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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#### **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.

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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.