**3. Sputum tests in TB diagnosis**

considered positive when the difference in a number of spots between the test sample and

Both IGRAs have the ability to diagnose latent mycobacterial infection in 24 h following the one-time patient visit and submission of the blood sample to a health center [11]. The sensitivity of the IGRAs in adults is 80–90% when mycobacterium standard culture is used as gold [13]. In contrast to tuberculin skin test, IGRAs do not cross-react with Bacille Calmette-Guérin (BCG) antigen hence prior BCG vaccination does not cause false positives [11]. IGRAs cannot discriminate active tuberculosis from LTBI and its sensitivity is low in immunocompromised patients and children under the age of 5 years [13]. The cost of mycobacterium infection diagnosis by interferon- γ release assays is more than 40 USD per sample, which is higher compared to 10.56–25.97 USD for tuberculin skin test [14, 15]. This factor may influence the availability of and accessibility to this diagnostic service, especially in low-income countries.

Whole blood transcriptional signature can assist in distinguishing latent from active tuberculosis by showing features, which are not present in asymptomatic patients. Moreover, the transition from latent infection to active TB can be predicted by assessing the blood parameter [9]. This approach is important because of the reported 10–20% of detectable tuberculosis in

TB risk signature or global gene expression based on whole blood RNA genotyping polymerase chain reaction (PCR and sequencing) can be used to predict the chance of progression from latent to active TB. It has been reported that the expression of signature genes (that is quantities of functional RNA) increases toward active TB development in contrast to non-TB progressor (latent mycobacterium infection). For instance, Zak and colleagues [17] have reported a 16-gene signature with a sensitivity of 71.2% and specificity of 80% (95% CI: 66·6–75·2) at a 6 month time before tuberculosis diagnosis. This approach shows promising future of TB diagnosis as early as possible when samples are submitted since PCR and

Transcriptional blood signatures studies have been conducted in many parts of the world including high-income countries (US, UK, and German), middle-income countries (Brazil, South Africa, and Indonesia) and low-income countries (Kenya, Gambia, and Malawi) [9]. However, the procedure is under research and there is hope that it will become a handy tool

Proteome, the entire set of proteins produced or modified by a cell, system or organism, vary with time, requirement, stress or disease state that cell, and system or organism experiences. Studies on types and levels of plasma or serum proteins show variations between individuals with LTBI, active TB and those under different stages of treatment. These features can be used to differentiate LTBI from active TB and other perplexing diseases. Plasma or serum protein peaks are identified using high-performance liquid chromatography (HPLC)-tandem matrix-assisted laser desorption/ionization-TOF-MS (MALDI-TOF-MS) and the results are

**2.2. Mycobacterium global gene signature/transcriptional blood signatures**

asymptomatic individuals diagnosed with latent *M. tuberculosis* infection [16].

the negative control is greater than eight (8) [13].

4 Basic Biology and Applications of Actinobacteria

sequencing can give diagnostic results within 24 h.

in TB diagnosis and treatment decision.

**2.3. Blood proteomic profiling/fingerprinting**

Sputum of patients with pulmonary TB contains mycobacterium pathogens, which serves a role in transmission. The presence of the pathogens and their features such as viability, staining, or release of odor compounds are useful in diagnosis. Staining and culture are two common procedures, which use sputum sample to diagnose tuberculosis, especially active form. An emerging third method, which also utilizes sputum, is sniffing by trained African giant pouched rats, the *Cricetomys gambianus*.

However, manipulation of suspected mycobacterial samples should be handled in a containment facility to minimize the danger of exposure to personnel and the surrounding environment. If samples are collected far from the culture laboratory, and to prevent contamination, transport in CPC is recommended. Culturing of such samples should be within 7–8 days of collection and storage has given accurate results compared to culture from non-CPC stored samples [25]. Recent reports have shown good yield from CPC stored sputum samples for up to 21 days [26]. This is relevant in low-income countries where focal sample processing and culture centers are far from collection sites. Transport and storage times are worth noting as positivity from culture has been reported to be significantly affected beyond 2 weeks [27].

Paralleling of Diagnostic Endeavor for Control of Mycobacterial Infections and Tuberculosis

http://dx.doi.org/10.5772/intechopen.77316

During sample processing, the sputum-CPC mixture is normally concentrated by centrifuging at 4000 rpm for 15 min, supernatants poured off into a splash proof container. The sediments can then be mixed with 20 ml of sterile distilled water, suspended by inverting the tubes several times, and then centrifuged at 3500 rpm for 15 min. The supernatant is then removed with the remaining pellets re-suspended in water for the inoculums ready for culture. Two Löwenstein-Jensen slants, one containing 0.75% glycerol and the other 0.6% pyruvate are used for inoculation of the sediments and incubated at 37°C. Growth of Mycobacterial inoculum is examined weekly for 8 weeks; at this juncture cultures with no growth after 8 weeks should

Several methods are available for extraction of mycobacterial DNA and follow pre-existing protocols depending on the purpose. However, boiling a loop full of bacteria in 100 μL H2

at 80°C for 60 min can suffice to provide sufficient DNA for subsequent analysis. The crude DNA extracted as per protocol is ready for subsequent analyses or storage at −20°C until typing is done. The most used genotyping methods for mycobacterium DNA are both polymerase chain reaction (PCR)-based spoligotyping; and mycobacterial interspaced repetitive units-variable number tandem repeat (MIRU-VNTR) typing. These techniques are useful and can characterize mycobacterial DNA to strain level and later differentiate into sub-lineages that despite enabling the establishment of transmission chains [28] can assist the mapping of TB strains across regions. This can be done by matching strains in question to existing mycobacterium database at *Mtb* complex (http://www.MIRU-VNTRPlus.org/). Whole genome sequencing can be done to further characterize mycobacteria pathogens. Clinically, whole genome sequencing for *M*. *tuberculosis* has an advantage in the sense that it provides a more rapid and comprehensive view of the genotype of the pathogen, and with the timely and reliable prediction of drug susceptibility including detection of resistance mutations in clinical samples [29]. Despite the robust software and database tools, Whole genome sequencing needs to be developed for its full potential; it provides the highest resolution when investigating transmission events in possible outbreak scenarios [30]. All these methods are geared at improving treatment outcomes when integrated into routine diagnostic workflows, early reporting of drug resistance rather than dependence on phenotypic drug-susceptibility tests

O

7

*3.3.1. Sputum sample processing, culture, and identification*

be considered negative.

*3.3.2. DNA extraction procedures*

#### **3.1. Sputum sample collection**

In TB diagnosis, a collection of right samples is necessary. First morning samples are preferred as the overnight accumulation of secretions give better results. Sputum samples are normally collected from self-reporting presumptive TB cases at TB clinic. For samples collected far away from the diagnostic center, the samples are collected in transport medium, cetyl-pyridinium chloride (CPC) [23] for best subsequent results.

#### **3.2. Ziehl Neelsen and fluorescent staining**

A smear is made by even spreading of sputum over the glass slide to make a 20 mm by 10 mm eclipse shape. It may be dried and fixed by heat.

Smears from sputum samples are commonly stained using Ziehl Neelsen (ZN) or by fluorescence staining technique and examined microscopically for the presence of AFB. AFB testing is done as a screening procedure at the point of sample collection and on arrival at TB reference laboratory before culture and molecular confirmation. The waxy mycobacterial cell wall of mycobacterium is resistant to stain by conventional dyes and in one way the phenol-carbolfuchsin stain is forced to penetrate the cell wall by gentle heating underneath the slide flooded with carbolfuchsin. Then acid decolorizing solution is applied to remove the dye in cells, tissue, and any organisms in the smear except mycobacteria, which retain the dye. After decolorization, the smear is counterstained with either malachite green or methylene blue, which stains the background material green or blue, respectively. The contrasting background color enables the red Acid-Fast Bacilli to be visualized under a light microscope using the 100x oil immersion objective.

On the other hand, sputum smear can be stained with Auramine O or Auramine rhodamine dye and decolorized by a decolorizing agent (containing ethanol and hydrochloric acid). Counterstaining of the background by potassium permanganate enables visualization of orange-yellow acid-fast mycobacteria against a black back ground under a fluorescence microscope.

On comparison, fluorescent microscopy is faster in giving results (1 min) compared to ZN stain (4 min) and more sensitive (up to 97%) than ZN staining (up to 94%) and generally the accuracy of fluorescent microscopy is higher (Area Under the curve (AUC) = 96) compared to ZN staining (AUC = 94). However, due to resources constraint, ZN staining is more popular in low-income TB endemic areas [24].

#### **3.3. Mycobacterial culture and molecular characterization**

Culture isolation of mycobacteria is the gold standard method for TB diagnosis. Sputum smear stain positive samples are a good candidate for culture and molecular characterization. However, manipulation of suspected mycobacterial samples should be handled in a containment facility to minimize the danger of exposure to personnel and the surrounding environment. If samples are collected far from the culture laboratory, and to prevent contamination, transport in CPC is recommended. Culturing of such samples should be within 7–8 days of collection and storage has given accurate results compared to culture from non-CPC stored samples [25]. Recent reports have shown good yield from CPC stored sputum samples for up to 21 days [26]. This is relevant in low-income countries where focal sample processing and culture centers are far from collection sites. Transport and storage times are worth noting as positivity from culture has been reported to be significantly affected beyond 2 weeks [27].

#### *3.3.1. Sputum sample processing, culture, and identification*

During sample processing, the sputum-CPC mixture is normally concentrated by centrifuging at 4000 rpm for 15 min, supernatants poured off into a splash proof container. The sediments can then be mixed with 20 ml of sterile distilled water, suspended by inverting the tubes several times, and then centrifuged at 3500 rpm for 15 min. The supernatant is then removed with the remaining pellets re-suspended in water for the inoculums ready for culture. Two Löwenstein-Jensen slants, one containing 0.75% glycerol and the other 0.6% pyruvate are used for inoculation of the sediments and incubated at 37°C. Growth of Mycobacterial inoculum is examined weekly for 8 weeks; at this juncture cultures with no growth after 8 weeks should be considered negative.

#### *3.3.2. DNA extraction procedures*

staining, or release of odor compounds are useful in diagnosis. Staining and culture are two common procedures, which use sputum sample to diagnose tuberculosis, especially active form. An emerging third method, which also utilizes sputum, is sniffing by trained African

In TB diagnosis, a collection of right samples is necessary. First morning samples are preferred as the overnight accumulation of secretions give better results. Sputum samples are normally collected from self-reporting presumptive TB cases at TB clinic. For samples collected far away from the diagnostic center, the samples are collected in transport medium,

A smear is made by even spreading of sputum over the glass slide to make a 20 mm by 10 mm

Smears from sputum samples are commonly stained using Ziehl Neelsen (ZN) or by fluorescence staining technique and examined microscopically for the presence of AFB. AFB testing is done as a screening procedure at the point of sample collection and on arrival at TB reference laboratory before culture and molecular confirmation. The waxy mycobacterial cell wall of mycobacterium is resistant to stain by conventional dyes and in one way the phenol-carbolfuchsin stain is forced to penetrate the cell wall by gentle heating underneath the slide flooded with carbolfuchsin. Then acid decolorizing solution is applied to remove the dye in cells, tissue, and any organisms in the smear except mycobacteria, which retain the dye. After decolorization, the smear is counterstained with either malachite green or methylene blue, which stains the background material green or blue, respectively. The contrasting background color enables the red Acid-Fast Bacilli to be visualized under a light microscope using the 100x oil immersion objective. On the other hand, sputum smear can be stained with Auramine O or Auramine rhodamine dye and decolorized by a decolorizing agent (containing ethanol and hydrochloric acid). Counterstaining of the background by potassium permanganate enables visualization of orange-yellow acid-fast mycobacteria against a black back ground under a fluorescence

On comparison, fluorescent microscopy is faster in giving results (1 min) compared to ZN stain (4 min) and more sensitive (up to 97%) than ZN staining (up to 94%) and generally the accuracy of fluorescent microscopy is higher (Area Under the curve (AUC) = 96) compared to ZN staining (AUC = 94). However, due to resources constraint, ZN staining is more popular

Culture isolation of mycobacteria is the gold standard method for TB diagnosis. Sputum smear stain positive samples are a good candidate for culture and molecular characterization.

giant pouched rats, the *Cricetomys gambianus*.

**3.2. Ziehl Neelsen and fluorescent staining**

eclipse shape. It may be dried and fixed by heat.

cetyl-pyridinium chloride (CPC) [23] for best subsequent results.

**3.1. Sputum sample collection**

6 Basic Biology and Applications of Actinobacteria

microscope.

in low-income TB endemic areas [24].

**3.3. Mycobacterial culture and molecular characterization**

Several methods are available for extraction of mycobacterial DNA and follow pre-existing protocols depending on the purpose. However, boiling a loop full of bacteria in 100 μL H2 O at 80°C for 60 min can suffice to provide sufficient DNA for subsequent analysis. The crude DNA extracted as per protocol is ready for subsequent analyses or storage at −20°C until typing is done. The most used genotyping methods for mycobacterium DNA are both polymerase chain reaction (PCR)-based spoligotyping; and mycobacterial interspaced repetitive units-variable number tandem repeat (MIRU-VNTR) typing. These techniques are useful and can characterize mycobacterial DNA to strain level and later differentiate into sub-lineages that despite enabling the establishment of transmission chains [28] can assist the mapping of TB strains across regions. This can be done by matching strains in question to existing mycobacterium database at *Mtb* complex (http://www.MIRU-VNTRPlus.org/). Whole genome sequencing can be done to further characterize mycobacteria pathogens. Clinically, whole genome sequencing for *M*. *tuberculosis* has an advantage in the sense that it provides a more rapid and comprehensive view of the genotype of the pathogen, and with the timely and reliable prediction of drug susceptibility including detection of resistance mutations in clinical samples [29]. Despite the robust software and database tools, Whole genome sequencing needs to be developed for its full potential; it provides the highest resolution when investigating transmission events in possible outbreak scenarios [30]. All these methods are geared at improving treatment outcomes when integrated into routine diagnostic workflows, early reporting of drug resistance rather than dependence on phenotypic drug-susceptibility tests [31]. In our context, we will discuss other characterization techniques, particularly spoligotyping, and MIRU-VNTR genotyping methods as our main focus rather than mycobacterium whole genome sequencing.

mainly in intergenic regions dispersed throughout the *M*. *tuberculosis* genome [28, 41]. The method combines the analysis of multiplex PCRs for the target loci on a fluorescence-based DNA analyzer with computerized automation of the genotyping [42]. Initially, the genotyping system used only 12 loci for epidemiological molecular studies in combination with spoligotyping, but now a more discriminatory 24 loci MIRU-VNTR is in place. This current genotyping system in combination with spoligotyping has been found to be 40% superior in a number of types among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci [28]. Nevertheless, researchers still continue to optimize the 24-loci MIRU-VNTR genotyping in an attempt to reduce the turnaround time of typing and financial burden [33] although the MIRU-VNTR typing manual [43] remains primary and

Paralleling of Diagnostic Endeavor for Control of Mycobacterial Infections and Tuberculosis

http://dx.doi.org/10.5772/intechopen.77316

9

The standardized 24 loci MIRU-VNTR typing protocol by Supply et al. [42] is performed using primers that amplify 24 polymorphic loci on the mycobacterial genome per DNA isolate. The number of tandem repeat units present at each locus is then calculated from the size of DNA fragments according to a standardized table (http://www.MIRU-VNTRplus.org). The results can be expressed in digital format where each number represents the number of repeat copies at a particular locus. Phylogenetic analysis and creation of dendrograms can then be done using MIRU-VNTR*plus* (http://www.MIRU-VNTRPlus.org/) to generate a categorical-based NJ-Tree dendrogram to enable comparison of strain genotypes [44]. Creation of dendrogram

In recent years diagnosis of pulmonary TB in humans has taken a turn to involve Trained African giant pouched rats (*Cricetomys gambianus*). The procedure takes advantage of the ability of the African giant pouched rats to detect and discriminate odor of volatile compounds produced by mycobacteria [45]. Weetjens and his colleges extended the dimension of research

The rats are trained to sniff heat-inactivated sputum from presumptive TB patients for the purpose of discriminating positive samples from negatives. According to Weetjens and colleagues [46], the rats are domesticated, bred and that training is done to the young rats. At the age of 4 weeks, young rats are identified and assigned to a specific trainer. This is accompanied by socialization and habituation learning. Then the rats are trained to locate the sniffing hole in the cage and sniffing at the age of 8 weeks. Discrimination of odor is introduced and multiple sample evaluation follows, at the age of 8 months the rats start operational training. During training, the rats learn to associate odor of TB volatiles with reward (food: peanut or banana). The trained rats sniff holes with sputum samples in the cage. The negative samples are sniffed for less than a second and ignored while for the positive samples, the rat will fix their nose at the hole for at least 5 seconds. In this exercise, each of positive sample detection

The rats can detect *M*. *tuberculosis*-specific volatiles such as nicotinate, methyl para-anisate, and ortho-phenylanisole from sputum infected with MTBC, *M*. *avium*, *M*. *intracellulare* and other NTM [47]. The detection has been reported to be at 80.4% sensitivity, 72.4% specificity,

from phylogenetic analysis enables the establishment of transmission links.

key guide to all MIRU-VNTR typing of MTBC strains.

**3.4. Sputum sniffing by African giant pouched rats**

from landmine detection to TB diagnosis [46].

is accompanied by a reward in form of food [46].

#### *3.3.3. Spacer oligonucleotide typing (Spoligotyping)*

In humans, TB infection is mostly due to MTBC, which comprises of a group of seven genetically similar species [32] namely *M*. *tuberculosis*, *M. bovis*, M. africanum, *M*. *microti*, *M*. *canettii*, *M*. *pinnipedii*, and *M*. *caprae*. To differentiate such genetically closely related species, special molecular techniques have been in place for quite some time now. Fortunately, despite the high genetic homogeneity among the members, they display divergent phenotypes, eliciting different pathologies, and while some show a degree of host specificities [33]. These qualities have made it possible to differentiate mycobacteria species to strain level and epidemiologically, determining their transmission chain using molecular techniques. Spoligotyping is among PCR-based technique that is used in combination with other molecular methods to establish strain variability in a given population. Using commercially available kits, spoligotyping can be done with reference to existing protocols described previously by Kamerbeek et al. [34]. This PCR-based fingerprinting method detects the presence or absence of 43 variable spacer sequences situated between short direct repeat (DR) sequences in the *M. tuberculosis* genome. The DNA from reference *M*. *tuberculosis* H37Rv and *M. bovis* BCG clones are commonly used as positive controls while autoclaved ultrapure water is used as a negative control. Visualization of presence (black squares) or absence (blank squares) of variable spacer sequences on film is achieved after incubation with streptavidin-peroxidase and detection of hybridized DNA using chemiluminescent detection liquid followed by exposure to radiography uses electromagnetic radiations (x-ray) film. Resulting spoligotypes are normally reported in octal and binary formats and compared to existing patterns in an international spoligotyping database profiles (SpolDB4.0) [35] available at http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/). Spoligotype patterns can then be grouped as spoligotype international types (SITs) if they share identical spoligotype patterns with patterns present in the existing database. Such strain comparisons using SITs are used to identify TB strains circulating in the population as well as new strains and assign names for new strains, which cannot be found in previous studies where possible [35, 36]. By using spoligotyping, it is possible to map TB strains with their diversity within and between regions [37], not only revealing differences in circulating *M*. *tuberculosis* strains [38] but also identification of new strains [39] even within the country in endemic areas. Further, molecular characterization has yielded molecular type patterns suggestive of similar strains in humans and in wild ungulates [40].

#### *3.3.4. Mycobacterial interspaced repetitive units-variable number tandem repeat (MIRU-VNTR) typing*

This is a PCR-based method that exploits the presence of interspaced repetitive DNA in mycobacterium and other genomes to characterize mycobacteria strains and within strains circulating in a given population. In combination with spoligotyping, the method forms a large-scale, high-throughput genotyping of *M*. *tuberculosis* [28]. The method is based on the presence of more extensive loci that contain variable number tandem repeat (VNTR) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) located mainly in intergenic regions dispersed throughout the *M*. *tuberculosis* genome [28, 41]. The method combines the analysis of multiplex PCRs for the target loci on a fluorescence-based DNA analyzer with computerized automation of the genotyping [42]. Initially, the genotyping system used only 12 loci for epidemiological molecular studies in combination with spoligotyping, but now a more discriminatory 24 loci MIRU-VNTR is in place. This current genotyping system in combination with spoligotyping has been found to be 40% superior in a number of types among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci [28]. Nevertheless, researchers still continue to optimize the 24-loci MIRU-VNTR genotyping in an attempt to reduce the turnaround time of typing and financial burden [33] although the MIRU-VNTR typing manual [43] remains primary and key guide to all MIRU-VNTR typing of MTBC strains.

The standardized 24 loci MIRU-VNTR typing protocol by Supply et al. [42] is performed using primers that amplify 24 polymorphic loci on the mycobacterial genome per DNA isolate. The number of tandem repeat units present at each locus is then calculated from the size of DNA fragments according to a standardized table (http://www.MIRU-VNTRplus.org). The results can be expressed in digital format where each number represents the number of repeat copies at a particular locus. Phylogenetic analysis and creation of dendrograms can then be done using MIRU-VNTR*plus* (http://www.MIRU-VNTRPlus.org/) to generate a categorical-based NJ-Tree dendrogram to enable comparison of strain genotypes [44]. Creation of dendrogram from phylogenetic analysis enables the establishment of transmission links.

#### **3.4. Sputum sniffing by African giant pouched rats**

[31]. In our context, we will discuss other characterization techniques, particularly spoligotyping, and MIRU-VNTR genotyping methods as our main focus rather than mycobacterium

In humans, TB infection is mostly due to MTBC, which comprises of a group of seven genetically similar species [32] namely *M*. *tuberculosis*, *M. bovis*, M. africanum, *M*. *microti*, *M*. *canettii*, *M*. *pinnipedii*, and *M*. *caprae*. To differentiate such genetically closely related species, special molecular techniques have been in place for quite some time now. Fortunately, despite the high genetic homogeneity among the members, they display divergent phenotypes, eliciting different pathologies, and while some show a degree of host specificities [33]. These qualities have made it possible to differentiate mycobacteria species to strain level and epidemiologically, determining their transmission chain using molecular techniques. Spoligotyping is among PCR-based technique that is used in combination with other molecular methods to establish strain variability in a given population. Using commercially available kits, spoligotyping can be done with reference to existing protocols described previously by Kamerbeek et al. [34]. This PCR-based fingerprinting method detects the presence or absence of 43 variable spacer sequences situated between short direct repeat (DR) sequences in the *M. tuberculosis* genome. The DNA from reference *M*. *tuberculosis* H37Rv and *M. bovis* BCG clones are commonly used as positive controls while autoclaved ultrapure water is used as a negative control. Visualization of presence (black squares) or absence (blank squares) of variable spacer sequences on film is achieved after incubation with streptavidin-peroxidase and detection of hybridized DNA using chemiluminescent detection liquid followed by exposure to radiography uses electromagnetic radiations (x-ray) film. Resulting spoligotypes are normally reported in octal and binary formats and compared to existing patterns in an international spoligotyping database profiles (SpolDB4.0) [35] available at http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/). Spoligotype patterns can then be grouped as spoligotype international types (SITs) if they share identical spoligotype patterns with patterns present in the existing database. Such strain comparisons using SITs are used to identify TB strains circulating in the population as well as new strains and assign names for new strains, which cannot be found in previous studies where possible [35, 36]. By using spoligotyping, it is possible to map TB strains with their diversity within and between regions [37], not only revealing differences in circulating *M*. *tuberculosis* strains [38] but also identification of new strains [39] even within the country in endemic areas. Further, molecular characterization has yielded molecular

type patterns suggestive of similar strains in humans and in wild ungulates [40].

*3.3.4. Mycobacterial interspaced repetitive units-variable number tandem repeat (MIRU-*

This is a PCR-based method that exploits the presence of interspaced repetitive DNA in mycobacterium and other genomes to characterize mycobacteria strains and within strains circulating in a given population. In combination with spoligotyping, the method forms a large-scale, high-throughput genotyping of *M*. *tuberculosis* [28]. The method is based on the presence of more extensive loci that contain variable number tandem repeat (VNTR) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) located

whole genome sequencing.

8 Basic Biology and Applications of Actinobacteria

*VNTR) typing*

*3.3.3. Spacer oligonucleotide typing (Spoligotyping)*

In recent years diagnosis of pulmonary TB in humans has taken a turn to involve Trained African giant pouched rats (*Cricetomys gambianus*). The procedure takes advantage of the ability of the African giant pouched rats to detect and discriminate odor of volatile compounds produced by mycobacteria [45]. Weetjens and his colleges extended the dimension of research from landmine detection to TB diagnosis [46].

The rats are trained to sniff heat-inactivated sputum from presumptive TB patients for the purpose of discriminating positive samples from negatives. According to Weetjens and colleagues [46], the rats are domesticated, bred and that training is done to the young rats. At the age of 4 weeks, young rats are identified and assigned to a specific trainer. This is accompanied by socialization and habituation learning. Then the rats are trained to locate the sniffing hole in the cage and sniffing at the age of 8 weeks. Discrimination of odor is introduced and multiple sample evaluation follows, at the age of 8 months the rats start operational training. During training, the rats learn to associate odor of TB volatiles with reward (food: peanut or banana). The trained rats sniff holes with sputum samples in the cage. The negative samples are sniffed for less than a second and ignored while for the positive samples, the rat will fix their nose at the hole for at least 5 seconds. In this exercise, each of positive sample detection is accompanied by a reward in form of food [46].

The rats can detect *M*. *tuberculosis*-specific volatiles such as nicotinate, methyl para-anisate, and ortho-phenylanisole from sputum infected with MTBC, *M*. *avium*, *M*. *intracellulare* and other NTM [47]. The detection has been reported to be at 80.4% sensitivity, 72.4% specificity, and 73.9% accuracy [47]. Sample evaluation results are potentiated by use of multiple rats (e.g. group of four) and positive indication by at least one rat criteria has been reported to be more sensitive than multiple positive indications [48].

due to the HIV or immunosuppressive chemotherapy, co-infections with viral, bacterial and fungal diseases affecting lymphoid organs (lymphoma, chronic lymphoid leukemia sarcoidosis), metabolic derangement (chronic renal failure), stress (surgery, burns, and mental illness), and live vaccination (measles, mumps, and polio) [53, 54]. False positive reactions are more commonly attributed to co-infection or pre-exposure to other related NTM in cattle and

Paralleling of Diagnostic Endeavor for Control of Mycobacterial Infections and Tuberculosis

http://dx.doi.org/10.5772/intechopen.77316

11

Despite of the several major limitations, TST has been, until recently, a useful procedure for detecting LTBI [57] and most widely used test for control of Bovine TB due to the low-cost, low logistical demands, well-documented use, and its ability to screen the entire cattle populations [52]. Due to the limitations of the sensitivity and specificity of the skin tests, newer ancillary in vitro diagnostic assays for latent tuberculosis has been developed, which mea-

Radiography uses electromagnetic radiations (x-rays) to visualize internal body organs. Chest radiography is used for imaging lungs, airways, ribs, heart, and diaphragm. The rays are allowed to pass through tissues and captured by a film. Variation of absorption of x-rays by different tissues enables contrasting visualization. Hard tissues, for example, bones absorb more rays and appear dense while soft tissues allow more passage of rays and appear light.

According to WHO [58] chest radiography is indicated as a pulmonary TB diagnostic tool for triaging, screening and as a diagnostic aid when clinical pulmonary TB cannot be confirmed bacteriologically. Chest radiography can also be used in the evaluation of TB treatment response. It is, therefore, part of an algorithm within health care system. It can be used to distinguish between active and inactive pulmonary TB based on a temporal evaluation of radiographs whereby the latter is characterized by stable radiographic pictures for 6 months [59].

Most common chest radiographic findings of TB include lymphadenopathy, parenchymal opacities, obstructive atelectasis, pleural effusion, cavitations, and tuberculomas [59]. Chest radiography has high-sensitivity (97%, 95% CI 0.90–1.00) and low-specificity (67%, 95% CI 0.64–0.70) [60]. It can detect any abnormality in the chest including those related to other non-TB pathological conditions. Other imaging techniques such as computed tomography (CT) and Magnetic resonance imaging (MRI) are superior to chest radiography and can be used for reference. It is important to note that besides the low specificity, chest radiography is expensive in terms of equipment and skilled labor, especially in low-income endemic areas.

Advocacy toward non-invasive, non-sputum simple TB diagnosis has pushed research into different directions including the use of urine sample from presumptive TB patients. There are a number of target TB biomarkers in urine including volatile compounds, proteins, and

sures the production of interferons in whole blood upon stimulation with PPD [52].

Knowledge of anatomy is important for diagnostic interpretation.

**5. Urine sample tests in TB diagnosis**

TB antigen.

human [55] and BCG vaccination in human [56].

**4.2. Imaging in TB diagnosis**

*Cricetomys gambianus* are resistant to TB infection [48]. The rats can detect mycobacteria in sputum in as low concentration as a few bacteria in less than 10 sec [49]. In contrast to a trained microscopist who can examine less than 50 samples a day, the rats can detect hundreds of samples per day before exhaustion [50]. These features make sputum sniffing by the giant African rats a potential rapid screening test. Such technique has been used in Tanzania and Mozambique as a second line screening after ZN staining sputum smear microscopy where 44% increase in TB case detection rate has been reported [48]. However, sputum sniffing by the African giant rats does not guarantee 100% accurate TB diagnosis. Although this disqualifies it as a stand-alone TB diagnostic or first line screening test for presumptive test it can be useful in increasing TB cases among smear-negatives especially in low-income TB endemic countries where diagnostics capabilities are limited [51].

Moreover, TB diagnosis by sniffing rats works in active TB, it may not be handy in latent TB cases. Furthermore, the rats are living creature whose health status needs attention and any anatomical, physiological, or functional deformity may impair TB diagnosis.
