**4.2. Molecular epidemiology of TB**

According to Cohn et al, 1997 [13], though the association of MDR-TB with AIDS has been well documented during outbreaks [22-24], the role of HIV infection as a risk factor for the devel‐ opment of drug-resistant TB in other settings was not clear [25]. In Kenya, Malawi, Tanzania, COte d'Ivoire, and France, drug resistance was not associated with HIV infection [26-30]. In contrast, in a survey of eight metropolitan areas of the United States, HIV infection was associated with resistance to antituberculous drugs, both within and outside the New York City area [31]. The acquired MDR-TB also occurs in largely immunocompetent hosts, which

The studies by Borrell and Gagneux [36] pointed out that, from a scientific point of view, the actual evidence for primary transmission of MDR -TB in HIV-negative individ‐ uals that has been confirmed by molecular methods is very limited, and that more stud‐ ies including molecular data are needed to know the true extent of primary MDR-TB &

Multidrug-resistant tuberculosis (MDR-TB) is a major challenge for TB control world‐ wide. Inadequate treatment of MDR-TB inevitably results in high mortality and the de‐ velopment of XDR-TB [37]. The study of Jeon et al, 2011 [38], shows how inadequate treatment has contributed to the high prevalence of MDR and XDR-TB in Korea. Accord‐ ing to Jeon et al, the three TB referral hospitals in the public sector are responsible for the management of MDR-TB in the public sector of Korea. This study showed poor out‐ come for patients with MDR-TB at the 3 TB hospitals in Korea: low treatment success rate (37.1%), high default rate (37.1%), and high all-cause mortality rate (31.2 %) during the 3-4 yr after treatment initiation. Since the National Tuberculosis Program (NTP) of Korea has focused on new cases, there have been limited nationwide data about the inci‐ dence and prevalence of MDR-TB and its treatment outcomes. Treatment success rate of

their study was the lowest ever reported among MDR-TB cohorts in Korea [38].

Many different definitions of molecular epidemiology have been published and all mention the use of molecular tools, but not all explicitly mention epidemiology. Molecular epidemiol‐ ogy is not just molecular taxonomy, phylogeny, or population genetics but the application of these techniques to epidemiologic problems [39]. Epidemiology attempts to identify factors that determine disease distribution in time and place, as well as factors that determine disease transmission, manifestation, and progression. Further, epidemiology is always motivated by an opportunity or possibility for intervention and prevention [39]. What distinguishes molecular epidemiology is both the "molecular," the use of the techniques of molecular biology to characterize nucleic acid- or amino acid-based content, and the "epidemiology," the study of the distribution and determinants of disease occurrence in human populations [39].

was seen in India, Korea, Nepal, and Bolivia [32-35].

190 Tuberculosis - Current Issues in Diagnosis and Management

**4. Molecular epidemiology of MDR-TB**

**4.1. Molecular epidemiology**

**3.4. Inadequate treatment and development of MDR and XDR-TB**

XDR –TB in a general population.

The molecular epidemiologic approach to studying tuberculosis epidemiology has identified several new observations that could not have been obtained by conventional epidemiologic or laboratory approaches [39]. Mycobacterial strain typing by means of molecular methods has become an important instrument for tuberculosis surveillance, control and prevention [40]. 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 [41], but other molecular typing techniques could be used as adjuncts in selected circumstances [42].

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 [43]. The direct-repeat locus in *M. tuberculosis* contains 10 to 50 copies of a 36-bp direct repeat, which are separated from one another by spacers that have different sequences. However, the spacer sequences between any two specific direct repeats are conserved among strains. Because strains differ in terms of the presence or absence of specific spacers, the pattern of spacers in a strain can be used for genotyping (spacer oligonu‐ cleotide typing, or "spoligotyping"). Spoligotyping has two advantages over IS*6110*-based genotyping. As small amounts of DNA are required, it can be performed on clinical samples or on strains of *M. tuberculosis* shortly after their inoculation into liquid culture. In addition the results of spoligotyping, which are expressed as positive or negative for each spacer, can be expressed in a digital format. However, spoligotyping has less power to discriminate among *M. tuberculosis* strains than does IS*6110*-based genotyping.

Mycobacterial interspersed repeat units (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. Two to eight alleles are at each of the 12 loci, yielding approximately 20 million possible combinations of alleles. The discriminatory power of MIRU genotyping is almost as great as that of IS*6110*-based geno‐ typing. 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.

The PGRS, the DR and the GTG repeated sequences have mainly been used for sub typ‐ ing strains for which differentiation by IS*6110* finger printing appeared insufficient. This is useful when *M. tuberculosis* strains contain no or lesser than six copies of IS*6110.* Ac‐ cording to a recorded study in Sri Lanka, 68% of the isolates had less than five copies which were similar to that of other countries in the Asian region, such as India, Malay‐ sia, Oman and Hong Kong [44].

strains (classifying into 10 clusters). Assuming that there was one source case in each cluster and the rest in the cluster were due to transmission, Hsu et al, concluded that 47.9% ([45 − 10]/

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193

To better understand the epidemiology of MDRTB, the New York City Tuberculosis Control Program began DNA genotyping of MDRTB strains from new cases in 1995 [48]. The objectives of the study were to provide descriptive molecular epidemiology of MDRTB cases in the city during 1995–1997 and to identify predominant MDR strains present during the three years, as well as the extent and risk factors for clustering among the tested cases. Genotyping results were available for 234 patients; 153 (65.4%) were clustered, 126 (82.3%) of them in eight clusters of >4 patients. Epidemiologic links were identified for 30 (12.8%) patients; most had been exposed to patients diagnosed before the study period. From the analysis, the largest cluster observed was from the "W" strain (59 patients) representing almost 25% of the 241 MDRTB patients during the 3 years. This strain caused a well-documented multi-institutional outbreak in New York City from 1990 through 1993 [49-53]. Strain "W1", which was isolated in seven patients, is a variant of the W strain. It had an additional IS*6110* copy and was a part of the W strain outbreak [52, 53]. Forty percent (12 of 30) of the epidemiologic links in this cohort were to patients with these two strains. According to Munsiff et al [48] these strains were likely transmitted in the early 1990s when MDRTB outbreaks and tuberculosis transmission were

To analyze the molecular epidemiology of *M. tuberculosis* strains at a hospital in Buenos Aires, Argentina, and mutations related to MDR and XDR-TB, Gonzalo et al [54], conducted a prospective case –control study. Spoligotyping identified predominance of the Haarlem family among the MDR TB cases (family responsible for the 1990s [55] outbreak) as well as the LAM and T families. A similar strain family distribution was reported for the French Departments of the Americas [56] and Turkey [57]. The Beijing family was seldom encountered in these areas, which is in line with recent observations in 7 countries in South America, including Argentina [58]. According to them [54] the MDR TB Haarlem2 strain appears to be more successful than other circulating MDR- TB strains and also than its susceptible counterpart (of

By genotyping all isolates and combining with the mutational results, Perdigão et al [59] were able to assess the isolates' genetic relatedness and determine possible transmission events. According to their study strains belonging to family Lisboa, characterized several years ago, were responsible for the majority of the MDR-TB. Even more alarming was the high prevalence of extensive drug-resistant tuberculosis (XDR-TB) among the MDR-TB isolates, which was

Mathematical models predict that the future of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) epidemic will depend to a large extent on the trans‐ mission efficiency or relative fitness of drug-resistant *Mycobacterium tuberculosis* compared to drug-susceptible strains. Molecular epidemiological studies comparing the spread of drug-

73) of the patients had MDR-TB due to recent transmission [47].

widespread in New York.

found to be 53%.

25 Haarlem2 strains, 20 were MDR TB).

**4.4. Transmission of MDR-TB and XDR-TB**

The study by Ghebremichael et al [45] determined the transmission pattern of TB strains in Sweden. By MIRU-VNTR 31 (45%) of the 69 patients with Beijing strains were found in altogether 7 clusters (2–11 per cluster), yielding 45 different patterns. Thus the MIRU-VNTR typing, with fewer and larger clusters, was less discriminatory than IS*6110* RFLP. The two strains where a possible epidemiological linkage was established differed in one allele and thus did not cluster in MIRU-VNTR. All strains that clustered by MIRU-VNTR were identical also by RD deletions, mutT gene polymorphism and Rv3135 gene analysis, but not by spoligotyping and IS1547. Four of the IS*6110* RFLP clusters contained isolates that differed by MIRU-VNTR. The combination of MIRUVNTR with RFLP resulted in the disappearance of two clusters, and a reduction of the number of isolates in two clusters, compared to the clustering observed with IS*6110* RFLP clustering alone. In this study they found that patients with DR Beijing strains have been diagnosed for more than a decade in Sweden. The majority of the patients were foreign born, and their country of origin reflects areas where the Beijing genotype is prevalent [45].

#### **4.3. Molecular epidemiology of MDR-TB**

A study by Calver et al [46], investigated an outbreak of tuberculosis using a molecular epidemiologic approach and clinical and epidemiologic data to identify inadequacies in the implemented DOTS-plus strategy that lead to the emergence of pre–XDR TB and XDR TB in South Africa. They genotyped the drug-resistant *M. tuberculosis* isolates using molecular techniques including insertion sequence (IS) *6110* RFLP, spoligotyping and MIRU typing (12 loci format). Genotyping results indicated an on-going transmission of drug-resistant TB, and contact tracing among case-patients in the largest cluster demonstrated multiple possible points of contact. Phylogenetic analysis demonstrated stepwise evolution of drug resistance, despite stringent treatment adherence. These findings suggested that existing TB control measures in South Africa were inadequate to control the spread of drug-resistant TB in their HIV co-infected population. Diagnosis delay and inappropriate therapy facilitated disease transmission and drug resistance.

Hsu et al, 2010 [47], investigated the transmission and predominant genotypes of MDR- TB in Eastern Taiwan using both spoligotyping and MIRU-VNTR. Of the tested MDR isolates of 73 (94%) Spoligotyping, identified the Beijing strain as the predominant genotype (n = 48, 66%), followed by Haarlem H3 (n = 15, 21%), T1 (n = 3, 4%) and East-African Indian 2 MANILLA (n = 1, 1%). Six (8%) isolates did not match any spoligotype in the SpolDB4 database. Using MIRU-VNTR typing, they observed a unique pattern in 27 isolates, and 46 had clustered pattern strains (10 clusters). According to them by MIRU-VNTR they observed an isolate in cluster 9, however from spoligotyping, it had a unique pattern and therefore they did not considered it as a clustered pattern strain. By considering both spoliotyping and MIRU-VNTR into account, 28 (38.4%) isolates were judged to have a unique pattern and 45 (61.6%) were clustered pattern strains (classifying into 10 clusters). Assuming that there was one source case in each cluster and the rest in the cluster were due to transmission, Hsu et al, concluded that 47.9% ([45 − 10]/ 73) of the patients had MDR-TB due to recent transmission [47].

To better understand the epidemiology of MDRTB, the New York City Tuberculosis Control Program began DNA genotyping of MDRTB strains from new cases in 1995 [48]. The objectives of the study were to provide descriptive molecular epidemiology of MDRTB cases in the city during 1995–1997 and to identify predominant MDR strains present during the three years, as well as the extent and risk factors for clustering among the tested cases. Genotyping results were available for 234 patients; 153 (65.4%) were clustered, 126 (82.3%) of them in eight clusters of >4 patients. Epidemiologic links were identified for 30 (12.8%) patients; most had been exposed to patients diagnosed before the study period. From the analysis, the largest cluster observed was from the "W" strain (59 patients) representing almost 25% of the 241 MDRTB patients during the 3 years. This strain caused a well-documented multi-institutional outbreak in New York City from 1990 through 1993 [49-53]. Strain "W1", which was isolated in seven patients, is a variant of the W strain. It had an additional IS*6110* copy and was a part of the W strain outbreak [52, 53]. Forty percent (12 of 30) of the epidemiologic links in this cohort were to patients with these two strains. According to Munsiff et al [48] these strains were likely transmitted in the early 1990s when MDRTB outbreaks and tuberculosis transmission were widespread in New York.

To analyze the molecular epidemiology of *M. tuberculosis* strains at a hospital in Buenos Aires, Argentina, and mutations related to MDR and XDR-TB, Gonzalo et al [54], conducted a prospective case –control study. Spoligotyping identified predominance of the Haarlem family among the MDR TB cases (family responsible for the 1990s [55] outbreak) as well as the LAM and T families. A similar strain family distribution was reported for the French Departments of the Americas [56] and Turkey [57]. The Beijing family was seldom encountered in these areas, which is in line with recent observations in 7 countries in South America, including Argentina [58]. According to them [54] the MDR TB Haarlem2 strain appears to be more successful than other circulating MDR- TB strains and also than its susceptible counterpart (of 25 Haarlem2 strains, 20 were MDR TB).

By genotyping all isolates and combining with the mutational results, Perdigão et al [59] were able to assess the isolates' genetic relatedness and determine possible transmission events. According to their study strains belonging to family Lisboa, characterized several years ago, were responsible for the majority of the MDR-TB. Even more alarming was the high prevalence of extensive drug-resistant tuberculosis (XDR-TB) among the MDR-TB isolates, which was found to be 53%.

#### **4.4. Transmission of MDR-TB and XDR-TB**

is useful when *M. tuberculosis* strains contain no or lesser than six copies of IS*6110.* Ac‐ cording to a recorded study in Sri Lanka, 68% of the isolates had less than five copies which were similar to that of other countries in the Asian region, such as India, Malay‐

The study by Ghebremichael et al [45] determined the transmission pattern of TB strains in Sweden. By MIRU-VNTR 31 (45%) of the 69 patients with Beijing strains were found in altogether 7 clusters (2–11 per cluster), yielding 45 different patterns. Thus the MIRU-VNTR typing, with fewer and larger clusters, was less discriminatory than IS*6110* RFLP. The two strains where a possible epidemiological linkage was established differed in one allele and thus did not cluster in MIRU-VNTR. All strains that clustered by MIRU-VNTR were identical also by RD deletions, mutT gene polymorphism and Rv3135 gene analysis, but not by spoligotyping and IS1547. Four of the IS*6110* RFLP clusters contained isolates that differed by MIRU-VNTR. The combination of MIRUVNTR with RFLP resulted in the disappearance of two clusters, and a reduction of the number of isolates in two clusters, compared to the clustering observed with IS*6110* RFLP clustering alone. In this study they found that patients with DR Beijing strains have been diagnosed for more than a decade in Sweden. The majority of the patients were foreign born, and their country of origin reflects areas where the Beijing

A study by Calver et al [46], investigated an outbreak of tuberculosis using a molecular epidemiologic approach and clinical and epidemiologic data to identify inadequacies in the implemented DOTS-plus strategy that lead to the emergence of pre–XDR TB and XDR TB in South Africa. They genotyped the drug-resistant *M. tuberculosis* isolates using molecular techniques including insertion sequence (IS) *6110* RFLP, spoligotyping and MIRU typing (12 loci format). Genotyping results indicated an on-going transmission of drug-resistant TB, and contact tracing among case-patients in the largest cluster demonstrated multiple possible points of contact. Phylogenetic analysis demonstrated stepwise evolution of drug resistance, despite stringent treatment adherence. These findings suggested that existing TB control measures in South Africa were inadequate to control the spread of drug-resistant TB in their HIV co-infected population. Diagnosis delay and inappropriate therapy facilitated disease

Hsu et al, 2010 [47], investigated the transmission and predominant genotypes of MDR- TB in Eastern Taiwan using both spoligotyping and MIRU-VNTR. Of the tested MDR isolates of 73 (94%) Spoligotyping, identified the Beijing strain as the predominant genotype (n = 48, 66%), followed by Haarlem H3 (n = 15, 21%), T1 (n = 3, 4%) and East-African Indian 2 MANILLA (n = 1, 1%). Six (8%) isolates did not match any spoligotype in the SpolDB4 database. Using MIRU-VNTR typing, they observed a unique pattern in 27 isolates, and 46 had clustered pattern strains (10 clusters). According to them by MIRU-VNTR they observed an isolate in cluster 9, however from spoligotyping, it had a unique pattern and therefore they did not considered it as a clustered pattern strain. By considering both spoliotyping and MIRU-VNTR into account, 28 (38.4%) isolates were judged to have a unique pattern and 45 (61.6%) were clustered pattern

sia, Oman and Hong Kong [44].

192 Tuberculosis - Current Issues in Diagnosis and Management

genotype is prevalent [45].

**4.3. Molecular epidemiology of MDR-TB**

transmission and drug resistance.

Mathematical models predict that the future of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) epidemic will depend to a large extent on the trans‐ mission efficiency or relative fitness of drug-resistant *Mycobacterium tuberculosis* compared to drug-susceptible strains. Molecular epidemiological studies comparing the spread of drugresistant to that of drug-susceptible strains have yielded conflicting results: MDR strains can be up to 10 times more or 10 times less transmissible than pan-susceptible strains [36].

appreciated [68] and because drug-resistant strains can exhibit heterogeneous fitness com‐

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195

An understanding of the epidemiology of multidrug resistant tuberculosis (MDR-TB) and the extensively drug-resistant tuberculosis (XDR-TB) is critical for effective control of the global burden of tuberculosis (TB). For a comprehensive study on epidemiology of multidrug

In all epidemiological studies it is essential to have a clear definition of a case of the disease being investigated by delineating the symptoms, signs or other characteristics indicating that a person has the disease. A clear definition of an exposed person is also necessary. This definition must include all the characteristics that identify a person as being exposed to the factor in question. In the absence of clear definitions of disease and exposure, it is very difficult

Future epidemiological studies on the transmission of drug-resistant TB should incorporate more comprehensive strain data, including specific drug resistance-conferring mutations and information on the strain genetic background. These variables, as well as their interaction, could play an important role in the transmission success of particular drug-resistant variants.

The investigators who conduct the studies on HIV/ TB need to consider other possible risk factors for drug resistance such as demographics; prior therapy, socioeconomic status, and

The research works on TB were supported by the grants, RG/2006/HS/07 NSF and 07-47 of NRC and by IFS, Sri Lanka. I am expressing my sincere gratitude to Professor Jennifer Perera and Dr. N.V. Chandrasekaran for their valuable guidance and Dr. D. Medagedara and Professor V. Thevanesam for their support in tuberculosis research and to Ms. R. P. Waniga‐

resistant tuberculosis (MDR-TB), please refer the reviews in the reference list.

pared to drug-susceptible strains [36].

**5. Conclusion**

**6. Future studies**

**Future Studies on Epidemiology**

to interpret the data from an epidemiological study.

tunge for technical support in preparation of manuscript.

**Future Studies on Transmission of TB**

**Future Studies on HIV/TB**

**Acknowledgements**

quality of TB control programs, etc.

Experimental work performed with model organisms has highlighted a level of complexity in the biology of bacterial drug resistance that is generally not considered during standard epidemiological studies of TB transmission. However, much more work is needed to under‐ stand the detailed molecular mechanisms and evolutionary forces that drive drug resistance in this pathogen. Such increased knowledge will allow for better epidemiological predictions and assist in the development of new tools and strategies to fight drug resistant TB [36].

In infectious disease epidemiology, the relevant measure that reflects the reproductive fitness of a pathogen is the number of secondary cases generated; this measure is also known as the basic reproductive rate, R0 [60]. In addition to the absolute number of secondary cases (i.e., absolute fitness), an often more useful measure is that of 'relative fitness', where the success of a particular pathogen variant is compared to the success of another. For example, the fitness of a drug-resistant bacterial strain can be expressed relative to the fitness of a drug-susceptible strain. In addition to epidemiological measures of relative fitness, differences in relative fitness can be measured experimentally [36].

The results of experimental studies performed with strains resistant to INH, SM or RMP suggested that, in clinical settings, there was a strong selection pressure for drug resistanceconferring mutations that cause minimal fitness defects [61]. Although these findings support the notion that virulence and competitive fitness assays can be predictive of the epidemiology of drug-resistant TB, they do not capture the overall complexity of the life cycle of *M. tuber‐ culosis* [36]. Although several mechanisms of compensatory evolution have been described in other bacteria [62] little work has been done on this topic in *M. tuberculosis.*

Various molecular tools have been developed to genotype *M. tuberculosis* strains [63]. These tools have been applied to molecular epidemiological investigation of TB transmission for many years. According to the standard concept, patient isolates sharing a particular genotype or DNA 'fingerprint' can be considered epidemiologically linked and represent cases of active TB transmission (i.e., they are clustered TB cases), whereas strains with distinct or 'unique' DNA patterns are thought to reflect reactivation of latent infections. They compared molecular epidemiological fitness estimates from two previous reviews and more recent studies [60, 64]. Overall, the relative fitness estimates for MDR-TB vary dramatically, ranging from an almost 10-fold increased fitness compared to fully drug-susceptible strains found in a study from Russia [65] to about 10-fold lower fitness in Mexico [66] other studies have reported that MDR strains do not cause any secondary cases at all [67]. The reasons for this high variability in relative fitness of MDR strains have likely to do with the differences in study design and setting, differences in sample size and different methodologies and also to the variation in the quality of the TB control programmes [36]. According to Borrell and Gagneux, in addition to methodological, socio-economic and environmental factors, the variation in MDR fitness also reflects biological heterogeneity. Current epidemiological evidence for transmission of MDRand XDR-TB, particularly compared to pan-susceptible TB, is very inconclusive. This can be partially explained by the fact that *M. tuberculosis* is more genetically diverse than is often appreciated [68] and because drug-resistant strains can exhibit heterogeneous fitness com‐ pared to drug-susceptible strains [36].
