**Part 3**

## **Improving Detection and Control of Resistances**

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

Zanini, M.S.; Moreira, E.C.; Lopes, M.T.P. & Salas, C.E. (1998). Detection of *Mycobacterium bovis* in milk by polymerase chain reaction. *J. Vet Med* 45, 1129-1132, 1998. Zumarraga, M.J.; Meickle, V.; Bernardelli, A.; Abdala, A.; Tarabla, H.; Romano, M. I. &

Janeiro, Vol. 96.

and Genotyping in Isolates from Southeast Brazil by Spolygotyping and Restriction Fragment Length Polymorphism. Mem Inst Oswaldo Cruz, Rio de

Cataldi, A. (2005). Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection. *J.Vet. Diagn. Invest.* 17, 232-238.

**21** 

*2Z-BioMed, Inc.,* 

*1China 2USA* 

**Survey and Molecular Characterization of Drug-**

Tuberculosis (TB) is one of the leading infectious disease killers in the world, especially in developing countries. The increasing frequency of human-to-human transmission of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of *Mycobacterium tuberculosis* poses challenges for effective therapeutic options and infection control. MDR-TB is defined as a form of TB that is resistant to at least isoniazid and rifampin, which are used to treat all TB patients. XDR-TB is a form of TB that is resistant to at least rifampicin and isoniazid in addition to any fluoroquinolone, and at least one of the three injectable secondline anti-TB drugs (amikacin, capreomycin and kanamycin). Based on the WHO Report 2010, China was ranked number one among high burden countries in terms of the estimated number of MDR-TB cases in 2008, and number two in terms of total numbers of TB cases in the world with the incidence rate per capita of 99 per 100,000 populations (WHO, 2011). Guizhou province is one of the highest-incidence-rate areas in China, and its prevalence of drug-resistant TB is higher than most of other provinces of China (Chen et al., 2011). The Affiliated Hospital of Zunyi Medical College is one of the specialized centers in Guizhou certified by the provincial government for the treatment of MDR-TB patients. In 2010, more than 15,000 TB patients were treated at the hospital. In order to determine the molecular characteristics of drug-resistant TB and assist in making informed TB treatment decisions for TB patients in the Zunyi area of Guizhou province, hundreds of *M. tuberculosis* clinical isolates were collected at the Affiliated Hospital of Zunyi Medical College and used for the systematic surveillance and other studies. We have made some progresses in the detection and molecular characterization of drug-resistant *M. tuberculosis* clinical isolates using different research methods such as *M. tuberculosis* culture, spoligotyping, gene sequencing, proteomics and drug susceptibility testing against first-line and second-line anti-

**1. Introduction** 

tuberculosis drugs.

Corresponding authors

 \*

**Resistant** *M. tuberculosis* **Clinical Isolates** 

Ling Chen1\*, Hong Zhang1,2\*, Jian-Yong Zhang1, Na-Na Li1, Mei Liu1,

**from Zunyi, Guizhou Province of China** 

Zhao-Jing Zong1, Jian-Hua Wang1 and Sheng-Lan Wang1 *1 Laboratory of Respiratory Medicine, Department of Respiratory Medicine* 

*Affiliated Hospital of Zunyi Medical College,* 

## **Survey and Molecular Characterization of Drug-Resistant** *M. tuberculosis* **Clinical Isolates from Zunyi, Guizhou Province of China**

Ling Chen1\*, Hong Zhang1,2\*, Jian-Yong Zhang1, Na-Na Li1, Mei Liu1, Zhao-Jing Zong1, Jian-Hua Wang1 and Sheng-Lan Wang1 *1 Laboratory of Respiratory Medicine, Department of Respiratory Medicine Affiliated Hospital of Zunyi Medical College, 2Z-BioMed, Inc., 1China 2USA* 

#### **1. Introduction**

Tuberculosis (TB) is one of the leading infectious disease killers in the world, especially in developing countries. The increasing frequency of human-to-human transmission of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of *Mycobacterium tuberculosis* poses challenges for effective therapeutic options and infection control. MDR-TB is defined as a form of TB that is resistant to at least isoniazid and rifampin, which are used to treat all TB patients. XDR-TB is a form of TB that is resistant to at least rifampicin and isoniazid in addition to any fluoroquinolone, and at least one of the three injectable secondline anti-TB drugs (amikacin, capreomycin and kanamycin). Based on the WHO Report 2010, China was ranked number one among high burden countries in terms of the estimated number of MDR-TB cases in 2008, and number two in terms of total numbers of TB cases in the world with the incidence rate per capita of 99 per 100,000 populations (WHO, 2011). Guizhou province is one of the highest-incidence-rate areas in China, and its prevalence of drug-resistant TB is higher than most of other provinces of China (Chen et al., 2011). The Affiliated Hospital of Zunyi Medical College is one of the specialized centers in Guizhou certified by the provincial government for the treatment of MDR-TB patients. In 2010, more than 15,000 TB patients were treated at the hospital. In order to determine the molecular characteristics of drug-resistant TB and assist in making informed TB treatment decisions for TB patients in the Zunyi area of Guizhou province, hundreds of *M. tuberculosis* clinical isolates were collected at the Affiliated Hospital of Zunyi Medical College and used for the systematic surveillance and other studies. We have made some progresses in the detection and molecular characterization of drug-resistant *M. tuberculosis* clinical isolates using different research methods such as *M. tuberculosis* culture, spoligotyping, gene sequencing, proteomics and drug susceptibility testing against first-line and second-line antituberculosis drugs.

<sup>\*</sup> Corresponding authors

Survey and Molecular Characterization of Drug- Resistant

unwanted microorganisms.

**2.1.1 Solutions for acid-fast staining** 

**2.1.2 Acid-fast staining procedure** 

slide.

Staining solution: 0.8% carbolfuchsin solution

Following is the acid-fast staining procedure:

7. Interpret acid-fast staining results

varies from 20% to 80% (Goodwin, 2007).

**2.2 Drug susceptibility testing against different drugs** 

conditions for direct testing of sputum speciments (WHO, 2010).

Destaining solution: 3% hydrochloric acid- alcohol solution Counterstaining solution: 0.3‰ methylene blue solution

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 437

specimen produced by a deep cough of the patient should have a volume of 5 to 10 ml, although less volumes are acceptable if the quality is good. All sputum specimens should be transported to the laboratory and processed as soon as possible after collection. If delay is unavoidable, the sputum specimens should be refrigerated to inhibit the growth of

The procedures for acid-fast staining were based on the modified method described by Hu et al. (Hu et al., 2008). To prepare a smear for AFB, appropriate specimens were spread uniformly on a microscope slide, which was then fixed at 80°C for 15 minutes. Smears were stained with a carbolfuchsin stain and were examined using a 100× oil immersion objective on a light microscope. In smears stained with carbolfuchsin, AFB typically appear as purple to red slightly curved, short or long rods (2-8μM). They may also appear beaded or banded.

1. Turn on the thermostat-controlled water-bath and set up the temperature to 56°C.

3. Add carbolfuchsin dye to the heat-fixed smear on slide for 15min, and take out the

4. Allow the slide to cool to room temperature, then, decolorize slide by adding the fresh

Smears should be carefully examined with a minimum of 300 fields, and three horizontal sweeps of a smear should be performed (Goodwin, 2007). The positive smear requires the cut-off of at least 5000 bacilli/ml. The overall sensitivity of the acid-fast staining method

WHO has endorsed commercial liquid culture systems and molecular line-probe assays as gold standards for rapid detection of MDR-TB; however, because of technical complexity, cost and the requirement of sophisticated lab infrastructure, their uses have been limited in many resource-constrained settings (WHO, 2010). Several noncommercial culture and DST methods have been developed for those resource-constrained settings, and assessed by WHO. Based on the testing results, WHO recently recommended MODS (microscopic observation of drug susceptibility) and NRA (nitrate reductase assay) under certain

2. Air dry smear on the slide, flame fix, and transfer it to the water-bath.

6. Examine the slide by a 100× oil immersion objective on a light microscope.

destaining solution, and wait until red color disappears. 5. Add counterstaining solution for 1 min, wash, and dry.

In this chapter, we will briefly review the recent advances in the area of MDR-TB research; provide detailed descriptions about our research methods and the summary of our current research progresses. This chapter is written for researchers, scientists and physicians in academic institutions, clinical laboratories, pharmaceutical companies and research hospitals. This chapter will also be suitable for readers such as undergraduate, graduate and medical students who wish to learn more about the drug-resistant *M. tuberculosis*, especially MDR/XDR-TB, and some of the current research methods used for the determination of the genetic diversity and anti-tuberculosis drug susceptibility profile of *M. tuberculosis* clinical isolates, and the detection of drug-resistant *M. tuberculosis*.

#### **2. Survey of drug-resistant** *M. tuberculosis* **clinical isolates**

Mycobacteria are aerobes and grow most successfully in tissues with high oxygen content. The suitable growth temperature for mycobacteria is 37°C. Mycobacteria are "acid-fast bacilli" (AFB) because of their lipid-rich cell walls, which are relatively impermeable to various basic dyes unless the dyes are combined with phenol. Once stained, mycobacteria are resistant to decolorization with acidified organic solvents. *M. tuberculosis* is an intracellular pathogen usually infecting mononuclear phagocytes, and slow-growing with a generation time of 18 to 22 hours. The diagnosis of tuberculosis usually requires the detection of acid-fast bacilli in sputum via the acid-fast staining method (Ziehl-Neelsen method), which uses carbolfuchsin as the stain, acid-alcohol as the destaining solution and methylene blue as the counterstain. The culture confirmation of *M. tuberculosis* is the gold standard for diagnosis of tuberculosis and the drug susceptibility testing (DST) provides the basis for surveillance of drug-resistant TB and for physicians to adjust chemotherapy.

Even though TB laboratory services formed an essential part of the DOTS (Directly Observed Treatment Short course) strategy for National Tuberculosis Programs, it was often the most neglected component of these programs because of the absence of standardized techniques which complicated the activities of new laboratory services. Based on those considerations, WHO prepared guidelines in 1998 for laboratory services for the framework of National Tuberculosis Programs (WHO, 1998). The guidelines included three detailed manuals, two of which focused on the technical aspects of TB microscopy and culture, and a third one dealt with laboratory management including lab safety and proficiency testing. These manuals were specifically developed for use in low- and middle-income countries with high TB prevalence and incidence rates (WHO, 1998). Most of the methods used in our laboratory at the Affiliated Hospital of Zunyi Medical College and described in this chapter are based on these three manuals.

#### **2.1 Collection of sputum specimens from patients**

*M. tuberculosis* may be isolated from various clinical specimens, including respiratory specimens such as sputum, body fluids and body tissues (Goodwin, 2007). TB clinical strains from sputum of patients with active pulmonary tuberculosis were collected at the Affiliated Hospital of Zunyi Medical College. Sputum specimens were collected into a sterile singlecollection Universal container (28ml) with a tightly fitted lid. A good sputum specimen is considered to be recently-discharged materials from the bronchial tree of the patient, with a minimum amount of oral or nasal materials (WHO, 1998). In an ideal situation, a sputum specimen produced by a deep cough of the patient should have a volume of 5 to 10 ml, although less volumes are acceptable if the quality is good. All sputum specimens should be transported to the laboratory and processed as soon as possible after collection. If delay is unavoidable, the sputum specimens should be refrigerated to inhibit the growth of unwanted microorganisms.

#### **2.1.1 Solutions for acid-fast staining**

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

In this chapter, we will briefly review the recent advances in the area of MDR-TB research; provide detailed descriptions about our research methods and the summary of our current research progresses. This chapter is written for researchers, scientists and physicians in academic institutions, clinical laboratories, pharmaceutical companies and research hospitals. This chapter will also be suitable for readers such as undergraduate, graduate and medical students who wish to learn more about the drug-resistant *M. tuberculosis*, especially MDR/XDR-TB, and some of the current research methods used for the determination of the genetic diversity and anti-tuberculosis drug susceptibility profile of *M. tuberculosis* clinical

Mycobacteria are aerobes and grow most successfully in tissues with high oxygen content. The suitable growth temperature for mycobacteria is 37°C. Mycobacteria are "acid-fast bacilli" (AFB) because of their lipid-rich cell walls, which are relatively impermeable to various basic dyes unless the dyes are combined with phenol. Once stained, mycobacteria are resistant to decolorization with acidified organic solvents. *M. tuberculosis* is an intracellular pathogen usually infecting mononuclear phagocytes, and slow-growing with a generation time of 18 to 22 hours. The diagnosis of tuberculosis usually requires the detection of acid-fast bacilli in sputum via the acid-fast staining method (Ziehl-Neelsen method), which uses carbolfuchsin as the stain, acid-alcohol as the destaining solution and methylene blue as the counterstain. The culture confirmation of *M. tuberculosis* is the gold standard for diagnosis of tuberculosis and the drug susceptibility testing (DST) provides the basis for surveillance of drug-resistant TB and for physicians to adjust chemotherapy.

Even though TB laboratory services formed an essential part of the DOTS (Directly Observed Treatment Short course) strategy for National Tuberculosis Programs, it was often the most neglected component of these programs because of the absence of standardized techniques which complicated the activities of new laboratory services. Based on those considerations, WHO prepared guidelines in 1998 for laboratory services for the framework of National Tuberculosis Programs (WHO, 1998). The guidelines included three detailed manuals, two of which focused on the technical aspects of TB microscopy and culture, and a third one dealt with laboratory management including lab safety and proficiency testing. These manuals were specifically developed for use in low- and middle-income countries with high TB prevalence and incidence rates (WHO, 1998). Most of the methods used in our laboratory at the Affiliated Hospital of Zunyi Medical College and described in this chapter

*M. tuberculosis* may be isolated from various clinical specimens, including respiratory specimens such as sputum, body fluids and body tissues (Goodwin, 2007). TB clinical strains from sputum of patients with active pulmonary tuberculosis were collected at the Affiliated Hospital of Zunyi Medical College. Sputum specimens were collected into a sterile singlecollection Universal container (28ml) with a tightly fitted lid. A good sputum specimen is considered to be recently-discharged materials from the bronchial tree of the patient, with a minimum amount of oral or nasal materials (WHO, 1998). In an ideal situation, a sputum

isolates, and the detection of drug-resistant *M. tuberculosis*.

are based on these three manuals.

**2.1 Collection of sputum specimens from patients** 

**2. Survey of drug-resistant** *M. tuberculosis* **clinical isolates** 

Staining solution: 0.8% carbolfuchsin solution

Destaining solution: 3% hydrochloric acid- alcohol solution

Counterstaining solution: 0.3‰ methylene blue solution

#### **2.1.2 Acid-fast staining procedure**

The procedures for acid-fast staining were based on the modified method described by Hu et al. (Hu et al., 2008). To prepare a smear for AFB, appropriate specimens were spread uniformly on a microscope slide, which was then fixed at 80°C for 15 minutes. Smears were stained with a carbolfuchsin stain and were examined using a 100× oil immersion objective on a light microscope. In smears stained with carbolfuchsin, AFB typically appear as purple to red slightly curved, short or long rods (2-8μM). They may also appear beaded or banded. Following is the acid-fast staining procedure:


Smears should be carefully examined with a minimum of 300 fields, and three horizontal sweeps of a smear should be performed (Goodwin, 2007). The positive smear requires the cut-off of at least 5000 bacilli/ml. The overall sensitivity of the acid-fast staining method varies from 20% to 80% (Goodwin, 2007).

#### **2.2 Drug susceptibility testing against different drugs**

WHO has endorsed commercial liquid culture systems and molecular line-probe assays as gold standards for rapid detection of MDR-TB; however, because of technical complexity, cost and the requirement of sophisticated lab infrastructure, their uses have been limited in many resource-constrained settings (WHO, 2010). Several noncommercial culture and DST methods have been developed for those resource-constrained settings, and assessed by WHO. Based on the testing results, WHO recently recommended MODS (microscopic observation of drug susceptibility) and NRA (nitrate reductase assay) under certain conditions for direct testing of sputum speciments (WHO, 2010).

Survey and Molecular Characterization of Drug- Resistant

drying of the medium (WHO, 1998).

**2.2.1.3 Digestion and decontamination** 

**2.2.2 Drug susceptibility testing** 

LVF, 2µg/ml.

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 439

Media containing different drugs were distributed to sterile universal screw-cap centrifuge tubes (7ml medium per tube); tubes were labeled and caps were tightened. The labeled tubes were placed in an oven at a slanted position (30° angle) and baked at 85°C for 50 min. Baked tubes were cooled down to room temperature and stored at 4°C refrigerator until use. Since the medium was prepared with sterile precautions, this heating process was to solidify the medium instead of sterilizing it. The quality of the egg-based media deteriorates when the baking temperature is too high or the baking time is too long. The L-J medium should be dated and stored in the refrigerator for up to 4 weeks if the caps are tightened to prevent

Drug Dissolvent Diluent Concentration (μg/ml)

*M. tuberculosis* grows slowly and takes four to eight weeks or longer to give visible colonies. Cultures are usually made in bottles because of the long incubation time required. The bottles are tightly capped to prevent drying of the cultures. The majority of clinical specimens submitted to the clinical laboratory are contaminated to varying degrees by more rapidly growing normal flora. These would rapidly overgrow the entire surface of the medium before the *M. tuberculosis* start to grow (Goodwin, 2007). Therefore, specimens must be subjected to digestion and decontamination that liquefies the organic debris and eliminates the unwanted normal flora. All currently available digesting and decontaminating methods are to some extent toxic to *M. tuberculosis*. Therefore, to ensure the survival of the maximum number of *M. tuberculosis* in the specimen, the digestion and

We have collected hundreds of TB culture samples from TB patients, and used them for the DNA extraction and drug-susceptibility testing against seven anti-TB drugs: rifampicin (RIF), isoniazid (INH), streptomycin (STR), ethambutol (EMB), capreomycin (CPM), ciprofloxacin (CIP) and levofloxacin (LVF). The proportion method on L-J media was used to perform the DST against different drugs in most of clinical *M. tuberculosis* isolates. The following drug concentrations in L-J media were used for the DST: RIF, 40μg/ml; INH, 0.2μg/ml; STR, 4µg/ml; EMB, 2µg/ml; CPM, 40µg/ml; CIP, 2µg/ml; and

Capreomycin dH2O dH2O 40000 40 Ciprofloxacin dH2O dH2O 2000 2 Ethambutol dH2O dH2O 2000 2 Isoniazid dH2O dH2O 200 0.2 Levofloxacin dH2O dH2O 2000 2 Rifampicin DMF dH2O 40000 40 Streptomycin dH2O dH2O 4000 4 Table 1. Recommended concentrations of anti-TB drugs used in the drug susceptibility

testing (WHO, 2009). dH2O, distilled water; DMF, dimethylformamide.

decontamination procedure must be precisely followed (WHO, 1998).

Stock solution Drug Medium

*M. tuberculosis* drug susceptibility testing methods using solid media include proportional, resistance ratio and absolute concentration, which are inexpensive and highly standardized for testing susceptibility to many drugs (WHO, 2009). The proportion method is the most commonly used worldwide. Sputum specimens were digested, decontaminated and used to inoculate egg-based media such as L-J slants for DST. The instructions for the preparation of the egg-based media such as L-J, and detailed protocols for performing standardized bacteriological services for detecting infectious cases of pulmonary tuberculosis, monitoring treatment progress and documenting cure at the end of treatment by means of microscopic examination were well described by WHO in Part III (Culture) of the Laboratory Services in Tuberculosis Control (WHO, 1998) and should be followed strictly.

#### **2.2.1 Preparation of culture media**

The recommended TB diagnostic laboratory procedures (WHO, 1998; Chinese Antituberculosis Association, 2006) were followed for the preparation of media and suspension, inoculation and mycobacterium culture in a biologic safety cabinet. Löwenstein-Jensen (L-J) solid media were prepared in the laboratory of the Affiliated Hospital of Zunyi Medical College. All the samples were cultured on L-J solid media and grown colonies were identified to the species level using TCH (2-thiophene carboxylic acid) and PNB (paranitrobenzoicacid) selective media or by standard biochemical procedures.

#### **2.2.1.1 Procedure for preparation of L-J culture medium (for 1632 ml)**

1. Dissolve salts from the following ingredients in order in about 300ml of distilled water by heating (WHO, 1998):



#### **2.2.1.2 Preparation of drug-containing solid media**

Drug stock solutions at recommended concentrations (Table 1) were made in distilled water. To make media containing the end concentration of different drugs (Table 1), 0.4ml of stock solution for each drug was diluted separately with 400ml of L-J mixture mentioned above.

*M. tuberculosis* drug susceptibility testing methods using solid media include proportional, resistance ratio and absolute concentration, which are inexpensive and highly standardized for testing susceptibility to many drugs (WHO, 2009). The proportion method is the most commonly used worldwide. Sputum specimens were digested, decontaminated and used to inoculate egg-based media such as L-J slants for DST. The instructions for the preparation of the egg-based media such as L-J, and detailed protocols for performing standardized bacteriological services for detecting infectious cases of pulmonary tuberculosis, monitoring treatment progress and documenting cure at the end of treatment by means of microscopic examination were well described by WHO in Part III (Culture) of the Laboratory Services in

The recommended TB diagnostic laboratory procedures (WHO, 1998; Chinese Antituberculosis Association, 2006) were followed for the preparation of media and suspension, inoculation and mycobacterium culture in a biologic safety cabinet. Löwenstein-Jensen (L-J) solid media were prepared in the laboratory of the Affiliated Hospital of Zunyi Medical College. All the samples were cultured on L-J solid media and grown colonies were identified to the species level using TCH (2-thiophene carboxylic acid) and PNB

1. Dissolve salts from the following ingredients in order in about 300ml of distilled water

5. Rinse fresh hen's eggs (not more than 7 days old) thoroughly in running water and soak

8. Mix the sterilized solution containing all the ingredients (600ml) with homogenized eggs (1000ml) in a large sterile beaker (2 L capacity) and let it stand at room

Drug stock solutions at recommended concentrations (Table 1) were made in distilled water. To make media containing the end concentration of different drugs (Table 1), 0.4ml of stock solution for each drug was diluted separately with 400ml of L-J mixture

(paranitrobenzoicacid) selective media or by standard biochemical procedures.

Potassium dihydrogen phosphate anhydrous (KH2PO4) 2.4g Magnesium sulfate (MgSO4·7H2O) 0.24g Magnesium citrate 0.6g Sodium glutamate 7.2g Glycerol (reagent grade) 12ml Malachite green (2% solution) 20ml Distilled water 600ml 2. Add glycerol, malachite green solution and make up to 600ml with distilled water.

6. Crack the eggs with the edge of the beaker and pour into a sterile beaker. 7. Stir them with an old-fashioned sterile egg beater until completely blended.

**2.2.1.1 Procedure for preparation of L-J culture medium (for 1632 ml)** 

3. Sterilize the solution by autoclaving at 121°C for 30 minutes.

Tuberculosis Control (WHO, 1998) and should be followed strictly.

**2.2.1 Preparation of culture media** 

by heating (WHO, 1998):

4. Cool the solution to room temperature.

them in 70% ethanol for 15 minutes.

**2.2.1.2 Preparation of drug-containing solid media** 

temperature for 1 hour.

mentioned above.

Media containing different drugs were distributed to sterile universal screw-cap centrifuge tubes (7ml medium per tube); tubes were labeled and caps were tightened. The labeled tubes were placed in an oven at a slanted position (30° angle) and baked at 85°C for 50 min. Baked tubes were cooled down to room temperature and stored at 4°C refrigerator until use. Since the medium was prepared with sterile precautions, this heating process was to solidify the medium instead of sterilizing it. The quality of the egg-based media deteriorates when the baking temperature is too high or the baking time is too long. The L-J medium should be dated and stored in the refrigerator for up to 4 weeks if the caps are tightened to prevent drying of the medium (WHO, 1998).


Table 1. Recommended concentrations of anti-TB drugs used in the drug susceptibility testing (WHO, 2009). dH2O, distilled water; DMF, dimethylformamide.

#### **2.2.1.3 Digestion and decontamination**

*M. tuberculosis* grows slowly and takes four to eight weeks or longer to give visible colonies. Cultures are usually made in bottles because of the long incubation time required. The bottles are tightly capped to prevent drying of the cultures. The majority of clinical specimens submitted to the clinical laboratory are contaminated to varying degrees by more rapidly growing normal flora. These would rapidly overgrow the entire surface of the medium before the *M. tuberculosis* start to grow (Goodwin, 2007). Therefore, specimens must be subjected to digestion and decontamination that liquefies the organic debris and eliminates the unwanted normal flora. All currently available digesting and decontaminating methods are to some extent toxic to *M. tuberculosis*. Therefore, to ensure the survival of the maximum number of *M. tuberculosis* in the specimen, the digestion and decontamination procedure must be precisely followed (WHO, 1998).

#### **2.2.2 Drug susceptibility testing**

We have collected hundreds of TB culture samples from TB patients, and used them for the DNA extraction and drug-susceptibility testing against seven anti-TB drugs: rifampicin (RIF), isoniazid (INH), streptomycin (STR), ethambutol (EMB), capreomycin (CPM), ciprofloxacin (CIP) and levofloxacin (LVF). The proportion method on L-J media was used to perform the DST against different drugs in most of clinical *M. tuberculosis* isolates. The following drug concentrations in L-J media were used for the DST: RIF, 40μg/ml; INH, 0.2μg/ml; STR, 4µg/ml; EMB, 2µg/ml; CPM, 40µg/ml; CIP, 2µg/ml; and LVF, 2µg/ml.

Survey and Molecular Characterization of Drug- Resistant

Table 3.

rpoB

katG

inhA

rpsl

rrs

gyrA

gyrB

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 441

streptomycin. Resistance to the first-line anti-TB drugs has been linked to mutations in at least 13 genes: *rpoB* for rifampicin resistance, *katG, inhA, kasA, ahpC, ndh, furA*, and *oxyR* for isoniazid resistance, *embCAB* for ethambutol resistance, *pncA* for pyrazinamide resistance, and *rpsL, rrs,* and *gidB* for streptomycin resistance. Resistance to the secondline anti-TB drugs has been linked to mutations in at least 10 genes (Banerjee, et al., 2008). For the detection of mutations in genes linked to rifampicin and isoniazid resistance, we amplified *rpoB* and *KatG* gene fragments from many drug-resistant *M. tuberculosis* clinical isolations and performed DNA sequencing analyses. We also detected mutations in 5 other genes associated with drug-resistance (*inhA, rpsl, rrs, gyrA and gyrB*). Nucleotide sequences of primers used for PCR amplification and DNA sequencing were listed in

Gene Primer Sequence (5'→3') Product Size (bp)

Reverse, GTCCATGTAGTCCACCTCAGACG 688

Reverse, CTCGTAGCCGTACAGGATCTCG 409

Reverse, CCAGCCGCTGTGCGATC 175

Reverse, TGTAGCGCACACCAGGCAGGT 211

Reverse, AATCCACATGCTCCGCCGCTTG 253

Reverse, GGGCTTCGGTGTACCTCAT 320

Reverse, GTTGTGCCAAAAACACATGC 346

The genomic DNA was extracted from *M. tuberculosis* clinical isolates using a Bacterial DNA Kit (Tiangen, China) following the manufacturer's instruction. DNA fragments for 7 drugrelated genes were amplified by PCR using synthetic oligonucleotide primers listed in Table 3. The following thermocycler parameters were applied with initial denaturation at 94°C for 5 min; 35-42 cycles of denaturation at 94°C for 30 sec; primer annealing at 58-62°C for 30 sec; extension at 72°C for 30-60 sec; and a final extension at 72°C for 7 min. The obtained DNA

Forward, TCAGACCACGATGACCGTTCC

Sequencing, TCGGCATGTCGCGGATGGAG

Forward, GCTCGGCGATGAGCGTTAC

Forward, TCGCAGCCACGTTACGCTC

Sequencing, GCTCGGCGATGAGCGTTAC

Sequencing, TCGCAGCCACGTTACGCTC

Sequencing, GGCAGCCCGCAGCGTCGTG

Forward, TCAGGAGGAACACCGGTGGCG

Forward, CAGCTACATCGACTATGCGA

**3.1 Determination of mutation profiles in drug-related genes** 

**3.1.1 DNA isolation and PCR amplification** 

Sequencing, TCAGGAGGAACACCGGTGGCG

Sequencing, CAGCTACATCGACTATGCGA

Forward, CGCAAGTCCGAACTGTATGTCGTAG

Sequencing, CGCAAGTCCGAACTGTATGTCGTAG Table 3. Primers used in this study for PCR amplification and DNA sequencing

Forward, GGCAGCCCGCAGCGTCGTG

#### **2.2.3 Quality control**

After coagulation, 5% of the slopes were picked up randomly and continued for incubation for 2 days at 37 °C to check for sterility. If no colony was grown on the solid medium after 48 hours of incubation at 37°C, the whole media batch should be good for DST.

#### **2.2.4 Results criteria**

The proportion testing results can be recorded at 28 days and again at 42 days as 3+ for confluent growth; 2+ for more than 100 colonies; and 1-99 colonies for the actual number of colonies. The percentage of drug resistance can be expressed as: Number of colonies on drug-containing medium/Number of colonies on L-J medium × 100%. If the percentage >1, the tested bacterium is considered to be drug resistant.

#### **2.2.5 Results and discussion**

We analyzed 316 clinical *M. tuberculosis* isolates for DST against four drugs (RIF, INH, STR and EMB). Results showed that 51.3% of isolates were resistant to one or more drugs, 19.0% were MDR, 20.9% were resistant to any single drug, 12.0% were resistant to any two drugs, 10.8% were resistant to any three drugs, and 7.6% were resistant to four drugs (Table 2). The prevalence of single drug-resistance was STR>RIF>EMB>INH in combined cases. There were 209 isolates in the first-treated (new TB cases) group, in which 42.1% were resistant to at least one drug, and 23.9% were resistant to any single drug. In addition, there were 107 isolates in the previously treated group, in which 69.2% were resistant to at least one drug, and 15.0% were resistant to any single drug. Our results indicated that the prevalence of drug-resistance in new tuberculosis cases was very high in the Zunyi area of Guizhou province, with 42.1% of the isolates resistant to at least one drug, and there were obvious differences in the drug susceptibility profiles between new and previously treated TB cases. These results also highlight the importance of surveillance of drug-resistant TB in order to improve the treatment outcomes of TB patients.


\*Drugs tested: isoniazid, rifampincin, ethambutol and streptomycin; PT, previously treated; FT, Firsttime treated (new cases).

Table 2. Drug susceptibility profiles of 316 clinical *M. tuberculosis* isolates.

#### **3. Molecular characterization of drug-resistant** *M. tuberculosis*

The recommended standard or first-line treatment of tuberculosis includes a combination of four drugs, rifampicin, isoniazid, ethambutol and pyrazinamide, with or without

After coagulation, 5% of the slopes were picked up randomly and continued for incubation for 2 days at 37 °C to check for sterility. If no colony was grown on the solid medium after

The proportion testing results can be recorded at 28 days and again at 42 days as 3+ for confluent growth; 2+ for more than 100 colonies; and 1-99 colonies for the actual number of colonies. The percentage of drug resistance can be expressed as: Number of colonies on drug-containing medium/Number of colonies on L-J medium × 100%. If the percentage >1,

We analyzed 316 clinical *M. tuberculosis* isolates for DST against four drugs (RIF, INH, STR and EMB). Results showed that 51.3% of isolates were resistant to one or more drugs, 19.0% were MDR, 20.9% were resistant to any single drug, 12.0% were resistant to any two drugs, 10.8% were resistant to any three drugs, and 7.6% were resistant to four drugs (Table 2). The prevalence of single drug-resistance was STR>RIF>EMB>INH in combined cases. There were 209 isolates in the first-treated (new TB cases) group, in which 42.1% were resistant to at least one drug, and 23.9% were resistant to any single drug. In addition, there were 107 isolates in the previously treated group, in which 69.2% were resistant to at least one drug, and 15.0% were resistant to any single drug. Our results indicated that the prevalence of drug-resistance in new tuberculosis cases was very high in the Zunyi area of Guizhou province, with 42.1% of the isolates resistant to at least one drug, and there were obvious differences in the drug susceptibility profiles between new and previously treated TB cases. These results also highlight the importance of surveillance of drug-resistant TB in order to

Drug Susceptibility\* PT (107 isolates) FT (209 isolates) Combined

Resistant to any 2 drugs 23 21.5 15 7.2 38 12.0 Resistant to any 3 drugs 22 20.6 12 5.7 34 10.8 Resistant to 4 drugs 13 12.1 11 5.3 24 7.6

\*Drugs tested: isoniazid, rifampincin, ethambutol and streptomycin; PT, previously treated; FT, First-

The recommended standard or first-line treatment of tuberculosis includes a combination of four drugs, rifampicin, isoniazid, ethambutol and pyrazinamide, with or without

Table 2. Drug susceptibility profiles of 316 clinical *M. tuberculosis* isolates.

**3. Molecular characterization of drug-resistant** *M. tuberculosis*

Total 74 69.2 88 42.1 162 51.3

No. of cases % No. of cases % No. of cases %

16 15.0 50 23.9 66 20.9

(316 isolates)

48 hours of incubation at 37°C, the whole media batch should be good for DST.

the tested bacterium is considered to be drug resistant.

improve the treatment outcomes of TB patients.

**2.2.3 Quality control** 

**2.2.4 Results criteria** 

**2.2.5 Results and discussion** 

Resistant to any single drug

time treated (new cases).

streptomycin. Resistance to the first-line anti-TB drugs has been linked to mutations in at least 13 genes: *rpoB* for rifampicin resistance, *katG, inhA, kasA, ahpC, ndh, furA*, and *oxyR* for isoniazid resistance, *embCAB* for ethambutol resistance, *pncA* for pyrazinamide resistance, and *rpsL, rrs,* and *gidB* for streptomycin resistance. Resistance to the secondline anti-TB drugs has been linked to mutations in at least 10 genes (Banerjee, et al., 2008). For the detection of mutations in genes linked to rifampicin and isoniazid resistance, we amplified *rpoB* and *KatG* gene fragments from many drug-resistant *M. tuberculosis* clinical isolations and performed DNA sequencing analyses. We also detected mutations in 5 other genes associated with drug-resistance (*inhA, rpsl, rrs, gyrA and gyrB*). Nucleotide sequences of primers used for PCR amplification and DNA sequencing were listed in Table 3.


Table 3. Primers used in this study for PCR amplification and DNA sequencing

#### **3.1 Determination of mutation profiles in drug-related genes**

#### **3.1.1 DNA isolation and PCR amplification**

The genomic DNA was extracted from *M. tuberculosis* clinical isolates using a Bacterial DNA Kit (Tiangen, China) following the manufacturer's instruction. DNA fragments for 7 drugrelated genes were amplified by PCR using synthetic oligonucleotide primers listed in Table 3. The following thermocycler parameters were applied with initial denaturation at 94°C for 5 min; 35-42 cycles of denaturation at 94°C for 30 sec; primer annealing at 58-62°C for 30 sec; extension at 72°C for 30-60 sec; and a final extension at 72°C for 7 min. The obtained DNA

Survey and Molecular Characterization of Drug- Resistant

1 r r r r TCG531T

3 r r r r CTG533C

4 r r r r CAC526T

5 r r r r CAC526G

8 r r r r CTG533C

10 r r r s TCG531T

11 r r r r TCG531T

12 r r r s CAC526G

7 r r r s

TG

CG

6 r r r s WT WT GCC5TC

9 r r r r WT WT GCC5TC

GAC516G TC

CG

TG

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 443

CGT16G GT GGA23C GA

2 r r r r WT WT WT WT WT NA WT

AC WT WT WT WT

T

T

AC WT WT AAG43A

GCC5TC C ACT6AG

GCC5TC

GCC5TC C GAA7G CA GGG8GC

CC WT AAG43A

AC WT WT WT WT NA WT

*rpoB katG inhA rpsL rrs gyrA gyrB* 

WT WT NA WT

GG WT NA WT

G

C

G

C

WT WT NA WT

C WT WT AGC95AC

NA WT

<sup>C</sup>NA NA NA NA

<sup>C</sup>WT WT NA WT

GG WT

C WT WT AGC95AC

GCG90GT

AGC95AC

GCG90GT

AGC95AC

<sup>C</sup>WT

<sup>C</sup>WT

GCG90GT

AGC95AC

WT

G

C

NA

WT

No. R H S E Mutations in specific resistant genes

AGC315A CC CCG232C AG GGC237G AG GAG340A AG

AGC315A

AGC315A CC

GTG230A TG

AGC315A CC

TG WT GCC5TC

fragments were analyzed by electrophoresis in 2.0% agarose gel and were visualized under UV light on a transilluminator.

#### **3.1.2 DNA sequencing analysis**

In order to determine mutation proles of 7 drug-related genes (*rpoB, katG, inhA, rpsl, rrs, gyrA* and *gyrB*), we purified PCR-amplied gene fragments and sent them to Shanghai Invitrogen for DNA sequencing using primers listed in Table 3. The FinchTV program was downloaded from Geospiza's website and used to view the original sequencing data. Sequence alignment analysis was conducted using the BLAST program at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to compare our sequencing results with those wild-type *M. tuberculosis* genes listed in the GenBank.

#### **3.1.3 Results and discussion**

In 32 rifampicin-resistant strains, we identified 13 different types of missense mutations at codons 509, 511, 516, 522, 526, 531, 533, 550 and 572 of *rpoB* gene, and compared the mutation profile with those of rifampicin-resistant *M. Tuberculosis* isolates from different geographical regions of the world. Comparison of the results showed that the *rpoB* gene mutation profile in rifampicin-resistant *M. Tuberculosis* clinical isolates from Guizhou province differed not only from other provinces of China but also from other countries of Asia, Europe and America (Chen et al., 2010). Two new mutations (Val550Leu and Ser509Arg) were identified and deposited in GenBank (GQ250580 and GQ250581). We concluded that mutation profiles of rifampicin-resistant *M. Tuberculosis* isolates were variable depending on the geographical locations, and further studies would be needed to determine the molecular basis for such variations (Chen et al., 2010).

In 30 isoniazid-resistant strains, 18 (60%) of them had mutations in *katG* and/or *inhA* genes and two newly identified mutations in *katG* gene (Val230Met and Pro232Gln) were deposited to GenBank (GQ250582 and GQ250583). Results from this study further confirmed that mutations in *katG* and *inhA* genes are related to the isoniazid resistance in *M. tuberculosis* (Chen Y et al., 2010).

To better compare drug resistance and mutation profiles of clinical *M. Tuberculosis* isolates collected at our hospital, we selected 23 representative isolates and combined the DST results against 4 drugs with mutation profiles in 7 genes related to drug resistance in the same table (Table 4). It is clear that the relationship between drug resistance and mutations in specific genes may be more complex than we expected. For example, no mutations were found in 6 of the 7 drug-related genes for isolate number 2, which was resistant to all 4 drugs tested, suggesting that there must be changes in other regions of the genes or other genes. Further studies will be needed to determine the molecular mechanism underlining the resistance of this kind of isolates to different anti-TB drugs. In another example, two mutations were identified in isolate number 17, one in *rpoB* gene (TCG531TTG) and another in *katG* gene (AGC315ACC). However, isolate number 17 was resistant only to rifampicin but sensitive to 3 other drugs. It will be necessary for us to repeat the DST for this isolate to confirm whether this isolate does have a mutation in *katG* gene but still sensitive to isoniazid. In summary, more studies are need to identify new genes related to MDR-TB.

fragments were analyzed by electrophoresis in 2.0% agarose gel and were visualized under

In order to determine mutation proles of 7 drug-related genes (*rpoB, katG, inhA, rpsl, rrs, gyrA* and *gyrB*), we purified PCR-amplied gene fragments and sent them to Shanghai Invitrogen for DNA sequencing using primers listed in Table 3. The FinchTV program was downloaded from Geospiza's website and used to view the original sequencing data. Sequence alignment analysis was conducted using the BLAST program at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to compare our sequencing results with those

In 32 rifampicin-resistant strains, we identified 13 different types of missense mutations at codons 509, 511, 516, 522, 526, 531, 533, 550 and 572 of *rpoB* gene, and compared the mutation profile with those of rifampicin-resistant *M. Tuberculosis* isolates from different geographical regions of the world. Comparison of the results showed that the *rpoB* gene mutation profile in rifampicin-resistant *M. Tuberculosis* clinical isolates from Guizhou province differed not only from other provinces of China but also from other countries of Asia, Europe and America (Chen et al., 2010). Two new mutations (Val550Leu and Ser509Arg) were identified and deposited in GenBank (GQ250580 and GQ250581). We concluded that mutation profiles of rifampicin-resistant *M. Tuberculosis* isolates were variable depending on the geographical locations, and further studies would be needed to

In 30 isoniazid-resistant strains, 18 (60%) of them had mutations in *katG* and/or *inhA* genes and two newly identified mutations in *katG* gene (Val230Met and Pro232Gln) were deposited to GenBank (GQ250582 and GQ250583). Results from this study further confirmed that mutations in *katG* and *inhA* genes are related to the isoniazid resistance in *M.* 

To better compare drug resistance and mutation profiles of clinical *M. Tuberculosis* isolates collected at our hospital, we selected 23 representative isolates and combined the DST results against 4 drugs with mutation profiles in 7 genes related to drug resistance in the same table (Table 4). It is clear that the relationship between drug resistance and mutations in specific genes may be more complex than we expected. For example, no mutations were found in 6 of the 7 drug-related genes for isolate number 2, which was resistant to all 4 drugs tested, suggesting that there must be changes in other regions of the genes or other genes. Further studies will be needed to determine the molecular mechanism underlining the resistance of this kind of isolates to different anti-TB drugs. In another example, two mutations were identified in isolate number 17, one in *rpoB* gene (TCG531TTG) and another in *katG* gene (AGC315ACC). However, isolate number 17 was resistant only to rifampicin but sensitive to 3 other drugs. It will be necessary for us to repeat the DST for this isolate to confirm whether this isolate does have a mutation in *katG* gene but still sensitive to isoniazid. In summary, more studies are need to identify new genes related to MDR-TB.

UV light on a transilluminator.

**3.1.2 DNA sequencing analysis** 

**3.1.3 Results and discussion** 

*tuberculosis* (Chen Y et al., 2010).

wild-type *M. tuberculosis* genes listed in the GenBank.

determine the molecular basis for such variations (Chen et al., 2010).


Survey and Molecular Characterization of Drug- Resistant

**3.2.1 Spoligotyping and its application in surveillance** 

**3.2 Genotyping** 

profiling.

IS6110 (Bauer et al., 1999).

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 445

Spacer oligonucleotide typing (Spoligotyping) is a molecular method used to differentiate *M. Tuberculosis Complex (MTC)* isolates. This method is based on PCR analysis of polymorphisms in the MTC direct repeat (DR) chromosomal region containing multiple 36bp DR loci. Each DR is interspersed by a unique spacer sequence of 35 to 41 bp. After PCR amplification, the fragment containing the whole DR region was hybridized to specific oligonucleotide probes designed according to the different spacer sequences, and genotypes were determined depending on the hybridization patterns. The most widely used 43 sites were developed in 1997 by Kamerbeek et al, including 37 derived from the reference strain H37Rv and 6 spacers derived from *M. bovis* BCG (Kamerbeek et al., 1997). This method is more rapid and easier to perform than the standard genotyping technique based on IS6110

(Missing Spacers)

Member of M. TB complex Characterization of Spoligotyping

Mycobacterium bovis 39~43 (Soini et al., 2000; Filliol et al., 2003)

Mycobacterium africanum 8, 9, 39 (Viana-Niero et al., 2001; Filliol et al., 2003)

MTC includes *M. tuberculosis*, *M. bovis*, *M. voles* and *Africa mycobacterium*. *M. tuberculosis* is one of the leading pathogens to human and animal, followed by *M. bovis*. *M. tuberculosis* and *M. bovis* can be distinguished from MTC by using L–J agar slants, PNB and TCH. *M. bovis* can be quickly identified by spoligotyping. Considering the difficulties in determining *M. vole* and *Africa mycobacterium* by traditional methods of bacteriology, Soolingen et al. developed the spoligotyping method to distinguish the *M. vole* from MTC (van Soolinger et al. 1998). Results from Niemann et al. proved that *M. bovis* could be differentiated from *M. Africa* by spoligotyping (Niemann et al., 2000). In addition, Viana-Niero et al. also indicated that the spoligotyping fingerprint of *M. Africa* was between *M. tuberculosis* and *M. bovis* (Viana-Niero et al., 2001). Spoligotyping can be used to distinguish MTC members based on their characteristic spoligotypes (Table 5). Until now, IS6110-RFLP has been the gold standard for genotyping of *M. tuberculosis* (Majeed et al., 2004). However, a comparison study using IS6110-RFLP and spoligotyping indicated that the genotyping capacity of spoligotyping was better than IS6110-RFLP to strains containing low-copy numbers of

To study the genotypic diversity, we choose spoligotyping for molecular typing of 100 clinical *M. tuberculosis* isolates collected at the Affiliated Hospital of Zunyi Medical College from 2008 to 2009. Bacterial growth and chromosomal DNA isolation were carried out by the method of Soolingen et al. (van Soolinger et al., 1991). The extracted DNA was used as a template for PCR amplification of the DR region of the genome with the biotinylated forward primer, 5'- GGTTTTGGGTCTGACGAC-3' and the reverse primer 5'-CCGAGAGGG GACGGAAAC-3'. The following thermocycler parameters were applied with initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s; primer annealing at 56°C for 30 s; extension at

Mycobacterium tuberculosis 33~36 (Viana-Niero et al., 2001)

Mycobacterium microti 37, 38 (Niemann et al., 2000) Table 5. Characterization of *M. tuberculosis complex* by Spoligotyping


Abbreviations: R, rifampicin; H, isoniazid; S, streptomycin; E, ethambutol; s, susceptible; r, resistant; WT, wild type; and NA, data not available.

Table 4. Drug susceptibility profiles and mutational patterns of 23 representative clinical isolates

#### **3.2 Genotyping**

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

CC WT WT WT

CC WT WT WT

CC WT AAG43A

WT WT AAG43A

CC WT AAG43A

Abbreviations: R, rifampicin; H, isoniazid; S, streptomycin; E, ethambutol; s, susceptible; r, resistant;

Table 4. Drug susceptibility profiles and mutational patterns of 23 representative clinical

*rpoB katG inhA rpsL rrs gyrA gyrB* 

CC WT WT WT AGC95AC

GG WT

CC WT NA NA NA NA

CC WT NA NA NA NA

Gene loss WT NA WT NA WT

CC WT WT WT AGC95AC

CC WT WT WT AGC95AC

GAC94GG

GAG528G CG

WT

ACC539C CC

AGC95AC

ACC80AT

AGC95AC

GAC94GG

AGC95AC

<sup>C</sup>WT

<sup>C</sup>WT

<sup>C</sup>WT

<sup>C</sup>WT

<sup>C</sup>WT

C

C

C

C

C

C

GG WT AGC95AC

GG WT AGC95AC

No. R H S E Mutations in specific resistant genes

AGC315A

AGC315A

AGC315A

AGC315A

AGC315A

AGC315A

AGC315A

AGC315A

AGC315A

13 r r r r CAC526T

15 r r r r TCG531T

16 r r r r TCG531T

17 r s s s TCG531T

18 r r r r TCG531T

20 s r r s CTG533C

22 r r r r TCG531T

19 r r r s

21 r r r r

23 r r s r

isolates

14 r r s r

AC

TG

TG

TG

TG

CG

TG

WT, wild type; and NA, data not available.

AGC509A GG CTG511C CG GAC516G TC

TCG522T TG GTG550T TG

CTG511C CG GAC516A AC

TCG522T TG GTG550T TG

#### **3.2.1 Spoligotyping and its application in surveillance**

Spacer oligonucleotide typing (Spoligotyping) is a molecular method used to differentiate *M. Tuberculosis Complex (MTC)* isolates. This method is based on PCR analysis of polymorphisms in the MTC direct repeat (DR) chromosomal region containing multiple 36bp DR loci. Each DR is interspersed by a unique spacer sequence of 35 to 41 bp. After PCR amplification, the fragment containing the whole DR region was hybridized to specific oligonucleotide probes designed according to the different spacer sequences, and genotypes were determined depending on the hybridization patterns. The most widely used 43 sites were developed in 1997 by Kamerbeek et al, including 37 derived from the reference strain H37Rv and 6 spacers derived from *M. bovis* BCG (Kamerbeek et al., 1997). This method is more rapid and easier to perform than the standard genotyping technique based on IS6110 profiling.


Table 5. Characterization of *M. tuberculosis complex* by Spoligotyping

MTC includes *M. tuberculosis*, *M. bovis*, *M. voles* and *Africa mycobacterium*. *M. tuberculosis* is one of the leading pathogens to human and animal, followed by *M. bovis*. *M. tuberculosis* and *M. bovis* can be distinguished from MTC by using L–J agar slants, PNB and TCH. *M. bovis* can be quickly identified by spoligotyping. Considering the difficulties in determining *M. vole* and *Africa mycobacterium* by traditional methods of bacteriology, Soolingen et al. developed the spoligotyping method to distinguish the *M. vole* from MTC (van Soolinger et al. 1998). Results from Niemann et al. proved that *M. bovis* could be differentiated from *M. Africa* by spoligotyping (Niemann et al., 2000). In addition, Viana-Niero et al. also indicated that the spoligotyping fingerprint of *M. Africa* was between *M. tuberculosis* and *M. bovis* (Viana-Niero et al., 2001). Spoligotyping can be used to distinguish MTC members based on their characteristic spoligotypes (Table 5). Until now, IS6110-RFLP has been the gold standard for genotyping of *M. tuberculosis* (Majeed et al., 2004). However, a comparison study using IS6110-RFLP and spoligotyping indicated that the genotyping capacity of spoligotyping was better than IS6110-RFLP to strains containing low-copy numbers of IS6110 (Bauer et al., 1999).

To study the genotypic diversity, we choose spoligotyping for molecular typing of 100 clinical *M. tuberculosis* isolates collected at the Affiliated Hospital of Zunyi Medical College from 2008 to 2009. Bacterial growth and chromosomal DNA isolation were carried out by the method of Soolingen et al. (van Soolinger et al., 1991). The extracted DNA was used as a template for PCR amplification of the DR region of the genome with the biotinylated forward primer, 5'- GGTTTTGGGTCTGACGAC-3' and the reverse primer 5'-CCGAGAGGG GACGGAAAC-3'. The following thermocycler parameters were applied with initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s; primer annealing at 56°C for 30 s; extension at

Survey and Molecular Characterization of Drug- Resistant

et al., 2004).

Kavallaris & Marshall, 2005; Drake et al., 2005; Stulik & Butaye, 2011).

development of potential therapeutics and new vaccines.

genes which might be associated with MDR or XDR.

**4.2 Sample preparation for MDR/XDR-TB isolates** 

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 447

Proteomics enables the qualitative and quantitative analyses of proteins in complex biological systems, such as cells, tissues, and body fluids under specific conditions or in response to different stimuli (Graham et al., 2011; Lim & Elenitoba-Johnson, 2004; List et al., 2008; Wu & Liu, 2009). Proteomics is an effective means to rapidly identify new proteins as diagnostic or prognostic markers and as therapeutic targets for various diseases including cancers, genetic diseases, and infectious diseases such as tuberculosis (Chung et al., 2007;

Proteomics has been applied to the research area of *M. tuberculosis* (Bahk et al., 2004; He et al., 2003; Kumar et al., 2010; Lee et al. 1999; Mattow et al. 2001; Mustafa, 2005; Pheiffer et al., 2005; Sharma et al., 2010; Wang et al., 2007; Zhu et al., 2003) and become an important tool to study functional genomics of the bacterium (Mattow et al., 2003; Jiang et al., 2006; Rison et al., 2007; Xie et al., 2009; Jungblut et al., 2001). Proteins from *M. tuberculosis* missing in attenuated *M. bovis* BCG strains were identified by using proteomics (Mattow et al., 2001). Comparative proteome analyses of culture supernatant proteins from virulent *M. tuberculosis* H37Rv and attenuated *M. bovis* BCG (Mattow et al., 2003) or H37Ra (He et al., 2003) were performed to identify new virulent factors. Proteomics was also used for genome-wide analysis of the host intracellular network regulating survival of *M. tuberculosis* (Kumar et al., 2010). It was demonstrated that protein expression by a Beijing strain was different from that of another clinical isolate and *M. tuberculosis* H37Rv (Pheiffer et al., 2005). The major membrane protein of virulent *M. tuberculosis* was characterized using proteomics technology (Lee et al., 1999). Xie et al. used proteomics to compare proteins of MDR-TB isolate with drug-sensitive isolate (Xie et al. 2009). Additionally, proteomics has been successfully used for vaccine design to improve protection against tuberculosis (Mollenkopf

The development of proteomic profiles for *M. tuberculosis* is needed to identify proteins that are differentially expressed in clinical strains with different drug susceptibility profiles such as MDR-TB and XDR-TB comparing to the standard strain, which can provide insights into the mechanisms by which the mycobacteria resistant to anti-TB drugs and the selection of suitable biomarkers for new diagnostics, new virulent factors and/targets for the

The most commonly used method for proteomic analysis is the two-dimensional polyacrylamide gel electrophoresis (2-DE), which allows the separation and display of thousands of proteins from a complex mixture by their charges (pI) and relative molecular mass (Mr). Gel-separated proteins can be identified rapidly by mass spectrometry (MS), and such analyses permit the systematic identification of the proteome if genomic information is available. In this preliminary study, we used 2-DE and mass spectrometry to compare protein expression profiles among different clinical *M. tuberculosis* strains such as MDR-TB, XDR-TB and the reference strain H37Rv, and identified some up-regulated, down-regulated

The sensitive strain used in this study was the standard *M. tuberculosis* strain H37Rv, which was obtained as a gift from the Chinese Centre for Disease Control and Prevention. Both MDR-TB and XDR-TB strains were clinical isolates collected at the Affiliated

72°C for 30 s; and a final extension at 72°C for 8 min. The PCR amplied products were hybridized to a membrane containing a set of 43 immobilized oligonucleotides, each corresponding to one of the unique spacer DNA sequences within the DR locus. DNAs isolated from *M. tuberculosis* H37Rv and *M. bovis* BCG were used as controls. The hybridized PCR products were incubated with 1:4000-diluted streptavidin-peroxidase conjugate (Boehringer) for 30min at 42°C. Detection of hybridizing DNA was done by using the chemiluminescent ECL (Amersham) detection system followed by exposure to X-ray lm (Hyperlm ECL; Amersham) in accordance with the manufacturer's instructions.

Spoligotypes in binary format were entered in the SITVIT database (Pasteur Institute of Guadeloupe). Twenty-nine distinct spoligotyping patterns were observed. In total, 20 orphan patterns were identified and the remaining 77 were contained within 9 superfamilies: Beijing, T1, T3, T2, MANU2, Beijing-like, U, H3 and H3-T3. Results showed that almost half of the isolates were clustered to the Beijing lineage, and 13 isolates were clustered to the T1 lineage.

To analyze the MTC population structure, evaluate the complicacy of the global TB transmission and provisional evolution of the TB genetic landscape, Institute Pasteur had built a genetic diversity database for the MTC DR locus in 1999. This database was updated to SpolDB4 (Spoligotyping Database 4) in 2006. The updated database contained 1939 shared-types (STs) of 39,295 strains from 122 countries, which were temporarily classified into 62 clades/lineages (Brudey et al., 2006). In this database, the 10 most prevalent clades are ST1 (Beijing family), ST53 (T1), ST190 (Beijing family), ST52 (T2), ST50 (H3), ST54 (MANU2), ST37 (T3), ST742 (H3), ST265 (Beijing family) and ST127 (H4).

The Beijing genotype family was originally identified in China (Brudey et al., 2006) and defined as strains that presence of at least three spacers 35-43 and absence of spacers 1-34 (van Soolinger et al., 1995). Beijing family strains represent at least 13% of strains worldwide and about half of strains in the East Asia. A total of 2,346 *M. tuberculosis* isolates from 13 provinces, excluding Guizhou province, in China were genotyped by spoligotyping, and results showed that 74.08% of the isolates belonged to the Beijing family (Dong et al., 2010). Other studies also examined the relationship of Beijing genotype with drug resistance, however, the association between them was still not clear. The possible reasons for this uncertainty might be: 1) the diversity of treatment programs; 2) compliance to treatment; 3) different quality of anti-TB drugs; and 4) spread of different and not yet distinguished sublineages of the Beijing strains (Parwati et al., 2010).

The "T" families were the most prevalent following Beijing family, belonged to modern TB strains, and ill-defined with more than 600 unclassified STs. The "T" families were further divided into 5 subclades (T1-T5) based on single spacer-differences (Brudey et al. 2006). The numbers of T1 and T2 families were ranked the second and third respectively after Beijing family in the updated SpolDB4.

#### **4. Application of proteomics in drug-resistant** *M. tuberculosis*

#### **4.1 Proteomics and its application in tuberculosis research**

Proteomics is defined as the large-scale study of proteins, their post-translational modifications, and their structures and functions underlying different biological processes.

72°C for 30 s; and a final extension at 72°C for 8 min. The PCR amplied products were hybridized to a membrane containing a set of 43 immobilized oligonucleotides, each corresponding to one of the unique spacer DNA sequences within the DR locus. DNAs isolated from *M. tuberculosis* H37Rv and *M. bovis* BCG were used as controls. The hybridized PCR products were incubated with 1:4000-diluted streptavidin-peroxidase conjugate (Boehringer) for 30min at 42°C. Detection of hybridizing DNA was done by using the chemiluminescent ECL (Amersham) detection system followed by exposure to X-ray lm

Spoligotypes in binary format were entered in the SITVIT database (Pasteur Institute of Guadeloupe). Twenty-nine distinct spoligotyping patterns were observed. In total, 20 orphan patterns were identified and the remaining 77 were contained within 9 superfamilies: Beijing, T1, T3, T2, MANU2, Beijing-like, U, H3 and H3-T3. Results showed that almost half of the isolates were clustered to the Beijing lineage, and 13 isolates were

To analyze the MTC population structure, evaluate the complicacy of the global TB transmission and provisional evolution of the TB genetic landscape, Institute Pasteur had built a genetic diversity database for the MTC DR locus in 1999. This database was updated to SpolDB4 (Spoligotyping Database 4) in 2006. The updated database contained 1939 shared-types (STs) of 39,295 strains from 122 countries, which were temporarily classified into 62 clades/lineages (Brudey et al., 2006). In this database, the 10 most prevalent clades are ST1 (Beijing family), ST53 (T1), ST190 (Beijing family), ST52 (T2), ST50 (H3), ST54

The Beijing genotype family was originally identified in China (Brudey et al., 2006) and defined as strains that presence of at least three spacers 35-43 and absence of spacers 1-34 (van Soolinger et al., 1995). Beijing family strains represent at least 13% of strains worldwide and about half of strains in the East Asia. A total of 2,346 *M. tuberculosis* isolates from 13 provinces, excluding Guizhou province, in China were genotyped by spoligotyping, and results showed that 74.08% of the isolates belonged to the Beijing family (Dong et al., 2010). Other studies also examined the relationship of Beijing genotype with drug resistance, however, the association between them was still not clear. The possible reasons for this uncertainty might be: 1) the diversity of treatment programs; 2) compliance to treatment; 3) different quality of anti-TB drugs; and 4) spread of different and not yet distinguished

The "T" families were the most prevalent following Beijing family, belonged to modern TB strains, and ill-defined with more than 600 unclassified STs. The "T" families were further divided into 5 subclades (T1-T5) based on single spacer-differences (Brudey et al. 2006). The numbers of T1 and T2 families were ranked the second and third respectively after Beijing

Proteomics is defined as the large-scale study of proteins, their post-translational modifications, and their structures and functions underlying different biological processes.

(Hyperlm ECL; Amersham) in accordance with the manufacturer's instructions.

(MANU2), ST37 (T3), ST742 (H3), ST265 (Beijing family) and ST127 (H4).

**4. Application of proteomics in drug-resistant** *M. tuberculosis*

**4.1 Proteomics and its application in tuberculosis research** 

sublineages of the Beijing strains (Parwati et al., 2010).

family in the updated SpolDB4.

clustered to the T1 lineage.

Proteomics enables the qualitative and quantitative analyses of proteins in complex biological systems, such as cells, tissues, and body fluids under specific conditions or in response to different stimuli (Graham et al., 2011; Lim & Elenitoba-Johnson, 2004; List et al., 2008; Wu & Liu, 2009). Proteomics is an effective means to rapidly identify new proteins as diagnostic or prognostic markers and as therapeutic targets for various diseases including cancers, genetic diseases, and infectious diseases such as tuberculosis (Chung et al., 2007; Kavallaris & Marshall, 2005; Drake et al., 2005; Stulik & Butaye, 2011).

Proteomics has been applied to the research area of *M. tuberculosis* (Bahk et al., 2004; He et al., 2003; Kumar et al., 2010; Lee et al. 1999; Mattow et al. 2001; Mustafa, 2005; Pheiffer et al., 2005; Sharma et al., 2010; Wang et al., 2007; Zhu et al., 2003) and become an important tool to study functional genomics of the bacterium (Mattow et al., 2003; Jiang et al., 2006; Rison et al., 2007; Xie et al., 2009; Jungblut et al., 2001). Proteins from *M. tuberculosis* missing in attenuated *M. bovis* BCG strains were identified by using proteomics (Mattow et al., 2001). Comparative proteome analyses of culture supernatant proteins from virulent *M. tuberculosis* H37Rv and attenuated *M. bovis* BCG (Mattow et al., 2003) or H37Ra (He et al., 2003) were performed to identify new virulent factors. Proteomics was also used for genome-wide analysis of the host intracellular network regulating survival of *M. tuberculosis* (Kumar et al., 2010). It was demonstrated that protein expression by a Beijing strain was different from that of another clinical isolate and *M. tuberculosis* H37Rv (Pheiffer et al., 2005). The major membrane protein of virulent *M. tuberculosis* was characterized using proteomics technology (Lee et al., 1999). Xie et al. used proteomics to compare proteins of MDR-TB isolate with drug-sensitive isolate (Xie et al. 2009). Additionally, proteomics has been successfully used for vaccine design to improve protection against tuberculosis (Mollenkopf et al., 2004).

The development of proteomic profiles for *M. tuberculosis* is needed to identify proteins that are differentially expressed in clinical strains with different drug susceptibility profiles such as MDR-TB and XDR-TB comparing to the standard strain, which can provide insights into the mechanisms by which the mycobacteria resistant to anti-TB drugs and the selection of suitable biomarkers for new diagnostics, new virulent factors and/targets for the development of potential therapeutics and new vaccines.

The most commonly used method for proteomic analysis is the two-dimensional polyacrylamide gel electrophoresis (2-DE), which allows the separation and display of thousands of proteins from a complex mixture by their charges (pI) and relative molecular mass (Mr). Gel-separated proteins can be identified rapidly by mass spectrometry (MS), and such analyses permit the systematic identification of the proteome if genomic information is available. In this preliminary study, we used 2-DE and mass spectrometry to compare protein expression profiles among different clinical *M. tuberculosis* strains such as MDR-TB, XDR-TB and the reference strain H37Rv, and identified some up-regulated, down-regulated genes which might be associated with MDR or XDR.

#### **4.2 Sample preparation for MDR/XDR-TB isolates**

The sensitive strain used in this study was the standard *M. tuberculosis* strain H37Rv, which was obtained as a gift from the Chinese Centre for Disease Control and Prevention. Both MDR-TB and XDR-TB strains were clinical isolates collected at the Affiliated

Survey and Molecular Characterization of Drug- Resistant

**4.5 Comparison of XDR and MDR patterns** 

strains.

the National Center for Biotechnology Information (NCBI) database.

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 449

mass ngerprint data were searched using the Mascot search (www.matrixscience.com) of

The comparison was repeated at least three times, and only those differences conrmed in all comparisons were accepted as strain specic. This study compared the proteome of XDR-TB clinical isolate to those of MDR-TB clinical isolate and the standard strain H37Rv, and demonstrated that the 2-DE protein expression patterns of XDR-TB and MDR-TB clinical isolates were highly correlated, but there were some visible differences between drug-resistant strains and the reference H37Rv strain. Results also showed that more basic proteins were expressed in drug-resistant *M. tuberculosis* isolates than in the standard H37Rv stain. Figure 1 shows differences among three different mycobacterium

Fig. 1. Protein expression patterns of XDR-TB and MDR-TB clinical isolates compared to the

standard strain H37Rv in two-dimensional electrophoresis

Hospital of Zunyi Medical College. Five to six inoculating loops of *M. tuberculosis* colonies were scraped from improved L-J culture media and transferred to 4 ml of purified water in a screw-top centrifuge tube. The mycobacterium suspension was heated at 80°C for 30 min in a water bath to inactivate *M. tuberculosis*, and centrifuged at 4000*g* for 10min for removing the supernatant. The precipitation was resuspended in the phosphate-buffered saline (pH7.4), and centrifuged at 4000*g* for 10min to remove the supernatant. The inactivated *M. tuberculosis* sample was resuspended in 0.3ml of lysis buffer, and 10l of proteinase inhibitors were added to the suspension followed by incubation at 4°C for 20 min. The suspension was then sonicated (200W, 1min×30 times), and centrifuged at 4000*g* for 30min at 4°C. Four-fold volume of cold dimethyl ketone were added to the suspension and the mixture was kept at -20°C overnight for precipitating proteins. The protein precipitates were centrifuged at 10000*g* for 5min at 4°C, and resuspended in 7M carbamide and 2M sulfourea solution to dissolve proteins followed by centrifugation at 4000*g* for 30min at 4°C. Concentration of protein samples was measured by the NanoDrop-1000 (Thermo, Germany) and protein samples were stored at -20°C until use.

#### **4.3 Two-dimensional electrophoresis (2-DE)**

Protein samples (200-300g) were resuspended in 480l of rehydration buffer and applied to pH 4-7 IPG strips (Amersham Biosciences) for rehydration. When IPG strips were rehydrated with the protein samples, isoelectric focusing (IEF) was performed in the following voltage mode: 0V 1h, 50V 10h fast voltage, 500V 1.5h linear voltage, 2000V 1.5h linear voltage, 5000V 1.5h linear voltage, 8000V 1.5h linear voltage, 8000V 70000VH fast voltage, and 500V 16h fast voltage.

Equilibration was performed immediately prior to the second-dimension run, in which step IPG strips should be laid in the strip equilibrium solution buffer I (6 mol/L Urea, 2% SDS, 0.375mol/L pH 8.8 Tris-HCl, 20%Glycerol, and 2% DTT), then strip equilibrium solution buffer II (mol/L Urea, 2% SDS, 0.375mol/L pH8.8 Tris-HCl, 20% Glycerol, and 2.5% Iodoacetamide) each for 14 minutes. The equilibrated IPG strip was located into prepared 13% SDS-PAGE with low-melting point agarose smother. Following electrophoresis, proteins were visualized by either silver staining (analytical gels) or Coomassie Brilliant Blue G-250 staining (preparative gels).

The pI and Mr gradient of the 2-DE gels were determined using an iterative calibration method after 2-DE gel images transferred to computer by means of the image scanner. Spot detection and image analysis were performed and compared using the program Progenesis SameSpots (Nonlinear Dynamics, UK) for 2-DE database construction. Images of MDR-TB and XDR-TB were compared to the gel image of the standard strain H37Rv, and protein spots showing differential expression levels of more than two-fold were analyzed.

#### **4.4 MALDI-MS and database**

Twenty protein spots of interest (5 spots from H37Rv, 10 spots from XDR-TB, and 5 upregulated protein spots from MDR-TB) were excised from Coomassie Brilliant Blue G-250 stained two dimensional gels (2-DE) and digested in gel using trypsin for 18h at 37°C. Masses of the peptides extracted from gel slices were applied to the sample plate of a matrix-assisted laser desorption ionization–time-of-ight MS (MALDI-TOF-MS). Peptide mass ngerprint data were searched using the Mascot search (www.matrixscience.com) of the National Center for Biotechnology Information (NCBI) database.

#### **4.5 Comparison of XDR and MDR patterns**

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

Hospital of Zunyi Medical College. Five to six inoculating loops of *M. tuberculosis* colonies were scraped from improved L-J culture media and transferred to 4 ml of purified water in a screw-top centrifuge tube. The mycobacterium suspension was heated at 80°C for 30 min in a water bath to inactivate *M. tuberculosis*, and centrifuged at 4000*g* for 10min for removing the supernatant. The precipitation was resuspended in the phosphate-buffered saline (pH7.4), and centrifuged at 4000*g* for 10min to remove the supernatant. The inactivated *M. tuberculosis* sample was resuspended in 0.3ml of lysis buffer, and 10l of proteinase inhibitors were added to the suspension followed by incubation at 4°C for 20 min. The suspension was then sonicated (200W, 1min×30 times), and centrifuged at 4000*g* for 30min at 4°C. Four-fold volume of cold dimethyl ketone were added to the suspension and the mixture was kept at -20°C overnight for precipitating proteins. The protein precipitates were centrifuged at 10000*g* for 5min at 4°C, and resuspended in 7M carbamide and 2M sulfourea solution to dissolve proteins followed by centrifugation at 4000*g* for 30min at 4°C. Concentration of protein samples was measured by the NanoDrop-1000 (Thermo, Germany) and protein samples were stored at -20°C until use.

Protein samples (200-300g) were resuspended in 480l of rehydration buffer and applied to pH 4-7 IPG strips (Amersham Biosciences) for rehydration. When IPG strips were rehydrated with the protein samples, isoelectric focusing (IEF) was performed in the following voltage mode: 0V 1h, 50V 10h fast voltage, 500V 1.5h linear voltage, 2000V 1.5h linear voltage, 5000V 1.5h linear voltage, 8000V 1.5h linear voltage, 8000V 70000VH fast

Equilibration was performed immediately prior to the second-dimension run, in which step IPG strips should be laid in the strip equilibrium solution buffer I (6 mol/L Urea, 2% SDS, 0.375mol/L pH 8.8 Tris-HCl, 20%Glycerol, and 2% DTT), then strip equilibrium solution buffer II (mol/L Urea, 2% SDS, 0.375mol/L pH8.8 Tris-HCl, 20% Glycerol, and 2.5% Iodoacetamide) each for 14 minutes. The equilibrated IPG strip was located into prepared 13% SDS-PAGE with low-melting point agarose smother. Following electrophoresis, proteins were visualized by either silver staining (analytical gels) or Coomassie Brilliant

The pI and Mr gradient of the 2-DE gels were determined using an iterative calibration method after 2-DE gel images transferred to computer by means of the image scanner. Spot detection and image analysis were performed and compared using the program Progenesis SameSpots (Nonlinear Dynamics, UK) for 2-DE database construction. Images of MDR-TB and XDR-TB were compared to the gel image of the standard strain H37Rv, and protein

Twenty protein spots of interest (5 spots from H37Rv, 10 spots from XDR-TB, and 5 upregulated protein spots from MDR-TB) were excised from Coomassie Brilliant Blue G-250 stained two dimensional gels (2-DE) and digested in gel using trypsin for 18h at 37°C. Masses of the peptides extracted from gel slices were applied to the sample plate of a matrix-assisted laser desorption ionization–time-of-ight MS (MALDI-TOF-MS). Peptide

spots showing differential expression levels of more than two-fold were analyzed.

**4.3 Two-dimensional electrophoresis (2-DE)** 

voltage, and 500V 16h fast voltage.

Blue G-250 staining (preparative gels).

**4.4 MALDI-MS and database** 

The comparison was repeated at least three times, and only those differences conrmed in all comparisons were accepted as strain specic. This study compared the proteome of XDR-TB clinical isolate to those of MDR-TB clinical isolate and the standard strain H37Rv, and demonstrated that the 2-DE protein expression patterns of XDR-TB and MDR-TB clinical isolates were highly correlated, but there were some visible differences between drug-resistant strains and the reference H37Rv strain. Results also showed that more basic proteins were expressed in drug-resistant *M. tuberculosis* isolates than in the standard H37Rv stain. Figure 1 shows differences among three different mycobacterium strains.

Fig. 1. Protein expression patterns of XDR-TB and MDR-TB clinical isolates compared to the standard strain H37Rv in two-dimensional electrophoresis

Survey and Molecular Characterization of Drug- Resistant

<sup>2994</sup>MMP=19 kda major membrane

**5. Conclusion** 

especially MDR/XDR-TB.

**6. Acknowledgments** 

**7. References** 

protein

Table 9. Description of three proteins identified by MALDI-TOF-MS

*M. tuberculosis* Clinical Isolates from Zunyi, Guizhou Province of China 451

1994 Ribosomal protein S3 Ribonucleoprotein, binding and

In this chapter, we have provided the detailed description about our current progresses in the field of survey and molecular characterization of drug-resistant *M. tuberculosis* clinical isolates using different research methods such as *M. tuberculosis* culture, spoligotyping, gene sequencing, proteomics and drug susceptibility testing against first-line and second-line anti-tuberculosis drugs. We hope this chapter will be useful for researchers, scientists and physicians in academic institutions, clinical laboratories, pharmaceutical companies and research hospitals who have interest in the research related to drug-resistant *M. tuberculosis*,

This project was funded in part with federal funds from the CDC, Department of Health and Human Services, under the contract no. 200-2007-M-22792, and by grants of International Cooperation Project from Guizhou Province of China [Qian Science co-G (2009) No. 700120], the Guizhou Science and Technology Fund Project [Qian Science co-J (2007) no. 2223] and Scientific Research Start Foundation from Zunyi Medical College in (F-327). The following people in the Affiliated Hospital of Zunyi Medical College made contributions to the research reported in this chapter: Xin Gan, Yang Chen, Kai-Lun Li, Yuan-Bo Lan, Zhen-Yong Chen, Lin-Mei Zhou, and Yi He. We would like to thank all health care staff in the Affiliated Hospital of Zunyi Medical College who participated in the research. We are also grateful to Professor Kanglin Wan, Zhiguang Liu, and Bing Lv in the Chinese Center for

Disease Control and Prevention for their support with the spoligotyping analyses.

and test for diagnostic marker. *Proteomics* 4(11): 3299-307.

cultured in Denmark. *J Clin Microbiol* 37(8): 2602-6.

*BMC Bioinformatics* 12: 224.

Bahk, Y.Y.; Kim, S.A.; Kim, J.S.; Euh, H.J.; Bai, G.H.; Cho, S.N. & Kim, Y.S. (2004). Antigens

Banerjee, R.; Schecter, G.F.; Flood, J. & Porco, T.C. (2008). Extensively drug-resistant tuberculosis: new strains, new challenges. *Expert Rev Anti Infect Ther* 6(5): 713-24. Bauer, J.; Andersen, A.B.; Kremer, K. & Miörner, H. (1999). Usefulness of spoligotyping To

Borile, C.; Labarre, M.; Franz, S.; Sola, C. & Refrégier, G. (2011). Using affinity propagation

secreted from Mycobacterium tuberculosis: identification by proteomics approach

discriminate IS6110 low-copy-number Mycobacterium tuberculosis complex strains

for identifying subspecies among clonal organisms: lessons from M. tuberculosis.

Lipomannan, overlapping peptide sequences

positioning mRNA for translation

Spot No. Protein Name Function

1813 YclD Unknown


Table 6. Expression levels of 19 proteins specifically overexpressed in the XDR-TB (protein expression level=number in the table×104)

There were 19 proteins specifically overexpressed in XDR-TB (Table 6), 3 proteins upregulated (Table 7), 13 proteins down-regulated (Table 8) and 10 proteins disappeared in the XDR-TB clinical isolate compared to the reference H37Rv strain.


Table 7. Expression levels of 3 up-regulated proteins in XDR-TB (protein expression level=number in the table×104)

Out of these forty-five protein spots, twenty were selected for the mass spectrometry analysis, and only three proteins were identified by the MALDI-TOF-MS (Table 9). The differential expression of the ribosomal protein S3 and 19 kda major membrane protein in the XDR-TB strain was validated by the real-time RT-PCR (data not shown). It is not clear how differential expression of these three proteins contributes to the drug resistance of the XDR-TB. Further function analysis of these three proteins will be conducted in our laboratory to determine their relationship with drug resistance of XDR-TB.


Table 8. Expression levels of 13 down-regulated proteins in XDR-TB (protein expression quantity=number in the table×104).


Table 9. Description of three proteins identified by MALDI-TOF-MS

#### **5. Conclusion**

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

2984 156.1 20.42 10.68 1328 36.14 3.39 6.39 1622 145.0 11.43 30.65 2418 31.63 3.22 3.04 2980 99.99 9.90 3.91 1440 31.49 3.43 3.28 1692 85.41 4.34 6.95 2679 26.51 5.71 3.25 1813 78.84 6.39 5.42 1600 26.19 1.36 3.61 1805 67.34 3.31 5.65 2379 13.16 1.821 1.12 2978 49.75 5.09 2.44 2787 11.91 1.79 0.85 1839 46.13 5.60 3.89 1994 9.77 0.90 2.11 1245 38.84 2.79 3.15 2449 5.37 0.72 0.74

Table 6. Expression levels of 19 proteins specifically overexpressed in the XDR-TB (protein

There were 19 proteins specifically overexpressed in XDR-TB (Table 6), 3 proteins upregulated (Table 7), 13 proteins down-regulated (Table 8) and 10 proteins disappeared in the

Out of these forty-five protein spots, twenty were selected for the mass spectrometry analysis, and only three proteins were identified by the MALDI-TOF-MS (Table 9). The differential expression of the ribosomal protein S3 and 19 kda major membrane protein in the XDR-TB strain was validated by the real-time RT-PCR (data not shown). It is not clear how differential expression of these three proteins contributes to the drug resistance of the XDR-TB. Further function analysis of these three proteins will be conducted in our

2394 58.18 159.8 122.2 2695 5.16 17.13 13.47 2994 43.26 11.03 92.87 2365 4.76 44.01 48.32 2991 26.93 41.37 65.52 2989 4.56 23.36 13.34 2499 18.92 49.45 49.02 2436 2.67 7.96 13.41 2637 16.45 84.32 53.78 1627 1.87 1.70 11.67 1279 11.77 18.13 36.18 2174 1.40 8.94 16.15

Table 8. Expression levels of 13 down-regulated proteins in XDR-TB (protein expression

No.

XDR-

TB MDR-TB H37Rv

v Spot No. XDR-

TB

MDR-

TB H37Rv

Spot

No. XDR-TB MDR-TB H37R

2726 36.46 3.16 3.07

XDR-TB clinical isolate compared to the reference H37Rv strain.

Spot No. XDR- TB MDR-TB H37Rv 2561 22.26 29.82 14.69 1228 46.98 4.657 20.38 2378 37.97 9.587 4.238 Table 7. Expression levels of 3 up-regulated proteins in XDR-TB (protein expression

laboratory to determine their relationship with drug resistance of XDR-TB.

No. XDR-TB MDR-TB H37Rv Spot

1616 7.60 22.65 12.33

quantity=number in the table×104).

expression level=number in the table×104)

level=number in the table×104)

Spot

In this chapter, we have provided the detailed description about our current progresses in the field of survey and molecular characterization of drug-resistant *M. tuberculosis* clinical isolates using different research methods such as *M. tuberculosis* culture, spoligotyping, gene sequencing, proteomics and drug susceptibility testing against first-line and second-line anti-tuberculosis drugs. We hope this chapter will be useful for researchers, scientists and physicians in academic institutions, clinical laboratories, pharmaceutical companies and research hospitals who have interest in the research related to drug-resistant *M. tuberculosis*, especially MDR/XDR-TB.

#### **6. Acknowledgments**

This project was funded in part with federal funds from the CDC, Department of Health and Human Services, under the contract no. 200-2007-M-22792, and by grants of International Cooperation Project from Guizhou Province of China [Qian Science co-G (2009) No. 700120], the Guizhou Science and Technology Fund Project [Qian Science co-J (2007) no. 2223] and Scientific Research Start Foundation from Zunyi Medical College in (F-327). The following people in the Affiliated Hospital of Zunyi Medical College made contributions to the research reported in this chapter: Xin Gan, Yang Chen, Kai-Lun Li, Yuan-Bo Lan, Zhen-Yong Chen, Lin-Mei Zhou, and Yi He. We would like to thank all health care staff in the Affiliated Hospital of Zunyi Medical College who participated in the research. We are also grateful to Professor Kanglin Wan, Zhiguang Liu, and Bing Lv in the Chinese Center for Disease Control and Prevention for their support with the spoligotyping analyses.

#### **7. References**


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**22** 

*India* 

**Molecular Biological Techniques for Detection** 

Tuberculosis (TB) is a contagious disease caused by *Mycobacterium tuberculosis* (*M. tuberculosis*). When people infected with tuberculosis cough, sneeze, talk or spit, the bacilli are propelled into the air. Each person with active TB disease will infect on average between 10 and 15 people every year. But people infected with TB bacilli will not necessarily become sick with the disease. The immune system "walls off" the TB bacilli which, protected by a thick waxy coat, can lie dormant for years. When the immune system is weakened, the

Overall, one-third of the world's population is currently infected with the TB bacillus. 5 - 10% of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life. People with HIV and TB infection are much

Globally, there were an estimated 14 million prevalent cases of TB in 2009, equivalent to 200 cases per 100 000 population. Most of the estimated number of cases in 2009 occurred in Asia (55%) and Africa (30%); 3 smaller proportions of cases occurred in the Eastern Mediterranean Region (7%), the European Region (4%) and the Region of the Americas (3%) [WHO, 2010]. In 2009, an estimated 1.3 million deaths (range: 1.2 – 1.5 million) occurred among HIV-negative cases of TB, including 0.38 million deaths (range: 0.3–0.5 million)

Until 50 years ago, there were no medicines to cure TB. Now, strains that are resistant to a single drug have been documented in every country surveyed and strains of TB resistant to all major anti-TB drugs have emerged. Drug-resistant TB is caused by inconsistent or partial treatment, when patients do not take all their medicines regularly for the required period because they start to feel better, or the doctors and health workers prescribe the wrong treatment regimens, or the drug supply is unreliable. A particularly dangerous form of drug-resistant TB is multidrug-resistant TB (MDR-TB), which is defined as the disease

among women. This is equivalent to 20 deaths per 100 000 population [WHO, 2010].

chances of the infection progressing to disease are higher.

more likely to develop TB [WHO, 2010].

**1. Introduction** 

**of Multidrug Resistant Tuberculosis** 

**(MDR) and Extremely Drug Resistant** 

**of** *Mycobacterium tuberculosis*

K.L. Therese, R. Gayathri and H.N. Madhavan

**Tuberculosis (XDR) in Clinical Isolates** 

*L&T Microbiology Research Centre, Vision Research Foundation, Chennai,* 


## **Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of** *Mycobacterium tuberculosis*

K.L. Therese, R. Gayathri and H.N. Madhavan *L&T Microbiology Research Centre, Vision Research Foundation, Chennai, India* 

#### **1. Introduction**

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

Soini, H.; Pan, X. ; Amin, A. ; Graviss, E.A. ; Siddiqui, A. & Musser, J.M. (2000).

Stulik, J. & Butaye, P. (2011). Introduction: application of proteomic technologies for the

van Soolingen, D.; Hermans, P.W.; de Haas, P.E.; Soll, D.R.; & van Embden, J.D. (1991).

van Soolingen, D.; Qian, L.; de Haas, P.E.; Douqlas, J.T.; Traore, H.; Portaels, F.; Qing, H.Z.;

van Soolingen, D.; van der Zanden, A.G.; de Haas, P.E.; Noordhoek, G.T.; Kiers, A.;

Viana-Niero, C.; Gutierrez, C.; Sola, C.; Filliol, L.; Boulahbal, F.; Vincent, V. & Rastoqi, N.

World Health Organization. (1998). Laboratory services in tuberculosis control. Part III:

World Health Organization. (2009). Guidelines for surveillance of drug resistance in tuberculosis. 4th Ed. who.int/tb/publications/mdr\_surveillance/en/index.html. World Health Organization. (2010). Noncommercial culture and drug-susceptibility testing

World Health Organization. WHO Report 2010. (2011). Global Tuberculosis Control 2010. http://www.who.int/tb/publications/global\_report/2010/en/index.html. Wu, X.Q. & Liu, Y.N. (2009). Advances in Mycobacterium tuberculosis proteomics.

Xie, Y.E.; Ren. B.X.; Hu, W.M.; Tang, E.J. & Jing, B.Q. (2009). Comparison of proteins of

Zhu, N.X.; Zheng, S.; Xu, R.Z. & Yu, R.X. (2003). Overexpression of S3 ribosomal protein

Culture. Geneva, WHO (document WHO/TB/98.258).

*Zhonghua Jie He He Hu Xi Za Zhi* 32(7): 527-9.

isolate. *Chin J Public Health* 25(5): 515-6.

variable number of tandem DNA repeats. *J Clin Microbiol* 39(1): 57-65. Wang, Q.; Yue, J.; Zhang, L.; Xu, Y.; Chen, J.; Zhang, M.; Zhu, B.; Wang, H. & Wang, H.

Texas, by spoligotyping. *J Clin Microbiol* 38(2): 669-76.

Robert G. Ulrich. 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

*Med Res* 132:400-8.

2578-86.

*Microbiol* 33(12): 3234-8.

*Clin Microbiol* 36(7): 1840-5.

*Proteome Res* 6(12): 4564-71.

s\_mar2011.pdf

24(3): 141-3.

tuberculosis by two-dimensional gel electrophoresis & mass spectrometry. *Indian J* 

Characterization of Mycobacterium tuberculosis isolates from patients in Houston,

analysis of microbial infections. *In BSL3 and BSL4 Agents: Proteomics, Glycomics, and Antigenicity*, First Edition. Edited by Jiri Stulik, Rudolf Toman, Patrick Butaye,

Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. *J Clin Microbiol* 29(11):

Enkhsaikan, D.; Nymadawa, P. & van Embden, J.D. (1995). Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. *J Clin* 

Foudraine, N.A.; Portaels, F.; Kolk, A.H. & Kremer, K. (1998). Diagnosis of Mycobacterium microti infections among humans by using novel genetic markers. *J* 

(2001). Genetic diversity of Mycobacterium africanum clinical isolates based on IS6110-restriction fragment length polymorphism analysis, spoligotyping, and

(2007). A newly identified 191A/C mutation in the Rv2629 gene that was significantly associated with rifampin resistance in Mycobacterium tuberculosis. *J* 

methods for screening patients at risk for multidrug-resistant tuberculosis. www.who.int/tb/laboratory/whopolicy\_noncommercialculture\_and\_dst\_method

multi-drug-resistant Mycobacterium tuberculosis isolate with drug-sensitive

gene is involved in drug resistance in K562/DOX cells. *Zhonghua Xue Ye Xue Za Zhi*

Tuberculosis (TB) is a contagious disease caused by *Mycobacterium tuberculosis* (*M. tuberculosis*). When people infected with tuberculosis cough, sneeze, talk or spit, the bacilli are propelled into the air. Each person with active TB disease will infect on average between 10 and 15 people every year. But people infected with TB bacilli will not necessarily become sick with the disease. The immune system "walls off" the TB bacilli which, protected by a thick waxy coat, can lie dormant for years. When the immune system is weakened, the chances of the infection progressing to disease are higher.

Overall, one-third of the world's population is currently infected with the TB bacillus. 5 - 10% of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life. People with HIV and TB infection are much more likely to develop TB [WHO, 2010].

Globally, there were an estimated 14 million prevalent cases of TB in 2009, equivalent to 200 cases per 100 000 population. Most of the estimated number of cases in 2009 occurred in Asia (55%) and Africa (30%); 3 smaller proportions of cases occurred in the Eastern Mediterranean Region (7%), the European Region (4%) and the Region of the Americas (3%) [WHO, 2010]. In 2009, an estimated 1.3 million deaths (range: 1.2 – 1.5 million) occurred among HIV-negative cases of TB, including 0.38 million deaths (range: 0.3–0.5 million) among women. This is equivalent to 20 deaths per 100 000 population [WHO, 2010].

Until 50 years ago, there were no medicines to cure TB. Now, strains that are resistant to a single drug have been documented in every country surveyed and strains of TB resistant to all major anti-TB drugs have emerged. Drug-resistant TB is caused by inconsistent or partial treatment, when patients do not take all their medicines regularly for the required period because they start to feel better, or the doctors and health workers prescribe the wrong treatment regimens, or the drug supply is unreliable. A particularly dangerous form of drug-resistant TB is multidrug-resistant TB (MDR-TB), which is defined as the disease

Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and

Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of *Mycobacterium tuberculosis* 457

surveillance data on MDR-TB: 42 perform continuous surveillance of anti-TB drug resistance based on routine testing of all TB patients; 72 rely on periodic surveys of representative samples of TB patients. The Russian Federation, which was able to provide high-quality continuous surveillance data from 12 of its oblasts and republics, reported proportions of 23.8–28.3% MDRTB among new TB cases in three of its oblasts in the northwest part of the country. Other Russian oblasts were found to have proportions of MDR-TB as low as 5.4% among new TB cases. Tajikistan, in its first ever survey, found proportions of 16.5% MDR-TB among new TB cases and 61.6% MDR-TB among previously treated TB patients in Dushanbe city and Rudaki district, the highest proportion ever reported among previously treated TB patients. To date, 12 countries have reported nationwide or subnational proportions of MDR-TB of 6% or more among new TB cases [WHO, November 2010].

Five of these countries also report MDR-TB proportions of 50% or more among previously treated cases. All of these settings are located in the eastern part of Europe or in Central

**2.2 Recently developed molecular techniques for the detection of drug resistance in** 

There are currently two commercially available solid-phase hybridization techniques: the Line Probe Assay (INNO-LiPA Rif TB Assay; Innogenetics, Ghent, Belgium) for the detection of rifampicin resistance and the GenoType MTBDR assay (Hain Lifesciences, Nehren, Germany) for the simultaneous detection of isoniazid and rifampicin resistance. The LiPA assay was introduced several years ago and is based on the hybridization of amplified DNA from cultured strains or clinical samples to 10 probes covering the core region of the *rpoB* gene of *M. tuberculosis*, immobilized on a nitrocellulose strip. The GenoType MTBDR on the other hand, detects resistance to isoniazid and rifampicin in culture samples based on the detection of the most common mutations in the *katG* and *rpoB* genes respectively. Both assays have now been evaluated in different settings, giving encouraging results. In a recent study Hillemann *et al* evaluated the GenoType MTBDR assay and found that 99% of MDR strains with mutations in the *rpoB* gene and 88.4% of strains with mutations in the codon 315 of the *katG* gene were correctly identified. Correlation with DNA sequencing was 100% and compared with conventional tests good sensitivity and specificity were also obtained. Both solid hybridization methods have shown to be relatively simple to perform although basic expertise in molecular biology and PCR techniques is required. As with other genotypic methods the sensitivity of the test depends on the amount of DNA present in the sample

**INNOLiPA Rif. TB kit:** INNOLiPA Rif. TB kit simultaneously detects the *M. tuberculosis* complex and the presence of mutations in the *rpoB* gene associated with resistance to rifampicin which is considered a marker for MDR-TB strains. The strip contains 5 probes for detection of sensitive genotypes (S1-S5) [Morgan et al, 2005; Makinen et al, 2006] and 4 probes for detection of resistance genotypes (R2,R4a, R4b and R5). Rifampicin resistance is indicated by the absence of one or more sensitive probes, possible accompanied by the

and also the presence of inhibitors could cause false-negative results.

appearance of one or more mutant probes.

Asia.

**tuberculosis** 

**2.2.1 Solid-phase Hybridization techniques** 

caused by TB bacilli resistant to isoniazid and rifampicin, the two most powerful anti-TB drugs. Rates of MDR-TB are high in some countries, especially in the former Soviet Union, and threaten TB control efforts [WHO, 2010].

While drug-resistant TB is generally treatable, it requires extensive chemotherapy (up to two years of treatment) with second-line anti-TB drugs which are more costly than first-line drugs, and which produce adverse drug reactions that are more severe, though manageable. Quality assured second-line anti-TB drugs are available at reduced prices for projects approved by the Green Light Committee [WHO, 2010].

The emergence of extensively drug-resistant (XDR) TB, particularly in settings where many TB patients are also infected with HIV, poses a serious threat to TB control, and confirms the urgent need to strengthen basic TB control and to apply the new WHO guidelines for the programmatic management of drug-resistant TB.

The important ramifications in the laboratory diagnosis of tuberculosis and drug resistant tuberculosis are: the delay in the isolation of the bacilli in culture, low sensitivity/detection limit of the direct smears and lack of technically trained personnel. Identification of tuberculosis using molecular techniques namely, polymerase chain reaction (PCR), PCR based restriction fragment length polymorphism (PCR-RFLP) and PCR based DNA sequencing are very rapid, more sensitive and reliable when compared to the conventional culture. There are several in house nested PCR (nPCR) standardized for the detection of *M. tuberculosis* from clinical specimens targeting MPB64 [Therese KL et al, 2005], IS6110 [Wang et al, 2004] and 16S rRNA [Ninet et al, 1996] genes. MPB64 is an immunogenic protein produced by *M. tuberculosis* and a few strains of *M. bovis* and BCG strain. IS6110 is an insertion element present in single or multiple copies in *M. tuberculosis* complex isolates. There are studies which reported on the lack of IS6110 insertion element in strains isolated from Asian population. Also there are strain variations among the *M. tuberculosis* isolates. Thus multiplex PCR or nPCR targeting more than one gene target will be a better tool for the detection of *M. tuberculosis* from direct clinical specimens.

Drug resistance in *M. tuberculosis* is caused by mutations in relatively restricted regions of the genome. Mutations associated with drug resistance occur in *rpoB* for rifampicin (RIF) *katG* and the promoter region of the *mabA (fabG*1)-*inhA* operon for Isoniazid (INH), *embB* for ethambutol (EMB), *pncA* for pyrazinamide (PZA), *rpsL* and *rrs* for streptomycin (STR) and *gyrA, gyrB* for fluoroquinolones (FQs) such as ofloxacin (OFX) and levofloxacin (LVX) [Musser et al, 1995; Zhang et al, 2000].

#### **2. MDR-TB**

#### **2.1 Worldwide reports on MDR-TB**

In 2008, there were an estimated 440,000 (range, 390 000-510 000) MDR-TB cases emerging worldwide. About 250,000 of these cases (range: 230 000–270 000) should have been reported to WHO, if countries had tested all the TB patients that they notified for drug resistance. However, only just over 30,000 MDR-TB cases (12%) were actually notified globally in 2009 [WHO, November 2010].

Almost 50% of MDR-TB cases worldwide are estimated to occur in China and India. In 2008, MDR-TB caused an estimated 150,000 deaths. Since 1994, 114 countries have reported surveillance data on MDR-TB: 42 perform continuous surveillance of anti-TB drug resistance based on routine testing of all TB patients; 72 rely on periodic surveys of representative samples of TB patients. The Russian Federation, which was able to provide high-quality continuous surveillance data from 12 of its oblasts and republics, reported proportions of 23.8–28.3% MDRTB among new TB cases in three of its oblasts in the northwest part of the country. Other Russian oblasts were found to have proportions of MDR-TB as low as 5.4% among new TB cases. Tajikistan, in its first ever survey, found proportions of 16.5% MDR-TB among new TB cases and 61.6% MDR-TB among previously treated TB patients in Dushanbe city and Rudaki district, the highest proportion ever reported among previously treated TB patients. To date, 12 countries have reported nationwide or subnational proportions of MDR-TB of 6% or more among new TB cases [WHO, November 2010].

Five of these countries also report MDR-TB proportions of 50% or more among previously treated cases. All of these settings are located in the eastern part of Europe or in Central Asia.

#### **2.2 Recently developed molecular techniques for the detection of drug resistance in tuberculosis**

#### **2.2.1 Solid-phase Hybridization techniques**

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

caused by TB bacilli resistant to isoniazid and rifampicin, the two most powerful anti-TB drugs. Rates of MDR-TB are high in some countries, especially in the former Soviet Union,

While drug-resistant TB is generally treatable, it requires extensive chemotherapy (up to two years of treatment) with second-line anti-TB drugs which are more costly than first-line drugs, and which produce adverse drug reactions that are more severe, though manageable. Quality assured second-line anti-TB drugs are available at reduced prices for projects

The emergence of extensively drug-resistant (XDR) TB, particularly in settings where many TB patients are also infected with HIV, poses a serious threat to TB control, and confirms the urgent need to strengthen basic TB control and to apply the new WHO guidelines for the

The important ramifications in the laboratory diagnosis of tuberculosis and drug resistant tuberculosis are: the delay in the isolation of the bacilli in culture, low sensitivity/detection limit of the direct smears and lack of technically trained personnel. Identification of tuberculosis using molecular techniques namely, polymerase chain reaction (PCR), PCR based restriction fragment length polymorphism (PCR-RFLP) and PCR based DNA sequencing are very rapid, more sensitive and reliable when compared to the conventional culture. There are several in house nested PCR (nPCR) standardized for the detection of *M. tuberculosis* from clinical specimens targeting MPB64 [Therese KL et al, 2005], IS6110 [Wang et al, 2004] and 16S rRNA [Ninet et al, 1996] genes. MPB64 is an immunogenic protein produced by *M. tuberculosis* and a few strains of *M. bovis* and BCG strain. IS6110 is an insertion element present in single or multiple copies in *M. tuberculosis* complex isolates. There are studies which reported on the lack of IS6110 insertion element in strains isolated from Asian population. Also there are strain variations among the *M. tuberculosis* isolates. Thus multiplex PCR or nPCR targeting more than one gene target will be a better tool for

Drug resistance in *M. tuberculosis* is caused by mutations in relatively restricted regions of the genome. Mutations associated with drug resistance occur in *rpoB* for rifampicin (RIF) *katG* and the promoter region of the *mabA (fabG*1)-*inhA* operon for Isoniazid (INH), *embB* for ethambutol (EMB), *pncA* for pyrazinamide (PZA), *rpsL* and *rrs* for streptomycin (STR) and *gyrA, gyrB* for fluoroquinolones (FQs) such as ofloxacin (OFX) and levofloxacin (LVX)

In 2008, there were an estimated 440,000 (range, 390 000-510 000) MDR-TB cases emerging worldwide. About 250,000 of these cases (range: 230 000–270 000) should have been reported to WHO, if countries had tested all the TB patients that they notified for drug resistance. However, only just over 30,000 MDR-TB cases (12%) were actually notified globally in 2009

Almost 50% of MDR-TB cases worldwide are estimated to occur in China and India. In 2008, MDR-TB caused an estimated 150,000 deaths. Since 1994, 114 countries have reported

and threaten TB control efforts [WHO, 2010].

approved by the Green Light Committee [WHO, 2010].

the detection of *M. tuberculosis* from direct clinical specimens.

[Musser et al, 1995; Zhang et al, 2000].

**2.1 Worldwide reports on MDR-TB** 

[WHO, November 2010].

**2. MDR-TB** 

programmatic management of drug-resistant TB.

There are currently two commercially available solid-phase hybridization techniques: the Line Probe Assay (INNO-LiPA Rif TB Assay; Innogenetics, Ghent, Belgium) for the detection of rifampicin resistance and the GenoType MTBDR assay (Hain Lifesciences, Nehren, Germany) for the simultaneous detection of isoniazid and rifampicin resistance. The LiPA assay was introduced several years ago and is based on the hybridization of amplified DNA from cultured strains or clinical samples to 10 probes covering the core region of the *rpoB* gene of *M. tuberculosis*, immobilized on a nitrocellulose strip. The GenoType MTBDR on the other hand, detects resistance to isoniazid and rifampicin in culture samples based on the detection of the most common mutations in the *katG* and *rpoB* genes respectively. Both assays have now been evaluated in different settings, giving encouraging results. In a recent study Hillemann *et al* evaluated the GenoType MTBDR assay and found that 99% of MDR strains with mutations in the *rpoB* gene and 88.4% of strains with mutations in the codon 315 of the *katG* gene were correctly identified. Correlation with DNA sequencing was 100% and compared with conventional tests good sensitivity and specificity were also obtained. Both solid hybridization methods have shown to be relatively simple to perform although basic expertise in molecular biology and PCR techniques is required. As with other genotypic methods the sensitivity of the test depends on the amount of DNA present in the sample and also the presence of inhibitors could cause false-negative results.

**INNOLiPA Rif. TB kit:** INNOLiPA Rif. TB kit simultaneously detects the *M. tuberculosis* complex and the presence of mutations in the *rpoB* gene associated with resistance to rifampicin which is considered a marker for MDR-TB strains. The strip contains 5 probes for detection of sensitive genotypes (S1-S5) [Morgan et al, 2005; Makinen et al, 2006] and 4 probes for detection of resistance genotypes (R2,R4a, R4b and R5). Rifampicin resistance is indicated by the absence of one or more sensitive probes, possible accompanied by the appearance of one or more mutant probes.

Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and

care.

**2.2.3 Microarray** 

*tuberculosis* 

type element.

various oligonucleotide probes.

Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of *Mycobacterium tuberculosis* 459

29 resistant *M. tuberculosis* isolates [Garcia de Viedma et al, 2002]. Since not all gene mutations conferring drug resistance are well characterized and are thus not amenable to PCR assay development, traditional culture-based susceptibility testing methods are still required. However, the ability to predict rifampin and isoniazid resistance up to 2 weeks sooner than current methods for some isolates should have significant benefit for patient

An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules [Heyman et al, 1999]. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots. Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane [Heyman et al, 1999]. The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot

Microarray technology is used in the detection of drug resistant *M. tuberculosis* rather than the detection of *M. tuberculosis* from clinical specimens. The TB Biochip oligonucleotide microarray is the most widely used Microarray for the detection of isoniazid and rifampicin

The TB-Biochip oligonucleotide microarray system is designed to detect and identify 29 codon substitutions and 1 codon deletion distributed over 10 codon positions (507, 511, 512, 513, 515, 516, 522, 526, 532, 533) within the rifampicin resistant determining region (RRDR). Each element of the microarray contains an immobilized oligonucleotide whose sequence matches that of either a wild-type or mutated segment of the RRDR. The use of acrylamide gel pads increases the robustness of the hybridization reaction. Hybridization of the microarray with fluorescently labeled target DNA produces a spatial pattern of fluorescence intensities corresponding to the efficiencies of hybridization of the labeled target DNA to the

In the TB-Biochip system, the fluorescence intensities are recorded using a charge-coupled device camera, and the relative intensities of fluorescence for the elements representing wildtype sequences and mutant sequences for each codon are compared using imaging software and automated computer-assisted interpretation of hybridization results [Garcia de Viedma D et al, 2003; Cavusoglu et al, 2004]. The isolate is designated **RIF susceptible** if the fluorescence of each of the wild-type elements is greater than the fluorescence of any of the corresponding mutant elements. The isolate is designated **RIF resistant** if the fluorescence of any one of the mutant elements is greater than the fluorescence of its corresponding wild-

**TB-Biochip oligonucleotide microarray for the detection of rifampicin resistant** *M.* 

on the array is used for the identification of a gene [Heyman et al, 1999].

resistance simultaneously [Garcia de Viedma D et al, 2003].

The **GenoType MTBDR** test is able to detect mutations in the *rpoB* gene for RIF resistance, and the most frequent mutation at codon 315 of the *katG* gene for INH resistance, either in isolates or clinical specimens. The specificity and sensitivity of the assay for RIF resistance were nearly 100%; for INH-resistance, despite a high specificity (approximately 100%), the sensitivity of the test ranged from 70% to 90%, depending on the prevalence of the particular mutation at the *katG* locus [Hilleman et al, 2007].

**GenoType MTBDRplus** (Hain Lifescience, Germany), an advanced version of the assay, includes probes for the identification of other mutations in the hotspot region of the *rpoB* gene for RIF resistance, and probes to detect mutations in the promoter region of the *inhA*  gene involved in INH resistance. These improvements facilitate the detection of another 10% to 20% of INH-resistant cases, with an enhancement in rapid MDR-TB diagnosis.

The line probe assays are accurate and useful for rapid detection of drug resistance directly in clinical specimens. However, the number of genes that can be analyzed remains limited and the test fails to distinguish insertion mutations. Furthermore, they retain a lower sensitivity among acid fast bacilli-negative samples. In general, line probe assays are expensive and require sophisticated laboratory infrastructure. Their role and utility in lowincome, high-burden countries needs to be evaluated in field studies.

#### **2.2.2 Real-time Polymerase Chain Reaction techniques**

Real-time PCR techniques have also been introduced for rapid detection of drug resistance. Different probes have been used like the TaqMan probe, fluorescence resonance energy transfer (FRET) probes, molecular beacons and bioprobes. The main advantages of real-time PCR techniques are the speed of the test and a lower risk of contamination. The main disadvantages would be the requirement for expensive equipment and reagents, and the need for skilled technical personnel. Real-time PCR was initially applied to *M. tuberculosis* strains but more recently it has been successfully applied directly in clinical samples. Results could be obtained in an average of 1.5-2.0 h after DNA extraction. Realtime PCR could eventually be implemented in reference laboratories with the required capacity to properly set up the technique and in settings where it can contribute to the management of TB patients.

Detection of antitubercular drug resistance is vital to effective patient management. Realtime PCR offers the potential to detect gene mutations responsible for drug resistance within hours from patient specimens compared with the average of 2 weeks required for traditional susceptibility test methods. The *rpoB* and *katG* genes are the most common *M. tuberculosis*  targets utilized in real-time PCR methods and well-known mutations in these genes correlate with resistance to rifampin and isoniazid, respectively [Edwards et al, 2001; El-Hajj et al, 2001; Garcia de Viedma et al, 2002; O'Mahony et al, 2002; Piatek et al, 2002; Torres et al, 2000; Torres et al, 2003; van Doorn et al, 2003]. The significance of other gene targets such as *kasA*, *ahpC*-*oxyR*, and *inhA* for the prediction of isoniazid resistance is somewhat controversial [Piatek et al, 2000]. Torres et al used two sets of FRET hybridization probes to detect *rpoB* mutations in 24 rifampin-resistant strains *of M. tuberculosis* and another set of FRET hybridization probes to detect *katG* mutations in 15 isoniazid-resistant *M. tuberculosis*  strains [Torres et al, 2000]. Additionally, Garcia de Viedma et al. used two sets of *rpoB*  probes and one set of *katG* probes to detect *rpoB* and *katG* mutations, but in a single tube, for 29 resistant *M. tuberculosis* isolates [Garcia de Viedma et al, 2002]. Since not all gene mutations conferring drug resistance are well characterized and are thus not amenable to PCR assay development, traditional culture-based susceptibility testing methods are still required. However, the ability to predict rifampin and isoniazid resistance up to 2 weeks sooner than current methods for some isolates should have significant benefit for patient care.

#### **2.2.3 Microarray**

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

The **GenoType MTBDR** test is able to detect mutations in the *rpoB* gene for RIF resistance, and the most frequent mutation at codon 315 of the *katG* gene for INH resistance, either in isolates or clinical specimens. The specificity and sensitivity of the assay for RIF resistance were nearly 100%; for INH-resistance, despite a high specificity (approximately 100%), the sensitivity of the test ranged from 70% to 90%, depending on the prevalence of the particular

**GenoType MTBDRplus** (Hain Lifescience, Germany), an advanced version of the assay, includes probes for the identification of other mutations in the hotspot region of the *rpoB* gene for RIF resistance, and probes to detect mutations in the promoter region of the *inhA*  gene involved in INH resistance. These improvements facilitate the detection of another 10%

The line probe assays are accurate and useful for rapid detection of drug resistance directly in clinical specimens. However, the number of genes that can be analyzed remains limited and the test fails to distinguish insertion mutations. Furthermore, they retain a lower sensitivity among acid fast bacilli-negative samples. In general, line probe assays are expensive and require sophisticated laboratory infrastructure. Their role and utility in low-

Real-time PCR techniques have also been introduced for rapid detection of drug resistance. Different probes have been used like the TaqMan probe, fluorescence resonance energy transfer (FRET) probes, molecular beacons and bioprobes. The main advantages of real-time PCR techniques are the speed of the test and a lower risk of contamination. The main disadvantages would be the requirement for expensive equipment and reagents, and the need for skilled technical personnel. Real-time PCR was initially applied to *M. tuberculosis* strains but more recently it has been successfully applied directly in clinical samples. Results could be obtained in an average of 1.5-2.0 h after DNA extraction. Realtime PCR could eventually be implemented in reference laboratories with the required capacity to properly set up the technique and in settings where it can contribute to the

Detection of antitubercular drug resistance is vital to effective patient management. Realtime PCR offers the potential to detect gene mutations responsible for drug resistance within hours from patient specimens compared with the average of 2 weeks required for traditional susceptibility test methods. The *rpoB* and *katG* genes are the most common *M. tuberculosis*  targets utilized in real-time PCR methods and well-known mutations in these genes correlate with resistance to rifampin and isoniazid, respectively [Edwards et al, 2001; El-Hajj et al, 2001; Garcia de Viedma et al, 2002; O'Mahony et al, 2002; Piatek et al, 2002; Torres et al, 2000; Torres et al, 2003; van Doorn et al, 2003]. The significance of other gene targets such as *kasA*, *ahpC*-*oxyR*, and *inhA* for the prediction of isoniazid resistance is somewhat controversial [Piatek et al, 2000]. Torres et al used two sets of FRET hybridization probes to detect *rpoB* mutations in 24 rifampin-resistant strains *of M. tuberculosis* and another set of FRET hybridization probes to detect *katG* mutations in 15 isoniazid-resistant *M. tuberculosis*  strains [Torres et al, 2000]. Additionally, Garcia de Viedma et al. used two sets of *rpoB*  probes and one set of *katG* probes to detect *rpoB* and *katG* mutations, but in a single tube, for

to 20% of INH-resistant cases, with an enhancement in rapid MDR-TB diagnosis.

income, high-burden countries needs to be evaluated in field studies.

**2.2.2 Real-time Polymerase Chain Reaction techniques** 

management of TB patients.

mutation at the *katG* locus [Hilleman et al, 2007].

An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules [Heyman et al, 1999]. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots. Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane [Heyman et al, 1999]. The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene [Heyman et al, 1999].

Microarray technology is used in the detection of drug resistant *M. tuberculosis* rather than the detection of *M. tuberculosis* from clinical specimens. The TB Biochip oligonucleotide microarray is the most widely used Microarray for the detection of isoniazid and rifampicin resistance simultaneously [Garcia de Viedma D et al, 2003].

#### **TB-Biochip oligonucleotide microarray for the detection of rifampicin resistant** *M. tuberculosis*

The TB-Biochip oligonucleotide microarray system is designed to detect and identify 29 codon substitutions and 1 codon deletion distributed over 10 codon positions (507, 511, 512, 513, 515, 516, 522, 526, 532, 533) within the rifampicin resistant determining region (RRDR). Each element of the microarray contains an immobilized oligonucleotide whose sequence matches that of either a wild-type or mutated segment of the RRDR. The use of acrylamide gel pads increases the robustness of the hybridization reaction. Hybridization of the microarray with fluorescently labeled target DNA produces a spatial pattern of fluorescence intensities corresponding to the efficiencies of hybridization of the labeled target DNA to the various oligonucleotide probes.

In the TB-Biochip system, the fluorescence intensities are recorded using a charge-coupled device camera, and the relative intensities of fluorescence for the elements representing wildtype sequences and mutant sequences for each codon are compared using imaging software and automated computer-assisted interpretation of hybridization results [Garcia de Viedma D et al, 2003; Cavusoglu et al, 2004]. The isolate is designated **RIF susceptible** if the fluorescence of each of the wild-type elements is greater than the fluorescence of any of the corresponding mutant elements. The isolate is designated **RIF resistant** if the fluorescence of any one of the mutant elements is greater than the fluorescence of its corresponding wildtype element.

Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and

Pipette 20l QIAGEN Proteinase k into the bottom of a 1.5ml microfuge tube.

Add 200l of AL buffer to the sample. Mix by pulse vortexing for 15 seconds

 Briefly centrifuge the micro centrifuge tube to remove drops from inside of the lid. Add 200l of ethanol (96-100%) to the sample, mix by pulse vortexing for 15 seconds. After mixing, briefly centrifuge the 1.5ml microfuge tube to remove drops from inside

serum, Buffy coat upto 5 X 106 lymphocytes in 200l PBS

i. Micropipettes (20-200µl) j. Filter barriers tips (20-200µl)

Incubate at 56° C for 10 minutes.

tube containing the filtrate.

empty spin at 14,000 rpm for 1min.

**Reagents and other accessories required:** 

1min. Discard the column and store the DNA at -20°C.

**Procedure** 

of the lid.

**2.3.2 PCR protocol** 

using MilliQ water. Forward and reverse primers *Taq* DNA polymerase (3 units)

 Sterile 0.5 ml or 0.2 ml vials Micropipettes (20-200µl, 0.5- 10µl) Filter barrier tips (20-200µl) Gel casting tray/trough

PCR Thermal cycler

Electrophoresis tank

Gel documentation system

Cyclomixer

Gel combs

Powerpack

Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of *Mycobacterium tuberculosis* 461

Add 200l of the sample to the microfuge tube. Use up to 200l whole blood, plasma,

 Carefully apply the mixture from step 6 to the QIAamp mini spin column (in a 2ml collecting tube) without wetting the rim. Close the cap, and centrifuge at 8000 rpm for 1 min. Place the QIAamp mini spin column in a clean 2 ml collecting tube and discard the

 Carefully open the QIAamp mini spin column and add 500l buffer AW1 without wetting the rim. Close the cap and centrifuge at 8000 rpm for 1min. Place the QIAamp mini spin column in a clean 2ml collecting tube and discard the tube containing filtrate. Carefully open the QIAamp mini spin column and add 500l buffer AW2 without wetting the rim. Close the cap and centrifuge at 14,000 rpm for 3min, followed by an

 Place the QIAamp mini spin column in a clean 1.5 ml microfuge tube and discard the tube containing filtrate. Carefully open the QIAamp mini spin column and add 200l AE Buffer. Incubate at room temperature for 1min, and then centrifuge at 8,000 rpm for

 Stock dNTPs dilution: 100 mM concentration of dNTPs- dATP, dCTP, dTTP and dGTP Working standard dNTP (200µM): 2 µl of each of the stock dNTP made up to 400 µl

#### **Advantange and disadvantage of TB- biochip system**

The complete TB-Biochip system may be suitable for use in clinical laboratories with molecular biology expertise because it requires relatively little hands-on time for experimental manipulations or data analysis, tests can be run individually or in batches, and specialized training is not required. The observed discrepancies between the results of conventional DST and the TB-Biochip system (all were falsely called susceptible with the TB-Biochip system) likely result from the large number of mutations found in RIF-resistant isolates and the limited range of mutations included on the biochip.

Although technically a solid-phase-type hybridization assay, microarrays, also known as biochips, have been proposed as new molecular methods for detecting drug resistance in *M. tuberculosis*. They are based on the hybridization of DNA obtained from clinical samples to oligonucleotides immobilized in a solid support, such as miniaturized glass slides. They have been mainly used to detect resistance to rifampicin. In a recent evaluation using oligonucleotide microarrays for analysis of drug resistance, Gryadunov *et al*. detected over 95% rifampicin resistant and almost 80% isoniazid resistant *M. tuberculosis* isolates within 12 h in a sample of drug-resistant isolates and clinical samples. For the time being and due to the high cost involved, the use of microarrays for detecting drug resistance in TB is still beyond the reach of most clinical mycobacteriology laboratories.

#### **2.3 DNA sequencing**

Sequencing DNA of PCR amplified products has been the most widely used method; it is accurate and reliable and it has become the gold standard for mutation detection. It has been performed by manual and automated procedures although the latter is now the most commonly used. It has been widely used for characterizing mutations in the *rpoB* gene in rifampicin-resistant strains and to detect mutations responsible for resistance to other antituberculosis drugs. It would be rather difficult, however, to implement it routinely for detection of drug resistance mutations for several drugs since it would involve several reactions for each isolate, making the cost high.

#### **2.3.1 DNA extraction**

DNA from clinical *M. tuberculosis* isolates can be extracted by using Qiagen kit (Germany) and also by keeping the MGIT suspension at 80C for 10 minutes. After 10 minutes, centrifuged at 3000 rpm and the supernatant can be used as template DNA for PCR. The procedure for extraction of DNA using Qiagen kit is mentioned below.

#### **Reagents and other accessories required**


#### **Procedure**

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

The complete TB-Biochip system may be suitable for use in clinical laboratories with molecular biology expertise because it requires relatively little hands-on time for experimental manipulations or data analysis, tests can be run individually or in batches, and specialized training is not required. The observed discrepancies between the results of conventional DST and the TB-Biochip system (all were falsely called susceptible with the TB-Biochip system) likely result from the large number of mutations found in RIF-resistant

Although technically a solid-phase-type hybridization assay, microarrays, also known as biochips, have been proposed as new molecular methods for detecting drug resistance in *M. tuberculosis*. They are based on the hybridization of DNA obtained from clinical samples to oligonucleotides immobilized in a solid support, such as miniaturized glass slides. They have been mainly used to detect resistance to rifampicin. In a recent evaluation using oligonucleotide microarrays for analysis of drug resistance, Gryadunov *et al*. detected over 95% rifampicin resistant and almost 80% isoniazid resistant *M. tuberculosis* isolates within 12 h in a sample of drug-resistant isolates and clinical samples. For the time being and due to the high cost involved, the use of microarrays for detecting drug resistance in TB is still

Sequencing DNA of PCR amplified products has been the most widely used method; it is accurate and reliable and it has become the gold standard for mutation detection. It has been performed by manual and automated procedures although the latter is now the most commonly used. It has been widely used for characterizing mutations in the *rpoB* gene in rifampicin-resistant strains and to detect mutations responsible for resistance to other antituberculosis drugs. It would be rather difficult, however, to implement it routinely for detection of drug resistance mutations for several drugs since it would involve several

DNA from clinical *M. tuberculosis* isolates can be extracted by using Qiagen kit (Germany) and also by keeping the MGIT suspension at 80C for 10 minutes. After 10 minutes, centrifuged at 3000 rpm and the supernatant can be used as template DNA for PCR. The

procedure for extraction of DNA using Qiagen kit is mentioned below.

**Advantange and disadvantage of TB- biochip system** 

isolates and the limited range of mutations included on the biochip.

beyond the reach of most clinical mycobacteriology laboratories.

reactions for each isolate, making the cost high.

**Reagents and other accessories required** 

**2.3 DNA sequencing** 

**2.3.1 DNA extraction** 

a. Proteinase K

c. Ethanol

b. Lysis buffer (AL buffer)

d. Washing buffer-1 (AW1 buffer) e. Washing buffer-2 (AW 2 Buffer) f. Elution buffer (AE Buffer) g. Minispin (eppendorff) h. Sterile 1.5ml vials


#### **2.3.2 PCR protocol**

#### **Reagents and other accessories required:**


Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and

**Target genes / Primer sequence** 

CGAGGAATTGGCCGACGAGTT CGGCGCCGCGGAGTTGAATGA

CCGGCACCTACCGCATCCAC GCCCCAATAGACCTCATCGG

CCTCGCTGCCCAGAAAGG A ATCCCCCGGTTTCCTCCGGT

GCTTGATGTCCGAGAGCAT GGTCGCGTAGGCAGTGCCCC

GGCCGACAAACAGAACGT GTTCACCAACTGGGTGAC

TTGGCCATGCTCTTGATGCCC TGCACACAGGCCACAAGGGA

GGCGTCATGGACCCTATATC CAACAGTTCATCCCGGTTC

GGTGGGCAGGATGAGGTAGT

CCGACCACGCTGAAACTGCTGGCGAT

*katG3* (targeting 138 codon) for isoniazid

*katG4* (targeting 315 codon) for isoniazid GAAACAGCGGCGCTGGATCGT GTTGTCCCATTTCGTCGGGG

CCACCCAGGACGTGGAGGCGATCACAC AGTGCGACGGGTGCACGTCGCGGACCT

**(5'-3' Direction)** 

*rpoB* for rifampicin

*katG1* for isoniazid GCCCGAGCAACACCC ATGTCCCGCGTCAGG

*katG2* for isoniazid

*inhA* for isoniazid

*oxyR-ahpC* for isoniazid

*rpsL* for streptomycin

*rrs* for streptomycin

*pncA* for pyrazinamide

*embB* for ethambutol

Sreevatsan et al 1997]

Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of *Mycobacterium tuberculosis* 463

**Thermal profile** 

95 ºC-5 min 95ºC-30sec 72ºC-1min 72ºC -5 min

94C - 1min 58C –1min 72C –2 min

94C - 1min 55C –1min 72C –2 min

95C - 1min 60C –30sec 72C –1 min

94C - 5min 94C - 1min 64C –1min 72C –2 min

94C - 2min 94C - 1min 60C –1min 72C –2 min

94C - 1min 94C - 1min 56C –1min 72C –1 min 72C – 7 min

94C - 1min 94C - 1min 56C –1min 72C –1 min 72C – 7 min

94C – 30 sec 60C – 30 sec 72C – 30 sec

95C – 5 sec 55C – 10 sec 72C – 10 sec

Table 2. Primer sets used in the study to sequence the different loci of target genes with their

thermal profile and expected amplicon sizes[Sekiguchi et al, 2007; Siddiqi et al, 2002;

**No. of Cycles** 

35 286

35 237

35 414

30 269

40 248

35 701

35 505

35 1140

35 670

35 937

Same as above 209

**Expected Amplicon Size** 

**(bp)** 

The PCR cocktail contained the following:


#### **2.3.3 PCR protocol for amplification of** *embB* **gene using primers targeting 640-1577 region**

PCR targeting *embB* gene was standardized with a specialized Taq DNA polymerase enzyme called "Z Taq" enzyme (Takara Bio, Ohtsu, Shiga, Japan). The *Z-Taq* polymerase offers unmatched PCR productivity, with a processing speed five times faster than those of other commercially available *Taq* polymerases. The total PCR cycle takes only 29 minutes. All the reagents for PCR (dNTP, 10X, Z Taq) will be provided along with the buffer. Each 50 µl reaction contained 2.5mM dNTP, 10X, 1pM of forward and reverse primers and 2.5 Units of Z taq enzyme. The details of primer targeting drug resistance genes, their thermal profile used and the expected amplicon size are given in the Table2.

**Detection Of Amplified Products:** The amplified product is subjected to electrophoresis on 2% agarose gel incorporated with 0.5 µg/ml ethidium bromide for visualization by UV transilluminator (Vilber Lourmat – France).

#### **2.3.4 DNA sequencing of amplified products**

The term **DNA sequencing** refers to sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA. DNA sequencing enables us to perform a thorough analysis of DNA because it provides us with the most basic information of all: the sequence of nucleotides. The classical chaintermination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. *Dye-terminator sequencing* utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which with different wavelengths of fluorescence and emission. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis. This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye

 dNTP 8 µl 10X buffer (15mM Mg2+,Tris, Kcl(500 mM)-pH 8.3) 5 µl Forward Primer (1 pM) 1 µl Reverse Primer (1 pM) 1 µl MilliQ water 30 µl  *Taq* polymerase 0.3 µl

**2.3.3 PCR protocol for amplification of** *embB* **gene using primers targeting** 

used and the expected amplicon size are given in the Table2.

transilluminator (Vilber Lourmat – France).

**2.3.4 DNA sequencing of amplified products** 

PCR targeting *embB* gene was standardized with a specialized Taq DNA polymerase enzyme called "Z Taq" enzyme (Takara Bio, Ohtsu, Shiga, Japan). The *Z-Taq* polymerase offers unmatched PCR productivity, with a processing speed five times faster than those of other commercially available *Taq* polymerases. The total PCR cycle takes only 29 minutes. All the reagents for PCR (dNTP, 10X, Z Taq) will be provided along with the buffer. Each 50 µl reaction contained 2.5mM dNTP, 10X, 1pM of forward and reverse primers and 2.5 Units of Z taq enzyme. The details of primer targeting drug resistance genes, their thermal profile

**Detection Of Amplified Products:** The amplified product is subjected to electrophoresis on 2% agarose gel incorporated with 0.5 µg/ml ethidium bromide for visualization by UV

The term **DNA sequencing** refers to sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA. DNA sequencing enables us to perform a thorough analysis of DNA because it provides us with the most basic information of all: the sequence of nucleotides. The classical chaintermination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. *Dye-terminator sequencing* utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which with different wavelengths of fluorescence and emission. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis. This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye

The PCR cocktail contained the following:

**640-1577 region** 


Table 2. Primer sets used in the study to sequence the different loci of target genes with their thermal profile and expected amplicon sizes[Sekiguchi et al, 2007; Siddiqi et al, 2002; Sreevatsan et al 1997]

Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and

Big Dye Terminator cycle sequencing Ready reaction kit (ABI prism, USA)


**Reaction Condition for Cycle Sequencing** 

 Holding Temp - 4C **Purification of Cycle Sequenced Product** 

before the samples are analyzed.

vial during pipetting.

**2.3.5 Loading into DNA sequencer** 

**Reagents Required**  500 mM EDTA 3M sodium acetate Chilled ethanol 70% ethanol

**Procedure** 

 Initial Denaturation - 96C for 1 minute. Denaturation - 96C for 10 seconds.

Extension - 60C for 4 minutes.

Take 0.5ml sterile PCR vial and add 10 µl of Milli Q water.

room temperature (22-28ºC) for 15 minutes Centrifuge at 12,000 rpm for 20 minutes.

**Reaction Protocol** 

Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of *Mycobacterium tuberculosis* 465

Annealing - 50C for 5 seconds. 25 cycles

The cycle sequenced products are purified to remove the unincorporated dye terminators

 Then add 2 µl of 125mM EDTA, followed by 10 µl of cycle sequenced product, 2 µl of 3M-sodium acetate (pH 4.6) and 50 µl of chilled ethanol. Vortex well and incubate at

 Pipette out the supernatant and wash the pellet twice with 250 µl of 70% ethanol at 12,000 rpm for 10 minutes. Care should be taken not to touch the sides of the eppendorf

 The vials are then dried at 37C (incubator) until ethanol completely evaporates. Presence of ethanol will prevent complete dissolving of DNA in formamaide.

Once the ethanol is completely dried, add 10 µl of formamide. This is denatured at 95C (Thermal cycler) for 3 minutes and immediately snap cooled in ice. The sequence of the PCR amplified DNA is deduced with the help of the ABI Prism 3100 AVANT (Applied Biosystems, USA) genetic analyzer that works based on the principle of Sanger dideoxy

blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.

DNA sequencing involves the following steps,


#### **Gel elution (Qiagen DNA Elution kit)**


#### **2.3.4 Cycle sequencing**

Cycle sequencing combines amplification and enzymatic DNA sequencing using 5' dye labeled terminators.

#### **Requirements for Cycle Sequencing**

Forward primer or Reverse primer at the concentration of 1 picomole/μl each.

Big Dye Terminator cycle sequencing Ready reaction kit (ABI prism, USA)

#### **Reaction Protocol**

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

blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.

DNA sequencing involves the following steps,

Elution of amplified products

Purification of extension products

**Gel elution (Qiagen DNA Elution kit)** 

(100 mg is approximately 100 l).

dissolved with intermittent vortexing).

Cycle sequencing

Sequence analysis

tube.

tube.

for 1 minute.

13,000 rpm.

for 1 minute.

**2.3.4 Cycle sequencing** 

labeled terminators.

17. Store the eluted products at -20 ºC.

**Requirements for Cycle Sequencing** 

Amplification of specific sequence from DNA

Electrophoresis of amplified products in 2% agarose gel

1. The amplified product (30 l) is run on 2% agarose gel

(similar to buffer QG without dissolved agarose).

pH 5.0, and mix. The color of the mixture will turn yellow.

15. Place the MinElute column into a clean 1.5ml micro centrifuge tube.

2. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

3. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel

4. Incubate at 50ºC for 10 mins in thermal cycler (or until the gel slice has completely

5. After the gel slice has dissolved completely, check that the color of the mixture is yellow

6. NOTE: If the color of the mixture is orange or violet, add 10l of 3 M Sodium acetate,

7. Add one gel volume of Isopropanol to the sample and mix by repeated pipetting. 8. Place a MinElute column in a 2ml collection tube provided in a suitable rack. 9. Apply the sample to MinElute column, and centrifuge at 13,000 rpm for 1 minute. 10. Discard the flow through and place the MinElute column back in the same collection

11. Add 500l of buffer QG to the spin column and centrifuge at 13,000 rpm for 1 minute. 12. Discard the flow through, and place the MinElute column back in the same collection

13. To wash, add 750l of buffer PE to the MinElute column and centrifuge at 13,000 rpm

14. Discard the flow through, and place the MinElute column for an additional 1 minute at

16. To elute DNA, add 10l of buffer EB (10mM Tris.Cl, pH 8.5) or water (pH 7.0-8.5) to the centre of the membrane, let the column stand for 1 minute, and centrifuge at 13,000 rpm

Cycle sequencing combines amplification and enzymatic DNA sequencing using 5' dye

Forward primer or Reverse primer at the concentration of 1 picomole/μl each.


#### **Reaction Condition for Cycle Sequencing**


#### **Purification of Cycle Sequenced Product**

The cycle sequenced products are purified to remove the unincorporated dye terminators before the samples are analyzed.

#### **Reagents Required**


#### **Procedure**


#### **2.3.5 Loading into DNA sequencer**

Once the ethanol is completely dried, add 10 µl of formamide. This is denatured at 95C (Thermal cycler) for 3 minutes and immediately snap cooled in ice. The sequence of the PCR amplified DNA is deduced with the help of the ABI Prism 3100 AVANT (Applied Biosystems, USA) genetic analyzer that works based on the principle of Sanger dideoxy

Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and

Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of *Mycobacterium tuberculosis* 467

Fig. 2. An example of multalin result targeting *katG* gene using forward primer showing the presence of most commonly reported mutation A**GC**→ACC (Ser315Thr). Inset enlarged

Table 3. Primer sets used in the study to sequence the different loci of target genes with their thermal profile and expected amplicon sizes [Sekiguchi et al, 2007; Siddiqi et al, 2002;

For PCR protocol and DNA sequencing protocol, please refer to procedures 2.3.1 to 2.3.7.

The expectation that molecular techniques would surpass conventional methods for diagnosis of TB or phenotypic susceptibility testing has not yet been realized. The genetic basis of resistance must be understood before achieving such a goal. However, the clinician now has a variety of new tools to improve the diagnosis of TB and drug resistance. Most of them still require detailed and systematic evaluations using standard techniques as references before their widespread application in clinical settings. Most of these techniques require trained personnel and specialized equipment, hindering their application in field conditions, but they can be used in reference laboratories as part of the TB control programs.

**Profile** 

95C – 1 min 60C – 1 min 72C – 1 min

95C – 1 min 60C – 1 min 72C – 1 min

94C – 1 min 52C – 1 min 72C – 1 min

94C – 1 min 57C – 1 min 72C – 1 min **No. of Cycles** 

30

30

40

40

**Target genes / Primer sequence (5'-3' Direction) Thermal** 

*thyA* (Amikacin, capreomycin, kanamycin)

*tlyA*(Amikacin, capreomycin, kanamycin)

*gyrA* for Moxifloxacin,Ofloxacin, Ciprofloxacin

*gyrB* for Moxifloxacin,Ofloxacin, Ciprofloxacin

ATCGTGTGCCCCATGGTGATCT CTCGGTGTATTCCCGTCGACT

CATCGCACGTCGTCTTTC AATACTTTTTCTACGCGCCG

CAGCTACATCGACTATGCGA GGGCTTCGGTGTTACCTCAT

CCACCGACATCGGTGGATT CTGCCACTTGAGTTTGTACA

Sreevatsan et al 1997]

**3. Conclusion** 

view of the mutation.

sequencing. The amplified products with the dye at the terminated 3'end is subjected to capillary electrophoresis by an automated sample injection. The emitted flurorescence from the dye labels on crossing the laser area are collected in the rate of one per second by cooled, charge-coupled device (CCD) camera at particular wavelength bands (virtual filters) and stored as digital signals on the computer for processing.

#### **2.3.6 Basic Local Alignment Search Tool (BLAST) analysis**

The sequences are analysed by sequence analysis softwares such as Bio Edit sequence alignment software or Chromas software. BLAST analysis, using pubmed, http://www.ncbi.nlm.nih.gov/BLAST is done to confirm the sequenced data with the standard strains and to determine the percentage homology.

#### **2.3.7 Multalin analysis**

Multalin analysis (http://multalin.toulouse.inra.fr/multalin/) to be done to identify the presence of polymorphism or mutation by comparing with the reference strain from genbank (Accession No. L27989 for *rpoB*, U41314 for *katG*, MTU16243 for *inhA* and *oxyRahpC*, X70995 for *rrs* and *rpsL*, AY743320 for *pncA* and MTU68480 for *embB*). An example of multalin analysis targeting *rpoB* and *katG* are shown in the figure 1&2.

Fig. 1. An example of multalin result targeting *rpoB* gene using forward primer showing the presence of most commonly reported mutation T**C**G→T**T**G (Ser531Leu). Inset enlarged view of the mutation.

#### **2.3.8 PCR based DNA sequencing for XDR-TB strains**

If an MDR-TB strain has the above-mentioned mutations, it should be screened for resistance to Amikacin, capreomycin, kanamycin and the fluoroquinolones by PCR based DNA sequencing targeting *rrs, tlyA, thyA*, *gyrA* and *gyrB*. The details of primer targeting drug resistance genes, their thermal profile used and the expected amplicon size are given in the Table 3.


Fig. 2. An example of multalin result targeting *katG* gene using forward primer showing the presence of most commonly reported mutation A**GC**→ACC (Ser315Thr). Inset enlarged view of the mutation.


Table 3. Primer sets used in the study to sequence the different loci of target genes with their thermal profile and expected amplicon sizes [Sekiguchi et al, 2007; Siddiqi et al, 2002; Sreevatsan et al 1997]

For PCR protocol and DNA sequencing protocol, please refer to procedures 2.3.1 to 2.3.7.

#### **3. Conclusion**

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

sequencing. The amplified products with the dye at the terminated 3'end is subjected to capillary electrophoresis by an automated sample injection. The emitted flurorescence from the dye labels on crossing the laser area are collected in the rate of one per second by cooled, charge-coupled device (CCD) camera at particular wavelength bands (virtual filters) and

The sequences are analysed by sequence analysis softwares such as Bio Edit sequence alignment software or Chromas software. BLAST analysis, using pubmed, http://www.ncbi.nlm.nih.gov/BLAST is done to confirm the sequenced data with the

Multalin analysis (http://multalin.toulouse.inra.fr/multalin/) to be done to identify the presence of polymorphism or mutation by comparing with the reference strain from genbank (Accession No. L27989 for *rpoB*, U41314 for *katG*, MTU16243 for *inhA* and *oxyRahpC*, X70995 for *rrs* and *rpsL*, AY743320 for *pncA* and MTU68480 for *embB*). An example of

Fig. 1. An example of multalin result targeting *rpoB* gene using forward primer showing the presence of most commonly reported mutation T**C**G→T**T**G (Ser531Leu). Inset enlarged view

If an MDR-TB strain has the above-mentioned mutations, it should be screened for resistance to Amikacin, capreomycin, kanamycin and the fluoroquinolones by PCR based DNA sequencing targeting *rrs, tlyA, thyA*, *gyrA* and *gyrB*. The details of primer targeting drug resistance genes, their thermal profile used and the expected amplicon size are given in

stored as digital signals on the computer for processing.

**2.3.6 Basic Local Alignment Search Tool (BLAST) analysis** 

standard strains and to determine the percentage homology.

**2.3.8 PCR based DNA sequencing for XDR-TB strains** 

multalin analysis targeting *rpoB* and *katG* are shown in the figure 1&2.

**2.3.7 Multalin analysis** 

of the mutation.

the Table 3.

The expectation that molecular techniques would surpass conventional methods for diagnosis of TB or phenotypic susceptibility testing has not yet been realized. The genetic basis of resistance must be understood before achieving such a goal. However, the clinician now has a variety of new tools to improve the diagnosis of TB and drug resistance. Most of them still require detailed and systematic evaluations using standard techniques as references before their widespread application in clinical settings. Most of these techniques require trained personnel and specialized equipment, hindering their application in field conditions, but they can be used in reference laboratories as part of the TB control programs.

Molecular Biological Techniques for Detection of Multidrug Resistant Tuberculosis (MDR) and

*J Microbiol Methods*, Vol. 51, No. 3, pp. 283–293.

Chemother. Vol. 44, No. 1, pp. 103–110.

*Microbiol*, Vol. 45, No. 1, pp.179-192.

*tuberculosis*. *Nat Biotechnol*, Vol. 16, No. 3, pp.359–363.

*Antimicrob Agents Chemother*, Vol. 41, No. 3, pp. 600-606.

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Extremely Drug Resistant Tuberculosis (XDR) in Clinical Isolates of *Mycobacterium tuberculosis* 469

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Piatek AS, Telenti A, Murray MR, El-Hajj H, Jacobs WR, KrameR FR and D. Alland. (2000).

Sekiguchi J, Miyoshi-Akiyama T, Augustynowicz-Kopeć E, Zwolska Z, Kirikae F, Toyota E,

Siddiqi N, Shamim M, Hussain S, Choudhary RK, Ahmed N, Prachee et al. (2002). Molecular

Sreevatsan S, Pan X, Zhang Y, Deretic V and Musser JM. (1997). Analysis of the *oxyR-ahpC*

TB India 2010. RNTCP status report. Central TB Division. Directorate General of Health

Therese KL, Jayanthi U, Madhavan HN. (2005). Application of nested Polymerase Chain

Torres M J, Criado A, Palomares JC and Aznar J. (2000). Use of real-time PCR and

van Doorn HR., Claas EC, Templeton KE, van der Zanden AG, te Koppele Vije A, de Jong

Wang JY, Lee LN, Chou CS, Huang CY, Wang SK, Lai HC, Hsueh PR, Luh KT. (2004).

Tuberculosis. Curr Opin Pulm Med. Vol. 12, No. 3, pp. 172-178.

16S rRNA genes in mycobacterial strains. J Clin Microbiol. Vol. 34, No. 10, pp. 2531-

*Mycobacterium avium subsp. paratuberculosis* using SYBR Green and the Light Cycler.

beacon sequence analysis for detecting drug resistance in *Mycobacterium* 

Genotypic analysis of *Mycobacterium tuberculosis* in two distinct populations using molecular beacons: implications for rapid susceptibility testing. Antimicrob. Agents

et al. (2007). Detection of multidrug resistance in *Mycobacterium tuberculosis*. *J Clin* 

characterization of multidrug resistant isolates of *Mycobacterium tuberculosis* from patients in North India. *Antimicrob. Agents Chemother*. 2002; Vol. 46, No. 2, pp. 443-

Region in Isoniazid-resistant and susceptible *Mycobacterium tuberculosis* complex organisms recovered from diseased humans and animals in diverse localities.

Services. Ministry of Health and Family Welfare . Nirman Bhawan, New Delhi - 110001. This can be obtained from the webste http://www.tbcindia.org. ISBN 81-

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fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in *Mycobacterium tuberculosis*. *J Clin Microbiol*, Vol. 38, No. 9, pp. 3194–

MD, Dankert J and Kuijper EJ. (2003). Detection of a point mutation associated with high-level isoniazid resistance in *Mycobacterium tuberculosis* by using real-time PCR technology with 3'-minor groove binder-DNA probes. *J Clin Microbiol*, Vol. 41, No.

Performance assessment of a nested-PCR assay (the RAPID BAP-MTB) and the BD

The physician must be cautious when using results obtained by these techniques, especially when diagnosing drug resistance. Although it is not recommended, these molecular methods might be used as a complement to the standard methods in situation of difficult diagnosis, but never should be used solely to base such decisions.

#### **4. Acknowledgement**

The authors gratefully acknowledge the infrastructure facility and support provided by Vision Research Foundation, Chennai and for the financial support through a research grant by Chennai Willingdon Corporate Foundation, Chennai, India.

#### **5. References**


The physician must be cautious when using results obtained by these techniques, especially when diagnosing drug resistance. Although it is not recommended, these molecular methods might be used as a complement to the standard methods in situation of difficult

The authors gratefully acknowledge the infrastructure facility and support provided by Vision Research Foundation, Chennai and for the financial support through a research grant

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Hillemann D, Rüsch-Gerdes S, Richter E. (2006). Application of the Genotype MTBDR assay directly on sputum specimens. *Int J Tuberc Lung Dis,* Vol. 10, No. 9, pp. 1057-9. Hillemann D, Rüsch-Gerdes S, Richter E. (2007). Evaluation of the GenoType MTBDRplus

strains and clinical specimens. *J Clin Microbiol*, Vol. 45, No. 8, pp. 2635-40. Morgan M, Kalantri S, Flores L, Pai M. (2005). A commercial line probe assay for the rapid

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*Mycobacterium tuberculosis*. *J Clin Microbiol.* Vol. 44, No. 2, pp.350-2.

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detection of rifampicin resistance in *Mycobacterium tuberculosis*: A systematic review

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diagnosis, but never should be used solely to base such decisions.

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*Clin Microbiol Infect*, Vol. 10, No. 7, pp. 662–665.

*Clin Microbiol*, Vol. 39, No. 11, pp. 4131–4137.

**4. Acknowledgement** 

3350–3352.

pp. 531–539.

**5. References** 


ProbeTec ET system for detection of Mycobacterium tuberculosis in clinical specimens. J Clin Microbiol. Vol. 42, No. 10, pp.4599-603.

**23** 

**Detection of** *Mycobacterium tuberculosis*

I. Chaoui1, M. Abid2, My. D. El Messaoudi2 and M. El Mzibri1 *1Unité de Biologie et Recherche Médicale, Centre National de l'Energie, des Sciences et Techniques Nucléaires (CNESTEN), Rabat, Maroc* 

Tuberculosis (TB) was responsible for millions of human deaths in the past, when there were no adequate treatment methods for infected patients. Introduction of chemotherapy and prophylactic measures led to drastic death reduction, which was maintained for various decades. However, "the good times" waned, as this disease became worldwide recognized as the one responsible for most human deaths caused by a single infectious agent: *Mycobacterium tuberculosi*s (MTB). TB resumption is basically a consequence of anthropic factors, such as the recent HIV/AIDS pandemic and the development of drug resistant

International attention has turned toward the evolving burden of multi-drug resistant tuberculosis (MDR TB) that has emerged in epidemic proportions in the wake of widespread HIV infection in the world's poorest populations, including sub-Saharan Africa. Extensively drug-resistant TB (XDR TB) was first reported in 2006 but has now been documented on six continents [WHO the global laboratory initiative]. These trends are critically important for global health, since drug-resistant TB mortality rates are high and second-line agents for the treatment of drug-resistant TB are less potent and less

Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Failure to quickly and effectively recognize and treat patients with drug-resistant tuberculosis (TB), particularly MDR and XDR tuberculosis, leads to increased mortality, nosocomial outbreaks and resistance to additional antituberculosis drugs. We believe that early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive

Therefore, there's a critical need for methods that can rapidly detect *M. tuberculosis* and

identify drug-resistant cases to optimize TB treatment with appropriate drugs.

strains (stemmed from inappropriate treatments and/or patient non-compliance).

**1. Introduction** 

tolerable than first-line therapies.

methods limit their accessibility and effect.

**and Drug Resistance: Opportunies** 

**and Challenges in Morocco** 

*2Institut Pasteur du Maroc* 

*Morocco* 


## **Detection of** *Mycobacterium tuberculosis* **and Drug Resistance: Opportunies and Challenges in Morocco**

I. Chaoui1, M. Abid2, My. D. El Messaoudi2 and M. El Mzibri1 *1Unité de Biologie et Recherche Médicale, Centre National de l'Energie, des Sciences et Techniques Nucléaires (CNESTEN), Rabat, Maroc 2Institut Pasteur du Maroc Morocco* 

#### **1. Introduction**

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

WHO Library Cataloguing-in-Publication Data. Multidrug and extensively drug-resistant

Zhang Y, Heym B, Young AD and Cole S. (1992). The catalase-peroxidase gene and

specimens. J Clin Microbiol. Vol. 42, No. 10, pp.4599-603.

WHO/HTM/TB/2010.3. ISBN 978 92 4 159919 1

591–593.

ProbeTec ET system for detection of Mycobacterium tuberculosis in clinical

TB (M/XDR-TB): 2010 global report on surveillance and response.

isoniazid resistance of *Mycobacterium tuberculosis*. *Nature*, Vol. 358, No. 6387, pp.

Tuberculosis (TB) was responsible for millions of human deaths in the past, when there were no adequate treatment methods for infected patients. Introduction of chemotherapy and prophylactic measures led to drastic death reduction, which was maintained for various decades. However, "the good times" waned, as this disease became worldwide recognized as the one responsible for most human deaths caused by a single infectious agent: *Mycobacterium tuberculosi*s (MTB). TB resumption is basically a consequence of anthropic factors, such as the recent HIV/AIDS pandemic and the development of drug resistant strains (stemmed from inappropriate treatments and/or patient non-compliance).

International attention has turned toward the evolving burden of multi-drug resistant tuberculosis (MDR TB) that has emerged in epidemic proportions in the wake of widespread HIV infection in the world's poorest populations, including sub-Saharan Africa. Extensively drug-resistant TB (XDR TB) was first reported in 2006 but has now been documented on six continents [WHO the global laboratory initiative]. These trends are critically important for global health, since drug-resistant TB mortality rates are high and second-line agents for the treatment of drug-resistant TB are less potent and less tolerable than first-line therapies.

Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Failure to quickly and effectively recognize and treat patients with drug-resistant tuberculosis (TB), particularly MDR and XDR tuberculosis, leads to increased mortality, nosocomial outbreaks and resistance to additional antituberculosis drugs. We believe that early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect.

Therefore, there's a critical need for methods that can rapidly detect *M. tuberculosis* and identify drug-resistant cases to optimize TB treatment with appropriate drugs.

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

a healthy diet.

air.

attenuated bacilli to children.

**2.3 Tuberculosis chemotherapy** 

discovered was Rifampin, in 1963.

a renaissance in TB research.

**2.4 Drug combinations** 

Opportunies and Challenges in Morocco 473

diagnosis. In the same period, Koch developed staining methods for the identification of the bacillus; these techniques were subsequently improved by the bacteriologist "Paul Ehrlich", whose method for detection of the bacillus provided the basis for the development of the Ziehl-Nielsen staining, which still is an important tool to diagnose TB. Koch's discovery allowed researchers to focus their efforts on the development of new and more efficient therapies to treat TB patients. One of the first attempts to fight the disease in 1884 was the introduction for TB patients of "sanatorium cure" concept, where the patients were isolated and taken care of, the treatment was based on rest, fresh air and

In 1896, the bacteriologist T. Smith demonstrated that bovine TB was not caused by *M. tuberculosis*, but rather by another species, *M. bovis*. Twelve years later, the scientist-couple A. Calmette and C. Guérin isolated the bovine variant from its host and grew the bacilli in dispersed culture. By the 39th passage they observed a morphological variant that was avirulent in several animal models and which conferred immunological protection against subsequent challenges with virulent *M. tuberculosis*. Thirteen years of experimentation led to the obtaining of the 231st passage, the variant that was administered for the first time in humans (orally), as an attempt to immunize a child whose mother died in childbirth victim of TB. Currently known as BCG (Bacille Calmette-Guérin), the "intradermal" vaccine has become widely used to combat TB; it relies on a prophylactic administration of live

Significant progress has been made in TB chemotherapy. In the pre-antibiotic era, before 1940, there were no drugs against the disease. The tuberculosis treatment at the time consisted mainly of cod liver oils (which by the way, include vitamin D), bed rest and fresh

The first TB drug, Streptomycin (SM), was discovered in 1944. It was followed by Paraaminosalicylic acid (PAS), which was discovered in 1946. Then, in 1952, two important firstline TB drugs were discovered, Isoniazid (INH) and Pyrazinamide (PZA). The last TB drug

Hence, the introduction of TB chemotherapy in 1950's led to a significant decline in the incidence of the disease, particularly in developed countries. This reduction has prompted some public health professionals, mainly in the US, to claim that tuberculosis no longer poses a problem in developed countries. They have even eliminated many TB control programmes in the country. However, in the late 1980's, a major outbreak of MDR-TB occurred in New York City, which has cost USD 1 billion to control. This outbreak has led to

It is noteworthy that the current TB therapy is based on the principle of drug combination. The first advantage of using drug combinations is that it reduces drug resistance. A second advantage of drug combinations is that they can enhance the efficacy of the therapy. This point is illustrated by the Mitchison hypothesis, also referred to as the Special Bacterial

The present review describes the main techniques used to detect TB and related resistant strains as well as the issues and challenges associated with implementing molecular techniques in Morocco to enhance the National Program against Tuberculosis.

#### **2. History of tuberculosis**

#### **2.1 Looking at past: Before the discovery of BCG**

First reference of a disease similar to TB in humans dates back to ancient Egypt. Examinations of mummies and tomb paintings reveal that tuberculosis was present at that time (around 5000 BC). The ancient Egyptian paintings portray spinal tuberculosis, indicating the presence of the disease. The reference of a disease called "phthisis" is found in ancient Greek literature by Hippocrates. In 1680, F. Sylvius carried out anatomic-pathologic studies in pulmonary nodules from TB patients, which he named as "tubercula", observing their evolution to lung ulcers (cavities). However, most of the great pathologists of his time believed that these knots were some type of tumor or abnormal gland, rejecting any probable infectious origin. The first credible speculation of the infectious nature of TB was performed by B. Marten, who proposed in 1722 that TB could be caused by microorganisms. R. Morton used the term "consumption" to specifically denote TB, and finally, in 1819, the inventor of the stethoscope, R. Laennec identified for the first time the TB manifestation unit. As the disease became completely established among every European social level, afflicting many of the intellectual and artists of the continent by the half of the XIX century, TB was romanticized, as typical symptoms like thin and pale faces of the infected ones became signs of beauty.

In 1865, JA Villemin demonstrated formally that TB is a contagious disease; although his experimentation could be effectively reproduced in rabbits, the finding was ignored by his contemporaries for a long time.

#### **2.2 BCG as prophylactic strategy**

One of the greatest works on TB was performed in 1882 by Robert Koch, who isolated and cultured *M. tuberculosis* from crushed tubercles. His experimental work identified the bacterium as the TB etiological agent (Bloom & Murray 1992, Daniel 1997). In August of 1890, during The First Ordinary Session of the International Medical Congress, in Berlin, he announced the discovery of a TB therapeutic drug. Three months later, M. Wochenschkift published a new statement of Koch, revealing that although interested in the therapeutic properties of his findings, he observed that the referred liquid, named tuberculin, could be useful as a diagnostic tool to detect the disease due to the intensified reaction developed by sick animals inoculated with this drug, as no measurable effect was ever observed in healthy ones. This concept was perpetuated for several years, until it was observed that even healthy animals could react to the drug. The veterinarians clarified the fact by demonstrating that the healthy ones could be simply infected, although not ill. As a result, it was established that *M. tuberculosis*-infected animals will react to tuberculin infusion, whereas the non-infected ones will not. This drug, the first industrialised one, was called old tuberculin; subsequently, other tuberculins were produced, such as purified protein derivate (PPD), PPD-S, and PPD RT23, among others (Vaccarezza 1965, Ruffino- Netto 1970). The tuberculin skin test became the principal tool for infection

The present review describes the main techniques used to detect TB and related resistant strains as well as the issues and challenges associated with implementing molecular

First reference of a disease similar to TB in humans dates back to ancient Egypt. Examinations of mummies and tomb paintings reveal that tuberculosis was present at that time (around 5000 BC). The ancient Egyptian paintings portray spinal tuberculosis, indicating the presence of the disease. The reference of a disease called "phthisis" is found in ancient Greek literature by Hippocrates. In 1680, F. Sylvius carried out anatomic-pathologic studies in pulmonary nodules from TB patients, which he named as "tubercula", observing their evolution to lung ulcers (cavities). However, most of the great pathologists of his time believed that these knots were some type of tumor or abnormal gland, rejecting any probable infectious origin. The first credible speculation of the infectious nature of TB was performed by B. Marten, who proposed in 1722 that TB could be caused by microorganisms. R. Morton used the term "consumption" to specifically denote TB, and finally, in 1819, the inventor of the stethoscope, R. Laennec identified for the first time the TB manifestation unit. As the disease became completely established among every European social level, afflicting many of the intellectual and artists of the continent by the half of the XIX century, TB was romanticized, as typical symptoms like thin and pale faces of the

In 1865, JA Villemin demonstrated formally that TB is a contagious disease; although his experimentation could be effectively reproduced in rabbits, the finding was ignored by his

One of the greatest works on TB was performed in 1882 by Robert Koch, who isolated and cultured *M. tuberculosis* from crushed tubercles. His experimental work identified the bacterium as the TB etiological agent (Bloom & Murray 1992, Daniel 1997). In August of 1890, during The First Ordinary Session of the International Medical Congress, in Berlin, he announced the discovery of a TB therapeutic drug. Three months later, M. Wochenschkift published a new statement of Koch, revealing that although interested in the therapeutic properties of his findings, he observed that the referred liquid, named tuberculin, could be useful as a diagnostic tool to detect the disease due to the intensified reaction developed by sick animals inoculated with this drug, as no measurable effect was ever observed in healthy ones. This concept was perpetuated for several years, until it was observed that even healthy animals could react to the drug. The veterinarians clarified the fact by demonstrating that the healthy ones could be simply infected, although not ill. As a result, it was established that *M. tuberculosis*-infected animals will react to tuberculin infusion, whereas the non-infected ones will not. This drug, the first industrialised one, was called old tuberculin; subsequently, other tuberculins were produced, such as purified protein derivate (PPD), PPD-S, and PPD RT23, among others (Vaccarezza 1965, Ruffino- Netto 1970). The tuberculin skin test became the principal tool for infection

techniques in Morocco to enhance the National Program against Tuberculosis.

**2. History of tuberculosis** 

infected ones became signs of beauty.

contemporaries for a long time.

**2.2 BCG as prophylactic strategy** 

**2.1 Looking at past: Before the discovery of BCG** 

diagnosis. In the same period, Koch developed staining methods for the identification of the bacillus; these techniques were subsequently improved by the bacteriologist "Paul Ehrlich", whose method for detection of the bacillus provided the basis for the development of the Ziehl-Nielsen staining, which still is an important tool to diagnose TB. Koch's discovery allowed researchers to focus their efforts on the development of new and more efficient therapies to treat TB patients. One of the first attempts to fight the disease in 1884 was the introduction for TB patients of "sanatorium cure" concept, where the patients were isolated and taken care of, the treatment was based on rest, fresh air and a healthy diet.

In 1896, the bacteriologist T. Smith demonstrated that bovine TB was not caused by *M. tuberculosis*, but rather by another species, *M. bovis*. Twelve years later, the scientist-couple A. Calmette and C. Guérin isolated the bovine variant from its host and grew the bacilli in dispersed culture. By the 39th passage they observed a morphological variant that was avirulent in several animal models and which conferred immunological protection against subsequent challenges with virulent *M. tuberculosis*. Thirteen years of experimentation led to the obtaining of the 231st passage, the variant that was administered for the first time in humans (orally), as an attempt to immunize a child whose mother died in childbirth victim of TB. Currently known as BCG (Bacille Calmette-Guérin), the "intradermal" vaccine has become widely used to combat TB; it relies on a prophylactic administration of live attenuated bacilli to children.

#### **2.3 Tuberculosis chemotherapy**

Significant progress has been made in TB chemotherapy. In the pre-antibiotic era, before 1940, there were no drugs against the disease. The tuberculosis treatment at the time consisted mainly of cod liver oils (which by the way, include vitamin D), bed rest and fresh air.

The first TB drug, Streptomycin (SM), was discovered in 1944. It was followed by Paraaminosalicylic acid (PAS), which was discovered in 1946. Then, in 1952, two important firstline TB drugs were discovered, Isoniazid (INH) and Pyrazinamide (PZA). The last TB drug discovered was Rifampin, in 1963.

Hence, the introduction of TB chemotherapy in 1950's led to a significant decline in the incidence of the disease, particularly in developed countries. This reduction has prompted some public health professionals, mainly in the US, to claim that tuberculosis no longer poses a problem in developed countries. They have even eliminated many TB control programmes in the country. However, in the late 1980's, a major outbreak of MDR-TB occurred in New York City, which has cost USD 1 billion to control. This outbreak has led to a renaissance in TB research.

#### **2.4 Drug combinations**

It is noteworthy that the current TB therapy is based on the principle of drug combination. The first advantage of using drug combinations is that it reduces drug resistance. A second advantage of drug combinations is that they can enhance the efficacy of the therapy. This point is illustrated by the Mitchison hypothesis, also referred to as the Special Bacterial

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

**3.1 Tuberculosis throughout the world: A catastrophic situation** 

is falling, but the rate of decline is very slow (less than 1%) (Wilson, 2011).

countries. The world is on track to achieve two TB targets set for 2015:

2009, ethambutol has replaced streptomycin in Category I regimen.

**3. Epidemiology of tuberculosis** 

comparison with 1990);

**3.2 Tuberculosis status in Morocco** 

1990).

2010).

tuberculosis.

Opportunies and Challenges in Morocco 475

More than two billion people, equal to one third of the world's total population, are infected with TB bacilli. A total of 1.7 million people died from TB in 2009 (including 380 000 people with HIV), equal to about 4700 deaths a day. TB is a disease of poverty, affecting mostly young adults in their most productive years. The vast majority of TB deaths are in the developing world, with more than half occurring in Asia. There were 9.4 million new TB cases in 2009, of which 80% were in just 22 countries. Per capita, the global TB incidence rate

TB is a worldwide pandemic. Among the 15 countries with the highest estimated TB incidence rates, 13 are in Africa, while a third of all new cases are in India and China. There were an estimated 440 000 new MDR-TB cases in 2008 with three countries accounting for over 50% of all cases globally: China, India and the Russian Federation (WHO, 2010). Extensively drug-resistant TB (XDR-TB) occurs when resistance to second-line drugs develops. It is extremely difficult to treat and cases have been confirmed in more than 58

The Millennium Development Goal, which aims to halt and reverse global incidence (in

The Stop TB Partnership target of halving deaths from TB (also in comparison with

During last years, the incidence of TB has stagnated and is reported to be 81 per 100,000 overall. However, the incidence was significantly higher in several urban areas, or "hot spots": Casablanca, Tangier and Rabat (together 43% of all notified cases in 2010). Statistical data show that 59% of TB patients are male and 65% are 15-34 years old. Of the roughly 28,000 new TB cases reported annually, 12% are re-treatment cases. Moreover, the prevalence of tubeculosis in HIV individuals is 1.7% in 2008 (Dooley, 2010, WHO, 2010). The prevalence of MDR TB is 0.5% within new cases and reaches 12.2% among previously treated patients with failure treatment, relapse or chronical cases (Othmani, 2003; WHO,

National TB treatment guidelines in 2007 and 2008 recommended a Category I treatment regimen – 2 months of INH, RIF, PZA, and SM followed by 4 months of RIF and INH (2SHRZ/4RH) – for new smear-positive cases and a Category II regimen – 2HRZES/1RHEZ/5RHE (E = Ethambutol) – for re-treatment cases. By the beginning of

The follow up of tuberculosis which lasts from 6 to 18 months is done in specialised centres of TB diagnosis of the ministry for Health. The late consultations and the no observance of treatments are responsible for TB resistance in Morocco. To strengthen its efforts, the ministry for Health is planning to carry a national plan of acceleration of the fight against

Populations Theory. According to this theory, TB bacteria found in the lesions consist of four different sub-populations. Population A, which is actively growing, is killed by Isoniazid. In case of Isoniazid resistance, it is killed by Rifampicin, Streptomycin, or Ethambutol. Population B, which has a slower metabolism, is killed by Rifampicin. Population C, which resides in an acidic environment, is killed by PZA. Finally, population D is a dormant population, and there are currently no drugs that can effectively kill this population (Paramasivan, 2005).

#### **2.5 Directly Observed Treatment Short course strategy**

TB persists as a global public health problem and the main focus for the twentieth century is firstly to cure the individual patient and secondly to minimise the transmission of *M. tuberculosis* to other persons (WHO, 2003; Blumberg, 2003). The ongoing TB problem has been due to the neglect of TB control by governments, inadequate access and infrastructure, poor patient adherence to medication, poor management of TB control programs, poverty, population growth and migration, and a significant rise in the number of TB cases in HIV infected individuals. Treatment of patients with TB is done according to the following five key components of the Directly Observed Treatment Short course (DOTS) strategy recommended by World Health Organization (WHO) (Walley, 1997):


Since the introduction of the DOTS strategy in the early '90s by the WHO, considerable progress has been made in global TB control (Sterling, 2003). In 1997, the estimated average treatment success rate worldwide was almost 80%. However, less than 25% of people who are sick with TB are treated through the DOTS strategy (Bastian, 2000). A total of 180 countries (including both developed and developing countries) had adopted and implemented the DOTS strategy by the end of 2002 and 69% of the global population are living in areas covered by the DOTS strategy (Blumberg, 2003). However, even though DOTS programs are in place, treatment success rates are very low in low income countries due to poor management of TB control programs and patient non-compliance (Lienhardt and Ogden, 2004; Bastian, 2003). Furthermore, the effectiveness of DOTS is facing new challenges with respect to the spread and increase of MDR-TB and the co-epidemic of TB/HIV (WHO, 2003). Since 1999, WHO and partners have addressed these new challenges and have developed DOTS-Plus strategy which prevent further development and spread of MDR-TB and help to manage MDR-TB using second line drugs in low- and middle-income countries within DOTS strategy. Morocco joined the global project in 2004 and carried out its first simultaneous survey on primary and acquired drug resistance in tuberculosis patients exactly according WHO/IUATLD recommendations.

Subsequently, 41 million of TB patients have been successfully treated in DOTS programs and up to 6 million lives saved since 1995. Moreover, 5 million more lives could be saved up to 2015 by fully funding and implementing The Global Plan to Stop TB 2011-2015.

### **3. Epidemiology of tuberculosis**

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

Populations Theory. According to this theory, TB bacteria found in the lesions consist of four different sub-populations. Population A, which is actively growing, is killed by Isoniazid. In case of Isoniazid resistance, it is killed by Rifampicin, Streptomycin, or Ethambutol. Population B, which has a slower metabolism, is killed by Rifampicin. Population C, which resides in an acidic environment, is killed by PZA. Finally, population D is a dormant population, and there are currently no drugs that can effectively kill this

TB persists as a global public health problem and the main focus for the twentieth century is firstly to cure the individual patient and secondly to minimise the transmission of *M. tuberculosis* to other persons (WHO, 2003; Blumberg, 2003). The ongoing TB problem has been due to the neglect of TB control by governments, inadequate access and infrastructure, poor patient adherence to medication, poor management of TB control programs, poverty, population growth and migration, and a significant rise in the number of TB cases in HIV infected individuals. Treatment of patients with TB is done according to the following five key components of the Directly Observed Treatment Short course (DOTS) strategy

Since the introduction of the DOTS strategy in the early '90s by the WHO, considerable progress has been made in global TB control (Sterling, 2003). In 1997, the estimated average treatment success rate worldwide was almost 80%. However, less than 25% of people who are sick with TB are treated through the DOTS strategy (Bastian, 2000). A total of 180 countries (including both developed and developing countries) had adopted and implemented the DOTS strategy by the end of 2002 and 69% of the global population are living in areas covered by the DOTS strategy (Blumberg, 2003). However, even though DOTS programs are in place, treatment success rates are very low in low income countries due to poor management of TB control programs and patient non-compliance (Lienhardt and Ogden, 2004; Bastian, 2003). Furthermore, the effectiveness of DOTS is facing new challenges with respect to the spread and increase of MDR-TB and the co-epidemic of TB/HIV (WHO, 2003). Since 1999, WHO and partners have addressed these new challenges and have developed DOTS-Plus strategy which prevent further development and spread of MDR-TB and help to manage MDR-TB using second line drugs in low- and middle-income countries within DOTS strategy. Morocco joined the global project in 2004 and carried out its first simultaneous survey on primary and acquired drug resistance in tuberculosis

Subsequently, 41 million of TB patients have been successfully treated in DOTS programs and up to 6 million lives saved since 1995. Moreover, 5 million more lives could be saved up

to 2015 by fully funding and implementing The Global Plan to Stop TB 2011-2015.

population (Paramasivan, 2005).

Government commitment

Case detection by sputum smear microscopy

A standard recording and reporting system.

 Standardised treatment regimen of six to eight months A regular, uninterrupted supply of all essential anti-TB drugs

patients exactly according WHO/IUATLD recommendations.

**2.5 Directly Observed Treatment Short course strategy** 

recommended by World Health Organization (WHO) (Walley, 1997):

#### **3.1 Tuberculosis throughout the world: A catastrophic situation**

More than two billion people, equal to one third of the world's total population, are infected with TB bacilli. A total of 1.7 million people died from TB in 2009 (including 380 000 people with HIV), equal to about 4700 deaths a day. TB is a disease of poverty, affecting mostly young adults in their most productive years. The vast majority of TB deaths are in the developing world, with more than half occurring in Asia. There were 9.4 million new TB cases in 2009, of which 80% were in just 22 countries. Per capita, the global TB incidence rate is falling, but the rate of decline is very slow (less than 1%) (Wilson, 2011).

TB is a worldwide pandemic. Among the 15 countries with the highest estimated TB incidence rates, 13 are in Africa, while a third of all new cases are in India and China. There were an estimated 440 000 new MDR-TB cases in 2008 with three countries accounting for over 50% of all cases globally: China, India and the Russian Federation (WHO, 2010). Extensively drug-resistant TB (XDR-TB) occurs when resistance to second-line drugs develops. It is extremely difficult to treat and cases have been confirmed in more than 58 countries. The world is on track to achieve two TB targets set for 2015:


#### **3.2 Tuberculosis status in Morocco**

During last years, the incidence of TB has stagnated and is reported to be 81 per 100,000 overall. However, the incidence was significantly higher in several urban areas, or "hot spots": Casablanca, Tangier and Rabat (together 43% of all notified cases in 2010). Statistical data show that 59% of TB patients are male and 65% are 15-34 years old. Of the roughly 28,000 new TB cases reported annually, 12% are re-treatment cases. Moreover, the prevalence of tubeculosis in HIV individuals is 1.7% in 2008 (Dooley, 2010, WHO, 2010). The prevalence of MDR TB is 0.5% within new cases and reaches 12.2% among previously treated patients with failure treatment, relapse or chronical cases (Othmani, 2003; WHO, 2010).

National TB treatment guidelines in 2007 and 2008 recommended a Category I treatment regimen – 2 months of INH, RIF, PZA, and SM followed by 4 months of RIF and INH (2SHRZ/4RH) – for new smear-positive cases and a Category II regimen – 2HRZES/1RHEZ/5RHE (E = Ethambutol) – for re-treatment cases. By the beginning of 2009, ethambutol has replaced streptomycin in Category I regimen.

The follow up of tuberculosis which lasts from 6 to 18 months is done in specialised centres of TB diagnosis of the ministry for Health. The late consultations and the no observance of treatments are responsible for TB resistance in Morocco. To strengthen its efforts, the ministry for Health is planning to carry a national plan of acceleration of the fight against tuberculosis.

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

**5. Molecular mechanisms of drug resistance** 

molecular basis of drug resistance in MTB.

(Ducati, 2006; Zhang and Yew, 2009).

toxicity to the patient during treatment.

not associated with INH resistance.

**5.1 First line drugs** 

**5.1.1 Isoniazid** 

Opportunies and Challenges in Morocco 477

As a consequence of the increase in DR TB and the relatively restricted number of therapeutic agents, there has been a renewed effort during the 2 past decades to define the

MTB acquires drug resistance by antibiotic selection of mutations that occur randomly at chromosomal loci. No plasmids or transposable elements (horizontal gene transfer) are involved in this process. Individual nucleotide changes (point mutations) confer resistance to single drugs, and the stepwise accumulation of these mutations leads to MDR TB. drug resistance strains emerge when chemotherapy is intermittent or otherwise inadequate

First-line drugs are mainly bactericidal and combine a high degree of efficacy with a relative

Currently, a five-drug regimen is used consisting of INH, RIF, SM, PZA and EMB. Resistance to first line anti-TB drugs has been linked to mutations in at least 10 genes; *katG, inhA, ahpC, kasA* and *ndh* for INH resistance; *rpoB* for RIF resistance, *embB* for EMB

*katG* **gene alterations**. INH or isonicotinic acid hydrazide, was synthesized in the early 1900s but its anti-TB action was first detected in 1951 (Heym, 1999; Slayden and Barry, 2000; Rattan, 1998). INH enters the cell as a prodrug that is activated by a catalase peroxidase encoded by *katG*. The peroxidase activity of the enzyme is necessary to activate INH to a toxic substance in the bacterial cell (Zhang, 1992). This toxic substance subsequently affects intracellular targets such as mycolic acid biosynthesis which are an important component of the cell wall. A lack of mycolic acid synthesis eventually results in loss of cellular integrity and the bacteria die (Barry, 1998)*.* Middlebrook *et al*. (1954) initially demonstrated that a loss of catalase activity can result in INH resistance. Subsequently, genetic studies demonstrated that transformation of INH-resistant *Mycobacterium smegmatis* and *M. tuberculosis* strains with a functional *katG* gene restored INH susceptibility and that *katG* deletions give rise to INH resistance (Zhang, 1992; Zhang, 1993). However, mutations in this gene are more frequent than deletions in clinical isolates and these can lower the activity of the enzyme.

Most mutations are found between codons 138 and 328 with the most commonly observed gene alteration being at codon 315 of the *katG* gene (Slayden and Barry, 2000). The Ser315Thr substitution is estimated to occur in 30–60% of INH resistant isolates (Ramaswamy and Musser, 1998; Musser, 1996; Slayden and Barry, 2000). The *katG* 463 (CGG-CTG / Arg-Leu) amino acid substitution is the most common polymorphism found in the *katG* gene and is

Resistance to INH could be also due to mutations in the promoter region of the *ahpC* gene. Indeed, it has been observed that a loss of *katG* activity due to the S315T amino acid substitution is often accompanied by an increase in expression of an alkyl hydroperoxide

resistance, *pncA* for PZA resistance and *rpsL* and *rrs* for STR resistance.

#### **4. Drug resistance**

#### **4.1 Drug resistance and global surveillance: History**

Shortly, after the first anti-tuberculosis (TB) drugs were introduced, streptomycin (STR), para-aminosalicylic acid (PAS) and isoniazid (INH), resistance to these drugs was observed in clinical isolates of *Mycobacterium tuberculosis* (Crofton and Mitchison, 1948). This led to the need to measure resistance accurately and easily. The Pasteur Institute introduced the critical proportion method in 1961 for drug susceptibility testing in TB and this method became the standard method of use (Espinal, 2000; 2003). Studies on drug resistance in various countries in the 1960s showed that developing countries had a much higher incidence of drug resistance than developed countries (Espinal, 2000; 2003). By the end of the 1960s rifampicin (RIF) was introduced and with the use of combination therapy, there was a decline in drug resistant and drug susceptible TB in developed countries. This led to a decline in funding and interest in TB control programs. As a result, no concrete monitoring of drug resistance was carried out for the following 20 years (Espinal, 2000; 2003). The arrival of HIV/AIDS in the 1980s resulted in an increase in transmission of TB associated with outbreaks of multidrug- resistant TB (MDR-TB) (Edlin, 1992; Fischl, 1992). In the early 1990s drug resistance surveillance was resumed in developed countries, but the true incidence remained unclear in the developing world (Cohn, 1997).

The emergence of MDR TB is the third epidemic, complicating the epidemics of acquired immune deficiency syndrome (AIDS) and tuberculosis, and is requiring urgent attention to achieve more rapid diagnosis, to develop new therapeutic regimens and to address the social and hospital environment to care for these patients ( Neville, 1994).

#### **4.2 Primary and acquired resistance: Definition and data**

WHO estimated that 50 million people were infected with drug resistant MTB. Single-drug resistance is defined as resistance to only one antituberculous agent.

MDR-TB, or multidrug-resistant TB, is a specific form of drug-resistant TB. It occurs when the TB bacteria are resistant to at least isoniazid and rifampicin, the two most powerful anti-TB drugs. XDR-TB is an MDR TB strain that is resistant to any fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin, kanamycin or amikacin).

Traditionally, patients with drug-resistant tuberculosis are classified as having primary or acquired drug resistance on the basis of a history of previous treatment. WHO criteria define acquired drug resistance as the isolation of drug-resistant *M. tuberculosis* from a patient with a record of previous treatment for 1 month, and primary drug resistance as the isolation of a drug-resistant strain from a patient without a history of previous treatment.

The classification of drug resistance as primary or acquired is used as an indicator of the efficiency of national tuberculosis programs and in the adjustment and development of these programs. The rate of primary drug resistance is interpreted as an epidemiological indicator for long-term surveillance of the quality of tuberculosis treatment in the community. The rate of acquired drug resistance reflects the efficacy of management of individual patients.

#### **5. Molecular mechanisms of drug resistance**

As a consequence of the increase in DR TB and the relatively restricted number of therapeutic agents, there has been a renewed effort during the 2 past decades to define the molecular basis of drug resistance in MTB.

MTB acquires drug resistance by antibiotic selection of mutations that occur randomly at chromosomal loci. No plasmids or transposable elements (horizontal gene transfer) are involved in this process. Individual nucleotide changes (point mutations) confer resistance to single drugs, and the stepwise accumulation of these mutations leads to MDR TB. drug resistance strains emerge when chemotherapy is intermittent or otherwise inadequate (Ducati, 2006; Zhang and Yew, 2009).

#### **5.1 First line drugs**

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

Shortly, after the first anti-tuberculosis (TB) drugs were introduced, streptomycin (STR), para-aminosalicylic acid (PAS) and isoniazid (INH), resistance to these drugs was observed in clinical isolates of *Mycobacterium tuberculosis* (Crofton and Mitchison, 1948). This led to the need to measure resistance accurately and easily. The Pasteur Institute introduced the critical proportion method in 1961 for drug susceptibility testing in TB and this method became the standard method of use (Espinal, 2000; 2003). Studies on drug resistance in various countries in the 1960s showed that developing countries had a much higher incidence of drug resistance than developed countries (Espinal, 2000; 2003). By the end of the 1960s rifampicin (RIF) was introduced and with the use of combination therapy, there was a decline in drug resistant and drug susceptible TB in developed countries. This led to a decline in funding and interest in TB control programs. As a result, no concrete monitoring of drug resistance was carried out for the following 20 years (Espinal, 2000; 2003). The arrival of HIV/AIDS in the 1980s resulted in an increase in transmission of TB associated with outbreaks of multidrug- resistant TB (MDR-TB) (Edlin, 1992; Fischl, 1992). In the early 1990s drug resistance surveillance was resumed in developed countries, but the true

The emergence of MDR TB is the third epidemic, complicating the epidemics of acquired immune deficiency syndrome (AIDS) and tuberculosis, and is requiring urgent attention to achieve more rapid diagnosis, to develop new therapeutic regimens and to address the

WHO estimated that 50 million people were infected with drug resistant MTB. Single-drug

MDR-TB, or multidrug-resistant TB, is a specific form of drug-resistant TB. It occurs when the TB bacteria are resistant to at least isoniazid and rifampicin, the two most powerful anti-TB drugs. XDR-TB is an MDR TB strain that is resistant to any fluoroquinolone, and at least

Traditionally, patients with drug-resistant tuberculosis are classified as having primary or acquired drug resistance on the basis of a history of previous treatment. WHO criteria define acquired drug resistance as the isolation of drug-resistant *M. tuberculosis* from a patient with a record of previous treatment for 1 month, and primary drug resistance as the isolation of a

The classification of drug resistance as primary or acquired is used as an indicator of the efficiency of national tuberculosis programs and in the adjustment and development of these programs. The rate of primary drug resistance is interpreted as an epidemiological indicator for long-term surveillance of the quality of tuberculosis treatment in the community. The rate of acquired drug resistance reflects the efficacy of management of

one of three injectable second-line drugs (capreomycin, kanamycin or amikacin).

drug-resistant strain from a patient without a history of previous treatment.

**4. Drug resistance** 

individual patients.

**4.1 Drug resistance and global surveillance: History** 

incidence remained unclear in the developing world (Cohn, 1997).

**4.2 Primary and acquired resistance: Definition and data** 

resistance is defined as resistance to only one antituberculous agent.

social and hospital environment to care for these patients ( Neville, 1994).

First-line drugs are mainly bactericidal and combine a high degree of efficacy with a relative toxicity to the patient during treatment.

Currently, a five-drug regimen is used consisting of INH, RIF, SM, PZA and EMB. Resistance to first line anti-TB drugs has been linked to mutations in at least 10 genes; *katG, inhA, ahpC, kasA* and *ndh* for INH resistance; *rpoB* for RIF resistance, *embB* for EMB resistance, *pncA* for PZA resistance and *rpsL* and *rrs* for STR resistance.

#### **5.1.1 Isoniazid**

*katG* **gene alterations**. INH or isonicotinic acid hydrazide, was synthesized in the early 1900s but its anti-TB action was first detected in 1951 (Heym, 1999; Slayden and Barry, 2000; Rattan, 1998). INH enters the cell as a prodrug that is activated by a catalase peroxidase encoded by *katG*. The peroxidase activity of the enzyme is necessary to activate INH to a toxic substance in the bacterial cell (Zhang, 1992). This toxic substance subsequently affects intracellular targets such as mycolic acid biosynthesis which are an important component of the cell wall. A lack of mycolic acid synthesis eventually results in loss of cellular integrity and the bacteria die (Barry, 1998)*.* Middlebrook *et al*. (1954) initially demonstrated that a loss of catalase activity can result in INH resistance. Subsequently, genetic studies demonstrated that transformation of INH-resistant *Mycobacterium smegmatis* and *M. tuberculosis* strains with a functional *katG* gene restored INH susceptibility and that *katG* deletions give rise to INH resistance (Zhang, 1992; Zhang, 1993). However, mutations in this gene are more frequent than deletions in clinical isolates and these can lower the activity of the enzyme.

Most mutations are found between codons 138 and 328 with the most commonly observed gene alteration being at codon 315 of the *katG* gene (Slayden and Barry, 2000). The Ser315Thr substitution is estimated to occur in 30–60% of INH resistant isolates (Ramaswamy and Musser, 1998; Musser, 1996; Slayden and Barry, 2000). The *katG* 463 (CGG-CTG / Arg-Leu) amino acid substitution is the most common polymorphism found in the *katG* gene and is not associated with INH resistance.

Resistance to INH could be also due to mutations in the promoter region of the *ahpC* gene. Indeed, it has been observed that a loss of *katG* activity due to the S315T amino acid substitution is often accompanied by an increase in expression of an alkyl hydroperoxide

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

has a mutation in this specific region (Telenti, 1993; Telenti, 1997).

507 | | 533

Codons are numbered according to the *rpoB* gene of *Escherichia Coli*

influence the accuracy of genotypic tests (Riska, 2000).

rifabutin (Zhang, 2005).

*rpoB*

**5.1.2 Rifampicin** 

507 Gly 508 Thr 509 Ser 510 Gln 511 Leu 512 Ser 513 Gln 514 Phe 515 Met 516 Asp 517 Gln 518 Asn 519 Asn 520 Pro 521 Leu 522 Ser 523 Gly 524 Leu 525 Thr 526 His 527 Lys 528 Arg 529 Arg 530 Leu 531 Ser 532 Ala 533 Leu

Opportunies and Challenges in Morocco 479

Rifampicin (RIF) is a major compound of anti-tuberculosis chemotherapy. A resistance to RIF is rarely found without associated resistance to other tuberculostatics. RIF resistance is a good marker for MDR-TB. Moreover, RIF resistance is a good predictor of poor treatment outcome. The mode of action of RIF is based on the inhibition of the elongation of transcripts by RNA polymerase in MTB, by covalent binding to the Beta sub-unit of RNA polymerase, thus leading to cell death. The RNA polymerase Beta sub-unit is encoded by the rpoB gene. RIF resistance is associated with a hotspot (codon 507 to 533) core region called RRDR, for "rifampicine resistance determining region" (81 bp) of the *rpoB* gene. More than 95% of RIFR *M. tuberculosis*

Asp Ala His Leu Thr Lys Leu Val Val Glu His Lys Leu Leu Asn Tyr Asn Pro Lys Leu Ala Pro Ser Val Arg Pro Tyr Ser Met Glu Asp His Trp Leu Pro Leu Gly Arg Phe Arg Glu Glu Leu Tyr

Fig. 1. Mutations of the *rpoB* gene associated with a resistance to rifampicin in *M. tuberculosis* Resistance to RIF occurs at a frequency of 1 out of 107 to 108 bacterial cells. Most RIFresistant strains show one mutation in the gene. Two to four mutations are rarely reported (Mani, 2001; Sekiguchi, 2007). The most prevalent mutations (81%) affect codons 531 and 526 and usually lead to a high level of phenotypical resistance (MIC > 64 µg/ml) as well as cross resistance to other rifamycins (Riska, 2000; Zhang, 2005). Mutations at codons 511, 516, 518 and 522 result in a low-level resistance to RIF and rifapentine; and some susceptibility to

At the same time, mutations in this hotspot region seem to confer low phenotypical resistance (deletion of codon 508-509, mutation at 515) (Taniguchi, 1996) or variable resistance (L533P) (Kim, 1997), which could lead to an overly hasty interpretation of resistance. The latest observations of Asian strains suggest a geographic variability that can

**Mutations of the** *rpoB* **gene associated with a resistance to rifampicin in** *M. tuberculosis*

Asn Asn

1% 2% 10% 2% 25% 56% 2%

Lys Cys Cys Ala Pro Gln

> Thr Gln Val Glu Gly

Glu

reductase (*ahpC*) protein that is capable of detoxifying damaging organic peroxides (Sherman, 1996). Five different nucleotide alterations have been identified in the promoter region of the *ahpC* gene, which lead to over expression of *ahpC* and INH resistance (Ramaswamy and Musser, 1998). *AhpC* overexpression exerts a detoxifying effect on organic peroxides within the cell and protects the bacteria against oxidative damage but does not provide protection against INH. *KatG* expression can also be up regulated under conditions of oxidative stress. The correlation between polymorphic sites in the *ahpC* regulatory region with INH resistance in *M. tuberculosis* requires further examination.

*inhA* **gene alterations**. One of the targets for activated INH is the protein encoded by the *inhA* locus. *InhA* is an enoyl–acyl carrier protein (ACP) reductase which is proposed to be the primary target for resistance to INH and ethionamide (ETH) (Banerjee, 1994). ETH, a second line drug, is a structural analogue of INH that is also thought to inhibit mycolic acid biosynthesis and several studies have suggested that low-level INH resistance is correlated with resistance to ETH. Activated INH binds to the InhA-NADH complex to form a ternary complex that results in inhibition of mycolic acid biosynthesis. Six point mutations associated with INH resistance within the structural *inhA* gene have been identified (Ile16Thr, Ile21Thr, Ile21Val, Ile47Thr, Val78Ala and Ile95Pro) (Ramaswamy and Musser, 1998; Basso and Blanchard, 1998). A Ser94Ala substitution results in a decreased binding affinity of *inhA* for NADH, resulting in mycolic acid synthesis inhibition. Although these mutations in the structural *InhA* gene are associated with INH resistance, it is not frequently reported in clinical isolates. *InhA* promoter mutations are more frequently seen and are present at positions -24(GT), -16(A-G), or -8(T-G/A) and -15(C-T). These promoter mutations result in over expression of *inhA* leading to low level INH resistance. To date approximately 70–80% of INH resistance in clinical isolates of *M. tuberculosis* can be attributed to mutations in the *katG* and *inhA* genes (Ramaswamy and Musser, 1998).

*kasA* **gene alterations***.* There seems to be considerable contreverse within the literature as to the role of *kasA* as a possible target for INH resistance (Sherman, 1996). This gene encodes a β-ketoacyl-ACP synthase involved in the synthesis of mycolic acids. Mutations have been described in this gene that confer low levels of INH resistance. Genotypic analysis of the *kas*A gene reveals 4 different amino acid substitutions involving codon 66 (GAT-AAT), codon 269 (GGT-AGT), codon 312 (GGC-AGC) and codon 413 (TTC-TTA) (Ramaswamy and Musser, 1998; Mdluli, 1998). However, similar mutations were also found in INH susceptible isolates (Lee, 1999; Piatek, 2000). Nevertheless, the possibility of *kasA*  constituting an additional resistance mechanism should not be completely excluded.

*Ndh* **gene alterations***.* In 1998 another mechanism for INH resistance in *M. smegmatis* was described by Miesel *et al*. (1998). The *ndh* gene encodes NADH dehydrogenase that is bound to the active site of *inhA* to form the ternary complex with activated INH. Structural studies have shown that a reactive form of INH attacks the NAD(H) co-factor and generates a covalent INH-NAD adduct. Mutations in the *ndh* gene, encoding NADH dehydrogenase, cause defects in the enzymatic activity. Thus, defects in the oxidation of NADH to NAD result in NADH accumulation and NAD depletion (Lee, 2001). These high levels of NADH can then inhibit the binding of the INH-NAD adduct to the active site of the InhA enzyme (Rozwarski, 1998; Miesel, 1998)*.* Prominent point mutations in the *ndh* gene at codons 110 and 268 (T110A and R268H) were detected in 9.5% of INH resistant samples. These similar mutations were not detected in the INH susceptible group (Lee, 2001).

#### **5.1.2 Rifampicin**

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

reductase (*ahpC*) protein that is capable of detoxifying damaging organic peroxides (Sherman, 1996). Five different nucleotide alterations have been identified in the promoter region of the *ahpC* gene, which lead to over expression of *ahpC* and INH resistance (Ramaswamy and Musser, 1998). *AhpC* overexpression exerts a detoxifying effect on organic peroxides within the cell and protects the bacteria against oxidative damage but does not provide protection against INH. *KatG* expression can also be up regulated under conditions of oxidative stress. The correlation between polymorphic sites in the *ahpC* regulatory region

*inhA* **gene alterations**. One of the targets for activated INH is the protein encoded by the *inhA* locus. *InhA* is an enoyl–acyl carrier protein (ACP) reductase which is proposed to be the primary target for resistance to INH and ethionamide (ETH) (Banerjee, 1994). ETH, a second line drug, is a structural analogue of INH that is also thought to inhibit mycolic acid biosynthesis and several studies have suggested that low-level INH resistance is correlated with resistance to ETH. Activated INH binds to the InhA-NADH complex to form a ternary complex that results in inhibition of mycolic acid biosynthesis. Six point mutations associated with INH resistance within the structural *inhA* gene have been identified (Ile16Thr, Ile21Thr, Ile21Val, Ile47Thr, Val78Ala and Ile95Pro) (Ramaswamy and Musser, 1998; Basso and Blanchard, 1998). A Ser94Ala substitution results in a decreased binding affinity of *inhA* for NADH, resulting in mycolic acid synthesis inhibition. Although these mutations in the structural *InhA* gene are associated with INH resistance, it is not frequently reported in clinical isolates. *InhA* promoter mutations are more frequently seen and are present at positions -24(GT), -16(A-G), or -8(T-G/A) and -15(C-T). These promoter mutations result in over expression of *inhA* leading to low level INH resistance. To date approximately 70–80% of INH resistance in clinical isolates of *M. tuberculosis* can be

attributed to mutations in the *katG* and *inhA* genes (Ramaswamy and Musser, 1998).

constituting an additional resistance mechanism should not be completely excluded.

mutations were not detected in the INH susceptible group (Lee, 2001).

*Ndh* **gene alterations***.* In 1998 another mechanism for INH resistance in *M. smegmatis* was described by Miesel *et al*. (1998). The *ndh* gene encodes NADH dehydrogenase that is bound to the active site of *inhA* to form the ternary complex with activated INH. Structural studies have shown that a reactive form of INH attacks the NAD(H) co-factor and generates a covalent INH-NAD adduct. Mutations in the *ndh* gene, encoding NADH dehydrogenase, cause defects in the enzymatic activity. Thus, defects in the oxidation of NADH to NAD result in NADH accumulation and NAD depletion (Lee, 2001). These high levels of NADH can then inhibit the binding of the INH-NAD adduct to the active site of the InhA enzyme (Rozwarski, 1998; Miesel, 1998)*.* Prominent point mutations in the *ndh* gene at codons 110 and 268 (T110A and R268H) were detected in 9.5% of INH resistant samples. These similar

*kasA* **gene alterations***.* There seems to be considerable contreverse within the literature as to the role of *kasA* as a possible target for INH resistance (Sherman, 1996). This gene encodes a β-ketoacyl-ACP synthase involved in the synthesis of mycolic acids. Mutations have been described in this gene that confer low levels of INH resistance. Genotypic analysis of the *kas*A gene reveals 4 different amino acid substitutions involving codon 66 (GAT-AAT), codon 269 (GGT-AGT), codon 312 (GGC-AGC) and codon 413 (TTC-TTA) (Ramaswamy and Musser, 1998; Mdluli, 1998). However, similar mutations were also found in INH susceptible isolates (Lee, 1999; Piatek, 2000). Nevertheless, the possibility of *kasA* 

with INH resistance in *M. tuberculosis* requires further examination.

Rifampicin (RIF) is a major compound of anti-tuberculosis chemotherapy. A resistance to RIF is rarely found without associated resistance to other tuberculostatics. RIF resistance is a good marker for MDR-TB. Moreover, RIF resistance is a good predictor of poor treatment outcome. The mode of action of RIF is based on the inhibition of the elongation of transcripts by RNA polymerase in MTB, by covalent binding to the Beta sub-unit of RNA polymerase, thus leading to cell death. The RNA polymerase Beta sub-unit is encoded by the rpoB gene. RIF resistance is associated with a hotspot (codon 507 to 533) core region called RRDR, for "rifampicine resistance determining region" (81 bp) of the *rpoB* gene. More than 95% of RIFR *M. tuberculosis* has a mutation in this specific region (Telenti, 1993; Telenti, 1997).

Codons are numbered according to the *rpoB* gene of *Escherichia Coli*

Fig. 1. Mutations of the *rpoB* gene associated with a resistance to rifampicin in *M. tuberculosis*

Resistance to RIF occurs at a frequency of 1 out of 107 to 108 bacterial cells. Most RIFresistant strains show one mutation in the gene. Two to four mutations are rarely reported (Mani, 2001; Sekiguchi, 2007). The most prevalent mutations (81%) affect codons 531 and 526 and usually lead to a high level of phenotypical resistance (MIC > 64 µg/ml) as well as cross resistance to other rifamycins (Riska, 2000; Zhang, 2005). Mutations at codons 511, 516, 518 and 522 result in a low-level resistance to RIF and rifapentine; and some susceptibility to rifabutin (Zhang, 2005).

At the same time, mutations in this hotspot region seem to confer low phenotypical resistance (deletion of codon 508-509, mutation at 515) (Taniguchi, 1996) or variable resistance (L533P) (Kim, 1997), which could lead to an overly hasty interpretation of resistance. The latest observations of Asian strains suggest a geographic variability that can influence the accuracy of genotypic tests (Riska, 2000).

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

**5.1.4 Pyrazinamide** 

**5.1.5 Ethambutol** 

of EMB resistance in clinical isolates.

Opportunies and Challenges in Morocco 481

Pyrazinamide (PZA) is a structural analog of nicotinamide that is used as a first –line TB drug. PZA kills semi-dormant tubercle bacilli under acidic conditions. PZA targets an enzyme involved in fatty-acid synthesis and is responsible for killing persistent tubercle bacilli in the initial intensive phase of chemotherapy (Somoskovi, 2001). However, during the first two days of treatment, PZA has no bactericidal activity against rapidly growing bacilli (Zhang and Mitchison, 2003). PZA on the other hand has effective sterilizing activity and shortens the chemotherapeutic regiment from 12 to 6 months. PZA is a prodrug which is converted to its active form, pyrazinoic acid (POA) by the pyrazinamidase (PZase) encoded by *pncA*. The activity of PZA is highly specific for *M. tuberculosis,* as it has no effect on other mycobacteria. *Mycobacterium bovis* is naturally resistant to PZA due to a unique C-G point mutation in codon 169 of the *pncA* gene. PZA is only active against *M. tuberculosis* at acidic pH where POA accumulates in the cytoplasm due to an ineffective efflux pump. Accumulation of POA results in the lowering of intracellular pH to a level that inactivates a vital fatty acid synthase (Zimhony, 2004). Cloning and characterization of the *M. tuberculosis pncA* gene by Scorpio *et al.* (1997) showed that some *pncA* mutations conferred PZA resistance. Various *pncA*  mutations have been identified in more than 70% of PZA resistant clinical isolates scattered throughout the *pncA* gene but thus far no mutational hot spot has been identified (Scorpio and Zhang, 1996; Sreevatsan, 1997b; Scorpio, 1997). PZA susceptibility testing is not done routinely in many countries due to technical difficulties. Thus the extent of PZA resistance globally is largely unknown. PZA resistant isolates had diverse nucleotide changes scattered throughout the *pncA* gene. However, PZA resistant isolates without *pncA* mutations were also observed suggesting that another mechanism may be involved in conferring PZA resistance in these isolates. In addition, not all mutations (e.g. Thr114Met) were associated with PZA resistance. In summary, the complexity of PZA resistance makes the development of molecular methods for rapid diagnosis difficult.

Ethambutol (EMB) is a very specific and effective drug that is used in combination with INH to treat *M. tuberculosis* infection (Ramaswamy and Musser, 1998). EMB inhibits an arabinosyl transferase (*embB*) involved in cell wall biosynthesis (Takayama and Kilburn, 1989).Three genes designated *emb*C, A and B (Telenti, 1997) encode homologous arabinosyl transferase enzymes involved in EMB resistance. Various studies have identified five mutations in codon 306 of *embB* gene [(ATG-GTG), (ATG-CTG), (ATG-ATA), (ATG-ATC) and (ATG-ATT)] which result in three different amino acid substitutions (Val, Leu and Ile) in EMBresistant isolates (Lee, 2002; Sreevatsan, 1997c; Mokrousov, 2002b; Ramaswamy, 2000). These five mutations are associated with 70–90% of all EMB resistant isolates (Ramaswamy and Musser, 1998). Missense mutations were identified in three additional codons: Phe285leu, Phe330Val and Thr630Ile in EMB resistant isolates. MIC's were generally higher for strains with Met306Leu, Met306Val, and Phe330Val and Thr630Ile substitutions than those organisms with Met306Ile substitutions. Mutations outside of codon 306 are present but quite rare. However a number of EMB phenotypic resistant isolates (about 30%) still lack an identified mutation in *embB*. There is therefore a need to fully understand the mechanism

Silent mutations (Leu511 and Leu521) have been reported in resistant strains. Interestingly, the L511L mutation is always associated with other mutations that confer resistance (Siddiqi, 2002). In rare cases, double mutations appear to have an additive effect on the degree of resistance. The role of mutations, combined with those known to confer resistance, is uncertain, as in the case of S509R described with H526R (Sekiguchi, 2007).

Finally, less than 5% of resistant strains do not show a mutation in the *rpoB* resistance region (Riska, 2000; Mani, 2001). Rare loci found outside the hotspot region of *rpoB* are associated with resistance without associated mutation known for conferring resistance (Taniguchi, 1996; Fang, 1999; Schilke, 1999; Yuen, 1999; Heep, 2001; Zhang, 2005; Rigouts, 2007; Prammananan, 2008).

Mutated strains in Val146Phe (Heep, 2001; Rigouts, 2007; Prammananan, 2008) show a lowlevel resistance (MIC 4 µg/ml) (Rigouts, 2007). The Ala381Val mutation (Taniguchi, 1996) is described on a strain of MIC 200, with no other mutation on the *rpoB* gene.

Most susceptible strains show no mutation, except for a few: seven susceptible Japanese strains are mutated in TCG Ser 450 Leu TTG, ATG Met511 Val GTG, CTG Leu 521 Pro CCG, CTG Leu 533 Pro CCG, GCC Ala 679 Ser TCC (two strains) and CGC Arg 687 Pro CCC (Taniguchi, 1996; Yang, 1998). The CTG Leu 533 Pro CCG mutation has been shown on two strains of low-level resistance (MIC 12.5 µg/ml) and on a strain that is susceptible according to phenotypic tests, yet clinically resistant (Riska, 2000). Other studies describe some sensitive strains with mutations as Ser 450 Leu (Sekiguchi, 2007), Leu 511 Arg or Ser 512 Thr (Moghazeh, 1996) or Gln CAA 513 Gln CAG (Kim, 1997). Genotypic detections would therefore be more sensitive in certain circumstances.

#### **5.1.3 Streptomycin**

SM is an aminocyclitol antibiotic that is one of the first drug used to treat TB, SM binds to 16S rRNA, inhibits translational initiation and detrimentally affects translation fidelity. Mutations associated with SM resistance in MTB have been identified mainly in *rpsL* gene encoding ribosomal protein S12 and in the 16S rRNA gene (*rrs*) in 65–67% of STR resistant isolates (Ramaswamy and Musser, 1998).

In the *rrs* gene a C-T transition at positions 491, 512 and 516, and a A-C/T transversion at position 513 were observed in the highly conserved 530 loop. The 530 loop region is part of the aminoacyl–tRNA binding site and is involved in the decoding process (Carter, 2000). The C-T transition at codon 491 is not responsible for resistance to STR as it occurs in both STR resistant and susceptible isolates but is strongly associated with the global spread of *M. tuberculosis* with a Western Cape F11 genotype (van Rie, 2001; Victor, 2001). Other mutations in the 915 loop [903 (C-A/G) and 904 (A-G)] have also been reported to have an association with STR resistance (Carter, 2000). Mutations in the *rpsL* gene at codon 43 (AAG-AGG/ACG) (Lys-Arg/Thr) and codon 88 (AAGAGG/ CAG) (Lys-Arg/Gln) are associated with STR resistance. MIC analysis of STR resistant isolates indicate that amino acid replacements in the *rpsL* genes correlate with a high level of resistance, whereas mutations in the *rrs* gene correlate with an intermediate level of resistance (Cooksey, 1996; Meier, 1996). In addition, it has been suggested that low levels of STR resistance are also associated with altered cell permeability or rare mutations which lie outside of the *rrs* and *rpsL* genes.

#### **5.1.4 Pyrazinamide**

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

Silent mutations (Leu511 and Leu521) have been reported in resistant strains. Interestingly, the L511L mutation is always associated with other mutations that confer resistance (Siddiqi, 2002). In rare cases, double mutations appear to have an additive effect on the degree of resistance. The role of mutations, combined with those known to confer resistance,

Finally, less than 5% of resistant strains do not show a mutation in the *rpoB* resistance region (Riska, 2000; Mani, 2001). Rare loci found outside the hotspot region of *rpoB* are associated with resistance without associated mutation known for conferring resistance (Taniguchi, 1996; Fang, 1999; Schilke, 1999; Yuen, 1999; Heep, 2001; Zhang, 2005; Rigouts, 2007;

Mutated strains in Val146Phe (Heep, 2001; Rigouts, 2007; Prammananan, 2008) show a lowlevel resistance (MIC 4 µg/ml) (Rigouts, 2007). The Ala381Val mutation (Taniguchi, 1996) is

Most susceptible strains show no mutation, except for a few: seven susceptible Japanese strains are mutated in TCG Ser 450 Leu TTG, ATG Met511 Val GTG, CTG Leu 521 Pro CCG, CTG Leu 533 Pro CCG, GCC Ala 679 Ser TCC (two strains) and CGC Arg 687 Pro CCC (Taniguchi, 1996; Yang, 1998). The CTG Leu 533 Pro CCG mutation has been shown on two strains of low-level resistance (MIC 12.5 µg/ml) and on a strain that is susceptible according to phenotypic tests, yet clinically resistant (Riska, 2000). Other studies describe some sensitive strains with mutations as Ser 450 Leu (Sekiguchi, 2007), Leu 511 Arg or Ser 512 Thr (Moghazeh, 1996) or Gln CAA 513 Gln CAG (Kim, 1997). Genotypic detections would

SM is an aminocyclitol antibiotic that is one of the first drug used to treat TB, SM binds to 16S rRNA, inhibits translational initiation and detrimentally affects translation fidelity. Mutations associated with SM resistance in MTB have been identified mainly in *rpsL* gene encoding ribosomal protein S12 and in the 16S rRNA gene (*rrs*) in 65–67% of STR resistant

In the *rrs* gene a C-T transition at positions 491, 512 and 516, and a A-C/T transversion at position 513 were observed in the highly conserved 530 loop. The 530 loop region is part of the aminoacyl–tRNA binding site and is involved in the decoding process (Carter, 2000). The C-T transition at codon 491 is not responsible for resistance to STR as it occurs in both STR resistant and susceptible isolates but is strongly associated with the global spread of *M. tuberculosis* with a Western Cape F11 genotype (van Rie, 2001; Victor, 2001). Other mutations in the 915 loop [903 (C-A/G) and 904 (A-G)] have also been reported to have an association with STR resistance (Carter, 2000). Mutations in the *rpsL* gene at codon 43 (AAG-AGG/ACG) (Lys-Arg/Thr) and codon 88 (AAGAGG/ CAG) (Lys-Arg/Gln) are associated with STR resistance. MIC analysis of STR resistant isolates indicate that amino acid replacements in the *rpsL* genes correlate with a high level of resistance, whereas mutations in the *rrs* gene correlate with an intermediate level of resistance (Cooksey, 1996; Meier, 1996). In addition, it has been suggested that low levels of STR resistance are also associated with altered cell permeability or rare mutations which lie outside of the *rrs* and *rpsL* genes.

is uncertain, as in the case of S509R described with H526R (Sekiguchi, 2007).

described on a strain of MIC 200, with no other mutation on the *rpoB* gene.

therefore be more sensitive in certain circumstances.

isolates (Ramaswamy and Musser, 1998).

Prammananan, 2008).

**5.1.3 Streptomycin** 

Pyrazinamide (PZA) is a structural analog of nicotinamide that is used as a first –line TB drug. PZA kills semi-dormant tubercle bacilli under acidic conditions. PZA targets an

enzyme involved in fatty-acid synthesis and is responsible for killing persistent tubercle bacilli in the initial intensive phase of chemotherapy (Somoskovi, 2001). However, during the first two days of treatment, PZA has no bactericidal activity against rapidly growing bacilli (Zhang and Mitchison, 2003). PZA on the other hand has effective sterilizing activity and shortens the chemotherapeutic regiment from 12 to 6 months. PZA is a prodrug which is converted to its active form, pyrazinoic acid (POA) by the pyrazinamidase (PZase) encoded by *pncA*. The activity of PZA is highly specific for *M. tuberculosis,* as it has no effect on other mycobacteria. *Mycobacterium bovis* is naturally resistant to PZA due to a unique C-G point mutation in codon 169 of the *pncA* gene. PZA is only active against *M. tuberculosis* at acidic pH where POA accumulates in the cytoplasm due to an ineffective efflux pump. Accumulation of POA results in the lowering of intracellular pH to a level that inactivates a vital fatty acid synthase (Zimhony, 2004). Cloning and characterization of the *M. tuberculosis pncA* gene by Scorpio *et al.* (1997) showed that some *pncA* mutations conferred PZA resistance. Various *pncA*  mutations have been identified in more than 70% of PZA resistant clinical isolates scattered throughout the *pncA* gene but thus far no mutational hot spot has been identified (Scorpio and Zhang, 1996; Sreevatsan, 1997b; Scorpio, 1997). PZA susceptibility testing is not done routinely in many countries due to technical difficulties. Thus the extent of PZA resistance globally is largely unknown. PZA resistant isolates had diverse nucleotide changes scattered throughout the *pncA* gene. However, PZA resistant isolates without *pncA* mutations were also observed suggesting that another mechanism may be involved in conferring PZA resistance in these isolates. In addition, not all mutations (e.g. Thr114Met) were associated with PZA resistance. In summary, the complexity of PZA resistance makes the development of molecular methods for rapid diagnosis difficult.

#### **5.1.5 Ethambutol**

Ethambutol (EMB) is a very specific and effective drug that is used in combination with INH to treat *M. tuberculosis* infection (Ramaswamy and Musser, 1998). EMB inhibits an arabinosyl transferase (*embB*) involved in cell wall biosynthesis (Takayama and Kilburn, 1989).Three genes designated *emb*C, A and B (Telenti, 1997) encode homologous arabinosyl transferase enzymes involved in EMB resistance. Various studies have identified five mutations in codon 306 of *embB* gene [(ATG-GTG), (ATG-CTG), (ATG-ATA), (ATG-ATC) and (ATG-ATT)] which result in three different amino acid substitutions (Val, Leu and Ile) in EMBresistant isolates (Lee, 2002; Sreevatsan, 1997c; Mokrousov, 2002b; Ramaswamy, 2000). These five mutations are associated with 70–90% of all EMB resistant isolates (Ramaswamy and Musser, 1998). Missense mutations were identified in three additional codons: Phe285leu, Phe330Val and Thr630Ile in EMB resistant isolates. MIC's were generally higher for strains with Met306Leu, Met306Val, and Phe330Val and Thr630Ile substitutions than those organisms with Met306Ile substitutions. Mutations outside of codon 306 are present but quite rare. However a number of EMB phenotypic resistant isolates (about 30%) still lack an identified mutation in *embB*. There is therefore a need to fully understand the mechanism of EMB resistance in clinical isolates.

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

**6. Availables tests for tuberculosis diagnosis** 

Barletta, 2003; Ramaswamy and Musser, 1998)*.* 

**5.2.5 Peptides** 

**6.1 History** 

tuberculosis management.

Opportunies and Challenges in Morocco 483

G→T transversion in the *alr* promoter may lead to the overexpression of *alr* (Feng and

Viomycin (VIO) and capreomycin (CAP) are basic peptide antibiotics that inhibit prokaryotic protein synthesis and have shown that resistance to VIO in *M. smegmatis* is caused by alterations in the 30S or 50S ribosomal subunits (Taniguchi, 1997). Mutations in the *rrs* gene that encodes the 16S rRNA is associated with resistance to VIO and CAP,

Since the 1880s with the development of the sputum smear microscopy, the most commonly used for TB diagnostic, several new and established methods were developed and implemented in many laboratory services worldwide to enhance MTB diagnosis and

The sputum smear microscopy has remained largely unchanged and is often described as a

Because microscopy is both cumbersome to implement and inherently insensitive, many patients remain undiagnosed and many non-TB patients are incorrectly treated with TB drugs on the basis of clinical suspicion alone. In endemic countries, simply obtaining an accurate diagnosis often takes weeks or months from the time a patient first visits a health centre. This delay prevents prompt treatment of TB and leads to continued disease

Mounting drug resistance, including MDR-TB and extensively drug-resistant (XDR) TB, coupled with a growing number of people co-infected with TB and HIV, have highlighted the urgent need for more accurate and rapid diagnostic tests. Many patients are never diagnosed and contribute to the astonishing number of yearly deaths from TB worldwide. The global control of tuberculosis remains a challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Thus, there recently has been a marked increase in the development and testing of novel assays designed to detect MTB complex and/or MDR MTB based either on conventional techniques or on molecular approaches. The Figure

Recently, the WHO has endorsed some of these novel methods, and they have been made available at discounted prices for procurement by the public health sector of high-burden countries. In addition, international and national laboratory partners and donors are currently evaluating other new diagnostics that will allow further and more rapid testing in point-of-care settings. While some techniques are simple, others have complex requirements, and therefore, it is important to carefully determine how to link these new tests and incorporate them within a country's national diagnostic algorithm. Finally, the successful implementation of these methods is dependent on key partnerships in the international laboratory community and ensuring that adequate quality assurance programs are inherent

specifically a G→A or G→T nucleotide change at codon 1473 (Taniguchi, 1997).

simple technology. However, it requires a high level of training and diligence.

transmission, at substantial cost to the individual and at huge cost to society.

2 summarise the main techniques used and under development for TB diagnosis.

#### **5.2 Second line drugs**

#### **5.2.1 Fluoroquinolones**

Ciproflaxin (CIP) and ofloxacin (OFL) are the two fluoroquinolones (FQs) used as secondline drugs in MDR-TB treatment (WHO, 2001). These FQs are bactericidal against MTB. Their target is the DNA gyrase, an ATP-dependent type II DNA topoisomerase that catalyses negative supercoiling of DNA. DNA gyrase is a tetrameric protein composed of two A and two B subunits encoded by the gyrA and gyrB genes, respectively. FQs bing to gyrase and inhibit supercoiling of DNA, thereby disrupting cellular processes dependent on DNA topology (Ramaswamy and Musser, 1998).

The quinolone resistance-determining region (QRDR) is a conserved region in the *gyrA*  (320bp) and *gyrB* (375bp) genes (Ginsburg, 2003) which is the point of interaction of FQ and gyrase (Ginsburg, 2003). Missense mutations in codon 90, 91, and 94 of *gyrA* are associated with resistance to FQs (Takiff, 1994; Xu, 1996). A 16-fold increase in resistance was observed for isolates with a Ala90Val substitution, a 30-fold increase for Asp94Asn or His94Tyr and a 60-fold increase for Asp94Gly (Xu, 1996). A polymorphism at *gyrA* codon 95 is not associated with FQ resistance, and is used, with the *katG*463 polymorphism, to classify *M. tuberculosis* into 3 phylogenetic groups (Sreevatsan, 1997a).

#### **5.2.2 Ethionamide**

Ethionamide (ETH) is a derivative of isonicotinic acid with potent activity against MTB and other mycobacteria. Like INH, ETH is also thought to be a prodrug that is activated by bacterial metabolism. The activated drug then disrupts cell wall biosynthesis by inhibiting mycolic acid synthesis. Mutations in the promoter of the *inhA* gene are associated with resistance to INH and ETH (Morlock, 2003). *EthA* catalyses a two step activation of ETH and gene alterations leading to reduced EthA activity lead to ETH resistance (Engohang- Ndong, 2004; Morlock, 2003; Vannelli, 2002). The expression of *ethA* is under the control of the neighbouring *ethR* gene encoding a repressor. *EthR* negatively regulates the expression of *ethA*, by binding upstream of *ethA* to suppress *ethA* expression (Engohang-Ndong, 2004).

#### **5.2.3 Kanamycine and amykacine**

KAN and AMY are aminoglycoside antibiotics that inhibits protein synthesis by inhibiting the normal function of ribosomes (Taniguchi, 1997; Ramaswamy and Musser, 1998). These drugs are used as second line anti-TB agents. Nucleotide substitutions in the region of *rrs* especially at position 1400 (between the *rrs* gene and 23S rRNA gene) are a major cause of resistance to KAN and AMY in *M.tuberculosis*. It seems that nucleotide substitutions at rrs position 1400 is implicated in high-level resistance to KAN and AMY (Taniguchi, 1997).

#### **5.2.4 D-Cycloserine**

D-cycloserine (DCS) is a cyclic analog of D-alanine which is one of the central molecules of the cross linking step of peptidoglycan assembly (Ramaswamy and Musser, 1998; Feng and Barletta, 2003). DCS inhibits cell wall synthesis by competing with D-Alanine for the enzymes D-alanyl-D-alanine synthetase (Ddl) and D-alanine racemase (Alr) and also inhibiting the synthesis of these proteins. Overexpression of *alr* causes DCS resistance. The G→T transversion in the *alr* promoter may lead to the overexpression of *alr* (Feng and Barletta, 2003; Ramaswamy and Musser, 1998)*.* 

#### **5.2.5 Peptides**

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

Ciproflaxin (CIP) and ofloxacin (OFL) are the two fluoroquinolones (FQs) used as secondline drugs in MDR-TB treatment (WHO, 2001). These FQs are bactericidal against MTB. Their target is the DNA gyrase, an ATP-dependent type II DNA topoisomerase that catalyses negative supercoiling of DNA. DNA gyrase is a tetrameric protein composed of two A and two B subunits encoded by the gyrA and gyrB genes, respectively. FQs bing to gyrase and inhibit supercoiling of DNA, thereby disrupting cellular processes dependent on

The quinolone resistance-determining region (QRDR) is a conserved region in the *gyrA*  (320bp) and *gyrB* (375bp) genes (Ginsburg, 2003) which is the point of interaction of FQ and gyrase (Ginsburg, 2003). Missense mutations in codon 90, 91, and 94 of *gyrA* are associated with resistance to FQs (Takiff, 1994; Xu, 1996). A 16-fold increase in resistance was observed for isolates with a Ala90Val substitution, a 30-fold increase for Asp94Asn or His94Tyr and a 60-fold increase for Asp94Gly (Xu, 1996). A polymorphism at *gyrA* codon 95 is not associated with FQ resistance, and is used, with the *katG*463 polymorphism, to classify *M.* 

Ethionamide (ETH) is a derivative of isonicotinic acid with potent activity against MTB and other mycobacteria. Like INH, ETH is also thought to be a prodrug that is activated by bacterial metabolism. The activated drug then disrupts cell wall biosynthesis by inhibiting mycolic acid synthesis. Mutations in the promoter of the *inhA* gene are associated with resistance to INH and ETH (Morlock, 2003). *EthA* catalyses a two step activation of ETH and gene alterations leading to reduced EthA activity lead to ETH resistance (Engohang- Ndong, 2004; Morlock, 2003; Vannelli, 2002). The expression of *ethA* is under the control of the neighbouring *ethR* gene encoding a repressor. *EthR* negatively regulates the expression of *ethA*, by binding upstream of *ethA* to suppress *ethA* expression (Engohang-Ndong, 2004).

KAN and AMY are aminoglycoside antibiotics that inhibits protein synthesis by inhibiting the normal function of ribosomes (Taniguchi, 1997; Ramaswamy and Musser, 1998). These drugs are used as second line anti-TB agents. Nucleotide substitutions in the region of *rrs* especially at position 1400 (between the *rrs* gene and 23S rRNA gene) are a major cause of resistance to KAN and AMY in *M.tuberculosis*. It seems that nucleotide substitutions at rrs position 1400 is implicated in high-level resistance to KAN and AMY (Taniguchi, 1997).

D-cycloserine (DCS) is a cyclic analog of D-alanine which is one of the central molecules of the cross linking step of peptidoglycan assembly (Ramaswamy and Musser, 1998; Feng and Barletta, 2003). DCS inhibits cell wall synthesis by competing with D-Alanine for the enzymes D-alanyl-D-alanine synthetase (Ddl) and D-alanine racemase (Alr) and also inhibiting the synthesis of these proteins. Overexpression of *alr* causes DCS resistance. The

**5.2 Second line drugs 5.2.1 Fluoroquinolones** 

**5.2.2 Ethionamide** 

**5.2.3 Kanamycine and amykacine** 

**5.2.4 D-Cycloserine** 

DNA topology (Ramaswamy and Musser, 1998).

*tuberculosis* into 3 phylogenetic groups (Sreevatsan, 1997a).

Viomycin (VIO) and capreomycin (CAP) are basic peptide antibiotics that inhibit prokaryotic protein synthesis and have shown that resistance to VIO in *M. smegmatis* is caused by alterations in the 30S or 50S ribosomal subunits (Taniguchi, 1997). Mutations in the *rrs* gene that encodes the 16S rRNA is associated with resistance to VIO and CAP, specifically a G→A or G→T nucleotide change at codon 1473 (Taniguchi, 1997).

#### **6. Availables tests for tuberculosis diagnosis**

#### **6.1 History**

Since the 1880s with the development of the sputum smear microscopy, the most commonly used for TB diagnostic, several new and established methods were developed and implemented in many laboratory services worldwide to enhance MTB diagnosis and tuberculosis management.

The sputum smear microscopy has remained largely unchanged and is often described as a simple technology. However, it requires a high level of training and diligence.

Because microscopy is both cumbersome to implement and inherently insensitive, many patients remain undiagnosed and many non-TB patients are incorrectly treated with TB drugs on the basis of clinical suspicion alone. In endemic countries, simply obtaining an accurate diagnosis often takes weeks or months from the time a patient first visits a health centre. This delay prevents prompt treatment of TB and leads to continued disease transmission, at substantial cost to the individual and at huge cost to society.

Mounting drug resistance, including MDR-TB and extensively drug-resistant (XDR) TB, coupled with a growing number of people co-infected with TB and HIV, have highlighted the urgent need for more accurate and rapid diagnostic tests. Many patients are never diagnosed and contribute to the astonishing number of yearly deaths from TB worldwide.

The global control of tuberculosis remains a challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Thus, there recently has been a marked increase in the development and testing of novel assays designed to detect MTB complex and/or MDR MTB based either on conventional techniques or on molecular approaches. The Figure 2 summarise the main techniques used and under development for TB diagnosis.

Recently, the WHO has endorsed some of these novel methods, and they have been made available at discounted prices for procurement by the public health sector of high-burden countries. In addition, international and national laboratory partners and donors are currently evaluating other new diagnostics that will allow further and more rapid testing in point-of-care settings. While some techniques are simple, others have complex requirements, and therefore, it is important to carefully determine how to link these new tests and incorporate them within a country's national diagnostic algorithm. Finally, the successful implementation of these methods is dependent on key partnerships in the international laboratory community and ensuring that adequate quality assurance programs are inherent

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

with either conventional light or fluorescent microscopy.

(mycobacterial growth indicator tube [MGIT 960]) media.

**6.2.2 Culture** 

in resource-limited settings.

''phased in as an alternative for conventional light microscopy''.

Opportunies and Challenges in Morocco 485

developed. This type of microscopy uses LED technology as a light source but still allows for the advantages of using a fluorescent stain while eliminating most of the disadvantages of fluorescent microscopy. LED microscopy is more sensitive and equally specific, compared

The World Health Organization (WHO) recommends that conventional fluorescence microscopy could be replaced by LED microscopy, and that LED microscopy should be

It's widely accepted that TB culture is substantially more sensitive, but is very slow, and results often do not get back to the health care workers until too late to be clinically useful. Culture is needed to screen for bacilli either on solid (Lowenstein-Jensen [LJ]) or liquid

In Morocco, as it's the case in many countries worldwide, culture on solid media using Lowenstein-Jensen is the main technique used to detect TB in samples. The culturedependent laboratory procedures may take 4 to 6 weeks to have MTB cultures both for TB

The liquid culture (LC) gives an alternative opportunity to enhance TB diagnosis by conventional techniques. In fact, LC is significantly faster: the average time-to-growth detection with liquid culture is 10 to 14 days. Controlled trials have demonstrated that the performance of liquid media culture (LMC) is superior to that of solid media culture for diagnosis of MTB (Srisuwanvilai, 2008), but there is limited evidence about its performance

TB culture on liquid media uses the Mycobacterium Growth Indicator Tube (MGIT). The MGIT is a commercial liquid culture system and the leading rapid culture method in the developed world. MGIT is manufactured in unbreakable plastic tubes containing enriched culture media. At the bottom of the tube is a silicone plug containing chemicals that become fluorescent when bacteria consume oxygen during the process of growth, making detection possible using either manual or automated systems. MGIT was approved by the World Health Organisation in 2007 and is already in use in high income countries and in the

The roll-out of MGIT for case detection is especially important for patients with low numbers of TB bacteria in their sputum, such as children and individuals infected with HIV.

Because traditional techniques have several limitations, considerable progress has recently been made in developing novel approaches and tools, especially molecular methods (commercial and 'in-house'), for direct detection and identification of *M. tuberculosis* in clinical specimens within a single day after sputum collection. The potential advantages of molecular assays are the ability to (1) design assays that are highly sensitive and specific; (2) manufacture some assays in large quantities, allowing for decreased cost and ease of standardization in field use; (3) yield rapid results; and (4) be used more widely, because

**6.3 Molecular tests for diagnosis and identification of mycobacterial species** 

diagnosis and for further analyses e.g. Drug susceptibility testing (Eing, 1998).

private sector. Its implementation in endemic countries is ongoing.

in each country's laboratory network. Moreover, it's widely accepted that if left untreated, each person with active TB infects an average of 10 to 15 people each year. Interrupting disease transmission will require early and accurate detection paired with appropriate treatment.

Fig. 2. Tuberculosis product deliverables 2007 2016

#### **6.2 Conventional tests**

Tuberculosis is generally diagnosed by traditional laboratory procedures, including microscopic examination of samples for the presence of acid-fast bacilli (AFB) and/ or isolation by culture followed by identification using biochemical tets.

#### **6.2.1 Microscopic examination**

The etiological diagnosis of tuberculosis is based on the appearance of bacilli in clinical samples (sputum). Direct sample examination according to Ziehl-Neelsen staining is used to screen for positive bacilli results. For pulmonary tuberculosis, direct examination of sputum is fast and reveals the quantity of acid-fast bacillus, and thus the risk of contagiousness. However, direct examination has a low susceptibility of 22% to 78% and can only detect a concentration of 103 bacilli / ml or more in the sample. In addition, it is not species specific. As a result, false-positives occur, particularly in heavily colonized samples from children patients with other chronic pulmonary infections and paucibacillary cases common among HIV-infected individuals. Nevertheless, many countries use direct examination of samples as a quick test for diagnosing tuberculosis. Early diagnosis can improve patient survival and reduces the spread of the *M. tuberculosis* strain.

Because of the limitations of conventional light microscopy using stains such as Ziehl-Neelsen, fluorochrome stains such as auramine were introduced that improve the sensitivity of the test and take less time to perform. However, fluorescence microscopy has the limitations of requiring a fluorescent microscope, a dark room, and an expensive light source.

Mercury vapor light sources used for this type of microscopy can also pose a hazard if bulbs are broken. To overcome these limitations, light emitting diode (LED) microscopy was developed. This type of microscopy uses LED technology as a light source but still allows for the advantages of using a fluorescent stain while eliminating most of the disadvantages of fluorescent microscopy. LED microscopy is more sensitive and equally specific, compared with either conventional light or fluorescent microscopy.

The World Health Organization (WHO) recommends that conventional fluorescence microscopy could be replaced by LED microscopy, and that LED microscopy should be ''phased in as an alternative for conventional light microscopy''.

#### **6.2.2 Culture**

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

in each country's laboratory network. Moreover, it's widely accepted that if left untreated, each person with active TB infects an average of 10 to 15 people each year. Interrupting disease transmission will require early and accurate detection paired with appropriate

Tuberculosis is generally diagnosed by traditional laboratory procedures, including microscopic examination of samples for the presence of acid-fast bacilli (AFB) and/ or

The etiological diagnosis of tuberculosis is based on the appearance of bacilli in clinical samples (sputum). Direct sample examination according to Ziehl-Neelsen staining is used to screen for positive bacilli results. For pulmonary tuberculosis, direct examination of sputum is fast and reveals the quantity of acid-fast bacillus, and thus the risk of contagiousness. However, direct examination has a low susceptibility of 22% to 78% and can only detect a concentration of 103 bacilli / ml or more in the sample. In addition, it is not species specific. As a result, false-positives occur, particularly in heavily colonized samples from children patients with other chronic pulmonary infections and paucibacillary cases common among HIV-infected individuals. Nevertheless, many countries use direct examination of samples as a quick test for diagnosing tuberculosis. Early diagnosis can improve patient survival and

Because of the limitations of conventional light microscopy using stains such as Ziehl-Neelsen, fluorochrome stains such as auramine were introduced that improve the sensitivity of the test and take less time to perform. However, fluorescence microscopy has the limitations of

Mercury vapor light sources used for this type of microscopy can also pose a hazard if bulbs are broken. To overcome these limitations, light emitting diode (LED) microscopy was

requiring a fluorescent microscope, a dark room, and an expensive light source.

treatment.

Fig. 2. Tuberculosis product deliverables 2007 2016

reduces the spread of the *M. tuberculosis* strain.

isolation by culture followed by identification using biochemical tets.

**6.2 Conventional tests** 

**6.2.1 Microscopic examination** 

It's widely accepted that TB culture is substantially more sensitive, but is very slow, and results often do not get back to the health care workers until too late to be clinically useful.

Culture is needed to screen for bacilli either on solid (Lowenstein-Jensen [LJ]) or liquid (mycobacterial growth indicator tube [MGIT 960]) media.

In Morocco, as it's the case in many countries worldwide, culture on solid media using Lowenstein-Jensen is the main technique used to detect TB in samples. The culturedependent laboratory procedures may take 4 to 6 weeks to have MTB cultures both for TB diagnosis and for further analyses e.g. Drug susceptibility testing (Eing, 1998).

The liquid culture (LC) gives an alternative opportunity to enhance TB diagnosis by conventional techniques. In fact, LC is significantly faster: the average time-to-growth detection with liquid culture is 10 to 14 days. Controlled trials have demonstrated that the performance of liquid media culture (LMC) is superior to that of solid media culture for diagnosis of MTB (Srisuwanvilai, 2008), but there is limited evidence about its performance in resource-limited settings.

TB culture on liquid media uses the Mycobacterium Growth Indicator Tube (MGIT). The MGIT is a commercial liquid culture system and the leading rapid culture method in the developed world. MGIT is manufactured in unbreakable plastic tubes containing enriched culture media. At the bottom of the tube is a silicone plug containing chemicals that become fluorescent when bacteria consume oxygen during the process of growth, making detection possible using either manual or automated systems. MGIT was approved by the World Health Organisation in 2007 and is already in use in high income countries and in the private sector. Its implementation in endemic countries is ongoing.

The roll-out of MGIT for case detection is especially important for patients with low numbers of TB bacteria in their sputum, such as children and individuals infected with HIV.

#### **6.3 Molecular tests for diagnosis and identification of mycobacterial species**

Because traditional techniques have several limitations, considerable progress has recently been made in developing novel approaches and tools, especially molecular methods (commercial and 'in-house'), for direct detection and identification of *M. tuberculosis* in clinical specimens within a single day after sputum collection. The potential advantages of molecular assays are the ability to (1) design assays that are highly sensitive and specific; (2) manufacture some assays in large quantities, allowing for decreased cost and ease of standardization in field use; (3) yield rapid results; and (4) be used more widely, because

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

respectively (Zakham, 2011).

sputum or with AFB-negative smears).

**6.3.3 Enhanced MTB direct test (E-MTD)** 

Ozkutuk, 2006).

Opportunies and Challenges in Morocco 487

showed a sensitivity of 81.13 % with specificity of 88, 24 % as compared with conventional techniques. Moreover, the positive and negative predictive values were 95.56 %, 60%

**6.3.2 The amplicor MTB test and its automated version the Cobas Amplicor MTB test**  The amplicor test is based on the PCR. In this assay, mycobacterial DNA is amplified with genus-specific primers formulated on the basis of the 16S rRNA gene. After denaturation, the amplicons are added to a microtiter plate containing a bound, *M tuberculosis* complexspecific oligonucleotide probe. An avidin-horseradish peroxidase conjugate then binds to the bound, biotin labelled amplicons. The conjugate then reacts with with peroxide and 3,39,5,59-tetramethylbenzidine in dimethylformamide to form a color complex. The results are measured with a photometer (D'Amato, 1995; Soini and Musser, 2001;

The Amplicor results are available in 6.5 h. An automated version of this test is available (Cobas Amplicor).The overall sensitivity of the Amplicor test (compared with culture) for respiratory specimens is 79.4 –91.9%, the specificity is 99.6 –99.8%. However, the sensitivity for smear negative specimens is somewhat lower, 40.0–73.1% (Bergmann, 1996; Stauffer, 1995; Tevere, 1996; Eing, 1998). Therefore, the Amplicor test has been approved by the Food and Drug Administration (FDA) only for direct detection of *M. tuberculosis* in AFB smearpositive respiratory specimens. Chin *et al.* (Chin, 1995) reported that the sensitivity of the Amplicor test was similar to that of culture (58% vs 56%) for detecting *M. tuberculosis* from respiratory specimens when the clinical case definition of TB was used as the reference standard. However, Al Zahrani *et al.* (2000) reported that although the Amplicor test had excellent specificity (100%), it was less sensitive than culture (42% vs 73%) for diagnosis of minimal active pulmonary TB (patients suspected of having TB but without spontaneous

The E-MTD test is based on the transcription-mediated amplification system developed by Kwoh *et al*. (1989). In this assay, rRNA is released from the target cells by sonication, and a promoter-primer binds to the rRNA target. Reverse transcriptase is then used to copy rRNA to a cDNA-RNA hybrid. The initial RNA strand is degraded, and a second primer binds to the cDNA and is extended, leading to the formation of double-stranded cDNA, which is then transcribed by DNA-directed RNA polymerase to produce more rRNA molecules. The new transcripts serve as templates for reverse transcription and further amplification. The RNA amplicons are detected with an acridinium ester-labeled DNA probe in a solution hybridization assay. Importantly, the amplification procedure is isothermal and the reaction is performed in a single tube, which helps to reduce carryover contamination. After standard

decontamination of the clinical specimen, the E-MTD test can be completed in 3.5 h.

The E-MTD test is FDA-approved for detection of *M. tuberculosis* in both AFB smear-positive and smear-negative specimens. The overall sensitivity (compared with culture) for respiratory specimens is 90.9 –95.2%, the specificity 98.8–100% (Bergmann, 1999; Gamboa, 1998; Smith, 1999).The performance of the E-MTD and the Cobas Amplicor is the same (Scarparo, 2000). However, it was noted that although the turnaround time is shorter for the

they require less training and infrastructure than do conventional mycobacterial cultures and AST.

These potential advantages must be weighed against the disadvantages of these assays, some of which are common to all molecular techniques and others specific to particular assays. Among the disadvantages of molecular assays are (1) a need for laboratory infrastructure that can accommodate molecular testing; (2) cost; (3) a continued need of cultures for AST; and (4) most work better with smear-positive than with smear-negative specimens.

These methods, based on nucleic acid amplification (NAA) of different targets, aim to identify the M. tuberculosis complex, and eventually drug resistant strains. In general, commercial methods are recommended since they have a better level of standardization, reproducibility and automation. Although some aspects such as cost-efficiency and the appropriate setting for the implementation of these techniques are not yet well established, organizations such as the WHO are strongly supporting the implementation and universal use of these new molecular methods (Moure, 2010).

The available molecular methods for direct detection of MTB from clinical samples include in house polymerase chain (PCR) using essentially IS6110, hsp65 and 16SrRNA as target. Two Commercial nucleic acid amplification ( NAA) tests for MTB detection in clinical specimens are available: the Enhanced MTB Direct Test (E-MTD), the Amplicor MTB test and its automated version the Cobas Amplicor MTB test, the BDProbe tec ET test, GeneXpert MTB/RIF Assay and the INNO-LiPA-Rif (Innogenetics, Ghent, Belgium).

#### **6.3.1 Classical PCR using essentially IS6110, Hsp 65 and 16SrRNA as targets**

The polymerase chain reaction (PCR) has been most widely used for the detection of *M. tuberculosis* in clinical specimens including sputum, blood, bone marrow, and biopsy samples.

The MTB Complex-specific insertion sequence 6110 is commonly used as a target for detecting MTB. The overall sensitivity and specificity of the assay range from 84.2-100% and 83-100%, respectively, for respiratory specimens (Shamputa, 2004).

The performance of the in house IS6110 PCR in direct detection of MTB on sputum has been evaluated in Morocco and showed promising results (under publication).

However, in non-respiratory samples, lower sensitivities were recorded in most studies with the exception of successful detection in pleural biopsy specimens in one study, and even in blood samples in another study.

The main advantage of the IS6110 targeted NAATs is the fact that most MTBC isolates carry more than five copies of this transposon, thereby increasing the sensitivity of the test. However, in some Asian regions MTBC isolates with no or few IS6110 copies are more prevalent.

The 16S rRNA and the Hsp65 genes have also been used to detect MTBC in respiratory and non-respiratory clinical specimens with high sensitivity and specificity values. In Morocco, the use of *hsp656* gene as a PCR target was evaluated as a direct method for the diagnosis of MTB in 70 clinical specimens (62 sputum, 6 cerebrospinal fluids, and 2 biopsies). Results

they require less training and infrastructure than do conventional mycobacterial cultures

These potential advantages must be weighed against the disadvantages of these assays, some of which are common to all molecular techniques and others specific to particular assays. Among the disadvantages of molecular assays are (1) a need for laboratory infrastructure that can accommodate molecular testing; (2) cost; (3) a continued need of cultures for AST; and (4) most work better with smear-positive than with smear-negative

These methods, based on nucleic acid amplification (NAA) of different targets, aim to identify the M. tuberculosis complex, and eventually drug resistant strains. In general, commercial methods are recommended since they have a better level of standardization, reproducibility and automation. Although some aspects such as cost-efficiency and the appropriate setting for the implementation of these techniques are not yet well established, organizations such as the WHO are strongly supporting the implementation and universal

The available molecular methods for direct detection of MTB from clinical samples include in house polymerase chain (PCR) using essentially IS6110, hsp65 and 16SrRNA as target. Two Commercial nucleic acid amplification ( NAA) tests for MTB detection in clinical specimens are available: the Enhanced MTB Direct Test (E-MTD), the Amplicor MTB test and its automated version the Cobas Amplicor MTB test, the BDProbe tec ET test,

The polymerase chain reaction (PCR) has been most widely used for the detection of *M. tuberculosis* in clinical specimens including sputum, blood, bone marrow, and biopsy

The MTB Complex-specific insertion sequence 6110 is commonly used as a target for detecting MTB. The overall sensitivity and specificity of the assay range from 84.2-100% and

The performance of the in house IS6110 PCR in direct detection of MTB on sputum has been

However, in non-respiratory samples, lower sensitivities were recorded in most studies with the exception of successful detection in pleural biopsy specimens in one study, and even in

The main advantage of the IS6110 targeted NAATs is the fact that most MTBC isolates carry more than five copies of this transposon, thereby increasing the sensitivity of the test. However, in some Asian regions MTBC isolates with no or few IS6110 copies are more

The 16S rRNA and the Hsp65 genes have also been used to detect MTBC in respiratory and non-respiratory clinical specimens with high sensitivity and specificity values. In Morocco, the use of *hsp656* gene as a PCR target was evaluated as a direct method for the diagnosis of MTB in 70 clinical specimens (62 sputum, 6 cerebrospinal fluids, and 2 biopsies). Results

GeneXpert MTB/RIF Assay and the INNO-LiPA-Rif (Innogenetics, Ghent, Belgium).

**6.3.1 Classical PCR using essentially IS6110, Hsp 65 and 16SrRNA as targets** 

83-100%, respectively, for respiratory specimens (Shamputa, 2004).

evaluated in Morocco and showed promising results (under publication).

use of these new molecular methods (Moure, 2010).

and AST.

specimens.

samples.

prevalent.

blood samples in another study.

showed a sensitivity of 81.13 % with specificity of 88, 24 % as compared with conventional techniques. Moreover, the positive and negative predictive values were 95.56 %, 60% respectively (Zakham, 2011).

#### **6.3.2 The amplicor MTB test and its automated version the Cobas Amplicor MTB test**

The amplicor test is based on the PCR. In this assay, mycobacterial DNA is amplified with genus-specific primers formulated on the basis of the 16S rRNA gene. After denaturation, the amplicons are added to a microtiter plate containing a bound, *M tuberculosis* complexspecific oligonucleotide probe. An avidin-horseradish peroxidase conjugate then binds to the bound, biotin labelled amplicons. The conjugate then reacts with with peroxide and 3,39,5,59-tetramethylbenzidine in dimethylformamide to form a color complex. The results are measured with a photometer (D'Amato, 1995; Soini and Musser, 2001; Ozkutuk, 2006).

The Amplicor results are available in 6.5 h. An automated version of this test is available (Cobas Amplicor).The overall sensitivity of the Amplicor test (compared with culture) for respiratory specimens is 79.4 –91.9%, the specificity is 99.6 –99.8%. However, the sensitivity for smear negative specimens is somewhat lower, 40.0–73.1% (Bergmann, 1996; Stauffer, 1995; Tevere, 1996; Eing, 1998). Therefore, the Amplicor test has been approved by the Food and Drug Administration (FDA) only for direct detection of *M. tuberculosis* in AFB smearpositive respiratory specimens. Chin *et al.* (Chin, 1995) reported that the sensitivity of the Amplicor test was similar to that of culture (58% vs 56%) for detecting *M. tuberculosis* from respiratory specimens when the clinical case definition of TB was used as the reference standard. However, Al Zahrani *et al.* (2000) reported that although the Amplicor test had excellent specificity (100%), it was less sensitive than culture (42% vs 73%) for diagnosis of minimal active pulmonary TB (patients suspected of having TB but without spontaneous sputum or with AFB-negative smears).

#### **6.3.3 Enhanced MTB direct test (E-MTD)**

The E-MTD test is based on the transcription-mediated amplification system developed by Kwoh *et al*. (1989). In this assay, rRNA is released from the target cells by sonication, and a promoter-primer binds to the rRNA target. Reverse transcriptase is then used to copy rRNA to a cDNA-RNA hybrid. The initial RNA strand is degraded, and a second primer binds to the cDNA and is extended, leading to the formation of double-stranded cDNA, which is then transcribed by DNA-directed RNA polymerase to produce more rRNA molecules. The new transcripts serve as templates for reverse transcription and further amplification. The RNA amplicons are detected with an acridinium ester-labeled DNA probe in a solution hybridization assay. Importantly, the amplification procedure is isothermal and the reaction is performed in a single tube, which helps to reduce carryover contamination. After standard decontamination of the clinical specimen, the E-MTD test can be completed in 3.5 h.

The E-MTD test is FDA-approved for detection of *M. tuberculosis* in both AFB smear-positive and smear-negative specimens. The overall sensitivity (compared with culture) for respiratory specimens is 90.9 –95.2%, the specificity 98.8–100% (Bergmann, 1999; Gamboa, 1998; Smith, 1999).The performance of the E-MTD and the Cobas Amplicor is the same (Scarparo, 2000). However, it was noted that although the turnaround time is shorter for the

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

(Minion, 2010).

reading results.

Gerdes, 1999).

**7.1.2 Drug susceptibility testing** 

**7.1.3 DST on liquid medium** 

**7.1.4 Colorimetric assays** 

Opportunies and Challenges in Morocco 489

The published sensitivity of the assay varies from 87.4% to 97.8%, although the assay was compared with different gold standards in these studies. The contamination rate for the MODS assay, although lower than that of solid media, is higher than that of liquid media

The standard methods using the Lowenstein-Jensen (LJ) or agar proportion method (PM) (Canetti 1963, 1969, Kent & Kubica 1985) and the radiometric method in BACTEC TB-460 system (Becton-Dickinson) (Roberts 1983) are the current standard methods recommended to perform susceptibility testing of *M. tuberculosis*. The absolute concentration method is also commonly used on account of its technical simplicity for inoculums preparation and for

In order to shorten the turnaround time and make it more convenient for case management, numerous new techniques have appeared, aiming to detect growth inhibition as early as possible. The most commonly used systems are detection of CO2 production, such as BACTEC 460 (Hawkins, 1991) or MB/Bact (Diaz-Infantes, 2000), and oxygen consumption, such as Mycobacteria Growth Indicator Tube (Bemer, 2002); there are others in developmental stage. However, many of those new techniques are difficult to implement in the developing countries where they are needed the most, because of high costs, technical

MGIT can also be used to perform drug susceptibility testing (DST), which is done by comparing the growth of mycobacteria with and without the addition of drugs used to treat TB. The combined use of MGIT for both TB detection and DST can shave months off the conventional process of identifying multidrug-resistant (MDR) TB (Hanna, 1999; Rusch-

Despite having been developed over a decade ago, the advantages of MGIT for TB detection were not reaching most endemic settings for several reasons. This was primarily due to the cost of the test, the lack of a simple means to confirm the growth of *M. tuberculosis* species in positive tubes, and the lack of data demonstrating that the use of liquid culture was feasible in resource-constrained settings. FIND has partnered with BD to overcome these obstacles and introduce MGIT as a solution for case detection and DST in developing countries.

A colorimetric method for detecting microbial growth in drug-resistant strains was described in 1998 and subsequently evaluated in a limited number of clinical trials (Martin, 2005; Abate, 2004; Montoro, 2005). The assay is based on the observation that growing tubercle bacilli convert a yellow dye [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide or MTT] to a purple color that can be detected visually or by use of a spectrophotometer. In field trials, the method has been shown to have a high degree of concordance with conventional AST (Martin, 2005; Abate, 2004; Montoro, 2005). The method has been compared with a nitrate reduction assay and a resazurin assay for detecting

complexity and absence of appropriately trained human resources.

E-MTD test, the Amplicor test can be fully automated and has an internal control for monitoring amplification inhibitors.

#### **6.3.4 BDProbeTec ET test**

The BDProbeTec ET system allows amplification and detection of *M*. *tuberculosis* complex (MTBC) DNA in 1 h and simultaneously detects the presence of inhibitors as well. The target of the BDProbeTec ET system is a 95-bp region of IS*6110*, a highly specific insertion element in the MTBC DNA where it is present in multiple copies. Nucleic acid amplification is isothermal and is based on homogeneous strand displacement amplification (SDA) (Spargo, 1993), while detection is based on real-time fluorescent energy transfer (Little, 1999). An internal amplification control (IAC) is run with each sample to confirm the validity of the amplification reaction and to identify potential inhibitory factors from the processed specimen.

Of the published studies on the BDProbeTec system, sensitivities and specificities for respiratory samples were ranging from 82.7% to 100% and from 96.5% to 99.8% respectively (Mazzarelli, 2003). The extrapulmonary specimens represent a major diagnostic problem, mainly as they are often paucibacillary and at times contain inhibitors. With such samples, the resolved sensitivity of the BDProbeTec ET system is lower (77.8%) than with pulmonary specimens (91.5%), but nevertheless higher than with microscopy (63.1%) (Mazzarelli, 2003; Cho, 2007).

Other tests, Xpert MTB/RIF Assay (Cepheid) and INNO-LiPA-Rif (Innogenetics, Ghent, Belgium), used both for TB diagnosis and TB drug resistance screening will be discussed in later.

#### **7. Diagnosis of drug resistant tuberculosis**

#### **7.1 Conventional tests**

#### **7.1.1 Microscopic Observation Drug Susceptibility (MODS) assay**

The MODS assay is a broth microtiter method designed to detect *M. tuberculosis* complex and to detect resistance to isoniazid and rifampin (Moore, 2006; Mello, 2007; Ha, 2010). The method uses standard microtiter plates and other materials that are readily available in larger diagnostic laboratories. The method is straightforward: microtiter plates are prepared that contain Middlebrook 7H9 broth medium, growth supplements, and antimicrobial agents to prevent overgrowth of bacterial contaminants. Anti-TB drugs, at different concentrations, are added to some of the wells (Wilson, 2011).

The performance characteristics of the MODS assay were summarized in a recent metaanalysis. For detecting lowlevel resistance to isoniazid the pooled sensitivity of the assay is 97.7% and specificity is 95.8%. For detecting high-level isoniazid resistance, the sensitivity decreases to 90.0%, but the specificity increases to 98.6%. For detection of rifampin resistance, the pooled sensitivity is 98.0% and the specificity is 99.4%. This meta-analysis did not summarize the ability of the assay to identify the presence of *M. tuberculosis* in sputum specimens (Ha, 2010; Minion, 2010).

The published sensitivity of the assay varies from 87.4% to 97.8%, although the assay was compared with different gold standards in these studies. The contamination rate for the MODS assay, although lower than that of solid media, is higher than that of liquid media (Minion, 2010).

#### **7.1.2 Drug susceptibility testing**

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

E-MTD test, the Amplicor test can be fully automated and has an internal control for

The BDProbeTec ET system allows amplification and detection of *M*. *tuberculosis* complex (MTBC) DNA in 1 h and simultaneously detects the presence of inhibitors as well. The target of the BDProbeTec ET system is a 95-bp region of IS*6110*, a highly specific insertion element in the MTBC DNA where it is present in multiple copies. Nucleic acid amplification is isothermal and is based on homogeneous strand displacement amplification (SDA) (Spargo, 1993), while detection is based on real-time fluorescent energy transfer (Little, 1999). An internal amplification control (IAC) is run with each sample to confirm the validity of the amplification reaction and to identify potential inhibitory factors from the processed

Of the published studies on the BDProbeTec system, sensitivities and specificities for respiratory samples were ranging from 82.7% to 100% and from 96.5% to 99.8% respectively (Mazzarelli, 2003). The extrapulmonary specimens represent a major diagnostic problem, mainly as they are often paucibacillary and at times contain inhibitors. With such samples, the resolved sensitivity of the BDProbeTec ET system is lower (77.8%) than with pulmonary specimens (91.5%), but nevertheless higher than with microscopy (63.1%) (Mazzarelli, 2003;

Other tests, Xpert MTB/RIF Assay (Cepheid) and INNO-LiPA-Rif (Innogenetics, Ghent, Belgium), used both for TB diagnosis and TB drug resistance screening will be discussed in

The MODS assay is a broth microtiter method designed to detect *M. tuberculosis* complex and to detect resistance to isoniazid and rifampin (Moore, 2006; Mello, 2007; Ha, 2010). The method uses standard microtiter plates and other materials that are readily available in larger diagnostic laboratories. The method is straightforward: microtiter plates are prepared that contain Middlebrook 7H9 broth medium, growth supplements, and antimicrobial agents to prevent overgrowth of bacterial contaminants. Anti-TB drugs, at different

The performance characteristics of the MODS assay were summarized in a recent metaanalysis. For detecting lowlevel resistance to isoniazid the pooled sensitivity of the assay is 97.7% and specificity is 95.8%. For detecting high-level isoniazid resistance, the sensitivity decreases to 90.0%, but the specificity increases to 98.6%. For detection of rifampin resistance, the pooled sensitivity is 98.0% and the specificity is 99.4%. This meta-analysis did not summarize the ability of the assay to identify the presence of *M. tuberculosis* in sputum

monitoring amplification inhibitors.

**7. Diagnosis of drug resistant tuberculosis** 

**7.1.1 Microscopic Observation Drug Susceptibility (MODS) assay** 

concentrations, are added to some of the wells (Wilson, 2011).

**6.3.4 BDProbeTec ET test** 

specimen.

Cho, 2007).

**7.1 Conventional tests** 

specimens (Ha, 2010; Minion, 2010).

later.

The standard methods using the Lowenstein-Jensen (LJ) or agar proportion method (PM) (Canetti 1963, 1969, Kent & Kubica 1985) and the radiometric method in BACTEC TB-460 system (Becton-Dickinson) (Roberts 1983) are the current standard methods recommended to perform susceptibility testing of *M. tuberculosis*. The absolute concentration method is also commonly used on account of its technical simplicity for inoculums preparation and for reading results.

In order to shorten the turnaround time and make it more convenient for case management, numerous new techniques have appeared, aiming to detect growth inhibition as early as possible. The most commonly used systems are detection of CO2 production, such as BACTEC 460 (Hawkins, 1991) or MB/Bact (Diaz-Infantes, 2000), and oxygen consumption, such as Mycobacteria Growth Indicator Tube (Bemer, 2002); there are others in developmental stage. However, many of those new techniques are difficult to implement in the developing countries where they are needed the most, because of high costs, technical complexity and absence of appropriately trained human resources.

#### **7.1.3 DST on liquid medium**

MGIT can also be used to perform drug susceptibility testing (DST), which is done by comparing the growth of mycobacteria with and without the addition of drugs used to treat TB. The combined use of MGIT for both TB detection and DST can shave months off the conventional process of identifying multidrug-resistant (MDR) TB (Hanna, 1999; Rusch-Gerdes, 1999).

Despite having been developed over a decade ago, the advantages of MGIT for TB detection were not reaching most endemic settings for several reasons. This was primarily due to the cost of the test, the lack of a simple means to confirm the growth of *M. tuberculosis* species in positive tubes, and the lack of data demonstrating that the use of liquid culture was feasible in resource-constrained settings. FIND has partnered with BD to overcome these obstacles and introduce MGIT as a solution for case detection and DST in developing countries.

#### **7.1.4 Colorimetric assays**

A colorimetric method for detecting microbial growth in drug-resistant strains was described in 1998 and subsequently evaluated in a limited number of clinical trials (Martin, 2005; Abate, 2004; Montoro, 2005). The assay is based on the observation that growing tubercle bacilli convert a yellow dye [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide or MTT] to a purple color that can be detected visually or by use of a spectrophotometer. In field trials, the method has been shown to have a high degree of concordance with conventional AST (Martin, 2005; Abate, 2004; Montoro, 2005). The method has been compared with a nitrate reduction assay and a resazurin assay for detecting

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

 Confirmation of PCR amplification by gel electrophoresis Blotting PCR product on a filter using dot blot apparatus

**7.2.2 Probe based hybridization methods** 

PCR amplification for target genes

during the development phase of this strategy.

**7.2.3 Reverse line probe assay** 

**DOT BLOT strategy** 

transferase. Dot Blot hybridization Autoradiography.

includes:

studied.

Opportunies and Challenges in Morocco 491

It is a technique which can detect any known or newly described mutations and which can fulfil the criteria of accuracy, speed and simplicity. The hybridization with wild type probes can be used to efficiently screen for all mutations conferring drug resistance. The method

 Labelling of probes for region of interest: the probes are 5' end-labelled by phosphorylation with [∂32P]-ATP or 3' end-labelled with digoxigenin by terminal

Although radioactive and non-radioactive detection procedures gave similar results, the radioactive procedure was favoured to empirically evaluate stringent hybridization washes

The dot –blot procedure may be specifically useful in countries with a high incidence of TBand where procedures such as automated DNA sequencing are not readily available. As the predictive value of any test is dependent on prevalence, a mutational screening strategy should initially focus on the mutations most frequently diagnosed in the geographic area

Codons 315 (KatG), 516,526 and 531 (rpoB), 43 (rpsL), 491,513 (rrs) and 306 (emb) are frequently altered in clinical isolates from many studies (Victor, 1999, Sabouni, 2008; Kourout, 2009; Chaoui, 2009). Such methods are needed to determine the most important mutations associated with drug resistance in different geographical regions, since it is known that drug resistant mutations may vary with the geographical origin of the sample. The wild type probe strategy is unable to provide a precise understanding of the different mutations occurring at a specific codon, however, it is known that 99% of mutations within these loci confer resistance and therefore the absence of a hybridization signal has been interpreted to directly reflect drug resistance. The application of specific mutant probes allows the identification or the confirmation of the nature of this mutational event. The method is reproducible, not technically demanding and it takes about two normal working days to obtain results. This technique could be adapted to amplify and detect drug resistant

This approach involves a combination of DNA amplification and reverse-line blot hybridisation. This home made and low cost test was first developed to detect RIF resistant isolates (RIFO): in this test , the DNA of *rpoB* gene of MTB is amplified by PCR using specific primers. Then, the PCR products are hybridized to oligonucleotides on a DNA membrane, encoding the consecutive parts of of the *rpoB* gene sequence and the consecutive parts of of

mutations directly from sputum samples or microscopy stained slides.

resistance to isoniazid, rifampin, ethambutol, and streptomycin; similar results were obtained for isoniazid and rifampin, but only the nitrate reduction assay showed a high level of concordance with all of the first-line drugs. Although these methods are conceptually straightforward, they are likely to be useful primarily in larger laboratories with the capacity to perform more complex assays (Montoro, 2005; Wilson, 2011).

#### **7.2 Molecular tests**

Several molecular detection methods for drug resistance are of great value; all these methods are based on the observation that resistance to anti-TB drugs develops through the sequential accumulation of mutations in mycobacterial genes targeted by different drugs. Mutations in specific codons can therefore be used to rapidly detect drug resistance, since drug susceptible samples lack the corresponding gene mutation. These Molecular methods are fast and reliable and can potentially reduce the diagnosis time from weeks to days, These methods include PCR-based sequencing, PCR-Restriction Fragment Length Polymorphism, PCR-Single Strand Conformation Polymorphism (PCR-SSCP), Heteroduplex Analysis, DNA Microarrays and Probe methods. Moreover, a commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) for detection of mutations conferring resistance to Rifampin (RMP) and Isoniazid (INH) in clinical *Mycobacterium tubercu*losis isolates is now widely used. GeneXpert MTB/RIF Assay and the INNO-LiPA-Rif (Innogenetics, Ghent, Belgium), are two rapid assays for simultaneous detection of MTBC and determination of the rifampicin (RIF) resistance profile (a marker for multidrug resistance).

#### **7.2.1 PCR-based sequencing**

PCR-based sequencing is the main technique used to elucidate the genetic mechanisms of drug resistance in *M. tuberculosis*. It is the most direct and reliable method for studying mutations and allows for detection of both previously recognized and unrecognized mutations. Unfortunately, the method is not as readily applicable for routine identification of drug resistance mutations as it is for identification of mycobacterial species because many different genes may be involved, as is the case in INH resistance, or the mutations may be scattered in a large segment of the gene. This means that several sequencing reactions need to be performed for each isolate. However, for targets such as *rpoB*, where mutations associated with RIF resistance are concentrated in a very short segment of the gene, PCR-based sequencing is a useful technique (Soini, 2001; Johnson, 2006; Kourout, 2009).

A previous study was done in Morocco to characterize mutations in rpoB gene associated with rifampicin (RMP) resistance in 47 RMP-resistant and 147 RMP-susceptible clinical strains of *Mycobacterium tuberculosis* by DNA sequencing. RMP-resistant mutations were identified in 85% of RMP-resistant isolates. Sequence analysis identified 10 alleles, including two deletions not previously reported: 514-515 Δ (Phe-Met → Leu) and 519-520 Δ (Asn-Pro). Nucleotide changes at codons 531, 526 and 516 were the most prominent, accounting for 74.4% of our RMP-resistant strains. These results demonstrate that DNA sequencing is an efficient tool for rapid detection of RMP resistance (Kourout, 2009).

#### **7.2.2 Probe based hybridization methods**

#### **DOT BLOT strategy**

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

resistance to isoniazid, rifampin, ethambutol, and streptomycin; similar results were obtained for isoniazid and rifampin, but only the nitrate reduction assay showed a high level of concordance with all of the first-line drugs. Although these methods are conceptually straightforward, they are likely to be useful primarily in larger laboratories

Several molecular detection methods for drug resistance are of great value; all these methods are based on the observation that resistance to anti-TB drugs develops through the sequential accumulation of mutations in mycobacterial genes targeted by different drugs. Mutations in specific codons can therefore be used to rapidly detect drug resistance, since drug susceptible samples lack the corresponding gene mutation. These Molecular methods are fast and reliable and can potentially reduce the diagnosis time from weeks to days, These methods include PCR-based sequencing, PCR-Restriction Fragment Length Polymorphism, PCR-Single Strand Conformation Polymorphism (PCR-SSCP), Heteroduplex Analysis, DNA Microarrays and Probe methods. Moreover, a commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) for detection of mutations conferring resistance to Rifampin (RMP) and Isoniazid (INH) in clinical *Mycobacterium tubercu*losis isolates is now widely used. GeneXpert MTB/RIF Assay and the INNO-LiPA-Rif (Innogenetics, Ghent, Belgium), are two rapid assays for simultaneous detection of MTBC and determination of the rifampicin (RIF) resistance profile (a marker for

PCR-based sequencing is the main technique used to elucidate the genetic mechanisms of drug resistance in *M. tuberculosis*. It is the most direct and reliable method for studying mutations and allows for detection of both previously recognized and unrecognized mutations. Unfortunately, the method is not as readily applicable for routine identification of drug resistance mutations as it is for identification of mycobacterial species because many different genes may be involved, as is the case in INH resistance, or the mutations may be scattered in a large segment of the gene. This means that several sequencing reactions need to be performed for each isolate. However, for targets such as *rpoB*, where mutations associated with RIF resistance are concentrated in a very short segment of the gene, PCR-based sequencing is a useful technique (Soini, 2001; Johnson,

A previous study was done in Morocco to characterize mutations in rpoB gene associated with rifampicin (RMP) resistance in 47 RMP-resistant and 147 RMP-susceptible clinical strains of *Mycobacterium tuberculosis* by DNA sequencing. RMP-resistant mutations were identified in 85% of RMP-resistant isolates. Sequence analysis identified 10 alleles, including two deletions not previously reported: 514-515 Δ (Phe-Met → Leu) and 519-520 Δ (Asn-Pro). Nucleotide changes at codons 531, 526 and 516 were the most prominent, accounting for 74.4% of our RMP-resistant strains. These results demonstrate that DNA sequencing is an

efficient tool for rapid detection of RMP resistance (Kourout, 2009).

with the capacity to perform more complex assays (Montoro, 2005; Wilson, 2011).

**7.2 Molecular tests** 

multidrug resistance).

2006; Kourout, 2009).

**7.2.1 PCR-based sequencing** 

It is a technique which can detect any known or newly described mutations and which can fulfil the criteria of accuracy, speed and simplicity. The hybridization with wild type probes can be used to efficiently screen for all mutations conferring drug resistance. The method includes:


Although radioactive and non-radioactive detection procedures gave similar results, the radioactive procedure was favoured to empirically evaluate stringent hybridization washes during the development phase of this strategy.

The dot –blot procedure may be specifically useful in countries with a high incidence of TBand where procedures such as automated DNA sequencing are not readily available. As the predictive value of any test is dependent on prevalence, a mutational screening strategy should initially focus on the mutations most frequently diagnosed in the geographic area studied.

Codons 315 (KatG), 516,526 and 531 (rpoB), 43 (rpsL), 491,513 (rrs) and 306 (emb) are frequently altered in clinical isolates from many studies (Victor, 1999, Sabouni, 2008; Kourout, 2009; Chaoui, 2009). Such methods are needed to determine the most important mutations associated with drug resistance in different geographical regions, since it is known that drug resistant mutations may vary with the geographical origin of the sample.

The wild type probe strategy is unable to provide a precise understanding of the different mutations occurring at a specific codon, however, it is known that 99% of mutations within these loci confer resistance and therefore the absence of a hybridization signal has been interpreted to directly reflect drug resistance. The application of specific mutant probes allows the identification or the confirmation of the nature of this mutational event. The method is reproducible, not technically demanding and it takes about two normal working days to obtain results. This technique could be adapted to amplify and detect drug resistant mutations directly from sputum samples or microscopy stained slides.

#### **7.2.3 Reverse line probe assay**

This approach involves a combination of DNA amplification and reverse-line blot hybridisation. This home made and low cost test was first developed to detect RIF resistant isolates (RIFO): in this test , the DNA of *rpoB* gene of MTB is amplified by PCR using specific primers. Then, the PCR products are hybridized to oligonucleotides on a DNA membrane, encoding the consecutive parts of of the *rpoB* gene sequence and the consecutive parts of of

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

and phenotypic or genotypic correlation (Kim, 1997).

is limited (Victor *et al*., 2002).

**7.2.7 Line-probe assays** 

**7.2.6 Microarrays** 

**7.2.5 PCR-Restriction Fragment Length Polymorphism** 

Opportunies and Challenges in Morocco 493

association with RIF resistance, which underlines the need for caution in interpreting results

Mutations associated with resistance can be identified by digestion of amplified PCR products with a restriction enzyme that cuts at the specific polymorphic DNA sequence followed by gel electrophoresis. Since not all mutations result in the gain or loss of a restriction site, general use of RFLP to screen for mutations associated with drug resistance

Although technically a solid-phase-type hybridization assay, microarrays, also known as biochips, have been proposed as new molecular methods for detecting drug resistance in *M. tuberculosis*. They are based on the hybridization of DNA obtained from clinical samples to oligonucleotides immobilized in a solid support, such as miniaturized glass slides. They have been mainly used to detect resistance to rifampicin. In a recent evaluation using oligonucleotide microarrays for analysis of drug resistance, Gryadunov et al. (2005) has detected over 95% rifampicin resistant and almost 80% isoniazid resistant *M. tuberculosis* isolates within 12 h in a sample of drug-resistant isolates and clinical samples. For the time being and due to the high cost involved, the use of microarrays for detecting drug resistance

This technology involves a series of steps including extraction of DNA from mycobacterial isolates or directly from clinical specimens, polymerase chain reaction (PCR) amplification of nucleic acid sequences, hybridization of labeled PCR products with oligonucleotide probes immobilized on a strip, and colorimetric development that allows for lines to be seen

In 2008, the WHO issued a policy statement regarding the molecular line-probe assays for

The first line-probe assay was the INNO-LiPA Rif TB (Innogenetics NV) (Rossau, 1997). The results of clinical evaluations of the assay indicated that it accurately detects resistance to rifampin, but some of the evaluations showed that the assay was less sensitive for the detection of *M. tuberculosis* complex. A meta-analysis performed in 2005 showed that 12 of 14 published studies showed 95% of sensitivity with a specificity of 100% but that, in studies in which the assay was applied to clinical specimens, the sensitivity ranged from 80% to 100%. One study showed that the assay could be used successfully in a resource-poor

The second line-probe assay was the GenoType MTBDR® developed by Hain Lifescience. Initially, this assay was developed as the GenoType MTBDR assay, but early evaluations showed that the assay did not detect drug resistance to a satisfactory degree, detecting only 90%–95% of isolates with rifampin or low-level isoniazid resistance. The assay was eventually modified to include detection of more rpoB and inhA mutations under the name

in TB is still beyond the reach of most clinical mycobacteriology laboratories.

where the probes are located (hence, the term ''line-probe'' assay) .

setting, compared with reference laboratories (Wilson, 2011).

use in detection on *M. tuberculosis* and for detection of drug resistance.

the *rpoB* gene sequence with the most frequently occurring mutations in rifampicin-resistant strains. The rpoB PCR products of in rifampicin-resistant strains will fail to hybridize to one or more of the wild type oligonucleotides, and will in most case show affinity to a mutant oligonucleotide. With this method, rifampicin resistance in MTB isolates can be detected within a few hours. In principle, the method can also be applied directly to clinical material and Ziehl-Neelsen (ZN) slides containing sufficient numbers of acid –fast bacilli, as has been demonstrated for spoligotyping, a PCR reverse-line blot assay to detect and type *M.tuberculosis.*

The accuracy, the high positive predictive value and the high sensitivity of the RIFO assay make it a useful tool for the early detection of MDR-TB cases (Morcillo, 2002; Senna, 2006). Even starting from early primary cultures, several important weeks can be saved with the application of the RIFO assay in comparaison with conventional laboratory methods.

The cost of the RIFO assay is 10 times lower than that of the commercially available kit to determine the RIF resistance of M.tuberculosis complex bacteria.

Later on, Mokrousov *et al* (1994) developed a home made reverse line blot (RLB) assay targeting a wide range of mutations in six genes (*rpoB*, *inhA*, *ahpC*, *rpsL*, *rrs*, *emb*B) associated with resistance to four first line anti-TB drugs (RIF, INH, SM and EMB).this macroarray based technique presnts in fact a rapid alternative to sequencing and may be recommended for use in TB reference laboratories. Its implementation can start with detection of RIF resistance as MDR marker and shoud focus on locallypredominant rpoB mutations. It is open to further development and it permits easy incorporation of new probes targeting mutations related either to newly uncovered mechanisms of resistance to the first-line anti-TB drugs, or to the second line drugs and newer anti-TB compounds. Analysis of the additional genes, such as, *gyrA* and *gyrB* (FQ resistance)*,* other *rrs* mutations in the 530 and 912 regions (SM resistance), and *rpoB* mutations outside RRDR, eventually using a multiple co-amplification/co-hubridisation approach, seems promising.

#### **7.2.4 PCR-Single Strand Conformation Polymorphism (PCR-SSCP)**

SSCP is a gel based method that can detect short stretches of DNA approximately 175–250bp in size. Small changes in a nucleotide sequence result in differences in secondary structures as well as measurable DNA mobility shifts that are detected on a non-denaturing polyacrylamide gel.

PCR-SSCP analysis is increasingly useful. To date various studies have applied PCR-SSCP to identify mutational changes associated with drug resistance in *M. tuberculosis* for frontline drugs like, RIF and INH (Kim, 2004; Cardoso, 2004; Fang, 1999; Heym, 1995; Pretorius, 1995) In particular, the development of nonisotopic PCR-SSCP analysis has simplified the procedure, enhancing its utility in routine laboratories (Kalia, 1997; Lee, 2003). However, PCR-SSCP analysis has been found to be technically demanding and not sufficiently sensitive. Furthermore SSCP conditions must be carefully evaluated since not all mutations will be detected under the same conditions. Also, results obtained with SSCP analysis should be interpreted with caution as the technique only detects mutations and gives no information on the nature of associated mutation. For example, silent mutations in the *rpo*B gene have been identified that give altered mobility patterns on SSCP analysis but have no association with RIF resistance, which underlines the need for caution in interpreting results and phenotypic or genotypic correlation (Kim, 1997).

#### **7.2.5 PCR-Restriction Fragment Length Polymorphism**

Mutations associated with resistance can be identified by digestion of amplified PCR products with a restriction enzyme that cuts at the specific polymorphic DNA sequence followed by gel electrophoresis. Since not all mutations result in the gain or loss of a restriction site, general use of RFLP to screen for mutations associated with drug resistance is limited (Victor *et al*., 2002).

#### **7.2.6 Microarrays**

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

the *rpoB* gene sequence with the most frequently occurring mutations in rifampicin-resistant strains. The rpoB PCR products of in rifampicin-resistant strains will fail to hybridize to one or more of the wild type oligonucleotides, and will in most case show affinity to a mutant oligonucleotide. With this method, rifampicin resistance in MTB isolates can be detected within a few hours. In principle, the method can also be applied directly to clinical material and Ziehl-Neelsen (ZN) slides containing sufficient numbers of acid –fast bacilli, as has been demonstrated for spoligotyping, a PCR reverse-line blot assay to detect and type

The accuracy, the high positive predictive value and the high sensitivity of the RIFO assay make it a useful tool for the early detection of MDR-TB cases (Morcillo, 2002; Senna, 2006). Even starting from early primary cultures, several important weeks can be saved with the

The cost of the RIFO assay is 10 times lower than that of the commercially available kit to

Later on, Mokrousov *et al* (1994) developed a home made reverse line blot (RLB) assay targeting a wide range of mutations in six genes (*rpoB*, *inhA*, *ahpC*, *rpsL*, *rrs*, *emb*B) associated with resistance to four first line anti-TB drugs (RIF, INH, SM and EMB).this macroarray based technique presnts in fact a rapid alternative to sequencing and may be recommended for use in TB reference laboratories. Its implementation can start with detection of RIF resistance as MDR marker and shoud focus on locallypredominant rpoB mutations. It is open to further development and it permits easy incorporation of new probes targeting mutations related either to newly uncovered mechanisms of resistance to the first-line anti-TB drugs, or to the second line drugs and newer anti-TB compounds. Analysis of the additional genes, such as, *gyrA* and *gyrB* (FQ resistance)*,* other *rrs* mutations in the 530 and 912 regions (SM resistance), and *rpoB* mutations outside RRDR, eventually using a multiple

SSCP is a gel based method that can detect short stretches of DNA approximately 175–250bp in size. Small changes in a nucleotide sequence result in differences in secondary structures as well as measurable DNA mobility shifts that are detected on a non-denaturing

PCR-SSCP analysis is increasingly useful. To date various studies have applied PCR-SSCP to identify mutational changes associated with drug resistance in *M. tuberculosis* for frontline drugs like, RIF and INH (Kim, 2004; Cardoso, 2004; Fang, 1999; Heym, 1995; Pretorius, 1995) In particular, the development of nonisotopic PCR-SSCP analysis has simplified the procedure, enhancing its utility in routine laboratories (Kalia, 1997; Lee, 2003). However, PCR-SSCP analysis has been found to be technically demanding and not sufficiently sensitive. Furthermore SSCP conditions must be carefully evaluated since not all mutations will be detected under the same conditions. Also, results obtained with SSCP analysis should be interpreted with caution as the technique only detects mutations and gives no information on the nature of associated mutation. For example, silent mutations in the *rpo*B gene have been identified that give altered mobility patterns on SSCP analysis but have no

application of the RIFO assay in comparaison with conventional laboratory methods.

determine the RIF resistance of M.tuberculosis complex bacteria.

co-amplification/co-hubridisation approach, seems promising.

**7.2.4 PCR-Single Strand Conformation Polymorphism (PCR-SSCP)** 

*M.tuberculosis.*

polyacrylamide gel.

Although technically a solid-phase-type hybridization assay, microarrays, also known as biochips, have been proposed as new molecular methods for detecting drug resistance in *M. tuberculosis*. They are based on the hybridization of DNA obtained from clinical samples to oligonucleotides immobilized in a solid support, such as miniaturized glass slides. They have been mainly used to detect resistance to rifampicin. In a recent evaluation using oligonucleotide microarrays for analysis of drug resistance, Gryadunov et al. (2005) has detected over 95% rifampicin resistant and almost 80% isoniazid resistant *M. tuberculosis* isolates within 12 h in a sample of drug-resistant isolates and clinical samples. For the time being and due to the high cost involved, the use of microarrays for detecting drug resistance in TB is still beyond the reach of most clinical mycobacteriology laboratories.

#### **7.2.7 Line-probe assays**

This technology involves a series of steps including extraction of DNA from mycobacterial isolates or directly from clinical specimens, polymerase chain reaction (PCR) amplification of nucleic acid sequences, hybridization of labeled PCR products with oligonucleotide probes immobilized on a strip, and colorimetric development that allows for lines to be seen where the probes are located (hence, the term ''line-probe'' assay) .

In 2008, the WHO issued a policy statement regarding the molecular line-probe assays for use in detection on *M. tuberculosis* and for detection of drug resistance.

The first line-probe assay was the INNO-LiPA Rif TB (Innogenetics NV) (Rossau, 1997). The results of clinical evaluations of the assay indicated that it accurately detects resistance to rifampin, but some of the evaluations showed that the assay was less sensitive for the detection of *M. tuberculosis* complex. A meta-analysis performed in 2005 showed that 12 of 14 published studies showed 95% of sensitivity with a specificity of 100% but that, in studies in which the assay was applied to clinical specimens, the sensitivity ranged from 80% to 100%. One study showed that the assay could be used successfully in a resource-poor setting, compared with reference laboratories (Wilson, 2011).

The second line-probe assay was the GenoType MTBDR® developed by Hain Lifescience. Initially, this assay was developed as the GenoType MTBDR assay, but early evaluations showed that the assay did not detect drug resistance to a satisfactory degree, detecting only 90%–95% of isolates with rifampin or low-level isoniazid resistance. The assay was eventually modified to include detection of more rpoB and inhA mutations under the name

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

Opportunies and Challenges in Morocco 495

Fig. 3. Schematic representation of results obtained with the GenoType MTBDRplus® test

This assay, usually called Xpert MTB/RIF, is a self-contained and fully automated technological platform that integrates sputum processing, DNA extraction and amplification, TB and MDR-TB diagnosis. This assay, which was CE (Conformité Européenne) marked in 2009, avoids many of the pitfalls of conventional nucleic acid amplification tests (Blakemore, 2010; Helb, 2010; Boehme, 2010; Hillemann, 2011). This is largely because the device is self-enclosed and, therefore, requires less sophisticated infrastructure in terms of laboratory facilities, user training, and supply chain


management.

I: Strain resistant only for isoniazid

**7.2.8 Cartridge-based automated NAAT** 

R+I: Strain resistant for both rifampicin and isoniazid,

GenoType MTBDRplus®. Although 2 evaluations of the new assay showed improvement of the detection of isoniazid resistance (Hillemann, 2007), 3 other evaluations showed that detection of isoniazid resistance remained suboptimal (particularly for strains with lowlevel resistance) (Helb, 2010).

GenoType MTBDRplus® is a DNA strip test that allows simultaneous molecular identification of tuberculosis and the most common genetic mutations causing resistance to rifampicin and isoniazid. This technology can diagnose MDR-TB directly from smearpositive sputum samples, providing results in just five hours - an enormous improvement on the 1 to 2 months needed for conventional DST (Figure 3).

A meta-analysis performed in 2008 confirmed these findings; the assay shows high sensitivity and specificity for detecting resistance to rifampin but variable results for detecting resistance to isoniazid (Barnard, 2008, Ling, 2008). A second meta-analysis performed the subsequent year showed similar results, although in this analysis, the pooled sensitivity of the GenoType MTBDRplus assay showed better sensitivity for detecting isoniazid resistance (Wilson, 2011). Overall, this genotyping kit is a rapid, manual nucleic acid amplification test (NAAT) and capable of both detecting *M. tuberculosis* and carrying out drug susceptibility testing (DST), however, results of studies' evaluations indicate that the assay is of limited use with smear-negative specimens and that detection of isoniazid resistance is more variable but generally lower than detection of rifampin resistance (Barnard, 2008).

More recently, another version, named GenoType MTBDRsl®, was developed to detect resistance to fluoroquinolones, ethambutol, kanamycin, amikacin and capreomycin. Two evaluations of this assay have shown promising but variable results for detection of resistance to the second-line drugs (Brossier, 2010).

As with any new diagnostic test, the impact of GenoType MTBDRplus® Assay will depend on the reproducibility of the results under actual field conditions, the manner and extent of their introduction, the strength of the laboratories and the degree to which access to appropriate therapy follows access to diagnosis.

LPA is now being rolled out by FIND and partners in 27 high MDR-TB burden countries under the EXPANDx-TB programme, supported by UNITAID.

Currently, the implementation of the GenoType MTBDRplus® as a tool for detection of MDR / XDR strains is under evaluation in two regions of Morocco and is supported by WHO under EMRO – COMSTECH projects. The first assay concerned Rabat and neighbours cities which is the highest rate of pulmonary tuberculosis in Morocco and containing the major and the reference hospital of pulmonary diseases. In fact, this hospital receives patients from hall the country and concentrate patients infected with MDR and XDR TB strains

The second assay concerned Tangier and neighbours cities, with a high prevalence of tuberculosis. Moreover, Tangier area has been the theater of a new phenomenon that can affect the epidemiology of tuberculosis in this region; immigration from the sub-Saharan region to attend Europe.

We believe that the implementation of the GenoType MTBDRplus® will be of a great interest to enhance drug resistant TB diagnosis and therefore to improve TB management in Morocco.

GenoType MTBDRplus®. Although 2 evaluations of the new assay showed improvement of the detection of isoniazid resistance (Hillemann, 2007), 3 other evaluations showed that detection of isoniazid resistance remained suboptimal (particularly for strains with low-

GenoType MTBDRplus® is a DNA strip test that allows simultaneous molecular identification of tuberculosis and the most common genetic mutations causing resistance to rifampicin and isoniazid. This technology can diagnose MDR-TB directly from smearpositive sputum samples, providing results in just five hours - an enormous improvement

A meta-analysis performed in 2008 confirmed these findings; the assay shows high sensitivity and specificity for detecting resistance to rifampin but variable results for detecting resistance to isoniazid (Barnard, 2008, Ling, 2008). A second meta-analysis performed the subsequent year showed similar results, although in this analysis, the pooled sensitivity of the GenoType MTBDRplus assay showed better sensitivity for detecting isoniazid resistance (Wilson, 2011). Overall, this genotyping kit is a rapid, manual nucleic acid amplification test (NAAT) and capable of both detecting *M. tuberculosis* and carrying out drug susceptibility testing (DST), however, results of studies' evaluations indicate that the assay is of limited use with smear-negative specimens and that detection of isoniazid resistance is more variable but generally lower than detection of rifampin resistance

More recently, another version, named GenoType MTBDRsl®, was developed to detect resistance to fluoroquinolones, ethambutol, kanamycin, amikacin and capreomycin. Two evaluations of this assay have shown promising but variable results for detection of

As with any new diagnostic test, the impact of GenoType MTBDRplus® Assay will depend on the reproducibility of the results under actual field conditions, the manner and extent of their introduction, the strength of the laboratories and the degree to which access to

LPA is now being rolled out by FIND and partners in 27 high MDR-TB burden countries

Currently, the implementation of the GenoType MTBDRplus® as a tool for detection of MDR / XDR strains is under evaluation in two regions of Morocco and is supported by WHO under EMRO – COMSTECH projects. The first assay concerned Rabat and neighbours cities which is the highest rate of pulmonary tuberculosis in Morocco and containing the major and the reference hospital of pulmonary diseases. In fact, this hospital receives patients from hall the

The second assay concerned Tangier and neighbours cities, with a high prevalence of tuberculosis. Moreover, Tangier area has been the theater of a new phenomenon that can affect the epidemiology of tuberculosis in this region; immigration from the sub-Saharan

We believe that the implementation of the GenoType MTBDRplus® will be of a great interest to enhance drug resistant TB diagnosis and therefore to improve TB management in

on the 1 to 2 months needed for conventional DST (Figure 3).

resistance to the second-line drugs (Brossier, 2010).

appropriate therapy follows access to diagnosis.

under the EXPANDx-TB programme, supported by UNITAID.

country and concentrate patients infected with MDR and XDR TB strains

level resistance) (Helb, 2010).

(Barnard, 2008).

region to attend Europe.

Morocco.

Fig. 3. Schematic representation of results obtained with the GenoType MTBDRplus® test - : Sensitive strain

R+I: Strain resistant for both rifampicin and isoniazid,

I: Strain resistant only for isoniazid

#### **7.2.8 Cartridge-based automated NAAT**

This assay, usually called Xpert MTB/RIF, is a self-contained and fully automated technological platform that integrates sputum processing, DNA extraction and amplification, TB and MDR-TB diagnosis. This assay, which was CE (Conformité Européenne) marked in 2009, avoids many of the pitfalls of conventional nucleic acid amplification tests (Blakemore, 2010; Helb, 2010; Boehme, 2010; Hillemann, 2011). This is largely because the device is self-enclosed and, therefore, requires less sophisticated infrastructure in terms of laboratory facilities, user training, and supply chain management.

Detection of *Mycobacterium tuberculosis* and Drug Resistance:

especially in extra-pulmonary tuberculosis.

of *Mycobacterium tuberculosis* (CDC, 2009).

NAA tests can provide substantial savings

optimum patient care);

isolation if TB is excluded);

infectiousness and improved patient outcomes.

infection control (respiratory isolation) decisions.

transmission, focused contact investigations).

Casablanca, Rabat and Tangier.

interventions.

Opportunies and Challenges in Morocco 497

laboratory confirmation of TB can lead to earlier treatment initiation, better patient care and outcomes, greater opportunities to interrupt transmission and improved public health

In Morocco, TB biological diagnosis is mainly limited to conventional techniques. These techniques (AFB smear, bacterial culture on solid and liquid media) are well established across the network of tuberculosis control laboratories. However, 2 laboratories, considered as reference laboratories, are authorized to perform drug susceptibility tests to antibiotics.

In some cases, molecular techniques based on PCR amplification are used to detect TB

Moreover, molecular techniques are well used in research and epidemiology (RFLP, spoligotyping, MIRUs,…). Currently, manual and automated techniques based on molecular approaches are achievable in some well-equipped laboratories in major centrs in

As in many developing countries, where the incidence of tuberculosis is declining slowly, molecular based tests should be introduced in the national program of Morocco; or at least tested for a period to verify the impact of such tests on the annual incidence of tuberculosis. In response to a request from many researchers in the field of tuberculosis, an advisory council should be set up to evaluate the place of NAA tests in the national program; against tuberculosis in Morocco. This committee must include TB clinicians, TB control officials; laboratory directors or supervisors from small, medium and large public health laboratories, hospital laboratories, and commercial laboratories and representatives from the Regional Training and Medical Consultation Centres. This suggestion is based on general observations for the use of NAA test to diagnose tuberculosis infections and drug resistance

NAA testing has significant potential added value for clinicians and TB control officials.

a. Earlier diagnosis leads to earlier initiation of treatment, a reduced period of

b. Earlier notification of TB cases to public health authorities should permit public health interventions sooner and may engage a TB expert sooner in the care of the TB patient. c. Earlier detection of *M. tuberculosis* bacteria in sputum specimens can facilitate earlier

d. Earlier differentiation of AFB-smear positive specimens containing *M. tuberculosis* from those containing other Mycobacteria can eliminate unnecessary contact investigations.

iii. for the hospital (less potential for nosocomial transmission, briefer period of respiratory

iv. for the public health program (interrupt transmission earlier, abbreviated period for

i. for the patient (earlier diagnosis, improved outcomes, reduced health-care costs); ii. for the health care provider (definitive diagnosis earlier, focused diagnostic testing,

According to WHO guidelines, the tested antibiotics are: INH, RIF, SM and EMB.

The assay has recently undergone performance evaluation to detect TB and rifampicin resistant strains on respiratory specimens (Wilson, 2011; Helb, 2010; Boehm, 2010; Rachow, 2011) as well as on non-respiratory samples (Rachow, 2011). The sensitivity of the test to detect smear-positive isolates reached 100%. However, the sensitivity for the identification of smear-negative culture positive isolates ranged from 71 to 72.5%. Xpert MTB/RIF test was shown to be specific in 99.2% of patients without TB. Moreover, its usefulness in detecting sputum smear and culture negative patients needs further studies (Helb, 2010; Marlowe, 2011).

On the other hand, and as compared with phenotypic drug-susceptibility testing, Xpert® MTB/RIF test was shown to be highly sensitive for detecting rifampin resistance, correctly identifying 97.6% of rifampicin-resistant isolates and 98.1% of rifampicin susceptible isolates (Boehme, 2010).

The main disadvantage of this system is the inability to test for and detect isoniazid resistance. Other potential disadvantages are related to the cost and a continued need for adequate laboratory infrastructure and training of personnel.

As for GenoType MTBDRplus® test, the Xpert MTB/RIF Assay will depend on the reproducibility of the results, the manner and extent of their introduction, the strength of the laboratories and the degree to which access to appropriate therapy follows access to diagnosis.

Thus, due to its high sensitivity and specificity, time consuming and the facility to use, the Wold Health Organisation endorsed in December 2010 Xpert® MTB/RIF technology and released a roadmap to guide its rapid adoption in endemic countries. In this context, the Xpert® MTB/RIF technology will be implemented in different cities of Morocco and should be used as the initial diagnostic test in individuals suspected of having MDR-TB or HIVassociated TB and may be considered as a follow-on test to microscopy in settings where MDR-TB or HIV is of lesser concern, especially in further testing of smear-negative specimens.

#### **8. The place of molecular approaches in the TB management policy in Morocco**

Guidelines for the use of nucleic acid amplification (NAA) tests for the diagnosis of tuberculosis (TB) were published in 1996 (CDC, 1996) and updated in 2000 (CDC, 2000). Since then, NAA testing has become a routine procedure in many institutions for the diagnosis of TB, because NAA tests can rapidly and reliably detect *Mycobacterium tuberculosis* bacteria directly in a specimen one or more weeks earlier than culture. Earlier laboratory confirmation of TB can lead to earlier treatment initiation, better patient care and outcomes, greater opportunities to interrupt transmission and improved public health interventions.

Guidelines for the use of nucleic acid amplification (NAA) tests for the diagnosis of tuberculosis (TB) were published in 1996 (CDC, 1996) and updated in 2000 (CDC, 2000). Since then, NAA testing has become a routine procedure in many institutions for the diagnosis of TB, because NAA tests can rapidly and reliably detect *Mycobacterium tuberculosis* bacteria directly in a specimen one or more weeks earlier than culture. Earlier

The assay has recently undergone performance evaluation to detect TB and rifampicin resistant strains on respiratory specimens (Wilson, 2011; Helb, 2010; Boehm, 2010; Rachow, 2011) as well as on non-respiratory samples (Rachow, 2011). The sensitivity of the test to detect smear-positive isolates reached 100%. However, the sensitivity for the identification of smear-negative culture positive isolates ranged from 71 to 72.5%. Xpert MTB/RIF test was shown to be specific in 99.2% of patients without TB. Moreover, its usefulness in detecting sputum smear and culture negative patients needs further studies (Helb, 2010;

On the other hand, and as compared with phenotypic drug-susceptibility testing, Xpert® MTB/RIF test was shown to be highly sensitive for detecting rifampin resistance, correctly identifying 97.6% of rifampicin-resistant isolates and 98.1% of rifampicin susceptible isolates

The main disadvantage of this system is the inability to test for and detect isoniazid resistance. Other potential disadvantages are related to the cost and a continued need for

As for GenoType MTBDRplus® test, the Xpert MTB/RIF Assay will depend on the reproducibility of the results, the manner and extent of their introduction, the strength of the laboratories and the degree to which access to appropriate therapy follows access to

Thus, due to its high sensitivity and specificity, time consuming and the facility to use, the Wold Health Organisation endorsed in December 2010 Xpert® MTB/RIF technology and released a roadmap to guide its rapid adoption in endemic countries. In this context, the Xpert® MTB/RIF technology will be implemented in different cities of Morocco and should be used as the initial diagnostic test in individuals suspected of having MDR-TB or HIVassociated TB and may be considered as a follow-on test to microscopy in settings where MDR-TB or HIV is of lesser concern, especially in further testing of smear-negative

**8. The place of molecular approaches in the TB management policy in** 

Guidelines for the use of nucleic acid amplification (NAA) tests for the diagnosis of tuberculosis (TB) were published in 1996 (CDC, 1996) and updated in 2000 (CDC, 2000). Since then, NAA testing has become a routine procedure in many institutions for the diagnosis of TB, because NAA tests can rapidly and reliably detect *Mycobacterium tuberculosis* bacteria directly in a specimen one or more weeks earlier than culture. Earlier laboratory confirmation of TB can lead to earlier treatment initiation, better patient care and outcomes, greater opportunities to interrupt transmission and improved public health

Guidelines for the use of nucleic acid amplification (NAA) tests for the diagnosis of tuberculosis (TB) were published in 1996 (CDC, 1996) and updated in 2000 (CDC, 2000). Since then, NAA testing has become a routine procedure in many institutions for the diagnosis of TB, because NAA tests can rapidly and reliably detect *Mycobacterium tuberculosis* bacteria directly in a specimen one or more weeks earlier than culture. Earlier

adequate laboratory infrastructure and training of personnel.

Marlowe, 2011).

(Boehme, 2010).

diagnosis.

specimens.

**Morocco** 

interventions.

laboratory confirmation of TB can lead to earlier treatment initiation, better patient care and outcomes, greater opportunities to interrupt transmission and improved public health interventions.

In Morocco, TB biological diagnosis is mainly limited to conventional techniques. These techniques (AFB smear, bacterial culture on solid and liquid media) are well established across the network of tuberculosis control laboratories. However, 2 laboratories, considered as reference laboratories, are authorized to perform drug susceptibility tests to antibiotics. According to WHO guidelines, the tested antibiotics are: INH, RIF, SM and EMB.

In some cases, molecular techniques based on PCR amplification are used to detect TB especially in extra-pulmonary tuberculosis.

Moreover, molecular techniques are well used in research and epidemiology (RFLP, spoligotyping, MIRUs,…). Currently, manual and automated techniques based on molecular approaches are achievable in some well-equipped laboratories in major centrs in Casablanca, Rabat and Tangier.

As in many developing countries, where the incidence of tuberculosis is declining slowly, molecular based tests should be introduced in the national program of Morocco; or at least tested for a period to verify the impact of such tests on the annual incidence of tuberculosis. In response to a request from many researchers in the field of tuberculosis, an advisory council should be set up to evaluate the place of NAA tests in the national program; against tuberculosis in Morocco. This committee must include TB clinicians, TB control officials; laboratory directors or supervisors from small, medium and large public health laboratories, hospital laboratories, and commercial laboratories and representatives from the Regional Training and Medical Consultation Centres. This suggestion is based on general observations for the use of NAA test to diagnose tuberculosis infections and drug resistance of *Mycobacterium tuberculosis* (CDC, 2009).

NAA testing has significant potential added value for clinicians and TB control officials.


NAA tests can provide substantial savings


Detection of *Mycobacterium tuberculosis* and Drug Resistance:

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For achieving this Implementation, that we believe will have benefic on the national program for TB in Morocco, research projects are needed to:


#### **9. Conclusion**

Truly rapid results for drug susceptibility tests are particularly important in the management of drug-resistant tuberculosis. Thus, the improvements made by molecular biology suggest that effective diagnostic strategies could be used to identify patients with or without MDR and even XDR TB strains. However, these techniques suffer from the problem that the genetic basis of resistance is not fully understood for any TB drug for all *M. tuberculosis* isolates.

Conventional techniques will yet be considered as a gold and reference methods. The currently available molecular methods may aid in rapid detection of mutations associated with drug resistance, but the test results must always be confirmed by phenotypic methods.

#### **10. References**


For achieving this Implementation, that we believe will have benefic on the national

1. Conduct operational, translational, and implementation research for developing, evaluating, and selecting the most effective and efficient NAA testing algorithms for

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4. Develop and evaluate optimal specimen collection, transport, and processing methods. 5. Determine the influences of specimen quality and quantity on NAA test performance. 6. Characterize the ability of NAA tests to detect *M. tuberculosis* bacteria in mixed

7. Develop, evaluate and deploy NAA tests with improved performance and ease-of-use.

Truly rapid results for drug susceptibility tests are particularly important in the management of drug-resistant tuberculosis. Thus, the improvements made by molecular biology suggest that effective diagnostic strategies could be used to identify patients with or without MDR and even XDR TB strains. However, these techniques suffer from the problem that the genetic basis of resistance is not fully understood for any TB drug for all *M.* 

Conventional techniques will yet be considered as a gold and reference methods. The currently available molecular methods may aid in rapid detection of mutations associated with drug resistance, but the test results must always be confirmed by phenotypic methods.

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**24** 

*Sri Lanka* 

**Pattern of Circulating** *Mycobacterium* 

Historically tuberculosis (TB) has been and today it remains the leading cause of mortality in adults due to an infectious agent, and with the increasing prevalence of TB's resistance to the drugs of choice the problem posed by TB to public health should not be underestimated. The strain classification or sub typing is important epidemiologically for recognizing outbreaks of infection, detecting the cross transmission of nosocomial pathogens, determining the source of infection, recognizing the particularly virulent strains of organisms and in monitoring vaccination programs (Olive and Bean, 1999). Sub typing has been accomplished by a number of different approaches, and if the method to be successful it has to satisfy several criteria. Mainly all the organisms within a species must be type able by the method used and secondly, it must have high differentiation power and the

Molecular epidemiology makes use of the genetic diversity within strains of infectious organisms to track the transmission of these organisms in human populations and to evaluate the host and parasite - specific risk factors for disease spread. In the past, efforts to type strains of *M. tuberculosis* in human hosts were hampered by the lack of a strain specific immune response and by an apparent lack of genetic polymorphism in the organism. However advances in the field of TB research paved the way in developing molecular techniques which

It is thought that the progenitor of the *M. tuberculosis* complex comprising *M. tuberculosis, M. bovis*, *M. bovis BCG, M .microti* and *M. africanum* arose from a soil bacterium and that the human bacillus may have been derived from the bovine form following the domestication of cattle. The complex lacks inter strain genetic diversity, and nucleotide changes are very rare. The H37Rv strain of *M. tuberculosis* was isolated in 1905, and since then it has found extensive, world - wide application in biomedical research. It has retained full virulence in animal models of tuberculosis and also susceptible to drugs and amenable to genetic manipulation (Cole et al., 1998). The genome comprises 4,411,529 base pairs (bp) with a G + C content of 65.6%, which is relatively constant throughout the genome and the genome is

methodologies should be reproducible (Olive and Bean, 1999).

**1.1 Molecular epidemiology of tuberculosis 1.1.1 Organization and sequence of genome** 

allowed the identification and tracking of individual strains of *M*. *tuberculosis*.

**1. Introduction** 

*tuberculosis* **Strains in Sri Lanka** 

Dhammika Magana-Arachchi *Institute of Fundamental Studies* 


## **Pattern of Circulating** *Mycobacterium tuberculosis* **Strains in Sri Lanka**

Dhammika Magana-Arachchi *Institute of Fundamental Studies Sri Lanka* 

#### **1. Introduction**

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

Zhang Y., C. V., and Jacobs W.R.Jr. 2005. Mechanisms of Drug Resistance in Mycobaterium

Zhang, Y., Garbe, T., and Young, D. 1993. Transformation with katG restores isoniazid-

Zhang. Y., B. Heym, B. Allen, D. Young and S. Cole. 1992. The catalase-peroxidase gene and isoniazid resistance of *Mycobacterium tuberculosis*. Nature 358: 591–593. Zimhony O, C. Vilcheze, and W.R.,Jr Jacobs. 2004. Characterization of Mycobacterium

C. e.) ASM Press, Washington, D.C., pp. 115-140.

concentrations. Mol. Microbiol. 8: 521–524.

gene. J. Bacteriol. 186: 4051–4055.

tuberculosis. In Tuberculosis and the Tubercle Bacillus, Vol. Chapter 8 (Ed, al., S. T.

sensitivity in *Mycobacterium tuberculosis* isolates resistant to a range of drug

smegmatis expressing the *Mycobacterium tuberculosis* fatty acid synthase I (fas1)

Historically tuberculosis (TB) has been and today it remains the leading cause of mortality in adults due to an infectious agent, and with the increasing prevalence of TB's resistance to the drugs of choice the problem posed by TB to public health should not be underestimated. The strain classification or sub typing is important epidemiologically for recognizing outbreaks of infection, detecting the cross transmission of nosocomial pathogens, determining the source of infection, recognizing the particularly virulent strains of organisms and in monitoring vaccination programs (Olive and Bean, 1999). Sub typing has been accomplished by a number of different approaches, and if the method to be successful it has to satisfy several criteria. Mainly all the organisms within a species must be type able by the method used and secondly, it must have high differentiation power and the methodologies should be reproducible (Olive and Bean, 1999).

Molecular epidemiology makes use of the genetic diversity within strains of infectious organisms to track the transmission of these organisms in human populations and to evaluate the host and parasite - specific risk factors for disease spread. In the past, efforts to type strains of *M. tuberculosis* in human hosts were hampered by the lack of a strain specific immune response and by an apparent lack of genetic polymorphism in the organism. However advances in the field of TB research paved the way in developing molecular techniques which allowed the identification and tracking of individual strains of *M*. *tuberculosis*.

#### **1.1 Molecular epidemiology of tuberculosis**

#### **1.1.1 Organization and sequence of genome**

It is thought that the progenitor of the *M. tuberculosis* complex comprising *M. tuberculosis, M. bovis*, *M. bovis BCG, M .microti* and *M. africanum* arose from a soil bacterium and that the human bacillus may have been derived from the bovine form following the domestication of cattle. The complex lacks inter strain genetic diversity, and nucleotide changes are very rare. The H37Rv strain of *M. tuberculosis* was isolated in 1905, and since then it has found extensive, world - wide application in biomedical research. It has retained full virulence in animal models of tuberculosis and also susceptible to drugs and amenable to genetic manipulation (Cole et al., 1998). The genome comprises 4,411,529 base pairs (bp) with a G + C content of 65.6%, which is relatively constant throughout the genome and the genome is

Pattern of Circulating *Mycobacterium tuberculosis* Strains in Sri Lanka 513

(Cohn and O'Brien, 1998). Das et al., 1995 studied the utility of a standardized IS*6110* / *Pvu* II, RFLP typing method for distinguishing between isolates of *M. tuberculosis*, and assess the potential for distinguishing between relapse versus re infection rates. They concluded that despite the high frequency of single and zero band isolates in the population, the discriminatory power of RFLP typing with IS*6110* is sufficiently high to be useful for clinical and epidemiological studies (Das et al*.*, 1995). Sahadevan et al., 1995 observed that *M. tuberculosis* isolates obtained from patients' sputa on diagnosis and during follow-up after short-course chemotherapy in Madras, had either no copy or only a single copy of IS*6110*. This posed a limitation for DNA fingerprinting with an IS*6110*-based probe to determine the frequency of exogenous re infection versus that of endogenous reactivation. They overcame this limitation by using an alternate probe, the direct-repeat element. Comparison of preand post treatment isolates by direct-repeat restriction fragment length polymorphism analysis indicated a high degree of endogenous reactivation among patients who had relapses after the successful completion of chemotherapy (Sahadevan et al*.*, 1995). van Duin et al., investigated an episode of laboratory cross contamination using IS*6110* RFLP typing and it proved to be a useful tool to trace the source of contamination (van Duin et al*.*, 1998).

Spoligotyping is a technique based on the polymorphism of the direct repeat (DR) locus present in *M. tuberculosis* DNA. The DR sequences are composed of multiple 36bp copies, interspersed by short non repetitive sequences (Kmerbeek et al., 1997). However, the spacer sequences between any two specific direct repeats are conserved among strains (Kmerbeek et al., 1997). The presence or absence of each non repetitive sequence creates a pattern for each strain when analyzed by spoligotyping. A database of spoligotypes of *M. tuberculosis* has been created (Sola et al., 2001) containing the global distribution and phylogenetic analysis of worldwide spoligotypes and this database is useful for comparing the patterns found in different regions of the world, enabling a better understanding of the dynamics of disease distribution. Simultaneous use of RFLP and spoligotyping methods increases understanding the epidemiological factors that facilitates the spread of tuberculosis inside a country. Studies have revealed that both transmission and reactivation are contributing to the spread of tuberculosis in the world. Another study result highlighted the importance of molecular epidemiology studies of tuberculosis in insufficiently studied regions with a high

TB burden, in order to uncover the true extent of genetic diversity of the pathogen.

Another genotyping technique which becoming popular is mycobacterial interspersed repeat units (MIRU). MIRU genotyping categorizes the number and size of the repeats in each of 12 independent MIRUs, with the use of a polymerase-chain-reaction (PCR) assay, followed by gel electrophoresis to categorize the number and size of repeats in 12 independent loci, each of which has a unique repeated sequence (Supply et al., 2001). The discriminatory power of MIRU genotyping is almost as great as that of IS*6110*-based genotyping (Supply et al., 2001). Unlike IS*6110*-based genotyping, MIRU analysis can be automated and can thus be used to evaluate large numbers of strains, yielding intrinsically digital results that can be easily catalogued on a computer data base (Supply et al., 2001). A Web site has been set up so that a worldwide data base of MIRU patterns can be created

**1.2.2 Spoligotyping** 

**1.2.3 MIRU** 

rich in repetitive DNA, and several regions show higher than average G + C content which corresponds to sequences belonging to a large gene family that includes the polymorphic G + C rich sequences (PGRSs) (Cole et al., 1998).

#### **1.1.2 Insertion sequences and prophages**

The genome of H37Rv contains 16 copies of the insertion sequence IS*6110*, and 6 copies of the insertion element IS*1081* (Philipp et al., 1996). Cole et al in 1998 found another 32 different insertion sequences, and of the 13E12 family of repetitive sequences which exhibit some of the characteristics of mobile genetic elements. Most of the insertion sequences belong to the IS*3* and IS*256* families but six of them form a new group (Cole et al*.,* 1998). Most of the insertion sequences in *M. tuberculosis* H37Rv are inserted in intergenic or non-coding regions (close to tRNA genes), with clustering suggesting the existence of insertion hot-spots that prevent genes from being inactivated (Cole et al*.*, 1998). The chromosomal distribution of the insertion sequences is informative and shows selection against insertions in the quadrant encompassing *oriC* and an overrepresentation in the direct repeat region that contains the prototype IS*6110* (Cole et al., 1998). According to Cole et al, two prophages, phiRv1 and phiRv2 (both – 10 kb in length) have been detected in the H37Rv genome sequence and only IS*1532* exhibited significant variability indicating that most of the prophages and insertion sequences are currently stable (Cole et al*.*, 1998).

#### **1.1.3 Insertion sequence IS***6110*

Insertion sequence IS*6110* is a 1361 base pair long sequence that was detected exclusively in members of the TB complex and differences of only a few nucleotides have been detected between the sequenced copies. The sequence is flanked by two 28 base pair repeats and has two open reading frames (ORF) that show homology with genes coding for putative transposases of other elements of the IS3 family, which are typical features of mobile elements (Suffys et al., 1997). Though the transposition of IS*6110* has not been experimentally demonstrated in *M. tuberculosis*, mobility of IS*986* has been observed. The number of copies of IS*6110* present in the genome is species and strain specific (Suffys et al., 1997) and most strains of *M. tuberculosis* carry between 8 to 15 copies in different positions of the genome. Several single copy strains of *M. tuberculosis* had been reported, while other studies found some *M. tuberculosis* isolates, which were devoid of the IS*6110* sequence.

#### **1.2 Genotyping methods**

Mycobacterial strain typing by means of molecular methods has become an important instrument for tuberculosis surveillance, control and prevention (van Soolingen, 1998).

#### **1.2.1 Restriction Fragment Length Polymorphism (RFLP)**

Among DNA fingerprinting methods which restriction fragment length polymorphism (RFLP) typing is the most common method used has permitted novel investigations of the epidemiology and pathogenesis of tuberculosis. The use of IS*6110*, an insertion sequence which is present in *Mycobacterium tuberculosis*, is generally considered to be the gold standard for tuberculosis molecular epidemiology studies (van Embden et al., 1993), but other molecular typing techniques could be used as adjuncts in selected circumstances (Cohn and O'Brien, 1998). Das et al., 1995 studied the utility of a standardized IS*6110* / *Pvu* II, RFLP typing method for distinguishing between isolates of *M. tuberculosis*, and assess the potential for distinguishing between relapse versus re infection rates. They concluded that despite the high frequency of single and zero band isolates in the population, the discriminatory power of RFLP typing with IS*6110* is sufficiently high to be useful for clinical and epidemiological studies (Das et al*.*, 1995). Sahadevan et al., 1995 observed that *M. tuberculosis* isolates obtained from patients' sputa on diagnosis and during follow-up after short-course chemotherapy in Madras, had either no copy or only a single copy of IS*6110*. This posed a limitation for DNA fingerprinting with an IS*6110*-based probe to determine the frequency of exogenous re infection versus that of endogenous reactivation. They overcame this limitation by using an alternate probe, the direct-repeat element. Comparison of preand post treatment isolates by direct-repeat restriction fragment length polymorphism analysis indicated a high degree of endogenous reactivation among patients who had relapses after the successful completion of chemotherapy (Sahadevan et al*.*, 1995). van Duin et al., investigated an episode of laboratory cross contamination using IS*6110* RFLP typing and it proved to be a useful tool to trace the source of contamination (van Duin et al*.*, 1998).

#### **1.2.2 Spoligotyping**

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

rich in repetitive DNA, and several regions show higher than average G + C content which corresponds to sequences belonging to a large gene family that includes the polymorphic G

The genome of H37Rv contains 16 copies of the insertion sequence IS*6110*, and 6 copies of the insertion element IS*1081* (Philipp et al., 1996). Cole et al in 1998 found another 32 different insertion sequences, and of the 13E12 family of repetitive sequences which exhibit some of the characteristics of mobile genetic elements. Most of the insertion sequences belong to the IS*3* and IS*256* families but six of them form a new group (Cole et al*.,* 1998). Most of the insertion sequences in *M. tuberculosis* H37Rv are inserted in intergenic or non-coding regions (close to tRNA genes), with clustering suggesting the existence of insertion hot-spots that prevent genes from being inactivated (Cole et al*.*, 1998). The chromosomal distribution of the insertion sequences is informative and shows selection against insertions in the quadrant encompassing *oriC* and an overrepresentation in the direct repeat region that contains the prototype IS*6110* (Cole et al., 1998). According to Cole et al, two prophages, phiRv1 and phiRv2 (both – 10 kb in length) have been detected in the H37Rv genome sequence and only IS*1532* exhibited significant variability indicating that most of the prophages and insertion

Insertion sequence IS*6110* is a 1361 base pair long sequence that was detected exclusively in members of the TB complex and differences of only a few nucleotides have been detected between the sequenced copies. The sequence is flanked by two 28 base pair repeats and has two open reading frames (ORF) that show homology with genes coding for putative transposases of other elements of the IS3 family, which are typical features of mobile elements (Suffys et al., 1997). Though the transposition of IS*6110* has not been experimentally demonstrated in *M. tuberculosis*, mobility of IS*986* has been observed. The number of copies of IS*6110* present in the genome is species and strain specific (Suffys et al., 1997) and most strains of *M. tuberculosis* carry between 8 to 15 copies in different positions of the genome. Several single copy strains of *M. tuberculosis* had been reported, while other studies found some *M. tuberculosis* isolates, which were devoid of the IS*6110* sequence.

Mycobacterial strain typing by means of molecular methods has become an important instrument for tuberculosis surveillance, control and prevention (van Soolingen, 1998).

Among DNA fingerprinting methods which restriction fragment length polymorphism (RFLP) typing is the most common method used has permitted novel investigations of the epidemiology and pathogenesis of tuberculosis. The use of IS*6110*, an insertion sequence which is present in *Mycobacterium tuberculosis*, is generally considered to be the gold standard for tuberculosis molecular epidemiology studies (van Embden et al., 1993), but other molecular typing techniques could be used as adjuncts in selected circumstances

**1.2.1 Restriction Fragment Length Polymorphism (RFLP)** 

+ C rich sequences (PGRSs) (Cole et al., 1998).

**1.1.2 Insertion sequences and prophages** 

sequences are currently stable (Cole et al*.*, 1998).

**1.1.3 Insertion sequence IS***6110* 

**1.2 Genotyping methods** 

Spoligotyping is a technique based on the polymorphism of the direct repeat (DR) locus present in *M. tuberculosis* DNA. The DR sequences are composed of multiple 36bp copies, interspersed by short non repetitive sequences (Kmerbeek et al., 1997). However, the spacer sequences between any two specific direct repeats are conserved among strains (Kmerbeek et al., 1997). The presence or absence of each non repetitive sequence creates a pattern for each strain when analyzed by spoligotyping. A database of spoligotypes of *M. tuberculosis* has been created (Sola et al., 2001) containing the global distribution and phylogenetic analysis of worldwide spoligotypes and this database is useful for comparing the patterns found in different regions of the world, enabling a better understanding of the dynamics of disease distribution. Simultaneous use of RFLP and spoligotyping methods increases understanding the epidemiological factors that facilitates the spread of tuberculosis inside a country. Studies have revealed that both transmission and reactivation are contributing to the spread of tuberculosis in the world. Another study result highlighted the importance of molecular epidemiology studies of tuberculosis in insufficiently studied regions with a high TB burden, in order to uncover the true extent of genetic diversity of the pathogen.

#### **1.2.3 MIRU**

Another genotyping technique which becoming popular is mycobacterial interspersed repeat units (MIRU). MIRU genotyping categorizes the number and size of the repeats in each of 12 independent MIRUs, with the use of a polymerase-chain-reaction (PCR) assay, followed by gel electrophoresis to categorize the number and size of repeats in 12 independent loci, each of which has a unique repeated sequence (Supply et al., 2001). The discriminatory power of MIRU genotyping is almost as great as that of IS*6110*-based genotyping (Supply et al., 2001). Unlike IS*6110*-based genotyping, MIRU analysis can be automated and can thus be used to evaluate large numbers of strains, yielding intrinsically digital results that can be easily catalogued on a computer data base (Supply et al., 2001). A Web site has been set up so that a worldwide data base of MIRU patterns can be created

Pattern of Circulating *Mycobacterium tuberculosis* Strains in Sri Lanka 515

In the first study RFLP analysis of the 131 isolates by Southern blotting and DNA hybridization with IS*6110* was performed according to the standard fingerprinting method (van Embden et al., 1993). In the second study DNA fingerprinting using IS*6110* as a probe was performed for 120 *M*. *tuberculosis* strains according to standardized protocol of van Embden et al., 1993. The software GeneDirectory from SYNGENE was used to compare RFLP hybridization patterns, using the Dice Coefficient of similarity and the UPGMA algorithm, with a 1% band position tolerance. A total of 110 *M. tuberculosis* isolates were subjected to standard spoligotyping and the spoligo patterns were analyzed using MS Excel data sheets and grouped together for any similarity. The data was further analyzed by

Genomic DNA (5 g) per each sample / isolate (obtained from above procedure) was digested with restriction enzyme *Pvu* II in a final volume of 25 l as recommended by the

The *Pvu* II digested chromosomal DNA from samples was size fractionated on 1% agarose gels. Along with the samples a DNA marker ( cleaved *Hind* III/PhiX 174-*Hae* III) and DNA

Gel was soaked in HCl (0.25 M) for 20 minutes, followed by several volumes of gel soak I solution (1.5 M NaCl, 0.5 M NaOH) for 30 minutes and next in several volumes of gel soak II solution (1 M Tris HCl, pH 8.0, 1.5 M NaCl) for 1 hr at room temperature with constant

The IS*6110* – specific DNA probe of 245 bp was amplified by PCR using the oligonucleotide primers INS-1 (5'-CGTGAGGGCATCGAGGTGGC-3') and INS- 2 (5'-GCGTAGGCGTCGGT GAC AAA-3') corresponding to bp 631 to 650 and 856 to 875 which are based on the

The probe was labelled by using a direct nucleic acid labelling and detection kit (ECL, Amersham, RPN 3001) according to the manufacturer's instructions. DNA to be labelled was diluted to a concentration of 10 ng/l using water. DNA was denatured by heating for 5 min in a boiling water bath. The DNA sample was immediately cooled on ice for 5 min and centrifuged (2 sec, 12000 g). An equivalent volume of DNA labelling reagent was added to the cooled DNA and mixed thoroughly. An equivalent volume of glutaraldehyde was added to the solution, and spun briefly in a micro centrifuge. Next the DNA was incubated for 10 min at 37 0C. The labelled DNA probe was stored in 50% (v/v) glycerol at –20 0C until

**2.2 IS***6110* **– RFLP and spoligotyping** 

comparing with the SPOTCLUST data base

manufacturer (Pharmacia Biotech).

**2.2.1.3 Southern blotting** 

used.

**2.2.1.1 Digestion of chromosomal DNA for RFLP** 

**2.2.1.2 Separation of DNA fragments by electrophoresis** 

shaking. The gel was then Southern blotted onto nylon filters.

**2.2.1.4 Preparation of DNA probe by PCR** 

positions of the IS*6110* sequence, respectively.

**2.2.1.5 Preparation of the labelled probe for RFLP** 

from the reference *M. tuberculosis* strain cleaved with *Pvu* II was included.

**2.2.1 IS***6110* **– RFLP** 

(Supply et al., 2001). MIRU genotyping is technically simpler than IS*6110*-based genotyping and can be applied directly to *M. tuberculosis* cultures without DNA purification (Barnes and Cave, 2003).

Sri Lanka is an island in the Indian Ocean, located in Southern Asia, southeast of India, in a strategic location near major Indian Ocean sea lanes. Although India accounts for nearly one-third of the global TB burden, with a population of 19 million Sri Lanka is among the low TB prevalence countries in the region. Only a few studies have been performed in Sri Lanka applying modern molecular DNA fingerprint techniques that are able to directly trace routes of TB transmission e.g., to analyze the epidemiology of resistant *M. tuberculosis*  strains in Sri Lanka.

Therefore, this chapter focus on Molecular Epidemiology of Tuberculosis including the two studies conducted in Sri Lanka on *M. tuberculosis* isolates with IS*6110* RFLP assays and spoligotyping.

#### **2. Genotyping by RFLP & spoligotyping**

#### **2.1 Study population**

One hundred and seventy sputum smear positive TB patients admitted for re-treatment to Chest Hospital, Welisara, Sri Lanka were enrolled for the first study. There were 24 patients among the chest clinic attendees having a history of imprisonment before being diagnosed as having TB (ex-prisoners). The study population consisted of 131 culture positive re treatment TB patients. Remaining patients were excluded, as their cultures were negative. The second study consisted of 121 mycobacterial isolates collected from first visit patients attending the Central Chest Clinic, Kandy, Sri Lanka who were positive for acid fast bacilli on direct examination of sputum by Ziehl- Neelsen stain and/or culture and/or had radiological findings suggestive of TB.

#### **2.1.1 Specimen processing, culture and isolation of genomic DNA from mycobacteria**

Sputum samples were decontaminated using the standard Sodium hydroxide – sodium citrate – N acetyl – L – cysteine method and were inoculated on Lowenstein-Jenson (LJ) medium and Middle brook 7H-10 agar medium to isolate the *M. tuberculosis* strains. The strains of *M. tuberculosis* obtained from these media were used for antibiotic sensitivity testing and RFLP analysis. Isolation of Genomic DNA was performed using standard protocols.

#### **2.1.2 Antibiotic sensitivity testing**

In the first study 12 drugs were tested and the criterion for resistance was based on the 1% survival level of the organism in comparison with a control medium without the drug. Resistance was defined as survival of the tubercle bacilli at the following drug concentrations (µg/ml); isoniazid (H), 0.2; rifampin (R), 1.0; streptomycin (S), 2.0; ethambutol (E), 5.0; pyrazinamide (Z), 25.0; *p*-amino salicylic acid (PASER), 2.0; ethionamide (Et), 5.0; cycloserine (Cs), 30.0; kanamycin (Km), 5.0; viomycin (Vm), 5.0; ciprofloxacin (Cx), 2.0 and rifabutin (Rb), 2.0 (Magana Arachchi et al., 2010). For the second study isoniazid and rifampin were tested.

#### **2.2 IS***6110* **– RFLP and spoligotyping**

In the first study RFLP analysis of the 131 isolates by Southern blotting and DNA hybridization with IS*6110* was performed according to the standard fingerprinting method (van Embden et al., 1993). In the second study DNA fingerprinting using IS*6110* as a probe was performed for 120 *M*. *tuberculosis* strains according to standardized protocol of van Embden et al., 1993. The software GeneDirectory from SYNGENE was used to compare RFLP hybridization patterns, using the Dice Coefficient of similarity and the UPGMA algorithm, with a 1% band position tolerance. A total of 110 *M. tuberculosis* isolates were subjected to standard spoligotyping and the spoligo patterns were analyzed using MS Excel data sheets and grouped together for any similarity. The data was further analyzed by comparing with the SPOTCLUST data base

#### **2.2.1 IS***6110* **– RFLP**

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

(Supply et al., 2001). MIRU genotyping is technically simpler than IS*6110*-based genotyping and can be applied directly to *M. tuberculosis* cultures without DNA purification (Barnes and

Sri Lanka is an island in the Indian Ocean, located in Southern Asia, southeast of India, in a strategic location near major Indian Ocean sea lanes. Although India accounts for nearly one-third of the global TB burden, with a population of 19 million Sri Lanka is among the low TB prevalence countries in the region. Only a few studies have been performed in Sri Lanka applying modern molecular DNA fingerprint techniques that are able to directly trace routes of TB transmission e.g., to analyze the epidemiology of resistant *M. tuberculosis* 

Therefore, this chapter focus on Molecular Epidemiology of Tuberculosis including the two studies conducted in Sri Lanka on *M. tuberculosis* isolates with IS*6110* RFLP assays and

One hundred and seventy sputum smear positive TB patients admitted for re-treatment to Chest Hospital, Welisara, Sri Lanka were enrolled for the first study. There were 24 patients among the chest clinic attendees having a history of imprisonment before being diagnosed as having TB (ex-prisoners). The study population consisted of 131 culture positive re treatment TB patients. Remaining patients were excluded, as their cultures were negative. The second study consisted of 121 mycobacterial isolates collected from first visit patients attending the Central Chest Clinic, Kandy, Sri Lanka who were positive for acid fast bacilli on direct examination of sputum by Ziehl- Neelsen stain and/or culture and/or had

**2.1.1 Specimen processing, culture and isolation of genomic DNA from mycobacteria**  Sputum samples were decontaminated using the standard Sodium hydroxide – sodium citrate – N acetyl – L – cysteine method and were inoculated on Lowenstein-Jenson (LJ) medium and Middle brook 7H-10 agar medium to isolate the *M. tuberculosis* strains. The strains of *M. tuberculosis* obtained from these media were used for antibiotic sensitivity testing and RFLP

In the first study 12 drugs were tested and the criterion for resistance was based on the 1% survival level of the organism in comparison with a control medium without the drug. Resistance was defined as survival of the tubercle bacilli at the following drug concentrations (µg/ml); isoniazid (H), 0.2; rifampin (R), 1.0; streptomycin (S), 2.0; ethambutol (E), 5.0; pyrazinamide (Z), 25.0; *p*-amino salicylic acid (PASER), 2.0; ethionamide (Et), 5.0; cycloserine (Cs), 30.0; kanamycin (Km), 5.0; viomycin (Vm), 5.0; ciprofloxacin (Cx), 2.0 and rifabutin (Rb), 2.0 (Magana Arachchi et al., 2010). For the second study isoniazid and

analysis. Isolation of Genomic DNA was performed using standard protocols.

Cave, 2003).

strains in Sri Lanka.

**2.1 Study population** 

**2. Genotyping by RFLP & spoligotyping** 

radiological findings suggestive of TB.

**2.1.2 Antibiotic sensitivity testing** 

rifampin were tested.

spoligotyping.

#### **2.2.1.1 Digestion of chromosomal DNA for RFLP**

Genomic DNA (5 g) per each sample / isolate (obtained from above procedure) was digested with restriction enzyme *Pvu* II in a final volume of 25 l as recommended by the manufacturer (Pharmacia Biotech).

#### **2.2.1.2 Separation of DNA fragments by electrophoresis**

The *Pvu* II digested chromosomal DNA from samples was size fractionated on 1% agarose gels. Along with the samples a DNA marker ( cleaved *Hind* III/PhiX 174-*Hae* III) and DNA from the reference *M. tuberculosis* strain cleaved with *Pvu* II was included.

#### **2.2.1.3 Southern blotting**

Gel was soaked in HCl (0.25 M) for 20 minutes, followed by several volumes of gel soak I solution (1.5 M NaCl, 0.5 M NaOH) for 30 minutes and next in several volumes of gel soak II solution (1 M Tris HCl, pH 8.0, 1.5 M NaCl) for 1 hr at room temperature with constant shaking. The gel was then Southern blotted onto nylon filters.

#### **2.2.1.4 Preparation of DNA probe by PCR**

The IS*6110* – specific DNA probe of 245 bp was amplified by PCR using the oligonucleotide primers INS-1 (5'-CGTGAGGGCATCGAGGTGGC-3') and INS- 2 (5'-GCGTAGGCGTCGGT GAC AAA-3') corresponding to bp 631 to 650 and 856 to 875 which are based on the positions of the IS*6110* sequence, respectively.

#### **2.2.1.5 Preparation of the labelled probe for RFLP**

The probe was labelled by using a direct nucleic acid labelling and detection kit (ECL, Amersham, RPN 3001) according to the manufacturer's instructions. DNA to be labelled was diluted to a concentration of 10 ng/l using water. DNA was denatured by heating for 5 min in a boiling water bath. The DNA sample was immediately cooled on ice for 5 min and centrifuged (2 sec, 12000 g). An equivalent volume of DNA labelling reagent was added to the cooled DNA and mixed thoroughly. An equivalent volume of glutaraldehyde was added to the solution, and spun briefly in a micro centrifuge. Next the DNA was incubated for 10 min at 37 0C. The labelled DNA probe was stored in 50% (v/v) glycerol at –20 0C until used.

Pattern of Circulating *Mycobacterium tuberculosis* Strains in Sri Lanka 517

incubated in 1:4000 diluted streptavidin-peroxidase conjugate: (2.5 l streptavidinperoxidase conjugate in 10 ml of 2x SSPE/ 0.5% SDS for 45-60 min in a sealed bag). Next membrane was washed twice in 250 ml 2x SSPE/ 0.5% SDS for 10 min at 42 0C. Then membrane was rinsed twice in 250 ml 2x SSPE for 5 min at room temperature. For chemiluminescence detection of hybridizing DNA the membrane was incubated for 1 min in 20 ml ECL detection liquid. Membrane was covered with a Saran-wrap and was exposed to an X-ray film overnight at room temperature. Finally, the X-ray film was developed using

In the first study the persistence of *M. tuberculosis* strains in a population was examined. To estimate the degree of transmission of TB within the general population (Colombo district) and among the prison population, analysis of the RFLP data were carried out in three ways: (I) determination of the degree of clustering of matching DNA types as a measure of transmission within the general population (ii) determination of the clustering and matching DNA types among the prisoners and (iii) degree of clustering and matching types among

The study showed that the majority of circulating *M. tuberculosis* strains in Sri Lanka belongs to a limited number of families, but the degree of IS*6110* DNA polymorphism among strains was high. Dendogram analysis showed 41 distinct IS*6110* banding patterns (Magana Arachchi et al., 2000). A close relationship between prison isolates and those from the general population was observed in this study (Magana Arachchi et al., 2000). Of the 20 strains isolated from prisoners, none of the strains displayed identical fingerprints (Magana Arachchi et al., 2000). In bacterial isolates of prisoners and ex-prisoners from the general population, there were two strains, which had identical banding patterns, while there were clear similarities between several isolates (Magana Arachchi et al., 2000). Comparative analysis of the study populations, observed five pairs showing identical banding patterns. One pair had a strain from prisoner and the other ex prisoner while another pair had identical banding patterns between an exprisoner and a patient from general population (Magana Arachchi et al., 2000). This indicates the spread of TB between prisoners and general population. Analysis of the data showed that ex-prisoners contributed to a substantial population of TB patients in the general population (Magana Arachchi, 2001). Therefore persons entering prison, carry the risk of being exposed to TB and when they leave could potentially carry the TB bacillus (Magana Arachchi, 2001). Reactivation of the latent disease among some will result in many new cases for many years to come. Compounding the tragedy is the fact that the prison is a perfect environment to produce drug resistant strains (MDR TB). Most prisoners in Sri Lanka do not receive follow up medical treatment, as they are lost for further follow up after discharge from prison. Inconsistent drug supplies can lead to strains of TB that are resistant to drugs. Thus inmates when released to their home communities pose a risk to public health as well as to themselves. Therefore

the prisoners , ex-prisoners and the patients from the general population.

continuity of medical care after release should take place in such instances.

Previous studies showed that *M. tuberculosis* strains carrying one or few IS*6110* copies are often difficult to differentiate by IS*6110* standard RFLP analysis because of a site specific preference for insertion of the IS element. Therefore to further differentiate the strains other

Kodak developer (1 min) and fixer (3 min).

**3.1 Pattern of TB transmission in first study** 

**3. Transmission of tuberculosis** 

#### **2.2.1.6 Hybridization and detection**

The nylon filter was pre hybridized with hybridization buffer (0.125 ml/cm2) in a sealed plastic bag for 1 hour at 42 0C. Labelled probe was mixed with the hybridization buffer, and was added to the solution containing the filter. The nylon filter was hybridized overnight at 42 0C with shaking. Next the hybridized filter was removed from the plastic bag and placed in a clean plastic box and the filter was washed twice (2x10 min) with the pre warmed (55 0C) primary wash buffer at 55 0C. Then the filter was placed in a clean plastic box and washed twice with the secondary wash buffer for 5 min at room temperature on a shaking platform**.** 

Next the filters were treated with detection reagents as per manufacturers instructions in a dark room and then exposed to Kodak XAR-5 film for overnight at room temperature. The nylon filters were stored under moist conditions at 4 0C for further use.

#### **2.2.2 Spoligotyping**

Spoligotyping was carried out as previously described by Kmerbeek et al., 1997.

#### **2.2.2.1 Preparation of the membrane containing the spacer-oligonucleotides**

Standard spacer oligonucleotides (n=43) were diluted to the optimized concentrations in 150 l 500 mM NaHCO3, pH 8.4. Next Biodyne C membrane was activated by 10 min incubation in 10 ml freshly prepared 16% (w/v) 1-ethyl -3-(3-dimethyl aminopropyl) carbodiimide (EDAC) in demineralized water, in a rolling bottle at room temperature. Membrane was rinsed with water for 2 min, placed on the mini blotter, and filled the slots with diluted oligonucleotides. Next membrane was incubated for 1 min at room temperature and then oligonucleotide solutions were removed by aspiration. Next blot was incubated in 100 mM NaOH for 10 min in a sealed bag to in activate the membrane. Membrane was washed in 250 ml 2x SSPE/ 0.1%SDS for 5 min at 60 0C and then in 100 ml 20 mM EDTA, pH 8.0 for 15 min at room temperature. Membrane was stored at 4 0C until used.

#### **2.2.2.2 PCR for DR**

PCR was performed with primers Dra (5'-GGTTTTGGGTCTGACGAC-3') (biotinylated 3'end) and Drb (5'-CCGAGAGGGGACGGAAAC-3'). The PCR reaction contained 10 ng of DNA, 1U of *Taq* DNA polymerase, 20 pmol of each primer and 200 m dNTPs. The cycling parameters were 3 min at 96 0C, followed by 1 min at 96 0C, 1 min at 55 0C and 30 sec at 72 0C for 30 cycles.

#### **2.2.2.3 Hybridization with PCR product and detection**

Membrane was washed in 250 ml 2x SSPE/ 0.1% SDS for 5 min at 60 0C and was placed in the mini blotter in a way that the slots were perpendicular to the line pattern of the applied oligonucleotides. 20 l of the PCR product was added into 150 l 2x SSPE/0.1% SDS and the diluted product was denatured by heating for 10 min at 100 0C and was immediately cooled on ice. Next residual fluid was removed from the slots and the slots were filled with diluted PCR product and incubated for 1hr at 60 0C. Next samples were removed and the membrane was washed twice in 250 ml 2x SSPE/ 0.5% SDS for 10 min at 60 0C. Membrane was placed in a sealed bag and allowed it cool to prevent inactivation of the peroxidase. Membrane was incubated in 1:4000 diluted streptavidin-peroxidase conjugate: (2.5 l streptavidinperoxidase conjugate in 10 ml of 2x SSPE/ 0.5% SDS for 45-60 min in a sealed bag). Next membrane was washed twice in 250 ml 2x SSPE/ 0.5% SDS for 10 min at 42 0C. Then membrane was rinsed twice in 250 ml 2x SSPE for 5 min at room temperature. For chemiluminescence detection of hybridizing DNA the membrane was incubated for 1 min in 20 ml ECL detection liquid. Membrane was covered with a Saran-wrap and was exposed to an X-ray film overnight at room temperature. Finally, the X-ray film was developed using Kodak developer (1 min) and fixer (3 min).

#### **3. Transmission of tuberculosis**

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

The nylon filter was pre hybridized with hybridization buffer (0.125 ml/cm2) in a sealed plastic bag for 1 hour at 42 0C. Labelled probe was mixed with the hybridization buffer, and was added to the solution containing the filter. The nylon filter was hybridized overnight at 42 0C with shaking. Next the hybridized filter was removed from the plastic bag and placed in a clean plastic box and the filter was washed twice (2x10 min) with the pre warmed (55 0C) primary wash buffer at 55 0C. Then the filter was placed in a clean plastic box and washed twice with the secondary wash buffer for 5 min at room temperature on a shaking

Next the filters were treated with detection reagents as per manufacturers instructions in a dark room and then exposed to Kodak XAR-5 film for overnight at room temperature. The

Standard spacer oligonucleotides (n=43) were diluted to the optimized concentrations in 150 l 500 mM NaHCO3, pH 8.4. Next Biodyne C membrane was activated by 10 min incubation in 10 ml freshly prepared 16% (w/v) 1-ethyl -3-(3-dimethyl aminopropyl) carbodiimide (EDAC) in demineralized water, in a rolling bottle at room temperature. Membrane was rinsed with water for 2 min, placed on the mini blotter, and filled the slots with diluted oligonucleotides. Next membrane was incubated for 1 min at room temperature and then oligonucleotide solutions were removed by aspiration. Next blot was incubated in 100 mM NaOH for 10 min in a sealed bag to in activate the membrane. Membrane was washed in 250 ml 2x SSPE/ 0.1%SDS for 5 min at 60 0C and then in 100 ml 20 mM EDTA, pH 8.0 for 15 min

PCR was performed with primers Dra (5'-GGTTTTGGGTCTGACGAC-3') (biotinylated 3'end) and Drb (5'-CCGAGAGGGGACGGAAAC-3'). The PCR reaction contained 10 ng of DNA, 1U of *Taq* DNA polymerase, 20 pmol of each primer and 200 m dNTPs. The cycling parameters were 3 min at 96 0C, followed by 1 min at 96 0C, 1 min at 55 0C and 30 sec at 72

Membrane was washed in 250 ml 2x SSPE/ 0.1% SDS for 5 min at 60 0C and was placed in the mini blotter in a way that the slots were perpendicular to the line pattern of the applied oligonucleotides. 20 l of the PCR product was added into 150 l 2x SSPE/0.1% SDS and the diluted product was denatured by heating for 10 min at 100 0C and was immediately cooled on ice. Next residual fluid was removed from the slots and the slots were filled with diluted PCR product and incubated for 1hr at 60 0C. Next samples were removed and the membrane was washed twice in 250 ml 2x SSPE/ 0.5% SDS for 10 min at 60 0C. Membrane was placed in a sealed bag and allowed it cool to prevent inactivation of the peroxidase. Membrane was

nylon filters were stored under moist conditions at 4 0C for further use.

at room temperature. Membrane was stored at 4 0C until used.

**2.2.2.3 Hybridization with PCR product and detection** 

Spoligotyping was carried out as previously described by Kmerbeek et al., 1997. **2.2.2.1 Preparation of the membrane containing the spacer-oligonucleotides** 

**2.2.1.6 Hybridization and detection** 

platform**.** 

**2.2.2 Spoligotyping** 

**2.2.2.2 PCR for DR** 

0C for 30 cycles.

#### **3.1 Pattern of TB transmission in first study**

In the first study the persistence of *M. tuberculosis* strains in a population was examined. To estimate the degree of transmission of TB within the general population (Colombo district) and among the prison population, analysis of the RFLP data were carried out in three ways: (I) determination of the degree of clustering of matching DNA types as a measure of transmission within the general population (ii) determination of the clustering and matching DNA types among the prisoners and (iii) degree of clustering and matching types among the prisoners , ex-prisoners and the patients from the general population.

The study showed that the majority of circulating *M. tuberculosis* strains in Sri Lanka belongs to a limited number of families, but the degree of IS*6110* DNA polymorphism among strains was high. Dendogram analysis showed 41 distinct IS*6110* banding patterns (Magana Arachchi et al., 2000). A close relationship between prison isolates and those from the general population was observed in this study (Magana Arachchi et al., 2000). Of the 20 strains isolated from prisoners, none of the strains displayed identical fingerprints (Magana Arachchi et al., 2000). In bacterial isolates of prisoners and ex-prisoners from the general population, there were two strains, which had identical banding patterns, while there were clear similarities between several isolates (Magana Arachchi et al., 2000). Comparative analysis of the study populations, observed five pairs showing identical banding patterns. One pair had a strain from prisoner and the other ex prisoner while another pair had identical banding patterns between an exprisoner and a patient from general population (Magana Arachchi et al., 2000). This indicates the spread of TB between prisoners and general population. Analysis of the data showed that ex-prisoners contributed to a substantial population of TB patients in the general population (Magana Arachchi, 2001). Therefore persons entering prison, carry the risk of being exposed to TB and when they leave could potentially carry the TB bacillus (Magana Arachchi, 2001). Reactivation of the latent disease among some will result in many new cases for many years to come. Compounding the tragedy is the fact that the prison is a perfect environment to produce drug resistant strains (MDR TB). Most prisoners in Sri Lanka do not receive follow up medical treatment, as they are lost for further follow up after discharge from prison. Inconsistent drug supplies can lead to strains of TB that are resistant to drugs. Thus inmates when released to their home communities pose a risk to public health as well as to themselves. Therefore continuity of medical care after release should take place in such instances.

Previous studies showed that *M. tuberculosis* strains carrying one or few IS*6110* copies are often difficult to differentiate by IS*6110* standard RFLP analysis because of a site specific preference for insertion of the IS element. Therefore to further differentiate the strains other

Pattern of Circulating *Mycobacterium tuberculosis* Strains in Sri Lanka 519

for the treatment failure in that patient (Magana Arachchi, et al unpublished data). In RFLP analysis all three strains did not produce any banding pattern with IS*6110* confirming their

The epidemiological analysis of TB using IS*6110* is based on the observation that the polymorphism of IS*6110* RFLP patterns among unrelated clinical isolates is high, where as epidemiologically related *M. tuberculosis* strains show identical or similar (one band variation) finger prints (Barnes and Cave, 2003). In this study RFLP analysis was successfully carried out to differentiate *Mycobacterium tuberculosis* complex from mycobacteria other than tuberculosis in 100 of 122 isolates from the first visit patients. A high degree of DNA polymorphism in both banding patterns and number of copies of IS*6110* among strains were observed. None of the isolates had an identical banding pattern except for the three strains with a single copy of IS*6110*. The number of IS*6110* DNA containing *Pvu* II fragments in strains varied between 1 and 17 indicating that these strains contain 1 to 17 copies of the IS*6110* element (Magana Arachchi et al., 2011). Table 2 summarizes the number of IS copies found in the strains that were investigated among the study group. Strains containing a single copy of IS*6110* were predominant among the study population (12) and except for three strains, the location of the bands in fingerprints were different and therefore the location of IS*6110* elements in the chromosomal DNA. Therefore *M. tuberculosis* strains carrying one or few IS*6110* copies were differentiated without difficulty (Magana Arachchi et al., 2011). In this study 52% of the isolates had five or less

Fig. 1. IS*6110* DNA finger prints of *M. tuberculosis* isolates from study population of Kandy

species variation.

**3.1.1.1 RFLP analysis** 

genetic markers such as polymorphic rich GC repetitive sequence (PGRS) and direct repeats (DR) have been used (van Soolingen et al., 1998). In the first study for DNA fingerprinting restriction enzyme *Pvu* II was used to cleave the chromosomal DNA of the mycobacterial strains (Magana Arachchi, 2001). The enzyme cleaves the 1.35 – kb IS*6110* element at a single site. In the first study the 541 bp DNA probe used for the hybridization corresponds to a piece of the IS*6110* element and the *Pvu* II site is located within that region. By using this DNA probe all of the possible IS*6110* containing restriction fragments were visualised (Magana Arachchi, 2001) and when analysing the fingerprints 2 bands were considered as a single copy. Therefore *M. tuberculosis* strains carrying one or few IS*6110* copies could be differentiated by the DNA probe used (Magana Arachchi, 2001). There were 6 strains among the prisoners who had a single copy of the IS*6110* and among the general population there were 13 strains which had a single IS copy (Magana Arachchi et al., 2010). In this study 68% of the isolates had less than five copies which were similar to that of other countries in the Asian region, such as India, Malaysia, Oman and Hong Kong (Magana Arachchi et al., 2010). According to previous studies, the strains from countries with a high prevalence of TB exhibited less DNA polymorphism than do strains in countries with a low prevalence of infection (van Soolingen et al., 1998). However strains analysed in study I showed an extensive polymorphism in the banding patterns even though the numbers of copies were less.


Table 1. Comparison of the IS element copies (IS*6110*) found among the different categories from Study I

#### **3.1.1 Pattern of TB transmission in second study**

The data included in this section are based on a study conducted over a period of 2 years in which total 121 *M. tuberculosis* isolates were analyzed from the first visit (n=178) and recurrent patients (n=12) who attended the Central Chest Clinic, Kandy, for pulmonary treatment. Two of the isolates from the first visit patients in this study (who were treated as having tuberculosis in the Central Chest Clinic, Kandy) were identified as mycobacteria other than tuberculosis (MOTT) biochemically. This finding is comparable to a previous study in which mycobacterial isolates obtained from patients throughout Sri Lanka where MOTT accounted for 3.27% of the total isolates. The isolate from the recurrent tuberculosis patient was found to be a MOTT strain with rifampin resistance, which explained the reason for the treatment failure in that patient (Magana Arachchi, et al unpublished data). In RFLP analysis all three strains did not produce any banding pattern with IS*6110* confirming their species variation.

#### **3.1.1.1 RFLP analysis**

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

genetic markers such as polymorphic rich GC repetitive sequence (PGRS) and direct repeats (DR) have been used (van Soolingen et al., 1998). In the first study for DNA fingerprinting restriction enzyme *Pvu* II was used to cleave the chromosomal DNA of the mycobacterial strains (Magana Arachchi, 2001). The enzyme cleaves the 1.35 – kb IS*6110* element at a single site. In the first study the 541 bp DNA probe used for the hybridization corresponds to a piece of the IS*6110* element and the *Pvu* II site is located within that region. By using this DNA probe all of the possible IS*6110* containing restriction fragments were visualised (Magana Arachchi, 2001) and when analysing the fingerprints 2 bands were considered as a single copy. Therefore *M. tuberculosis* strains carrying one or few IS*6110* copies could be differentiated by the DNA probe used (Magana Arachchi, 2001). There were 6 strains among the prisoners who had a single copy of the IS*6110* and among the general population there were 13 strains which had a single IS copy (Magana Arachchi et al., 2010). In this study 68% of the isolates had less than five copies which were similar to that of other countries in the Asian region, such as India, Malaysia, Oman and Hong Kong (Magana Arachchi et al., 2010). According to previous studies, the strains from countries with a high prevalence of TB exhibited less DNA polymorphism than do strains in countries with a low prevalence of infection (van Soolingen et al., 1998). However strains analysed in study I showed an extensive polymorphism in the banding patterns even though the numbers of copies were

> Number of strains among ex-prisoners,

Number of strains among general population, n=106 (%)

n=24 (%)

1 6 (30) 6 (25) 13 (10.32) 2 4 (20) 5 (20.83) 11 (8.73) 3 2 (10) 5 (20.83) 24 (19.05) 4 4 (20) 2 (8.33) 22 (17.46) 5 3 (15) 4 (16.67) 20 (15.87) 6 1 (5) 2 (8.33) 10 (7.94) 7 0 (0) 0 (0) 06 (4.76)

Table 1. Comparison of the IS element copies (IS*6110*) found among the different categories

The data included in this section are based on a study conducted over a period of 2 years in which total 121 *M. tuberculosis* isolates were analyzed from the first visit (n=178) and recurrent patients (n=12) who attended the Central Chest Clinic, Kandy, for pulmonary treatment. Two of the isolates from the first visit patients in this study (who were treated as having tuberculosis in the Central Chest Clinic, Kandy) were identified as mycobacteria other than tuberculosis (MOTT) biochemically. This finding is comparable to a previous study in which mycobacterial isolates obtained from patients throughout Sri Lanka where MOTT accounted for 3.27% of the total isolates. The isolate from the recurrent tuberculosis patient was found to be a MOTT strain with rifampin resistance, which explained the reason

less.

Number of IS*6110* copies

from Study I

Number of strains among prisoners,

n= 20 (%)

**3.1.1 Pattern of TB transmission in second study** 

The epidemiological analysis of TB using IS*6110* is based on the observation that the polymorphism of IS*6110* RFLP patterns among unrelated clinical isolates is high, where as epidemiologically related *M. tuberculosis* strains show identical or similar (one band variation) finger prints (Barnes and Cave, 2003). In this study RFLP analysis was successfully carried out to differentiate *Mycobacterium tuberculosis* complex from mycobacteria other than tuberculosis in 100 of 122 isolates from the first visit patients. A high degree of DNA polymorphism in both banding patterns and number of copies of IS*6110* among strains were observed. None of the isolates had an identical banding pattern except for the three strains with a single copy of IS*6110*. The number of IS*6110* DNA containing *Pvu* II fragments in strains varied between 1 and 17 indicating that these strains contain 1 to 17 copies of the IS*6110* element (Magana Arachchi et al., 2011). Table 2 summarizes the number of IS copies found in the strains that were investigated among the study group. Strains containing a single copy of IS*6110* were predominant among the study population (12) and except for three strains, the location of the bands in fingerprints were different and therefore the location of IS*6110* elements in the chromosomal DNA. Therefore *M. tuberculosis* strains carrying one or few IS*6110* copies were differentiated without difficulty (Magana Arachchi et al., 2011). In this study 52% of the isolates had five or less

Fig. 1. IS*6110* DNA finger prints of *M. tuberculosis* isolates from study population of Kandy

Pattern of Circulating *Mycobacterium tuberculosis* Strains in Sri Lanka 521

*Mycobacterium africanum, M. bovis,* East African-Indian (EAI) Beijing, Haarlem, Latin American and Mediterranean (LAM), Central and Middle Eastern Asian (CAS), a European family X, and a default family T (Table 3). The most predominant group among the isolates of *M. tuberculosis* corresponded to Family33. In this family, only spacers 33-34 are absent and recently described clade MANU of Indian origin belongs to the same family (Magana Arachchi et al., 2011). When compared to the single publication of spoligotyping patterns from Sri Lanka similarity was observed in only five clades namely Beijing, T1, EAI5, T2 and T3 (Magana Arachchi et al., 2011). According to the analysis, bacterial strains were distributed among all three principal genetic groups PGG1, PGG2 and PGG3. Segregation of *M. tuberculosis* into 'ancestral' versus 'modern' lineages based on PGG indicates that isolates from Kandy have originated from both lineages. In the spoligotyping patterns high strain diversity was observed and except for two strains 000000000003771 (ST1) and 000000000000031(ST 585) the tested strains were not defined in the latest spoligotype data bases SpolDB4/SITVIT (Magana Arachchi et al., 2011). The cluster analysis on spoligotyping are being carried out and after completing it in due course identifying the risk factors associated with TB transmission as

Table 3. Spoligotyping – based families that observed in *M. tuberculosis* isolates in patients

with tuberculosis in Kandy by SPOTCLUST (n=110)

**Family T**

Family 33 Family36

*M. africanum*  Family 35

*M.tuberculosis*EAI1 *M. tuberculosis* Beijing

*M. tuberculosis*LAM7 *M. tuberculosis* T3 *M. bovis* – BCG *M. tuberculosis* T1 *M. microti M. tuberculosis* T2 *M. tuberculosis* CAS *M. tuberculosis* LAM8 *M. tuberculosis* Haarlem3 *M. tuberculosis* Haarlem1 *M. tuberculosis* X3 *M. tuberculosis* H37Rv *M. tuberculosis* LAM 3 *M. tuberculosis* LAM 1 *M. tuberculosis* X2 *M. tuberculosis* EAI-5 *M. tuberculosis* T4 *M. tuberculosis* Haarlem2

well as the evolution of *M. tuberculosis* in Sri Lanka could be achieved.

1 5 10 15 20 25 30 35 40 43

**Spacer** 


a = MTC */* MTb *Mycobacterium tuberculosis,* b = MOTT confirmed, c = to be identified

Table 2. IS element copies observed in *M. tuberculosis* isolates from Kandy

than five copies and pattern is similar to the previous study in which 68% was recorded from recurrent TB patients (Magana Arachchi et al., 2011).

The fingerprints of the 73 strains were subjected to similarity analysis by using the software programme GeneDirectory from SYNGENE. This study showed that the majority of circulating *M. tuberculosis* strains in Kandy belongs to a single family, but the degree of IS*6110* DNA polymorphism among strains was high. In total 71 distinct IS*6110* patterns were found with strains clustering into one main family (63) and 10 distinct strains. Within the main family three isolates were grouped into one cluster, with closely related isolates while rests of the bacterial strains (60) were grouped into one. Sub clustering pattern of the main family was interesting with total 57 bacterial strains clustering into 3 main groups with 19, 27 and 11 strains respectively (Magana Arachchi et al., 2011). Interpretation of the clustering of the isolates in the family is complex and the explanation for the high degree of polymorphism in DNA fingerprints can be due to the different origins. Without performing DNA sequencing analysis definite conclusions cannot be made whether the isolates underwent any genetic changes within a given time (Magana Arachchi et al., 2011).

#### **3.1.1.2 Spoligotyping**

In this study, the used the algorithm SPOTCLUST incorporates biological information on spoligotype evolution, without attempting to derive the full phylogeny of *M. tuberculosis* complex. A total of 110 *M. tuberculosis* isolates were analyzed by spoligotyping. When spoligo patterns were compared from SPOTCLUST which was based on the SpolDB3 model, 24 distinct families were identified including the nine major spoligotyping-based families;

**Number of IS***6110* 

a = MTC */* MTb *Mycobacterium tuberculosis,* b = MOTT confirmed, c = to be identified Table 2. IS element copies observed in *M. tuberculosis* isolates from Kandy

from recurrent TB patients (Magana Arachchi et al., 2011).

**3.1.1.2 Spoligotyping** 

than five copies and pattern is similar to the previous study in which 68% was recorded

The fingerprints of the 73 strains were subjected to similarity analysis by using the software programme GeneDirectory from SYNGENE. This study showed that the majority of circulating *M. tuberculosis* strains in Kandy belongs to a single family, but the degree of IS*6110* DNA polymorphism among strains was high. In total 71 distinct IS*6110* patterns were found with strains clustering into one main family (63) and 10 distinct strains. Within the main family three isolates were grouped into one cluster, with closely related isolates while rests of the bacterial strains (60) were grouped into one. Sub clustering pattern of the main family was interesting with total 57 bacterial strains clustering into 3 main groups with 19, 27 and 11 strains respectively (Magana Arachchi et al., 2011). Interpretation of the clustering of the isolates in the family is complex and the explanation for the high degree of polymorphism in DNA fingerprints can be due to the different origins. Without performing DNA sequencing analysis definite conclusions cannot be made whether the isolates

underwent any genetic changes within a given time (Magana Arachchi et al., 2011).

In this study, the used the algorithm SPOTCLUST incorporates biological information on spoligotype evolution, without attempting to derive the full phylogeny of *M. tuberculosis* complex. A total of 110 *M. tuberculosis* isolates were analyzed by spoligotyping. When spoligo patterns were compared from SPOTCLUST which was based on the SpolDB3 model, 24 distinct families were identified including the nine major spoligotyping-based families; *Mycobacterium africanum, M. bovis,* East African-Indian (EAI) Beijing, Haarlem, Latin American and Mediterranean (LAM), Central and Middle Eastern Asian (CAS), a European family X, and a default family T (Table 3). The most predominant group among the isolates of *M. tuberculosis* corresponded to Family33. In this family, only spacers 33-34 are absent and recently described clade MANU of Indian origin belongs to the same family (Magana Arachchi et al., 2011). When compared to the single publication of spoligotyping patterns from Sri Lanka similarity was observed in only five clades namely Beijing, T1, EAI5, T2 and T3 (Magana Arachchi et al., 2011). According to the analysis, bacterial strains were distributed among all three principal genetic groups PGG1, PGG2 and PGG3. Segregation of *M. tuberculosis* into 'ancestral' versus 'modern' lineages based on PGG indicates that isolates from Kandy have originated from both lineages. In the spoligotyping patterns high strain diversity was observed and except for two strains 000000000003771 (ST1) and 000000000000031(ST 585) the tested strains were not defined in the latest spoligotype data bases SpolDB4/SITVIT (Magana Arachchi et al., 2011). The cluster analysis on spoligotyping are being carried out and after completing it in due course identifying the risk factors associated with TB transmission as well as the evolution of *M. tuberculosis* in Sri Lanka could be achieved.


Table 3. Spoligotyping – based families that observed in *M. tuberculosis* isolates in patients with tuberculosis in Kandy by SPOTCLUST (n=110)

Pattern of Circulating *Mycobacterium tuberculosis* Strains in Sri Lanka 523

for the high degree of polymorphism in DNA fingerprints can be due to the different origins. Molecular epidemiologic studies have shown that the dynamics of the transmission of TB vary greatly geographically (Barnes and Cave, 2003). Findings of the two studies indicate the differences observed in two provinces in both banding patterns and number of copies of IS*6110* among strains with Colombo district having 0-7 IS copies while Kandy

Although the number of copies of IS*6110* can range from 0-25, population based molecular epidemiological studies report that most strains contain 8-18 copies a number sufficient to discrimination between the majority of strains (Burgos and Pym, 2002) and the findings of the two studies clearly emphasize the value of RFLP and spoligotyping in molecular epidemiology. However both studies included only culture-positive patients to enhance the possibility of typing actively transmitting strains. Although the exclusion of culture-negative cases could potentially have introduced a bias in the strain composition, due to study constraints RFLP and spoligotyping were not performed on all patients. Additionally by performing both RFLP and spoligotyping on culture positives high strain diversity was observed with a large number of small clusters, as well as a significant proportion of strains hitherto unreported in the global databases. But performing spoligotyping alone has advantageous over IS*6110* RFLP typing. As the technique needs only small amounts of DNA the test can be performed on clinical samples directly or on strains of *M. tuberculosis* shortly after their inoculation into liquid cultures (Kmerbeek et al., 1997). Presently the gold standard for molecular epidemiological studies on tuberculosis is changing towards MIRU-VNTR typing because this technique generates easily analyzed numerical results and it is less labour intensive and has a discriminative power comparable to that of IS*6110*-based

Genotyping has been used to study the transmission dynamics of TB in both developed and developing countries. However, only the developed nations are using it to guide tuberculosis control efforts (Barnes and Cave, 2003). This is the first study in Sri Lanka in which both the RFLP pattern of *M. tuberculosis* strains and the spoligotyping in a population has been examined. In this study the feasibility of establishing molecular typing methods in a developing country like Sri Lanka has been demonstrated specially in spoligotyping

Typing of *Mycobacterium tuberculosis* isolates is of great potential value for basic and epidemiological studies on tuberculosis. Results obtained from restriction fragment length polymorphism typing and spoligotyping show that the majority of circulating *Mycobacterium tuberculosis* strains in Sri Lanka belong to a limited number of families, but the degree of IS*6110* DNA polymorphism among strains were high. By using the genetic marker of IS*6110* it was possible to differentiate most of the *M. tuberculosis* isolates. The preliminary inferences from these studies plead for a more extensive analysis of the data, to study the variability of *M. tuberculosis* strains and their transmission dynamics. The goal of molecular epidemiology is to quantify the extent of ongoing transmission of infectious

having 0-17 copies of IS*6110* (Tables 1 and 2).

**4. Future use of genotyping** 

RFLP (Barnes and Cave, 2003).

without using any commercial kits.

**5. Conclusions** 

#### **3.2 Evaluation of drug susceptibility**

It has also been noted that the DNA polymorphism could be made use of to identify transmission rates of drug resistance and drug sensitive strains. RFLP typing can be carried out on primary isolates to determine drug resistance. By comparison of these isolates with the existing RFLP patterns of the drug resistance isolates the time taken for determining drug resistance may be much shorter compared to the conventional antibiotic sensitivity testing which takes more than four weeks. Genotyping also permits the evaluation of isolates with different patterns of drug susceptibility. Such an evaluation may helpful in cases in which the original organism developed resistance during or after antituberculosis therapy, the patient was reinfected with a different *M*. *tuberculosis* strain or cross contamination is suspected (Barnes and Cave, 2003). According to literature higher number of susceptible *M. tuberculosis* strains tends to be in clusters, where as only 22% of the isoniazid (INH) mono resistant strains were found to be clustered. But some studies did not find differences in clustering between susceptible and streptomycin mono-resistant strains (Soolingen et al., 1993). In the first study no difference in clustering was observed among the drug resistance and susceptible isolates, while analysis is being performed for the second study. Most studies have shown that acquisition of drug resistance of *M. tuberculosis in vivo*  did not result in any observable IS mediated genetic rearrangements. But in contrast to the findings of others the relative, instability of IS*6110* was found in one of two MDR out break strains, and also four of the nine tested IS*6110* RFLP patterns showed a minor and different alteration. According to them the transposition rate may be strongly related to the *M. tuberculosis* genotype represented. In study I there were 2 pairs of isolates, which had identical banding patterns. However the pattern of drug resistance in the two strains was different and these isolates were collected from patients coming from different districts but from same Western province. Although they come to the same hospital for treatment, the strains were unlikely to be epidemiologically related. The findings of other research showed that non-random association of IS*6110* with *M .tuberculosis* could result in false positive clustering in unselected collections of isolates.

#### **3.3 IS***6110* **as a diagnostic tool**

Among the strains tested in the first study there were two strains (one strain from general population and the other from DNA amplification studies) that lacked the IS*6110* element. In the second study among the strains tested there were 25 strains that lacked the IS*6110* element. Among these, 15 strains were confirmed as *M. tuberculosis* while three were identified as MOTT with DNA sequencing and biochemical analysis. This has implications for diagnosis of infection when IS*6110* is used as the sequence for DNA amplification.

#### **3.4 Insights into transmission of TB**

Clustered cases of TB are defined as those in which have identical or closely related genotypes with recent transmission while isolates with distinct genotypes generally represent a reactivation or infection acquired in the distant past (Barnes and Cave, 2003). However there are limitations to this concept (Barnes and Cave, 2003). Both studies showed that the majority of circulating *M. tuberculosis* strains in Sri Lanka belongs to a limited number of families, but the degree of IS*6110* DNA polymorphism among strains was high. Interpretation of the clustering of the isolates in the family is complex and the explanation for the high degree of polymorphism in DNA fingerprints can be due to the different origins. Molecular epidemiologic studies have shown that the dynamics of the transmission of TB vary greatly geographically (Barnes and Cave, 2003). Findings of the two studies indicate the differences observed in two provinces in both banding patterns and number of copies of IS*6110* among strains with Colombo district having 0-7 IS copies while Kandy having 0-17 copies of IS*6110* (Tables 1 and 2).

#### **4. Future use of genotyping**

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

It has also been noted that the DNA polymorphism could be made use of to identify transmission rates of drug resistance and drug sensitive strains. RFLP typing can be carried out on primary isolates to determine drug resistance. By comparison of these isolates with the existing RFLP patterns of the drug resistance isolates the time taken for determining drug resistance may be much shorter compared to the conventional antibiotic sensitivity testing which takes more than four weeks. Genotyping also permits the evaluation of isolates with different patterns of drug susceptibility. Such an evaluation may helpful in cases in which the original organism developed resistance during or after antituberculosis therapy, the patient was reinfected with a different *M*. *tuberculosis* strain or cross contamination is suspected (Barnes and Cave, 2003). According to literature higher number of susceptible *M. tuberculosis* strains tends to be in clusters, where as only 22% of the isoniazid (INH) mono resistant strains were found to be clustered. But some studies did not find differences in clustering between susceptible and streptomycin mono-resistant strains (Soolingen et al., 1993). In the first study no difference in clustering was observed among the drug resistance and susceptible isolates, while analysis is being performed for the second study. Most studies have shown that acquisition of drug resistance of *M. tuberculosis in vivo*  did not result in any observable IS mediated genetic rearrangements. But in contrast to the findings of others the relative, instability of IS*6110* was found in one of two MDR out break strains, and also four of the nine tested IS*6110* RFLP patterns showed a minor and different alteration. According to them the transposition rate may be strongly related to the *M. tuberculosis* genotype represented. In study I there were 2 pairs of isolates, which had identical banding patterns. However the pattern of drug resistance in the two strains was different and these isolates were collected from patients coming from different districts but from same Western province. Although they come to the same hospital for treatment, the strains were unlikely to be epidemiologically related. The findings of other research showed that non-random association of IS*6110* with *M .tuberculosis* could result in false positive

Among the strains tested in the first study there were two strains (one strain from general population and the other from DNA amplification studies) that lacked the IS*6110* element. In the second study among the strains tested there were 25 strains that lacked the IS*6110* element. Among these, 15 strains were confirmed as *M. tuberculosis* while three were identified as MOTT with DNA sequencing and biochemical analysis. This has implications for diagnosis of infection when IS*6110* is used as the sequence for DNA amplification.

Clustered cases of TB are defined as those in which have identical or closely related genotypes with recent transmission while isolates with distinct genotypes generally represent a reactivation or infection acquired in the distant past (Barnes and Cave, 2003). However there are limitations to this concept (Barnes and Cave, 2003). Both studies showed that the majority of circulating *M. tuberculosis* strains in Sri Lanka belongs to a limited number of families, but the degree of IS*6110* DNA polymorphism among strains was high. Interpretation of the clustering of the isolates in the family is complex and the explanation

**3.2 Evaluation of drug susceptibility** 

clustering in unselected collections of isolates.

**3.3 IS***6110* **as a diagnostic tool** 

**3.4 Insights into transmission of TB** 

Although the number of copies of IS*6110* can range from 0-25, population based molecular epidemiological studies report that most strains contain 8-18 copies a number sufficient to discrimination between the majority of strains (Burgos and Pym, 2002) and the findings of the two studies clearly emphasize the value of RFLP and spoligotyping in molecular epidemiology. However both studies included only culture-positive patients to enhance the possibility of typing actively transmitting strains. Although the exclusion of culture-negative cases could potentially have introduced a bias in the strain composition, due to study constraints RFLP and spoligotyping were not performed on all patients. Additionally by performing both RFLP and spoligotyping on culture positives high strain diversity was observed with a large number of small clusters, as well as a significant proportion of strains hitherto unreported in the global databases. But performing spoligotyping alone has advantageous over IS*6110* RFLP typing. As the technique needs only small amounts of DNA the test can be performed on clinical samples directly or on strains of *M. tuberculosis* shortly after their inoculation into liquid cultures (Kmerbeek et al., 1997). Presently the gold standard for molecular epidemiological studies on tuberculosis is changing towards MIRU-VNTR typing because this technique generates easily analyzed numerical results and it is less labour intensive and has a discriminative power comparable to that of IS*6110*-based RFLP (Barnes and Cave, 2003).

Genotyping has been used to study the transmission dynamics of TB in both developed and developing countries. However, only the developed nations are using it to guide tuberculosis control efforts (Barnes and Cave, 2003). This is the first study in Sri Lanka in which both the RFLP pattern of *M. tuberculosis* strains and the spoligotyping in a population has been examined. In this study the feasibility of establishing molecular typing methods in a developing country like Sri Lanka has been demonstrated specially in spoligotyping without using any commercial kits.

#### **5. Conclusions**

Typing of *Mycobacterium tuberculosis* isolates is of great potential value for basic and epidemiological studies on tuberculosis. Results obtained from restriction fragment length polymorphism typing and spoligotyping show that the majority of circulating *Mycobacterium tuberculosis* strains in Sri Lanka belong to a limited number of families, but the degree of IS*6110* DNA polymorphism among strains were high. By using the genetic marker of IS*6110* it was possible to differentiate most of the *M. tuberculosis* isolates. The preliminary inferences from these studies plead for a more extensive analysis of the data, to study the variability of *M. tuberculosis* strains and their transmission dynamics. The goal of molecular epidemiology is to quantify the extent of ongoing transmission of infectious

Pattern of Circulating *Mycobacterium tuberculosis* Strains in Sri Lanka 525

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agents and to identify host and strain specific risk factors for disease spread. Molecular methods in epidemiology require the development of both appropriate epidemiologic study design and analytical tools to yield meaningful assessments of disease transmission. Therefore the interpretation of molecular epidemiological studies should largely depend on the study question, the geographical area under study and the typing methods used to prevent the sampling bias in the molecular epidemiology of TB.

#### **6. Acknowledgement**

The research works on TB were supported by three grants RG/95/BT/08 NARESA, RG/2006/HS/07 NSF and 07-47 of NRC Sri Lanka. I am expressing my sincere gratitude to Professor Jennifer Perera and Dr. N.V. Chandrasekharan for their valuable guidance and Dr. D. Medagedara and Professor V. Thevanesam for their support in tuberculosis research and to Ms. R. P. Wanigatunge and Ms. S. Maheswaran for technical support in preparation of manuscript.

#### **7. References**


agents and to identify host and strain specific risk factors for disease spread. Molecular methods in epidemiology require the development of both appropriate epidemiologic study design and analytical tools to yield meaningful assessments of disease transmission. Therefore the interpretation of molecular epidemiological studies should largely depend on the study question, the geographical area under study and the typing methods used to

The research works on TB were supported by three grants RG/95/BT/08 NARESA, RG/2006/HS/07 NSF and 07-47 of NRC Sri Lanka. I am expressing my sincere gratitude to Professor Jennifer Perera and Dr. N.V. Chandrasekharan for their valuable guidance and Dr. D. Medagedara and Professor V. Thevanesam for their support in tuberculosis research and to Ms. R. P. Wanigatunge and Ms. S. Maheswaran for technical support in preparation of

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Das, S., Paramasivan, C.N., Lowrie, D.B., Prabhakar, R., & Narayanan, P.R. (1995). IS*6110*

Kamerbeek, J., Schouls, L., Kolk, A., Van Agterveld, M., Van Soolingen, D., Kuijper, S.,

and epidemiology. *Journal of Clinical Microbiology*, 35, 4, pp. 907-914. Magana Arachchi, D.N., Perera. J., Gamage, S., & Chandrasekharan, N.V. (2000). DNA

South India. *Tubercle and Lung Disease*, 76, 6, pp. 550-554.

Eiglmeier, K., Gas, S., Barry III, C.E., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M.A., Rajandream, M.-A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor, K., Whitehead, S., & Barrell, B.G. (1998). Deciphering the biology of *Mycobacterium* 

(RFLP) analysis for epidemiological studies of tuberculosis in developing countries.

restriction fragment length polymorphism typing of clinical isolates of *Mycobacterium tuberculosis* from patients with pulmonary tuberculosis in Madras,

Bunschoten, A., Molhuizen, H., Shaw, R., Goyal, M., & Van Embden, J. (1997). Simultaneous detection and strain differentiation of *M. tuberculosis* for diagnosis

fingerprinting of *M*. *tuberculosis* using restriction fragment length polymorphism (RFLP) with special reference to the prison population. *Proceedings of the Sri Lanka* 

prevent the sampling bias in the molecular epidemiology of TB.

Suppl. 36, pp. 54-65, ISSN 0904-1850.

*Int J Tuberc Lung Dis*, 2, 1, pp. 16-26.

349, pp. 1149-1156.

**6. Acknowledgement** 

manuscript.

**7. References** 

*Association for the Advancement of Science, 56th Annual Session*, 1391-023X, Colombo, Sri Lanka, 27th November- 1st December, p. 34.


Recommendation for a standardized methodology. *J Clin Microbiol*, 31, 2, pp. 406- 409.

**25** 

*Brazil* 

**Health Interventions to Improve** 

Fernando Rogério Pavan and Luiz Antonio Dutra *Universidade Estadual Paulista "Júlio de Mesquita Filho" Faculdade de Ciências Farmacêuticas – UNESP Araraquara* 

Jean Leandro dos Santos, Chung Man Chin, Ednir de Oliveira Vizioli,

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. According to the World Health Organization (WHO), there are an estimated of 8.8 million new cases annually -including 200.000 HIV-infected individuals- and 1.6 million deaths (WHO, 2008). Despite some reports demonstrates reduction of new cases, it is extensive in literature data showing the problem of drug-resistance and the consequence of this one to the effective treatment (FAUCI, 2008). The drug resistance can be caused by several factors such as: 1) antibiotic selective pressure characterized by inadequate selection or dosage; 2) immune status of the individuals; 3) No compliance by patients; 4) natural selection or any pre-

Nowadays, HIV co-morbidity treatment is a challenge. It has been reported that HIV is a potent risk factor to TB disease development. Some studies showed that HIV/TB coinfection increase 100 times the risk to develop TB disease when compared to people infected only with TB (HAVLIR & BARNES, 1999; PITCHENIK et al., 1988). In addition, the probability to develop drug resistance is higher in patients under HIV infection treatment

One of the first randomized studies about antitubercular drug was performed with streptomycin. This drug reduces 50% the mortality of infected patients after 6 months of treatment. However, it was observed high rate of resistance in cases of monotherapy. The combination of drugs reduces the resistance to TB drugs, therefore the current treatment of

WHO recommends the use of rifampicin (or rifabutin), isoniazid, pyrazinamide and ethambutol by two months followed by rifampicin and isoniazid for four months. The first phase of the treatment aims eliminates the bacilli in mutiplicate and semi dormant stage. The second stage called the maintenance phase aims eliminate dormant bacilli reducing the number of failures and relapses (Bisaglia, 2003). The scheme of treatment is prolonged (6

existing resistances in the infecting clone, among others (Figure 1).

tuberculosis involves multidrug therapy (Herzog, 1998).

months) contributing to no-therapy compliance by some patients.

due mainly drug related problems (Frieden et al. 1993; Gordin et al., 1996).

**1. Introduction** 

**the Medication Efficacy in** 

**Tuberculosis Treatment** 

van Soolingen, D. (1998). Utility of molecular epidemiology of tuberculosis. *Eur Respir J*, 11, 795-797.

## **Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment**

Jean Leandro dos Santos, Chung Man Chin, Ednir de Oliveira Vizioli, Fernando Rogério Pavan and Luiz Antonio Dutra *Universidade Estadual Paulista "Júlio de Mesquita Filho" Faculdade de Ciências Farmacêuticas – UNESP Araraquara Brazil* 

#### **1. Introduction**

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

van Soolingen, D. (1998). Utility of molecular epidemiology of tuberculosis. *Eur Respir J*, 11,

409.

795-797.

Recommendation for a standardized methodology. *J Clin Microbiol*, 31, 2, pp. 406-

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. According to the World Health Organization (WHO), there are an estimated of 8.8 million new cases annually -including 200.000 HIV-infected individuals- and 1.6 million deaths (WHO, 2008). Despite some reports demonstrates reduction of new cases, it is extensive in literature data showing the problem of drug-resistance and the consequence of this one to the effective treatment (FAUCI, 2008). The drug resistance can be caused by several factors such as: 1) antibiotic selective pressure characterized by inadequate selection or dosage; 2) immune status of the individuals; 3) No compliance by patients; 4) natural selection or any preexisting resistances in the infecting clone, among others (Figure 1).

Nowadays, HIV co-morbidity treatment is a challenge. It has been reported that HIV is a potent risk factor to TB disease development. Some studies showed that HIV/TB coinfection increase 100 times the risk to develop TB disease when compared to people infected only with TB (HAVLIR & BARNES, 1999; PITCHENIK et al., 1988). In addition, the probability to develop drug resistance is higher in patients under HIV infection treatment due mainly drug related problems (Frieden et al. 1993; Gordin et al., 1996).

One of the first randomized studies about antitubercular drug was performed with streptomycin. This drug reduces 50% the mortality of infected patients after 6 months of treatment. However, it was observed high rate of resistance in cases of monotherapy. The combination of drugs reduces the resistance to TB drugs, therefore the current treatment of tuberculosis involves multidrug therapy (Herzog, 1998).

WHO recommends the use of rifampicin (or rifabutin), isoniazid, pyrazinamide and ethambutol by two months followed by rifampicin and isoniazid for four months. The first phase of the treatment aims eliminates the bacilli in mutiplicate and semi dormant stage. The second stage called the maintenance phase aims eliminate dormant bacilli reducing the number of failures and relapses (Bisaglia, 2003). The scheme of treatment is prolonged (6 months) contributing to no-therapy compliance by some patients.

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 529

According to WHO new patients is recommended the treatment to receive a daily intensive phase of the two months of the isoniazid (H), rifampicin (R), pyrazinamid (P) and ethambutol (E); followed by 4 months for the maintenance phase of the H and R [2HRZE/4HR] (WHO, 2009). This treatment is highly efficient for the drug-susceptible TB patients, but, the questions is what about the latent and MDR (multi-drug resistant) or XDR

Latent tuberculosis is individuals infected with M. tuberculosis but has no active disease. Although this state not is completely clear, there are two main hypothesis to explain this condition: 1) M. tuberculosis persists in a lazy state within granulomatous lesions, but periodically recrudesces; 2) the bacterium persisting in a dormant state resides within alveolar epithelial cells in the lung apices and adipocytes (Ma et al., 2010). Epidemiologically latent infection is responsible to contaminated 1/3 of the world population, and there is no specific treatment for it (Koul et al., 2011). However H has been used as preventive therapy (IPT) has long been known to markedly reduce the risk of reactivation of latent M. tuberculosis infection, but this affirmation not is completely proved and clear (Golub et al.,

MDR-TB occurs when the TB mycobacteria are resistant to at least H, R and XDR-TB occur when the mycobacteria has the same resistant characteristic than MDR-TB plus resistant to any fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin, kanamycin, and amikacin). The resistant bacteria are selected due to mainly to failure of the treatment. Failures attributed mainly to the desistance of treatment by the patient. To understand how the bacteria become resistant is not completely understood, but various biochemical pathways to escape the lethal action of drugs can be assigned: (i) decreased intracellular accumulation of the antibiotic by an alteration of outer membrane permeability, diminished transport across the inner membrane, (ii) alteration of the target by mutation or enzymatic modification; (iii) enzymatic detoxification of the drug; and (iv) bypass of the drug target. The coexistence of several of these mechanisms in the same host can lead to MDR and XDR-TB. (Piddock et al., 2006; De Rossi et al., 2006). Another important question that needs to be highlighted is the association of these biochemical resistant pathways with the efflux pump system. Efux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic and eukaryotic cells. M. tuberculosis presents one of the largest numbers of putative drug efux pumps compared with its genome size. Antimicrobial resistance in an efflux mutant is due to one of two mechanisms: (i) expression of the efflux pump protein is increased or (ii) the protein contains an amino acid

substitution(s) that makes the protein more efficient at export (Piddock, 2006).

MDR treatment, anti-TB drugs are grouped according to efficacy, experience of use and drug class. There are five groups and only the group 1 can receive the first line drugs, all others groups will receive the second-line drugs namely: kanamycin (Km), amikacin (Am), capreomycin (Cm), streptomycin (S), Levofloxacin (Lfx), Moxifloxacin (Mfx), Ofloxacin (Ofx), para-aminosalicylic acid (PAS), cycloserine (Cs), terizidone (Trd), ethionamide (Eto), protionamide (Pto), clofazimine (Cfz), linezolid (Lzd), amoxicillin/clavulanate (Amx/Clv), thioacetazone (Thz), imipenem/cilastatin (Ipm/Cln), clarithromycin (Clr) (WHO, 2009).

TB Treatment

2008).

(extremely drug-resistant) TB treatment?

Fig. 1. Some causes of TB drug resistance.

The lack of therapy adherence is a serious problem to eliminate TB mainly in developing countries. Several aspects are related to this problem such as: adverse effects of TB drugs, long-term therapy, damage to certain organs (i.e. liver), lack of available drugs and comorbidities.

Another important aspect to be considered in tuberculosis treatment is the unavailability of new drugs. The last discovery drug to TB was rifampicin in the 60's. Despite TB drug development efforts have emerged in the last years few advances were reached. Currently, there are some drugs, obtained by molecular modification, being evaluated in clinical trials. An ideal drug against TB must possess some characteristics that include: broad spectrum of action with possibility to be use in resistant strains; adequate pharmacokinetic profile increasing the concentration in some tissues target and reduce drug-drug interaction; adequate and shorten treatment duration reducing pill burden in order to reduce numbers of pills taken increasing the patients compliance (Koul et al, 2011).

The combination of all these factors discussed above makes difficult TB treatment. Not only strategies to discovery new drugs, but rational approach to improve the use of old drugs are essential to improve the efficacy of the treatment. This chapter aiming discusses some factors to improve medication efficacy in tuberculosis treatment. Some aspects of TB such as disease development, resistance and treatment will be discussed. Furthermore therapeutical aspects such as drug-drug interaction, patient compliance and some health interventions will be present.

#### TB Treatment

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

The lack of therapy adherence is a serious problem to eliminate TB mainly in developing countries. Several aspects are related to this problem such as: adverse effects of TB drugs, long-term therapy, damage to certain organs (i.e. liver), lack of available drugs and co-

Another important aspect to be considered in tuberculosis treatment is the unavailability of new drugs. The last discovery drug to TB was rifampicin in the 60's. Despite TB drug development efforts have emerged in the last years few advances were reached. Currently, there are some drugs, obtained by molecular modification, being evaluated in clinical trials. An ideal drug against TB must possess some characteristics that include: broad spectrum of action with possibility to be use in resistant strains; adequate pharmacokinetic profile increasing the concentration in some tissues target and reduce drug-drug interaction; adequate and shorten treatment duration reducing pill burden in order to reduce numbers

The combination of all these factors discussed above makes difficult TB treatment. Not only strategies to discovery new drugs, but rational approach to improve the use of old drugs are essential to improve the efficacy of the treatment. This chapter aiming discusses some factors to improve medication efficacy in tuberculosis treatment. Some aspects of TB such as disease development, resistance and treatment will be discussed. Furthermore therapeutical aspects such as drug-drug interaction, patient compliance and some health interventions

of pills taken increasing the patients compliance (Koul et al, 2011).

Fig. 1. Some causes of TB drug resistance.

morbidities.

will be present.

According to WHO new patients is recommended the treatment to receive a daily intensive phase of the two months of the isoniazid (H), rifampicin (R), pyrazinamid (P) and ethambutol (E); followed by 4 months for the maintenance phase of the H and R [2HRZE/4HR] (WHO, 2009). This treatment is highly efficient for the drug-susceptible TB patients, but, the questions is what about the latent and MDR (multi-drug resistant) or XDR (extremely drug-resistant) TB treatment?

Latent tuberculosis is individuals infected with M. tuberculosis but has no active disease. Although this state not is completely clear, there are two main hypothesis to explain this condition: 1) M. tuberculosis persists in a lazy state within granulomatous lesions, but periodically recrudesces; 2) the bacterium persisting in a dormant state resides within alveolar epithelial cells in the lung apices and adipocytes (Ma et al., 2010). Epidemiologically latent infection is responsible to contaminated 1/3 of the world population, and there is no specific treatment for it (Koul et al., 2011). However H has been used as preventive therapy (IPT) has long been known to markedly reduce the risk of reactivation of latent M. tuberculosis infection, but this affirmation not is completely proved and clear (Golub et al., 2008).

MDR-TB occurs when the TB mycobacteria are resistant to at least H, R and XDR-TB occur when the mycobacteria has the same resistant characteristic than MDR-TB plus resistant to any fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin, kanamycin, and amikacin). The resistant bacteria are selected due to mainly to failure of the treatment. Failures attributed mainly to the desistance of treatment by the patient. To understand how the bacteria become resistant is not completely understood, but various biochemical pathways to escape the lethal action of drugs can be assigned: (i) decreased intracellular accumulation of the antibiotic by an alteration of outer membrane permeability, diminished transport across the inner membrane, (ii) alteration of the target by mutation or enzymatic modification; (iii) enzymatic detoxification of the drug; and (iv) bypass of the drug target. The coexistence of several of these mechanisms in the same host can lead to MDR and XDR-TB. (Piddock et al., 2006; De Rossi et al., 2006). Another important question that needs to be highlighted is the association of these biochemical resistant pathways with the efflux pump system. Efux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic and eukaryotic cells. M. tuberculosis presents one of the largest numbers of putative drug efux pumps compared with its genome size. Antimicrobial resistance in an efflux mutant is due to one of two mechanisms: (i) expression of the efflux pump protein is increased or (ii) the protein contains an amino acid substitution(s) that makes the protein more efficient at export (Piddock, 2006).

MDR treatment, anti-TB drugs are grouped according to efficacy, experience of use and drug class. There are five groups and only the group 1 can receive the first line drugs, all others groups will receive the second-line drugs namely: kanamycin (Km), amikacin (Am), capreomycin (Cm), streptomycin (S), Levofloxacin (Lfx), Moxifloxacin (Mfx), Ofloxacin (Ofx), para-aminosalicylic acid (PAS), cycloserine (Cs), terizidone (Trd), ethionamide (Eto), protionamide (Pto), clofazimine (Cfz), linezolid (Lzd), amoxicillin/clavulanate (Amx/Clv), thioacetazone (Thz), imipenem/cilastatin (Ipm/Cln), clarithromycin (Clr) (WHO, 2009).

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 531

Hepatitis (do not show tolerance to treatment);

diagnosis of HIV, or vice versa (53% abandonment).

Health related problems Fractures, improvement of symptoms, mental Illness;

*Patients with co-morbidity*

Familiar Death and other diseases in the family, motivation,

Living away from family;

Absence of familiar support.

(Gonçalves et al., 1999).

Risk groups A community in contact with contagious TB or focus intra-

Alcoholic: 24% abandonment;

Drug addict: 12% abandonment;

Others Patient does not trust in the treatment, doctors or the health

Wait for the health service; Distance from health service; System health bureaucracy.

1997) abandoned;

system itself.

Table 1. Main reasons related to treatment abandonment.

contaminating and seek treatment lately;

in same bed);

recommendation to stop (by others);

Social behavior and sex An anthropological phenomenon is observed with male younger,

2001).

Non-acceptance of diagnosis, riot with the illness. No knowledge of the existence of extra-pulmonary forms. No symptoms after initial treatment interpreted by patients as a cure (Uplekar et al

Diabetes / TB = TB infections have more severe, with treatment failure rate of 8.5 x higher, which generates most abandoned, because this patient needs a longer treatment (Gupta et al, 2011). HIV / TB: The patient focuses on the treatment of HIV, at least tolerate the associated treatment. It is discouraged when after

Overpopulation in a single family home (sleeping more than two

particular way of life believing that they are not susceptible to TB disease. This group has not a tendency to modify some habits to contribute for the TB treatment during the 6 months of treatment

household is recommended prophylaxis with isoniazid, however, is not accepted by individuals. Typically, these individuals end up

Smokers: 40% (Mendes & Fensterseifer, 2004) 48.8% (Christmas

Drinker and smoker: 86.6% abandonment (Lima et al., 2001);

unmarried and/or separated which seek to preserve their

Disease related problems

#### **2. TB therapy problems**

TB treatment presents several challenges to be overcome. The treatment abandonment is one of them which contribute to development of resistance by mycobacterium to available drugs. The abandonment or lack of adherence occurs when the patient does not attend to receive medication for a month or more (Pablós-Méndez et al., 1997). Several factors contribute to this situation such as socio-economic factors, adverse effects of drugs, comorbidities, environmental factors (familiar, social behavior). In developing countries the access to adequate health system is one of the most problems to TB therapy. It is very common situation that the population has not medical service neither education level to comprehend the therapy. There is a relation between poverty and predisposition to disease difficult the TB control. Malnutrition, increased expensive to take medicines and stigmatization are some intrinsic factors related to the inadequate control of TB in poor countries (Cegielski & McMurray, 2004; Atre et al., 2009; Dhingra et al., 2010; WHO, 2005; Hargreaves et al., 2011). Educational levels and employment are conditions related to abandonment of therapy. It has been reported that a worker, provider of family without adequate financial support present high abandonment rate (64%). An interesting relation is also observed when someone analyses the educational level of the patients. Those which possess higher education (university) demonstrate high level of adherence despite those with low educational level. Low level of knowledge is directed relate to inadequate treatment (Grace & Chenhall, 2006). The Table 1 shows the main reasons related to treatment abandonment.

The adverse effect of antitubercular drugs is one the main causes of therapy abandonment. Some adverse effects of antitubercular drugs are described above:


TB treatment presents several challenges to be overcome. The treatment abandonment is one of them which contribute to development of resistance by mycobacterium to available drugs. The abandonment or lack of adherence occurs when the patient does not attend to receive medication for a month or more (Pablós-Méndez et al., 1997). Several factors contribute to this situation such as socio-economic factors, adverse effects of drugs, comorbidities, environmental factors (familiar, social behavior). In developing countries the access to adequate health system is one of the most problems to TB therapy. It is very common situation that the population has not medical service neither education level to comprehend the therapy. There is a relation between poverty and predisposition to disease difficult the TB control. Malnutrition, increased expensive to take medicines and stigmatization are some intrinsic factors related to the inadequate control of TB in poor countries (Cegielski & McMurray, 2004; Atre et al., 2009; Dhingra et al., 2010; WHO, 2005; Hargreaves et al., 2011). Educational levels and employment are conditions related to abandonment of therapy. It has been reported that a worker, provider of family without adequate financial support present high abandonment rate (64%). An interesting relation is also observed when someone analyses the educational level of the patients. Those which possess higher education (university) demonstrate high level of adherence despite those with low educational level. Low level of knowledge is directed relate to inadequate treatment (Grace & Chenhall, 2006). The Table 1 shows the main reasons related to

The adverse effect of antitubercular drugs is one the main causes of therapy abandonment.

Socio-economic Worker, a provider of family, lack of financial sources for feeding and locomotion: 64% abandonment.

Therapeutical Adverse effects relate to antitubercular drugs: nausea, vomiting,


E – ethambutol; Et – ethionamide.

hyperthermia and edema. Denial and oblivion.

Unemployed: 36% abandonment (finantial problems and low self

Education: illiteracy (20% abandonment), less than 8 years of study (72 % abandonment), between 8 and 12 years of study (8% abandonment) and more than 13 years (0% abandonment).


R – rifampicin; I – isoniazid; P- pyrazinamide; S- streptomycin;

Some adverse effects of antitubercular drugs are described above:

steem)

Regimens:

**2. TB therapy problems** 

treatment abandonment.


Table 1. Main reasons related to treatment abandonment.

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 533

Environment factors are determinants for TB treatment. The family has a crucial role to the treatment. The knowledge of the disease by all familiar members is an important factor to control TB. After the first phase of the treatment, when some symptoms decrease, is common identify problems with adherence by patients. So, educational interventions by health professionals are important to improve the TB treatment management. Some studies demonstrated that previous cases in the family increase the knowledge and the adherence by patients (Costa et al., 2011). However, some factors such live away from family, absence of familiar support and overpopulation in a single family home (with more than two people

Gender is another factor relate to TB. Worldwide, more men than women are diagnosed with TB. This higher TB notification in men is relate to epidemiological differences such as risk of exposure, infection and progression to disease. However, it has been reported that women have higher case of fatalities in the early reproductive ages and higher rates of

The association of TB with co-morbidities is a complicated factors related to increase of adverse effects and high rate of abandonment treatment by patients. It has been reported that treatment fail is increased 8.5 times in diabetic patients with TB, general relate to abandonment. In diabetic group infection with TB seems to be more severe that in people

The abandonment of HIV co-infection patients could reach 53% (Table 1). The recommended TB treatment of HIV-negative people is the same of HIV-positive people but sometimes the therapy is extended to 9 months or more in patients with extra-pulmonar TB. However, some management of the therapy is complicated due to paradoxical reaction, drug interactions and the difficulty to ingest a large number of tablets (Yew, 2002). The paradoxical reactions, is an exacerbation of TB symptoms, due to due to immune recovery, called immune reconstitution syndrome. Although the mechanism is not totally clear it is presumed to represent an interaction between the host responses and effects produced by mycobacterial products leading to inflammatory lesions (Orlovic & Smego, 2001). Some

sleeping in the same bed) is a risk factor to abandon TB treatment.

progression from infection to disease (Holmes et al., 1998).

no-diabetic (Gupta et al., 2011).

Fig. 2. Some TB therapy problems.


In general, the adverse effects related to first line antitubercular drugs include skin rash, itch, nausea and vomiting, thrombocytopenia, symptoms influenza-símile, arthralgias and neuropsychiatric manifestations (Yee et al., 2003; Fekih et al., 2011; Fountain et al., 2005).

It has been reported that the rate of adverse effects during the treatment could reach 30% of the patients or 7,3 per 100 patients/month. Therapy with four drugs increase the incidence to 23,3 events by 100-patients/month. In addition, the adverse effects are used to appear during the first 100 days of treatment and the most common effects observed were hepatitis (28%), gastrointestinal disorders (19%), skin rash (15%), weakness or tiredness (7%) and joint pain (6%) (Yee et al., 2003).

The hepatotoxicity is a deleterious adverse effect responsible to determine changes in therapy. TB first line drugs such as rifampicin, rifabutin, isoniazid and pyrazinamide can cause hepatotoxicity, alone isoniazid is responsible to 20% of reported cases. The combination of drugs increases the probability to develop hepatotoxicity. Risk factors such as HIV co-infection, hepatitis B and/or C, alcohol abuse or the use of some medicines (i.e. anticonvulsant) should be taken in consideration due the due to the increased likelihood of causing liver toxicity.

In situation which patients present previous diagnosis of advance liver disease, when the doctor wants to keep only one hepatotoxic drug, rifampicin is usually selected. However, other agents should be added the therapy such as fluorquinolone, cycloserine and aminoglycoside. The treatment time of these schemes can vary from 12-18 months (American Thoracic Society, 2003).

It is important to note that the assessment of adverse effects should be performed throughout therapy, and compared with pre-treatment parameters. The patient should be prepared to identify adverse effects related to the use of anti-TB drugs.

 Isoniazid: Peripheral neuritis (prevent with the use of pyridoxine); may occur again, optic neuritis, ataxia, mental disturbances and incoordination. Hypersensitivity to isoniazid can cause fever, various skin rashes, hepatitis and skin rashes, hematologic reactions may occur (agranulocytosis, eosinophilia, thrombocytopenia, anemia); neuritis and optic atrophy. Twitches, dizziness, ataxia, paresthesias, numbness, toxic encephalopathy are other manifestations of neurotoxicity of isoniazid. It may also appear several mental abnormalities. May precipitate seizures in patients with previous history of seizures. Rifampicin: Facial flushing, generalized itching and skin rash, purpura, epistaxis, menorrhagia, gingival bleeding and hemolytic anemia. Pseudogripal syndrome with fever, malaise, headache, chills and myalgia, which may progress to interstitial nephritis, acute tubular necrosis, thrombocytopenia and shock. In the digestive tract:

Malaise, loss of appetite, nausea, vomiting, jaundice, liver failure and diarrhea. Pirazinamide: Liver damage with elevation of plasmatic AST and ALT is the main adverse effect. Furthermore it is observed arthralgia, anorexia, nausea and vomiting,

 Ethambutol: The observed adverse reactions include itching, joint pain, gastrointestinal disorders, abdominal pain, malaise, headache, dizziness, mental confusion, disorientation and possible hallucinations, acute gout or hyperuricemia. Although rare the peripheral neuritis and retrobulbar optic neuritis (blurred vision, eye pain, red-gray images, decreased vision) are reported. The retrobulbar optic neuritis is dosedependent, occurring more frequently with daily doses of 25 mg / kg and after two months of therapy, in many cases is reversible after several weeks or several months. In general, the adverse effects related to first line antitubercular drugs include skin rash, itch, nausea and vomiting, thrombocytopenia, symptoms influenza-símile, arthralgias and neuropsychiatric manifestations (Yee et al., 2003; Fekih et al., 2011; Fountain et al., 2005). It has been reported that the rate of adverse effects during the treatment could reach 30% of the patients or 7,3 per 100 patients/month. Therapy with four drugs increase the incidence to 23,3 events by 100-patients/month. In addition, the adverse effects are used to appear during the first 100 days of treatment and the most common effects observed were hepatitis (28%), gastrointestinal disorders (19%), skin rash (15%), weakness or tiredness (7%) and joint

The hepatotoxicity is a deleterious adverse effect responsible to determine changes in therapy. TB first line drugs such as rifampicin, rifabutin, isoniazid and pyrazinamide can cause hepatotoxicity, alone isoniazid is responsible to 20% of reported cases. The combination of drugs increases the probability to develop hepatotoxicity. Risk factors such as HIV co-infection, hepatitis B and/or C, alcohol abuse or the use of some medicines (i.e. anticonvulsant) should be taken in consideration due the due to the increased likelihood of

In situation which patients present previous diagnosis of advance liver disease, when the doctor wants to keep only one hepatotoxic drug, rifampicin is usually selected. However, other agents should be added the therapy such as fluorquinolone, cycloserine and aminoglycoside. The treatment time of these schemes can vary from 12-18 months

It is important to note that the assessment of adverse effects should be performed throughout therapy, and compared with pre-treatment parameters. The patient should be

prepared to identify adverse effects related to the use of anti-TB drugs.

dysuria, malaise and fever.

pain (6%) (Yee et al., 2003).

causing liver toxicity.

(American Thoracic Society, 2003).

Environment factors are determinants for TB treatment. The family has a crucial role to the treatment. The knowledge of the disease by all familiar members is an important factor to control TB. After the first phase of the treatment, when some symptoms decrease, is common identify problems with adherence by patients. So, educational interventions by health professionals are important to improve the TB treatment management. Some studies demonstrated that previous cases in the family increase the knowledge and the adherence by patients (Costa et al., 2011). However, some factors such live away from family, absence of familiar support and overpopulation in a single family home (with more than two people sleeping in the same bed) is a risk factor to abandon TB treatment.

Gender is another factor relate to TB. Worldwide, more men than women are diagnosed with TB. This higher TB notification in men is relate to epidemiological differences such as risk of exposure, infection and progression to disease. However, it has been reported that women have higher case of fatalities in the early reproductive ages and higher rates of progression from infection to disease (Holmes et al., 1998).

The association of TB with co-morbidities is a complicated factors related to increase of adverse effects and high rate of abandonment treatment by patients. It has been reported that treatment fail is increased 8.5 times in diabetic patients with TB, general relate to abandonment. In diabetic group infection with TB seems to be more severe that in people no-diabetic (Gupta et al., 2011).

Fig. 2. Some TB therapy problems.

The abandonment of HIV co-infection patients could reach 53% (Table 1). The recommended TB treatment of HIV-negative people is the same of HIV-positive people but sometimes the therapy is extended to 9 months or more in patients with extra-pulmonar TB. However, some management of the therapy is complicated due to paradoxical reaction, drug interactions and the difficulty to ingest a large number of tablets (Yew, 2002). The paradoxical reactions, is an exacerbation of TB symptoms, due to due to immune recovery, called immune reconstitution syndrome. Although the mechanism is not totally clear it is presumed to represent an interaction between the host responses and effects produced by mycobacterial products leading to inflammatory lesions (Orlovic & Smego, 2001). Some

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 535

↓50% ↓58% ↓68% The standard dose of

neveriapine should be used among patients taking rifampin (200 mg daily for 2 weeks, followed by 200 mg

\*Increasing nevirapine dose by 50% to 300 mg twice/daily would achieve therapeutic concentration but safety has not been adequately explored as a routine clinical practice

twice daily

and my result in

thrice weekly

↓24% ↓25% ↓22% Rifampin 600 mg once/day

(600 mg once daily) in

hypersensivity reactions.

300 mg rifabutin daily or

**no changes recommended** 

200 mg efanvirenz twice/day No changes recommended

combination with an anti-TB regimen containing rifampin (480 to 720 mg/day based on body weight) for 7 days

Cmax AUC Cmin

Decrease plasma Concentration Caution: Sub-therapeutic dosage found

Not significant changes in plasma concentration

not significant decrease plasma concentration

toxicity : anxiety, depression, hepatitis (mainly in African descendent) \* efavirenz has been associated

Increase clinical

with

hepatotoxicity during

metformin

postmarketing use. -**co-administration of drugs with caution**: drugs that induces liver damages such as acetaminophen, kava-kava, statins,

nevirapine **+ RIFAMPIN**

nevirapine **+ RIFABUTIN**

Efavirenz + **RIFAMPIN**

symptoms include restart or worsening of fever, lymphadenopathy, dyspnea, worsening of brain injury (Navas et al., 2002).

#### **3. Anti-TB drug interactions**

Concurrent treatment of TB and HIV is associated with a higher risk of adverse reactions compared to treatment of either infection alone. The first-line anti-TB drugs isoniazid, rifampin, and pyrazinamide may each cause hepatotoxicity, which may be compounded by concomitant use of protease inhibitors (PI) and nonnucleoside reverse transcriptase inhibitors (NRTI). Pharmacokinetic interactions between HIV and TB regimens can have a significant impact on the therapeutic efficacy of each regimen.

Rifamycin antibiotics, the main drug in TB therapy induce the synthesis of drug metabolizing enzymes (cytochrome P450 emzyme system, particulary the CYP 3A4, CYP 2C8/9 isoenzymes and to a lesser extent CYP2C19 and CYPD6 isozymes; the rifamycins vary in their potential as CYP450 inducers. Potency of induction: Rifampin > rifapentine > rifabutin. Rifampin also upregulates the synthesis of cytosolic drug-metabolizing enzymes, including glucuronosyl transferase, an enzyme involved in the metabolism of zidovudine and raltegravir. Potent induction of the cytochrome p450 system by rifampin can lead to subtherapeutic levels of the protease inhibitors accompanied by virological failure

Antiretroviral treatment (ART) improves survival in co-infected TB patients and vice-versa, however, these concomitant drug therapy in the early stage can increase risk of paradoxal TB- immune reconstitution inflammatory syndrome, risk of overlapping drug toxicities with possibility in drug treatment interruption, high pill burden that can impact in adherence and increased potential drug-drug interactions. The delayed treatment can promote the advancing immunosuppression and the development of others opportunist conditions that may increase mortality.

The estimated cumulative probability to develop an adverse event was significantly higher in HIV/TB co-infected in Ruanda patients: 20.9% within the first month of antituberculous treatment (vs. 3.0% in HIV-uninfected) and up to 29.9% at two months of treatment (vs. 6.9% in HIV-uninfected) (Gordin et al., 1996)

Rifamycin is related to interact with four classes of anti-HIV drugs (protease inhibitors, nonnucleoside reverse-transcriptase inhibitors [NNRTI], CCR5-receptor antagonistis and integrase inhibitors. Zidovudine, the nucleoside analogues and enfuvirtide do not have significant interactions with rifamycins.

The initial ATR regimens in areas with high rates of TB use efavirenz (in combination with nucleoside analogues), because of its potency and durability on randomized clinical trials.

The co-adminstration rifampin decrease plasma concentration of efavirenz but not in a significant way. Some expertise suggested an increase of efavirenz to 800 mg in TB/HIV concomitant treatment to patients weighing more than 60 kg (WHO guidelines). However, Cohen and Meinttjes (2010), do not recommend these proceedings based on the fact that the CYP 450 2B6 516 G>T polymorphism, which impairs the function of the primary pathway of efavirenz metabolism (2B6) is present in African populations. So, the consequence of even

symptoms include restart or worsening of fever, lymphadenopathy, dyspnea, worsening of

Concurrent treatment of TB and HIV is associated with a higher risk of adverse reactions compared to treatment of either infection alone. The first-line anti-TB drugs isoniazid, rifampin, and pyrazinamide may each cause hepatotoxicity, which may be compounded by concomitant use of protease inhibitors (PI) and nonnucleoside reverse transcriptase inhibitors (NRTI). Pharmacokinetic interactions between HIV and TB regimens can have a

Rifamycin antibiotics, the main drug in TB therapy induce the synthesis of drug metabolizing enzymes (cytochrome P450 emzyme system, particulary the CYP 3A4, CYP 2C8/9 isoenzymes and to a lesser extent CYP2C19 and CYPD6 isozymes; the rifamycins vary in their potential as CYP450 inducers. Potency of induction: Rifampin > rifapentine > rifabutin. Rifampin also upregulates the synthesis of cytosolic drug-metabolizing enzymes, including glucuronosyl transferase, an enzyme involved in the metabolism of zidovudine and raltegravir. Potent induction of the cytochrome p450 system by rifampin can lead to

Antiretroviral treatment (ART) improves survival in co-infected TB patients and vice-versa, however, these concomitant drug therapy in the early stage can increase risk of paradoxal TB- immune reconstitution inflammatory syndrome, risk of overlapping drug toxicities with possibility in drug treatment interruption, high pill burden that can impact in adherence and increased potential drug-drug interactions. The delayed treatment can promote the advancing immunosuppression and the development of others opportunist conditions that

The estimated cumulative probability to develop an adverse event was significantly higher in HIV/TB co-infected in Ruanda patients: 20.9% within the first month of antituberculous treatment (vs. 3.0% in HIV-uninfected) and up to 29.9% at two months of treatment (vs. 6.9%

Rifamycin is related to interact with four classes of anti-HIV drugs (protease inhibitors, nonnucleoside reverse-transcriptase inhibitors [NNRTI], CCR5-receptor antagonistis and integrase inhibitors. Zidovudine, the nucleoside analogues and enfuvirtide do not have

The initial ATR regimens in areas with high rates of TB use efavirenz (in combination with nucleoside analogues), because of its potency and durability on randomized clinical

The co-adminstration rifampin decrease plasma concentration of efavirenz but not in a significant way. Some expertise suggested an increase of efavirenz to 800 mg in TB/HIV concomitant treatment to patients weighing more than 60 kg (WHO guidelines). However, Cohen and Meinttjes (2010), do not recommend these proceedings based on the fact that the CYP 450 2B6 516 G>T polymorphism, which impairs the function of the primary pathway of efavirenz metabolism (2B6) is present in African populations. So, the consequence of even

subtherapeutic levels of the protease inhibitors accompanied by virological failure

brain injury (Navas et al., 2002).

**3. Anti-TB drug interactions** 

may increase mortality.

trials.

in HIV-uninfected) (Gordin et al., 1996)

significant interactions with rifamycins.

significant impact on the therapeutic efficacy of each regimen.


Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 537

**+ food** ↓50% Food increase bioavailabitily

of etravirine (unknown mechanism, but ranging from 345 kilocalories containing 17 grams fat to 1160 kilocalories containing 70 grams fat did not impact on etravirine

(300 mg rifabutin daily or 3 x

**No changes recommended** 

atazanavir, with or without low-dose ritonavir as a pharmacokinetic booster. The mechanism is etravirine induction of CYP450 3A4, the isoenzyme responsible for the metabolic clearance of

atazanavir and other protease

dose to 150 mg/day or 3 x

meal ↑increased AUC of a single 400 mg dose of atazanavir relative to the

induction of CYP450 3A4, the isoenzyme responsible for the metabolic clearance of PIs.

bioavailability

↓ 45% No clinical experience.

weekly)

inhibitors

↑250% Recommendation: ↓ribatutin

fasting state.

↓75-95% the mechanism is rifampin

week

↑57% ↑70% administration with a light

↓95% \* with etravirine may ↓

**Etravirine** + **RIFAMPIN** 

**Etravirine + RIFABUTIN**

**Atazanavir \*** 

**RIFAMPIN** 

**Atazanavir** 

RIFABUTIN

+

**Fos-**

+

**amprenavir** 

**RIFAMPIN** 

+

Decrease etravirine

atazanavir plasma concentration

**RECOMMEND CONCOMITANT** 

Increase rifabutin

**Recommendation:** To ensure maximal oral absorption of atazanavir, it should be

administered with or immediately after a meal

Strongly decrease fosamprenavir

concentration –

RECOMMEND CONCOMITANT

plasma

NOT

USE

Cmin

Strongly ↓

**NOT** 

**USE**

plasma concentration

+ food


↓ 38% **Recommendation:** ↑ to 450-

↓90%, ↓97% ↓100% rifampin (600 mg once daily

↓72% ↓82% ↓94% 300 mg once daily for 15 days

Predicted (etravirine is a

**+ ETHABUTOL** 

intermittent)

600 mg of rifabutin (daily or

for 15 days) ↓ plasma concentration of delavirdine (400 mg 3x/ day for 30 days)

substrate of CYP450 2C19,

**(same effect can be observed when use ethambutol + isoniazide only)** 

2C9, and 3A4).

**+ ISONIAZIDE** 

Efavirenz + RIFABUTIN

**Delavirdine** 

**RIFAMPIN**

**Delavirdine**

**RIFABUTIN**

**Etravirine** + **RIFAMPIN** 

+

+

↓rifabutin plasma concentration

delavirdine plasma concentration

**RECOMMEND CONCOMITANT** 

delavirdine plasma concentration and

**RECOMMEND CONCOMITANT** 

etravirine plasma concentration

**RECOMMEND CONCOMITANT** 

irreversible despite discontinuation of the medications

Strongly ↓

**NOT** 

**USE**

**USE** 

**NOT** 

**USE** 

The risk of peripheral neuropathy may be increased during concurrent use of two or more agents that are associated with this adverse effect. In some cases, the neuropathy may progress or become

Strongly ↓

Strongly ↓

↑rifabutin concentration by 100% (AUC) **NOT** 


Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 539

Lopinavir / ritonavir– 2

Increase the dose of lopinavir

303% **Recommendation**: ↓ribatutin

week

week

The mechanism is rifampin

tablets (200 mg of lopinavir with 50 mg of ritonavir) + 300 mg of ritonavir twice-daily + 600 mg rifampin/day Have favorable

pharmacokinetic and clinical data among young children

/ ritonavir to 4 tablets (200 mg of lopinavir with 50 mg of ritonavir) twice-daily. This combination resulted in hepatitis in all adult healthy volunteers in an initial study.

dose to 150 mg/day or 3 x

induction of CYP450 3A4, the isoenzyme responsible for the metabolic clearance of PIs.

**Recommendation:** ↓ribatutin dose to 150 mg/day or 3 x

**Ritonavir + lopinavir + rifampin** 

**Ritonavir + lopinavir + rifampin** 

**Ritonavir + lopinavir Rifabutin** 

**atazanavir/ tipranavir or darunavir + ritonavir + rifampin** 

**Ritonavir + saquinavir/i ndinavir/am prenavir/fos**

**amprenavir/ atazanavir/ti pranavir or darunavir + RIFABUTIN**

**-**

Hepatotoxicity. **Use with caution**

Rifampin decrease

concentration of lopinavir

**Use with caution:** 

↑ RIFABUTIN and 25-o-desacetil rifabutin (47,5 fold) concentration

Rifampin strongly

concentration –

**RECOMMEND CONCOMITANT** 

↑ RIFABUTIN and 25-o-desacetil rifabutin concentration (varyng degreee)

decrease of protease inhibitors (PIs)

plasma

**NOT** 

**USE** 

plasma


↓75% to 95%

↑119% ↑193% ↑271% The mechanism is

amprenavir inhibition of CYP450 3A4, the isoenzyme responsible for the metabolic clearance of rifabutin and 25-

O-desacetylrifabutin

The mechanism is rifampin

↑170% **Recommendation:** ↓ribatutin

week

↑207% Not significant change in

week.

transporter

ritonavir

Drug-induced hepatitis with

↓35% no change in rifampin

week

**Recommendation:** ↓ribatutin dose to 150 mg/day or 3 x

induction of CYP450 3A4, the isoenzyme responsible for the metabolic clearance of PIs.

dose to 150 mg/day or 3 x

nelfinavir concentration. **Recommendation:** ↓ribatutin dose to 150 mg/day or 3 x

concentration (600 mg/day) rifampin will decrease the level or effect of ritonavir by P-glycoprotein (MDR1) efflux

**Recommedantion:** Monitor for antiretroviral activity of

marked ↑ transaminase has been observed in healthy volunteers receiving rifampin 600 mg once daily with ritonavir 100 mg and saquinavir 1000 mg twice daily (i.e., ritonavir-boosted saquinavir). The mechanism has not been described.

**Fos-**

**+** 

**amprenavir** 

Increase significantly rifabutin plasma

and 25-)

plasma concentration **NOT** 

**USE** 

**RIFAMPIN Use with caution** 

%)

in

desacetylrifabutin

7.39 fold(Cmax) 13.35 fold (AUC) 32.9 fold (Cmin) **Use with caution** 

Strongly decrease of protease inhibitors (PIs)

**RECOMMEND CONCOMITANT** 

↑ RIFABUTIN concentration and ↓ indinavir by 34%

↑ RIFABUTIN concentration

Hepatotoxicity. Transaminase elevations up to or even exceeding 20 times the upper limit of normal.(38

**Use with caution**

**RIFABUTIN**

**saquinavir /indinavir or nelfinavir + RIFAMPIN** 

**Indinavir + RIFABUTIN**

**nelfinavir + RIFABUTIN**

**Ritonavir +** 

**Ritonavir + saquinavir + RIFAMPIN** 


Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 541

No changes were observed when use nevirapine and rifabutin. Others some important

*TB/HIV treatment need to avoid co-administration*: with potent inducers of CYP450 isoenzymes such as carbamazepine, phenobarbital, phenytoin, rifampin and rifapentine due the risk of reduced viral susceptibility and resistance development associated with sub-therapeutic

Coadministration with inhibitors of CYP450 3A4 may increase the plasma concentrations of lopinavir, which is metabolized by the

the risk is low in patients with adequate renal function receiving the normally recommended dosage but may increase in patients with underlying renal impairment

rifampin (600 mg orally once

recomemmendation Monitor for antiretroviral activity of

a day for 14 days)

zidovudine

isoenzyme

**Isoniazid + lopinavir** 

**Streptomyci**

**Zidovudine + rifampin** 

**n + tenofovir**  The magnitude and clinical significance of this interaction are unknown

Coadministration of tenofovir with other nephrotoxic agents may increase the risk and severity of renal impairment due to additive effects on the kidney.

Additionally, renal impairment secondary to the use of these agents may reduce the clearance of tenofovir, which is

primarily eliminated by renal excretion.

↓ zidovudine plasma

Table 2. Antitubercular Drug Interactions.

considerations area presented below:

antiretroviral drug levels.

concentration not significantly rifabutin also reportedly decreased zidovudine AUC by 32% and increased its clearance by 43%


↓ 40- 61%

↑ ↑13% The mechanism probably is

↓66% ↓63% ↓ 78% No clinical experience with

No studies have been

rifampin

mg/day)

these patients.

No clinical experience with

in rifampin dose (600 mg/day)

+ rifampin

increased dose of raltegravir

Recommendation: no changes

competitive inhibition of isoniazid metabolism **Recommendation:** monitor isoniazide-related toxicity

increased dose of maraviroc +

Recommendation: no changes

performed in subjects with severe renal impairment or ESRD co-treated with maraviroc and potent

CYP450 3A4 inducers. Hence, no dosage recommendation for maraviroc is available for

in rifampin dose (600

**Maraviroc + rifampin** 

**Maraviroc + rifampin** 

**Maraviroc + rifabutin** 

**Raltegravir + rifampin** 

**Raltegravir + rifabutin** 

**Isoniazid + indinavir** 

Not studied

↓ **Raltegravir**  plasma concentration

No studied

↓ maraviroc plasma concentration

Patients with severe renal impairment or end-stage renal disease (CrCl <30 mL/min) given maraviroc may have an increased risk of postural hypotension due to increased maraviroc exposure. Moreover, these patients often have cardiovascular comorbidities that could predispose them to adverse cardiovascular events triggered by postural hypotension.


Table 2. Antitubercular Drug Interactions.

No changes were observed when use nevirapine and rifabutin. Others some important considerations area presented below:

*TB/HIV treatment need to avoid co-administration*: with potent inducers of CYP450 isoenzymes such as carbamazepine, phenobarbital, phenytoin, rifampin and rifapentine due the risk of reduced viral susceptibility and resistance development associated with sub-therapeutic antiretroviral drug levels.

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 543

*The use of Isoniazid in HIV/TB treatment with ritonavir/lopinavir and others PIs*: The isoniazid will increase the level or effect of PIs by affecting hepatic/intestinal enzyme CYP3A4 metabolism. Avoid co-administration with others inhibitors of the hepatic/intestinal enzyme CYP3A4 metabolism such as macrolide antibiotics, itraconazole, ketoconazole,

*Isoniazid + ethambutol*: increase the risk of peripheral neuropathy: burning, tingling, pain, or

*Isoniazid + rifampin/rifabutin*: The risk of hepatotoxicity is greater when rifampin and isoniazid are given concomitantly than when either drug is given alone. Rifampin alters the metabolism of isoniazid and increase the amount of toxic metabolites. Similar reaction may occur with rifabutin and isoniazid. Patients who are elderly, have hepatic impairment, are slow acetylators of isoniazid, drink alcohol daily, are female, or are taking other CYP450 inducing agents may be at greater risk of hepatotoxicity. Recommend the monitoring for clinical or laboratory evidence of hepatic function during the treatment. Discontinuation of either or both drugs may be necessary when simptoms of hepatite such as fatigue,

*Isoniazid + food:* Food significantly reduces isoniazid absorption, increasing the risk of therapeutic failure or resistance. The mechanism is unknown. In addition, the ingestion of certain histamine-rich fish (e.g., tuna) and cheeses during isoniazid therapy may cause a flushing reaction in some patients. The proposed mechanism is inhibition of monoamine oxidase and histaminase by isoniazid, resulting in histamine intoxication. The associated symptoms is: flushing, tachycardia, chills, headache, nausea, vomiting, diarrhea, burning sensation, sweating, or shortness of breath after eating certain foods. Isoniazid cause depletion of B6 vitamin during the treatment. Since HIV-infected persons are at increased risk for isoniazid-induced peripheral neuropathy, these patients should take vitamin B6 and avoid antiretroviral drugs with potential peripheral neurotoxicity (e.g., stavudine and

**4. Adherence to therapy – Process to improve medication efficacy** 

It has been reported that treatment abandonment rate reaches until 25% of all patients during the treatment (Veronesi & Focaccia, 1996). In developing countries such as Brazil, this rates decreased in the last years to 4,5%, but it is possible to find out in some regions values until 20,3% (Costa, Gonçalves & Menezes, 1998; Lima et al., 2001). The WHO's recommendation is that abandonment rates must be until 5%, however, it is possible to find in some countries such as Colombia abandonment rates of 65,6% (Mateus-Solarte &

In order to reduce the abandonment of therapy and ensure the correct treatment of patients some strategies have been adopted to improve adherence to therapy. According to WHO "Adherence corresponds to the behavior of one person - in terms of taking a drug, a diet or executing lifestyle changes - that is in accordance with recommendations arising from health

Adherence to therapy is a serious problem responsible for the evolution of diseases and complications, loss of quality of life and even death. In the United States it is estimated that

nefazodone, fluconazole, verapamil, diltiazen, grapefruit juice.

weakness, malaise, anorexia, nausea, or vomiting appears.

numbness in the hands and feet

didanosine).

Carvajal-Barona, 2008).

professionals" (WHO, 2003).

*The use of tenofovir in HIV/TB need avoid the co-administration:* with other potentially nephrotoxic agents (e.g., aminoglycosides; polypeptide, glycopeptide, and polymyxin antibiotics; amphotericin B; adefovir; cidofovir; foscarnet; cisplatin; deferasirox; gallium nitrate; lithium; mesalazine; certain immunosuppressants; intravenous bisphosphonates; intravenous pentamidine; high intravenous dosages of methotrexate; high dosages and/or chronic use of nonsteroidal anti-inflammatory agents. Renal function should be evaluated prior to and during therapy with tenofovir. Patients with renal insufficiency at baseline or during treatment may require dosage adjustment in accordance with the manufacturer's product labeling.

*The use of efavirenz in HIV/TB need avoid the co-administration:* with potentially hepatotoxicity agents: pyrazinamide, isoniazid, acetaminophen; alcohol; androgens and anabolic steroids; azole antifungal agents; ACE inhibitors; endothelin receptor antagonists; interferons; other nucleoside reverse transcriptase inhibitors; retinoids; thiazolidinediones; anticonvulsants such as carbamazepine, hydantoins, felbamate, and valproic acid; lipid-lowering medications such as fenofibrate, HMG-CoA reductase inhibitors, and niacin; herbals and nutritional supplements such as chaparral, comfrey, DHEA, kava, pennyroyal oil, and red yeast rice. Patients should be advised to seek medical attention if they experience potential signs and symptoms of hepatotoxicity such as fever, rash, itching, anorexia, nausea, vomiting, fatigue, right upper quadrant pain, dark urine, light-colored stools, and jaundice. Monitoring of liver function should occur before and during treatment, especially in patients with other hepatic disease (including hepatitis B or C co-infection) or marked transaminase elevations. The benefit of continued therapy with efavirenz should be considered against the unknown risks of significant liver toxicity in patients who develop persistent elevations of serum transaminases greater than five times the upper limit of normal.

*The use of tenofovir in HIV/TB treatment with streptomycin*: Caution for renal function impairment due to additive effects on the kidney. The deleterious effect on the kindney can occur with concomitant use of others nephrotoxic drugs such as aminoglycosides; polypeptide, glycopeptide, and polymyxin antibiotics; amphotericin B; adefovir; cidofovir; foscarnet; cisplatin; deferasirox; gallium nitrate; lithium; mesalamine; certain immunosuppressants; intravenous bisphosphonates; intravenous pentamidine; high intravenous dosages of methotrexate; high dosages and/or chronic use of nonsteroidal antiinflammatory agents. Renal function should be evaluated prior to and during therapy. Patients with renal insufficiency at baseline or during treatment may require dosage adjustment in accordance with the manufacturer's product labeling.

*The use of maraviroc in HIV/TB treatment with rifampin and others CYP 450 3A4 inhibitors:* maraviroc should be administered at a dosage of 600 mg twice daily during coadministration with potent CYP450 3A4 inducers such as efavirenz, rifampin, carbamazepine, phenobarbital, and phenytoin. However, if a potent CYP450 3A4 inhibitor such as itraconazole, ketoconazole, delavirdine, clarithromycin, telithromycin, nefazodone, or any protease inhibitor (except tipranavir + ritonavir) is also used in combination with the inducer, then maraviroc dosage should be reduced to 150 mg twice daily. Maraviroc is contraindicated for use in combination with potent CYP450 3A4 inducers in patients with severe renal impairment or end-stage renal disease (CrCl <30 mL/min).

*The use of tenofovir in HIV/TB need avoid the co-administration:* with other potentially nephrotoxic agents (e.g., aminoglycosides; polypeptide, glycopeptide, and polymyxin antibiotics; amphotericin B; adefovir; cidofovir; foscarnet; cisplatin; deferasirox; gallium nitrate; lithium; mesalazine; certain immunosuppressants; intravenous bisphosphonates; intravenous pentamidine; high intravenous dosages of methotrexate; high dosages and/or chronic use of nonsteroidal anti-inflammatory agents. Renal function should be evaluated prior to and during therapy with tenofovir. Patients with renal insufficiency at baseline or during treatment may require dosage adjustment in accordance with the manufacturer's

*The use of efavirenz in HIV/TB need avoid the co-administration:* with potentially hepatotoxicity agents: pyrazinamide, isoniazid, acetaminophen; alcohol; androgens and anabolic steroids; azole antifungal agents; ACE inhibitors; endothelin receptor antagonists; interferons; other nucleoside reverse transcriptase inhibitors; retinoids; thiazolidinediones; anticonvulsants such as carbamazepine, hydantoins, felbamate, and valproic acid; lipid-lowering medications such as fenofibrate, HMG-CoA reductase inhibitors, and niacin; herbals and nutritional supplements such as chaparral, comfrey, DHEA, kava, pennyroyal oil, and red yeast rice. Patients should be advised to seek medical attention if they experience potential signs and symptoms of hepatotoxicity such as fever, rash, itching, anorexia, nausea, vomiting, fatigue, right upper quadrant pain, dark urine, light-colored stools, and jaundice. Monitoring of liver function should occur before and during treatment, especially in patients with other hepatic disease (including hepatitis B or C co-infection) or marked transaminase elevations. The benefit of continued therapy with efavirenz should be considered against the unknown risks of significant liver toxicity in patients who develop persistent elevations of serum transaminases greater than five times the upper limit of

*The use of tenofovir in HIV/TB treatment with streptomycin*: Caution for renal function impairment due to additive effects on the kidney. The deleterious effect on the kindney can occur with concomitant use of others nephrotoxic drugs such as aminoglycosides; polypeptide, glycopeptide, and polymyxin antibiotics; amphotericin B; adefovir; cidofovir; foscarnet; cisplatin; deferasirox; gallium nitrate; lithium; mesalamine; certain immunosuppressants; intravenous bisphosphonates; intravenous pentamidine; high intravenous dosages of methotrexate; high dosages and/or chronic use of nonsteroidal antiinflammatory agents. Renal function should be evaluated prior to and during therapy. Patients with renal insufficiency at baseline or during treatment may require dosage

*The use of maraviroc in HIV/TB treatment with rifampin and others CYP 450 3A4 inhibitors:* maraviroc should be administered at a dosage of 600 mg twice daily during coadministration with potent CYP450 3A4 inducers such as efavirenz, rifampin, carbamazepine, phenobarbital, and phenytoin. However, if a potent CYP450 3A4 inhibitor such as itraconazole, ketoconazole, delavirdine, clarithromycin, telithromycin, nefazodone, or any protease inhibitor (except tipranavir + ritonavir) is also used in combination with the inducer, then maraviroc dosage should be reduced to 150 mg twice daily. Maraviroc is contraindicated for use in combination with potent CYP450 3A4 inducers in patients with

adjustment in accordance with the manufacturer's product labeling.

severe renal impairment or end-stage renal disease (CrCl <30 mL/min).

product labeling.

normal.

*The use of Isoniazid in HIV/TB treatment with ritonavir/lopinavir and others PIs*: The isoniazid will increase the level or effect of PIs by affecting hepatic/intestinal enzyme CYP3A4 metabolism. Avoid co-administration with others inhibitors of the hepatic/intestinal enzyme CYP3A4 metabolism such as macrolide antibiotics, itraconazole, ketoconazole, nefazodone, fluconazole, verapamil, diltiazen, grapefruit juice.

*Isoniazid + ethambutol*: increase the risk of peripheral neuropathy: burning, tingling, pain, or numbness in the hands and feet

*Isoniazid + rifampin/rifabutin*: The risk of hepatotoxicity is greater when rifampin and isoniazid are given concomitantly than when either drug is given alone. Rifampin alters the metabolism of isoniazid and increase the amount of toxic metabolites. Similar reaction may occur with rifabutin and isoniazid. Patients who are elderly, have hepatic impairment, are slow acetylators of isoniazid, drink alcohol daily, are female, or are taking other CYP450 inducing agents may be at greater risk of hepatotoxicity. Recommend the monitoring for clinical or laboratory evidence of hepatic function during the treatment. Discontinuation of either or both drugs may be necessary when simptoms of hepatite such as fatigue, weakness, malaise, anorexia, nausea, or vomiting appears.

*Isoniazid + food:* Food significantly reduces isoniazid absorption, increasing the risk of therapeutic failure or resistance. The mechanism is unknown. In addition, the ingestion of certain histamine-rich fish (e.g., tuna) and cheeses during isoniazid therapy may cause a flushing reaction in some patients. The proposed mechanism is inhibition of monoamine oxidase and histaminase by isoniazid, resulting in histamine intoxication. The associated symptoms is: flushing, tachycardia, chills, headache, nausea, vomiting, diarrhea, burning sensation, sweating, or shortness of breath after eating certain foods. Isoniazid cause depletion of B6 vitamin during the treatment. Since HIV-infected persons are at increased risk for isoniazid-induced peripheral neuropathy, these patients should take vitamin B6 and avoid antiretroviral drugs with potential peripheral neurotoxicity (e.g., stavudine and didanosine).

#### **4. Adherence to therapy – Process to improve medication efficacy**

It has been reported that treatment abandonment rate reaches until 25% of all patients during the treatment (Veronesi & Focaccia, 1996). In developing countries such as Brazil, this rates decreased in the last years to 4,5%, but it is possible to find out in some regions values until 20,3% (Costa, Gonçalves & Menezes, 1998; Lima et al., 2001). The WHO's recommendation is that abandonment rates must be until 5%, however, it is possible to find in some countries such as Colombia abandonment rates of 65,6% (Mateus-Solarte & Carvajal-Barona, 2008).

In order to reduce the abandonment of therapy and ensure the correct treatment of patients some strategies have been adopted to improve adherence to therapy. According to WHO "Adherence corresponds to the behavior of one person - in terms of taking a drug, a diet or executing lifestyle changes - that is in accordance with recommendations arising from health professionals" (WHO, 2003).

Adherence to therapy is a serious problem responsible for the evolution of diseases and complications, loss of quality of life and even death. In the United States it is estimated that

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 545

therapy is not immediately improve the main symptoms, should be established an accord with the patients which should be aware of the goals of therapy. Treatment should be simplified to suit the patient's situation. Complex dosing regimens tend have higher dropout rates. The health system has an important role in the adherence. Access to professional and medicines is a determining factor for treatment. There is no possibility to discuss adherence even if the patients do not have access to health care. In this case, it is essential to adopt policies to control diseases such as tuberculosis ensuring access to the

Several strategies have been described in the literature to promote adherence to therapy. Educational interventions may involve pedagogical strategies (oral or written) aiming increase the knowledge. This strategy can be used individually or collectively. The uses of

Behavioral interventions aimed changing certain habits. They are guided by theories about behavior change. In these strategies the role of health professionals is essential in promoting adherence. This one can work with combinations of rewards and communication strategies, such as reminders to join the proposed therapy. Praise for the efforts is the reward commonly used. The possibility of monitoring the condition at home is also very useful and

Interventions with affective character take into account social relationships and may be done with the patient or the family. Family visits provide a good opportunity for this type of intervention. The professional can intervene, in educational way, with all the family doing them to understand the situation and help the patient. In TB intervention the combination of

Some care should be taken into account in monitoring patients for tuberculosis to detect adverse effects or ineffective therapy since the beginning of treatment. After 3 months of treatment, most patients (90-95%) will have negative smears (American Thoracic Society, 2003). Thus, situations of therapeutical inefficacy will be characterized with smear-positive after this period. Among factors responsible for ineffective treatment can be mentioned: bacterial resistance, poor absorption of drugs, inadequate adherence to treatment and drug

Some adverse effects such as peripheral neuritis induced by isoniazid can be prevented by supplementation with vitamin B6 (pyridoxine). This approach should be considered in special situations such as malnutrition, pregnancy, HIV co-infection and alcohol abuse.

Monitoring liver function is especially important for patients with any risk factor. About 20% of patients will have elevated levels of liver transaminases without symptoms. In such cases, after treatment the enzymes return to levels considered normal. Hepatitis should be suspected only when the elevation of transaminases is three to five times higher than considered normal associated with symptoms such as nausea, vomiting and abdominal pain. Some risk factors for hepatotoxicity are: chronic liver disease or hepatitis, alcohol abuse, use of isoniazid, female, HIV infection, low body mass index and age over 60 years

written or audiovisual materials help the patient to understand the strategy.

strategies seems to be more appropriate to increase therapy adherence.

enables patient involvement in the action plan proposed.

**5. Pharmacotherapeutic monitoring** 

interactions (Figure 3).

entire population.

the cost of low compliance reach over \$ 147 billion (Council on Patient Information and Education, 2007). WHO estimate that rate of adherence of patients which treat chronic diseases is only 50% (WHO, 2003). These same values were found in a study with TB patients (Cuneo, 1989).

Literature reports show that interventions to promote adherence are effective in short treatments, but these same interventions are less effective in prolonged treatments. In the latter situation it requires a combination of strategies, becoming more complex the interventions performed by health professionals (Haynes et al., 2009).

Several methods are available in the literature to assess adherence to treatment. These methods can be divided into direct and indirect. In direct methods is possible to quantify the drug in the body. These methods are expensive, invasive and not available for all drugs, however are suitable methods to say whether or not adherence to therapy allowing include adjust the dose. The indirect methods allow assessing the use of medicines by health professionals. These methods are usually overestimated and with low sensitivity. Among the indirect methods can include: patient's self-report; prescriber's report; pill count; measurement of prescription replacement (allowing indirect evaluation of non-compliance by the lack of access to medication). Strategies using devices of separation and counting of medicines may be useful for patients who do not understand the regimen. These devices can be purchased at pharmacies and separate properly in accordance with the therapy the patient by the pharmacist.

However, in the therapy of tuberculosis, one of the strategies most used and recommended by the World Health Organization (WHO) treatment involves supervised therapy (DOTS - Directly Observed Therapy). DOTS strategy includes the delivery of a short course of standard drugs, lasting 6 months for new Patients (and 8 months for retreatment Patients). The delivery includes the direct observation of therapy (DOT), either by a health worker or by caregiver (WHO, 2002).

During the promotion of strategies to increase adherence is important to consider some factors related to therapy adherence that may be related to: patient, health professional, the disease condition, treatment and health care system (and the policies government).

With regard to the patient an important strategy to be adopted is empowerment. This strategy is an educational intervention that aims to transfer responsibilities to patients which is tone of the most important person responsible to conduct the therapy, based in the principle that the decision to follow or not the treatment is only taken by the patient. The patient must know about the disease and treatment to participate in the process of using the medicine. Factors such as social stigma are common in patients with tuberculosis, however, this factor must be worked in the intervention. Economic factors and access to health services are factors that also interfere with the patient's decision to adhere to therapy or not.

With regard to health professionals, active participation is required in treating patients, because is often the situation that them cannot understand the proper way to use the antitubercular drugs. The treatment cannot be seen as the sole responsibility of the patient. Health professionals have a large portion of the contribution of adherence to treatment. With regard to health status, disease severity and the observation of improvement with the medicines use is important for maintenance adherence. In situations where the goal of

the cost of low compliance reach over \$ 147 billion (Council on Patient Information and Education, 2007). WHO estimate that rate of adherence of patients which treat chronic diseases is only 50% (WHO, 2003). These same values were found in a study with TB

Literature reports show that interventions to promote adherence are effective in short treatments, but these same interventions are less effective in prolonged treatments. In the latter situation it requires a combination of strategies, becoming more complex the

Several methods are available in the literature to assess adherence to treatment. These methods can be divided into direct and indirect. In direct methods is possible to quantify the drug in the body. These methods are expensive, invasive and not available for all drugs, however are suitable methods to say whether or not adherence to therapy allowing include adjust the dose. The indirect methods allow assessing the use of medicines by health professionals. These methods are usually overestimated and with low sensitivity. Among the indirect methods can include: patient's self-report; prescriber's report; pill count; measurement of prescription replacement (allowing indirect evaluation of non-compliance by the lack of access to medication). Strategies using devices of separation and counting of medicines may be useful for patients who do not understand the regimen. These devices can be purchased at pharmacies and separate properly in accordance with the therapy the

However, in the therapy of tuberculosis, one of the strategies most used and recommended by the World Health Organization (WHO) treatment involves supervised therapy (DOTS - Directly Observed Therapy). DOTS strategy includes the delivery of a short course of standard drugs, lasting 6 months for new Patients (and 8 months for retreatment Patients). The delivery includes the direct observation of therapy (DOT), either by a health worker or

During the promotion of strategies to increase adherence is important to consider some factors related to therapy adherence that may be related to: patient, health professional, the

With regard to the patient an important strategy to be adopted is empowerment. This strategy is an educational intervention that aims to transfer responsibilities to patients which is tone of the most important person responsible to conduct the therapy, based in the principle that the decision to follow or not the treatment is only taken by the patient. The patient must know about the disease and treatment to participate in the process of using the medicine. Factors such as social stigma are common in patients with tuberculosis, however, this factor must be worked in the intervention. Economic factors and access to health services are factors that also interfere with the patient's decision to adhere to therapy or not. With regard to health professionals, active participation is required in treating patients, because is often the situation that them cannot understand the proper way to use the antitubercular drugs. The treatment cannot be seen as the sole responsibility of the patient. Health professionals have a large portion of the contribution of adherence to treatment. With regard to health status, disease severity and the observation of improvement with the medicines use is important for maintenance adherence. In situations where the goal of

disease condition, treatment and health care system (and the policies government).

interventions performed by health professionals (Haynes et al., 2009).

patients (Cuneo, 1989).

patient by the pharmacist.

by caregiver (WHO, 2002).

therapy is not immediately improve the main symptoms, should be established an accord with the patients which should be aware of the goals of therapy. Treatment should be simplified to suit the patient's situation. Complex dosing regimens tend have higher dropout rates. The health system has an important role in the adherence. Access to professional and medicines is a determining factor for treatment. There is no possibility to discuss adherence even if the patients do not have access to health care. In this case, it is essential to adopt policies to control diseases such as tuberculosis ensuring access to the entire population.

Several strategies have been described in the literature to promote adherence to therapy. Educational interventions may involve pedagogical strategies (oral or written) aiming increase the knowledge. This strategy can be used individually or collectively. The uses of written or audiovisual materials help the patient to understand the strategy.

Behavioral interventions aimed changing certain habits. They are guided by theories about behavior change. In these strategies the role of health professionals is essential in promoting adherence. This one can work with combinations of rewards and communication strategies, such as reminders to join the proposed therapy. Praise for the efforts is the reward commonly used. The possibility of monitoring the condition at home is also very useful and enables patient involvement in the action plan proposed.

Interventions with affective character take into account social relationships and may be done with the patient or the family. Family visits provide a good opportunity for this type of intervention. The professional can intervene, in educational way, with all the family doing them to understand the situation and help the patient. In TB intervention the combination of strategies seems to be more appropriate to increase therapy adherence.

#### **5. Pharmacotherapeutic monitoring**

Some care should be taken into account in monitoring patients for tuberculosis to detect adverse effects or ineffective therapy since the beginning of treatment. After 3 months of treatment, most patients (90-95%) will have negative smears (American Thoracic Society, 2003). Thus, situations of therapeutical inefficacy will be characterized with smear-positive after this period. Among factors responsible for ineffective treatment can be mentioned: bacterial resistance, poor absorption of drugs, inadequate adherence to treatment and drug interactions (Figure 3).

Some adverse effects such as peripheral neuritis induced by isoniazid can be prevented by supplementation with vitamin B6 (pyridoxine). This approach should be considered in special situations such as malnutrition, pregnancy, HIV co-infection and alcohol abuse.

Monitoring liver function is especially important for patients with any risk factor. About 20% of patients will have elevated levels of liver transaminases without symptoms. In such cases, after treatment the enzymes return to levels considered normal. Hepatitis should be suspected only when the elevation of transaminases is three to five times higher than considered normal associated with symptoms such as nausea, vomiting and abdominal pain. Some risk factors for hepatotoxicity are: chronic liver disease or hepatitis, alcohol abuse, use of isoniazid, female, HIV infection, low body mass index and age over 60 years

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 547

postpone antiretroviral therapy until TB treatment is complete. However, the optimal time for starting ATR therapy should be individualized based on initial response to TB treatment

A patient who cannot take efavirenz, and when rifabutin is not available, the alternative is nevirapine - with rifampin. The pharmacokinetic effect of the rifamycin is moderate in this regimen. When used with isoniazid, rifampin, and pyrazinamide, there is some concern about hepatotoxicity. However, given the risk of reduced viral susceptibility and resistance development associated with subtherapeutic antiretroviral drug levels of nevirapine, some experts recommend that alternative antimycobacterial agents be considered in patients

Other alternatives for patients who cannot take efavirenz, and when rifabutin is not available, are as follows: rifampin with a) zidovudine/lamivudine/abacavir/tenofovir treatment; b) zidovudine/lamivudine/tenofovir or c) zidovudine/lamivudine/abacavir. The toxicity of these alternative regimens is primarily anemia. Pharmacokinetic concerns are a 50% decrease in zidovudine and the effect on abacavir is not yet evaluated. For the treatment of latent TB infection, a nine-month regimen of isoniazid may be

In general, treatment of TB /HIV in the context of ATR therapy is complex and requires an individualized approach. Experts in the treatment of HIV-related tuberculosis should be consulted, and TB and HIV care providers should work in close coordination throughout

Additional care should be conducted with patients who have resistant infection. To decrease the resistance some principles should be adopted: a) the scheme more effective should be adopted, b) the therapeutic regimen should include at least three antimycobacterial drugs, c) the treatment should be daily and preferably monitorate by health professional and d)

In situations of resistance to isoniazid, one possibility is the use of rifampicin, pyrazinamide, ethambutol (and some fluoroquinolone) for a period of 6 months. For resistance to rifampicin and isoniazid regimen can be use fluoroquinolone, pyrazinamide and ethambutol associated with some other drug for a period of 18-24 months. In situations of resistance to rifampin, isoniazid, pyrazinamide (or ethambutol) the scheme would be to use a fluoquinolone (ethambutol or pyrazinamide depending on the susceptibility) and two

In cases of latent infection, correctly detected, should be assessed the risk and benefit of treatment. People living with HIV, immunocompromised, malnourished, drug users and from endemic regions with positive test to TB are considered at risk of developing tuberculosis. A study by Wilkinson and colleagues (1998) demonstrated that treatment for latent infection in HIV people decreased by 43% the risk of developing tuberculosis (Wilkinson et al., 1998). Among the schemes that have been proposed that it should be noted: a) isoniazid for 6-9 months in the absence of active infection b) rifampicin and

isoniazid for 3 months and others (Balcells et al. 2006; Ena et al., 2005).

already receiving effective nevirapine-containing antiretroviral therapy.

and occurrence of adverse effects such as IRIS.

the best treatment considering patient quality of life.

medicines should not be left for possible "future use".

alternative agents for a period of 24 months.

considered.

Fig. 3. Some factors responsible to inefficacy.

The action plan for monitoring patients with hepatotoxicity during the treatment includes withdrawal of all medicines. After the reduction of transaminases levels, reintroduction of individual drug must be re-thinking. It is recommended to start treatment with rifampicin (less hepatotoxic), and between 3-7 days to introduce a new drug. Treatment regimens without isoniazid are possible. In this last case, one possibility is treating the patient with rifampicin, ethambutol and pyrazinamide for six months.

Drug interactions should be identified and prevented. Beyond the hepatotoxic effect already discussed, some antituberculosis drugs like rifampicin are metabolic enzymatic inducers and may interfere with the metabolism of many drugs. One example is the oral contraceptive whose effectiveness can be compromised with concomitant use of rifampicin. In these situations alternative reproductive control methods should be used. Isoniazid may increase serum levels of drugs of narrow therapeutic index such as theophylline used by asthma patients.

Regimen that includes rifabutin is generally preferred, as rifabutin appears to be as effective as rifampin but is a much less potent inducer of CYP450 3A4. Non rifamycin-containing regimens is related to higher rates of treatment relapse and failure; longer treatment duration with increased adverse effects and higher mortality rates. This regimen is only recommended to patients who are intolerant of rifamycins or are infected with rifamycinresistant strains.

Usual dosages of rifampin may be used in patients receiving ritonavir 600 mg twice/day or ritonavir 400 mg twice/day in combination with another PI at reduced dosage (e.g., saquinavir 400 mg twice/ day). If the patients have not begun antiretroviral therapy at the time TB treatment is initiated, clinicians may also consider using rifampin and postponing ATR therapy, because in the early HIV stage of the disease, there is low risk of HIV disease progression or death. During this period the physician may monitor CD4 cell count and

The action plan for monitoring patients with hepatotoxicity during the treatment includes withdrawal of all medicines. After the reduction of transaminases levels, reintroduction of individual drug must be re-thinking. It is recommended to start treatment with rifampicin (less hepatotoxic), and between 3-7 days to introduce a new drug. Treatment regimens without isoniazid are possible. In this last case, one possibility is treating the patient with

Drug interactions should be identified and prevented. Beyond the hepatotoxic effect already discussed, some antituberculosis drugs like rifampicin are metabolic enzymatic inducers and may interfere with the metabolism of many drugs. One example is the oral contraceptive whose effectiveness can be compromised with concomitant use of rifampicin. In these situations alternative reproductive control methods should be used. Isoniazid may increase serum levels of drugs of narrow therapeutic index such as theophylline used by

Regimen that includes rifabutin is generally preferred, as rifabutin appears to be as effective as rifampin but is a much less potent inducer of CYP450 3A4. Non rifamycin-containing regimens is related to higher rates of treatment relapse and failure; longer treatment duration with increased adverse effects and higher mortality rates. This regimen is only recommended to patients who are intolerant of rifamycins or are infected with rifamycin-

Usual dosages of rifampin may be used in patients receiving ritonavir 600 mg twice/day or ritonavir 400 mg twice/day in combination with another PI at reduced dosage (e.g., saquinavir 400 mg twice/ day). If the patients have not begun antiretroviral therapy at the time TB treatment is initiated, clinicians may also consider using rifampin and postponing ATR therapy, because in the early HIV stage of the disease, there is low risk of HIV disease progression or death. During this period the physician may monitor CD4 cell count and

Fig. 3. Some factors responsible to inefficacy.

asthma patients.

resistant strains.

rifampicin, ethambutol and pyrazinamide for six months.

postpone antiretroviral therapy until TB treatment is complete. However, the optimal time for starting ATR therapy should be individualized based on initial response to TB treatment and occurrence of adverse effects such as IRIS.

A patient who cannot take efavirenz, and when rifabutin is not available, the alternative is nevirapine - with rifampin. The pharmacokinetic effect of the rifamycin is moderate in this regimen. When used with isoniazid, rifampin, and pyrazinamide, there is some concern about hepatotoxicity. However, given the risk of reduced viral susceptibility and resistance development associated with subtherapeutic antiretroviral drug levels of nevirapine, some experts recommend that alternative antimycobacterial agents be considered in patients already receiving effective nevirapine-containing antiretroviral therapy.

Other alternatives for patients who cannot take efavirenz, and when rifabutin is not available, are as follows: rifampin with a) zidovudine/lamivudine/abacavir/tenofovir treatment; b) zidovudine/lamivudine/tenofovir or c) zidovudine/lamivudine/abacavir. The toxicity of these alternative regimens is primarily anemia. Pharmacokinetic concerns are a 50% decrease in zidovudine and the effect on abacavir is not yet evaluated. For the treatment of latent TB infection, a nine-month regimen of isoniazid may be considered.

In general, treatment of TB /HIV in the context of ATR therapy is complex and requires an individualized approach. Experts in the treatment of HIV-related tuberculosis should be consulted, and TB and HIV care providers should work in close coordination throughout the best treatment considering patient quality of life.

Additional care should be conducted with patients who have resistant infection. To decrease the resistance some principles should be adopted: a) the scheme more effective should be adopted, b) the therapeutic regimen should include at least three antimycobacterial drugs, c) the treatment should be daily and preferably monitorate by health professional and d) medicines should not be left for possible "future use".

In situations of resistance to isoniazid, one possibility is the use of rifampicin, pyrazinamide, ethambutol (and some fluoroquinolone) for a period of 6 months. For resistance to rifampicin and isoniazid regimen can be use fluoroquinolone, pyrazinamide and ethambutol associated with some other drug for a period of 18-24 months. In situations of resistance to rifampin, isoniazid, pyrazinamide (or ethambutol) the scheme would be to use a fluoquinolone (ethambutol or pyrazinamide depending on the susceptibility) and two alternative agents for a period of 24 months.

In cases of latent infection, correctly detected, should be assessed the risk and benefit of treatment. People living with HIV, immunocompromised, malnourished, drug users and from endemic regions with positive test to TB are considered at risk of developing tuberculosis. A study by Wilkinson and colleagues (1998) demonstrated that treatment for latent infection in HIV people decreased by 43% the risk of developing tuberculosis (Wilkinson et al., 1998). Among the schemes that have been proposed that it should be noted: a) isoniazid for 6-9 months in the absence of active infection b) rifampicin and isoniazid for 3 months and others (Balcells et al. 2006; Ena et al., 2005).

Health Interventions to Improve the Medication Efficacy in Tuberculosis Treatment 549

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All these factors must be taken into account during the pharmacotherapeutic monitoring of tuberculosis patients to adequate the therapy and improve quality of life of patients with tuberculosis.

#### **6. Conclusion**

The current TB treatment presents several challenges. The absence of development new drugs is the cruelest face of this disease. This fact associated with inadequate treatment and TB-drug resistance contributes to survival of the disease today. Anti-TB therapy has several problems relate to noncompliance, lack of adherence to therapy, patients with comorbidity, socio-economic factors, environmental factors and problems related to drugs such as adverse effects and drug interactions. Health interventions can improve the medication efficacy, reduce the resistance and improve patients quality of life. Pharmacotherapeutic follow-up and adequate strategies to increase therapy adherence are important factors to be monitored during TB therapy. It is important to keep in mind that interventions that guarantee the adequate use of the medication are the first step to improve TB treatment.

#### **7. References**


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### *Edited by Pere-Joan Cardona*

Mycobacterium tuberculosis is a disease that is transmitted through aerosol. This is the reason why it is estimated that a third of humankind is already infected by Mycobacterium tuberculosis. The vast majority of the infected do not know about their status. Mycobacterium tuberculosis is a silent pathogen, causing no symptomatology at all during the infection. In addition, infected people cannot cause further infections. Unfortunately, an estimated 10 per cent of the infected population has the probability to develop the disease, making it very difficult to eradicate. Once in this stage, the bacilli can be transmitted to other persons and the development of clinical symptoms is very progressive. Therefore the diagnosis, especially the discrimination between infection and disease, is a real challenge. In this book, we present the experience of worldwide specialists on the diagnosis, along with its lights and shadows.

Understanding Tuberculosis

Global Experiences and Innovative

Approaches to the Diagnosis

*Edited by Pere-Joan Cardona*

ISBN 978-953-307-938-7

ISBN 978-953-51-4360-4

Understanding Tuberculosis - Global Experiences and Innovative Approaches to the Diagnosis

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