.25 TBtype=PTB} = 462 
[20] {HIV=Negative TBtype=PTB} = 465 
[21] {chroniccough(weeks)<39.0 HIV=Negative TBtype=PTB} = 459 
[22] {intermittentfever(days)<91.25 HIV=Negative TBtype=PTB} = 455 
[23] {intermittentfever(days)<91.25 Bloodcough=null HIV=Negative TBtype=PTB} = 442 
[24] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 Bloodcough=null 
HIV=Negative TBtype=PTB} = 437
```
Fig. 3. Frequent Itemsets with 60%support and 80% confidence

#### **b. Discovered TB Association rules**

Several medically important association rules are obtained after applying apriori algorithm to the normalized table. It takes the frequent itemsets in figure 2 and 3 and generates rules as shown in figure 4 and 5 respectively. Each association rule shows the relation between one symptom with the other. Data set was first tested by fixing support=70% and confidence=90%.We could get very few association rules, some are listed in figure 4. Rule 7 in figure 4 describes that if weightloss equals null and intermittent fever is less than 91 days implies Bloodcough is null with 97.5% confidence. Most of the rules show the relationship between only few attributes like weightloss, intermittent fever, Bloodcough and chest pain. All the attributes were not shown here. Next with 60% support and 80% confidence we could get large number of association rules, few listed in figure 5 that provides more relationship with many frequent attributes. Rule 1 in figure 5 says if HIV status is negative their TBtype is Pulmonary Tuberculosis (PTB) with 100% confidence. Rule 5 shows the relationship between intermittent fever, Bloodcough, HIV and PTB. Though all the rules

80%confidence. The Format of the rule is: [N] {I} = S, where N is a sequential number, I is the item set converted from normalized numerical value to schema text (symptoms) and S the

Fig. 3. Frequent Itemsets with 60%support and 80% confidence

[21] {chroniccough(weeks)<39.0 HIV=Negative TBtype=PTB} = 459 [22] {intermittentfever(days)<91.25 HIV=Negative TBtype=PTB} = 455

Several medically important association rules are obtained after applying apriori algorithm to the normalized table. It takes the frequent itemsets in figure 2 and 3 and generates rules as shown in figure 4 and 5 respectively. Each association rule shows the relation between one symptom with the other. Data set was first tested by fixing support=70% and confidence=90%.We could get very few association rules, some are listed in figure 4. Rule 7 in figure 4 describes that if weightloss equals null and intermittent fever is less than 91 days implies Bloodcough is null with 97.5% confidence. Most of the rules show the relationship between only few attributes like weightloss, intermittent fever, Bloodcough and chest pain. All the attributes were not shown here. Next with 60% support and 80% confidence we could get large number of association rules, few listed in figure 5 that provides more relationship with many frequent attributes. Rule 1 in figure 5 says if HIV status is negative their TBtype is Pulmonary Tuberculosis (PTB) with 100% confidence. Rule 5 shows the relationship between intermittent fever, Bloodcough, HIV and PTB. Though all the rules

[23] {intermittentfever(days)<91.25 Bloodcough=null HIV=Negative TBtype=PTB} = 442

[24] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 Bloodcough=null

[14] {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 HIV=Negative} = 450

**b. Discovered TB Association rules** 

HIV=Negative TBtype=PTB} = 437

[1] {chroniccough(weeks)<39.0} = 692

[4] {intermittentfever(days)<91.25} = 669

[7] {weightloss=null Bloodcough=null} = 534

[10] {weightloss=null chestpain=null} = 454

[15] {Bloodcough=null HIV=Negative} = 452

[20] {HIV=Negative TBtype=PTB} = 465

[3] {chroniccough(weeks)<39.0 weightloss=null} = 544

[6] {chroniccough(weeks)<39.0 Bloodcough=null} = 663

[9] {chroniccough(weeks)<39.0 chestpain=null} = 564

[12] {chroniccough(weeks)<39.0 HIV=Negative} = 459 [13] {intermittentfever(days)<91.25 HIV=Negative} = 455

[18] {chroniccough(weeks)<39.0 TBtype=PTB} = 466 [19] {intermittentfever(days)<91.25 TBtype=PTB} = 462

[2] {weightloss=null} = 550

[5] {Bloodcough=null} = 671

[8] {chestpain=null} = 570

[11] {HIV=Negative} = 465

[16] {Sputum=yes} = 422 [17] {TBtype=PTB} = 472

support.

```
(1) {intermittentfever(days)<91.25 chestpain=null} -> {chroniccough(weeks)<39.0} 
99.44 
(2) {intermittentfever(days)<91.25 Bloodcough=null chestpain=null} -> 
{chroniccough(weeks)<39.0} 99.42 
(3) {nightsweats=null} -> {chroniccough(weeks)<39.0} 98.99 
(4) {chestpain=null} -> {chroniccough(weeks)<39.0} 98.94 
(5) {weightloss=null} -> {chroniccough(weeks)<39.0} 98.9 
(6) {Bloodcough=null} -> {chroniccough(weeks)<39.0} 98.8 
(7) {weightloss=null intermittentfever(days)<91.25} -> {Bloodcough=null} 97.54 
(8) {chroniccough(weeks)<39.0 weightloss=null intermittentfever(days)<91.25} -> 
{Bloodcough=null} 97.52 
(9) {chroniccough(weeks)<39.0 weightloss=null Bloodcough=null} -> 
{intermittentfever(days)<91.25} 97.15 
(10) {weightloss=null} -> {Bloodcough=null} 97.09 
(11) {Bloodcough=null chestpain=null} -> {chroniccough(weeks)<39.0 
intermittentfever(days)<91.25} 95.05 
(12) {chestpain=null} -> {chroniccough(weeks)<39.0 Bloodcough=null} 94.73 
(13) {chroniccough(weeks)<39.0 chestpain=null} -> {intermittentfever(days)<91.25 
Bloodcough=null} 92.02 
(14) {chestpain=null} -> {intermittentfever(days)<91.25 Bloodcough=null} 91.57 
(15) {chestpain=null} -> {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 
Bloodcough=null} 91.05
```
Fig. 4. Rule generation with 70%support and 90% confidence

(1) {HIV=Negative} -> {TBtype=PTB} 100.0 (2) {chroniccough(weeks)<39.0 HIV=Negative} -> {TBtype=PTB} 100.0 (3) {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 HIV=Negative} -> {TBtype=PTB} 100.0 (4) {Bloodcough=null HIV=Negative} -> {TBtype=PTB} 100.0 (5) {intermittentfever(days)<91.25 Bloodcough=null HIV=Negative} -> {TBtype=PTB} 100.0 (6) {HIV=Negative TBtype=PTB} -> {chroniccough(weeks)<39.0} 98.7 (7) {Bloodcough=null TBtype=PTB} -> {chroniccough(weeks)<39.0} 98.69 (8) {chroniccough(weeks)<39.0 nightsweats=null Bloodcough=null} -> {intermittentfever(days)<91.25} 98.1 (9) {chroniccough(weeks)<39.0 TBtype=PTB} -> {intermittentfever(days)<91.25} 98.06 (10) {TBtype=PTB} -> {chroniccough(weeks)<39.0 intermittentfever(days)<91.25} 96.82 (11) {weightloss=null Bloodcough=null} -> {intermittentfever(days)<91.25} 96.81 (12) {Bloodcough=null TBtype=PTB} -> {chroniccough(weeks)<39.0 intermittentfever(days)<91.25 HIV=Negative} 95.2 (13) {chroniccough(weeks)<39.0 weightloss=null} -> {chestpain=null} 82.53

Data Mining Techniques in the Diagnosis of Tuberculosis 345

appear as the class label. In order to perform AC, a classifier will first mine CARs from a given transaction and later select the most predictive rule to perform a classifier (Chien and Chen, 2010). AC generates CARs depending on the frequent item generation technique in mining rules. Despite its benefit, AC does propose challenges in its classification performance. The most important thing is to the approach in mining appropriate CARs for the classification and its pruning technology since AC will generate large number of frequent item sets due to its pruning algorithm. Its prominent pitfalls are in its incapability

Different approaches have been proposed for associative classification that has been found to outperform traditional classification algorithms. Some of AC algorithms include Classification based on Association (CBA), Classification based on Multiple Association Rules (CMAR), and Classification based on Predictive Association Rules (CPAR-Chien and Chen, 2010). Generally, AC consists of three main phases, which are rule generation, rule

of handling numerical data**.**

Fig. 6. Associative classification procedure

**4.1 Associative Classification Algorithms** 

may not be interesting to users, only few rules like explained above gives very good description and some hidden relationship may also be found.

We could see from the following output that left side (Antecedent) and right side (consequent) of the rule keep on interchanging repeatedly, which can be pruned by applying some conditions on both antecedent and consequent of a rule.

The format is: (N) ANTECEDENT -> CONSEQUENT CONFIDENCE (%)

#### **4. Associative classification**

Association Rule Mining (ARM) as explained in section 3 is one of the most popular approaches in data mining and if used in the medical domain has a great potential to improve disease prediction. This results in large number of descriptive rules. Therefore ARM can be integrated within classification task to generate a single system called as Associative classification (AC) which is a better alternative for predictive analytics.

Classification based on association rules has been proved as very competitive (Liu.B et al., 1998). The general idea is to generate a set of association rules with a fixed consequent (involving the class attribute) and then use subsets of these rules to classify new examples. This approach has the advantage of searching a larger portion of the rule version space, since no search heuristics are employed, in contrast to Decision Tree and traditional classification rule induction. The extra search is done in a controlled manner enabled by the good computational behaviour of association rule discovery algorithms. Another advantage is that the produced rich rule set can be used in a variety of ways without relearning, which can be used to improve the classification accuracy ( Jorge and Azevedo, 2005).

The procedure of associative classification rule mining as shown in figure 6 is not much different from that of general association rule mining. A typical associative classification system is constructed in two stages: 1) discovering all the event association rules (in which the frequency of occurrences is significant according to some tests); 2) generating classification rules from the association patterns to build a classifier. In the first stage, the learning target is to discover the association rules inherent in a database, but generating frequent itemsets may prove to be quite expensive. The number of rules generated from association rule discovery is quite large. Hence rule pruning is required. Moreover, to avoid the problem of overfitting, proper rule pruning method is to be employed. Ranking of the rules is also important. When a test instance has more than one potentially applicable rules, rule ranking is necessary to prefer one rule over the others. In the second stage, the task is to select a set of relevant association rules discovered to construct a classifier given the predicting attribute.

For example given a rule X -> Y, AC will only consider rules having a target class as the consequent. This means the new integration focuses on a subset of association rules, whose right hand-sides are restricted to the classification class attribute. This type of rule is called Class Association Rules (CARs). While normal association rule allows more than one condition as its consequent and any item from *X* can be the consequent, CARs generated in AC limit the consequent to one fixed target class for each rule and item from *X* are forbid to

may not be interesting to users, only few rules like explained above gives very good

We could see from the following output that left side (Antecedent) and right side (consequent) of the rule keep on interchanging repeatedly, which can be pruned by

Association Rule Mining (ARM) as explained in section 3 is one of the most popular approaches in data mining and if used in the medical domain has a great potential to improve disease prediction. This results in large number of descriptive rules. Therefore ARM can be integrated within classification task to generate a single system called as

Classification based on association rules has been proved as very competitive (Liu.B et al., 1998). The general idea is to generate a set of association rules with a fixed consequent (involving the class attribute) and then use subsets of these rules to classify new examples. This approach has the advantage of searching a larger portion of the rule version space, since no search heuristics are employed, in contrast to Decision Tree and traditional classification rule induction. The extra search is done in a controlled manner enabled by the good computational behaviour of association rule discovery algorithms. Another advantage is that the produced rich rule set can be used in a variety of ways without relearning, which can be used to improve the classification accuracy ( Jorge and

The procedure of associative classification rule mining as shown in figure 6 is not much different from that of general association rule mining. A typical associative classification system is constructed in two stages: 1) discovering all the event association rules (in which the frequency of occurrences is significant according to some tests); 2) generating classification rules from the association patterns to build a classifier. In the first stage, the learning target is to discover the association rules inherent in a database, but generating frequent itemsets may prove to be quite expensive. The number of rules generated from association rule discovery is quite large. Hence rule pruning is required. Moreover, to avoid the problem of overfitting, proper rule pruning method is to be employed. Ranking of the rules is also important. When a test instance has more than one potentially applicable rules, rule ranking is necessary to prefer one rule over the others. In the second stage, the task is to select a set of relevant association rules discovered to construct a classifier given the

For example given a rule X -> Y, AC will only consider rules having a target class as the consequent. This means the new integration focuses on a subset of association rules, whose right hand-sides are restricted to the classification class attribute. This type of rule is called Class Association Rules (CARs). While normal association rule allows more than one condition as its consequent and any item from *X* can be the consequent, CARs generated in AC limit the consequent to one fixed target class for each rule and item from *X* are forbid to

Associative classification (AC) which is a better alternative for predictive analytics.

description and some hidden relationship may also be found.

**4. Associative classification** 

Azevedo, 2005).

predicting attribute.

applying some conditions on both antecedent and consequent of a rule. The format is: (N) ANTECEDENT -> CONSEQUENT CONFIDENCE (%) appear as the class label. In order to perform AC, a classifier will first mine CARs from a given transaction and later select the most predictive rule to perform a classifier (Chien and Chen, 2010). AC generates CARs depending on the frequent item generation technique in mining rules. Despite its benefit, AC does propose challenges in its classification performance. The most important thing is to the approach in mining appropriate CARs for the classification and its pruning technology since AC will generate large number of frequent item sets due to its pruning algorithm. Its prominent pitfalls are in its incapability of handling numerical data**.**

Fig. 6. Associative classification procedure

#### **4.1 Associative Classification Algorithms**

Different approaches have been proposed for associative classification that has been found to outperform traditional classification algorithms. Some of AC algorithms include Classification based on Association (CBA), Classification based on Multiple Association Rules (CMAR), and Classification based on Predictive Association Rules (CPAR-Chien and Chen, 2010). Generally, AC consists of three main phases, which are rule generation, rule

Data Mining Techniques in the Diagnosis of Tuberculosis 347

Next, the subset of matching rules are used to classify a test instance instead of one rule, and

The CMAR algorithm generates and evaluates rules in a similar way as CBA, but uses a more efficient FPtree structure. A major difference is that it uses multiple rules in prediction

The CMAR algorithm (as described in Li et al., 2001) uses an FP-growth algorithm (Han & Kamber, 2000) to produce a set of CARs and uses CBA method for rule ranking. It prunes rules using high confidence, highly related rules and analyzes the correlation among them using Chi-Squared testing. To test the resulting classifier Li et al. propose the following

1. Collect all rules that satisfy r, and if consequents of all rules are all identical, or only one

2. Else group rules according to classifier and determine the combined effect of the rules in each group, the classifier associated with the "strongest group" is then selected. The strength of a group is calculated using a *Weighted Chi Squared* (WCS) measure.

3. While both the training data set and rule set are not empty, for each rule R in rank descending order, find all data objects matching rule R. If R can correctly classify at least one object then select R and increase the cover-count of those objects matching R

Nonetheless, when the datasets are large, both rule generation and rule selection in CBA and CMAR are time consuming. The CPAR and other predictive mining algorithms overcome this problem by generating a small set of predictive rules directly from the dataset based on the rule prediction and coverage analysis, as opposed to generating candidate

CPAR is an improvement to CBA and CMAR (Thabtah et al., 2005; Thabtah, 2007). It is proposed by Chen, Yin and Huang in 2005.The core of CPAR and other predictive mining algorithms is the predictive rule mining capability, whereby after an instance has been correctly covered by a rule, instead of removing it, its weight is decreased by multiplying a factor. This is essentially a greedy approach in rule generation, which is more efficient than

CPAR may choose a number of attributes if those attributes have similar best gain. This is done by first calculating the gain and applying a GAIN\_SIMILARITY\_RATIO to this. All attributes with gain better than Local Gain Threshold (LGT) are then selected for further

by 1. A data object is removed if its cover-count passes coverage threshold δ;

this in turn produces better accuracy.

with associated weights**.**

Given a record r in the test set:

Selecting rules based on database:

1. Sort rules in the rank descending order;

rule, classify record according to the consequents.

Following algorithm shows steps for rule pruning.

2. For each data object in the training data set, set its cover-count to 0;

process.

rules.

**4.1.3 CPAR** 

processing.

generating all candidate rules.

pruning, and classification (Do et al., 2009; Tang and Liao, 2007).The performance, however, might differ depending on the algorithm employed in any of these three phases.

#### **4.1.1 CBA**

The first AC algorithm was introduced by (Liu. B et al., 1998), namely CBA. The algorithm is based on the Apriori association rule algorithm in generating CARs. These rules are later pruned and only one most suitable rule will be used to classify the test set. Essentially, the CBA algorithm performs three tasks. First, it mines all CARs. Second, it produces a classifier from CARs, and finally, it mines normal association rules**.**

1. Generation of CARs

In CBA, the classification Association rules (CARs) are found iteratively in an apriori algorithm-like fashion. At first, frequent 1-rule itemsets are generated and are pruned. Using this iteratively, other frequent rule itemsets are also found. They are then pruned to get complete set of Classification association rules.

2. Building classifier (Ranking and Pruning Rules)

To prune the rules, CBA uses pessimistic error based pruning method in C4.5. The rule ranking is defined as below:

Given two rules ri and rj, ri > rj (i.e., ri precedes rj or ri has higher precedence over rj ) if one of the following holds good:


After rule ranking, each training instance is covered by the rule having highest precedence among the rules that can cover the case. Every rule correctly classifies at least one training instance. The rules that do not cover any training instance are removed. The training instances that do not fall into any of the observed classes are added to a default class.

The multiple capabilities in CBA solve a number of problems in traditional classification systems. Since traditional classifiers only generate a small subset of rules that exists in data to form a classifier, the discovered rules may not be interesting. Also, to generate more rules we would need the classification system to load the entire database into the main memory. But because CBA generate all rules, the algorithm is more successful in finding interesting rules and the system also allows the data to reside on disk. However, in CBA, the rule generation process might degrade the accuracy of the classifier due to its randomness in selecting the most suitable rule to form the classifier model. CBA inherits Apriori multiple scan features that generates large number of rules, which is costly in terms of large computational time.

#### **4.1.2 CMAR**

CMAR is later introduced as the extension to CBA (Li et al., 2001). The CMAR algorithm implements FP-Growth algorithm instead of Apriori in generating its frequent itemset. Next, the subset of matching rules are used to classify a test instance instead of one rule, and this in turn produces better accuracy.

The CMAR algorithm generates and evaluates rules in a similar way as CBA, but uses a more efficient FPtree structure. A major difference is that it uses multiple rules in prediction with associated weights**.**

The CMAR algorithm (as described in Li et al., 2001) uses an FP-growth algorithm (Han & Kamber, 2000) to produce a set of CARs and uses CBA method for rule ranking. It prunes rules using high confidence, highly related rules and analyzes the correlation among them using Chi-Squared testing. To test the resulting classifier Li et al. propose the following process.

Given a record r in the test set:

346 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

pruning, and classification (Do et al., 2009; Tang and Liao, 2007).The performance, however,

The first AC algorithm was introduced by (Liu. B et al., 1998), namely CBA. The algorithm is based on the Apriori association rule algorithm in generating CARs. These rules are later pruned and only one most suitable rule will be used to classify the test set. Essentially, the CBA algorithm performs three tasks. First, it mines all CARs. Second, it produces a classifier

In CBA, the classification Association rules (CARs) are found iteratively in an apriori algorithm-like fashion. At first, frequent 1-rule itemsets are generated and are pruned. Using this iteratively, other frequent rule itemsets are also found. They are then pruned to get

To prune the rules, CBA uses pessimistic error based pruning method in C4.5. The rule

Given two rules ri and rj, ri > rj (i.e., ri precedes rj or ri has higher precedence over rj ) if one

3. Both the confidences and supports of ri and rj are the same, but ri is generated before rj After rule ranking, each training instance is covered by the rule having highest precedence among the rules that can cover the case. Every rule correctly classifies at least one training instance. The rules that do not cover any training instance are removed. The training

The multiple capabilities in CBA solve a number of problems in traditional classification systems. Since traditional classifiers only generate a small subset of rules that exists in data to form a classifier, the discovered rules may not be interesting. Also, to generate more rules we would need the classification system to load the entire database into the main memory. But because CBA generate all rules, the algorithm is more successful in finding interesting rules and the system also allows the data to reside on disk. However, in CBA, the rule generation process might degrade the accuracy of the classifier due to its randomness in selecting the most suitable rule to form the classifier model. CBA inherits Apriori multiple scan features that generates large number of rules, which is costly in terms of large

CMAR is later introduced as the extension to CBA (Li et al., 2001). The CMAR algorithm implements FP-Growth algorithm instead of Apriori in generating its frequent itemset.

instances that do not fall into any of the observed classes are added to a default class.

2. Their confidences are the same but support of ri is greater than that of rj

might differ depending on the algorithm employed in any of these three phases.

from CARs, and finally, it mines normal association rules**.**

complete set of Classification association rules.

1. The confidence of ri is greater than that of rj

2. Building classifier (Ranking and Pruning Rules)

**4.1.1 CBA** 

1. Generation of CARs

ranking is defined as below:

of the following holds good:

computational time.

**4.1.2 CMAR** 


Selecting rules based on database:


Nonetheless, when the datasets are large, both rule generation and rule selection in CBA and CMAR are time consuming. The CPAR and other predictive mining algorithms overcome this problem by generating a small set of predictive rules directly from the dataset based on the rule prediction and coverage analysis, as opposed to generating candidate rules.

#### **4.1.3 CPAR**

CPAR is an improvement to CBA and CMAR (Thabtah et al., 2005; Thabtah, 2007). It is proposed by Chen, Yin and Huang in 2005.The core of CPAR and other predictive mining algorithms is the predictive rule mining capability, whereby after an instance has been correctly covered by a rule, instead of removing it, its weight is decreased by multiplying a factor. This is essentially a greedy approach in rule generation, which is more efficient than generating all candidate rules.

CPAR may choose a number of attributes if those attributes have similar best gain. This is done by first calculating the gain and applying a GAIN\_SIMILARITY\_RATIO to this. All attributes with gain better than Local Gain Threshold (LGT) are then selected for further processing.

Data Mining Techniques in the Diagnosis of Tuberculosis 349

extracting relationships and patterns hidden in this data and can provide a new medical

Association Rule Mining (ARM) is one of the most popular approaches in data mining and if used in the medical domain has a great potential to improve disease prediction. It shows doctor the hidden disease symptoms associated with one another. There are many algorithms associated with ARM and the most popular is Apiori. It works in two phasesfirst is frequent itemset generation where all the items in a database above some minimum specified threshold called support will be generated. Second one is rule generation which generates from the frequent sets, an association rule of the form X->Y based on some minimum confidence. We can say that whenever X appears there is a chance that Y also appears along with it with minimum confidence threshold. These concepts are applied on TB dataset which reveals important association between the symptoms. But this method

Associative classification (AC) is another data mining approach that integrates association rule mining and classification. It uses association rule mining algorithm, such as Apriori or Frequent pattern growth , to generate the complete set of association rules. Then it selects a small set of high quality rules and uses this rule set for prediction. This method results in

Three important algorithms of AC such as CBA, CMAR and CPAR have been discussed in the chapter. Almost every algorithm contains two major data mining steps, an association rule (AR) mining stage- rules generated here are called as classification association rules (CARs) and a classification stage which uses the mined rules from the first stage directly. The second stage chooses rules with high priority from the CARs to cover training set. The difference between them is based on the priority evaluation of rules which usually depends on the confidence, support, rule length or common quality standard of classification rules. CPAR is better in rule generation compared to others. TB rules and accuracy are compared

Though the entire rules may not help doctors, few rules may describe the relationship between one symptom with the other and also sometimes it can reveal hidden

Agrawal, R., Imielinski, T. and Swami, A. "Mining association rules between sets of items

Ali. A. El-Solh, M.D., Chiu-Bin Hsiao, M.D., Susan Goodnough RN, et al " Predicting Active

Antonie, M.-L., Za¨ane, O. R. and Holte, R.C. "Learning to use a learned model: A two-

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pulmonary Tuberculosis using an Artificial Neural network ," CHEST journal

stage approach to classification", In *Proceedings of the Sixth International Conference* 

knowledge to doctors in their treatment procedure.

results in large number of repetitive rules.

smaller number rules compared to ARM.

for every associative classification algorithm

relationship.

**6. References** 

216.1993.

116(4), 968-973,1999.

The Local Gain Threshold (LGT) is given by the formula:

LGT = bestGain \* GAIN\_SIMILARITY\_RATIO

Where, GAIN\_SIMILARITY\_RATIO is a constant whose value is 0.99.

CPAR takes as input a (space separated) binary valued data set R and produces a set of CARs. The resulting classifier comprises a linked-list of rules ordered according to Laplace accuracy. CPAR also uses a dynamic programming approach to avoid repeated calculation in rule generation, which in turn is more economical. More importantly, CPAR selects best *k*  rules in prediction.

#### **4.2 Predictive accuracy and rules of associative classifiers**

Difference between ARM and AC with reference to results is that the former generates only large number of descriptive rules whereas the latter generate fewer rules along with their performance measure thru accuracy.

CBA generates around 81 rules once it is pruned we get only two rules with an accuracy of 81.14%.


CMAR generated about 1091 rules and the pruned output is only 38 rules with an accuracy of 99.1428%. Few are listed below:


CPAR after pruning could produce only 4 rules with an accuracy of 99.14%.


When compared to both ARM and AC rules, it can be seen that AC rules are smaller and better in description and also CPAR provides better rules compared to all algorithms.

#### **5. Summary**

In this chapter two data mining techniques which help in the diagnosis of Tuberculosis have been discussed. Medical databases have accumulated large quantities of information about patients and their clinical conditions and digital era has provided the availability of these information in abundance. Data mining is a knowledge discovery process that helps in

LGT = bestGain \* GAIN\_SIMILARITY\_RATIO

CPAR takes as input a (space separated) binary valued data set R and produces a set of CARs. The resulting classifier comprises a linked-list of rules ordered according to Laplace accuracy. CPAR also uses a dynamic programming approach to avoid repeated calculation in rule generation, which in turn is more economical. More importantly, CPAR selects best *k* 

Difference between ARM and AC with reference to results is that the former generates only large number of descriptive rules whereas the latter generate fewer rules along with their

CBA generates around 81 rules once it is pruned we get only two rules with an accuracy of

CMAR generated about 1091 rules and the pruned output is only 38 rules with an accuracy

3. {chroniccough(weeks) <= 22 Bloodcough = {null} HIV = {Negative} } ->TBtype=PTB

6. {Age>36 chroniccough(weeks)<=22 chestpain={null} HIV={positive} } ->

When compared to both ARM and AC rules, it can be seen that AC rules are smaller and better in description and also CPAR provides better rules compared to all algorithms.

In this chapter two data mining techniques which help in the diagnosis of Tuberculosis have been discussed. Medical databases have accumulated large quantities of information about patients and their clinical conditions and digital era has provided the availability of these information in abundance. Data mining is a knowledge discovery process that helps in

The Local Gain Threshold (LGT) is given by the formula:

rules in prediction.

81.14%.

performance measure thru accuracy.

1. { chroniccough(weeks)>23} ->{ TBtype=PTB}

2. { HIV = {Negative}} -> {TBtype=PTB}

1. {HIV = {Negative} } -> { TBtype=PTB}

2. {HIV = {Negative} } -> {TBtype=PTB}

4. {HIV = {positive}} -> {TBtype=retroviralPTB}

of 99.1428%. Few are listed below:

{TBtype=retroviralPTB}

**5. Summary** 

Where, GAIN\_SIMILARITY\_RATIO is a constant whose value is 0.99.

**4.2 Predictive accuracy and rules of associative classifiers** 

2. {Bloodcough = {null} HIV = {Negative} } -> {TBtype=PTB}

4. {HIV = {positive} Sputum = {null}} -> {TBtype=retroviralPTB}

1. {wheezing = {yes} HIV = {positive}} -> TBtype=retroviralPTB

3. {weightloss = {yes} HIV = {positive}} -> {TBtype=retroviralPTB}

5. {Age>36 HIV = {positive} Sputum = {null}} -> {TBtype=retroviralPTB}

CPAR after pruning could produce only 4 rules with an accuracy of 99.14%.

extracting relationships and patterns hidden in this data and can provide a new medical knowledge to doctors in their treatment procedure.

Association Rule Mining (ARM) is one of the most popular approaches in data mining and if used in the medical domain has a great potential to improve disease prediction. It shows doctor the hidden disease symptoms associated with one another. There are many algorithms associated with ARM and the most popular is Apiori. It works in two phasesfirst is frequent itemset generation where all the items in a database above some minimum specified threshold called support will be generated. Second one is rule generation which generates from the frequent sets, an association rule of the form X->Y based on some minimum confidence. We can say that whenever X appears there is a chance that Y also appears along with it with minimum confidence threshold. These concepts are applied on TB dataset which reveals important association between the symptoms. But this method results in large number of repetitive rules.

Associative classification (AC) is another data mining approach that integrates association rule mining and classification. It uses association rule mining algorithm, such as Apriori or Frequent pattern growth , to generate the complete set of association rules. Then it selects a small set of high quality rules and uses this rule set for prediction. This method results in smaller number rules compared to ARM.

Three important algorithms of AC such as CBA, CMAR and CPAR have been discussed in the chapter. Almost every algorithm contains two major data mining steps, an association rule (AR) mining stage- rules generated here are called as classification association rules (CARs) and a classification stage which uses the mined rules from the first stage directly. The second stage chooses rules with high priority from the CARs to cover training set. The difference between them is based on the priority evaluation of rules which usually depends on the confidence, support, rule length or common quality standard of classification rules. CPAR is better in rule generation compared to others. TB rules and accuracy are compared for every associative classification algorithm

Though the entire rules may not help doctors, few rules may describe the relationship between one symptom with the other and also sometimes it can reveal hidden relationship.

#### **6. References**


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#### **Books**

[1] Ian H Witten and Eibe Frank. 2001. Data mining practical machine learning tools and techniques. Morgan Kaufmann publishers.

**17** 

*Japan* 

**Immunological Diagnosis** 

*Tohoku University, Graduate School of Medicine* 

Beata Shiratori, Hiroki Saitoh, Umme Ruman Siddiqi, Jingge Zhao, Haorile Chagan-Yasutan, Motoki Usuzawa, Chie Nakajima1, Yasuhiko Suzuki1 and Toshio Hattori

*1Hokkaido University, Research Center for Zoonosis Control* 

Immunological diagnosis of tuberculosis (TB) is based on the immune responses against Mycobacterium tuberculosis (MTB). Immunological diagnosis can detect both active and latent TB, and can detect not only pulmonary TB but also extra-pulmonary TB. Compared to conventional diagnosis, immunological diagnostic tests have eminent advantages. On the other hand, there are still some limitations. As is known, various mycobacteria share homologous proteins, that lead to immunological cross reaction. To correctly detect TB infection, we need to choose a method that initiates the anti-MTB immune response properly. Human immunodeficiency virus (HIV) infection weakens the immune response, which may lead to false-negative results. HIV infection accompanied by TB is another urgent issue in global health. In this chapter, we will explain the immunological responses to MTB and the immunological interaction between HIV and TB. We will then introduce each diagnosis from the immunological point of view, and describe novel assays which we

In this section, we describe the immune response to MTB and MTB/HIV co-infection.

When the immune system encounters foreign organisms, it works to eliminate them and both innate immunity and adaptive immunity are engaged in this process (Murphy, 2011). In this section, we will briefly describe the immune responses to give the theoretical basis

Innate immunity is a non-specific response to pathogen and is the first line of defence against microorganisms. When macrophages recognize foreign organisms, the cells ingest and digest them. Receptors on the cell surface, especially those of toll-like receptor family,

**1. Introduction** 

are now developing.

**2. Immunological response to MTB** 

for the immunological diagnosis of TB.

**2.1 Innate immunity and adaptive immunity** 

**of Active and Latent TB** 

[2] J. Han and M. Kamber 2006 Data mining: concepts and techniques. Morgan Kaufmann publishers, Sanfrancisco, 47-97.

## **Immunological Diagnosis of Active and Latent TB**

Beata Shiratori, Hiroki Saitoh, Umme Ruman Siddiqi, Jingge Zhao, Haorile Chagan-Yasutan, Motoki Usuzawa, Chie Nakajima1, Yasuhiko Suzuki1 and Toshio Hattori *Tohoku University, Graduate School of Medicine 1Hokkaido University, Research Center for Zoonosis Control Japan* 

#### **1. Introduction**

352 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

[1] Ian H Witten and Eibe Frank. 2001. Data mining practical machine learning tools and

[2] J. Han and M. Kamber 2006 Data mining: concepts and techniques. Morgan Kaufmann

techniques. Morgan Kaufmann publishers.

publishers, Sanfrancisco, 47-97.

**Books** 

Immunological diagnosis of tuberculosis (TB) is based on the immune responses against Mycobacterium tuberculosis (MTB). Immunological diagnosis can detect both active and latent TB, and can detect not only pulmonary TB but also extra-pulmonary TB. Compared to conventional diagnosis, immunological diagnostic tests have eminent advantages. On the other hand, there are still some limitations. As is known, various mycobacteria share homologous proteins, that lead to immunological cross reaction. To correctly detect TB infection, we need to choose a method that initiates the anti-MTB immune response properly. Human immunodeficiency virus (HIV) infection weakens the immune response, which may lead to false-negative results. HIV infection accompanied by TB is another urgent issue in global health. In this chapter, we will explain the immunological responses to MTB and the immunological interaction between HIV and TB. We will then introduce each diagnosis from the immunological point of view, and describe novel assays which we are now developing.

#### **2. Immunological response to MTB**

In this section, we describe the immune response to MTB and MTB/HIV co-infection.

#### **2.1 Innate immunity and adaptive immunity**

When the immune system encounters foreign organisms, it works to eliminate them and both innate immunity and adaptive immunity are engaged in this process (Murphy, 2011). In this section, we will briefly describe the immune responses to give the theoretical basis for the immunological diagnosis of TB.

Innate immunity is a non-specific response to pathogen and is the first line of defence against microorganisms. When macrophages recognize foreign organisms, the cells ingest and digest them. Receptors on the cell surface, especially those of toll-like receptor family,

Immunological Diagnosis of Active and Latent TB 355

probably develop symptoms homogeneous with Immune Reconstitution Inflammatory Syndrome (IRIS) (Murdoch et al., 2007; Shelburne et al., 2005). Therefore, exploring the

Since HIV virus can weaken the immune system, LTBI can be activated resulting in pulmonary or extrapulmonary TB. A variety of immune cells and immune cytokines are involved in the reactivation of LTBI. Some cytokines, such as interleukin-8 (IL-8) and interleukin-12β (IL-12β), may be used as biomarkers to monitor the immune reaction to LTBI (Wu et al., 2007) and possibly shed light on preventing of LTBI progression in HIV patients (Walzl et al., 2011). So far, various biomarkers to characterize LTBI and TB in HIV patients have been proposed. Without medical intervention, HIV infection will progress to acquired immune deficiency syndrome (AIDS), accompanied by multi-microbial infections,

Infection with HIV enhances the susceptibility to MTB infection. Because the occurrence of these two diseases is heavily dependent on the immune system, their interactions are more complex than previously understood. Previously, we studied the plasma levels of two matricelluar proteins such as galectin-9 (GAL-9) and osteopontin (OPN) in AIDS patients complicated with various opprotunistic infections (Chagan-Yasutan et al., 2009). The levels of both molecules were high in all the patients but only the level of GAL-9 decreased and that of OPN remained high after Highly Active Antiretroviral Therapy (HAART). Also, As Figure 1 shows, it was noted that the GAL-9 level was exceptionally high in acute HIV infected individuals (Chagan-Yasutan et al., 2009). The cellular receptor for GAL-9 is the T-cell immunoglobulin domain and mucin domain 3 (Tim-3) and Tim-3 is expressed on Th1 cells. Mycobacterium tuberculosis-infected macrophages express GAL-9 and the Tim-3 GAL-9 interaction leads to macrophage activation and stimulates the bactericidal activity by inducing caspase-1–dependent interleukin-1β (IL-1β) secretion. Therefore, Th1 cell surface molecule Tim-3 may have evolved to inhibit the growth of intracellular pathogens via its ligand GAL-9, which is also known to inhibit the expansion of effector Th1 cells (Jayaraman et al., 2010). Therefore, only

In contrast, chronic HIV infected individuals succumb to various opportunistic infections and pulmonary TB is known to occur when CD4+ T cell numbers are still high, indicating that the immune system plays a role in the development of pulmonary TB (Holmes et al., 2005). Similarly, T cell epitopes of different strains representative of global diversity are highly conserved in MTB (Comas et al., 2010). Due to the conserved epitopes, the host can maintain MTB for a long time as latent infection and can transmit it to the next generation. It is also suspected that CD4+ T cells have an essential role in tissue damage that results in cavity formation, which enhances aerosol infection. In HIV endemic areas, the situation becomes more complex if the CD4+ T cells numbers increase after HAART and then

A. Healthy Individuals. For immunologically potent people, the reaction to invading microbes consists of innate and adaptive immune mechanisms. Genome research has

relationship between TB and HIV become necessary.

including TB infection, which often proves lethal.

recovery of the immune system is variable (Fig 2).

**2.3.2 Network among LTBI and HIV** 

**2.3.1 Mechanism of immunological interaction of HIV and TB** 

one case of MTB associated with acute HIV was reported (Crowley et al., 2011).

are involved in this process. Then macrophages express digested fragments on their surface promoting the initiation of the secondary response, the adaptive immunity.

Adaptive immunity is antigen specific and creates immunological memory. Responding T cells are functionally divided into T-helper cells 1 (Th1) and T-helper cells 2 (Th2), which are activated by antigen presentation through major histocompatibility complex class II (MHC II) on macrophages. Th1 cells stimulate T cell populations that secrete interferon gamma (IFN-) and interleukin-2 (IL-2) to activate cytotoxic T cells and, finally, to eliminate foreign organisms. This response is known as cell mediated immunity. Th2 cells secrete interleukin-4 (IL-4) to stimulate B cells, which produce antibody against the pathogen. This response is known as humoral immunity. Immunological diagnosis is based on the adaptive immune response to a targeted pathogen. If we detect or measure the activation of the immune response induced by MTB, we can diagnose MTB infection.

Recently, Th17 cells, which are responsible for inflammation, and T regulatory (Treg) populations, which suppress a variety of immune responses, have received much attention.

#### **2.2 Immunological responses to MTB**

MTB enters our body through the airway in droplet nuclei and is phagocytosed by alveolar macrophages. Macrophages digest MTB to connect MHC II molecules in the cell and fragments of MTB, then present their complex on the cell surface. Antigen presentation generates adaptive immunity(Walzl et al., 2011).

Th1 cells are activated and produce cytokines, such as IFN- and IL-2. These cytokines activate cytotoxic T cells and macrophages. IFN- enhances the anti-microbial activity of macrophages.

Th2 cells, producing IL-4, promote TB specific antibody production by B cells. These interactions lead to the generation of memory cells. There are two main populations of memory cells, effector memory T cells (which may be transiently present in the blood if bacteria are cleared) and central memory T cells (which may remain for life but may not provide protection in all individuals). The recently developed IGRA test measures IFN produced by effector memory T cells (Horsburgh & Rubin, 2011).

Despite these sophiscated immune responses, they often fail to eliminate MTB from the body and the bacteria may exist in a quiescent state for a prolonged period. Such a state is called latent tuberculosis infection (LTBI). In 10% of infected individuals, active TB develops and more than 80% of new cases of TB result from reactivation of the primary infection. The increase of HIV rates facilitates the reactivation of TB due to the imunosuppression.

#### **2.3 TB with HIV infection**

This lethal combination of TB/HIV is anything but rare; demographic analyses have estimated that over 60 % of the population in the sub-Saharan region has been infected by MTB, which has become a leading cause of mortality among HIV patients. At the end of 2009, TB infection was reported to be responsible for 13% of HIV deaths (Science Daily, 2009). Retrospective studies concluded that, among HIV/TB patients, 7% to 45% of them

are involved in this process. Then macrophages express digested fragments on their surface

Adaptive immunity is antigen specific and creates immunological memory. Responding T cells are functionally divided into T-helper cells 1 (Th1) and T-helper cells 2 (Th2), which are activated by antigen presentation through major histocompatibility complex class II (MHC II) on macrophages. Th1 cells stimulate T cell populations that secrete interferon gamma (IFN-) and interleukin-2 (IL-2) to activate cytotoxic T cells and, finally, to eliminate foreign organisms. This response is known as cell mediated immunity. Th2 cells secrete interleukin-4 (IL-4) to stimulate B cells, which produce antibody against the pathogen. This response is known as humoral immunity. Immunological diagnosis is based on the adaptive immune response to a targeted pathogen. If we detect or measure the activation of the immune

Recently, Th17 cells, which are responsible for inflammation, and T regulatory (Treg) populations, which suppress a variety of immune responses, have received much attention.

MTB enters our body through the airway in droplet nuclei and is phagocytosed by alveolar macrophages. Macrophages digest MTB to connect MHC II molecules in the cell and fragments of MTB, then present their complex on the cell surface. Antigen presentation

Th1 cells are activated and produce cytokines, such as IFN- and IL-2. These cytokines activate cytotoxic T cells and macrophages. IFN- enhances the anti-microbial activity of

Th2 cells, producing IL-4, promote TB specific antibody production by B cells. These interactions lead to the generation of memory cells. There are two main populations of memory cells, effector memory T cells (which may be transiently present in the blood if bacteria are cleared) and central memory T cells (which may remain for life but may not provide protection in all individuals). The recently developed IGRA test measures IFN-

Despite these sophiscated immune responses, they often fail to eliminate MTB from the body and the bacteria may exist in a quiescent state for a prolonged period. Such a state is called latent tuberculosis infection (LTBI). In 10% of infected individuals, active TB develops and more than 80% of new cases of TB result from reactivation of the primary infection. The

This lethal combination of TB/HIV is anything but rare; demographic analyses have estimated that over 60 % of the population in the sub-Saharan region has been infected by MTB, which has become a leading cause of mortality among HIV patients. At the end of 2009, TB infection was reported to be responsible for 13% of HIV deaths (Science Daily, 2009). Retrospective studies concluded that, among HIV/TB patients, 7% to 45% of them

increase of HIV rates facilitates the reactivation of TB due to the imunosuppression.

promoting the initiation of the secondary response, the adaptive immunity.

response induced by MTB, we can diagnose MTB infection.

**2.2 Immunological responses to MTB** 

macrophages.

**2.3 TB with HIV infection** 

generates adaptive immunity(Walzl et al., 2011).

produced by effector memory T cells (Horsburgh & Rubin, 2011).

probably develop symptoms homogeneous with Immune Reconstitution Inflammatory Syndrome (IRIS) (Murdoch et al., 2007; Shelburne et al., 2005). Therefore, exploring the relationship between TB and HIV become necessary.

Since HIV virus can weaken the immune system, LTBI can be activated resulting in pulmonary or extrapulmonary TB. A variety of immune cells and immune cytokines are involved in the reactivation of LTBI. Some cytokines, such as interleukin-8 (IL-8) and interleukin-12β (IL-12β), may be used as biomarkers to monitor the immune reaction to LTBI (Wu et al., 2007) and possibly shed light on preventing of LTBI progression in HIV patients (Walzl et al., 2011). So far, various biomarkers to characterize LTBI and TB in HIV patients have been proposed. Without medical intervention, HIV infection will progress to acquired immune deficiency syndrome (AIDS), accompanied by multi-microbial infections, including TB infection, which often proves lethal.

#### **2.3.1 Mechanism of immunological interaction of HIV and TB**

Infection with HIV enhances the susceptibility to MTB infection. Because the occurrence of these two diseases is heavily dependent on the immune system, their interactions are more complex than previously understood. Previously, we studied the plasma levels of two matricelluar proteins such as galectin-9 (GAL-9) and osteopontin (OPN) in AIDS patients complicated with various opprotunistic infections (Chagan-Yasutan et al., 2009). The levels of both molecules were high in all the patients but only the level of GAL-9 decreased and that of OPN remained high after Highly Active Antiretroviral Therapy (HAART). Also, As Figure 1 shows, it was noted that the GAL-9 level was exceptionally high in acute HIV infected individuals (Chagan-Yasutan et al., 2009). The cellular receptor for GAL-9 is the T-cell immunoglobulin domain and mucin domain 3 (Tim-3) and Tim-3 is expressed on Th1 cells. Mycobacterium tuberculosis-infected macrophages express GAL-9 and the Tim-3 GAL-9 interaction leads to macrophage activation and stimulates the bactericidal activity by inducing caspase-1–dependent interleukin-1β (IL-1β) secretion. Therefore, Th1 cell surface molecule Tim-3 may have evolved to inhibit the growth of intracellular pathogens via its ligand GAL-9, which is also known to inhibit the expansion of effector Th1 cells (Jayaraman et al., 2010). Therefore, only one case of MTB associated with acute HIV was reported (Crowley et al., 2011).

In contrast, chronic HIV infected individuals succumb to various opportunistic infections and pulmonary TB is known to occur when CD4+ T cell numbers are still high, indicating that the immune system plays a role in the development of pulmonary TB (Holmes et al., 2005). Similarly, T cell epitopes of different strains representative of global diversity are highly conserved in MTB (Comas et al., 2010). Due to the conserved epitopes, the host can maintain MTB for a long time as latent infection and can transmit it to the next generation. It is also suspected that CD4+ T cells have an essential role in tissue damage that results in cavity formation, which enhances aerosol infection. In HIV endemic areas, the situation becomes more complex if the CD4+ T cells numbers increase after HAART and then recovery of the immune system is variable (Fig 2).

#### **2.3.2 Network among LTBI and HIV**

A. Healthy Individuals. For immunologically potent people, the reaction to invading microbes consists of innate and adaptive immune mechanisms. Genome research has

Immunological Diagnosis of Active and Latent TB 357

 also enhances MHC II on macrophages. Other cytokines such as IL-2 and IL-4 could act in synergy with IFN- to control LTBI. However, 10% of them eventually progress to active TB

The horizontal plane indicates the make up of inflammatory CD4+ cells and TB effective CD4 + T cells (Effector memory T cells), which are coloured in blue and yellow, respectively. The overall reaction of different cells to TB is described according to the volumes of the different colours, the vertical lengths of which indicate biomarkers that rise up or descend resulting from TB reaction. Such schema and many exceptional cases are present in

C. Pulmonary TB. A pulmonary cavity is a typical sign of pulmonary TB. A recent paper reported that CD4+ T cells could be an essential factor for TB cavity formation (Russell et al., 2010). Patients of pulmonary TB have been reported to have mainstream TNF- TB specific CD4+ T cells, which can lead to an inflammatory reaction (Indicated by blue colour in vertical direction of C) in active TB patients. A recent study based on 101 TB and LTBI individuals described that TNF- specific CD4+ T cells might be an important biomarker for diagnosing active TB. In the majority of active TB patients TNF- can be detected in the MTB antigen stimulated cells. Flow-cytometry showed that inflammatory-related CD4+ T cells represent 37.4% of total CD4+ T cells (indicated by area between blue and yellow colour in C) is the cut-off of LTBI becoming active TB (Harari et al., 2011). And such TNF- TB specific CD4+ T cells activate other inflammatory cells. However, IFN-inducible neutrophils may also play an essential role in eliciting inflammatory CD4+ T cells.

(Day et al., 2011).

Fig. 2. Schematic network among LTBI and HIV.

HIV/AIDS and tuberculosis.

Fig. 1. Proposed biological effects of GAL-9 in HIV or HIV/TB infection. The plasma levels of GAL-9 were elevated in chronic AIDS patients as well as in TB patients (non-published data), but were exceptionally high in acute HIV infection (Chagan-Yasutan et al., 2009). It was reported that GAL-9 interacts with its Tim-3 ligand to regulate the overexpansion of Th1 cells to induce apoptosis (Zhu et al., 2005; Kashio et al., 2003). In TB, however it was speculated that GAL-9 contributes to the activation of macrophage cells (Mø) and then inhibits intracellular bacterial infection by caspase-1 dependent IL-1β production (Jayaraman et al., 2010).

revealed that TB epitopes binding to human CD4 + T cells are conserved (Comas et al., 2010). Accordingly, during the long history of fighting against TB, human beings have evolved a spectrum of potential TB-specific naive CD4+ T cells, which can be activated as soon as TB invades and transformed to TB effective CD4+ T cells to fight against TB bacilli, some of which would transform into central memory T cells (CMT) after TB is controlled. Such CMT cells are capable of quick proliferation once they confront TB. During infection, these TB antigen specific CD4 + T cells make up a certain percentage of CD4+ T cells; the percentage could be affected by the volume of CMT and individual differences in gene expression profiles (Maertzdorf et al., 2011).

B. LTBI (Latent tuberculosis infection) Individuals. Thirty percents of the population in the world is infected by MTB. However, effector CD4+ T cells protect LTBI individuals from developing active TB. In this case CMT act as a backup to proliferate of effector CD4+ T cells. Observation of this TB-specific reaction can be simplified by monitoring the IFN- level in IGRA (Rueda et al., 2010). Apart from IFN-, other biomarkers including CD154 and CD107 also indicate a TB-specific reaction (Streitz et al., 2011). IFN- has multiple effects on the immune system to control TB. As IFN- is secreted by TB effector CD4+ Th1 cells, it could mediate cytotoxic T cells to recognize and damage TB-infected macrophage cells. IFN-

Fig. 1. Proposed biological effects of GAL-9 in HIV or HIV/TB infection.

production (Jayaraman et al., 2010).

expression profiles (Maertzdorf et al., 2011).

The plasma levels of GAL-9 were elevated in chronic AIDS patients as well as in TB patients (non-published data), but were exceptionally high in acute HIV infection (Chagan-Yasutan et al., 2009). It was reported that GAL-9 interacts with its Tim-3 ligand to regulate the overexpansion of Th1 cells to induce apoptosis (Zhu et al., 2005; Kashio et al., 2003). In TB, however it was speculated that GAL-9 contributes to the activation of macrophage cells (Mø) and then inhibits intracellular bacterial infection by caspase-1 dependent IL-1β

revealed that TB epitopes binding to human CD4 + T cells are conserved (Comas et al., 2010). Accordingly, during the long history of fighting against TB, human beings have evolved a spectrum of potential TB-specific naive CD4+ T cells, which can be activated as soon as TB invades and transformed to TB effective CD4+ T cells to fight against TB bacilli, some of which would transform into central memory T cells (CMT) after TB is controlled. Such CMT cells are capable of quick proliferation once they confront TB. During infection, these TB antigen specific CD4 + T cells make up a certain percentage of CD4+ T cells; the percentage could be affected by the volume of CMT and individual differences in gene

B. LTBI (Latent tuberculosis infection) Individuals. Thirty percents of the population in the world is infected by MTB. However, effector CD4+ T cells protect LTBI individuals from developing active TB. In this case CMT act as a backup to proliferate of effector CD4+ T cells. Observation of this TB-specific reaction can be simplified by monitoring the IFN- level in IGRA (Rueda et al., 2010). Apart from IFN-, other biomarkers including CD154 and CD107 also indicate a TB-specific reaction (Streitz et al., 2011). IFN- has multiple effects on the immune system to control TB. As IFN- is secreted by TB effector CD4+ Th1 cells, it could mediate cytotoxic T cells to recognize and damage TB-infected macrophage cells. IFN-

 also enhances MHC II on macrophages. Other cytokines such as IL-2 and IL-4 could act in synergy with IFN- to control LTBI. However, 10% of them eventually progress to active TB (Day et al., 2011).

Fig. 2. Schematic network among LTBI and HIV.

The horizontal plane indicates the make up of inflammatory CD4+ cells and TB effective CD4 + T cells (Effector memory T cells), which are coloured in blue and yellow, respectively. The overall reaction of different cells to TB is described according to the volumes of the different colours, the vertical lengths of which indicate biomarkers that rise up or descend resulting from TB reaction. Such schema and many exceptional cases are present in HIV/AIDS and tuberculosis.

C. Pulmonary TB. A pulmonary cavity is a typical sign of pulmonary TB. A recent paper reported that CD4+ T cells could be an essential factor for TB cavity formation (Russell et al., 2010). Patients of pulmonary TB have been reported to have mainstream TNF- TB specific CD4+ T cells, which can lead to an inflammatory reaction (Indicated by blue colour in vertical direction of C) in active TB patients. A recent study based on 101 TB and LTBI individuals described that TNF- specific CD4+ T cells might be an important biomarker for diagnosing active TB. In the majority of active TB patients TNF- can be detected in the MTB antigen stimulated cells. Flow-cytometry showed that inflammatory-related CD4+ T cells represent 37.4% of total CD4+ T cells (indicated by area between blue and yellow colour in C) is the cut-off of LTBI becoming active TB (Harari et al., 2011). And such TNF- TB specific CD4+ T cells activate other inflammatory cells. However, IFN-inducible neutrophils may also play an essential role in eliciting inflammatory CD4+ T cells.

Immunological Diagnosis of Active and Latent TB 359

et al., 2011). Notably, HAART unmasking TB manifestation can be found in some cases and C-reactive protein (CRP) was reported to be helpful to detect the development of unmasking

Over decades, there have been attempts to find new diagnostic tools, that are sensitive and specific, simple, inexpensive and able to distinguish latent tuberculosis from active

Tuberculin skin test, also called as Mantoux skin test, has been used for the diagnosis of tuberculosis for more than a century. Despite the numbers of logistic and performance problems and poor specificity, TST is still performed as a routine diagnostic method. The purified protein derivative (PPD) antigens, that are used for TST are highly homologous to antigens of Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine and nontuberculosis mycobacteria (NTM) antigens. These and other factors may lead to false-

Although other antigens have been evaluated as a skin test reagents e.g.molybdopterin-64 (MPT-64) and molybdopterin-59 (MPT-59), none of them proved superior to tuberculin skin

Because IFN- is a cytokine that plays a critical role in resistance to Mycobacterium tuberculosis infection and MTB infected individuals respond to MTB antigen stimulation by releasing increased amounts of this cytokine from effector memory cells, methods based on measuring the IFN- production by antigen stimulated human T lymphocytes have been developed. There are two new blood tests on the market, the enzyme-linked immunospot assay (ELISpot) (T-SPOT®.TB, Oxford Immunotec, Oxford, UK) and the enzyme-linked immunosorbent assay (ELISA) (QuantiFERON-TB Gold In-Tube, QFT-GIT, Cellestis, Carnegie, Australia). Both IGRAs have high sensitivity and specificity, for QFT-GIT 81%-

 Cutaneus anergy Recent TB infection Very old TB infection

old)

Very young age (less than 6 months

 Recent live-virus vaccination (e.g., measles and chicken pox) Incorrect method of TST administration

Incorrect interpretation of reaction

tuberculosis as well as MTB infected individuals from uninfected ones.

False-positive reactions **False-negative reactions** 

Table 1. False-positive and false-negative TST reactions. (CDC, 2010)

TB-IRIS cases (Haddow et al., 2011).

positive or false negative TST results.

 Incorrect interpretation of reaction Incorrect bottle of antigen used

**3.2 Interferon Gamma Releasing Assay (IGRA)** 

**3. Immunological diagnosis** 

**3.1 Tuberculin skin test** 

test (Wilcke et al., 1996).

 Infection with NTM Previous BCG vaccination Incorrect method of TST administration

Neutrophils corresponding to IFN-, and modulate an inflammatory reaction in active TB patients (Berry et al., 2010).

D. Extrapulmonary TB. Due to the small number of inflammatory CD4+ T cells, TB lacks the ability to form cavities in the lungs and lead to multi-organ or systemic infection. Such patients are frequently found among those with AIDS and patients on immunosuppressive therapy. Interestingly, extrapulmonary TB is a prominent risk factor for IRIS. (Manosuthi et al., 2006)

E. The stage between LTBI and active TB infection. It features opacities in the lungs, but sputum and IGRAs test are negative.

F1, G1. HIV-infected Patients*.* In Sub-Saharan region, frequently seen cases are LTBI individuals infected by HIV. Asymptomatic HIV can last by 2 years to 10 years in normal individuals after infection. As a result of HIV infection, CD4+ cells drop progressively. After HIV infection, the virus targets all CD4+ T cells including effector, inflammatory and CMT CD4+ T cells and anti-HIV drugs restores their function. F1 indicates those patients who didn't carry larger number of CMT cells and could not produce enough effector CD4+ T cells after TB stimulation. G1 indicates that those patients with a large pool of TB CMTs or who has been infected by LTBI prior to HIV infection and then carried more LTBI stimulating specific effector CD4+ T cells or memory T cells (Mueller et al., 2008). F1, G1 will finally process to extrapulmonary TB (indicated by E), if no medication intervenes.

F1-F2. HIV Patients during HAART treatment. In macaque experiment, SHIV was found to preferentially infect CMT cells (He et al., 2011). When HAART treatment is applied, CD4+ T cells count will rise. Occasionally, such rise causes IRIS, because CMT cell count will bounce up and effector CD4+ T cells will show increased activity. However MTB specific inflammatory CD4+ T cells will dominate their large number. (Indicated by proportional volume between blue and yellow). As TB Inflammatory CD4+ T cells lead the reaction, patients have similar prognoses as pulmonary TB (Indicated by C) (Worodria et al., 2011).

G1-G2. HIV Patients after HAART treatment. HAART will rapidly reconstitute the immune surveillance (indicated by elevation of yellow column) (Hua et al., 2011). The occurrence of TB therefore decreases (Sant'Anna et al., 2009).

Net work

C-F1-F2. TB and HIV stimulate specific inflammatory T cells to produce TNF- which, in turn, help them to progress at faster rate (Sorathiya et al., 2010). The patients can be treated by anti-TB therapy followed by HAART. Occasionally, paradoxical TB IRIS1 occurs, probably caused by MTB specific inflammatory CD4+ T cells. MCP-1 (monocyte chemotactic protein 1) was found to be a reliable candidate biomarker to screen patients who may develop to paradoxical IRIS (Haddow et al., 2011). B-G1-G2. HAART strengthen immune responses against MTB and can decrease the occurrence of TB (Middelkoop et al., 2011, Hua

<sup>1</sup> Definition of TB IRIS: 'paradoxical' worsening of symptoms of known disease, either at a new body site or at the original body site, with an incidence of 8–43% of TB-co-infected individuals starting ART; or 'unmasking' of occult Mycobacterium tuberculosis infection, in which infection was not clinically apparent prior to ART but presents floridly during ART, affecting around 5% among those starting ART without known TB infection in South Africa.

et al., 2011). Notably, HAART unmasking TB manifestation can be found in some cases and C-reactive protein (CRP) was reported to be helpful to detect the development of unmasking TB-IRIS cases (Haddow et al., 2011).

### **3. Immunological diagnosis**

Over decades, there have been attempts to find new diagnostic tools, that are sensitive and specific, simple, inexpensive and able to distinguish latent tuberculosis from active tuberculosis as well as MTB infected individuals from uninfected ones.

#### **3.1 Tuberculin skin test**

358 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Neutrophils corresponding to IFN-, and modulate an inflammatory reaction in active

D. Extrapulmonary TB. Due to the small number of inflammatory CD4+ T cells, TB lacks the ability to form cavities in the lungs and lead to multi-organ or systemic infection. Such patients are frequently found among those with AIDS and patients on immunosuppressive therapy. Interestingly, extrapulmonary TB is a prominent risk factor for IRIS. (Manosuthi et

E. The stage between LTBI and active TB infection. It features opacities in the lungs, but

F1, G1. HIV-infected Patients*.* In Sub-Saharan region, frequently seen cases are LTBI individuals infected by HIV. Asymptomatic HIV can last by 2 years to 10 years in normal individuals after infection. As a result of HIV infection, CD4+ cells drop progressively. After HIV infection, the virus targets all CD4+ T cells including effector, inflammatory and CMT CD4+ T cells and anti-HIV drugs restores their function. F1 indicates those patients who didn't carry larger number of CMT cells and could not produce enough effector CD4+ T cells after TB stimulation. G1 indicates that those patients with a large pool of TB CMTs or who has been infected by LTBI prior to HIV infection and then carried more LTBI stimulating specific effector CD4+ T cells or memory T cells (Mueller et al., 2008). F1, G1 will

finally process to extrapulmonary TB (indicated by E), if no medication intervenes.

F1-F2. HIV Patients during HAART treatment. In macaque experiment, SHIV was found to preferentially infect CMT cells (He et al., 2011). When HAART treatment is applied, CD4+ T cells count will rise. Occasionally, such rise causes IRIS, because CMT cell count will bounce up and effector CD4+ T cells will show increased activity. However MTB specific inflammatory CD4+ T cells will dominate their large number. (Indicated by proportional volume between blue and yellow). As TB Inflammatory CD4+ T cells lead the reaction, patients have similar prognoses as pulmonary TB (Indicated by C) (Worodria et al., 2011).

G1-G2. HIV Patients after HAART treatment. HAART will rapidly reconstitute the immune surveillance (indicated by elevation of yellow column) (Hua et al., 2011). The occurrence of

C-F1-F2. TB and HIV stimulate specific inflammatory T cells to produce TNF- which, in turn, help them to progress at faster rate (Sorathiya et al., 2010). The patients can be treated by anti-TB therapy followed by HAART. Occasionally, paradoxical TB IRIS1 occurs, probably caused by MTB specific inflammatory CD4+ T cells. MCP-1 (monocyte chemotactic protein 1) was found to be a reliable candidate biomarker to screen patients who may develop to paradoxical IRIS (Haddow et al., 2011). B-G1-G2. HAART strengthen immune responses against MTB and can decrease the occurrence of TB (Middelkoop et al., 2011, Hua

1 Definition of TB IRIS: 'paradoxical' worsening of symptoms of known disease, either at a new body site or at the original body site, with an incidence of 8–43% of TB-co-infected individuals starting ART; or 'unmasking' of occult Mycobacterium tuberculosis infection, in which infection was not clinically apparent prior to ART but presents floridly during ART, affecting around 5% among those starting ART

TB patients (Berry et al., 2010).

sputum and IGRAs test are negative.

TB therefore decreases (Sant'Anna et al., 2009).

without known TB infection in South Africa.

al., 2006)

Net work

Tuberculin skin test, also called as Mantoux skin test, has been used for the diagnosis of tuberculosis for more than a century. Despite the numbers of logistic and performance problems and poor specificity, TST is still performed as a routine diagnostic method. The purified protein derivative (PPD) antigens, that are used for TST are highly homologous to antigens of Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine and nontuberculosis mycobacteria (NTM) antigens. These and other factors may lead to falsepositive or false negative TST results.

Although other antigens have been evaluated as a skin test reagents e.g.molybdopterin-64 (MPT-64) and molybdopterin-59 (MPT-59), none of them proved superior to tuberculin skin test (Wilcke et al., 1996).


Table 1. False-positive and false-negative TST reactions. (CDC, 2010)

#### **3.2 Interferon Gamma Releasing Assay (IGRA)**

Because IFN- is a cytokine that plays a critical role in resistance to Mycobacterium tuberculosis infection and MTB infected individuals respond to MTB antigen stimulation by releasing increased amounts of this cytokine from effector memory cells, methods based on measuring the IFN- production by antigen stimulated human T lymphocytes have been developed. There are two new blood tests on the market, the enzyme-linked immunospot assay (ELISpot) (T-SPOT®.TB, Oxford Immunotec, Oxford, UK) and the enzyme-linked immunosorbent assay (ELISA) (QuantiFERON-TB Gold In-Tube, QFT-GIT, Cellestis, Carnegie, Australia). Both IGRAs have high sensitivity and specificity, for QFT-GIT 81%-

Immunological Diagnosis of Active and Latent TB 361

Immunotec, 2011). The important step is the accurate adjustment of the cell count. However, if the cells are not well adjusted and the nil control contains more spots than indicated in the instructions, the test needs to be repeated. What is more, to run the ELISpot assay, a trained professional should be engaged and other special tools such as a plate reader are needed. Exact interpretation of the results is crucial, so the manufacturer prepared a training manual for distinguishing between valid and invalid spots. The ELISpot assay`s intermediate result rate is 3-4% (Dosanjh et al., 2008), which is significantly lower than that for QFT assays (11-

QFT-GIT test has recently become routinely used as a diagnostic tool for MTB, but the T-SPOT®.TB test is employed only in few countries, mostly because of the high cost of this assay. In conclusion, IGRA seems to be beneficial tool for TB diagnosis, especially for people

In addition to ESAT-6 and CFP-10, other antigens have been proposed for ELISpot assay but have not been implemented for commercial use. Addition of the novel antigen Rv3879c increased the diagnostic sensitivity of the standard ELISpot assay and, in combination with TST, reached a sensitivity of 99% (Dosanjh et al., 2008). Other researchers showed that antigen heparin-binding-hemagglutinin was significantly more sensitive than ESAT-6 and

There are various conditions such as oncologic disease, HIV infection, anti-TNF-, corticoid or other immunomodulatory therapy, diabetes mellitus, renal failure or other immunocompromising conditions that are responsible for intermediate or false negative results (Schoepfer et al., 2008; Matulis et al., 2008; Kim et al., 2009). Particularly, the anti-TNF- treatment has increased recently, which hampers the activation of the innate immune responses, T cell mediated adaptive immune response and production of protective IFN-. Similarly, metabolic diseases such as diabetes mellitus are known to affect chemotaxis, phagocytosis, activation, and antigen presentation by phagocytes in response to MTB, and this defect does not improve with insulin (Moutschen et al., 1992). Importantly, IFN production was found to be impared in hyperglycemia (Yamashiro et al., 2005), but another study showed that both IGRA results were not affected in diabetes mellitus patients (Walsh et al., 2011). Intermediate results have been found to be also associated with lower serum

Since TB is a chronic disease in which bacilli evade the immune system to persist in the host organism, scientists are trying to find and understand the mechanism of MTB immunopathology. It is still unclear what conditions prone to MTB infection, what factors are involved in TB latency, activation or masking of the disease and what causes the imbalance of the immune responses that finally lead to the failure of MTB eradication. The immune system posses a regulatory mechanism in which Treg cells play essential roles in

more specific than PPD for the detection of LTBI (Hougardy et al., 2007a).

albumin and double immunomodulatory therapy (Papay et al.,2011).

**3.3 Role of regulatory T cells in diagnosis of MTB** 

21%) (Ferrara et al., 2006; Piana et al., 2006).

with a high-risk of developing active TB.

**3.2.4 Conditions altering IGRA results** 

**3.2.3 Other approaches of LTB diagnosis** 

92.6% and 98.8%-99.2% and for T-SPOT®.TB 87.5%-95.6% and 86.3%-99.9%, respectively (Diel et al., 2008; Harada et al., 2008; Oxford Immunotec, 2011). Firstly, it should be mentioned that using IGRA it is impossible to distinguish between latent and active TB for which no such a method yet exists. However the detection of both latent and active TB has been markedly improved by employing IGRA methods. The factor, that increased the sensitivity and specificity of IGRA was the discovery and use of antigens encoded by Regions of Difference 1 (RD1) in the MTB genome, which is absent in BCG vaccination or NTB. Among the nine antigens encoded by RD1, early secreted antigenic target 6kDa (ESAT-6) and culture filtrate protein 10kDa (CFP-10) are used as a stimulatory antigens. However, ESAT-6 and CFP-10 antigens are also present in NTM, namely M. leprae, wild type M. bovis, M. marium, M. kansasii, M. sulgai, M. flavescens, in NTM endemic areas, IGRA results might be false positive and make it difficult to distinguish MTB between NTM.

#### **3.2.1 QuantiFERON-TB Gold In Tube assay**

In comparison to the previous form of QuantiFERON Gold, the QuantiFERON Gold In Tube (QFT-GIT) version enables immediate antigen stimulation of lymphocytes within whole blood. In addition to ESAT-6 and CFP-10, QFT-GIT contains a peptide from the internal section of TB 7.7 (Rv2654), which may increase the sensitivity of the test (Aagaard et al. 2004; Brock et al., 2004), though it is arguable whether specificity is improved. All three antigens are present on the wall surface of the blood collection tubes. Besides the immunity status and the absolute and relative lymphocyte number there are external factors that may influence the QFT results, e.g. drawing an adequate volume of blood, appropriate attachment of lymphocytes to the antigens, sample handling and the ELISA assay procedure. Another important issue is the interpretation of the results. There are two cutoffs given by the manufacture, 0.35 IU/l and 0.1 IU/l. A value of more than 0.35 IU/l seems to be appropriate for good discrimination of truly infected individuals (Harada et al., 2008). On the other hand, the values between 0.35 IU/l and 0.1 IU/l, the intermediate result, should take into account the individual patient`s condition (Harada et al., 2008; Liote H & Liote F, 2011). In Japan, the interpretation criteria differ from those anywhere else. Intermediate results in Japanese people are suspected to be positive and are flagged for follow-up observation (Prevention Committee, Japanese Society of Tuberculosis, 2006). Depending on the MTB infection prevalence, it has been suggested to use different cut-offs (Harada et al., 2008). In areas where the MTB infection prevalence is low, the specificity is probably of great importance. However, in high-risk TB screening situations, identification of LTBI is likely to be more important than the potential side effects of the MTB treatment and a cut-off value of 0.1 IU/l should be employed (Yew & Leung, 2006).

#### **3.2.2 T-SPOT® .TB test (ELISpot)**

Using the ELISpot assay it is possible to visualize and count MTB-specific memory T cells producing IFN-. The great advantage of this test is that each test well contains the same number of peripheral blood mononuclear cells (PBMCs). Especially in the patients with low T cell counts from HIV or other immune disorders, it enables the objective evaluation by adjusting the designed cell number. While in QFT-GIT all antigens are present in the same tube, in the ELISpot assay ESAT-6 and CFP-10 antigens are added and read separately. The manufacture claims that this assay has very low cross-reactivity with NTM (Oxford

92.6% and 98.8%-99.2% and for T-SPOT®.TB 87.5%-95.6% and 86.3%-99.9%, respectively (Diel et al., 2008; Harada et al., 2008; Oxford Immunotec, 2011). Firstly, it should be mentioned that using IGRA it is impossible to distinguish between latent and active TB for which no such a method yet exists. However the detection of both latent and active TB has been markedly improved by employing IGRA methods. The factor, that increased the sensitivity and specificity of IGRA was the discovery and use of antigens encoded by Regions of Difference 1 (RD1) in the MTB genome, which is absent in BCG vaccination or NTB. Among the nine antigens encoded by RD1, early secreted antigenic target 6kDa (ESAT-6) and culture filtrate protein 10kDa (CFP-10) are used as a stimulatory antigens. However, ESAT-6 and CFP-10 antigens are also present in NTM, namely M. leprae, wild type M. bovis, M. marium, M. kansasii, M. sulgai, M. flavescens, in NTM endemic areas, IGRA results might be false positive and make it difficult to distinguish MTB between NTM.

In comparison to the previous form of QuantiFERON Gold, the QuantiFERON Gold In Tube (QFT-GIT) version enables immediate antigen stimulation of lymphocytes within whole blood. In addition to ESAT-6 and CFP-10, QFT-GIT contains a peptide from the internal section of TB 7.7 (Rv2654), which may increase the sensitivity of the test (Aagaard et al. 2004; Brock et al., 2004), though it is arguable whether specificity is improved. All three antigens are present on the wall surface of the blood collection tubes. Besides the immunity status and the absolute and relative lymphocyte number there are external factors that may influence the QFT results, e.g. drawing an adequate volume of blood, appropriate attachment of lymphocytes to the antigens, sample handling and the ELISA assay procedure. Another important issue is the interpretation of the results. There are two cutoffs given by the manufacture, 0.35 IU/l and 0.1 IU/l. A value of more than 0.35 IU/l seems to be appropriate for good discrimination of truly infected individuals (Harada et al., 2008). On the other hand, the values between 0.35 IU/l and 0.1 IU/l, the intermediate result, should take into account the individual patient`s condition (Harada et al., 2008; Liote H & Liote F, 2011). In Japan, the interpretation criteria differ from those anywhere else. Intermediate results in Japanese people are suspected to be positive and are flagged for follow-up observation (Prevention Committee, Japanese Society of Tuberculosis, 2006). Depending on the MTB infection prevalence, it has been suggested to use different cut-offs (Harada et al., 2008). In areas where the MTB infection prevalence is low, the specificity is probably of great importance. However, in high-risk TB screening situations, identification of LTBI is likely to be more important than the potential side effects of the MTB treatment

and a cut-off value of 0.1 IU/l should be employed (Yew & Leung, 2006).

Using the ELISpot assay it is possible to visualize and count MTB-specific memory T cells producing IFN-. The great advantage of this test is that each test well contains the same number of peripheral blood mononuclear cells (PBMCs). Especially in the patients with low T cell counts from HIV or other immune disorders, it enables the objective evaluation by adjusting the designed cell number. While in QFT-GIT all antigens are present in the same tube, in the ELISpot assay ESAT-6 and CFP-10 antigens are added and read separately. The manufacture claims that this assay has very low cross-reactivity with NTM (Oxford

**.TB test (ELISpot)** 

**3.2.1 QuantiFERON-TB Gold In Tube assay** 

**3.2.2 T-SPOT®**

Immunotec, 2011). The important step is the accurate adjustment of the cell count. However, if the cells are not well adjusted and the nil control contains more spots than indicated in the instructions, the test needs to be repeated. What is more, to run the ELISpot assay, a trained professional should be engaged and other special tools such as a plate reader are needed. Exact interpretation of the results is crucial, so the manufacturer prepared a training manual for distinguishing between valid and invalid spots. The ELISpot assay`s intermediate result rate is 3-4% (Dosanjh et al., 2008), which is significantly lower than that for QFT assays (11- 21%) (Ferrara et al., 2006; Piana et al., 2006).

QFT-GIT test has recently become routinely used as a diagnostic tool for MTB, but the T-SPOT®.TB test is employed only in few countries, mostly because of the high cost of this assay. In conclusion, IGRA seems to be beneficial tool for TB diagnosis, especially for people with a high-risk of developing active TB.

#### **3.2.3 Other approaches of LTB diagnosis**

In addition to ESAT-6 and CFP-10, other antigens have been proposed for ELISpot assay but have not been implemented for commercial use. Addition of the novel antigen Rv3879c increased the diagnostic sensitivity of the standard ELISpot assay and, in combination with TST, reached a sensitivity of 99% (Dosanjh et al., 2008). Other researchers showed that antigen heparin-binding-hemagglutinin was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI (Hougardy et al., 2007a).

#### **3.2.4 Conditions altering IGRA results**

There are various conditions such as oncologic disease, HIV infection, anti-TNF-, corticoid or other immunomodulatory therapy, diabetes mellitus, renal failure or other immunocompromising conditions that are responsible for intermediate or false negative results (Schoepfer et al., 2008; Matulis et al., 2008; Kim et al., 2009). Particularly, the anti-TNF- treatment has increased recently, which hampers the activation of the innate immune responses, T cell mediated adaptive immune response and production of protective IFN-. Similarly, metabolic diseases such as diabetes mellitus are known to affect chemotaxis, phagocytosis, activation, and antigen presentation by phagocytes in response to MTB, and this defect does not improve with insulin (Moutschen et al., 1992). Importantly, IFN production was found to be impared in hyperglycemia (Yamashiro et al., 2005), but another study showed that both IGRA results were not affected in diabetes mellitus patients (Walsh et al., 2011). Intermediate results have been found to be also associated with lower serum albumin and double immunomodulatory therapy (Papay et al.,2011).

#### **3.3 Role of regulatory T cells in diagnosis of MTB**

Since TB is a chronic disease in which bacilli evade the immune system to persist in the host organism, scientists are trying to find and understand the mechanism of MTB immunopathology. It is still unclear what conditions prone to MTB infection, what factors are involved in TB latency, activation or masking of the disease and what causes the imbalance of the immune responses that finally lead to the failure of MTB eradication. The immune system posses a regulatory mechanism in which Treg cells play essential roles in

Immunological Diagnosis of Active and Latent TB 363

study. A TST result ≥ 10 mm was considered to be positive. Both individuals were vaccinated with BCG when young. The protocol was approved by the ethical commitee of Tohoku University Medical School. Written informed consent was obtained. Both

From each donor 20 ml of Ethylenediamine tetraacetic acid (EDTA) treated peripheral venous blood was obtained, centrifuged and the peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll separation (Ficoll-Paque PLUS,GE Healthcare Bio-Science AB, Uppsala, Sweden). After washing twice in Phosphate Buffered Saline (PBS), PBMCs were resuspended in complete RPMI 1640 medium (consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2% glutamine and 1% penicilin/streptomycin) at

The cells designed for depletion were centrifuged and resusupended in Running buffer (MACS Separation Buffer, Miltenyi Biotec). To avoid unspecific binding of the antibody, 100 ul of FcR Blocking Reagent (Miltenyi Biotec) was added to the PBMCs which were then incubated for 15 minutes on ice. After adding 25 ul of CD25-Biotin monoclonal antibody (Miltenyi Biotec) and 15 minutes incubation on ice, the cells were washed with Running buffer. To cells resuspended in Running buffer, 25 ul of anti-Biotin Microbeads (Miltenyi Biotec) were added, followed by incubation for 15 minutes on ice. Subsequently, the cells were washed and resuspended in Rising Solution (Miltenyi Biotec) and CD4+CD25+ T cells were depleted by positive selection using a magnetic-activated cell sorter (MACS)-assisted cell sorting system (Miltenyi Biotec, Auburn, CA) according to the manufacturer´s

To visualize the biotinylated mAb on CD25+ cells, streptavidin-allophycocyanin (BD PharMingen) was used and the CD4+ cells were stained by anti-CD4 FITC antibody. Flow cytometric analyses were performed with a FACSCalibur flow cytometer (Becton Dickinson) using the CELL quest program (Becton Dickinson). More than 90% of CD4+CD25+ cells

Freshly isolated, undepleted and Treg depleted PBMCs were cultured in triplicate in 96-well plates at a concentration of 2x105 cells per well in 200 ul of complete RPMI 1640 medium and incubated for 24 hours at 37 °C with 5% CO2. The cells were stimulated with 1 ug/ml of PPD (Statens Serum Institute, Copenhagen, Denmark), 500nM of recombinant CFP-10 and ESAT-6 protein antigen. The cell culture supernatant was harvested from each well for ELISA analysis. IFN-gamma and IL-10 production was determined by using human IFNgamma and IL-10 BD Opt EIATM Set according to the manufacturer´s instructions (BD Bioscience). Optical densities were read at 450 nm on an ELISA plate reader (VersaMax-KT, Molecular Devices Corp., CA, USA ) and the concentrations were calculated from standard

Two-sided paired *t*-test was used to analyze the effect of Treg on cytokine production.

individuals were HIV, hepatitis B and C virus negative.

a concentration 5x106 cells/ml. **Depletion of CD4+CD25+ cells** 

**Cell phenotype determination** 

were depleted from the PMBCs of both individuals.

curves using the Soft Max Pro program (Molecular Devices Corp.).

Differences were considered significant when the *p* value was less than 0.05.

**Cultivation and cytokine determination** 

instructions.

**Statistical analysis** 

establishing and sustaining self tolerance and immune homeostasis as well as regulate the host response to infection.

#### **3.3.1 Role of Treg in mycobacterium tuberculosis infection**

The first demonstration of the suppressive capacity of T cells was performed in 1973 in an animal model of bacillus BCG vaccination. Thymocytes from BCG-injectected rats were harvested and transfected to alive normal recipients. Subsequently, recipients were challenged with the same antigen and the inhibition of their skin reaction was observed (Ha & Waksman, 1973).

The number of CD4+CD25+FoxP3+ Treg cells was found to be increased in the blood or at the site of infection in active tuberculosis patients (Guyot-Revol et al., 2005) and the frequency of CD4+CD25+FoxP3+ T lymphocytes was inversely collated with the local MTBspecific immunity, and both blood and pleural Treg cells were able to suppress IFN- and IL-10 production in TB patients (Chen et al., 2007). This mechanism is thought to contribute to the pathogenesis of human TB (Guyot-Revol et al., 2005; Chen et al., 2007). Treg cell expansion is believed to predispose or be a marker of the progression of latent TB to active disease (Hougardy et al., 2007). What is more, it was found that depletion of CD4+CD25+ T cells enhanced the protective IFN- production in TB patients (Guyot-Revol et al., 2005) and transiently reduced the bacterial load and granuloma formation (Ozeki et al., 2010).

#### **3.3.2 Objectives of our Treg study**

The majority of individuals vaccinated with BCG or infected with MTB develop a delayedtype hypersensitivity which is manifested as a positive response of intradermal injection to a purified protein derivative from MTB. But about 15% of active TB patients show false negative results and are considered to be anergic TB (Bloom & Small,1998). Similarly, in HIV infected individuals, TST results are often found to be false-negative. Concerning the high frequencies of Treg cells in both diseases (Chen et al., 2007; Bi et al., 2009), it is highly probable that Treg cells may play central role in the anergy mechanism. Cutaneus anergy in active TB is associated with the absence of granuloma formation and poor clinical outcome (Boussiotis et al., 2000). Another study showed that, in an anergic patient, sustained MTB stimulation led to enhanced IL-10 production and the generation of anergic MTB-specific T regulatory cells with the Tr-1 phenotype (Boussitis, 2000). In certain closed populations, a high percentage of TB anergy and high prevalence of active TB were observed, which led to the speculation that innate genetic factors may play a role (Delgado & Ganea, 2001). We observed several cases of anergy in health care workers (HCW), who had been in close contact with active TB patients. Therefore, we questioned whether Treg cell may mask latent TB infection.

#### **3.3.3 Materials and methods**

#### **Human subjects and samples**

According to previously obtained TST results, one TST positive (27 years old) and one TST negative (42 years old) healthy (X-ray and sputum smear negative) health care individuals with no history of previous MTB infection or other chronic disease were recruited in this

establishing and sustaining self tolerance and immune homeostasis as well as regulate the

The first demonstration of the suppressive capacity of T cells was performed in 1973 in an animal model of bacillus BCG vaccination. Thymocytes from BCG-injectected rats were harvested and transfected to alive normal recipients. Subsequently, recipients were challenged with the same antigen and the inhibition of their skin reaction was observed (Ha

The number of CD4+CD25+FoxP3+ Treg cells was found to be increased in the blood or at the site of infection in active tuberculosis patients (Guyot-Revol et al., 2005) and the frequency of CD4+CD25+FoxP3+ T lymphocytes was inversely collated with the local MTBspecific immunity, and both blood and pleural Treg cells were able to suppress IFN- and IL-10 production in TB patients (Chen et al., 2007). This mechanism is thought to contribute to the pathogenesis of human TB (Guyot-Revol et al., 2005; Chen et al., 2007). Treg cell expansion is believed to predispose or be a marker of the progression of latent TB to active disease (Hougardy et al., 2007). What is more, it was found that depletion of CD4+CD25+ T cells enhanced the protective IFN- production in TB patients (Guyot-Revol et al., 2005) and

transiently reduced the bacterial load and granuloma formation (Ozeki et al., 2010).

The majority of individuals vaccinated with BCG or infected with MTB develop a delayedtype hypersensitivity which is manifested as a positive response of intradermal injection to a purified protein derivative from MTB. But about 15% of active TB patients show false negative results and are considered to be anergic TB (Bloom & Small,1998). Similarly, in HIV infected individuals, TST results are often found to be false-negative. Concerning the high frequencies of Treg cells in both diseases (Chen et al., 2007; Bi et al., 2009), it is highly probable that Treg cells may play central role in the anergy mechanism. Cutaneus anergy in active TB is associated with the absence of granuloma formation and poor clinical outcome (Boussiotis et al., 2000). Another study showed that, in an anergic patient, sustained MTB stimulation led to enhanced IL-10 production and the generation of anergic MTB-specific T regulatory cells with the Tr-1 phenotype (Boussitis, 2000). In certain closed populations, a high percentage of TB anergy and high prevalence of active TB were observed, which led to the speculation that innate genetic factors may play a role (Delgado & Ganea, 2001). We observed several cases of anergy in health care workers (HCW), who had been in close contact with active TB patients. Therefore, we questioned whether Treg cell may mask latent

According to previously obtained TST results, one TST positive (27 years old) and one TST negative (42 years old) healthy (X-ray and sputum smear negative) health care individuals with no history of previous MTB infection or other chronic disease were recruited in this

**3.3.1 Role of Treg in mycobacterium tuberculosis infection** 

host response to infection.

& Waksman, 1973).

TB infection.

**3.3.3 Materials and methods Human subjects and samples** 

**3.3.2 Objectives of our Treg study** 

study. A TST result ≥ 10 mm was considered to be positive. Both individuals were vaccinated with BCG when young. The protocol was approved by the ethical commitee of Tohoku University Medical School. Written informed consent was obtained. Both individuals were HIV, hepatitis B and C virus negative.

From each donor 20 ml of Ethylenediamine tetraacetic acid (EDTA) treated peripheral venous blood was obtained, centrifuged and the peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll separation (Ficoll-Paque PLUS,GE Healthcare Bio-Science AB, Uppsala, Sweden). After washing twice in Phosphate Buffered Saline (PBS), PBMCs were resuspended in complete RPMI 1640 medium (consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2% glutamine and 1% penicilin/streptomycin) at a concentration 5x106 cells/ml.

#### **Depletion of CD4+CD25+ cells**

The cells designed for depletion were centrifuged and resusupended in Running buffer (MACS Separation Buffer, Miltenyi Biotec). To avoid unspecific binding of the antibody, 100 ul of FcR Blocking Reagent (Miltenyi Biotec) was added to the PBMCs which were then incubated for 15 minutes on ice. After adding 25 ul of CD25-Biotin monoclonal antibody (Miltenyi Biotec) and 15 minutes incubation on ice, the cells were washed with Running buffer. To cells resuspended in Running buffer, 25 ul of anti-Biotin Microbeads (Miltenyi Biotec) were added, followed by incubation for 15 minutes on ice. Subsequently, the cells were washed and resuspended in Rising Solution (Miltenyi Biotec) and CD4+CD25+ T cells were depleted by positive selection using a magnetic-activated cell sorter (MACS)-assisted cell sorting system (Miltenyi Biotec, Auburn, CA) according to the manufacturer´s instructions.

#### **Cell phenotype determination**

To visualize the biotinylated mAb on CD25+ cells, streptavidin-allophycocyanin (BD PharMingen) was used and the CD4+ cells were stained by anti-CD4 FITC antibody. Flow cytometric analyses were performed with a FACSCalibur flow cytometer (Becton Dickinson) using the CELL quest program (Becton Dickinson). More than 90% of CD4+CD25+ cells were depleted from the PMBCs of both individuals.

#### **Cultivation and cytokine determination**

Freshly isolated, undepleted and Treg depleted PBMCs were cultured in triplicate in 96-well plates at a concentration of 2x105 cells per well in 200 ul of complete RPMI 1640 medium and incubated for 24 hours at 37 °C with 5% CO2. The cells were stimulated with 1 ug/ml of PPD (Statens Serum Institute, Copenhagen, Denmark), 500nM of recombinant CFP-10 and ESAT-6 protein antigen. The cell culture supernatant was harvested from each well for ELISA analysis. IFN-gamma and IL-10 production was determined by using human IFNgamma and IL-10 BD Opt EIATM Set according to the manufacturer´s instructions (BD Bioscience). Optical densities were read at 450 nm on an ELISA plate reader (VersaMax-KT, Molecular Devices Corp., CA, USA ) and the concentrations were calculated from standard curves using the Soft Max Pro program (Molecular Devices Corp.).

#### **Statistical analysis**

Two-sided paired *t*-test was used to analyze the effect of Treg on cytokine production. Differences were considered significant when the *p* value was less than 0.05.

Immunological Diagnosis of Active and Latent TB 365

Fig. 3. Immunossuppresive effect of Treg on (A) IFN- production and (B) IL-10 production.

Antigen presenting cells matured in the presence of BCG are able to instruct naive T cells to develop into cytokine-producing T cells that can be categorized into Th1 (IFN producing), Th2 (IL-4-producing) or Tr1 (IL-10-producing) cells (Larsen et al. 2007). We can speculate that upon first contact with BCG vaccine in early childhood, the naive T cell development may lead to polarization of the immune response in favor of Th2 and Tr1 IL-10-producing cells. An anergic individual, in comparison to a TST positive person, have elevated levels of IL-10 upon MTB antigen stimulation and it might be possible that his immune responses are switched towards an IL-10 immune response. The anergizing effect and antiinflammatory properties of IL-10 might be one of the factors maintaining the

Even if this is a sporadic finding, we believe that these results enable a better understanding of the immune mechanisms involved in anergy and LTBI in adult, healthy, BCG-vaccinated individuals. It is necessary to confirm these data in larger numbers of volunteers. It might be

In summary, Treg cells play a role in masking LTBI by suppressing the specific MTB

disputed whether the in vitro conditions mimic MTB infection in vivo.

immune response through altering IFN- and IL-10 production.

Statistically significant (\*) p < 0.05

anergy and LTBI chronicity.

#### **3.3.4 Results**

To investigate the immunosuppressive effect of Treg cells on the anti-TB immune response, CD25+ T cells were depleted from PBMCs of TST positive and TST negative healthy HCW. IFN- and IL-10 production upon PPD, CFP-10 and ESAT-6 stimulation was assayed.

The positive response to PPD stimulation in the TST-positive individual agreed with the TST result. Similarly, the low IFN- response to PPD in the TST anergic person supported the unresponsiveness of PBMCs to PPD stimulation. Both individuals had low levels of IFN on CFP-10 stimulation, but the TST anergic person, in contrast to the TST positive one, responded to ESAT-6 stimulation, what is suspectious for LTBI. Depletion of Treg cells significantly enhanced the IFN- production in the TST anergic person, but in the TST positive person it influenced the IFN- level only after PPD stimulation (Fig. 3A). Depletion of Treg cells significantly influenced the IL-10 production only by PPD and CFP-10, but not by ESAT-6 stimulation (Fig. 3B).

#### **3.3.5 Discussion**

PPD is the prototypical mycobacterial antigen, which is included also in the Mantoux skin test antigens. The result with PPD stimulation demonstrated a T cell antigen recognition level consistent with TST results. PPD unresponsiveness in anergic TB patients might be due to the inability of their antigen presenting cells to present antigens or to the inability of their T cells to respond to antigen-specific stimulation (Boussiotis et al., 2000). Our results showed that PPD anergy might be due to an impaired T cell response as an effect of the Treg cell activity.

Recombinant antigens such as ESAT-6 and CFP-10 have been reported to be strong IFN inducers. CFP-10 was reported to be less reactive in comparison to ESAT-6 in TB patients as well as in healthy controls (Oliveira et al., 2007), which was also observed in our assay. The anergic subject in our study showed a strong response to ESAT-6 stimulation, which made him highly suspected to be TB infected.

CD4+CD25+FoxP3+ T cells have been found to be increased in MTB infection and to suppress the MTB-specific immunity (Hourgady et al., 2007; Chen et al., 2007). It has been found that elimination of CD4+CD25+ T cells significantly increased the BCG-induced production of IFN- and IL-10 by PBMCs from patients with active TB, but not by those from healthy volunteers (Chen et al. 2007), but there is no report of Treg functions in TST anergic LTBI. We have demonstrated for the first time that Treg cell depletion in anergic individual led to enhanced IFN- production upon MTB specific ESAT-6 and CFP-10 antigen stimulation, while in the TST positive healthy individual we did not observed such a phenomenon. These results support the argument that CD4+CD25+ T cells suppress the Th1 immune response.

IL-10 is considered a soluble factor that plays a central role in controlling inflammatory processes, suppressing T cell responses, and maintaining immunological tolerance (Moore et al., 2001). In the condition of MTB infection, mycobacteria-induced IL-10 production by macrophages allow mycobacteria-infected cells to elude immune surveillance (Larsen et al., 2007).

To investigate the immunosuppressive effect of Treg cells on the anti-TB immune response, CD25+ T cells were depleted from PBMCs of TST positive and TST negative healthy HCW.

The positive response to PPD stimulation in the TST-positive individual agreed with the TST result. Similarly, the low IFN- response to PPD in the TST anergic person supported the unresponsiveness of PBMCs to PPD stimulation. Both individuals had low levels of IFN on CFP-10 stimulation, but the TST anergic person, in contrast to the TST positive one, responded to ESAT-6 stimulation, what is suspectious for LTBI. Depletion of Treg cells significantly enhanced the IFN- production in the TST anergic person, but in the TST positive person it influenced the IFN- level only after PPD stimulation (Fig. 3A). Depletion of Treg cells significantly influenced the IL-10 production only by PPD and CFP-10, but not

PPD is the prototypical mycobacterial antigen, which is included also in the Mantoux skin test antigens. The result with PPD stimulation demonstrated a T cell antigen recognition level consistent with TST results. PPD unresponsiveness in anergic TB patients might be due to the inability of their antigen presenting cells to present antigens or to the inability of their T cells to respond to antigen-specific stimulation (Boussiotis et al., 2000). Our results showed that PPD anergy might be due to an impaired T cell response as an effect of the Treg cell

Recombinant antigens such as ESAT-6 and CFP-10 have been reported to be strong IFN inducers. CFP-10 was reported to be less reactive in comparison to ESAT-6 in TB patients as well as in healthy controls (Oliveira et al., 2007), which was also observed in our assay. The anergic subject in our study showed a strong response to ESAT-6 stimulation, which made

CD4+CD25+FoxP3+ T cells have been found to be increased in MTB infection and to suppress the MTB-specific immunity (Hourgady et al., 2007; Chen et al., 2007). It has been found that elimination of CD4+CD25+ T cells significantly increased the BCG-induced production of IFN- and IL-10 by PBMCs from patients with active TB, but not by those from healthy volunteers (Chen et al. 2007), but there is no report of Treg functions in TST anergic LTBI. We have demonstrated for the first time that Treg cell depletion in anergic individual led to enhanced IFN- production upon MTB specific ESAT-6 and CFP-10 antigen stimulation, while in the TST positive healthy individual we did not observed such a phenomenon. These results support the argument that CD4+CD25+ T cells suppress the Th1

IL-10 is considered a soluble factor that plays a central role in controlling inflammatory processes, suppressing T cell responses, and maintaining immunological tolerance (Moore et al., 2001). In the condition of MTB infection, mycobacteria-induced IL-10 production by macrophages allow mycobacteria-infected cells to elude immune surveillance (Larsen et al.,

IFN- and IL-10 production upon PPD, CFP-10 and ESAT-6 stimulation was assayed.

**3.3.4 Results** 

by ESAT-6 stimulation (Fig. 3B).

him highly suspected to be TB infected.

**3.3.5 Discussion** 

immune response.

2007).

activity.

Fig. 3. Immunossuppresive effect of Treg on (A) IFN- production and (B) IL-10 production. Statistically significant (\*) p < 0.05

Antigen presenting cells matured in the presence of BCG are able to instruct naive T cells to develop into cytokine-producing T cells that can be categorized into Th1 (IFN producing), Th2 (IL-4-producing) or Tr1 (IL-10-producing) cells (Larsen et al. 2007). We can speculate that upon first contact with BCG vaccine in early childhood, the naive T cell development may lead to polarization of the immune response in favor of Th2 and Tr1 IL-10-producing cells. An anergic individual, in comparison to a TST positive person, have elevated levels of IL-10 upon MTB antigen stimulation and it might be possible that his immune responses are switched towards an IL-10 immune response. The anergizing effect and antiinflammatory properties of IL-10 might be one of the factors maintaining the anergy and LTBI chronicity.

Even if this is a sporadic finding, we believe that these results enable a better understanding of the immune mechanisms involved in anergy and LTBI in adult, healthy, BCG-vaccinated individuals. It is necessary to confirm these data in larger numbers of volunteers. It might be disputed whether the in vitro conditions mimic MTB infection in vivo.

In summary, Treg cells play a role in masking LTBI by suppressing the specific MTB immune response through altering IFN- and IL-10 production.

Immunological Diagnosis of Active and Latent TB 367

**38kDa antigen**: The 38-kDa antigen (also known as Antigen 5) is a major lipoprotein antigen of M. tuberculosis. As reviewed by Steigart et. al in a meta-analysis, it yielded a sensitivity of 47% and a specificity of 94% in smear positive tuberculosis patients. The increased sensitivity is found to be associated with smear-positive than negative tuberculosis (Wilkinsonson et al. 1997; Julian et al., 2000). Use of native or recombinant protein did not show any difference in term of diagnostic efficacy. But, the sensitivity of IgG detection was relatively higher than that of IgA against 38 kDa antigen (Verma & Jain, 2007). Several commercially available antibody detection kits were also developed using this antigen including Pathozyme Myco (LAM+38kDa), Pathozyme TB complex plus (38kDa+16kDa). The sensitivity varies in both Pathozyme Myco (21-46%) and Pathozyme TB complex plus (29-76%). However, the tests are highly specific (94-100%) for tuberculosis diagnosis (Steingart et al., 2007). The sensitivity was less than 70% and the specificity varies from 70% to 94.9% in different studies for the diagnosis of smear-positive tuberculosis patients coinfected with HIV (Abebe et al., 2007). The sensitivity of Pathozyme Myco and Pathozyme TB complex plus varies from 11% to 51% respectively, although the specificities were more than 90% by both the kits for the diagnosis of extra-pulmonary tuberculosis (Steigart et. al.,

**Antigen 85B:** It is a member of a family of Ag85 protein complex (Ag85A, Ag85B and Ag85C). It is a major fraction of secreted proteins in the MTB culture filtrate and cell wall. A relatively higher sensitivity in HIV-positive tuberculosis patients (62%) than HIV-negative tuberculosis patients (53%) with a high specificity (>95%) in both groups were reported for Ag85B (Steingart et al., 2009). MPT51 is an antigen also related to the protein family of Ag85 complex. MPT51 provided comparable diagnostic efficacy in both HIV-negative (sensitivity: 59%) and HIV-positive tuberculosis patients (sensitivity: 58%) and the specificities of 94%

**ESAT-6 and CFP-10:** These are two low molecular weight secreted proteins, encoded within the RD1 region of M. tuberculosis and highly associated with the virulence of the organism. It is known to induce strong cell mediated immune response. Although the use of ESAT-6 and CFP-10 in IFN- based immunological methods for tuberculosis is widely acceptable for the diagnosis of active and latent tuberculosis infection, the potential use of this antigens in antibody-based diagnostic methods were also evaluated. Association of antibody responses against ESAT-6 was described to be related with inactive stage of tuberculosis (Davidow et al., 2005; Doherty et al., 2002), although increased antibody in progressive tuberculosis was also demonstrated in other report (Demissie et al., 2006). In addition, CFP-10 showed a sensitivity of 48% and a specificity of 96% for the diagnosis of active pulmonary tuberculosis. The use of CFP-10/ESAT-6 fusion antigen obtained a relatively higher sensitivity of 60.4% and a specificity of 73.8% in HIV-seronegative tuberculosis patients (Wu

**Antigen 60 (A60):** It is a heat-stable component of PPD extracted from BCG that can be recognized by the sera of tuberculosis patients (Abebe et al., 2007). Anda TB (Anda Biologicals, Strasbourg, France), a commercially available ELISA kit was developed using A60 and its diagnostic ability was evaluated by many investigators for the diagnosis of pulmonary and extra-pulmonary tuberculosis. The sensitivity was variable in pulmonary

**4.1 Serodiagnostic markers** 

and 97% respectively (Steingart et al., 2009).

2007).

et al., 2010).

#### **4. Serological diagnosis of tuberculosis**

The diagnosis of tuberculosis infection remain unchanged or with very limited progress for many decades. Until recent years, diagnosis of tuberculosis primarily depends on traditional sputum microscopy for acid fast bacilli (AFB) in low and middle income countries where the disease is heavily concentrated, although the sensitivity of the method is variable (20~60%) (Steingart et al., 2006). In HIV/AIDS infection, frequent occurrence of non-cavitary pulmonary lesion can cause sputum negative tuberculosis disease. Extra-pulmonary involvement is 10-20% of all tuberculosis case and can occur relatively frequently in children than adults and in HIV/AIDS infection than healthy. Since enhancement of B cell immunity and production of antibody along with protective cell-mediated immunity may play an important role in the immunopathogenesis of tuberculosis, detection of specific antibodies against various mycobacterial derived antigens could also play a significant role in the diagnosis of tuberculosis. The value of various mycobacterial native or recombinant protein, lipid or different combinations of purified antigens or commercially available kits as a potential candidate of active tuberculosis sero-marker was evaluated by the ELISA method in many attempts (Abebe et al., 2007; Verma & Jain, 2007; Steingart et al., 2009). Development of a serological test with sufficient diagnostic efficacy for tuberculosis diagnosis could be very much useful tool in resource-limited countries, as the procedures are simple, relatively cost effective and can be performed rapidly.

Characteristics of commonly used antigens have been listed in the following table (Table 2). Until recently, various mycobacterial culture filtrate and surface exposed proteins including 38kDa, Ag85B, MPT51, Ag60 antigens, malate synthase, heat shock protein, RD1 antigens were investigated to determine their diagnostic efficacy.


Table 2. Characteristic of mycobacterial antigens commonly assessed for the serodiagnosis of tuberculosis.

LAM: lipoarabinomannan; TDM: trehalose 6,6 dimycolate; DAT: 2,3-diacyl trehalose; TAT: 2,3,6-triacyl trehalose; SL-I: 2,3,6,6-tetraacyl trehalose 2́ ́ ́ '-sulphate (Sulfolipid-I); Ag85B: Antigen85B; ESAT-6: early secreted antigenic target-6; CFP-10: culture filtrate protein-10

#### **4.1 Serodiagnostic markers**

366 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

The diagnosis of tuberculosis infection remain unchanged or with very limited progress for many decades. Until recent years, diagnosis of tuberculosis primarily depends on traditional sputum microscopy for acid fast bacilli (AFB) in low and middle income countries where the disease is heavily concentrated, although the sensitivity of the method is variable (20~60%) (Steingart et al., 2006). In HIV/AIDS infection, frequent occurrence of non-cavitary pulmonary lesion can cause sputum negative tuberculosis disease. Extra-pulmonary involvement is 10-20% of all tuberculosis case and can occur relatively frequently in children than adults and in HIV/AIDS infection than healthy. Since enhancement of B cell immunity and production of antibody along with protective cell-mediated immunity may play an important role in the immunopathogenesis of tuberculosis, detection of specific antibodies against various mycobacterial derived antigens could also play a significant role in the diagnosis of tuberculosis. The value of various mycobacterial native or recombinant protein, lipid or different combinations of purified antigens or commercially available kits as a potential candidate of active tuberculosis sero-marker was evaluated by the ELISA method in many attempts (Abebe et al., 2007; Verma & Jain, 2007; Steingart et al., 2009). Development of a serological test with sufficient diagnostic efficacy for tuberculosis diagnosis could be very much useful tool in resource-limited countries, as the procedures

Characteristics of commonly used antigens have been listed in the following table (Table 2). Until recently, various mycobacterial culture filtrate and surface exposed proteins including 38kDa, Ag85B, MPT51, Ag60 antigens, malate synthase, heat shock protein, RD1 antigens

LAM Lipoglycan Cell wall Immunomodulation,

DAT Glycolipid Cell wall Immunmodulation TAT Glycolipid Cell wall Immunomodulation SL-I Glycolipid Cell wall Related to MTB virulence 38kDa Protein Rv0934 Immunogenic protein Ag85B Protein Rv1886c Immunogenic protein

synthase Protein Rv1837c Immunogenic protein MPT51 Protein 3803c Immunogenic protein ESAT-6 Protein 3875 Immunogenic protein CFP-10 Protein 3874 Immunogenic protein Antigen 60 Glycopeptidolipid Immunomodulation

Table 2. Characteristic of mycobacterial antigens commonly assessed for the serodiagnosis

LAM: lipoarabinomannan; TDM: trehalose 6,6 dimycolate; DAT: 2,3-diacyl trehalose; TAT:

Antigen85B; ESAT-6: early secreted antigenic target-6; CFP-10: culture filtrate protein-10

́ ́

location/Rv region Biological effects

Anti-inflammatory

Immunomodulation, Enhance inflammation and granuloma formation

'-sulphate (Sulfolipid-I); Ag85B:

**4. Serological diagnosis of tuberculosis** 

are simple, relatively cost effective and can be performed rapidly.

were investigated to determine their diagnostic efficacy.

antigen Type of antigen Mycobacterial

2,3,6-triacyl trehalose; SL-I: 2,3,6,6-tetraacyl trehalose 2́

TDM Glycolipid Cell wall

Target

Malate

of tuberculosis.

**38kDa antigen**: The 38-kDa antigen (also known as Antigen 5) is a major lipoprotein antigen of M. tuberculosis. As reviewed by Steigart et. al in a meta-analysis, it yielded a sensitivity of 47% and a specificity of 94% in smear positive tuberculosis patients. The increased sensitivity is found to be associated with smear-positive than negative tuberculosis (Wilkinsonson et al. 1997; Julian et al., 2000). Use of native or recombinant protein did not show any difference in term of diagnostic efficacy. But, the sensitivity of IgG detection was relatively higher than that of IgA against 38 kDa antigen (Verma & Jain, 2007). Several commercially available antibody detection kits were also developed using this antigen including Pathozyme Myco (LAM+38kDa), Pathozyme TB complex plus (38kDa+16kDa). The sensitivity varies in both Pathozyme Myco (21-46%) and Pathozyme TB complex plus (29-76%). However, the tests are highly specific (94-100%) for tuberculosis diagnosis (Steingart et al., 2007). The sensitivity was less than 70% and the specificity varies from 70% to 94.9% in different studies for the diagnosis of smear-positive tuberculosis patients coinfected with HIV (Abebe et al., 2007). The sensitivity of Pathozyme Myco and Pathozyme TB complex plus varies from 11% to 51% respectively, although the specificities were more than 90% by both the kits for the diagnosis of extra-pulmonary tuberculosis (Steigart et. al., 2007).

**Antigen 85B:** It is a member of a family of Ag85 protein complex (Ag85A, Ag85B and Ag85C). It is a major fraction of secreted proteins in the MTB culture filtrate and cell wall. A relatively higher sensitivity in HIV-positive tuberculosis patients (62%) than HIV-negative tuberculosis patients (53%) with a high specificity (>95%) in both groups were reported for Ag85B (Steingart et al., 2009). MPT51 is an antigen also related to the protein family of Ag85 complex. MPT51 provided comparable diagnostic efficacy in both HIV-negative (sensitivity: 59%) and HIV-positive tuberculosis patients (sensitivity: 58%) and the specificities of 94% and 97% respectively (Steingart et al., 2009).

**ESAT-6 and CFP-10:** These are two low molecular weight secreted proteins, encoded within the RD1 region of M. tuberculosis and highly associated with the virulence of the organism. It is known to induce strong cell mediated immune response. Although the use of ESAT-6 and CFP-10 in IFN- based immunological methods for tuberculosis is widely acceptable for the diagnosis of active and latent tuberculosis infection, the potential use of this antigens in antibody-based diagnostic methods were also evaluated. Association of antibody responses against ESAT-6 was described to be related with inactive stage of tuberculosis (Davidow et al., 2005; Doherty et al., 2002), although increased antibody in progressive tuberculosis was also demonstrated in other report (Demissie et al., 2006). In addition, CFP-10 showed a sensitivity of 48% and a specificity of 96% for the diagnosis of active pulmonary tuberculosis. The use of CFP-10/ESAT-6 fusion antigen obtained a relatively higher sensitivity of 60.4% and a specificity of 73.8% in HIV-seronegative tuberculosis patients (Wu et al., 2010).

**Antigen 60 (A60):** It is a heat-stable component of PPD extracted from BCG that can be recognized by the sera of tuberculosis patients (Abebe et al., 2007). Anda TB (Anda Biologicals, Strasbourg, France), a commercially available ELISA kit was developed using A60 and its diagnostic ability was evaluated by many investigators for the diagnosis of pulmonary and extra-pulmonary tuberculosis. The sensitivity was variable in pulmonary

Immunological Diagnosis of Active and Latent TB 369

were found in aged (>40 yrs) healthy control people (17%) in Japan (Maekura et al., 2001) and high titers of both TBGL IgG (46%) and IgA (36%) in healthy adults was also observed in our recent study in Thailand (Siddiqi et. al.; in press). But, elevated titers of TBGL IgG are not related to BCG vaccination (Nabeshima et al., 2005). Very recently, significant association between the TBGL IgG and Quatiferon-TB Gold IT assay responses in diagnosis of latent tuberculosis infection in healthy healthcare workers was also found (Siddiqi et. al., unpublished data) that represents enhancement of humoral immune responses along with T cell mediated immunity in latent tuberculosis infection. Increased synthesis of TBGL IgA titers that was related to serum IgA were observed in HIV carriers with low CD4+ T cells counts (less than 350/µl) compared to high CD4+ T cells counts (more than 350/µl). However, the sensitivities of TBGL IgG and IgA were very low (10% and 8% respectively) although their specificities were more than 90% for the diagnosis of tuberculosis in pediatric

The performance of various purified antigens and commercially available kits for the serodiagnosis of active pulmonary tuberculosis with or without associated HIV/AIDS coinfection were evaluated in many studies and were reviewed extensively (Abebe et al., 2007; Steingart et al., 2006, 2007, 2009; Varma & Jain, 2007). Protein antigens were reported to have high specificity (>95%) than lipid antigens. In relation to use of single antigen, relatively higher sensitivities can be achieved by using multiple antigens. Cord factor (TDM), among the lipid antigens had the best overall performance. In addition, higher rate of sensitivity can be obtained by evaluation of IgG and/or IgA than IgM. Maes and colleagues has been conceptualized the human immune responses against mycobacteria into four different stages based on BCG vaccination and tuberculosis disease and treatment, initiated from innate response followed by intermingled innate and adaptive response against low molecular weight oligopeptiic and nonpeptidic, as muramyldipeptide and trehalose 6,6 dimycolate and high molecular weight nonpeptidic antigens such as lipoarabinomannan. The final response is directed against protein antigens (Maes et al., 1999). Although it is not clearly understood, enhancement of humoral immune response can be dependent on disease pathogenesis and different stage of infection can influence different subclasses of immunoglobulins. Enhanced IgM expression can usually occur in the early stage of infection that can subsequently be diminished on progression of disease. Therefore, detection of IgM may show limited value in the sero-diagnostic assay. The reason of low sensitivity of IgA antibody is not clear. It is possible that the generation of IgA antibody needs larger amounts of antigens and related with degree of disease pathogenesis than do IgG responses and

However, until now, any performance was not successful to show the, stable, consistent and acceptable sero-diagnostic efficacy with a sensitivity of at least more than 85% and a specificity of more than 95% and to replace the traditional sputum microscopy as a reliable diagnostic tool in different groups of tuberculosis patients including HIV-positive, negative, extra-pulmonary tuberculosis or in pediatric tuberculosis detection. However, extensive study for evaluation of humoral immune responses in different stages of tuberculosis infection and disease and their association with the disease pathogenesis should be consider to clarify the variable antibody responses against different antigens. As

cases (Siddiqi et. al., 2009, unpublished data).

indicate the heterogeneity of tuberculosis infection.

**4.2 Discussion** 

tuberculosis (29%-85%) as well as in extra-pulmonary tuberculosis (0%-100%). However, the specificities ranges 70%-100% for both types of tuberculosis (Steingart et al., 2007).

Non-peptidic antigens from the mycobacterial cell wall grasp the main focus of comprehensive research for the determination of their potential role in the protective immunity or marker of TB disease.

**Lipoarabinomannan (LAM)**: LAM, a complex glycolipid antigen forming is a major part of cell wall of MTB. Evaluation of anti-LAM-IgG against purified LAM from MTB for the serodiagnosis of tuberculosis was reported to have good diagnostic ability (sensitivity: 91%, specificity: 72%) in detection of both pulmonary, pleural and miliary tuberculosis as well as tubercular lymphadenitis (Sada et al., 1990). However, lower rate of sensitivity (50.5%) and comparable specificity (78.3%) for the diagnosis of pulmonary tuberculosis patients were reported by Tessema et al. (2002). In addition, MycoDot, (Genelabs, Switzerland), a commercially available kit for the detection of antibodies against MTB specific LAM was also evaluated in many reports (Steingart et al., 2007; Verma & Jain, 2007). Although the specificities were high (84-100%), low rate of sensitivities (16-56%) were obtained and low sensitivities were mostly related to HIV/TB co-infection (Verma & Jain, 2007)

**DAT, TAT and SL-I:** Assessment of antibody responses using DAT, TAT, and SL-I antigens in ELISA for serodiagnosis of tuberculosis revealed variable results in terms of diagnostic efficacy. Widely variable ranges of sensitivity of 11 to 88% by DAT antigen were reported in several studies. A similar rate of sensitivity by MTB (44.5%) and M. fortuitum (48.6%) infection were also demonstrated. In addition, the test sensitivities of TAT anigen also vary from 51-93% (Julian et al., 2002). Julian et. al. reported the best performance of IgG (Sensitivity: 81% and specificity: 77.6%) and IgA (sensitivity: 66% and specificity: 87%) antibodies by SL-I among four trehalose contacting glycolipids (DAT, TAT, SL-I, TDM). Although, several reasons including variation in the ELISA protocol, using of different antigen concentrations and population from different subgroups were described as possible reason of such variability, the effectiveness of theses antigens are still uncertain.

**TDM (also known as cord factor):** It composes a major part of the mycobacterial cell wall, was identified as the most immunogenic glycolipid. Clinical evaluation of serodiagnosis of pulmonary tuberculosis using of TDM purified from Mycobacterium tuberculosis H37Rv, in ELISA reported its sensitivity of 81% and its specificity of 96% (Mizusawa et. al. 2008). Best performance by TDM (sensitivity 69%, specificity: 91%) among other lipid antigens including DAT, TAT, SL-I for the serodiagnosis of tuberculosis were also reviewed by Steingart et al. (2009). IgG antibody against TDM can also recognize mycolic acid sub-classes and highly active against methoxy-mycolic acid in the cord factor of M. tuberculosis than keto-mycolic acid in M. avium complex (Pan et al., 1999). By combining TDM with more hydrophobic glycolipids, a new tuberculous glycolipid (TBGL) antigen was designed and a more sensitive serodiagnostic kit for TB, an anti-TBGL IgG test was developed (Kyowa Medex Co, Japan). Anti-TBGL IgG antibody (TBGL IgG) has been proposed as a useful serodiagnostic marker of active pulmonary tuberculosis (PTB) (sensitivity: 84% and specificity: 95% in young adults) in Japan (Maekura et al., 2001). Strong association between TBGL IgG and IgA was also revealed in active pulmonary tuberculosis cases and increased TBGL IgG and IgA was found to be associated with CRP and cavity formation indicating their involvement in the disease pathogenesis (Mizusawa et al., 2008). Elevated titers of TBGL IgG were found in aged (>40 yrs) healthy control people (17%) in Japan (Maekura et al., 2001) and high titers of both TBGL IgG (46%) and IgA (36%) in healthy adults was also observed in our recent study in Thailand (Siddiqi et. al.; in press). But, elevated titers of TBGL IgG are not related to BCG vaccination (Nabeshima et al., 2005). Very recently, significant association between the TBGL IgG and Quatiferon-TB Gold IT assay responses in diagnosis of latent tuberculosis infection in healthy healthcare workers was also found (Siddiqi et. al., unpublished data) that represents enhancement of humoral immune responses along with T cell mediated immunity in latent tuberculosis infection. Increased synthesis of TBGL IgA titers that was related to serum IgA were observed in HIV carriers with low CD4+ T cells counts (less than 350/µl) compared to high CD4+ T cells counts (more than 350/µl). However, the sensitivities of TBGL IgG and IgA were very low (10% and 8% respectively) although their specificities were more than 90% for the diagnosis of tuberculosis in pediatric cases (Siddiqi et. al., 2009, unpublished data).

#### **4.2 Discussion**

368 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

tuberculosis (29%-85%) as well as in extra-pulmonary tuberculosis (0%-100%). However, the

Non-peptidic antigens from the mycobacterial cell wall grasp the main focus of comprehensive research for the determination of their potential role in the protective

**Lipoarabinomannan (LAM)**: LAM, a complex glycolipid antigen forming is a major part of cell wall of MTB. Evaluation of anti-LAM-IgG against purified LAM from MTB for the serodiagnosis of tuberculosis was reported to have good diagnostic ability (sensitivity: 91%, specificity: 72%) in detection of both pulmonary, pleural and miliary tuberculosis as well as tubercular lymphadenitis (Sada et al., 1990). However, lower rate of sensitivity (50.5%) and comparable specificity (78.3%) for the diagnosis of pulmonary tuberculosis patients were reported by Tessema et al. (2002). In addition, MycoDot, (Genelabs, Switzerland), a commercially available kit for the detection of antibodies against MTB specific LAM was also evaluated in many reports (Steingart et al., 2007; Verma & Jain, 2007). Although the specificities were high (84-100%), low rate of sensitivities (16-56%) were obtained and low

**DAT, TAT and SL-I:** Assessment of antibody responses using DAT, TAT, and SL-I antigens in ELISA for serodiagnosis of tuberculosis revealed variable results in terms of diagnostic efficacy. Widely variable ranges of sensitivity of 11 to 88% by DAT antigen were reported in several studies. A similar rate of sensitivity by MTB (44.5%) and M. fortuitum (48.6%) infection were also demonstrated. In addition, the test sensitivities of TAT anigen also vary from 51-93% (Julian et al., 2002). Julian et. al. reported the best performance of IgG (Sensitivity: 81% and specificity: 77.6%) and IgA (sensitivity: 66% and specificity: 87%) antibodies by SL-I among four trehalose contacting glycolipids (DAT, TAT, SL-I, TDM). Although, several reasons including variation in the ELISA protocol, using of different antigen concentrations and population from different subgroups were described as possible

**TDM (also known as cord factor):** It composes a major part of the mycobacterial cell wall, was identified as the most immunogenic glycolipid. Clinical evaluation of serodiagnosis of pulmonary tuberculosis using of TDM purified from Mycobacterium tuberculosis H37Rv, in ELISA reported its sensitivity of 81% and its specificity of 96% (Mizusawa et. al. 2008). Best performance by TDM (sensitivity 69%, specificity: 91%) among other lipid antigens including DAT, TAT, SL-I for the serodiagnosis of tuberculosis were also reviewed by Steingart et al. (2009). IgG antibody against TDM can also recognize mycolic acid sub-classes and highly active against methoxy-mycolic acid in the cord factor of M. tuberculosis than keto-mycolic acid in M. avium complex (Pan et al., 1999). By combining TDM with more hydrophobic glycolipids, a new tuberculous glycolipid (TBGL) antigen was designed and a more sensitive serodiagnostic kit for TB, an anti-TBGL IgG test was developed (Kyowa Medex Co, Japan). Anti-TBGL IgG antibody (TBGL IgG) has been proposed as a useful serodiagnostic marker of active pulmonary tuberculosis (PTB) (sensitivity: 84% and specificity: 95% in young adults) in Japan (Maekura et al., 2001). Strong association between TBGL IgG and IgA was also revealed in active pulmonary tuberculosis cases and increased TBGL IgG and IgA was found to be associated with CRP and cavity formation indicating their involvement in the disease pathogenesis (Mizusawa et al., 2008). Elevated titers of TBGL IgG

specificities ranges 70%-100% for both types of tuberculosis (Steingart et al., 2007).

sensitivities were mostly related to HIV/TB co-infection (Verma & Jain, 2007)

reason of such variability, the effectiveness of theses antigens are still uncertain.

immunity or marker of TB disease.

The performance of various purified antigens and commercially available kits for the serodiagnosis of active pulmonary tuberculosis with or without associated HIV/AIDS coinfection were evaluated in many studies and were reviewed extensively (Abebe et al., 2007; Steingart et al., 2006, 2007, 2009; Varma & Jain, 2007). Protein antigens were reported to have high specificity (>95%) than lipid antigens. In relation to use of single antigen, relatively higher sensitivities can be achieved by using multiple antigens. Cord factor (TDM), among the lipid antigens had the best overall performance. In addition, higher rate of sensitivity can be obtained by evaluation of IgG and/or IgA than IgM. Maes and colleagues has been conceptualized the human immune responses against mycobacteria into four different stages based on BCG vaccination and tuberculosis disease and treatment, initiated from innate response followed by intermingled innate and adaptive response against low molecular weight oligopeptiic and nonpeptidic, as muramyldipeptide and trehalose 6,6 dimycolate and high molecular weight nonpeptidic antigens such as lipoarabinomannan. The final response is directed against protein antigens (Maes et al., 1999). Although it is not clearly understood, enhancement of humoral immune response can be dependent on disease pathogenesis and different stage of infection can influence different subclasses of immunoglobulins. Enhanced IgM expression can usually occur in the early stage of infection that can subsequently be diminished on progression of disease. Therefore, detection of IgM may show limited value in the sero-diagnostic assay. The reason of low sensitivity of IgA antibody is not clear. It is possible that the generation of IgA antibody needs larger amounts of antigens and related with degree of disease pathogenesis than do IgG responses and indicate the heterogeneity of tuberculosis infection.

However, until now, any performance was not successful to show the, stable, consistent and acceptable sero-diagnostic efficacy with a sensitivity of at least more than 85% and a specificity of more than 95% and to replace the traditional sputum microscopy as a reliable diagnostic tool in different groups of tuberculosis patients including HIV-positive, negative, extra-pulmonary tuberculosis or in pediatric tuberculosis detection. However, extensive study for evaluation of humoral immune responses in different stages of tuberculosis infection and disease and their association with the disease pathogenesis should be consider to clarify the variable antibody responses against different antigens. As

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most of the investigations for the determination of sero-diagnostic ability of various antigens were carried out in tuberculosis endemic countries, determination of antibody responses in latent tuberculosis infection could be helpful to some extent for explaining the reason of low specificities and the possibility of influence by tuberculosis endemicity in such countries. The immune response in HIV/AIDS patients co-infected with tuberculosis is more complex than single infection. Antibodies against several single or multiple antigens were detected in HIV/AIDS patients with active tuberculosis and even months to year's prior development of tuberculosis related symptoms in some prospective studies. More investigation with diverse antigens for the sero-diagnosis of subclinical and active tuberculosis particularly sputum-negative and extra-pulmonary tuberculosis especially in those with HIV/AIDS co-infection and in pediatric cases is an urgent necessity.

It is generally believed that, T helper immunity and elaboration of IFN- offer vital role in protective immunity to clear or containment of the intracellular MTB infection. However, BCG-induced antibodies was shown to potentiate IFN- production by mycobacteriumspecific CD4+ T cells and also can cause enhancement of mycobacterial phagocytosis probably by inducing opsonization by antibodies and that might be related to mucosal immunity (Abebe & Bjune, 2009). Protective role of antibodies against several TB antigens in mice model was also review by Glatman-Freedman ( 2009). Therefore, antibody-mediated immunity against diverse mycobacterial antigens in synergy with cell mediated immunity can play a vital role in the protection and immunopathogenesis of tuberculosis infection and disease. Frequent detection of antibodies in latent or progressive stages of latent to active tuberculosis and their relation to immune responses especially with mucosal immunity needs to be clarified further.

#### **5. Acknowledgment**

This work is supported the Scientific Research Expenses for Health and Welfare program from the Ministry of Health, Labour and Welfare, Japan (TH) and Science and Technology Research Partnership for Sustainable Development from Japan Science and Technology Agency, Japan (YS). This work was supported by collaborative funding from the Research Centre for Zoonosis Control, Hokkaido University. We are grateful to prof. Ishii N. (Tohoku University) and Dr. Mousavi S.F. (Pasteur Institute of Iran) for the help with Treg depletion experiment. We would like to thank to Mr. Brent Bell for reading the manuscript.

#### **6. References**


most of the investigations for the determination of sero-diagnostic ability of various antigens were carried out in tuberculosis endemic countries, determination of antibody responses in latent tuberculosis infection could be helpful to some extent for explaining the reason of low specificities and the possibility of influence by tuberculosis endemicity in such countries. The immune response in HIV/AIDS patients co-infected with tuberculosis is more complex than single infection. Antibodies against several single or multiple antigens were detected in HIV/AIDS patients with active tuberculosis and even months to year's prior development of tuberculosis related symptoms in some prospective studies. More investigation with diverse antigens for the sero-diagnosis of subclinical and active tuberculosis particularly sputum-negative and extra-pulmonary tuberculosis especially in

It is generally believed that, T helper immunity and elaboration of IFN- offer vital role in protective immunity to clear or containment of the intracellular MTB infection. However, BCG-induced antibodies was shown to potentiate IFN- production by mycobacteriumspecific CD4+ T cells and also can cause enhancement of mycobacterial phagocytosis probably by inducing opsonization by antibodies and that might be related to mucosal immunity (Abebe & Bjune, 2009). Protective role of antibodies against several TB antigens in mice model was also review by Glatman-Freedman ( 2009). Therefore, antibody-mediated immunity against diverse mycobacterial antigens in synergy with cell mediated immunity can play a vital role in the protection and immunopathogenesis of tuberculosis infection and disease. Frequent detection of antibodies in latent or progressive stages of latent to active tuberculosis and their relation to immune responses especially with mucosal immunity

This work is supported the Scientific Research Expenses for Health and Welfare program from the Ministry of Health, Labour and Welfare, Japan (TH) and Science and Technology Research Partnership for Sustainable Development from Japan Science and Technology Agency, Japan (YS). This work was supported by collaborative funding from the Research Centre for Zoonosis Control, Hokkaido University. We are grateful to prof. Ishii N. (Tohoku University) and Dr. Mousavi S.F. (Pasteur Institute of Iran) for the help with Treg depletion

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the serological diagnosis of tuberculosis, *Journal of clinical microbiology,* Vol.28,

C.; Grinsztejn, B. & Rolla, V. C. (2009). Effectiveness of Highly Active Antiretroviral Therapy (Haart) Used Concomitantly with Rifampicin in Patients with Tuberculosis and Aids, *The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases*, Vol.13, No.5, (October 2009),

Discriminating IBD from IBS: comparison of the test performance of fecal markers, blood leukocytes, CRP, and IBD antibodies, *Inflammatory Bowel Diseases,* Vol.14,

Jr. & Hamill, R. J. (2005). Incidence and Risk Factors for Immune Reconstitution Inflammatory Syndrome During Highly Active Antiretroviral Therapy, *AIDS*,

Chotpittayasunondh, T.; & Hattori, T. Elevated anti-tubercular glycolipid antibody titers in healthy adults as well as pulmonary tuberculosis patients in Thailand. *The* 

A.; Pai, M. & Laal, S. (2009). Performance of purified antigens for serodiagnosis of pulmonary tuberculosis: a meta-analysis, *Clinical and vaccine immunology,* Vol.16,

J.; Weldingh, K. & Pai, M. (2007). A systematic review of commercial serological antibody detection tests for the diagnosis of extrapulmonary tuberculosis, *Thorax,*

R.; Perkins, M.; Aziz, M. A. & Pai, M. (2006). Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review, *The Lancet* 


MTB48, and CFP-10/ESAT-6 antigens in tuberculosis, *Clinical and vaccine immunology,* Vol.17, No.3, (March 2010), pp.372-375, ISSN 1556-679X

**18** 

*México* 

**Immune Diagnosis of Tuberculosis** 

Miguel A. Arroyo-Ornelas1, Ma. Concepción Arenas-Arrocena1, Horacio V. Estrada2, Victor M. Castaño1 and Luz M. López-Marín1

Due to its highly contagious nature, the control of tuberculosis (TB) is strongly dependent on the efficiency of diagnosis. Natural history of TB comprises two main stages: a latent, non-infective form, in which bacilli are efficiently controlled by the human defense system and, active TB disease, whose pulmonary form is the most common and infective variant in humans (Figure 1). Diagnosis of TB is needed at different stages: the detection of latent TB, a condition estimated to be present in one third of the world population (Russell et al 2010), screening tests for active TB in large populations and confirmatory/drugsusceptibility diagnostic tools aimed to select appropriate chemotherapy regimes. In particular, tools to screen active, contagious TB cases are critical to overcome diagnosis delays, transmission and spread of the disease, and represent one of the top priorities for TB control. For more than a century, diagnosis of active TB has been essentially based on sputum smear microscopy (SSM). Simple and rapid, considered a low-cost control tool, SSM fails however to detect about half of active pulmonary cases since its sensitivity is compromised by low bacterial loads: only above 104 bacilli per ml of sputum are detectable by SSM (Abebe et al, 2007). Moreover, detection of positive smears needs to be confirmed in three independent samples so that mycobacteria other than *M. tuberculosis*, which may be present as normal flora do not generate false positive diagnosis results. When suspected cases still remain, time-consuming and high-cost *M. tuberculosis* cultures are required. For many decades, a myriad of alternative tools have been explored to replace SSM for screening active TB. After growth of the pathogen inside a body, exposition to bacterial components is followed by the secretion of specific antibodies. Antibodies associated to active TB are not correlated to protective immunity, but their presence may be exploited as biomarker for active TB. In this chapter, the potential of antigen-antibody measurements to screen active TB will be pointed up, with a special emphasis in the need for controlling particular but large populations. The basis, challenges and opportunities of immune diagnosis will be described, putting an emphasis

**1. Introduction** 

on our work involving novel technologies.

**Through Novel Technologies** 

*1Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Querétaro,* 

*2Centro Nacional de Metrología, Querétaro,* 


## **Immune Diagnosis of Tuberculosis Through Novel Technologies**

Miguel A. Arroyo-Ornelas1, Ma. Concepción Arenas-Arrocena1, Horacio V. Estrada2, Victor M. Castaño1 and Luz M. López-Marín1 *1Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Querétaro, 2Centro Nacional de Metrología, Querétaro, México* 

#### **1. Introduction**

378 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Yew, W. W. & Leung, C. C. (2006). Antituberculosis drugs and hepatotoxicity, *Respirology,*

Zhu, C.; Anderson, A. C.; Schubart, A.; Xiong, H.; Imitola, J.; Khoury, S. J.; Zheng, X. X.;

Vol.11, No.6, (November 2006), pp.699-707, ISSN 1323-7799

*immunology,* Vol.17, No.3, (March 2010), pp.372-375, ISSN 1556-679X Yamashiro, S.; Kawakami, K.; Uezu, K.; Kinjo, T.; Miyagi, K.; Nakamura, K. & Saito, A.

(January 2005), pp.57-64, ISSN 0009-9104

2005), pp.1245-1252, ISSN 1529-2908

MTB48, and CFP-10/ESAT-6 antigens in tuberculosis, *Clinical and vaccine* 

(2005). Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis, *Clinical and experimental immunology,* Vol.139, No.1,

Strom, T. B. & Kuchroo, V. K. (2005). The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity, *Nature immunology,Vol.*6, No.12, (December

> Due to its highly contagious nature, the control of tuberculosis (TB) is strongly dependent on the efficiency of diagnosis. Natural history of TB comprises two main stages: a latent, non-infective form, in which bacilli are efficiently controlled by the human defense system and, active TB disease, whose pulmonary form is the most common and infective variant in humans (Figure 1). Diagnosis of TB is needed at different stages: the detection of latent TB, a condition estimated to be present in one third of the world population (Russell et al 2010), screening tests for active TB in large populations and confirmatory/drugsusceptibility diagnostic tools aimed to select appropriate chemotherapy regimes. In particular, tools to screen active, contagious TB cases are critical to overcome diagnosis delays, transmission and spread of the disease, and represent one of the top priorities for TB control. For more than a century, diagnosis of active TB has been essentially based on sputum smear microscopy (SSM). Simple and rapid, considered a low-cost control tool, SSM fails however to detect about half of active pulmonary cases since its sensitivity is compromised by low bacterial loads: only above 104 bacilli per ml of sputum are detectable by SSM (Abebe et al, 2007). Moreover, detection of positive smears needs to be confirmed in three independent samples so that mycobacteria other than *M. tuberculosis*, which may be present as normal flora do not generate false positive diagnosis results. When suspected cases still remain, time-consuming and high-cost *M. tuberculosis* cultures are required. For many decades, a myriad of alternative tools have been explored to replace SSM for screening active TB. After growth of the pathogen inside a body, exposition to bacterial components is followed by the secretion of specific antibodies. Antibodies associated to active TB are not correlated to protective immunity, but their presence may be exploited as biomarker for active TB. In this chapter, the potential of antigen-antibody measurements to screen active TB will be pointed up, with a special emphasis in the need for controlling particular but large populations. The basis, challenges and opportunities of immune diagnosis will be described, putting an emphasis on our work involving novel technologies.

Immune Diagnosis of Tuberculosis Through Novel Technologies 381

making those methods unaffordable. Thus, novel approaches must be focused in the diagnosis of TB at high-burden settings. A few years ago, the World Health Organization (WHO) has prompted for the development of tests for active TB optimally fulfilling seven

Rapid (to enable treatment at first visit and Robust (does not require refrigerated

Currently, the development of ASSURED tests has been considered for the control of a variety of diseases, including TB, malaria, syphilis and dengue. Considering the number of affected people and the feasibility for its development, the availability of an ASSURED test for the screening of active TB would have a tremendous, positive impact in World Public Health (Mabey et al, 2004). Before the emergence of human immune deficiency virus (HIV) epidemy, TB became "invisible to international donors and taken to be a fact of life in the most-affected parts of the world" (Dye & Williams, 2010). Thereby, the search for better vaccines, therapeutic and diagnostic tools was neglected for decades, even for more than a century in some cases (Kaufmann & Parida 2007). At present, this lack of technological developments has made TB control tools virtually inaccessible for most endemic settings. Manipulation of *M. tuberculosis* specimens represents a high level biological risk, requiring high-cost, sophisticated facilities. For these reasons, diagnosis based on the immune response to *M. tuberculosis* represents an alternative to cover the main indications for an ASSURED test, including minimal handling requirements, rapidity and adaptability to close-to- the-patient formats. In this chapter we will describe the biological basis, challenges and opportunities related to immunological tests, with an emphasis on point-of-care (POC) tests. In addition to rapid immunochromatography formats that have been explored by various groups, we herein propose the development of friendly, label-free platforms, using

**3. Human immune response to** *Mycobacterium tuberculosis* **infection** 

It is estimated that one third of the World population is currently infected by *M. tuberculosis* (Dye & Williams, 2010). After infection, *M. tuberculosis* is promptly recognized by the innate immune system. Pattern-recognition receptors located at the surface of myeloid cells, such as mannose recognizing receptors located in macrophages or dendritic cells, recognize molecular patterns commonly associated to pathogens and immediately respond through pro-inflammatory signals (Dorhoi et al, 2011). Whether this process is able to control the spread of the bacilli is still controversial. Supporting the hypothesis is the fact that a number of healthy contacts have been reported with no apparent sign of acquired immunity, thus suggesting that innate mediators were able to stop the infection before the establishment of adaptive, memory-derived responses (Dorhoi et al, 2011). In most people, however, the establishment of innate immunity allows a set of specific defence mechanisms to be

requirements, the so-called ASSURED diagnostic tests:

User-friendly (simple to perform and requiring minimal training)

Affordable by those at risk of infection

 Sensitive (few false-negatives) Specific (few false-positives)

Delivered to those who need them

Micro-Electro-Mechanical Systems (MEMS).

storage) Equipment-free

Fig. 1. Natural history of *Mycobacterium tuberculosis* infection. *M. tuberculosis* is an obligate pathogen normally acquired through respiratory tract. After infection, establishment of human response is able to contain microbial growth in 90 – 95% cases; this process is characterized by activation, recruitment and/or proliferation of distinct cells in the infectious foci, growth control and confinement of the pathogen inside a granuloma, a condition called as latent TB (Inlet a). In contrast, immune compromised-associated conditions lead to active disease, characterized by microbial growth, tissue damage and antibody production (Inlet b). Case finding of active cases is critical to stop TB transmission. Antibodies, depicted using the symbol Y, and antigens liberated from bacteria may serve as biomarkers at this infection stage.

#### **2. The need of rapid tests for diagnosing active TB**

Efficient treatment is available for most cases of TB. However, thousands of deaths are reported every day due to TB. Due to the fact that healthy immune systems are a condition for TB containment, malnourishment, poverty and a fail in Public Health coverage have been strongly associated to the development of active TB cases. The burdens of TB morbidity and mortality have a tremendous impact in young adults, children and women, and result in the loss of potentially healthy and productive life. It is estimated that active TB cases result in a strong economical impact through loss of work, absence from school and public health expenses. Accentuating this scenario, TB active cases also represent the major cause of death during human immunodeficiency virus (HIV) infection. To deal with this problem, the availability of simple diagnostic tools for the detection of tuberculosis is essential, as it is the basis to treat and control infective cases. During the last years, a set of novel diagnostic methodologies have been developed. Some popular examples are tools based on nucleic acid amplification, such as the polymerase chain reaction (PCR), or the introduction of radioactive probes to speed bacterial culture detection (WHO, 2009). Many new diagnostic technologies had been based on sophisticated equipment and highly specialized training, but up to 90% TB cases occur in low-income settings (WHO 2006), making those methods unaffordable. Thus, novel approaches must be focused in the diagnosis of TB at high-burden settings. A few years ago, the World Health Organization (WHO) has prompted for the development of tests for active TB optimally fulfilling seven requirements, the so-called ASSURED diagnostic tests:


380 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Fig. 1. Natural history of *Mycobacterium tuberculosis* infection. *M. tuberculosis* is an obligate pathogen normally acquired through respiratory tract. After infection, establishment of human response is able to contain microbial growth in 90 – 95% cases; this process is characterized by activation, recruitment and/or proliferation of distinct cells in the infectious foci, growth control and confinement of the pathogen inside a granuloma, a condition called as latent TB (Inlet a). In contrast, immune compromised-associated conditions lead to active disease, characterized by microbial growth, tissue damage and antibody production (Inlet b). Case finding of active cases is critical to stop TB transmission. Antibodies, depicted using the symbol Y, and antigens liberated from bacteria may serve as

Efficient treatment is available for most cases of TB. However, thousands of deaths are reported every day due to TB. Due to the fact that healthy immune systems are a condition for TB containment, malnourishment, poverty and a fail in Public Health coverage have been strongly associated to the development of active TB cases. The burdens of TB morbidity and mortality have a tremendous impact in young adults, children and women, and result in the loss of potentially healthy and productive life. It is estimated that active TB cases result in a strong economical impact through loss of work, absence from school and public health expenses. Accentuating this scenario, TB active cases also represent the major cause of death during human immunodeficiency virus (HIV) infection. To deal with this problem, the availability of simple diagnostic tools for the detection of tuberculosis is essential, as it is the basis to treat and control infective cases. During the last years, a set of novel diagnostic methodologies have been developed. Some popular examples are tools based on nucleic acid amplification, such as the polymerase chain reaction (PCR), or the introduction of radioactive probes to speed bacterial culture detection (WHO, 2009). Many new diagnostic technologies had been based on sophisticated equipment and highly specialized training, but up to 90% TB cases occur in low-income settings (WHO 2006),

biomarkers at this infection stage.

**2. The need of rapid tests for diagnosing active TB** 

Delivered to those who need them

Currently, the development of ASSURED tests has been considered for the control of a variety of diseases, including TB, malaria, syphilis and dengue. Considering the number of affected people and the feasibility for its development, the availability of an ASSURED test for the screening of active TB would have a tremendous, positive impact in World Public Health (Mabey et al, 2004). Before the emergence of human immune deficiency virus (HIV) epidemy, TB became "invisible to international donors and taken to be a fact of life in the most-affected parts of the world" (Dye & Williams, 2010). Thereby, the search for better vaccines, therapeutic and diagnostic tools was neglected for decades, even for more than a century in some cases (Kaufmann & Parida 2007). At present, this lack of technological developments has made TB control tools virtually inaccessible for most endemic settings. Manipulation of *M. tuberculosis* specimens represents a high level biological risk, requiring high-cost, sophisticated facilities. For these reasons, diagnosis based on the immune response to *M. tuberculosis* represents an alternative to cover the main indications for an ASSURED test, including minimal handling requirements, rapidity and adaptability to close-to- the-patient formats. In this chapter we will describe the biological basis, challenges and opportunities related to immunological tests, with an emphasis on point-of-care (POC) tests. In addition to rapid immunochromatography formats that have been explored by various groups, we herein propose the development of friendly, label-free platforms, using Micro-Electro-Mechanical Systems (MEMS).

#### **3. Human immune response to** *Mycobacterium tuberculosis* **infection**

It is estimated that one third of the World population is currently infected by *M. tuberculosis* (Dye & Williams, 2010). After infection, *M. tuberculosis* is promptly recognized by the innate immune system. Pattern-recognition receptors located at the surface of myeloid cells, such as mannose recognizing receptors located in macrophages or dendritic cells, recognize molecular patterns commonly associated to pathogens and immediately respond through pro-inflammatory signals (Dorhoi et al, 2011). Whether this process is able to control the spread of the bacilli is still controversial. Supporting the hypothesis is the fact that a number of healthy contacts have been reported with no apparent sign of acquired immunity, thus suggesting that innate mediators were able to stop the infection before the establishment of adaptive, memory-derived responses (Dorhoi et al, 2011). In most people, however, the establishment of innate immunity allows a set of specific defence mechanisms to be

Immune Diagnosis of Tuberculosis Through Novel Technologies 383

foreign entities (as antigens). Two lymphocyte families, the T and B-cells, are activated after encountering with their matching antigen, then develop into the effectors of adaptive immunity. For T cells, presentation of foreign entities by antigen-presenting cells is mandatory for recognition. In contrast, B cells recognize their cognate antigens through direct interaction via the B-cell receptor. Once activated, B and T cells trigger a variety of functions, mainly including: (a) secretion of cytokines and chemoattractants by CD4+ helper T-cells, (b) lysis of infected cells via the release of lytic enzymes by CD8+ cytotoxic T-cells, (c) secretion of antibodies by plasma B-cells that have been derived from activated Blymphocytes, (d) production of a number of long-lived memory T- and B-cells, which last for many years circulating in the bloodstream and monitor for infection (either newly

As depicted in Figure 2, some responses related to cell-mediated immunity are restricted to active infection. One illustrative example is the release of some pro-inflammatory cytokines by T-cells, such as interferon-gamma. The secretion of specific antibodies by plasma Blymphocytes also occurs during active or newly acquired TB, where antigen-presenting cells are charged with bacterial moieties. Some of the immune responses during active infection are known to be critical for arresting the growth of *M. tuberculosis*. In contrast, antibody secretion has been considered irrelevant, although some recent studies suggest a role for antibodies on the fate of *M. tuberculosis* infection (Glatman-Freedman et al, 2010). Yet, secretion of antibodies during bacterial growth (*i.e*. during active infection) can be exploited

Isolated specific antigens have been used for detecting antibodies in human plasma, through the measurement of antigen-antibody reactions *in vitro*. Alternatively, antibodies may be produced in the lab and used to capture mycobacterial antigens that have been secreted to body fluids. Because free mycobacterial antigens are not encountered in many biological fluids, our work has been dedicated to the detection of antibodies. Importantly, antibodies associated to *M. tuberculosis* disease are found in sera from individuals affected by both pulmonary and extra-pulmonary TB, thus allowing the detection of difficult-to-detect TB pathologies (Daniel, 1989). Challenges related to the production of antigens for diagnostic

Specific activation of immune cells against *M. tuberculosis* occurs after the recognition of bacterial components as foreign entities. With about 4,000 genes (TubercuList web page), *M. tuberculosis* synthesizes a complex array of molecular products, mainly composed of proteins, lipids and carbohydrates. Box 1 summarizes the highlights of different antigens

A few years after Robert Koch discovered *M. tuberculosis*, his work was largely dedicated to look for a cure against TB disease, which was the major health threat in Europe by the time. His work allowed the description of *M. tuberculosis* extracts, obtained by glycerol extraction of liquid cultures of the bacilli (Kaufmann & Schaible 2005). Although this material, called tuberculin, was found unable to inhibit the growth of *M. tuberculosis* in guinea pigs or

to detect infectious cases, thus contributing to stop TB transmission.

developed or endogenously re-activated).

tests will be presented in the following sections.

**4. Antigen repertoire of** *M. tuberculosis*

**5. Immunodiagnosis of TB: From Koch to POC tests** 

from the bacillus.

initiated. This process involves destruction of bacilli by professional phagocytes, and presentation of pathogen molecular fragments (antigens) to lymphocytes, followed by activation and proliferative steps. This response, also known as adaptive immunity, may give rise to either protection via the containment of the bacilli inside a granuloma, or an exacerbated inflammatory process associated to destructive pathology. In both cases, lymphocytes that specifically recognize their cognate antigen are activated, leading to the production of cytokines or antibodies, the destruction of infected cells and, the formation of memory lymphocyte clones. Since these processes involve mechanisms specifically originated during *M. tuberculosis* infection, the associated biomarkers have been largely exploited for diagnostic purposes.

An overview of adaptive, cell-mediated responses to *M. tuberculosis* is schematized in Fig. 2. Adaptive responses take place when lymphocytes recognize mycobacterial molecules as

Fig. 2. Cell-mediated immunity against *M. tuberculosis* infection. After recognition and engulfment of the pathogen by phagocytic cells (macrophages, dendritic cells), bacterial components (antigens) are processed into small fragments and presented to lymphocytes. Thymus-derived T-lymphocytes recognize antigens presented by antigen-presenting cells, such as macrophages, dendritic cells and B-lymphocytes. After antigen recognition, T-cells are activated and develop into cytokine-producing (CD4+) cells or cytolytic (CD8+) mediators. B-cells are directly activated by antigens. However, fully activation of B-cells may be coordinated by interactions with CD4+ T-cells, in the case of peptide antigens, or through thymus-independent, poorly described pathways for non-protein antigens, such as lipids. Figure is out of scale.

initiated. This process involves destruction of bacilli by professional phagocytes, and presentation of pathogen molecular fragments (antigens) to lymphocytes, followed by activation and proliferative steps. This response, also known as adaptive immunity, may give rise to either protection via the containment of the bacilli inside a granuloma, or an exacerbated inflammatory process associated to destructive pathology. In both cases, lymphocytes that specifically recognize their cognate antigen are activated, leading to the production of cytokines or antibodies, the destruction of infected cells and, the formation of memory lymphocyte clones. Since these processes involve mechanisms specifically originated during *M. tuberculosis* infection, the associated biomarkers have been largely

An overview of adaptive, cell-mediated responses to *M. tuberculosis* is schematized in Fig. 2. Adaptive responses take place when lymphocytes recognize mycobacterial molecules as

Fig. 2. Cell-mediated immunity against *M. tuberculosis* infection. After recognition and engulfment of the pathogen by phagocytic cells (macrophages, dendritic cells), bacterial components (antigens) are processed into small fragments and presented to lymphocytes. Thymus-derived T-lymphocytes recognize antigens presented by antigen-presenting cells, such as macrophages, dendritic cells and B-lymphocytes. After antigen recognition, T-cells are activated and develop into cytokine-producing (CD4+) cells or cytolytic (CD8+) mediators. B-cells are directly activated by antigens. However, fully activation of B-cells may be coordinated by interactions with CD4+ T-cells, in the case of peptide antigens, or through thymus-independent, poorly described pathways for non-protein antigens, such as

exploited for diagnostic purposes.

lipids. Figure is out of scale.

foreign entities (as antigens). Two lymphocyte families, the T and B-cells, are activated after encountering with their matching antigen, then develop into the effectors of adaptive immunity. For T cells, presentation of foreign entities by antigen-presenting cells is mandatory for recognition. In contrast, B cells recognize their cognate antigens through direct interaction via the B-cell receptor. Once activated, B and T cells trigger a variety of functions, mainly including: (a) secretion of cytokines and chemoattractants by CD4+ helper T-cells, (b) lysis of infected cells via the release of lytic enzymes by CD8+ cytotoxic T-cells, (c) secretion of antibodies by plasma B-cells that have been derived from activated Blymphocytes, (d) production of a number of long-lived memory T- and B-cells, which last for many years circulating in the bloodstream and monitor for infection (either newly developed or endogenously re-activated).

As depicted in Figure 2, some responses related to cell-mediated immunity are restricted to active infection. One illustrative example is the release of some pro-inflammatory cytokines by T-cells, such as interferon-gamma. The secretion of specific antibodies by plasma Blymphocytes also occurs during active or newly acquired TB, where antigen-presenting cells are charged with bacterial moieties. Some of the immune responses during active infection are known to be critical for arresting the growth of *M. tuberculosis*. In contrast, antibody secretion has been considered irrelevant, although some recent studies suggest a role for antibodies on the fate of *M. tuberculosis* infection (Glatman-Freedman et al, 2010). Yet, secretion of antibodies during bacterial growth (*i.e*. during active infection) can be exploited to detect infectious cases, thus contributing to stop TB transmission.

Isolated specific antigens have been used for detecting antibodies in human plasma, through the measurement of antigen-antibody reactions *in vitro*. Alternatively, antibodies may be produced in the lab and used to capture mycobacterial antigens that have been secreted to body fluids. Because free mycobacterial antigens are not encountered in many biological fluids, our work has been dedicated to the detection of antibodies. Importantly, antibodies associated to *M. tuberculosis* disease are found in sera from individuals affected by both pulmonary and extra-pulmonary TB, thus allowing the detection of difficult-to-detect TB pathologies (Daniel, 1989). Challenges related to the production of antigens for diagnostic tests will be presented in the following sections.

#### **4. Antigen repertoire of** *M. tuberculosis*

Specific activation of immune cells against *M. tuberculosis* occurs after the recognition of bacterial components as foreign entities. With about 4,000 genes (TubercuList web page), *M. tuberculosis* synthesizes a complex array of molecular products, mainly composed of proteins, lipids and carbohydrates. Box 1 summarizes the highlights of different antigens from the bacillus.

#### **5. Immunodiagnosis of TB: From Koch to POC tests**

A few years after Robert Koch discovered *M. tuberculosis*, his work was largely dedicated to look for a cure against TB disease, which was the major health threat in Europe by the time. His work allowed the description of *M. tuberculosis* extracts, obtained by glycerol extraction of liquid cultures of the bacilli (Kaufmann & Schaible 2005). Although this material, called tuberculin, was found unable to inhibit the growth of *M. tuberculosis* in guinea pigs or

Immune Diagnosis of Tuberculosis Through Novel Technologies 385

agglutination and electrophoretic immune precipitation techniques (Zykov et al 1966), to lab-on-chip formats, in which a set of laboratory procedures are automatically performed within a microfluidics technology-based chip (Schulte et al 2002). Contrasting to cellmediated responses, the assessment of antigen-antibody reactions does not require special conditions, such as regulated temperature or specific environment. In addition, no exposition to high level biological risk bacilli is involved to obtain plasma or serum samples. The search for antibodies in body fluids may be performed using a variety of technological platforms. Currently, Enzyme-Linked ImmunoSorbent Assay (ELISA) is the most common method for analyzing antigen-antibody reactions. The typical format used for screening antibodies in sera, an indirect ELISA, is schematized in Figure 3. ELISA has been widely used since the late 80's to screen for active TB (Daniel, 1989). Crude bacterial extracts, including tuberculin, represented the first bacterial materials explored as reagents. One of the most popular mixtures was Antigen-5, a preparation composed of various proteins and lipoarabinomannan, a specific cell-wall glycolipid (Daniel, T. M. et al, 1985). As modern Biochemistry tools have been developed, more purified, specific antigens have been obtained. Up to now, many proteins, post-translationally modified peptides, glycolipids and saccharides have been proposed as antigens for immunodiagnosis of active TB (Steingart et

Fig. 3. The Enzyme-Linked ImmunoSorbent Assay for the detection of antibodies. A selected antigen is used to coat a plate surface (1); a solution of non-reacting protein, such as bovine serum albumin, is used to block non-occupied sites at the plate surface, avoiding nonspecific binding between serum proteins and the plate (2); serum is added and antibodies bind to matching epitopes of immobilized antigens (3); non-reactive antibodies from sera are eliminated by washings (4); a secondary antibody linked to an enzyme is used to bind the primary, tuberculosis-associated antibody (5); the substrate of the enzyme is added allowing

to enzyme-dependent colorimetric changes, which are quantified through

al, 2009).

spectrophotometry (6).

#### *M. tuberculosis* **is a bacterial pathogen of atypical molecular composition:**


#### Box 1. **Antigens from** *Mycobacterium tuberculosis*

humans, Koch reported for the first time that previously-infected individuals developed a local inflammation at the site where tuberculin was injected, whereas healthy controls did not present such a response (Kaufmann & Schaible 2005). This hypersensitivity reaction was later related to the activation of memory T-cells, which are able to recognize tuberculincontaining antigens. Named the delayed-type hypersensitivity (DTH) test, the intradermal reaction to tuberculin constituted the first diagnostic tool based on the immune response against *M. tuberculosis*, and has been used for more than a century to diagnose latent TB (WHO 2006). During latent TB, memory T-lymphocytes may be searched through either DTH tests performed *in-vivo*, or more rapid, *in-vitro* cytokine analyses (Lalvani 2007, Hanekom et al, 2004). In any case, crude mixtures of mycobacterial antigens, such as tuberculin, often produce unspecific, false-positive results. In fact, *M. tuberculosis* shares a number of antigens with other microorganisms, including vaccine strains against TB. To circumvent this problem, the use of single or a small cocktail of antigens has been proposed. Two secreted protein antigens, ESAT-6 and CFP-10, are worth of mention since a high specificity and abundant T-cell responses have been associated to them (Fox et al, 2007). More recently, a non-protein, lipid antigen has also been proposed as a reagent to look for DTH responses (Komori et al 2011). Considering the lack of gene polymorphism of lipidpresenting molecules in humans (De Libero & Mori 2010), introduction of lipid antigens for diagnosing latent TB appears very promising.

In contrast to memory T-cell responses, production of antibodies requires the presence of plasma circulating B-lymphocytes, a phenomenon associated to active infection (see Figure 2). Therefore, the search for antigen-antibody reactions has been largely explored to diagnose active TB. Such a reaction may be measured in a wide set of platforms, from old

 Protein molecular patterns include stress-inducible proteins of wide distribution within bacteria, enzymes presenting homology with many other human pathogens (bacteria and parasites), and newly described or putative gene products devoid of homology vis-à-vis any other peptide annotated so far (16% of *M. tuberculosis* open

 Cellular wall is unusually thick, conferring unique properties of tinction (*M. tuberculosis* is not Gram+ nor Gram-) and atypical antibiotic susceptibility patterns. Genes related to lipid metabolism are especially abundant (5-fold more genes in *M. tuberculosis* genome compared to that of *Escherichia coli*). In accordance, the Koch bacillus produces lipids with amazing structural features and sizes (fatty acids up to 80 carbons length). Many of these lipids are recognized as antigens during active TB. Carbohydrate products have distinctive structures. Polysaccharides and glycoconjugates (glycolipids, glycoproteins) comprise molecular motifs recognized by innate immune cells and important virulence factors. In contrast to findings in model animals, sera from

*M. tuberculosis* **is a bacterial pathogen of atypical molecular composition:** 

*M. tuberculosis*-infected humans strongly recognize sugar structures.

humans, Koch reported for the first time that previously-infected individuals developed a local inflammation at the site where tuberculin was injected, whereas healthy controls did not present such a response (Kaufmann & Schaible 2005). This hypersensitivity reaction was later related to the activation of memory T-cells, which are able to recognize tuberculincontaining antigens. Named the delayed-type hypersensitivity (DTH) test, the intradermal reaction to tuberculin constituted the first diagnostic tool based on the immune response against *M. tuberculosis*, and has been used for more than a century to diagnose latent TB (WHO 2006). During latent TB, memory T-lymphocytes may be searched through either DTH tests performed *in-vivo*, or more rapid, *in-vitro* cytokine analyses (Lalvani 2007, Hanekom et al, 2004). In any case, crude mixtures of mycobacterial antigens, such as tuberculin, often produce unspecific, false-positive results. In fact, *M. tuberculosis* shares a number of antigens with other microorganisms, including vaccine strains against TB. To circumvent this problem, the use of single or a small cocktail of antigens has been proposed. Two secreted protein antigens, ESAT-6 and CFP-10, are worth of mention since a high specificity and abundant T-cell responses have been associated to them (Fox et al, 2007). More recently, a non-protein, lipid antigen has also been proposed as a reagent to look for DTH responses (Komori et al 2011). Considering the lack of gene polymorphism of lipidpresenting molecules in humans (De Libero & Mori 2010), introduction of lipid antigens for

 Shared with other bacteria, some small phosphorus-containing compounds, phosphoantigens, constitute a separate group of antigens in *M. tuberculosis*. Phosphoantigens activate a set of unusual T-cells and possess interesting therapeutic

In contrast to memory T-cell responses, production of antibodies requires the presence of plasma circulating B-lymphocytes, a phenomenon associated to active infection (see Figure 2). Therefore, the search for antigen-antibody reactions has been largely explored to diagnose active TB. Such a reaction may be measured in a wide set of platforms, from old

Box 1. **Antigens from** *Mycobacterium tuberculosis* 

effects against specific lymphomas.

reading frames are novel sequences).

diagnosing latent TB appears very promising.

agglutination and electrophoretic immune precipitation techniques (Zykov et al 1966), to lab-on-chip formats, in which a set of laboratory procedures are automatically performed within a microfluidics technology-based chip (Schulte et al 2002). Contrasting to cellmediated responses, the assessment of antigen-antibody reactions does not require special conditions, such as regulated temperature or specific environment. In addition, no exposition to high level biological risk bacilli is involved to obtain plasma or serum samples.

The search for antibodies in body fluids may be performed using a variety of technological platforms. Currently, Enzyme-Linked ImmunoSorbent Assay (ELISA) is the most common method for analyzing antigen-antibody reactions. The typical format used for screening antibodies in sera, an indirect ELISA, is schematized in Figure 3. ELISA has been widely used since the late 80's to screen for active TB (Daniel, 1989). Crude bacterial extracts, including tuberculin, represented the first bacterial materials explored as reagents. One of the most popular mixtures was Antigen-5, a preparation composed of various proteins and lipoarabinomannan, a specific cell-wall glycolipid (Daniel, T. M. et al, 1985). As modern Biochemistry tools have been developed, more purified, specific antigens have been obtained. Up to now, many proteins, post-translationally modified peptides, glycolipids and saccharides have been proposed as antigens for immunodiagnosis of active TB (Steingart et al, 2009).

Fig. 3. The Enzyme-Linked ImmunoSorbent Assay for the detection of antibodies. A selected antigen is used to coat a plate surface (1); a solution of non-reacting protein, such as bovine serum albumin, is used to block non-occupied sites at the plate surface, avoiding nonspecific binding between serum proteins and the plate (2); serum is added and antibodies bind to matching epitopes of immobilized antigens (3); non-reactive antibodies from sera are eliminated by washings (4); a secondary antibody linked to an enzyme is used to bind the primary, tuberculosis-associated antibody (5); the substrate of the enzyme is added allowing to enzyme-dependent colorimetric changes, which are quantified through spectrophotometry (6).

Immune Diagnosis of Tuberculosis Through Novel Technologies 387

**6.2 The structural nature of various sensitive, specific antigens is not addressable via** 

According to serodiagnostic analyses performed in TB endemic settings (including ours), antigens able to achieve high diagnostics performances represent mycobacterial components non-addressable by genetic engineering. Some of the highest specificities and sensitivities have been found using glycolipids. These are fatty acyl-containing carbohydrate, secondary genetic products, whose biosynthesis involves multiple enzymatic steps, some of them still undefined. Lipoarabinomannan (LAM), di-*O*-acyl trehalose (DAT) and cord factor (CF) belong to this group of difficult-to-obtain highly performance antigens (Barihuta et al, 1993; Escamilla et al, 1996; López-Marín et al, 2003; Maekura et al, 1993; Julián et al, 2001; Simonney et al, 1996). Besides, some of the best protein antigens to diagnose active TB are post-translationally modified products, namely glycosylated proteins such as the 38-kDa, antigen (Espitia et al, 1989). Worth noting, the best diagnostic performances obtained with this protein have been found when it has been obtained from glycosylating mycobacterial cells. In contrast, a non-glycosylated 38-kDa antigen obtained by recombinant technology in

The isolation of glycosylated antigens involves cumbersome steps, making difficult their use in large-scale applications. The involvement of complex, methyl-containing fatty acyl structures in antibody recognition may limit the use of synthetic approaches for these antigens. In view of the structural similarity of glycolipids from *M. tuberculosis* and some other mycobacterial species, we looked for surrogate sources of glycolipids as antigens for TB diagnosis. Structural and serologic studies of glycosylated lipids from mycobacteria allowed the identification of *Mycobacterium fortuitum*, a species of rapidly growing nontuberculous mycobacteria, as surrogate source for two promising antigens: di-*O*-acyl trehalose (DAT) (Escamilla et al, 1996), and cord factor (López-Marín et al, 2003). Interestingly, *M. fortuitum* synthesizes abundant quantities of DAT and cord factor. The antigens afford specific reactivities vis-à-vis healthy controls and patients infected with other pathogenic actinomycetes (López-Marín et al, 2003). In addition, glycolipids from *M. fortuitum* are not longer recognized by individuals with healed TB and do not present cross-

Glycosylation has been detected as a critical factor for antibody recognition during active TB. For instance, the ability of antigens from *M. tuberculosis* to bind antibodies in sera from infected people is strongly decreased after periodate treatments, indicating that antibodies predominantly react with carbohydrate determinants (Udaykumar & Saxena, 1991). Sugar antigens in *M. tuberculosis* comprise protein and lipid glycoconjugates. At present, recombinant production of mycobacterial sugars is unfeasible, since biosynthetic pathways are still poorly described. A few decades ago, phage-display based technologies emerged as a powerful method to look for structurally diverse unknown ligands. Through recombinant technology, *E. coli* phages are modified to obtain combinatorial peptides libraries displayed on the virion surface (Smith, 1985). Through this technology, phage displayed peptides with affinity to any ligand can be identified by in-vitro screening (Figure 4). Using a phage

*Escherichia coli* has shown poor efficiencies (Gennaro, 2000).

**6.2.1 Surrogate microbial sources of secondary-genetic products** 

reactivities with vaccinated healthy controls (Escamilla et al, 1996).

**6.2.2 Peptide mimicry and combinatorial strategies** 

**recombinant technology** 

The development of ELISA represented a breakthrough for the detection of antigenantibody responses. Before enzyme-linked tests, cumbersome and/or hazardous methods, including radioimmunoassays (RIA), importantly limited the application of immune diagnostic tests for large populations (Lequin, 2005). Yet, an ELISA presents important limitations for large-scale applications. Major drawbacks associated to this technique include: the need of large amounts of samples, a multistep procedure poorly suitable for high throughput scales, and high costs related to the use of enzymatic markers, specialized equipment and trained personnel. Recently, the development of more friendly, equipmentfree technologies has been largely explored, importantly including the search for miniaturized analytical devices. In particular, the integration of currently existing detection methods, such as those based on antigen-antibody responses, into microdevices represents a promise for bringing diagnostics closer to those who need it. The introduction of microtechnologies may afford automated systems for the detection of TB at the site of patient care. Such devices have been called Point-Of-Care (POC) tests.

#### **6. Current challenges for the development of POC tests for TB diagnosis**

TB is considered one of the most complex diseases ever established in humankind. Active TB has been related to multiple processes but not to any single pathogenic or host factor. Therefore, the demand of technological innovations for the development of POC tests is accompanied by a particular challenge regarding the selection of bioreagents. In the following paragraphs, we enumerate the major current limitations and possible technological solutions for the development of POC tests to diagnose active TB.

#### **6.1 The heterogeneous human response to** *M. tuberculosis* **antigens**

Up to now, few systematic studies to address the antibody response during tuberculosis in humans have been performed. The presence of different antibody secretion patterns in humans has been largely observed. HLA genes encoding for molecules presenting protein antigens, namely the Major Histocompatibility Complex (MHC) molecules, are known to be highly polymorphic in humans, and some data indicate this gene polymorphism as a source of variability to recognize peptide motifs by immune cells (Bothamley et al, 1989). On another side, the spectrum of responses at different stage of the disease may account for important variability. In this way, protein antigens that had proved high sensitivities in some trials, have given unsatisfactory results when tested in a different setting (Gennaro, 2000). The use of species-specific antigens, not present in mycobacteria other than *M. tuberculosis,* is known to be required to avoid false-positive results, and antigens synthesized by vaccine strains are also precluded. According to a meta-analysis reported by Steingart et al. (2009), antibody detection methods could achieve high efficiencies only if a mixture of multiple antigens is used. Most analyzed trials failed, however, to include appropriate healthy controls, thus limiting the results in regard to test specificity. Therefore, in spite of the need to perform better diagnostic trials including appropriate controls, a conclusive remark is the convenience to use a cocktail of antigens. With this in mind, antibodies associated to active TB seem largely elicited by unusual, difficult to obtain mycobacterial antigens.

The development of ELISA represented a breakthrough for the detection of antigenantibody responses. Before enzyme-linked tests, cumbersome and/or hazardous methods, including radioimmunoassays (RIA), importantly limited the application of immune diagnostic tests for large populations (Lequin, 2005). Yet, an ELISA presents important limitations for large-scale applications. Major drawbacks associated to this technique include: the need of large amounts of samples, a multistep procedure poorly suitable for high throughput scales, and high costs related to the use of enzymatic markers, specialized equipment and trained personnel. Recently, the development of more friendly, equipmentfree technologies has been largely explored, importantly including the search for miniaturized analytical devices. In particular, the integration of currently existing detection methods, such as those based on antigen-antibody responses, into microdevices represents a promise for bringing diagnostics closer to those who need it. The introduction of microtechnologies may afford automated systems for the detection of TB at the site of

patient care. Such devices have been called Point-Of-Care (POC) tests.

**6. Current challenges for the development of POC tests for TB diagnosis** 

technological solutions for the development of POC tests to diagnose active TB.

**6.1 The heterogeneous human response to** *M. tuberculosis* **antigens** 

antigens.

TB is considered one of the most complex diseases ever established in humankind. Active TB has been related to multiple processes but not to any single pathogenic or host factor. Therefore, the demand of technological innovations for the development of POC tests is accompanied by a particular challenge regarding the selection of bioreagents. In the following paragraphs, we enumerate the major current limitations and possible

Up to now, few systematic studies to address the antibody response during tuberculosis in humans have been performed. The presence of different antibody secretion patterns in humans has been largely observed. HLA genes encoding for molecules presenting protein antigens, namely the Major Histocompatibility Complex (MHC) molecules, are known to be highly polymorphic in humans, and some data indicate this gene polymorphism as a source of variability to recognize peptide motifs by immune cells (Bothamley et al, 1989). On another side, the spectrum of responses at different stage of the disease may account for important variability. In this way, protein antigens that had proved high sensitivities in some trials, have given unsatisfactory results when tested in a different setting (Gennaro, 2000). The use of species-specific antigens, not present in mycobacteria other than *M. tuberculosis,* is known to be required to avoid false-positive results, and antigens synthesized by vaccine strains are also precluded. According to a meta-analysis reported by Steingart et al. (2009), antibody detection methods could achieve high efficiencies only if a mixture of multiple antigens is used. Most analyzed trials failed, however, to include appropriate healthy controls, thus limiting the results in regard to test specificity. Therefore, in spite of the need to perform better diagnostic trials including appropriate controls, a conclusive remark is the convenience to use a cocktail of antigens. With this in mind, antibodies associated to active TB seem largely elicited by unusual, difficult to obtain mycobacterial

#### **6.2 The structural nature of various sensitive, specific antigens is not addressable via recombinant technology**

According to serodiagnostic analyses performed in TB endemic settings (including ours), antigens able to achieve high diagnostics performances represent mycobacterial components non-addressable by genetic engineering. Some of the highest specificities and sensitivities have been found using glycolipids. These are fatty acyl-containing carbohydrate, secondary genetic products, whose biosynthesis involves multiple enzymatic steps, some of them still undefined. Lipoarabinomannan (LAM), di-*O*-acyl trehalose (DAT) and cord factor (CF) belong to this group of difficult-to-obtain highly performance antigens (Barihuta et al, 1993; Escamilla et al, 1996; López-Marín et al, 2003; Maekura et al, 1993; Julián et al, 2001; Simonney et al, 1996). Besides, some of the best protein antigens to diagnose active TB are post-translationally modified products, namely glycosylated proteins such as the 38-kDa, antigen (Espitia et al, 1989). Worth noting, the best diagnostic performances obtained with this protein have been found when it has been obtained from glycosylating mycobacterial cells. In contrast, a non-glycosylated 38-kDa antigen obtained by recombinant technology in *Escherichia coli* has shown poor efficiencies (Gennaro, 2000).

#### **6.2.1 Surrogate microbial sources of secondary-genetic products**

The isolation of glycosylated antigens involves cumbersome steps, making difficult their use in large-scale applications. The involvement of complex, methyl-containing fatty acyl structures in antibody recognition may limit the use of synthetic approaches for these antigens. In view of the structural similarity of glycolipids from *M. tuberculosis* and some other mycobacterial species, we looked for surrogate sources of glycolipids as antigens for TB diagnosis. Structural and serologic studies of glycosylated lipids from mycobacteria allowed the identification of *Mycobacterium fortuitum*, a species of rapidly growing nontuberculous mycobacteria, as surrogate source for two promising antigens: di-*O*-acyl trehalose (DAT) (Escamilla et al, 1996), and cord factor (López-Marín et al, 2003). Interestingly, *M. fortuitum* synthesizes abundant quantities of DAT and cord factor. The antigens afford specific reactivities vis-à-vis healthy controls and patients infected with other pathogenic actinomycetes (López-Marín et al, 2003). In addition, glycolipids from *M. fortuitum* are not longer recognized by individuals with healed TB and do not present crossreactivities with vaccinated healthy controls (Escamilla et al, 1996).

#### **6.2.2 Peptide mimicry and combinatorial strategies**

Glycosylation has been detected as a critical factor for antibody recognition during active TB. For instance, the ability of antigens from *M. tuberculosis* to bind antibodies in sera from infected people is strongly decreased after periodate treatments, indicating that antibodies predominantly react with carbohydrate determinants (Udaykumar & Saxena, 1991). Sugar antigens in *M. tuberculosis* comprise protein and lipid glycoconjugates. At present, recombinant production of mycobacterial sugars is unfeasible, since biosynthetic pathways are still poorly described. A few decades ago, phage-display based technologies emerged as a powerful method to look for structurally diverse unknown ligands. Through recombinant technology, *E. coli* phages are modified to obtain combinatorial peptides libraries displayed on the virion surface (Smith, 1985). Through this technology, phage displayed peptides with affinity to any ligand can be identified by in-vitro screening (Figure 4). Using a phage

Immune Diagnosis of Tuberculosis Through Novel Technologies 389

structures are distinct from mammalian carbohydrates. Thus, expression of *M. tuberculosis* glycoproteins in other actinomycetes, such as rapidly growing *Mycobacterium smegmatis*  (Garbe et al, 1993) and *Streptomyces* (Lara et al, 2003) appears as an interesting tool towards

Screening tests to detect TB are essential to overcome the epidemic. In particular POC tests may be implemented at lower levels of health services, contributing to stop TB transmission. The use of microfluidic technologies seem of special interest since they are associated to small volumes of samples. For instance, POC tests have been already developed to monitor biomarkers in a few blood drops. Two platforms for the study of antigen-antibody interactions are described below, immunochromatography tests and microchip-based

One of the most advantageous platforms for the study of antigen-antibody reactions is lateral-flow immunochromatography. The basis of this method is schematized in Figure 5. Immunochromatography tests enable added value to antigen-antibody reactions since they allow higher throughput, reduced volume of samples, as well as lower costs than traditional immunoassays, since no specialized equipment or skilled personnel are necessary. Although this format has been used in many endemic settings, antigen evaluations vis-à-vis healthy controls or related pathologies require further studies. To our knowledge, highly specific glycolipid antigens, hydrophobic in nature, have not been included in

Based on resistant but flexible silicon platforms constructed through microchip-based technologies, MicroElectroMechanical Systems (MEMS) are micrometric devices which include mechanical parts, such as actuators, sensors or integrated microfluidic systems. MEMS have been widely used in aerospace and automotive industry. Examples of some popular MEMS are microaccelerometers in crash air-bag systems or micromirrors for projection systems. Some of the most attractive features in MEMS are reliability and lowcost, which are associated to their large batch processing. Interestingly, a MEMS is able to perform automated analyses, including transport, separations, chemical reactions and sensing. All necessary instruments can be integrated in a single device, the MEMS, so that new terms, micro Total Analysis Systems (μTAS), and "Lab-On-a-Chip" (LOC) technologies have been coined for such instruments. Over the course of the past fifteen years, MEMS have also been explored for a set of biomedical applications, including metabolite analyses, drug testing, drug discovery, combinatorial assays for DNA screening and, obviously, immunodiagnosis (Hedlund, 2009). Figure 6 shows a scanning electron micrography of a MEMS. A detailed description of MEMS is out of the scope of this chapter. However, we enumerate the key features allowing this technology to be a promising tool for the

**6.3 Currently available platforms for POC immunodiagnostics** 

**6.3.1 Lateral-flow immunochromatography tests** 

**6.3.2 Micro-Electro-Mechanical Systems (MEMS)** 

development of ASSURED tests for diagnosis (Box 2).

the application of serodiagnostics.

immunochromatography formats.

devices.

displayed dodecapeptide library, we have selected phages with specific binding to a serum directed towards *M. tuberculosis* carbohydrate antigens (Gevorkian et al, 2005). This approach resulted in the identification of peptides that mimic mannose-containing molecules of *M. tuberculosis*. A set of peptides were readily recognized by antibodies raised against mycobacterial sugars. More surprisingly, one of these peptides induced, in rabbits, the production of antibodies recognizing mannan. More recently, Bua and coworkers used this technology to obtain phages as useful reagents for the serodiagnosis of tuberculosis (Bua et al, 2009).

Fig. 4. Phage Display technology for the identification of biological reagents. Combinatorial peptide libraries displayed in virion surfaces can be obtained through genetic engineering. These libraries have proved to be useful for the identification of antibody ligands, even for those associated to poorly defined antigens. In this method, antigens are immobilized on a plate (1) and incubated in the presence of phage suspension (2); non bound phages are removed by washings (3), and selected phage clones are obtained through pH-mediated elution (4); finally, selected clones are amplified (5). The selection cycle is usually repeated 3 times.

Oligopeptides have been found to mimic a set of 3D structures, including sugars, linear and conformational peptide epitopes. Therefore, phage display represents a promising technology for the identification of peptides able to replace difficult-to-obtain or previously unknown antigens. At present, our work is focused to find optimal conditions in order to use peptide mimotopes as immunodiagnostic reagents.

#### **6.2.3 New vectors for the recombinant production of post-translational modified antigens**

A different approach to get reagents for diagnosis of TB has been the production of glycosylated proteins of *M. tuberculosis* in novel expression systems. Traditionally, recombinant mycobacterial proteins have been produced in *E. coli*. Mycobacterial glycosyl

displayed dodecapeptide library, we have selected phages with specific binding to a serum directed towards *M. tuberculosis* carbohydrate antigens (Gevorkian et al, 2005). This approach resulted in the identification of peptides that mimic mannose-containing molecules of *M. tuberculosis*. A set of peptides were readily recognized by antibodies raised against mycobacterial sugars. More surprisingly, one of these peptides induced, in rabbits, the production of antibodies recognizing mannan. More recently, Bua and coworkers used this technology to obtain phages as useful reagents for the serodiagnosis of tuberculosis

Fig. 4. Phage Display technology for the identification of biological reagents. Combinatorial peptide libraries displayed in virion surfaces can be obtained through genetic engineering. These libraries have proved to be useful for the identification of antibody ligands, even for those associated to poorly defined antigens. In this method, antigens are immobilized on a plate (1) and incubated in the presence of phage suspension (2); non bound phages are removed by washings (3), and selected phage clones are obtained through pH-mediated elution (4); finally, selected clones are amplified (5). The selection cycle is usually repeated

Oligopeptides have been found to mimic a set of 3D structures, including sugars, linear and conformational peptide epitopes. Therefore, phage display represents a promising technology for the identification of peptides able to replace difficult-to-obtain or previously unknown antigens. At present, our work is focused to find optimal conditions in order to

**6.2.3 New vectors for the recombinant production of post-translational modified** 

A different approach to get reagents for diagnosis of TB has been the production of glycosylated proteins of *M. tuberculosis* in novel expression systems. Traditionally, recombinant mycobacterial proteins have been produced in *E. coli*. Mycobacterial glycosyl

use peptide mimotopes as immunodiagnostic reagents.

(Bua et al, 2009).

3 times.

**antigens** 

structures are distinct from mammalian carbohydrates. Thus, expression of *M. tuberculosis* glycoproteins in other actinomycetes, such as rapidly growing *Mycobacterium smegmatis*  (Garbe et al, 1993) and *Streptomyces* (Lara et al, 2003) appears as an interesting tool towards the application of serodiagnostics.

#### **6.3 Currently available platforms for POC immunodiagnostics**

Screening tests to detect TB are essential to overcome the epidemic. In particular POC tests may be implemented at lower levels of health services, contributing to stop TB transmission. The use of microfluidic technologies seem of special interest since they are associated to small volumes of samples. For instance, POC tests have been already developed to monitor biomarkers in a few blood drops. Two platforms for the study of antigen-antibody interactions are described below, immunochromatography tests and microchip-based devices.

#### **6.3.1 Lateral-flow immunochromatography tests**

One of the most advantageous platforms for the study of antigen-antibody reactions is lateral-flow immunochromatography. The basis of this method is schematized in Figure 5. Immunochromatography tests enable added value to antigen-antibody reactions since they allow higher throughput, reduced volume of samples, as well as lower costs than traditional immunoassays, since no specialized equipment or skilled personnel are necessary. Although this format has been used in many endemic settings, antigen evaluations vis-à-vis healthy controls or related pathologies require further studies. To our knowledge, highly specific glycolipid antigens, hydrophobic in nature, have not been included in immunochromatography formats.

#### **6.3.2 Micro-Electro-Mechanical Systems (MEMS)**

Based on resistant but flexible silicon platforms constructed through microchip-based technologies, MicroElectroMechanical Systems (MEMS) are micrometric devices which include mechanical parts, such as actuators, sensors or integrated microfluidic systems. MEMS have been widely used in aerospace and automotive industry. Examples of some popular MEMS are microaccelerometers in crash air-bag systems or micromirrors for projection systems. Some of the most attractive features in MEMS are reliability and lowcost, which are associated to their large batch processing. Interestingly, a MEMS is able to perform automated analyses, including transport, separations, chemical reactions and sensing. All necessary instruments can be integrated in a single device, the MEMS, so that new terms, micro Total Analysis Systems (μTAS), and "Lab-On-a-Chip" (LOC) technologies have been coined for such instruments. Over the course of the past fifteen years, MEMS have also been explored for a set of biomedical applications, including metabolite analyses, drug testing, drug discovery, combinatorial assays for DNA screening and, obviously, immunodiagnosis (Hedlund, 2009). Figure 6 shows a scanning electron micrography of a MEMS. A detailed description of MEMS is out of the scope of this chapter. However, we enumerate the key features allowing this technology to be a promising tool for the development of ASSURED tests for diagnosis (Box 2).

Immune Diagnosis of Tuberculosis Through Novel Technologies 391

Including mechanical and optical parts, such as actuators, motors or sensors, MEMS

A variety of materials in MEMS technology, including some new advanced materials

 Some developments for biomedical applications already follow clinical tests for their introduction to the market. These include bioMEMS to sense glucose during diabetes

MEMS are machines with sizes ranging from a micrometer to a few milimetres

Batch processing determines very low-costs for an individual machine.

Metal surfaces able to immobilize a set of molecules, by keeping bioactivity

Fig. 7. Label-free measurement systems in MEMS include cantilevers. If antigens are immobilized onto a quartz crystal, fine mass changes, as those produced by antibody capture, will result in measurable alterations in the quartz resonance frequency, an optical property that can be precisely detected combining the use of a laser microbeam and a

A rapid and affordable test for detecting contagious TB patients is the cornerstone of current strategies for TB control. Although a set of clever methods has been proposed for this objective, the search for new tools adaptable to low-resource endemic settings is still a demand. The search of antigen-antibody responses for diagnosing active TB represents a promising alternative, since the associated methods may fulfill the major diagnostic requirements, namely minimal handling, rapidity and adaptability to point-of-care formats. Future success of immunodiagnosis tools for detecting TB will depend on both basic and technological advances: (1) The identification of specific, affordable biological reagents for large scale production (antigens, antibodies or fragments) and (2) The development of immunological tests into low-cost, friendly formats. According to different studies

Box 2. MicroElectroMechanical Systems (MEMS) at a glance

make possible the application of MEMS in Biomedicine

are automated Lab-On-Chip machines

or antibodies levels to detect dengue

photodetector

**7. Conclusion** 

Fig. 5. Immunochromatography platform for assessment of antigen-antibody reactions. Immunochromatography strips contain a sample pad, in which biological fluids are deposited. Capillarity induces a lateral flow of the sample throughout the pad. Frequently, nanocolloidal gold particles are functionalized for conjugation to total antibodies. Antigen immobilized as a line in the nitrocellulose strip captures the antibody-colloidal gold complex. A control line reacting with colloidal gold is also present. After developing, the test can be read since purple lines develop if colloidal gold is hold.

To illustrate the suitability of MEMS technology for clinical POC tests, a good example is the possibility to sense biological markers in a sample using label-free technologies. In MEMS, physical properties, such as conductivity or mass changes, are usually analyzed through electric or optical low-cost systems (Battiston et al, 2001; Fischer, 2011). For immune diagnostic systems, the capture of antibodies by antigen-displaying surfaces will result in changes of mass or conductivity parameters, both of them addressable through MEMS standard technologies (Figure 7).

Fig. 6. Panoramic image (a) and scanning electron micrographs (b, c) of a MicroElectroMechanical System (MEMS). Constructed using microchip technology, such as lithography on silica supports, MEMS also contain mechanical components, including beams, gears, diaphragms, grooves, orifices, springs or suspensions and optical systems. Three dimensional fabrication processes allow the design of automated systems able to reproduce any laboratory procedure.

Fig. 5. Immunochromatography platform for assessment of antigen-antibody reactions. Immunochromatography strips contain a sample pad, in which biological fluids are deposited. Capillarity induces a lateral flow of the sample throughout the pad. Frequently, nanocolloidal gold particles are functionalized for conjugation to total antibodies. Antigen immobilized as a line in the nitrocellulose strip captures the antibody-colloidal gold complex. A control line reacting with colloidal gold is also present. After developing, the

To illustrate the suitability of MEMS technology for clinical POC tests, a good example is the possibility to sense biological markers in a sample using label-free technologies. In MEMS, physical properties, such as conductivity or mass changes, are usually analyzed through electric or optical low-cost systems (Battiston et al, 2001; Fischer, 2011). For immune diagnostic systems, the capture of antibodies by antigen-displaying surfaces will result in changes of mass or conductivity parameters, both of them addressable through MEMS

test can be read since purple lines develop if colloidal gold is hold.

Fig. 6. Panoramic image (a) and scanning electron micrographs (b, c) of a

MicroElectroMechanical System (MEMS). Constructed using microchip technology, such as lithography on silica supports, MEMS also contain mechanical components, including beams, gears, diaphragms, grooves, orifices, springs or suspensions and optical systems. Three dimensional fabrication processes allow the design of automated systems able to

standard technologies (Figure 7).

reproduce any laboratory procedure.


Box 2. MicroElectroMechanical Systems (MEMS) at a glance

Fig. 7. Label-free measurement systems in MEMS include cantilevers. If antigens are immobilized onto a quartz crystal, fine mass changes, as those produced by antibody capture, will result in measurable alterations in the quartz resonance frequency, an optical property that can be precisely detected combining the use of a laser microbeam and a photodetector

#### **7. Conclusion**

A rapid and affordable test for detecting contagious TB patients is the cornerstone of current strategies for TB control. Although a set of clever methods has been proposed for this objective, the search for new tools adaptable to low-resource endemic settings is still a demand. The search of antigen-antibody responses for diagnosing active TB represents a promising alternative, since the associated methods may fulfill the major diagnostic requirements, namely minimal handling, rapidity and adaptability to point-of-care formats. Future success of immunodiagnosis tools for detecting TB will depend on both basic and technological advances: (1) The identification of specific, affordable biological reagents for large scale production (antigens, antibodies or fragments) and (2) The development of immunological tests into low-cost, friendly formats. According to different studies

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addressing antigen-antibody responses in different populations, the search for antibodies in TB patients must include specific peptides, but also non-protein (lipid) and posttranslational modified antigens, such as glycosylated proteins. In this regard, strategies to obtain complex antigens are critical. The development of novel expression systems and phage-display technologies could be the answer. Finally, we herein suggest that, in addition to further improvements of the already explored immunochromatography strips for TB diagnosis, Micro-ElectroMechanical Systems (MEMS) deserve a particular attention to develop better POC tests for diagnosing active, but also to look for tests addressing the detection of inactive, latent TB.

#### **8. Acknowledgments**

This work has been supported by Grant IA102611 from Universidad Nacional Autónoma de México (IACOD-DGAPA). We thank the Micro and Nanotechnology Department, ITESI (Guanajuato, Mexico) for providing MEMS devices for Figure 6. Alicia del Real performed SEM analyses of MEMS.

#### **9. References**


addressing antigen-antibody responses in different populations, the search for antibodies in TB patients must include specific peptides, but also non-protein (lipid) and posttranslational modified antigens, such as glycosylated proteins. In this regard, strategies to obtain complex antigens are critical. The development of novel expression systems and phage-display technologies could be the answer. Finally, we herein suggest that, in addition to further improvements of the already explored immunochromatography strips for TB diagnosis, Micro-ElectroMechanical Systems (MEMS) deserve a particular attention to develop better POC tests for diagnosing active, but also to look for tests addressing the

This work has been supported by Grant IA102611 from Universidad Nacional Autónoma de México (IACOD-DGAPA). We thank the Micro and Nanotechnology Department, ITESI (Guanajuato, Mexico) for providing MEMS devices for Figure 6. Alicia del Real performed

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SEM analyses of MEMS.

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**19** 

*USA* 

*1Colorado State University 2University of Notre Dame* 

**Exosomes: New Tuberculosis Biomarkers –** 

Biomarkers, derived from either the host (e.g. immunological markers, such as cytokines) or infectious agent (e.g. exported products, such as lipoarabinomannan), are indicative not only of disease but also of disease stage, severity and drug failure. Discovering new biomarkers from easily attainable bodily fluids is essential if we are to control tuberculosis, a diseases that kills 1.5 to 2 million individuals a year. The ideal biomarker could be used in the diagnosis and prognosis of disease from any suspect patient regardless of age, immune status or vaccination history. One potential biomarker source is exosomes; small membranebound vesicles released from cells which can be found circulating in the blood, and can be readily harvested for diagnostic testing. Exosomes shed from *Mycobacterium tuberculosis* (*Mtb*) infected samples (from *in vitro* produced to *in vivo* models and clinical samples) could provide an ideal reservoir of tuberculosis biomarkers. While it is possible to detect and monitor host and other bacterial components on exosomes, the scope of the following experiments are focused specifically on the *Mtb* proteins that are incorporated into

New diagnostic measures for the detection of tuberculosis are needed to identify and distinguish individuals with active tuberculosis from those harboring the dormant *Mtb* bacillus. The established method of TB detection for over seventy years is the tuberculin skin test (TST) or Mantoux test. This approach monitors the presence and diameter of induration as an indicator of the delayed-type hypersensitivity reaction to an intradermal injection of purified protein derivative (PPD) of *Mtb*. Despite its worldwide use, there are several fundamental problems with the TST. First, while it can be used as an indicator of both the active disease and the asymptomatic, latent tuberculosis infection (LTBI), the test cannot differentiate between the two. Second, false positive results are frequent, most commonly due to a history of Bacille Calmette Guérin (BCG) vaccination. Similarly, due to the dominance of several conserved heat-shock protein in PPD, and their influence on the antigenicity of PPD (Yang, Troudt et al.), there is frequent cross-reactivity between infections with nontuberculosis mycobacterium (NTM) species (such as *M. avium*). Most

**1. Introduction** 

exosomes.

**1.1 Detection of tuberculosis** 

**Prospects From the Bench to the Clinic** 

Nicole A. Kruh-Garcia1, Jeff S. Schorey2 and Karen M. Dobos1


## **Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic**

Nicole A. Kruh-Garcia1, Jeff S. Schorey2 and Karen M. Dobos1 *1Colorado State University 2University of Notre Dame USA* 

#### **1. Introduction**

394 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Lalvani, A. (2007). Diagnosing Tuberculosis Infection in the 21st Century: New Tools to

Lara, M., Servín-González, L., Singh, M., Moreno, C., Cohen, I., Nimtz, M., Espitia, C. (2004).

*Microbiology*, Vol.70, No.2, (February 2004), pp. 679-685, ISSN 1098-5336. López-Marín, L.M., Segura, E., Hermida-Escobedo, C., Lemassu, A., Salinas-Carmona, M.C.

*Microbiology*, Vol.36, No.1-2, (May 2003), pp. 47-54, ISSN 1574-695X. Mabey, D., Peeling, R.W., Ustianowski, A., Perkins, M.D. (2004). Diagnostics for the

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Expression, Secretion, and Glycosylation of the 45- and 47-kDa Glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans. *Applied and Environmental* 

(2003). 6,6'-dimycoloyl trehalose from a rapidly growing Mycobacterium: an alternative antigen for tuberculosis serodiagnosis. *FEMS Immunology and Medical* 

Developing World. *Nature Reviews in Microbiology*, Vol.2, No.3, (March 2004), pp.

Yano, S., He, H., Oka, S., Kashima, K., Yano, I. (1993). Clinical evaluation of rapid serodiagnosis of pulmonary tuberculosis by ELISA with cord factor (trehalose 6,6' dimycolate) as antigen purified from Mycobacterium tuberculosis. *American Review of Respiratory Diseases*, Vol.148, Pt.1, (October 1993), pp. 997-1001, ISSN0003-0805 Russell, D.G., Barry, C.E. 3rd, Flynn, J.L. (2010). Tuberculosis: What we don't know can, and does, hurt us. *Science*, Vol.328, No.5980*,* (May 2010), pp. 852-855, ISSN 1095-9203. Schulte, T.H., Bardell, R.L., Weigl, B.H. (2002). Microfluidic technologies in clinical

diagnostics, *Clinica Chimica Acta*, Vol.321, No.1-2, (July 2002), pp. 1-10, ISSN 0009-

antigens on the virion surface. *Science*, Vol.228, No. 4705, (June 1985), pp. 1315-1317,

A., Pai, M., Laal, S. (2009). Performance of Purified Antigens for Serodiagnosis of Pulmonary Tuberculosis: a Meta-Analysis. *Clinical and Vaccine Immunology*, Vol.16,

Comparison of A60 and three glycolipid antigens in an ELISA test for tuberculosis. *Clinical Microbiology and Infections*, Vol.2, No.3 (February 1996), pp. 214-222, ISSN

recognized by antibodies in tuberculosis and mouse antisera. *FEMS Microbiology* 

Trial of the Kaolin-Agglutination Test (KAT) for Detection of Tuberculosis Antibodies. *Bulletin of the World Health Organization*, Vol.35, No.4, pp. 581-592, ISSN

Smith, G. P. (1985).Filamentous fusion phage: novel expression vectors that display cloned

Steingart, K. R., Dendukuri, N., Henry, M., Schiller, I., Nahid, P., Hopewell, P. C., Ramsay,

Simonney, N., Molina, J.M., Molimard, M., Oksenhendler, E., Lagrange, P.H. (1996).

The New Diagnostics Working Group of the Stop TB Partnership, WHO (2009). *Pathways to better diagnostics for tuberculosis,* WHO, ISBN 9778 92 4 159881 1, Switzerland. Udaykumar, Saxena, R.K. (1991). Antigenic epitopes on Mycobacterium tuberculosis

*and Immunology*, Vol.3, No.1, (February 1991), pp. 7-12, ISSN 0920-8534. World Health Organization/Tropical Diseases (2006). *Diagnostics for Tuberculosis. Global Demand and Market Potential,* WHO, ISBN 978 92 4 156330 7, Switzerland. Zykov, M.P., Geser, A., Egsmose, T. et al. (1966). A serological test of tuberculosis. A "Blind"

No.2, (February 2009), pp. 260-276, ISSN 1556-6811.

Maekura, R., Nakagawa, M., Nakamura, Y., Hiraga, T., Yamamura, Y., Ito, M., Ueda, E.,

Biomarkers, derived from either the host (e.g. immunological markers, such as cytokines) or infectious agent (e.g. exported products, such as lipoarabinomannan), are indicative not only of disease but also of disease stage, severity and drug failure. Discovering new biomarkers from easily attainable bodily fluids is essential if we are to control tuberculosis, a diseases that kills 1.5 to 2 million individuals a year. The ideal biomarker could be used in the diagnosis and prognosis of disease from any suspect patient regardless of age, immune status or vaccination history. One potential biomarker source is exosomes; small membranebound vesicles released from cells which can be found circulating in the blood, and can be readily harvested for diagnostic testing. Exosomes shed from *Mycobacterium tuberculosis* (*Mtb*) infected samples (from *in vitro* produced to *in vivo* models and clinical samples) could provide an ideal reservoir of tuberculosis biomarkers. While it is possible to detect and monitor host and other bacterial components on exosomes, the scope of the following experiments are focused specifically on the *Mtb* proteins that are incorporated into exosomes.

#### **1.1 Detection of tuberculosis**

New diagnostic measures for the detection of tuberculosis are needed to identify and distinguish individuals with active tuberculosis from those harboring the dormant *Mtb* bacillus. The established method of TB detection for over seventy years is the tuberculin skin test (TST) or Mantoux test. This approach monitors the presence and diameter of induration as an indicator of the delayed-type hypersensitivity reaction to an intradermal injection of purified protein derivative (PPD) of *Mtb*. Despite its worldwide use, there are several fundamental problems with the TST. First, while it can be used as an indicator of both the active disease and the asymptomatic, latent tuberculosis infection (LTBI), the test cannot differentiate between the two. Second, false positive results are frequent, most commonly due to a history of Bacille Calmette Guérin (BCG) vaccination. Similarly, due to the dominance of several conserved heat-shock protein in PPD, and their influence on the antigenicity of PPD (Yang, Troudt et al.), there is frequent cross-reactivity between infections with nontuberculosis mycobacterium (NTM) species (such as *M. avium*). Most

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic 397

with case detection rates (CDR) of only 63% (even lower in Africa) of pulmonary and other forms of the disease which is primarily due to undiagnosed and unreported cases (WHO 2010). Of those individuals newly infected with *Mtb*, greater than one-tenth involved coinfection with HIV. Uncovering LTBI in at-risk individuals (e.g.. HIV positive, household contacts, those on immunosuppressive drugs) is critical so that the proper medical treatments can be administered to prevent future activation of the disease. Expeditious detection of smear negative cases, of which there were an estimated 2 million instances

There are two major classes of biomarkers – host response and pathogen generated. The majority of the research being conducted focuses on host biomarkers – including INF- (as measured through the IGRA kits), other immunological markers (e.g.. cytokines, like IL-6 and TNF), host protein profiling, as well as, the production of antibodies to dominant *Mtb* antigens (such as, malate synthase and MPT51 (Wanchu, Dong et al. 2008)) (Walzl, Ronacher et al.). One of the primary concerns with using immune response as biomarkers of disease is the variation observed among the patient population due to numerous factors including secondary disease (causing increased/decreased inflammation) and altered response due to drug therapy. Alternatively, products made and released by *Mtb* during infection can also serve as biomarkers. Bacterial molecules including DNA (Cannas, Goletti et al. 2008) and lipoarabinomannan (LAM) (Minion, Leung et al.) can both be detected in urine during infection. Unlike most immunological biomarkers, bacterial products are specific to the tuberculosis. Moreover, identification and quantification of multiple *Mtb* products secreted/released during infection has the potential to be developed into a multiplex assay. Further, by monitoring changes in a panel of biomolecules, one could generate a fingerprint that could be used to indicate the different stages of disease (such as active versus latent).

To identify a combination of novel biomarkers for the generation of a point-of-care diagnostic test, we have begun screening human exosomes as a source of *Mtb* proteins. Preliminary data has proven that these vesicles are an untapped supply of pathogen derived biomarkers. There are several advantages to using exosomes: first, they can be isolated in a facile and expeditious manner. Second, they can be harvested from blood and urine – which is beneficial when sputum collection is not feasible (from children or in MDR cases). Third, our data indicates that *Mtb* proteins are not only contained, but also concentrated in these vesicles – allowing for greater sensitivity than can be achieved using unpurified whole

Exosomes were first described in mid-1980's by Johnstone *et al.* and Stahl *et al*. in reference to the small vesicles that bud from reticulocytes during maturation (Harding, Heuser et al. 1984; Johnstone, Adam et al. 1987). It was hypothesized that this process, was required to remove membrane-bound proteins such as the transferrin receptor from the maturing reticulocyte as these cells lack the lysosomes for protein degradation (Pan and Johnstone 1983). In a series of elegant electron microscopy studies, it was determined that the transferrin receptor (TR) was endocytosed and trafficked to the MVB where it was observed

(WHO 2010), could potentially allow for treatment 4-6 weeks sooner.

**1.2 Identifying diagnostic biomarkers of TB** 

blood or urine.

**1.3 Exosomes: Initial discoveries** 

disconcerting are false negative readings typically associated with impaired immune function (attributed to infections with HIV or with drug use).

The development of the interferon-gamma release assay (IGRA) has addressed some of the problems associated with the TST. Two types of IGRA test are currently commercially available, QuantiFERON-TB (Cellestis Limited) and T-SPOT-TB (Oxford Immunotec). Both measure the host biomarker, interferon-gamma (INF-) in whole-blood after stimulation with the mycobacterial antigens ESAT-6 and CFP10, by either ELISA or ELISpot assay, respectively. The T-SPOT-TB appears to be the most comprehensive diagnostic test to date (Table 1). Lastly, the Xpert MTB/RIF assay (Cephid) is a nucleic acid amplification based diagnostic and has the advantage of identifying resistance to rifampicin. However, since none of the aforementioned tests can distinguish between active and latent infections, sputum collection followed by microscopy (AFB smear) and culture, as well as chest radiography must be performed. The AFB smear test is simple, cost-effective and achieves high specificity; however this test suffers from a lack of sensitivity as on average it only detects 50% of active TB patients and even less in HIV positive patients and children.


1Provided that sputum can be obtained.

2This method can discriminate between most NTM (exceptions are *M. gordonae*, *M. kansasii*, *M. szulgai*  and *M. marinum*); but it cannot distinguish between other members of the *Mtb* complex (*M. bovis*, *M. africanum*, *M. microti* and *M. canetti*). 3Data is limited.

Table 1. Comparison of a sampling of tuberculosis detection methods.

The ideal diagnostic would minimize false positives due to BCG vaccination and infections with NTM, eliminate false negatives associated with the immunocompromised and be able to distinguish between active and latent infections while permitting increased case detection via a robust, cheap, fast and sensitive assay for its use in high-burden, low-income countries. According to the World Health Organization (WHO), there were 9.4 million new cases of active tuberculosis (TB) with a prevalence of 14 million cases in 2009, which accounted for about 1.7 million deaths (WHO 2010). A serious issue is the inaccuracy of these estimations, with case detection rates (CDR) of only 63% (even lower in Africa) of pulmonary and other forms of the disease which is primarily due to undiagnosed and unreported cases (WHO 2010). Of those individuals newly infected with *Mtb*, greater than one-tenth involved coinfection with HIV. Uncovering LTBI in at-risk individuals (e.g.. HIV positive, household contacts, those on immunosuppressive drugs) is critical so that the proper medical treatments can be administered to prevent future activation of the disease. Expeditious detection of smear negative cases, of which there were an estimated 2 million instances (WHO 2010), could potentially allow for treatment 4-6 weeks sooner.

#### **1.2 Identifying diagnostic biomarkers of TB**

396 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

disconcerting are false negative readings typically associated with impaired immune

The development of the interferon-gamma release assay (IGRA) has addressed some of the problems associated with the TST. Two types of IGRA test are currently commercially available, QuantiFERON-TB (Cellestis Limited) and T-SPOT-TB (Oxford Immunotec). Both measure the host biomarker, interferon-gamma (INF-) in whole-blood after stimulation with the mycobacterial antigens ESAT-6 and CFP10, by either ELISA or ELISpot assay, respectively. The T-SPOT-TB appears to be the most comprehensive diagnostic test to date (Table 1). Lastly, the Xpert MTB/RIF assay (Cephid) is a nucleic acid amplification based diagnostic and has the advantage of identifying resistance to rifampicin. However, since none of the aforementioned tests can distinguish between active and latent infections, sputum collection followed by microscopy (AFB smear) and culture, as well as chest radiography must be performed. The AFB smear test is simple, cost-effective and achieves high specificity; however this test suffers from a lack of sensitivity as on average it only

detects 50% of active TB patients and even less in HIV positive patients and children.

AFB Smear Yes No Yes Yes No Yes1

culture Yes No Yes Yes Yes Yes1 TST Yes Yes No No No No

FERON Yes Yes No **Yes** Yes 2 3 T-SPOT-TB Yes Yes No **Yes** Yes 2 **Yes** 

MTB/RIF Yes Yes 3 **Yes Yes Yes** 

TB ELISA Yes No Yes **No Yes** 

2This method can discriminate between most NTM (exceptions are *M. gordonae*, *M. kansasii*, *M. szulgai*  and *M. marinum*); but it cannot distinguish between other members of the *Mtb* complex (*M. bovis*, *M.* 

The ideal diagnostic would minimize false positives due to BCG vaccination and infections with NTM, eliminate false negatives associated with the immunocompromised and be able to distinguish between active and latent infections while permitting increased case detection via a robust, cheap, fast and sensitive assay for its use in high-burden, low-income countries. According to the World Health Organization (WHO), there were 9.4 million new cases of active tuberculosis (TB) with a prevalence of 14 million cases in 2009, which accounted for about 1.7 million deaths (WHO 2010). A serious issue is the inaccuracy of these estimations,

Table 1. Comparison of a sampling of tuberculosis detection methods.

**Distinguishes between BCG vaccination and** *Mtb*  **infection** 

**Distinguishes between** *Mtb* **and NTM infection** 

**Reliable results in immunecompromised patients?** 

**Distinguishes between active and latent TB** 

function (attributed to infections with HIV or with drug use).

Test

AFB

Quanti-

Xpert

Clearview

**Detects active TB** 

1Provided that sputum can be obtained.

*africanum*, *M. microti* and *M. canetti*). 3Data is limited.

**Detects latent TB** 

There are two major classes of biomarkers – host response and pathogen generated. The majority of the research being conducted focuses on host biomarkers – including INF- (as measured through the IGRA kits), other immunological markers (e.g.. cytokines, like IL-6 and TNF), host protein profiling, as well as, the production of antibodies to dominant *Mtb* antigens (such as, malate synthase and MPT51 (Wanchu, Dong et al. 2008)) (Walzl, Ronacher et al.). One of the primary concerns with using immune response as biomarkers of disease is the variation observed among the patient population due to numerous factors including secondary disease (causing increased/decreased inflammation) and altered response due to drug therapy. Alternatively, products made and released by *Mtb* during infection can also serve as biomarkers. Bacterial molecules including DNA (Cannas, Goletti et al. 2008) and lipoarabinomannan (LAM) (Minion, Leung et al.) can both be detected in urine during infection. Unlike most immunological biomarkers, bacterial products are specific to the tuberculosis. Moreover, identification and quantification of multiple *Mtb* products secreted/released during infection has the potential to be developed into a multiplex assay. Further, by monitoring changes in a panel of biomolecules, one could generate a fingerprint that could be used to indicate the different stages of disease (such as active versus latent).

To identify a combination of novel biomarkers for the generation of a point-of-care diagnostic test, we have begun screening human exosomes as a source of *Mtb* proteins. Preliminary data has proven that these vesicles are an untapped supply of pathogen derived biomarkers. There are several advantages to using exosomes: first, they can be isolated in a facile and expeditious manner. Second, they can be harvested from blood and urine – which is beneficial when sputum collection is not feasible (from children or in MDR cases). Third, our data indicates that *Mtb* proteins are not only contained, but also concentrated in these vesicles – allowing for greater sensitivity than can be achieved using unpurified whole blood or urine.

#### **1.3 Exosomes: Initial discoveries**

Exosomes were first described in mid-1980's by Johnstone *et al.* and Stahl *et al*. in reference to the small vesicles that bud from reticulocytes during maturation (Harding, Heuser et al. 1984; Johnstone, Adam et al. 1987). It was hypothesized that this process, was required to remove membrane-bound proteins such as the transferrin receptor from the maturing reticulocyte as these cells lack the lysosomes for protein degradation (Pan and Johnstone 1983). In a series of elegant electron microscopy studies, it was determined that the transferrin receptor (TR) was endocytosed and trafficked to the MVB where it was observed

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic 399

blood has been key to biomarker discovery in several diseases (Bard, Hegmans et al. 2004; Mears, Craven et al. 2004; Thomas, Sexton et al. 2010). However, the mining of exosomederived proteins related to infectious diseases has yet to be exploited. The discovery that mycobacterial products could be found in exosomes release from infected animals led to the first full-scale attempt to characterize the *Mtb* exo-proteome (comprehensive collection of proteins capable of being secreted from *Mtb*-infected cells within exosomes) (Giri, Kruh et al. 2010). The objective of this research is to fuel biomarker discovery utilizing mass

Based on the hypothesis that exosomes are a novel source of bacterial components which could be exploited for biomarker identification, we set out to define the mycobacterial proteins contained in exosomes released from *Mtb*-infected macrophages. Exosomes were purified from *Mtb* infected macrophages using differential centrifugation and sucrose gradient (Griffiths, Heesom et al. 2007; Giri, Kruh et al. 2010). The exosomes contained the

 Fig. 1. Electron micrograph of exosomes purified from *Mtb* infected J774a.1 cells at (A) 3000x (bar = 0.5 m) and (B) 8000x magnification (bar = 0.1 m). The vesicles showed the size (30 –

Consistent with previous publications (Bhatnagar, Shinagawa et al. 2007), the host exosomal marker LAMP-1, as well as mycobacterial LAM and 19 kDa antigen (Rv3763) were detected via western blot (data not shown). Several known secreted proteins previously undetected in exosomes, including KatG (Rv1908c), the Ag85 complex (Rv3804c, Rv1886c and Rv0129c), GroES (Rv3418c) and CFP10 (Rv3874), were also demonstrated by western blot analysis (Giri, Kruh et al. 2010). In addition to these proteins, we identified roughly forty mycobacterial proteins by MS, of which 95% were previously defined either experimentally or through predictive algorithms to be secreted (Table 2) (Rosenkrands, King et al. 2000; Malen, Berven et al. 2007; Giri, Kruh et al. 2010). One can hypothesize that the mycobacterial

spectrometry based approaches.

**2. The** *Mtb* **exo-proteome:** *in vitro* 

characteristic size and shape as defined by EM (Figure 1).

100 nm) and spherical or cup-like shape expected of exosomes.

**2.1 Proteomics of exosomes released from** *Mtb***-infected macrophages** 

on ~50 nm interluminal vesicles. MVBs containing the TR were shown to fuse with the plasma membrane and release the interluminal vesicles (Pan, Teng et al. 1985). These released vesicles were called exosomes.

Additional studies have shown that exosome release is not specific to maturing reticulocytes, as they are constitutively shed from most mammalian cells types including Bcells, macrophages and dendritic cells (Raposo, Nijman et al. 1996). Exosomes can be readily obtained from most bodily fluids, including but not limited to urine, plasma, breast milk, bronchoalveolar lavage (BAL) fluid and saliva (Admyre, Grunewald et al. 2003; Pisitkun, Shen et al. 2004; Caby, Lankar et al. 2005; Admyre, Johansson et al. 2007; Ogawa, Kanai-Azuma et al. 2008) Exosomes released from antigen presenting cells contain MHC-I and MHC-II as well as CD86 and therefore are capable of antigen presentation (Zitvogel, Fernandez et al. 1999). Interestingly, exosomes derived from tumor cells have also been shown to induce an anti-tumor immune response, implicating a future utility in vaccine development (Zitvogel, Regnault et al. 1998).

#### **1.4 The advent of exosomes in tuberculosis research**

The *Mtb* cell wall is composed of a myriad of proteins and lipids, including the highly studied glycolipid, LAM. This molecule has been shown to be readily liberated from the mycobacteria during infection and has been correlated with several immunomodulatory functions (such as interactions with TLRs (Bhatnagar and Schorey 2007)). Beatty et al was the first to recognize LAM accumulation in MVBs and this report is consistent with subsequent exosomal release from infected cells (Beatty, Rhoades et al. 2000). Building upon initial evidence, Schorey et al characterized exosomes released from BCG-infected macrophages. Flow cytometry allowed for the identification of vesicles positive for MHC-II, Hsp70, and LAMP1, as well as mycobacterial pathogen-associated molecular patterns (PAMPs) (Bhatnagar, Shinagawa et al. 2007; Giri and Schorey 2008). They went on to demonstrate the exosomes released from mycobacterial-infected macrophages could activate both the inate and acquired immune responses *in vitro* and *in vivo* (Blood, 2007 and PLoS One 2008). In addition, it was shown that exosomes isolated from the BAL fluid of *M. bovis BCG* infected mice contained LAM and the 19kDa antigen (Bhatnagar, Shinagawa et al. 2007).

#### **1.5 Concentration and siphoning of biomarkers in exosomes**

Exosome biology is a relatively new field with roughly 1000 primary journal articles and over 200 reviews (Raimondo, Morosi et al. 2011). Exosomes have been studied with techniques such as western blotting, microscopy, FACS and mass spectrometry. Characterization of exosomal contents, resulted in the identification of a number of host, as well as pathogen derived products, including: lipids (Laulagnier, Motta et al. 2004) and nucleic acids (miRNA and mRNA) (Valadi, Ekstrom et al. 2007), in addition to proteins. However, only a few of these studies utilize proteomics to decipher the contents of these vesicles and their relationship to various diseases, such as cancers, diabetes, prenatal disorders and renal diseases (Ben Ameur, Molina et al. 2010; Diaz, Pinto et al. 2011; Moon, You et al. 2011). Proteomics was performed on purified exosomes as early as 2004 (Bard, Hegmans et al. 2004). Defining the proteomic content of exosomes derived from urine and blood has been key to biomarker discovery in several diseases (Bard, Hegmans et al. 2004; Mears, Craven et al. 2004; Thomas, Sexton et al. 2010). However, the mining of exosomederived proteins related to infectious diseases has yet to be exploited. The discovery that mycobacterial products could be found in exosomes release from infected animals led to the first full-scale attempt to characterize the *Mtb* exo-proteome (comprehensive collection of proteins capable of being secreted from *Mtb*-infected cells within exosomes) (Giri, Kruh et al. 2010). The objective of this research is to fuel biomarker discovery utilizing mass spectrometry based approaches.

### **2. The** *Mtb* **exo-proteome:** *in vitro*

398 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

on ~50 nm interluminal vesicles. MVBs containing the TR were shown to fuse with the plasma membrane and release the interluminal vesicles (Pan, Teng et al. 1985). These

Additional studies have shown that exosome release is not specific to maturing reticulocytes, as they are constitutively shed from most mammalian cells types including Bcells, macrophages and dendritic cells (Raposo, Nijman et al. 1996). Exosomes can be readily obtained from most bodily fluids, including but not limited to urine, plasma, breast milk, bronchoalveolar lavage (BAL) fluid and saliva (Admyre, Grunewald et al. 2003; Pisitkun, Shen et al. 2004; Caby, Lankar et al. 2005; Admyre, Johansson et al. 2007; Ogawa, Kanai-Azuma et al. 2008) Exosomes released from antigen presenting cells contain MHC-I and MHC-II as well as CD86 and therefore are capable of antigen presentation (Zitvogel, Fernandez et al. 1999). Interestingly, exosomes derived from tumor cells have also been shown to induce an anti-tumor immune response, implicating a future utility in vaccine

The *Mtb* cell wall is composed of a myriad of proteins and lipids, including the highly studied glycolipid, LAM. This molecule has been shown to be readily liberated from the mycobacteria during infection and has been correlated with several immunomodulatory functions (such as interactions with TLRs (Bhatnagar and Schorey 2007)). Beatty et al was the first to recognize LAM accumulation in MVBs and this report is consistent with subsequent exosomal release from infected cells (Beatty, Rhoades et al. 2000). Building upon initial evidence, Schorey et al characterized exosomes released from BCG-infected macrophages. Flow cytometry allowed for the identification of vesicles positive for MHC-II, Hsp70, and LAMP1, as well as mycobacterial pathogen-associated molecular patterns (PAMPs) (Bhatnagar, Shinagawa et al. 2007; Giri and Schorey 2008). They went on to demonstrate the exosomes released from mycobacterial-infected macrophages could activate both the inate and acquired immune responses *in vitro* and *in vivo* (Blood, 2007 and PLoS One 2008). In addition, it was shown that exosomes isolated from the BAL fluid of *M. bovis BCG* infected mice contained LAM and the 19kDa antigen (Bhatnagar, Shinagawa et al.

Exosome biology is a relatively new field with roughly 1000 primary journal articles and over 200 reviews (Raimondo, Morosi et al. 2011). Exosomes have been studied with techniques such as western blotting, microscopy, FACS and mass spectrometry. Characterization of exosomal contents, resulted in the identification of a number of host, as well as pathogen derived products, including: lipids (Laulagnier, Motta et al. 2004) and nucleic acids (miRNA and mRNA) (Valadi, Ekstrom et al. 2007), in addition to proteins. However, only a few of these studies utilize proteomics to decipher the contents of these vesicles and their relationship to various diseases, such as cancers, diabetes, prenatal disorders and renal diseases (Ben Ameur, Molina et al. 2010; Diaz, Pinto et al. 2011; Moon, You et al. 2011). Proteomics was performed on purified exosomes as early as 2004 (Bard, Hegmans et al. 2004). Defining the proteomic content of exosomes derived from urine and

released vesicles were called exosomes.

development (Zitvogel, Regnault et al. 1998).

2007).

**1.4 The advent of exosomes in tuberculosis research** 

**1.5 Concentration and siphoning of biomarkers in exosomes** 

Based on the hypothesis that exosomes are a novel source of bacterial components which could be exploited for biomarker identification, we set out to define the mycobacterial proteins contained in exosomes released from *Mtb*-infected macrophages. Exosomes were purified from *Mtb* infected macrophages using differential centrifugation and sucrose gradient (Griffiths, Heesom et al. 2007; Giri, Kruh et al. 2010). The exosomes contained the characteristic size and shape as defined by EM (Figure 1).

Fig. 1. Electron micrograph of exosomes purified from *Mtb* infected J774a.1 cells at (A) 3000x (bar = 0.5 m) and (B) 8000x magnification (bar = 0.1 m). The vesicles showed the size (30 – 100 nm) and spherical or cup-like shape expected of exosomes.

#### **2.1 Proteomics of exosomes released from** *Mtb***-infected macrophages**

Consistent with previous publications (Bhatnagar, Shinagawa et al. 2007), the host exosomal marker LAMP-1, as well as mycobacterial LAM and 19 kDa antigen (Rv3763) were detected via western blot (data not shown). Several known secreted proteins previously undetected in exosomes, including KatG (Rv1908c), the Ag85 complex (Rv3804c, Rv1886c and Rv0129c), GroES (Rv3418c) and CFP10 (Rv3874), were also demonstrated by western blot analysis (Giri, Kruh et al. 2010). In addition to these proteins, we identified roughly forty mycobacterial proteins by MS, of which 95% were previously defined either experimentally or through predictive algorithms to be secreted (Table 2) (Rosenkrands, King et al. 2000; Malen, Berven et al. 2007; Giri, Kruh et al. 2010). One can hypothesize that the mycobacterial

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic 401

**Abundance (%)** 

 **Rv # Protein Name Relative** 

Rv2220 GlnA1 13.05 Rv1860 Mpt32/APA 12.34 Rv1886c Antigen 85-B 9.58 Rv3804c Antigen 85-A 8.92 Rv1980c Mpt64 6.89 Rv1908c KatG 4.43 Rv3418c GroES 3.59 Rv0934 PstS1 3.41 Rv3248c SahH 3.17 Rv0462 LpdC 2.87 Rv1926c Mpt63 2.46 Rv1355c MoeY 2.22 Rv2244 AcpM 2.16 Rv2031c HspX 1.86 Rv1932 Cfp20 1.80

Table 2. Sampling of proteins identified in exosomes from H37Rv-infected J774a.1 cells by LC-MS-MS. See Giri, Kruh et al. 2010, for the extended list. The fifteen most abundant mycobacterial proteins are listed, based on the total number of spectral counts from both insolution and in-gel processing (compiled in the aforementioned paper plus additional unpublished experiments). Relative abundance was calculated using the following formula: (total # of spectral counts for a given protein/cumulative total # of mycobacterial spectral

The primary analysis from *in vitro* derived exosomes, provides an extended candidate list for the study of *Mtb* biomarkers. Our next step in the biomarker discovery pipeline is to

Since tuberculosis is primarily a respiratory affliction, the initial *in vivo* investigation was performed utilizing exosomes isolated from BAL fluid to determine if mycobacterial proteins could be identified from an infected animal model. The hypothesis is that the protein composition of BAL fluid exosomes will reflect the initial *in vitro* findings and the proteins identified will change over the course of the infection. This may provide the starting point for making correlations between exosomal content and stage of infection. The end goal is find "peptide fingerprints" which represent the mycobacterial proteins

most commonly present in exosomes of patients with active or latent tuberculosis.

**3.1 Proteomics of exosomes released into the bronchoalveolar lavage fluid of** *Mtb***-**

BAL fluid was collected from mice infected via aerosol with either wild-type (H37Rv) or a *relA* deficient mutant of *Mtb*. Both the concentration and *Mtb* protein content of the exosomes were monitored over time. To accomplish this, we employed similar MS methodology as in our *in vitro* analysis (Giri, Kruh et al. 2010). MS analysis of BAL fluid

verify that these results translate to an *in vivo* system.

**3. The** *Mtb* **exo-proteome:** *in vivo*

counts) \* 100.

**infected mice** 

proteins released into the phagosome or cytoplasm are transported to an MVB, incorporated into the interluminal vesicles and released from the infected cell via exosomes into various bodily fluids (Figure 2). As indicated earlier, the exosomes containing both host and mycobacterial components can modulate the host immune response. Similar experiments were performed with irradiated (dead) *Mtb* and only 1 mycobacterial protein was identified. Whether this difference between live and dead *Mtb* was due to differential transport of the mycobacteria within the macrophage or a requirement for mycobacterial metabolic activity requires further investigation.

Fig. 2. Hypothesized pathway of exosome secretion in *Mtb*-infected cells. Based on experimental evidence, antigenic proteins are secreted within the *Mtb*-containing host compartment, packaged and shuttled into multivesicular bodies. The MVBs can fuse with the plasma membrane to release the exosomes into the peripheral fluids.

In addition, J774a.1 macrophages were exposed to culture filtrate proteins (CFP), a complex mixture of over 200 proteins secreted from *Mtb* during *in vitro* growth (Sonnenberg and Belisle 1997). Exosomes released during this experiment were collected and analyzed after in-gel digestion. The mycobacterial proteins identified from the CFP-treated exosomes included many well-characterized immunedominant antigens and were remarkably similar (>85%) to those isolated from J774a.1 cells infected with live *Mtb*. As previously mentioned, exosomes are being evaluated as tumor vaccines and the presence of highly antigenic mycobacterial proteins on exosomes from CFP-treated macrophages suggests that they too may be a viable vaccine candidates for TB. This hypothesis is presently under investigtion.

These aforementioned *in vitro* experiments afforded us with enough exosomal material to build our methodology and lay the foundation for the analysis of exosomes isolated from *Mtb*-infected mice, where samples more limited. In prior studies of exosomes, mycobacterial proteins were identified only by western blot. However, mass spectrometry allows for the identification of proteins without the use of antibodies (which are only available to a small subset of proteins). Moreover, while ~8 µg of exosomes is required for each western blot (identifying only one protein per blot), a parallel sample subject to MS analysis, allows for the discrimination of hundreds of proteins of both host and bacterial origin.

proteins released into the phagosome or cytoplasm are transported to an MVB, incorporated into the interluminal vesicles and released from the infected cell via exosomes into various bodily fluids (Figure 2). As indicated earlier, the exosomes containing both host and mycobacterial components can modulate the host immune response. Similar experiments were performed with irradiated (dead) *Mtb* and only 1 mycobacterial protein was identified. Whether this difference between live and dead *Mtb* was due to differential transport of the mycobacteria within the macrophage or a requirement for mycobacterial metabolic activity

Fig. 2. Hypothesized pathway of exosome secretion in *Mtb*-infected cells. Based on experimental evidence, antigenic proteins are secreted within the *Mtb*-containing host compartment, packaged and shuttled into multivesicular bodies. The MVBs can fuse with

In addition, J774a.1 macrophages were exposed to culture filtrate proteins (CFP), a complex mixture of over 200 proteins secreted from *Mtb* during *in vitro* growth (Sonnenberg and Belisle 1997). Exosomes released during this experiment were collected and analyzed after in-gel digestion. The mycobacterial proteins identified from the CFP-treated exosomes included many well-characterized immunedominant antigens and were remarkably similar (>85%) to those isolated from J774a.1 cells infected with live *Mtb*. As previously mentioned, exosomes are being evaluated as tumor vaccines and the presence of highly antigenic mycobacterial proteins on exosomes from CFP-treated macrophages suggests that they too may be a viable vaccine candidates for TB. This hypothesis is presently under investigtion. These aforementioned *in vitro* experiments afforded us with enough exosomal material to build our methodology and lay the foundation for the analysis of exosomes isolated from *Mtb*-infected mice, where samples more limited. In prior studies of exosomes, mycobacterial proteins were identified only by western blot. However, mass spectrometry allows for the identification of proteins without the use of antibodies (which are only available to a small subset of proteins). Moreover, while ~8 µg of exosomes is required for each western blot (identifying only one protein per blot), a parallel sample subject to MS analysis, allows for

the plasma membrane to release the exosomes into the peripheral fluids.

the discrimination of hundreds of proteins of both host and bacterial origin.

requires further investigation.


Table 2. Sampling of proteins identified in exosomes from H37Rv-infected J774a.1 cells by LC-MS-MS. See Giri, Kruh et al. 2010, for the extended list. The fifteen most abundant mycobacterial proteins are listed, based on the total number of spectral counts from both insolution and in-gel processing (compiled in the aforementioned paper plus additional unpublished experiments). Relative abundance was calculated using the following formula: (total # of spectral counts for a given protein/cumulative total # of mycobacterial spectral counts) \* 100.

The primary analysis from *in vitro* derived exosomes, provides an extended candidate list for the study of *Mtb* biomarkers. Our next step in the biomarker discovery pipeline is to verify that these results translate to an *in vivo* system.

#### **3. The** *Mtb* **exo-proteome:** *in vivo*

Since tuberculosis is primarily a respiratory affliction, the initial *in vivo* investigation was performed utilizing exosomes isolated from BAL fluid to determine if mycobacterial proteins could be identified from an infected animal model. The hypothesis is that the protein composition of BAL fluid exosomes will reflect the initial *in vitro* findings and the proteins identified will change over the course of the infection. This may provide the starting point for making correlations between exosomal content and stage of infection. The end goal is find "peptide fingerprints" which represent the mycobacterial proteins most commonly present in exosomes of patients with active or latent tuberculosis.

#### **3.1 Proteomics of exosomes released into the bronchoalveolar lavage fluid of** *Mtb***infected mice**

BAL fluid was collected from mice infected via aerosol with either wild-type (H37Rv) or a *relA* deficient mutant of *Mtb*. Both the concentration and *Mtb* protein content of the exosomes were monitored over time. To accomplish this, we employed similar MS methodology as in our *in vitro* analysis (Giri, Kruh et al. 2010). MS analysis of BAL fluid

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic 403

Fig. 4. CFUs versus *Mtb* proteins identified over time in (A) H37Rv *Mtb*-infected mice and (B) ∆RelA-infected mice. The red lines indicate the Log CFU in the lung over time, as indicated on the right y-axius. CFUs are representative of the bacterial load of the lungs of 5 mice per time point. The ble line represents the number of proteins identified by MS over

the course of the infection, as measured on the left y-axis.

derived exosomes from H37Rv *Mtb*-infected mice over the different time points revealed a significant overlap (50%) with the mycobacterial proteins previously identified in the *in vitro* tissue culture experiments (Figure 3). No mycobacterial proteins were identified in exosomes purified from BAL fluid on day 1 post-infection or as expected, in naïve mice. In all other time points, the number of mycobacterial spectral counts (data not shown) as well as the proteins identified changed over time (Figure 4). In exosomes from both H37Rv and the *relA* mutant infections, the largest number of proteins were identified early in the infection (day 14), however the number of proteins identified from the *relA* mutant was half that of the wt strain. Interestingly, during the wt infection, the exosomes that yielded the highest total mycobacterial spectral count was at day 56. Moreover, the exosomes from wtinfected mice showed on average three times as many spectral counts per time point than the *relA* mutant (data not shown). This is likely, at least in part, due to the differences in bacterial load between wt and ∆RelA-infected mice (10 to 100 fold difference in lung colony forming units at different time points). In both wt and ∆RelA-infected mice there were some proteins present on exosomes which were there throughout the course of disease, while others were transiently expressed.

Fig. 3. Venn diagram depicting the number of mycobacterial proteins identified in exosomes from H37Rv-infected macrophages (yellow), the BAL fluid of H37Rv-infected mice (blue), and both (overlap).

derived exosomes from H37Rv *Mtb*-infected mice over the different time points revealed a significant overlap (50%) with the mycobacterial proteins previously identified in the *in vitro* tissue culture experiments (Figure 3). No mycobacterial proteins were identified in exosomes purified from BAL fluid on day 1 post-infection or as expected, in naïve mice. In all other time points, the number of mycobacterial spectral counts (data not shown) as well as the proteins identified changed over time (Figure 4). In exosomes from both H37Rv and the *relA* mutant infections, the largest number of proteins were identified early in the infection (day 14), however the number of proteins identified from the *relA* mutant was half that of the wt strain. Interestingly, during the wt infection, the exosomes that yielded the highest total mycobacterial spectral count was at day 56. Moreover, the exosomes from wtinfected mice showed on average three times as many spectral counts per time point than the *relA* mutant (data not shown). This is likely, at least in part, due to the differences in bacterial load between wt and ∆RelA-infected mice (10 to 100 fold difference in lung colony forming units at different time points). In both wt and ∆RelA-infected mice there were some proteins present on exosomes which were there throughout the course of disease, while

Fig. 3. Venn diagram depicting the number of mycobacterial proteins identified in exosomes from H37Rv-infected macrophages (yellow), the BAL fluid of H37Rv-infected mice (blue),

others were transiently expressed.

and both (overlap).

Fig. 4. CFUs versus *Mtb* proteins identified over time in (A) H37Rv *Mtb*-infected mice and (B) ∆RelA-infected mice. The red lines indicate the Log CFU in the lung over time, as indicated on the right y-axius. CFUs are representative of the bacterial load of the lungs of 5 mice per time point. The ble line represents the number of proteins identified by MS over the course of the infection, as measured on the left y-axis.

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic 405

Fig. 5. Kinetics of various proteins during the course of the wild-type H37Rv (blue) compared to the *relA* mutant (red) infection, including GlnA, Antigen 85-A, HspX and Mpt63. These graphs emphasize the range of spectral counts and the pattern of individual protein expression. Transposon mutants effecting the expression of most of these proteins have led to the hypothesis that many of these proteins (HspX, Ag-85a, Mpt63) are "nonessential" (Sassetti, Boyd et al. 2003) – but their presence in exosomes is an indicator that their expression may be triggered during infection (HspX, as seen by transcipts (Hu,

Mangan et al. 2000; Dubnau, Fontan et al. 2002) and not in a simple culture.

Tuberculosis research in diagnostics has suffered from a lack of resources for the discovery and down-stream development of a cheap and facile clinical assay for the detection of active TB and to distinguish active from latent infection. This is due in part to the reliance on the TST; which for decades provided a cheap assay for detection of *Mtb* infection, and by our reliance on sputum smear tests to detect active disease. Most would argue that neither are optimal means to detect and diagnose TB nor evaluate drug treatment. However, in the absence of assays that could replace these tests without increases in cost, these remain the gold standard. The *Mtb* proteome has been mined for decades in pursuit of a serodiagnostic or celluar assay for tuberculosis, and the development and use of IGRA tests represent a hallmark of success. The outlook for a serodiagnostic assay appears more dismal, due to the requirement of either a predictable antibody response or high bacterial product shedding into the blood or BAL fluid. Our initial identification of *Mtb* proteins in BAL fluid exosomes

**4. Conclusions: Future of biomarkers in tuberculosis** 

While majority of the 15 most dominant mycobacterial proteins found in exosomes released from infected J774a.1 cells were seen in the mouse exosomes (Table 3), many of them were less abundant (e.g.. KatG, SahH and Cfp20) and several of these proteins were absent entirely (e.g. LpdC and MoeY). Most interesting are several striking differences in the mycobacterial protein identified on exosomes from BALfluid when mice were infected with wt or *relA* mutant. The *relA* gene is annotated as being a non-essential gene, based on viability of the strain subsequent to the insertion of the *Himar1* transposon (Lamichhane, Zignol et al. 2003). Biochemical characterization of the RelA protein suggests that this enzyme has both 3'-pyrophosphoryltransferase and pyrophosphohydrolase activities. Functional deletion of this protein results in the depletion of hyperphophorylated guanosine ((p)ppGpp), which can decrease protein synthesis leading to effects on long term growth and the ability to transition in and out of dormancy (Avarbock, Salem et al. 1999; Primm, Andersen et al. 2000; Klinkenberg, Lee et al. 2010). Dahl et al. has reported that the gene expression level of several secreted proteins (including the Antigen 85 complex and GroES) are increased in the *relA* mutant (Dahl, Kraus et al. 2003), however, based on spectral counts, the majority of the secreted proteins identified in exosomes display the opposite trend (Figure 5). While a more quantitative approach is necessary to truly hone in on the differences between the wt and *relA* mutant, this preliminary data suggest that there is a qualitative and quantitative difference in proteins targeted to MVB/exosomes when macrophages are infected with wt compared to *relA* mutant *in vivo*. Future experiments, including multiple reaction monitoring (MRM)-MS analysis will allow for the monitoring of the intensity of specific peptides in each sample, while allowing one to ignore the m/z's corresponding to host peptides.


Table 3. Comparison of the fifteen most abundant proteins originally identified in exosomes from H37Rv-infected J774a.1 cells (Table 2) to their presence (+) or absence (-) in BAL fluid exosomes at various time-points during the infection of Balc/c mice. + = protein detected in both H37Rv and *relA* mutant exosomes; \* = presence in H37Rv exosomes only; ∆ = only in *relA* mutant exosomes.

While majority of the 15 most dominant mycobacterial proteins found in exosomes released from infected J774a.1 cells were seen in the mouse exosomes (Table 3), many of them were less abundant (e.g.. KatG, SahH and Cfp20) and several of these proteins were absent entirely (e.g. LpdC and MoeY). Most interesting are several striking differences in the mycobacterial protein identified on exosomes from BALfluid when mice were infected with wt or *relA* mutant. The *relA* gene is annotated as being a non-essential gene, based on viability of the strain subsequent to the insertion of the *Himar1* transposon (Lamichhane, Zignol et al. 2003). Biochemical characterization of the RelA protein suggests that this enzyme has both 3'-pyrophosphoryltransferase and pyrophosphohydrolase activities. Functional deletion of this protein results in the depletion of hyperphophorylated guanosine ((p)ppGpp), which can decrease protein synthesis leading to effects on long term growth and the ability to transition in and out of dormancy (Avarbock, Salem et al. 1999; Primm, Andersen et al. 2000; Klinkenberg, Lee et al. 2010). Dahl et al. has reported that the gene expression level of several secreted proteins (including the Antigen 85 complex and GroES) are increased in the *relA* mutant (Dahl, Kraus et al. 2003), however, based on spectral counts, the majority of the secreted proteins identified in exosomes display the opposite trend (Figure 5). While a more quantitative approach is necessary to truly hone in on the differences between the wt and *relA* mutant, this preliminary data suggest that there is a qualitative and quantitative difference in proteins targeted to MVB/exosomes when macrophages are infected with wt compared to *relA* mutant *in vivo*. Future experiments, including multiple reaction monitoring (MRM)-MS analysis will allow for the monitoring of the intensity of specific peptides in each sample, while allowing one to ignore the m/z's

corresponding to host peptides.

*relA* mutant exosomes.

**Rv # Protein Name Day**

**14** 

Rv2220 GlnA1 + \* \* \* Rv1860 Mpt32/APA + \* \* ∆ Rv1886c Antigen 85-B + + + + Rv3804c Antigen 85-A + + + + Rv1980c Mpt64 + + + \* Rv1908c KatG \* \* \* - Rv3418c GroES + + \* \* Rv0934 PstS1 + + \* - Rv3248c SahH \* + \* - Rv0462 LpdC - - - - Rv1926c Mpt63 \* + \* - Rv1355c MoeY - - - - Rv2244 AcpM + + + ∆ Rv2031c HspX + + + \* Rv1932 Cfp20 \* \* \* - Table 3. Comparison of the fifteen most abundant proteins originally identified in exosomes from H37Rv-infected J774a.1 cells (Table 2) to their presence (+) or absence (-) in BAL fluid exosomes at various time-points during the infection of Balc/c mice. + = protein detected in both H37Rv and *relA* mutant exosomes; \* = presence in H37Rv exosomes only; ∆ = only in

**Day 28** 

**Day 56** 

**Day 112** 

Fig. 5. Kinetics of various proteins during the course of the wild-type H37Rv (blue) compared to the *relA* mutant (red) infection, including GlnA, Antigen 85-A, HspX and Mpt63. These graphs emphasize the range of spectral counts and the pattern of individual protein expression. Transposon mutants effecting the expression of most of these proteins have led to the hypothesis that many of these proteins (HspX, Ag-85a, Mpt63) are "nonessential" (Sassetti, Boyd et al. 2003) – but their presence in exosomes is an indicator that their expression may be triggered during infection (HspX, as seen by transcipts (Hu, Mangan et al. 2000; Dubnau, Fontan et al. 2002) and not in a simple culture.

#### **4. Conclusions: Future of biomarkers in tuberculosis**

Tuberculosis research in diagnostics has suffered from a lack of resources for the discovery and down-stream development of a cheap and facile clinical assay for the detection of active TB and to distinguish active from latent infection. This is due in part to the reliance on the TST; which for decades provided a cheap assay for detection of *Mtb* infection, and by our reliance on sputum smear tests to detect active disease. Most would argue that neither are optimal means to detect and diagnose TB nor evaluate drug treatment. However, in the absence of assays that could replace these tests without increases in cost, these remain the gold standard. The *Mtb* proteome has been mined for decades in pursuit of a serodiagnostic or celluar assay for tuberculosis, and the development and use of IGRA tests represent a hallmark of success. The outlook for a serodiagnostic assay appears more dismal, due to the requirement of either a predictable antibody response or high bacterial product shedding into the blood or BAL fluid. Our initial identification of *Mtb* proteins in BAL fluid exosomes

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic 407

The use of quantitative proteomics and MRM technology will be key in narrowing down the list of candidates. While this method is not necessarily suitable for the clinical lab (especially those in resource-limited areas) or is it cost effective – it is a mechanism to define biomarkers that can be implemented in a more cost effective diagnostic tool, such as an ELISA, or micro-

Another important consideration is the method used for exosome purification. At present the gold standard calls for differential centrifugation followed by sucrose gradient. Although this results is highly purified exosomes it is not suitable for high throughput analysis of serum/urine samples and cannot be used in the setting where TB diagnostics is most needed. Recently there have been a number of new purification techniques that have been developed include microfiltration using low protein-binding size exclusion filters, sizeexclusion chromatography, and microfluidics. ExoQuickTM, a recently developed commercial product from *System Biosciences,* allows for rapid isolation of exosomes based on a simple precipitation process and shows promise as a purification technique amenable to a

This work was supported through grants AI056979 and AI052439 (J. S. S.) and HHSN266200400091c (K.M.D) from the National Institute of Allergy and Infectious Diseases (NIAID). We would like to thank Pramod Giri for isolation of exosomes from mouse BAL fluid and EM photos. The antibodies to the various mycobacterial proteins as well as the *Mtb* culture filtrate proteins were provided from Colorado State University as part of NIH, NIAID Contract No. HHSN266200400091C, entitled ''Tuberculosis Vaccine Testing and Research Materials''. Mouse infections and BAL fluid collection were performed under William Bishai's laboratory at Johns Hopkins School of Medicine and funded under the "Tuberculosis Animal Research and Gene Evaluation Taskforce", NIH/NIAID N01-

AI30036. The authors graciously thank Lisa M. Wolfe for critique of this manuscript.

present in human breast milk." J Immunol 179(3): 1969-1978.

Admyre, C., J. Grunewald, et al. (2003). "Exosomes with major histocompatibility complex

Admyre, C., S. M. Johansson, et al. (2007). "Exosomes with immune modulatory features are

Anderson, L. and C. L. Hunter (2006). "Quantitative mass spectrometric multiple

Avarbock, D., J. Salem, et al. (1999). "Cloning and characterization of a bifunctional

Bard, M. P., J. P. Hegmans, et al. (2004). "Proteomic analysis of exosomes isolated from human malignant pleural effusions." Am J Respir Cell Mol Biol 31(1): 114-121.

class II and co-stimulatory molecules are present in human BAL fluid." Eur Respir J

reaction monitoring assays for major plasma proteins." Mol Cell Proteomics

RelA/SpoT homologue from Mycobacterium tuberculosis." Gene 233(1-2): 261-

fluidic "dip-test" that could be performed in a resource poor setting.

point-of-care diagnostics.

**5. Acknowledgements** 

**6. References** 

22(4): 578-583.

5(4): 573-588.

269.

was a stepping-stone in the process of novel discovery of resources for development of second-generation diagnostic and prognostic assays. While BAL fluid is not the most ideal fluid to collect from patients, its isolation from mice and the use of it in the discovery phase of biomarker identification is suitable if these *Mtb* exo-proteome findings translate to other bodily fluids. Exosomes can be isolated from urine and serum, both of which are easier to collect than sputum. Harvesting and characterization of sera and urine exosomes are commonplace in diseases such as cancer. Consistent with our findings in the mouse model, our preliminary evidence from a panel of five TB patients and five household contacts suggests that sera analyzed from patients with active disease contain on average over 3000 µg of exosomes per mL, which is over 30 times greater than the concentration of exosomes in the control sera. This makes exosomes an attractive target for pathogen-associated diagnostic biomarkers. Since many diagnostics are unreliable in immune compromised individuals, the allure of an exosome-based diagnostic will be enhanced if the exosomes undergo a similar rise in individuals co-infected with HIV.

Bodily fluids such as urine and blood are highly complex (with an estimate of >106 protein products in human plasma (Anderson and Hunter 2006)). The purification of exosomes from these samples reduces the amount of biocomponents resulting in a drastic enrichment. Numerous attempts have been made using MS to search for mycobacterial proteins in whole blood or urine. Unfortunately the results from these studies were not encouraging as even immunodepletion (with a MARS column) and various fractionation techniques (e.g. SCX and mRP on an HPLC), did not lead to the identification of a significant number of mycobacterial proteins in blood or urine. One major benefit to isolating exosomes from these fluids prior to proteomic studies is that these vesicles not only serve as a mechanism of concentrating the mycobacterial proteins, but they are also an effective method of removing major contaminants found in bodily fluids.

Urine is an established, readily attainable source of exosomes (Pisitkun, Shen et al. 2004). As previously mentioned, LAM (and mycobacterial DNA) has been found in urine of *Mtb*infected patients, including those with HIV. One kit currently on the market for the detection of LAM in urine is the Clearview TB ELISA (Inverness Medical Innovations Inc.). How LAM is secreted into the urine is questionable, however one can hypothesize that transport from the site of infection into the urine can occur through exosomes – either by encapsulation or incorporation into the lipid membrane of the vesicle. It is possible that LAM could be an additional marker in combination with *Mtb* proteins in a future clinical assay. Although at this point, like all proteins identified in exosomes, the concentration of LAM per volume of exosome would need to be quantified at various time-points during infection.

The discovery phase of identifying biomarkers from exosomes is in its infancy. While we have the potential to study fluctuations in exosomal host proteins too, deciphering these differences can be tricky due to additional changes in immune response and reactions to secondary infections or inflammatory disease. In the end, the best diagnostic may involve monitoring numerous proteins, perhaps both host and pathogen derived. The content of exosomes has the potential not only for use in diagnostics, but also additional predictive value in the classification of disease status, assessment of therapeutics and overall prognosis.

The use of quantitative proteomics and MRM technology will be key in narrowing down the list of candidates. While this method is not necessarily suitable for the clinical lab (especially those in resource-limited areas) or is it cost effective – it is a mechanism to define biomarkers that can be implemented in a more cost effective diagnostic tool, such as an ELISA, or microfluidic "dip-test" that could be performed in a resource poor setting.

Another important consideration is the method used for exosome purification. At present the gold standard calls for differential centrifugation followed by sucrose gradient. Although this results is highly purified exosomes it is not suitable for high throughput analysis of serum/urine samples and cannot be used in the setting where TB diagnostics is most needed. Recently there have been a number of new purification techniques that have been developed include microfiltration using low protein-binding size exclusion filters, sizeexclusion chromatography, and microfluidics. ExoQuickTM, a recently developed commercial product from *System Biosciences,* allows for rapid isolation of exosomes based on a simple precipitation process and shows promise as a purification technique amenable to a point-of-care diagnostics.

#### **5. Acknowledgements**

406 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

was a stepping-stone in the process of novel discovery of resources for development of second-generation diagnostic and prognostic assays. While BAL fluid is not the most ideal fluid to collect from patients, its isolation from mice and the use of it in the discovery phase of biomarker identification is suitable if these *Mtb* exo-proteome findings translate to other bodily fluids. Exosomes can be isolated from urine and serum, both of which are easier to collect than sputum. Harvesting and characterization of sera and urine exosomes are commonplace in diseases such as cancer. Consistent with our findings in the mouse model, our preliminary evidence from a panel of five TB patients and five household contacts suggests that sera analyzed from patients with active disease contain on average over 3000 µg of exosomes per mL, which is over 30 times greater than the concentration of exosomes in the control sera. This makes exosomes an attractive target for pathogen-associated diagnostic biomarkers. Since many diagnostics are unreliable in immune compromised individuals, the allure of an exosome-based diagnostic will be enhanced if the exosomes

Bodily fluids such as urine and blood are highly complex (with an estimate of >106 protein products in human plasma (Anderson and Hunter 2006)). The purification of exosomes from these samples reduces the amount of biocomponents resulting in a drastic enrichment. Numerous attempts have been made using MS to search for mycobacterial proteins in whole blood or urine. Unfortunately the results from these studies were not encouraging as even immunodepletion (with a MARS column) and various fractionation techniques (e.g. SCX and mRP on an HPLC), did not lead to the identification of a significant number of mycobacterial proteins in blood or urine. One major benefit to isolating exosomes from these fluids prior to proteomic studies is that these vesicles not only serve as a mechanism of concentrating the mycobacterial proteins, but they are also an effective method of removing

Urine is an established, readily attainable source of exosomes (Pisitkun, Shen et al. 2004). As previously mentioned, LAM (and mycobacterial DNA) has been found in urine of *Mtb*infected patients, including those with HIV. One kit currently on the market for the detection of LAM in urine is the Clearview TB ELISA (Inverness Medical Innovations Inc.). How LAM is secreted into the urine is questionable, however one can hypothesize that transport from the site of infection into the urine can occur through exosomes – either by encapsulation or incorporation into the lipid membrane of the vesicle. It is possible that LAM could be an additional marker in combination with *Mtb* proteins in a future clinical assay. Although at this point, like all proteins identified in exosomes, the concentration of LAM per volume of exosome would need to be quantified at various time-points during

The discovery phase of identifying biomarkers from exosomes is in its infancy. While we have the potential to study fluctuations in exosomal host proteins too, deciphering these differences can be tricky due to additional changes in immune response and reactions to secondary infections or inflammatory disease. In the end, the best diagnostic may involve monitoring numerous proteins, perhaps both host and pathogen derived. The content of exosomes has the potential not only for use in diagnostics, but also additional predictive value in the classification of disease status, assessment of therapeutics and overall

undergo a similar rise in individuals co-infected with HIV.

major contaminants found in bodily fluids.

infection.

prognosis.

This work was supported through grants AI056979 and AI052439 (J. S. S.) and HHSN266200400091c (K.M.D) from the National Institute of Allergy and Infectious Diseases (NIAID). We would like to thank Pramod Giri for isolation of exosomes from mouse BAL fluid and EM photos. The antibodies to the various mycobacterial proteins as well as the *Mtb* culture filtrate proteins were provided from Colorado State University as part of NIH, NIAID Contract No. HHSN266200400091C, entitled ''Tuberculosis Vaccine Testing and Research Materials''. Mouse infections and BAL fluid collection were performed under William Bishai's laboratory at Johns Hopkins School of Medicine and funded under the "Tuberculosis Animal Research and Gene Evaluation Taskforce", NIH/NIAID N01- AI30036. The authors graciously thank Lisa M. Wolfe for critique of this manuscript.

#### **6. References**


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**20** 

*Cuiabá/MT, Brasil* 

**Molecular Techniques for Identification of** 

**Complex: The use of Multiplex PCR and** 

**of** *Mycobacterium bovis* **and** 

Eduardo Eustáquio de Souza Figueiredo et al. \*

**Diagnosis of Bovine Tuberculosis** 

**Species of the** *Mycobacterium tuberculosis* 

**an Adapted HPLC Method for Identification** 

*Departamento de Alimentos e Nutrição, Universidade Federal de Mato Grosso,* 

*Mycobacterium bovis* is a member of the *M. tuberculosis* complex (MTC), a group of species (*Mycobacterium tuberculosis, M. bovis, M. africanum, M. microti*, and *M. canetti*) with a high genetic homology. *M. bovis* is the causative agent of tuberculosis in a range of animal species and humans, with worldwide annual losses to agriculture of \$3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. *M. bovis* was also the progenitor for the *M. bovis* bacillus Calmette–Guérin vaccine strain, the most widely used human vaccine. Garnier et al. (2003) described the 4,345,492-bp genome sequence of *M. bovis* AF2122/97 and compared it with the genomes of *Mycobacterium tuberculosis* and *Mycobacterium leprae*. Strikingly, the genome sequence of *M. bovis* is >99.95% identical to that of *M. tuberculosis*, but deletion of genetic information

Bovine tuberculosis (BTB) is a major infectious disease of cattle in many countries. Although cattle are the main host and reservoir of this chronic infection, other mammalian species, including humans, are also susceptible to *Mycobacterium bovis* (Romano et al., 1996). Considering that more than 94% of the world population lives in countries where the

Carlos Adam Conte Júnior2, Leone Vinícius Furlanetto3, Flávia Galindo Silvestre Silva4,

*2Faculdade de Veterinária, Universidade Federal Fluminense, Niterói/RJ, Brasil 3Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brasil 4Universidade de Cuiabá, Cuiabá/MT, Brasil* 

*5Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brasil 6Instituto Biomédico, Universidade Federal Fluminense, Niterói/RJ, Brasil* 

Rafael Silva Duarte5, Joab Trajano Silva3, Walter Lilenbaum6 and Vânia Margaret Flosi Paschoalin3\* *1Departamento de Alimentos e Nutrição, Universidade Federal de Mato Grosso, Cuiabá/MT, Brasil* 

**1. Introduction** 

has reduced the genome size.

 \*


## **Molecular Techniques for Identification of Species of the** *Mycobacterium tuberculosis*  **Complex: The use of Multiplex PCR and an Adapted HPLC Method for Identification of** *Mycobacterium bovis* **and Diagnosis of Bovine Tuberculosis**

Eduardo Eustáquio de Souza Figueiredo et al. \*

*Departamento de Alimentos e Nutrição, Universidade Federal de Mato Grosso, Cuiabá/MT, Brasil* 

#### **1. Introduction**

410 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

Wanchu, A., Y. Dong, et al. (2008). "Biomarkers for clinical and incipient tuberculosis:

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responses indistinguishable from that elicited by purified protein derivative in the guinea pig model of Mycobacterium tuberculosis infection." Infect Immun 79(2):

a novel cell-free vaccine: dendritic cell-derived exosomes." Nat Med 4(5): 594-

performance in a TB-endemic country." PLoS One 3(4): e2071.

WHO (2010). Global Tuberculosis Report, World Health Organization.

biotherapies of cancer." Eur J Cancer 35 Suppl 3: S36-38.

716-723.

600.

*Mycobacterium bovis* is a member of the *M. tuberculosis* complex (MTC), a group of species (*Mycobacterium tuberculosis, M. bovis, M. africanum, M. microti*, and *M. canetti*) with a high genetic homology. *M. bovis* is the causative agent of tuberculosis in a range of animal species and humans, with worldwide annual losses to agriculture of \$3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. *M. bovis* was also the progenitor for the *M. bovis* bacillus Calmette–Guérin vaccine strain, the most widely used human vaccine. Garnier et al. (2003) described the 4,345,492-bp genome sequence of *M. bovis* AF2122/97 and compared it with the genomes of *Mycobacterium tuberculosis* and *Mycobacterium leprae*. Strikingly, the genome sequence of *M. bovis* is >99.95% identical to that of *M. tuberculosis*, but deletion of genetic information has reduced the genome size.

Bovine tuberculosis (BTB) is a major infectious disease of cattle in many countries. Although cattle are the main host and reservoir of this chronic infection, other mammalian species, including humans, are also susceptible to *Mycobacterium bovis* (Romano et al., 1996). Considering that more than 94% of the world population lives in countries where the

<sup>\*</sup> Carlos Adam Conte Júnior2, Leone Vinícius Furlanetto3, Flávia Galindo Silvestre Silva4,

Rafael Silva Duarte5, Joab Trajano Silva3, Walter Lilenbaum6 and Vânia Margaret Flosi Paschoalin3\*

*<sup>1</sup>Departamento de Alimentos e Nutrição, Universidade Federal de Mato Grosso, Cuiabá/MT, Brasil* 

*<sup>2</sup>Faculdade de Veterinária, Universidade Federal Fluminense, Niterói/RJ, Brasil 3Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brasil 4Universidade de Cuiabá, Cuiabá/MT, Brasil* 

*<sup>5</sup>Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brasil 6Instituto Biomédico, Universidade Federal Fluminense, Niterói/RJ, Brasil* 

quantity (Medeiros, 2010).

endemic area of BTB (CDC, 2005).

presence of clinical signs of the disease is directly associated with their distribution and

Airborne infection is the most common transmission route, and more than 15% of cattle with BTB shed the mycobacteria, mainly early in the course of the infection (Cassidy et al., 1998). Studies with molecular markers have shown that infected cattle are a potential source of tuberculosis transmission to humans (Serrano-Moreno et al., 2008). Milk can be an important transmission route, resulting in extra-lung presentation of the illness (Wedlock et al., 2002). This was demonstrated in New York City, where 35 cases of infection by *M. bovis* were reported from 2001 to 2004; when they were traced back, some of the cases were associated with the consumption of cheese made with non-pasteurized milk, imported from an

Using bacteriological culture methods, it has been calculated that only about 5% of tuberculin-reacting cattle (TRC) can eliminate *M. bovis* in milk. In such animals, the incidence of visible gross lesions in the mammary gland (MG) or supramammary lymph nodes (LN) is less than 0.5% (Goodchild and Clifton-Hadley, 2001). *M. bovis* has been isolated from milk samples from storage tanks, inadequately pasteurized milk, and milk samples from tuberculin non-reacting cattle (Pardo et al., 2001; Leite et al., 2003). This situation dramatizes the need for sensitive and accurate procedures for rapid identification of the bacteria in milk, to assist in the control of this zoonosis. PCR techniques offer high sensitivity, and have been successfully used for diagnosing BTB in several types of naturally infected organic materials such as tissue, blood and nasal exudates (Figueiredo et al., 2010; Cardoso et al., 2009; Romero et al., 1999). However, PCR techniques have been seldom

According to OIE, the proportion of zoonotic TB cases in Brazil is unknown (OIE, 2007), since bacteriological culturing for diagnosing TB is not employed routinely for all samples (Sequeira, 2005). The most recently published data estimating the incidence of zoonotic TB in Brazil (Kantor et al., 2008) reported the occurrence of only one occasion in 20 years (1987– 2006), where *M. bovis* was confirmed as the etiological agent of human tuberculosis at the Hélio Fraga National Reference Laboratory (NRL) at the Federal University Hospital, Rio de Janeiro. In a two-year study, nearly 8,000 clinical samples were cultured for detection of mycobacteria, and no *M. bovis* isolate was obtained (Sobral, 2009). In the São Paulo laboratory network (Adolfo Lutz Institute), a total of 355,383 cultures were performed in the period 2001–2005, and only two *M. bovis* strains were recovered from two patients, one in 2001 from a lymph-node biopsy, and another in 2002 from a cerebrospinal-fluid sample. At the State Reference Laboratory in Rio Grande do Sul (1997–2005), of approximately 5,000

mycobacterial isolates phenotyped, no *M. bovis* was confirmed (Kantor et al., 2008).

Despite the presence of the disease in Brazil, there is a lack of official data concerning the current prevalence of bovine tuberculosis in the country. From 1989 to 1998, data from official reports indicate a national mean prevalence of 1.3% of infected cows (BRASIL, 2008). Since the implementation of PNCEBT in Brazil, the few studies reporting on the prevalence of the disease have provided estimates ranging from 0.7% to 3.3% (Baptista et al., 2004; Oliveira, 2007; Poletto et al., 2004; Ribeiro et al., 2003). According to the epidemiology of the disease, the higher incidence in dairy than in beef herds will also determine the geographical distribution. Roxo (2005) reported the rate of infection in different areas in

tested for use in milk, particularly in Brazil (Zanini et al., 1998)

control of bovine tuberculosis is limited or absent (Vordemeier, 2006), there is a consensus about the risks regarding human health.

Humans can develop latent TB infection, active TB or reactivation of latent TB infection. In veterinary medicine, the distinction between latent TB infection and active TB disease is not as important as it is in human beings, since in both cases the animal must be slaughtered. According to the National Control Program implemented in Brazil, treatment of reactive animals is not allowed, and all reactive animals must be slaughtered (Brasil, 2004).

Zoonotic TB is a recognized public health threat in the developing world. In some countries, control measures against bovine tuberculosis are limited or nonexistent (Cosivi, 1998; Thoen, 2006). Human infection by *M. bovis* can occur by the aerogenous route, ingestion of infected milk (WHO, 1994), or, less frequently, by contact with mucous membranes and broken skin. The disease caused by *M. bovis* is clinically, radiologically and pathologically indistinguishable from that caused by *M. tuberculosis*, while, differently from the typical tuberculosis and due to the infection route*,* the non-pulmonary presentation is most frequent (Grange, 2001). Considering the standard treatment for *M. tuberculosis* infection, the lack of differentiation between *M. bovis* and *M. tuberculosis* is a problem. *M. bovis* is naturally resistant to pyrazinamide, a drug that is frequently used to treat TB in humans. Thus, individuals infected by *M. bovis* may present a treatment failure, which makes them potential transmitters of these resistant strains to other people and animals (Abrahão, 2005).

In industrialized countries, human infection with *M. bovis* has been largely controlled by pasteurization of cow's milk, inspection in slaughterhouses, and culling of cattle reacting to the compulsory diagnosis (Romero et al., 2006). In Brazil, despite the existence of a National Eradication Plan, clandestine meat and milk are marketed without sanitary control, which is a threat to public health; the ingestion of these products is a possible route of infection to humans (Abrahão, 2005). In some developing countries with uncontrolled bovine tuberculosis, most human cases occur in young persons, and result from drinking or handling contaminated milk (Cosivi, 1998).

Zoonotic TB can also be considered a socio-economic disease; it causes direct economic losses in agricultural areas and hampers the commercial exchange of animal products (Zumárraga et al., 1999). Many countries around the world support the control or eradication of bovine tuberculosis by national control programs, based on a test-andslaughter policy. Brazilian policies regarding the control and eradication of bovine tuberculosis include the National Plan for Control and Eradication of Bovine Brucellosis and Tuberculosis (PNCEBT), written in 2001 and revised in 2004, which is based on the slaughtering of all animals reactive to the tuberculin tests. However, this traditional policy has not been fully successful in many countries, and new tools, including additional diagnostic tests and new vaccines, are urgently required (Pollock et al., 2005).

In cattle, tuberculous lesions are most often found in organs rich in reticuloendothelial tissue, particularly the lungs and associated lymph nodes, and the liver (Corner, 1990). Other studies conducted on naturally and experimentally infected cattle have demonstrated that the lesions are most commonly observed in the lower respiratory tract; however, the upper respiratory tract and associated tissues may also display disease in a significant number of cases. Although tubercles are not a pathognomonic lesion of cattle TB, the

control of bovine tuberculosis is limited or absent (Vordemeier, 2006), there is a consensus

Humans can develop latent TB infection, active TB or reactivation of latent TB infection. In veterinary medicine, the distinction between latent TB infection and active TB disease is not as important as it is in human beings, since in both cases the animal must be slaughtered. According to the National Control Program implemented in Brazil, treatment of reactive

Zoonotic TB is a recognized public health threat in the developing world. In some countries, control measures against bovine tuberculosis are limited or nonexistent (Cosivi, 1998; Thoen, 2006). Human infection by *M. bovis* can occur by the aerogenous route, ingestion of infected milk (WHO, 1994), or, less frequently, by contact with mucous membranes and broken skin. The disease caused by *M. bovis* is clinically, radiologically and pathologically indistinguishable from that caused by *M. tuberculosis*, while, differently from the typical tuberculosis and due to the infection route*,* the non-pulmonary presentation is most frequent (Grange, 2001). Considering the standard treatment for *M. tuberculosis* infection, the lack of differentiation between *M. bovis* and *M. tuberculosis* is a problem. *M. bovis* is naturally resistant to pyrazinamide, a drug that is frequently used to treat TB in humans. Thus, individuals infected by *M. bovis* may present a treatment failure, which makes them potential transmitters of these resistant strains to other people and animals (Abrahão, 2005). In industrialized countries, human infection with *M. bovis* has been largely controlled by pasteurization of cow's milk, inspection in slaughterhouses, and culling of cattle reacting to the compulsory diagnosis (Romero et al., 2006). In Brazil, despite the existence of a National Eradication Plan, clandestine meat and milk are marketed without sanitary control, which is a threat to public health; the ingestion of these products is a possible route of infection to humans (Abrahão, 2005). In some developing countries with uncontrolled bovine tuberculosis, most human cases occur in young persons, and result from drinking or

Zoonotic TB can also be considered a socio-economic disease; it causes direct economic losses in agricultural areas and hampers the commercial exchange of animal products (Zumárraga et al., 1999). Many countries around the world support the control or eradication of bovine tuberculosis by national control programs, based on a test-andslaughter policy. Brazilian policies regarding the control and eradication of bovine tuberculosis include the National Plan for Control and Eradication of Bovine Brucellosis and Tuberculosis (PNCEBT), written in 2001 and revised in 2004, which is based on the slaughtering of all animals reactive to the tuberculin tests. However, this traditional policy has not been fully successful in many countries, and new tools, including additional

In cattle, tuberculous lesions are most often found in organs rich in reticuloendothelial tissue, particularly the lungs and associated lymph nodes, and the liver (Corner, 1990). Other studies conducted on naturally and experimentally infected cattle have demonstrated that the lesions are most commonly observed in the lower respiratory tract; however, the upper respiratory tract and associated tissues may also display disease in a significant number of cases. Although tubercles are not a pathognomonic lesion of cattle TB, the

diagnostic tests and new vaccines, are urgently required (Pollock et al., 2005).

animals is not allowed, and all reactive animals must be slaughtered (Brasil, 2004).

about the risks regarding human health.

handling contaminated milk (Cosivi, 1998).

presence of clinical signs of the disease is directly associated with their distribution and quantity (Medeiros, 2010).

Airborne infection is the most common transmission route, and more than 15% of cattle with BTB shed the mycobacteria, mainly early in the course of the infection (Cassidy et al., 1998). Studies with molecular markers have shown that infected cattle are a potential source of tuberculosis transmission to humans (Serrano-Moreno et al., 2008). Milk can be an important transmission route, resulting in extra-lung presentation of the illness (Wedlock et al., 2002). This was demonstrated in New York City, where 35 cases of infection by *M. bovis* were reported from 2001 to 2004; when they were traced back, some of the cases were associated with the consumption of cheese made with non-pasteurized milk, imported from an endemic area of BTB (CDC, 2005).

Using bacteriological culture methods, it has been calculated that only about 5% of tuberculin-reacting cattle (TRC) can eliminate *M. bovis* in milk. In such animals, the incidence of visible gross lesions in the mammary gland (MG) or supramammary lymph nodes (LN) is less than 0.5% (Goodchild and Clifton-Hadley, 2001). *M. bovis* has been isolated from milk samples from storage tanks, inadequately pasteurized milk, and milk samples from tuberculin non-reacting cattle (Pardo et al., 2001; Leite et al., 2003). This situation dramatizes the need for sensitive and accurate procedures for rapid identification of the bacteria in milk, to assist in the control of this zoonosis. PCR techniques offer high sensitivity, and have been successfully used for diagnosing BTB in several types of naturally infected organic materials such as tissue, blood and nasal exudates (Figueiredo et al., 2010; Cardoso et al., 2009; Romero et al., 1999). However, PCR techniques have been seldom tested for use in milk, particularly in Brazil (Zanini et al., 1998)

According to OIE, the proportion of zoonotic TB cases in Brazil is unknown (OIE, 2007), since bacteriological culturing for diagnosing TB is not employed routinely for all samples (Sequeira, 2005). The most recently published data estimating the incidence of zoonotic TB in Brazil (Kantor et al., 2008) reported the occurrence of only one occasion in 20 years (1987– 2006), where *M. bovis* was confirmed as the etiological agent of human tuberculosis at the Hélio Fraga National Reference Laboratory (NRL) at the Federal University Hospital, Rio de Janeiro. In a two-year study, nearly 8,000 clinical samples were cultured for detection of mycobacteria, and no *M. bovis* isolate was obtained (Sobral, 2009). In the São Paulo laboratory network (Adolfo Lutz Institute), a total of 355,383 cultures were performed in the period 2001–2005, and only two *M. bovis* strains were recovered from two patients, one in 2001 from a lymph-node biopsy, and another in 2002 from a cerebrospinal-fluid sample. At the State Reference Laboratory in Rio Grande do Sul (1997–2005), of approximately 5,000 mycobacterial isolates phenotyped, no *M. bovis* was confirmed (Kantor et al., 2008).

Despite the presence of the disease in Brazil, there is a lack of official data concerning the current prevalence of bovine tuberculosis in the country. From 1989 to 1998, data from official reports indicate a national mean prevalence of 1.3% of infected cows (BRASIL, 2008). Since the implementation of PNCEBT in Brazil, the few studies reporting on the prevalence of the disease have provided estimates ranging from 0.7% to 3.3% (Baptista et al., 2004; Oliveira, 2007; Poletto et al., 2004; Ribeiro et al., 2003). According to the epidemiology of the disease, the higher incidence in dairy than in beef herds will also determine the geographical distribution. Roxo (2005) reported the rate of infection in different areas in

this infection in countries where the disease still occurs.

**from the field with a dairy herd in Brazil** 

HPLC and m-PCR.

**(Figueiredo et al., 2009)** 

of multiplex PCR and HPLC methods, which have been used to decrease the prevalence of

**2. Molecular methods in the diagnosis of bovine tuberculosis: Experiences** 

**2.1 Identification of** *Mycobacterium bovis* **isolates by a multiplex PCR** 

*bovis* isolates from the other members of this complex.

Our studies were conducted on a dairy herd comprised of 270 adult crossbred Holstein and Gir cows, located in the Municipality of Macaé, state of Rio de Janeiro in southeast Brazil. Prior to the study, 21 adult cows had positive reactions to a Single Intradermal Tuberculin Test (SITT) and were kept in quarantine for 90 days, awaiting confirmatory tests. After 90 days, a Comparative Intradermal Tuberculin Test (CITT) was performed on these 21 cows (Group A), plus 29 selected cows that were negative for the first SITT test, including those with inconclusive results in the first test (Group B). A total of 34 animals reacted in the CITT (21/21 from Group A and 13/29 from Group B). From all 34 cows, milk samples and nasal swabs were collected and subjected to bacteriological culture, and the isolates were identified by the HPLC method and m-PCR, and also direct detection by m-PCR. All 34 cows were slaughtered 30 days after the injection of PPD, and thorough necropsies were performed. Mediastinal, scapular and retropharyngeal lymph nodes, as well as samples from the lungs were collected and also were analyzed by bacteriological tests, as well as

Several PCR systems have been developed for the detection of species belonging to the *M. tuberculosis* complex (MTC). The most commonly used system is based on primers that amplify segments of the IS*6110* element, particularly targeting the 123-bp (Eisenach et al., 1990) and 245-bp fragments (Hermans et al., 1990). Another PCR system that has yielded successful identification of *M. bovis* isolates is focused on the amplification of a 500-bp DNA fragment in the RvD1Rv2031c genomic sequence (Rodríguez et al., 1999). A combination of conventional culture and biochemical techniques is the gold-standard method currently used for the identification of *M. bovis*, combining the isolation of the etiological agent and the unequivocal identification of the isolate. Mycobacteria were isolated form suggestive bovine tuberculous lesions, and the pure cultures of acid-fast bacilli (AFB) were identified by molecular analysis. The molecular assay consists of a single-step multiplex PCR (m-PCR), based on two set primers already tested and proved to be reliable, but not yet combined in a single PCR system. The combined PCR assay targets simultaneously the RvD1Rv2031c and IS6110 sequences, aiming to identify bacteria as MTC members as well as to distinguish *M.* 

Among the 50 adult cows from this herd that were tested by the intradermal tuberculin test according to official standards (Brasil, 2004), 34 animals were reactive, and were euthanized and necropsied. During the necropsy, a total of 91 samples of lymph nodes and lungs were collected, although not all the animals presented typical lesions. Samples were maintained under refrigeration, and tissues of each animal were processed together as one pooled sample per animal, totaling 34 samples. Samples were decontaminated using the Petroff method, inoculated on slopes of Lowenstein- Jensen medium with sodium pyruvate and

Brazil, and not surprisingly, the region with the lowest rate of infection is the one where beef herds are most predominant. Nevertheless, these data represent only particular regions, and cannot be used for estimates in the national context. It is important to keep in mind the enormous size of the Brazilian herd, which comprises approximately 200 million bovines (PAHO/WHO, 2006).

There is a growing perception that no single method is sufficient for detecting all cattle infected with BTB (Salfinger et al., 1994); therefore, a multidisciplinary approach must be employed, based on currently available methods. Some of the diagnostic methods and combinations of methods that are regularly used for diagnosing BTB are shown in Figure 1.

Bovine tuberculosis infection in cattle is usually diagnosed in the live animal. The diagnosis is based on delayed hypersensitivity reactions (intradermal tuberculin tests), a method that may lack both sensitivity and specificity. However, a definitive diagnosis is still established by isolation and identification of the etiological agent (*M. bovis*) from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the "gold standard". These methods are slow, cumbersome, unreliable, and time-consuming (it may take more than 4 weeks to grow the microorganism, and an additional 2 weeks to identify it). Several alternative approaches have been attempted for the rapid and specific diagnosis of tuberculosis, but molecular methods, especially polymerase chain reaction (PCR) assays, are the most promising for diagnoses in live cattle (Serrano-Moreno et al., 2008; Figueiredo et al., 2010) and directdetection *post mortem* diagnosis in bovine tissue samples (Cardoso et al., 2009; Liebana et al., 1995; Meickle et al., 2001; Romero et al., 1999; Vitale et al., 1998; Wards et al., 1995; Zanini et al., 1998; Zanini et al., 2001;).

Fig. 1. Methods currently used to diagnose bovine tuberculosis (Medeiros et al., 2010).

The purpose of this chapter is to present new diagnostic approaches for the *Mycobacterium tuberculosis* complex in particular. We focus on discriminating *Mycobacterium bovis* by the use

Brazil, and not surprisingly, the region with the lowest rate of infection is the one where beef herds are most predominant. Nevertheless, these data represent only particular regions, and cannot be used for estimates in the national context. It is important to keep in mind the enormous size of the Brazilian herd, which comprises approximately 200 million bovines

There is a growing perception that no single method is sufficient for detecting all cattle infected with BTB (Salfinger et al., 1994); therefore, a multidisciplinary approach must be employed, based on currently available methods. Some of the diagnostic methods and combinations of methods that are regularly used for diagnosing BTB are shown in Figure 1. Bovine tuberculosis infection in cattle is usually diagnosed in the live animal. The diagnosis is based on delayed hypersensitivity reactions (intradermal tuberculin tests), a method that may lack both sensitivity and specificity. However, a definitive diagnosis is still established by isolation and identification of the etiological agent (*M. bovis*) from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the "gold standard". These methods are slow, cumbersome, unreliable, and time-consuming (it may take more than 4 weeks to grow the microorganism, and an additional 2 weeks to identify it). Several alternative approaches have been attempted for the rapid and specific diagnosis of tuberculosis, but molecular methods, especially polymerase chain reaction (PCR) assays, are the most promising for diagnoses in live cattle (Serrano-Moreno et al., 2008; Figueiredo et al., 2010) and directdetection *post mortem* diagnosis in bovine tissue samples (Cardoso et al., 2009; Liebana et al., 1995; Meickle et al., 2001; Romero et al., 1999; Vitale et al., 1998; Wards et al., 1995;

Fig. 1. Methods currently used to diagnose bovine tuberculosis (Medeiros et al., 2010).

The purpose of this chapter is to present new diagnostic approaches for the *Mycobacterium tuberculosis* complex in particular. We focus on discriminating *Mycobacterium bovis* by the use

(PAHO/WHO, 2006).

Zanini et al., 1998; Zanini et al., 2001;).

of multiplex PCR and HPLC methods, which have been used to decrease the prevalence of this infection in countries where the disease still occurs.

#### **2. Molecular methods in the diagnosis of bovine tuberculosis: Experiences from the field with a dairy herd in Brazil**

Our studies were conducted on a dairy herd comprised of 270 adult crossbred Holstein and Gir cows, located in the Municipality of Macaé, state of Rio de Janeiro in southeast Brazil. Prior to the study, 21 adult cows had positive reactions to a Single Intradermal Tuberculin Test (SITT) and were kept in quarantine for 90 days, awaiting confirmatory tests. After 90 days, a Comparative Intradermal Tuberculin Test (CITT) was performed on these 21 cows (Group A), plus 29 selected cows that were negative for the first SITT test, including those with inconclusive results in the first test (Group B). A total of 34 animals reacted in the CITT (21/21 from Group A and 13/29 from Group B). From all 34 cows, milk samples and nasal swabs were collected and subjected to bacteriological culture, and the isolates were identified by the HPLC method and m-PCR, and also direct detection by m-PCR. All 34 cows were slaughtered 30 days after the injection of PPD, and thorough necropsies were performed. Mediastinal, scapular and retropharyngeal lymph nodes, as well as samples from the lungs were collected and also were analyzed by bacteriological tests, as well as HPLC and m-PCR.

#### **2.1 Identification of** *Mycobacterium bovis* **isolates by a multiplex PCR (Figueiredo et al., 2009)**

Several PCR systems have been developed for the detection of species belonging to the *M. tuberculosis* complex (MTC). The most commonly used system is based on primers that amplify segments of the IS*6110* element, particularly targeting the 123-bp (Eisenach et al., 1990) and 245-bp fragments (Hermans et al., 1990). Another PCR system that has yielded successful identification of *M. bovis* isolates is focused on the amplification of a 500-bp DNA fragment in the RvD1Rv2031c genomic sequence (Rodríguez et al., 1999). A combination of conventional culture and biochemical techniques is the gold-standard method currently used for the identification of *M. bovis*, combining the isolation of the etiological agent and the unequivocal identification of the isolate. Mycobacteria were isolated form suggestive bovine tuberculous lesions, and the pure cultures of acid-fast bacilli (AFB) were identified by molecular analysis. The molecular assay consists of a single-step multiplex PCR (m-PCR), based on two set primers already tested and proved to be reliable, but not yet combined in a single PCR system. The combined PCR assay targets simultaneously the RvD1Rv2031c and IS6110 sequences, aiming to identify bacteria as MTC members as well as to distinguish *M. bovis* isolates from the other members of this complex.

Among the 50 adult cows from this herd that were tested by the intradermal tuberculin test according to official standards (Brasil, 2004), 34 animals were reactive, and were euthanized and necropsied. During the necropsy, a total of 91 samples of lymph nodes and lungs were collected, although not all the animals presented typical lesions. Samples were maintained under refrigeration, and tissues of each animal were processed together as one pooled sample per animal, totaling 34 samples. Samples were decontaminated using the Petroff method, inoculated on slopes of Lowenstein- Jensen medium with sodium pyruvate and

500 bp 245 bp

number of bacilli hinders identification by classical methods. It also can be a valuable tool for the rapid identification of acid-fast bacilli isolated from suggestive bovine TB lesions.

Fig. 2. **Identification of ABF isolates by m-PCR.** DNA extracted from 17 different acid-fast bacilli isolates was used as a template for m-PCR amplification of the RvD1Rv2031c and the IS6110 sequences. Amplification products were separated by electrophoresis on 1.5% agarose gel and stained with ethidium bromide (10 μg/mL). Lane M: 100-bp DNA ladder (Fermentas®); lanes 1-17: m-PCR products of acid-fast bacilli isolated from suggestive BT lesions; lane 18: negative control. Arrows indicate the positions of the fragments of 500 bp

**2.2 Detection of M***ycobacterium bovis* **DNA in nasal swabs from tuberculous cattle by** 

The multiplex PCR-based method for the simultaneous detection of mycobacteria belonging to MTC and the specific identification of *M. bovis* was adapted to screen nasal swabs collected from live cows, suspected to be tuberculous. A total of 50 adult cows from a dairy herd with a previous history of bovine tuberculosis, including clinical cases, from Macaé were tested by the cervical comparative intradermal tuberculin test (ITT) with PPD (purified protein derivative) according to official standards (Brasil, 2004). In parallel, samples of nasal mucus were collected using sterile swabs and submitted to both microbiological culture and m-PCR. All 34 ITT-reactive animals (68% of the total of cows examined) were slaughtered; the lungs and lymph nodes were removed and processed for bacteriology according to the OIE Terrestrial Manual (OIE, 2009). Briefly, after decontamination by the Petroff method, samples from lungs, lymph nodes and nasal swabs were inoculated on Lowenstein-Jensen and Stonebrink agar slopes and the tubes were incubated at 37ºC for up to 12 weeks. The presence of *M. bovis* and other mycobacteria belonging to MTC in nasal mucus was investigated by a single-step multiplex PCR (m-PCR) using two sets of primers, as previously described (Figueiredo et al., 2009), that targets simultaneously the *RvD1-Rv2031c* (specific for *M. bovis*) and IS*6110* (present in all MTC species) genomic sequences, but that to date had not yet been combined together in a single m-PCR assay. DNA was extracted from nasal swabs by a modification of a QIAamp Blood and Tissue Kit (Qiagen). The bacterial pellet was suspended in 180 μl of 20 mg/mL lysozyme in 20 mM Tris·HCl, pH 8.0; 2 mM EDTA; 1.2% Triton and incubated for 30 min at 37°C prior to the proteinase K treatment, in order to improve the process of bacterial lysis. DNA eluted from the QIAamp Mini spin columns was concentrated by precipitation with absolute ethanol at -80ºC. m-PCR was performed in a reaction mix (50 µL) as described by Figueiredo et al. (2009). No mycobacterial growth was observed on agar slopes inoculated with nasal swab samples collected either from ITT-reactive or ITT-negative cows. On the other hand, mycobacterial colonies were observed in cultures from lung or lymph-node samples isolated from 17 of 34

(diagnostic for *M. bovis*) and 245 bp (diagnostic for MTBC members).

**a multiplex PCR (Figueiredo et al., 2010)** 

incubated for three months at 37ºC. After growth, AFB-positive colonies were screened by m-PCR. Briefly, the mycobacterial DNA was extracted as described previously (Meickle et al., 20073). m-PCR was performed in a reaction mixture (50 μL) containing 5 μl of 10 × PCR buffer (Invitrogen®), 200 μM dNTP (GE Healthcare®), 2.5 U of recombinant *Taq* polymerase (Invitrogen®), 0.2 μM of each primer (Invitrogen®) JB21 (5´- TCGTCCGCTGATGCAAGTGC-3´) and JB22 (5´-CGTCCGCTGACCTCAAGAAAG-3´) (4) and INS1 (5'-CGTGAGGGCATCGAGGTGGC-3') and INS2 (5'- GCGTAGGCGTCGGTGACAAA-3') (10), 2.0 mM MgCl2, and 5 μL of purified DNA template. Amplification was carried out in a GeneAmp PCR System 9600 (Applied Biosystems®) with the following cycling parameters: 94ºC for 5 min, followed by 30 cycles of 1 min at 94ºC, 1 min at 68ºC and 1 min at 72ºC, with a final extension at 72ºC for 7 min. PCR products were checked by electrophoresis on 1.5% agarose gels stained with ethidium bromide (10 μg/mL). Negative samples were analyzed by PCR restriction analysis (PRA), using primers Tb11 (5'-ACCAACGATGGTGTGTCCA T-3') and Tb12 (5'- CTTGTCGAACCGCATACCCT-3') targeting for the *hsp*65 gene (Telenti et al., 1993). The amplification products were digested with *Bst*E II and *Hae*III and the resulting fragments were fractionated by agarose gel electrophoresis and stained with ethidium bromide.

*Mycobacteria* colonies were isolated in Lowenstein-Jensen medium with sodium pyruvate from 17 of 34 (50%) processed samples, therefore confirming the infection. This herd had been TB-free in the last test, performed six months before the study. Therefore, we believe that the reactive cows had a recent infection, where visible lesions are not always present and the bacterial load is low. Considering the decontamination method used, it is not surprising that not all cultures yielded *M. bovis*. Nevertheless, it is noteworthy that the presence of some positive cultures is sufficient to characterize the outbreak of TB in this herd.

In these 17 isolates, m-PCR successfully amplified both target regions (the 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTBC) in 15 of them (88.24%) (Figure 2, lanes 1-15). The two (11.76%) m-PCR-negative isolates (Figure 2, lanes 16 and 17) were confirmed by PCR-restriction analysis as *Mycobacterium* sp., but were not included in the *Mycobacterium tuberculosis* complex (results not shown).

PCR assays using primers JB21/JB22 have been considered to be highly reliable in identifying *M. bovis* isolates, showing 100% concordance with the conventional microbiological method (Rodríguez et al., 1999). However, the absolute specificity of JB21/JB22 primers for *M.* bovis has been disputed by another study, which reported that 13.3% (4/30) of *M. bovis* isolates failed to produce the 500-bp fragment (Sechi et al., 2000). Using specific primers for the IS6110 sequence, the 500-bp negative isolates were identified as belonging to the MTC, leading the authors to suggest that these isolates may lack the genomic target for JB21/JB22 primers. As this genotypic characteristic may not be infrequent, the use of a single primer pair can produce false negative results. On the other hand, an additional primer pair targeting for a different sequence, as in m-PCR, minimizes the occurrence of such false-negative results. The two sets of primers, although already described in the literature (Hermans et al., 1990; Rodríguez et al., 1999), as explained before, for the first time were combined to optimize a mPCR assay able to identify unequivocally *M. bovis* among mycobacterial isolates. The mPCR method was fast, reproducible and useful for the study of slow-growing mycobacteria, particularly in cultures where the small

incubated for three months at 37ºC. After growth, AFB-positive colonies were screened by m-PCR. Briefly, the mycobacterial DNA was extracted as described previously (Meickle et al., 20073). m-PCR was performed in a reaction mixture (50 μL) containing 5 μl of 10 × PCR buffer (Invitrogen®), 200 μM dNTP (GE Healthcare®), 2.5 U of recombinant *Taq* polymerase (Invitrogen®), 0.2 μM of each primer (Invitrogen®) JB21 (5´- TCGTCCGCTGATGCAAGTGC-3´) and JB22 (5´-CGTCCGCTGACCTCAAGAAAG-3´) (4) and INS1 (5'-CGTGAGGGCATCGAGGTGGC-3') and INS2 (5'- GCGTAGGCGTCGGTGACAAA-3') (10), 2.0 mM MgCl2, and 5 μL of purified DNA template. Amplification was carried out in a GeneAmp PCR System 9600 (Applied Biosystems®) with the following cycling parameters: 94ºC for 5 min, followed by 30 cycles of 1 min at 94ºC, 1 min at 68ºC and 1 min at 72ºC, with a final extension at 72ºC for 7 min. PCR products were checked by electrophoresis on 1.5% agarose gels stained with ethidium bromide (10 μg/mL). Negative samples were analyzed by PCR restriction analysis (PRA), using primers Tb11 (5'-ACCAACGATGGTGTGTCCA T-3') and Tb12 (5'- CTTGTCGAACCGCATACCCT-3') targeting for the *hsp*65 gene (Telenti et al., 1993). The amplification products were digested with *Bst*E II and *Hae*III and the resulting fragments

were fractionated by agarose gel electrophoresis and stained with ethidium bromide.

herd.

*Mycobacteria* colonies were isolated in Lowenstein-Jensen medium with sodium pyruvate from 17 of 34 (50%) processed samples, therefore confirming the infection. This herd had been TB-free in the last test, performed six months before the study. Therefore, we believe that the reactive cows had a recent infection, where visible lesions are not always present and the bacterial load is low. Considering the decontamination method used, it is not surprising that not all cultures yielded *M. bovis*. Nevertheless, it is noteworthy that the presence of some positive cultures is sufficient to characterize the outbreak of TB in this

In these 17 isolates, m-PCR successfully amplified both target regions (the 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTBC) in 15 of them (88.24%) (Figure 2, lanes 1-15). The two (11.76%) m-PCR-negative isolates (Figure 2, lanes 16 and 17) were confirmed by PCR-restriction analysis as *Mycobacterium* sp., but were not included in

PCR assays using primers JB21/JB22 have been considered to be highly reliable in identifying *M. bovis* isolates, showing 100% concordance with the conventional microbiological method (Rodríguez et al., 1999). However, the absolute specificity of JB21/JB22 primers for *M.* bovis has been disputed by another study, which reported that 13.3% (4/30) of *M. bovis* isolates failed to produce the 500-bp fragment (Sechi et al., 2000). Using specific primers for the IS6110 sequence, the 500-bp negative isolates were identified as belonging to the MTC, leading the authors to suggest that these isolates may lack the genomic target for JB21/JB22 primers. As this genotypic characteristic may not be infrequent, the use of a single primer pair can produce false negative results. On the other hand, an additional primer pair targeting for a different sequence, as in m-PCR, minimizes the occurrence of such false-negative results. The two sets of primers, although already described in the literature (Hermans et al., 1990; Rodríguez et al., 1999), as explained before, for the first time were combined to optimize a mPCR assay able to identify unequivocally *M. bovis* among mycobacterial isolates. The mPCR method was fast, reproducible and useful for the study of slow-growing mycobacteria, particularly in cultures where the small

the *Mycobacterium tuberculosis* complex (results not shown).

number of bacilli hinders identification by classical methods. It also can be a valuable tool for the rapid identification of acid-fast bacilli isolated from suggestive bovine TB lesions.

Fig. 2. **Identification of ABF isolates by m-PCR.** DNA extracted from 17 different acid-fast bacilli isolates was used as a template for m-PCR amplification of the RvD1Rv2031c and the IS6110 sequences. Amplification products were separated by electrophoresis on 1.5% agarose gel and stained with ethidium bromide (10 μg/mL). Lane M: 100-bp DNA ladder (Fermentas®); lanes 1-17: m-PCR products of acid-fast bacilli isolated from suggestive BT lesions; lane 18: negative control. Arrows indicate the positions of the fragments of 500 bp (diagnostic for *M. bovis*) and 245 bp (diagnostic for MTBC members).

#### **2.2 Detection of M***ycobacterium bovis* **DNA in nasal swabs from tuberculous cattle by a multiplex PCR (Figueiredo et al., 2010)**

The multiplex PCR-based method for the simultaneous detection of mycobacteria belonging to MTC and the specific identification of *M. bovis* was adapted to screen nasal swabs collected from live cows, suspected to be tuberculous. A total of 50 adult cows from a dairy herd with a previous history of bovine tuberculosis, including clinical cases, from Macaé were tested by the cervical comparative intradermal tuberculin test (ITT) with PPD (purified protein derivative) according to official standards (Brasil, 2004). In parallel, samples of nasal mucus were collected using sterile swabs and submitted to both microbiological culture and m-PCR. All 34 ITT-reactive animals (68% of the total of cows examined) were slaughtered; the lungs and lymph nodes were removed and processed for bacteriology according to the OIE Terrestrial Manual (OIE, 2009). Briefly, after decontamination by the Petroff method, samples from lungs, lymph nodes and nasal swabs were inoculated on Lowenstein-Jensen and Stonebrink agar slopes and the tubes were incubated at 37ºC for up to 12 weeks. The presence of *M. bovis* and other mycobacteria belonging to MTC in nasal mucus was investigated by a single-step multiplex PCR (m-PCR) using two sets of primers, as previously described (Figueiredo et al., 2009), that targets simultaneously the *RvD1-Rv2031c* (specific for *M. bovis*) and IS*6110* (present in all MTC species) genomic sequences, but that to date had not yet been combined together in a single m-PCR assay. DNA was extracted from nasal swabs by a modification of a QIAamp Blood and Tissue Kit (Qiagen). The bacterial pellet was suspended in 180 μl of 20 mg/mL lysozyme in 20 mM Tris·HCl, pH 8.0; 2 mM EDTA; 1.2% Triton and incubated for 30 min at 37°C prior to the proteinase K treatment, in order to improve the process of bacterial lysis. DNA eluted from the QIAamp Mini spin columns was concentrated by precipitation with absolute ethanol at -80ºC. m-PCR was performed in a reaction mix (50 µL) as described by Figueiredo et al. (2009). No mycobacterial growth was observed on agar slopes inoculated with nasal swab samples collected either from ITT-reactive or ITT-negative cows. On the other hand, mycobacterial colonies were observed in cultures from lung or lymph-node samples isolated from 17 of 34

**2.3 Detection of** *Mycobacterium bovis* **DNA in milk by m-PCR** 

eradication program.

Rv2031c.

some cases for several months. In conclusion, we have successfully used m-PCR assay to detect *M. bovis* in nasal exudates of naturally infected cattle, as previously reported (Meickle et al., 2007; Tejada et al., 2006; Vitale et al.,1998). Indeed, Vitale et al. (1998) reported high specificity and positive predictive value in the detection of MTC in nasal swabs by PCR, and Romero et al. (1999) demonstrated that nasal-mucus samples work better for the *in vivo* PCR-based detection of the microorganism than other fluids such as blood or milk. However, all these previous reports utilized primers to detect MTC species, and the identification of *M. bovis* was presumptive. The mPCR used here has the advantage of being specific for *M. bovis,* but simultaneously identifies the presence of *M. bovis* and other non-*M. bovis* mycobacterial species belonging to MTC. Although limited by the natural evolution of the infection, since shedding of mycobacteria in nasal mucus is required, the use of m-PCR for detecting live tuberculous animals by testing the nasal mucus could be an effective and highly specific *ante-mortem* ancillary method for surveillance of bovine tuberculosis in herds, if a periodic sampling scheme is followed; or as a confirmatory method for animals with inconclusive intradermal testing, thus assisting the bovine tuberculosis control and

Another valuable tool in confirming tuberculous cows is the identification of *M. bovis* in milk produced by the suspected animals. A PCR assay was developed for direct detection of *M. bovis* DNA in artificially and naturally contaminated milk. The assay used a pair of primers that were previously tested and proved reliable in targeting putative gene RvD1-

Milk previously seeded with *M. bovis* was used as the starting material. The procedure involved DNA extraction by enzymatic lysing (proteinase K and lysozyme) and phenol:chloroform:isoamyl alcohol, followed by ethanol precipitation and m-PCR. The m-PCR was performed according to Figueiredo et al. (2010), and allowed us to detect *M. bovis* 

The use of the PCR method in spiked milk samples does not guarantee that it would perform equally effectively in the analysis of naturally infected samples. One could expect that in the latter, the interaction between the bacilli and the milk matrix could be more complex, and even that bacilli in milk might have already been killed by mammary macrophages (Zumarraga et al., 2005) and the DNA partially degraded. Therefore, the mPCR described here was evaluated for detection of *M. bovis* DNA in fresh unprocessed milk from CITT-reactive cows. A total of 50 adult cows from a dairy herd with a previous history of bovine tuberculosis, including clinical cases, from Macaé were tested by the cervical comparative intradermal tuberculin test (CITT) with PPD (purified protein derivative) according to official standards (Brasil, 2004). Thirty-four animals were CITTreactive, and from all 50 cows, milk samples were collected (on the day that PPD was

No mycobacterium growth was observed in CITT-negative cows (0/16). but in five milk samples collected from CITT-reactive cows (5/34) mycobacterial growth was observed. Only one isolate was confirmed as *M. bovis* by m-PCR (Figure 1, lane 1). Mycobacterial colonies were also observed in cultures from lung or lymph-node samples isolated from 17

DNA in artificially contaminated milk, with a detection limit of 100 CFU/mL.

injected) and were subjected to bacteriological culture and m-PCR assay.

PPD-positive cows (50% of the total of positive cows). In parallel, nasal swabs were examined for the presence of mycobacteria by m-PCR. DNA was extracted from nasal swabs collected from 34 ITT-reactive and 16 ITT-negative cows, using a modification of a QIAamp blood and tissue kit (Qiagen) that was devised to improve bacterial lysis and concentrate DNA. The nucleic acids isolated from all samples using the above-modified procedure showed high quality in terms of integrity and purity, and were suitable for use as templates in the m-PCR. The 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTC were simultaneously observed in 2 of 34 (5.9%) of m-PCR reactions performed using nasal-swab DNA from ITT-reactive cows. Importantly, neither the 500-bp band nor the 245-bp band was found as m-PCR reaction products when swabs from ITT-negative cows were tested (results not shown).

*M. bovis* has been recovered from nasal exudates collected from cattle in naturally infected herds by using conventional culture techniques (de Kantor & Roswurm, 1978; McIlroy et al., 1986; Meickle et al., 2007). In these reports, recovery efficiencies varied from 8.7 (4) to 28.5%, when solely ITT-reactive animals were assessed (Meickle et al., 2007), regardless of the difficulty of the procedure, since it requires the presence of 10-100 viable organisms in the sample for a positive result, a condition attained only in advanced stages of the disease (Barry et al., 1993). Using PCR-based methods, the presence of species of the MTC group in nasal exudates of ITT-reactive animals was detected in 26% of the tested samples (Tejada et al., 2006), with some studies reporting detection rates of 50 or 58% (13,23), even though some PCR techniques may detect *M. bovis* using as little as 5 fg of DNA, which is equivalent to the amount of nucleic acid in a single mycobacterial genome (Estrada-Chávez et al., 2004).

The number of positive animals was smaller than expected, which was probably caused by limitations in the current PCR protocols for detection of mycobacteria in nasal exudates, such as intermittent shedding, inefficient DNA extraction, or the presence of PCR inhibitors in the samples (de la Rua-Domenech et al., 2006). None of the nasal-exudate samples from 34 ITT-reactive cows were found to be positive for the growth of *M. bovis*. Furthermore, 2 of 34 nasal-exudate samples (5.9%) were positive by m-PCR, a more sensitive and specific method than culturing (Meickle et al., 2007; Zanini et al., 1998; Zumárraga et al., 2005). These figures are lower than those previously obtained by using culture- or PCR-based methods to evaluate the presence of *M. bovis* in nasal exudates (de la Rua-Domenech et al., 2006; Meickle et al., 2007; Tejada et al., 2006; Vitale et al., 1998). The low rate of positive results may possibly be a consequence of the small numbers of viable bacteria present in nasal-swab samples, because the growth of the etiological agent was observed in cultures of lung and lymph-node samples from 17 of these cows, using the same procedure.

It has been well documented that in cattle experimentally infected with *M. bovis,* after each infection there is a lag period during which the etiological agent cannot be isolated from nasal mucus (Neill et al., 1998; McCorry et al., 2005; Kao et al., 2007**)**. In a previously reported study, all experimentally infected animals shed *M. bovis* in the nasal mucus (Neill et al., 1998); but failure of some experimental animals to shed mycobacteria has also been reported (McCorry et al., 2005; Kao et al., 2007). Importantly, differences in the shedding profiles were observed, where those animals shedding *M. bovis* in nasal exudates were classified as either intermittent or as persistent shedders. It also appears that the overall level of shedding increases during the first four weeks after exposure and then begins to decline (Kao et al., 2007), although shedding can still be detected for many weeks, and in

PPD-positive cows (50% of the total of positive cows). In parallel, nasal swabs were examined for the presence of mycobacteria by m-PCR. DNA was extracted from nasal swabs collected from 34 ITT-reactive and 16 ITT-negative cows, using a modification of a QIAamp blood and tissue kit (Qiagen) that was devised to improve bacterial lysis and concentrate DNA. The nucleic acids isolated from all samples using the above-modified procedure showed high quality in terms of integrity and purity, and were suitable for use as templates in the m-PCR. The 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTC were simultaneously observed in 2 of 34 (5.9%) of m-PCR reactions performed using nasal-swab DNA from ITT-reactive cows. Importantly, neither the 500-bp band nor the 245-bp band was found as m-PCR reaction products when swabs from ITT-negative

*M. bovis* has been recovered from nasal exudates collected from cattle in naturally infected herds by using conventional culture techniques (de Kantor & Roswurm, 1978; McIlroy et al., 1986; Meickle et al., 2007). In these reports, recovery efficiencies varied from 8.7 (4) to 28.5%, when solely ITT-reactive animals were assessed (Meickle et al., 2007), regardless of the difficulty of the procedure, since it requires the presence of 10-100 viable organisms in the sample for a positive result, a condition attained only in advanced stages of the disease (Barry et al., 1993). Using PCR-based methods, the presence of species of the MTC group in nasal exudates of ITT-reactive animals was detected in 26% of the tested samples (Tejada et al., 2006), with some studies reporting detection rates of 50 or 58% (13,23), even though some PCR techniques may detect *M. bovis* using as little as 5 fg of DNA, which is equivalent to the amount of nucleic acid in a single mycobacterial genome (Estrada-Chávez et al., 2004). The number of positive animals was smaller than expected, which was probably caused by limitations in the current PCR protocols for detection of mycobacteria in nasal exudates, such as intermittent shedding, inefficient DNA extraction, or the presence of PCR inhibitors in the samples (de la Rua-Domenech et al., 2006). None of the nasal-exudate samples from 34 ITT-reactive cows were found to be positive for the growth of *M. bovis*. Furthermore, 2 of 34 nasal-exudate samples (5.9%) were positive by m-PCR, a more sensitive and specific method than culturing (Meickle et al., 2007; Zanini et al., 1998; Zumárraga et al., 2005). These figures are lower than those previously obtained by using culture- or PCR-based methods to evaluate the presence of *M. bovis* in nasal exudates (de la Rua-Domenech et al., 2006; Meickle et al., 2007; Tejada et al., 2006; Vitale et al., 1998). The low rate of positive results may possibly be a consequence of the small numbers of viable bacteria present in nasal-swab samples, because the growth of the etiological agent was observed in cultures of lung and

lymph-node samples from 17 of these cows, using the same procedure.

It has been well documented that in cattle experimentally infected with *M. bovis,* after each infection there is a lag period during which the etiological agent cannot be isolated from nasal mucus (Neill et al., 1998; McCorry et al., 2005; Kao et al., 2007**)**. In a previously reported study, all experimentally infected animals shed *M. bovis* in the nasal mucus (Neill et al., 1998); but failure of some experimental animals to shed mycobacteria has also been reported (McCorry et al., 2005; Kao et al., 2007). Importantly, differences in the shedding profiles were observed, where those animals shedding *M. bovis* in nasal exudates were classified as either intermittent or as persistent shedders. It also appears that the overall level of shedding increases during the first four weeks after exposure and then begins to decline (Kao et al., 2007), although shedding can still be detected for many weeks, and in

cows were tested (results not shown).

some cases for several months. In conclusion, we have successfully used m-PCR assay to detect *M. bovis* in nasal exudates of naturally infected cattle, as previously reported (Meickle et al., 2007; Tejada et al., 2006; Vitale et al.,1998). Indeed, Vitale et al. (1998) reported high specificity and positive predictive value in the detection of MTC in nasal swabs by PCR, and Romero et al. (1999) demonstrated that nasal-mucus samples work better for the *in vivo* PCR-based detection of the microorganism than other fluids such as blood or milk. However, all these previous reports utilized primers to detect MTC species, and the identification of *M. bovis* was presumptive. The mPCR used here has the advantage of being specific for *M. bovis,* but simultaneously identifies the presence of *M. bovis* and other non-*M. bovis* mycobacterial species belonging to MTC. Although limited by the natural evolution of the infection, since shedding of mycobacteria in nasal mucus is required, the use of m-PCR for detecting live tuberculous animals by testing the nasal mucus could be an effective and highly specific *ante-mortem* ancillary method for surveillance of bovine tuberculosis in herds, if a periodic sampling scheme is followed; or as a confirmatory method for animals with inconclusive intradermal testing, thus assisting the bovine tuberculosis control and eradication program.

#### **2.3 Detection of** *Mycobacterium bovis* **DNA in milk by m-PCR**

Another valuable tool in confirming tuberculous cows is the identification of *M. bovis* in milk produced by the suspected animals. A PCR assay was developed for direct detection of *M. bovis* DNA in artificially and naturally contaminated milk. The assay used a pair of primers that were previously tested and proved reliable in targeting putative gene RvD1- Rv2031c.

Milk previously seeded with *M. bovis* was used as the starting material. The procedure involved DNA extraction by enzymatic lysing (proteinase K and lysozyme) and phenol:chloroform:isoamyl alcohol, followed by ethanol precipitation and m-PCR. The m-PCR was performed according to Figueiredo et al. (2010), and allowed us to detect *M. bovis*  DNA in artificially contaminated milk, with a detection limit of 100 CFU/mL.

The use of the PCR method in spiked milk samples does not guarantee that it would perform equally effectively in the analysis of naturally infected samples. One could expect that in the latter, the interaction between the bacilli and the milk matrix could be more complex, and even that bacilli in milk might have already been killed by mammary macrophages (Zumarraga et al., 2005) and the DNA partially degraded. Therefore, the mPCR described here was evaluated for detection of *M. bovis* DNA in fresh unprocessed milk from CITT-reactive cows. A total of 50 adult cows from a dairy herd with a previous history of bovine tuberculosis, including clinical cases, from Macaé were tested by the cervical comparative intradermal tuberculin test (CITT) with PPD (purified protein derivative) according to official standards (Brasil, 2004). Thirty-four animals were CITTreactive, and from all 50 cows, milk samples were collected (on the day that PPD was injected) and were subjected to bacteriological culture and m-PCR assay.

No mycobacterium growth was observed in CITT-negative cows (0/16). but in five milk samples collected from CITT-reactive cows (5/34) mycobacterial growth was observed. Only one isolate was confirmed as *M. bovis* by m-PCR (Figure 1, lane 1). Mycobacterial colonies were also observed in cultures from lung or lymph-node samples isolated from 17

dairy industry, to prevent contaminated milk from entering the food supply.

**2.4 Detection of** *Mycobacterium bovis* **DNA in bovine tissues by m-PCR** 

test and conventional culture for *M. bovis*.

200 µL of the buffer.

The PCR assay allowed us to detect *M. bovis* DNA in artificially contaminated milk, with a detection limit of 100 CFU/mL, and also proved to be able to detect the bacilli in naturally infected milk. This method could be useful to assist the *in vivo* diagnosis for BTB, complementing the serological or microbiological tests, and is an alternative option in cases of mammary tuberculosis where the efficiency of serological diagnosis is nil. The method will be useful in epidemiological studies of BTB transmission and in quality control for the

We adapted the m-PCR assay targeting the RvD1Rv2031c and IS6110 sequences, which are specific for *M. bovis* and MTC respectively, to identify *M. bovis* DNA in tissues from slaughtered positive-skin-test animals. The results are compared with those from the skin

Of 270 adult crossbred Holstein and Gir cows in a herd located in Macaé, 34 cows were considered CITT-reactive and also infected, by IFN assay (Marasi et al., 2010). At 30 days after CIIT, all 34 reactive animals were slaughtered and necropsied. Tissue samples were collected and analyzed by bacteriological methodology and m-PCR. DNA was extracted from lymph nodes, lung and udder tissues taken from the slaughtered animals, by a modification of a QIAamp Blood and Tissue Kit (Qiagen). One sample was selected per animal. A small piece of tissue (1-2 g) was macerated and an aliquot of 1 mL was taken. The pellet was suspended in 180 μl of 20 mg/mL lysozyme in 20 mM Tris·HCl, pH 8.0; 2 mM EDTA; and 1.2% Triton, and incubated for 1 h at 37°C prior to proteinase K treatment, in order to improve the process of bacterial lysis. DNA eluted from the QIAamp mini spin columns was concentrated by precipitation with absolute ethanol at -80ºC and eluted with

The m-PCR was performed according to Figueiredo et al. (2010). In 17/34 (50%) samples *Mycobacterium* sp. isolates were obtained, and 15/17 were confirmed as *M. bovis* by m-PCR (Figueiredo et al., 2009). Direct m-PCR on tissue samples from CITT-reactive cows was positive for *M. bovis* DNA in 25/34 (73.5%) of the samples. All positive-culture specimens were also positive for m-PCR; and 10 (59%) samples that were negative by culturing yielded a positive result after m-PCR assay. It should be mentioned that the PCR was sensitive enough to detect *M. bovis* in a large proportion (59%) of those samples that failed to grow in culture, as also reported by Liebana et al. (1995), Zanini et al. (2001) and Araújo et al. (2005). The efficiency of the culture method used as a first criterion for *M. bovis* identification is low, because of the small number of live bacilli present in some tissues. Small numbers of live bacilli may be a consequence of a short delay in getting tissues to the laboratory, or may be

The improved identification shown here can be attributed to the removal of unwanted inhibitors. Ward et al. (1995) and Liebana et al. (1995) stated that "mycobacteria are difficult organisms from which to extract DNA and because they often exist as intracellular pathogens, may also be difficult organisms to purify from clinical samples, particularly tissues". Some compounds present in tissues, such as eukaryotic DNA or blood-originated inhibitory substances such as hemoglobin, lactoferrin and undegraded nucleic-acid samples from inflamed tissue can inhibit DNA amplification (Cardoso et al., 2009). On the other

due to the sensitivity of mycobacteria to the NaOH used in the Petroff method.

of the 34 PPD-positive cows, and were confirmed as *M. bovis* by m-PCR (Figueiredo et al., 2009). In parallel, milk samples were tested for the presence of mycobacterium DNA using the m-PCR. The 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTC were simultaneously observed in 4 of 34 (12%) m-PCR reactions performed with milk DNA templates from CITT-reactive cows (Figure 4). Importantly, neither the 500-bp nor the 245-bp amplicons were found when milk from CITT-negative cows was tested.

Similarly, in analyzing milk samples of cows from infected herds, previous studies have not detected any positive animal (Perez et al., 2002), while others targeting for the RvD1- Rv2031c, IS6110 sequence and MPB70 gene have reported amplifications from 2% to 28% of the cows (Romero et al., 1999; Zumárraga et al., 2005). Other studies using nested PCR (Serrano-Moreno et al., 2008; Vitale et al., 1998) also showed that the presence of *M. bovis* in milk is heterogeneous. The variable PCR results can be explained since the bacilli shed may be associated with cell-mediated immunity (CMI) in tuberculous cows (Pollock et al., 2001; Romero et al., 2006), as well as with epidemiological factors such as viral immunosuppression, metabolic imbalance, corticosteroids and peripartum (Doherth et al., 1995, 1996; Sordillo et al., 1997; Piccinini et al., 2006).

Thirty of the milk samples from CIIT-reactive cows were negative by PCR. This suggests that some periods of bacterial excretion might have been missed, due to the design of the study, which included only one sampling. The intermittent character of bacilli secretion after a short constant post-infection period was documented by Menzies and Neill (2000). Another important point is that the 500-bp band was not found as a PCR product when milk from CITT-negative cows was tested, and mycobacterial colonies could not be isolated by culturing. The lack of recovery of *Mycobacterium* sp. could be due to the small number of excreted bacteria, or to the presence of dead or non-viable bacilli due to the action of macrophages, or even to the use of the Petroff decontamination method and reduced sensitivity of culturing compared with PCR (Zumárraga et al., 2005).

Fig. 3. **Direct Detection of** *M. bovis* **DNA in milk samples from CITT-reactive cows.** DNA templates obtained from 1 mL of milk samples were amplified by m-PCR of the RvD1Rv2031c and the IS6110 sequences. Amplicons were resolved on a 1.5% agarose gel stained with ethidium bromide. Lane M: 100-bp DNA ladder (Promega®); lane 1: positive control, *M. bovis* IP; lanes 2-5: milk samples from CITT-reactive cows; lane 6: negative control (water); lane 7: negative control (DNA template from *M. fortuitum* ATCC 6841). From each cow, three samples were analyzed and three independent experiments were performed.

of the 34 PPD-positive cows, and were confirmed as *M. bovis* by m-PCR (Figueiredo et al., 2009). In parallel, milk samples were tested for the presence of mycobacterium DNA using the m-PCR. The 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTC were simultaneously observed in 4 of 34 (12%) m-PCR reactions performed with milk DNA templates from CITT-reactive cows (Figure 4). Importantly, neither the 500-bp nor the

Similarly, in analyzing milk samples of cows from infected herds, previous studies have not detected any positive animal (Perez et al., 2002), while others targeting for the RvD1- Rv2031c, IS6110 sequence and MPB70 gene have reported amplifications from 2% to 28% of the cows (Romero et al., 1999; Zumárraga et al., 2005). Other studies using nested PCR (Serrano-Moreno et al., 2008; Vitale et al., 1998) also showed that the presence of *M. bovis* in milk is heterogeneous. The variable PCR results can be explained since the bacilli shed may be associated with cell-mediated immunity (CMI) in tuberculous cows (Pollock et al., 2001; Romero et al., 2006), as well as with epidemiological factors such as viral immunosuppression, metabolic imbalance, corticosteroids and peripartum (Doherth et al.,

Thirty of the milk samples from CIIT-reactive cows were negative by PCR. This suggests that some periods of bacterial excretion might have been missed, due to the design of the study, which included only one sampling. The intermittent character of bacilli secretion after a short constant post-infection period was documented by Menzies and Neill (2000). Another important point is that the 500-bp band was not found as a PCR product when milk from CITT-negative cows was tested, and mycobacterial colonies could not be isolated by culturing. The lack of recovery of *Mycobacterium* sp. could be due to the small number of excreted bacteria, or to the presence of dead or non-viable bacilli due to the action of macrophages, or even to the use of the Petroff decontamination method and reduced

Fig. 3. **Direct Detection of** *M. bovis* **DNA in milk samples from CITT-reactive cows.** DNA

RvD1Rv2031c and the IS6110 sequences. Amplicons were resolved on a 1.5% agarose gel stained with ethidium bromide. Lane M: 100-bp DNA ladder (Promega®); lane 1: positive control, *M. bovis* IP; lanes 2-5: milk samples from CITT-reactive cows; lane 6: negative control (water); lane 7: negative control (DNA template from *M. fortuitum* ATCC 6841). From each cow, three samples were analyzed and three independent experiments were

templates obtained from 1 mL of milk samples were amplified by m-PCR of the

performed.

245-bp amplicons were found when milk from CITT-negative cows was tested.

1995, 1996; Sordillo et al., 1997; Piccinini et al., 2006).

sensitivity of culturing compared with PCR (Zumárraga et al., 2005).

The PCR assay allowed us to detect *M. bovis* DNA in artificially contaminated milk, with a detection limit of 100 CFU/mL, and also proved to be able to detect the bacilli in naturally infected milk. This method could be useful to assist the *in vivo* diagnosis for BTB, complementing the serological or microbiological tests, and is an alternative option in cases of mammary tuberculosis where the efficiency of serological diagnosis is nil. The method will be useful in epidemiological studies of BTB transmission and in quality control for the dairy industry, to prevent contaminated milk from entering the food supply.

#### **2.4 Detection of** *Mycobacterium bovis* **DNA in bovine tissues by m-PCR**

We adapted the m-PCR assay targeting the RvD1Rv2031c and IS6110 sequences, which are specific for *M. bovis* and MTC respectively, to identify *M. bovis* DNA in tissues from slaughtered positive-skin-test animals. The results are compared with those from the skin test and conventional culture for *M. bovis*.

Of 270 adult crossbred Holstein and Gir cows in a herd located in Macaé, 34 cows were considered CITT-reactive and also infected, by IFN assay (Marasi et al., 2010). At 30 days after CIIT, all 34 reactive animals were slaughtered and necropsied. Tissue samples were collected and analyzed by bacteriological methodology and m-PCR. DNA was extracted from lymph nodes, lung and udder tissues taken from the slaughtered animals, by a modification of a QIAamp Blood and Tissue Kit (Qiagen). One sample was selected per animal. A small piece of tissue (1-2 g) was macerated and an aliquot of 1 mL was taken. The pellet was suspended in 180 μl of 20 mg/mL lysozyme in 20 mM Tris·HCl, pH 8.0; 2 mM EDTA; and 1.2% Triton, and incubated for 1 h at 37°C prior to proteinase K treatment, in order to improve the process of bacterial lysis. DNA eluted from the QIAamp mini spin columns was concentrated by precipitation with absolute ethanol at -80ºC and eluted with 200 µL of the buffer.

The m-PCR was performed according to Figueiredo et al. (2010). In 17/34 (50%) samples *Mycobacterium* sp. isolates were obtained, and 15/17 were confirmed as *M. bovis* by m-PCR (Figueiredo et al., 2009). Direct m-PCR on tissue samples from CITT-reactive cows was positive for *M. bovis* DNA in 25/34 (73.5%) of the samples. All positive-culture specimens were also positive for m-PCR; and 10 (59%) samples that were negative by culturing yielded a positive result after m-PCR assay. It should be mentioned that the PCR was sensitive enough to detect *M. bovis* in a large proportion (59%) of those samples that failed to grow in culture, as also reported by Liebana et al. (1995), Zanini et al. (2001) and Araújo et al. (2005). The efficiency of the culture method used as a first criterion for *M. bovis* identification is low, because of the small number of live bacilli present in some tissues. Small numbers of live bacilli may be a consequence of a short delay in getting tissues to the laboratory, or may be due to the sensitivity of mycobacteria to the NaOH used in the Petroff method.

The improved identification shown here can be attributed to the removal of unwanted inhibitors. Ward et al. (1995) and Liebana et al. (1995) stated that "mycobacteria are difficult organisms from which to extract DNA and because they often exist as intracellular pathogens, may also be difficult organisms to purify from clinical samples, particularly tissues". Some compounds present in tissues, such as eukaryotic DNA or blood-originated inhibitory substances such as hemoglobin, lactoferrin and undegraded nucleic-acid samples from inflamed tissue can inhibit DNA amplification (Cardoso et al., 2009). On the other

literature to enable identification of MTC species.

Butler and Guthertz (2001).

separation capabilities and the methodology specificity. Mycolic acids from 35 different reference *Mycobacterium* strains were saponified, extracted, derivatized, analyzed and successfully identified by the adapted HPLC method. The identification of mycobacteria was based on the relative retention times (RRT) of the chromatograms, comparing the profile obtained from the reference strains with profiles available in external databases. Although an internal standard was not used to align the chromatograms, the method showed good reproducibility and standardization, using the range of the relative standard deviation (RSD) of absolute retention time (ART) and the RRT, which varied from 0.68% to 0.97% and from 0.39% to 0.72%, respectively. The adapted method improved the identification of *Mycobacterium* species of clinical and veterinary interest, by comparing the new isolates with a database of mycolic acid chromatogram patterns from 35 reference mycobacteria strains, and comparing those profiles with those previously reported in the

A suspension of acid-fast bacteria grown in LJ medium was removed with a swab and saponified with 2 ml KOH 25% in methanol:H2O (v:v) autoclaved for 1 h at 121°C, 15 psi, to cleave the mycolic acids bound to the cell wall (Butler et al., 1991). Mycolic acids were then separated by acidification with HCl:H2O (v:v) and extraction in chloroform. After conversion to ultraviolet (UV)-absorbing *p*-bromophenacyl esters (Pircen®) (Butler & Guthertz, 2001) and clearing with HCl:H2O:Methanol (1:1:2, v:v:v), the mycolic acids were analyzed on a reverse-phase C18 100 x 4.6 mm column (Kromasil®) using high-performance liquid chromatography (Cage, 1994; Duffey et al., 1996). A gradient of methanol and dichloromethane (methylene chloride) generated by microprocessor-controlled pumps was used to separate the mycolic acid esters (Butler et al., 1991, Viader-Salvadó et al., 2007), which were detected with a UV detector at 260 nm (Du et al., 2008). Reproducible chromatographic patterns containing combinations of different diagnostic peaks (Butler et al., 1991, Glickman et al., 1994) were obtained by using reference strains (*M. abscessus* ATCC 19977, *M. africanum* ATCC 25420, *M. agri* ATCC 27406, *M. aichiense* ATCC 27280, *M. asiaticum* ATCC 25276, *M. aurum* ATCC 23366, *M. avium* ATCC 25291, *M. bovis* ATCC 19210, *M. bovis* BCG INCQS 00062, *M. chelonae* ATCC 35752, *M. flavescens* ATCC 14474, *M. fortuitum* ATCC 6841, *M. gastri* ATCC 15754, *M. godornae* ATCC 141470, *M. intracellulare* ATCC 13950, *M. malmoense* ATCC 29571, *M. mucogenicum* ATCC 49650, *M. scrofulaceum* ATCC 19981, *M. simiae* ATCC 25275, *M. terrae* ATCC 15755, *M. tuberculosis* ATCC 25177, *M. vaccae*  ATCC15483 and *M. triviale* ATCC23292). Pattern recognition was done by visual comparison of the results for the reference strains with mycolic acid patterns from species of known mycobacteria (CDC, 1996, 1999). Identification of mycobacterial species by mycolic acid analysis was performed by visually comparing the UV patterns obtained from the samples with UV patterns from reference species, following recommendations of Butler and collaborators (Butler et al., 1991; Butler and Guthertz, 2001). Chromatographic patterns for each strain were examined for differences in the heights for pairs of peaks. HPLC patterns were grouped according to species, and the calculated values for each ratio were combined, sorted in numerical order, and examined for their ability to discriminate species, using the range of the relative standard deviation (RSD) of absolute retention time (ART) and the relative retention time (RRT). The 35 species were grouped into three general patterns (single, double and triple clusters) and divided accordingly into subgroups, according to

hand, the use of the QIAamp Blood and Tissue Kit (Qiagen) circumvented those problems and supplied DNA templates suitable for amplification.

The nine remaining CITT-reactive cows were negative by both the culturing and m-PCR assays. Those results could be attributed to an inhibitory effect in the PCR assay (Al-Soud and Radstrom, 2001; Cardoso et al., 2009), and additional inquires are needed with regard to DNA extraction methods. In addition, it should be considered that the tissue samples collected from those animals contained a low pathogen load, characterizing paucibacillary lesions that are commonly observed in recent infections occurring intra-herd. Two previous studies (Zanini et al., 2001; Cardoso et al., 2009) also reported a decreased efficiency in detecting mycobacteria in paucibacillary tissue samples. It is generally accepted that the CITT is a correlate of *M. bovis* infection and not necessarily of disease (Neill et al. 1994). In this study, CIIT-reactive animals developed disease, as demonstrated by the presence of lesions.

The results presented here indicate that m-PCR can detect *M. bovis* DNA in tissue samples, and represents a valid additional tool for the *post-mortem* diagnosis of BTB. Multiplex PCR is faster than culture-based detection, reducing diagnosis time from 120 to approximately 2 days, even when automated culturing with broth medium is used. Moreover, m-PCR is useful when the bacilli are non-viable and cannot be detected by culture methods. It can be of valuable help during sanitary inspection at slaughterhouses for condemnation of carcasses that show suspected lesions, or slaughtered animals suspected of having the disease. It is also important to note that a detailed inspection of bovine organs performed during necropsy in the field is more efficient than a rapid inspection at the slaughterhouse, because in the latter situation small lesions may be not detected.

#### **2.5 Identification of species of the** *Mycobacterium tuberculosis* **complex by adapted High-Performance Liquid Chromatography (HPLC)**

Complex high-molecular-weight β-hydroxyl fatty acids with a 22- or 24-carbon alkyl chain at the α-position are structural characteristics of mycolic acids, a fatty acid found in the *Mycobacterium* cell wall. By using several methods of fatty acid analysis, mycolic acids have been considered to be species- or group-specific (Butler et al., 1991). High-performance liquid chromatography (HPLC) analysis of mycolic acids has emerged as a reliable method for the diagnosis of mycobacteria, due to its rapid and reproducible nature, and because the mycolic-acid elution pattern observed for each mycobacterial species has generally been found to be unique, except for a few species that share the same pattern profile (Hagen & Thompson, 1995). The HPLC method has been considered a standard test for chemotaxonomic classification and rapid identification of *Mycobacterium* species by the Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov), since 1990, and has been reported to achieve accuracy above 96% compared with DNA probe tests (Butler & Guthertz, 2001). A dedicated database, using adapted local protocols, must be developed in order to obtain chromatogram profiles from reference strains in the new analytic conditions, accrediting the local methodology and allowing for the correct analysis of clinical samples.

An HPLC method to identify *Mycobacterium* species, originally developed on a short column (CDC, 1996), was transferred to a longer column with similar stationary phase properties, but with a length of at most 33% of the initial one. Protocol modifications improved the

hand, the use of the QIAamp Blood and Tissue Kit (Qiagen) circumvented those problems

The nine remaining CITT-reactive cows were negative by both the culturing and m-PCR assays. Those results could be attributed to an inhibitory effect in the PCR assay (Al-Soud and Radstrom, 2001; Cardoso et al., 2009), and additional inquires are needed with regard to DNA extraction methods. In addition, it should be considered that the tissue samples collected from those animals contained a low pathogen load, characterizing paucibacillary lesions that are commonly observed in recent infections occurring intra-herd. Two previous studies (Zanini et al., 2001; Cardoso et al., 2009) also reported a decreased efficiency in detecting mycobacteria in paucibacillary tissue samples. It is generally accepted that the CITT is a correlate of *M. bovis* infection and not necessarily of disease (Neill et al. 1994). In this study, CIIT-reactive animals developed disease, as demonstrated by the presence of

The results presented here indicate that m-PCR can detect *M. bovis* DNA in tissue samples, and represents a valid additional tool for the *post-mortem* diagnosis of BTB. Multiplex PCR is faster than culture-based detection, reducing diagnosis time from 120 to approximately 2 days, even when automated culturing with broth medium is used. Moreover, m-PCR is useful when the bacilli are non-viable and cannot be detected by culture methods. It can be of valuable help during sanitary inspection at slaughterhouses for condemnation of carcasses that show suspected lesions, or slaughtered animals suspected of having the disease. It is also important to note that a detailed inspection of bovine organs performed during necropsy in the field is more efficient than a rapid inspection at the slaughterhouse,

**2.5 Identification of species of the** *Mycobacterium tuberculosis* **complex by adapted** 

Complex high-molecular-weight β-hydroxyl fatty acids with a 22- or 24-carbon alkyl chain at the α-position are structural characteristics of mycolic acids, a fatty acid found in the *Mycobacterium* cell wall. By using several methods of fatty acid analysis, mycolic acids have been considered to be species- or group-specific (Butler et al., 1991). High-performance liquid chromatography (HPLC) analysis of mycolic acids has emerged as a reliable method for the diagnosis of mycobacteria, due to its rapid and reproducible nature, and because the mycolic-acid elution pattern observed for each mycobacterial species has generally been found to be unique, except for a few species that share the same pattern profile (Hagen & Thompson, 1995). The HPLC method has been considered a standard test for chemotaxonomic classification and rapid identification of *Mycobacterium* species by the Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov), since 1990, and has been reported to achieve accuracy above 96% compared with DNA probe tests (Butler & Guthertz, 2001). A dedicated database, using adapted local protocols, must be developed in order to obtain chromatogram profiles from reference strains in the new analytic conditions, accrediting the local methodology and allowing for the correct analysis of clinical samples. An HPLC method to identify *Mycobacterium* species, originally developed on a short column (CDC, 1996), was transferred to a longer column with similar stationary phase properties, but with a length of at most 33% of the initial one. Protocol modifications improved the

and supplied DNA templates suitable for amplification.

because in the latter situation small lesions may be not detected.

**High-Performance Liquid Chromatography (HPLC)** 

lesions.

separation capabilities and the methodology specificity. Mycolic acids from 35 different reference *Mycobacterium* strains were saponified, extracted, derivatized, analyzed and successfully identified by the adapted HPLC method. The identification of mycobacteria was based on the relative retention times (RRT) of the chromatograms, comparing the profile obtained from the reference strains with profiles available in external databases. Although an internal standard was not used to align the chromatograms, the method showed good reproducibility and standardization, using the range of the relative standard deviation (RSD) of absolute retention time (ART) and the RRT, which varied from 0.68% to 0.97% and from 0.39% to 0.72%, respectively. The adapted method improved the identification of *Mycobacterium* species of clinical and veterinary interest, by comparing the new isolates with a database of mycolic acid chromatogram patterns from 35 reference mycobacteria strains, and comparing those profiles with those previously reported in the literature to enable identification of MTC species.

A suspension of acid-fast bacteria grown in LJ medium was removed with a swab and saponified with 2 ml KOH 25% in methanol:H2O (v:v) autoclaved for 1 h at 121°C, 15 psi, to cleave the mycolic acids bound to the cell wall (Butler et al., 1991). Mycolic acids were then separated by acidification with HCl:H2O (v:v) and extraction in chloroform. After conversion to ultraviolet (UV)-absorbing *p*-bromophenacyl esters (Pircen®) (Butler & Guthertz, 2001) and clearing with HCl:H2O:Methanol (1:1:2, v:v:v), the mycolic acids were analyzed on a reverse-phase C18 100 x 4.6 mm column (Kromasil®) using high-performance liquid chromatography (Cage, 1994; Duffey et al., 1996). A gradient of methanol and dichloromethane (methylene chloride) generated by microprocessor-controlled pumps was used to separate the mycolic acid esters (Butler et al., 1991, Viader-Salvadó et al., 2007), which were detected with a UV detector at 260 nm (Du et al., 2008). Reproducible chromatographic patterns containing combinations of different diagnostic peaks (Butler et al., 1991, Glickman et al., 1994) were obtained by using reference strains (*M. abscessus* ATCC 19977, *M. africanum* ATCC 25420, *M. agri* ATCC 27406, *M. aichiense* ATCC 27280, *M. asiaticum* ATCC 25276, *M. aurum* ATCC 23366, *M. avium* ATCC 25291, *M. bovis* ATCC 19210, *M. bovis* BCG INCQS 00062, *M. chelonae* ATCC 35752, *M. flavescens* ATCC 14474, *M. fortuitum* ATCC 6841, *M. gastri* ATCC 15754, *M. godornae* ATCC 141470, *M. intracellulare* ATCC 13950, *M. malmoense* ATCC 29571, *M. mucogenicum* ATCC 49650, *M. scrofulaceum* ATCC 19981, *M. simiae* ATCC 25275, *M. terrae* ATCC 15755, *M. tuberculosis* ATCC 25177, *M. vaccae*  ATCC15483 and *M. triviale* ATCC23292). Pattern recognition was done by visual comparison of the results for the reference strains with mycolic acid patterns from species of known mycobacteria (CDC, 1996, 1999). Identification of mycobacterial species by mycolic acid analysis was performed by visually comparing the UV patterns obtained from the samples with UV patterns from reference species, following recommendations of Butler and collaborators (Butler et al., 1991; Butler and Guthertz, 2001). Chromatographic patterns for each strain were examined for differences in the heights for pairs of peaks. HPLC patterns were grouped according to species, and the calculated values for each ratio were combined, sorted in numerical order, and examined for their ability to discriminate species, using the range of the relative standard deviation (RSD) of absolute retention time (ART) and the relative retention time (RRT). The 35 species were grouped into three general patterns (single, double and triple clusters) and divided accordingly into subgroups, according to Butler and Guthertz (2001).

Fig. 5. Characteristic HPLC chromatograms of *Mycobacterium* species with late-emerging, simple, single-cluster peak patterns. **A**) *M. tuberculosis* H37Ra (ATCC 25177) and H37Rv (ATCC 27294) and *M. bovis* (ATCC 19210). **B**) *M. gordonae* chromotype I (ATCC 14470), *M. kansasii* (ATCC 12478) and *M. triviale* (ATCC 23292). \*peaks showing a high degree of separation (appearing as a "double peak"), named according to Butler et al. (1991). *M. triviale* strain: xpeaks showing a high degree of separation (appearing as a "double peak"),

Fig. 6. Representative reverse-phase HPLC chromatograms of mycolic acid methylesters from reference strains and isolates: (A) *M. bovis* ATCC 19210; (B) 21 clinical *M. bovis* isolates

from dairy herds in Brazil.

compared to the chromatogram profile described by Butler & Guthertz (2001).

A total of 21 *M. bovis* isolates from tissue, milk and nasal-swab samples from a dairy herd comprised of 270 adult crossbred Holstein and Gir cows, located in Macaé were confirmed by multiplex PCR (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, which are specific for the *M. bovis* and *M. tuberculosis* complexes, respectively (Figueiredo et al., 2009). Spoligotyping (Kamerbeek et al., 1997) was used to validate the HPLC methodology.

It has been reported that BCG-attenuated strains of *M. bovis* could be successfully differentiated from the MTC by HPLC (Floyd et al., 1992). This observation was confirmed in the present study, by comparing the chromatograms obtained from reference strains (Fig. 4). Other members of the complex, such as *M. bovis* and *M. tuberculosis*, were known to produce very similar chromatogram patterns, making it impossible to discriminate between them by this methodology. However, although requiring further work, the chromatogram profiles generated by the adapted elution protocol showed discrete and consistent differences in their chromatograms that could be used to discriminate them (Fig. 5-A). The simple and late-emerging single-cluster peak pattern group also included *M. asiaticum*, *M. gordonae* chromotype I (Fig. 5-B) and *M. kansasii* (Fig. 5-B). *M. triviale* was the only mycobacterium species present in this group, and it can be easily recognized (Fig. 5-B).

In these 21 isolates, m-PCR successfully amplified both target regions (the 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTBC) in all isolates. A total of four spoligotypes were identified among the 21 *M. bovis* isolates. Two spoligotypes (SB0120 and SB0833) were described in the *M. bovis* spoligotype database (Brudey et al., 2006; www.mbovis.org). The other two represent novel, previously undescribed spoligotypes. The HPLC assay also identified the clinical *M. bovis* isolates as members of the *Mycobacterium tuberculosis* complex (Figure 6).

Fig. 4. Mycolic acid chromatograms from *M. bovis* BCG (INCQS0062) and *M. bovis* (ATCC 19210). \* peaks showing a high degree of separation (appearing as a "double peak"), named according to Butler et al. (1991).

424 Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis

A total of 21 *M. bovis* isolates from tissue, milk and nasal-swab samples from a dairy herd comprised of 270 adult crossbred Holstein and Gir cows, located in Macaé were confirmed by multiplex PCR (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, which are specific for the *M. bovis* and *M. tuberculosis* complexes, respectively (Figueiredo et al., 2009). Spoligotyping (Kamerbeek et al., 1997) was used to validate the HPLC

It has been reported that BCG-attenuated strains of *M. bovis* could be successfully differentiated from the MTC by HPLC (Floyd et al., 1992). This observation was confirmed in the present study, by comparing the chromatograms obtained from reference strains (Fig. 4). Other members of the complex, such as *M. bovis* and *M. tuberculosis*, were known to produce very similar chromatogram patterns, making it impossible to discriminate between them by this methodology. However, although requiring further work, the chromatogram profiles generated by the adapted elution protocol showed discrete and consistent differences in their chromatograms that could be used to discriminate them (Fig. 5-A). The simple and late-emerging single-cluster peak pattern group also included *M. asiaticum*, *M. gordonae* chromotype I (Fig. 5-B) and *M. kansasii* (Fig. 5-B). *M. triviale* was the only mycobacterium species present in this group,

In these 21 isolates, m-PCR successfully amplified both target regions (the 500-bp fragment specific for *M. bovis* and the 245-bp fragment diagnostic for MTBC) in all isolates. A total of four spoligotypes were identified among the 21 *M. bovis* isolates. Two spoligotypes (SB0120 and SB0833) were described in the *M. bovis* spoligotype database (Brudey et al., 2006; www.mbovis.org). The other two represent novel, previously undescribed spoligotypes. The HPLC assay also identified the clinical *M. bovis* isolates as members of the

Fig. 4. Mycolic acid chromatograms from *M. bovis* BCG (INCQS0062) and *M. bovis* (ATCC 19210). \* peaks showing a high degree of separation (appearing as a "double peak"), named

methodology.

and it can be easily recognized (Fig. 5-B).

*Mycobacterium tuberculosis* complex (Figure 6).

according to Butler et al. (1991).

Fig. 5. Characteristic HPLC chromatograms of *Mycobacterium* species with late-emerging, simple, single-cluster peak patterns. **A**) *M. tuberculosis* H37Ra (ATCC 25177) and H37Rv (ATCC 27294) and *M. bovis* (ATCC 19210). **B**) *M. gordonae* chromotype I (ATCC 14470), *M. kansasii* (ATCC 12478) and *M. triviale* (ATCC 23292). \*peaks showing a high degree of separation (appearing as a "double peak"), named according to Butler et al. (1991). *M. triviale* strain: xpeaks showing a high degree of separation (appearing as a "double peak"), compared to the chromatogram profile described by Butler & Guthertz (2001).

Fig. 6. Representative reverse-phase HPLC chromatograms of mycolic acid methylesters from reference strains and isolates: (A) *M. bovis* ATCC 19210; (B) 21 clinical *M. bovis* isolates from dairy herds in Brazil.

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Identification of mycobacterium species using HPLC for mycolic acid analysis has proven to be rapid, reproducible and easily executed by several laboratories, making this approach one of the most appropriate methods to distinguish among the species. The separation capability using the modified method was superior to CDC patterns, and could be an alternative to allow discrimination between species with homologous HPLC chromatogram profiles.

#### **3. Conclusion**

Despite all efforts to control BTB, the disease persists, with serious implications for human health and the economy, particularly in the context of global trade. Because of the particular and complex characteristics of BTB, there is a growing realization that no single method by itself is sufficient to detect all the reactive animals in every stage of infection. Therefore, a multidisciplinary approach must be employed, using various categories of currently available methods. In a modern approach to the diagnosis and control of BTB, bacteriological, molecular, histopathological, and immunological assays must be employed, considering the indications, advantages, and disadvantages of each method. In this study we found that molecular diagnosis, combined with *ante mortem* and *post mortem* inspection, appeared to be a promising technique to improve the surveillance of BTB in herds, slaughterhouses, and the dairy industry, contributing to the success of the bovine tuberculosis eradication program.

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Identification of mycobacterium species using HPLC for mycolic acid analysis has proven to be rapid, reproducible and easily executed by several laboratories, making this approach one of the most appropriate methods to distinguish among the species. The separation capability using the modified method was superior to CDC patterns, and could be an alternative to allow discrimination between species with homologous HPLC chromatogram

Despite all efforts to control BTB, the disease persists, with serious implications for human health and the economy, particularly in the context of global trade. Because of the particular and complex characteristics of BTB, there is a growing realization that no single method by itself is sufficient to detect all the reactive animals in every stage of infection. Therefore, a multidisciplinary approach must be employed, using various categories of currently available methods. In a modern approach to the diagnosis and control of BTB, bacteriological, molecular, histopathological, and immunological assays must be employed, considering the indications, advantages, and disadvantages of each method. In this study we found that molecular diagnosis, combined with *ante mortem* and *post mortem* inspection, appeared to be a promising technique to improve the surveillance of BTB in herds, slaughterhouses, and the dairy industry, contributing to the success of the bovine

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**Part 3** 

**Improving Detection and Control of Resistances** 

