**3.1 IS***6110* **in the detection of members of the MTB complex**

TB is a major public health problem in humans affecting many countries and large numbers of people. There are many reasons to explain the global relevance of this disease, including poverty, the limited vaccine efficacy and the persistence of the pathogen itself. One crucial factor is the difficulty in diagnosis TB. Currently, the main impediment is the lack of adequately sensitive, specificity and rapid tests. Culture and smear microscopy are probably the most common tools used worldwide for confirming the identification of TB in clinical samples. But culture is time consuming and smear microscopy is not specific enough. This has led to its gradual replacement in the developed world by more sensible, specific and rapid methods, such as PCR.

Recently, Sankar and cols (2011b) have been suggested variations into the sequence of IS*6110* from different strains of *M. tuberculosis*, which could have implications in its

IS*6110* does not have a known target or consensus sequence, it has been found within ORFs and intergenic regions (see part 4.1). It may be present up to 25 copies per genome in *M. tuberculosis* (Brosch et al., 2000), only a few number of strains have no copies of this IS (see

Many functions have been shown by the IS*6110*: (i) activation of genes during infection (Safi et al., 2004) (ii) participation in the evolution as an epidemiological marker (van Embden et al., 1993) (iii) activation of downstream genes with an activity promoter orientationdependent (Soto et al., 2004). Finally, it has been suggested that the presence of IS*6110* in *M. bovis* could participate in the adaptation of this bacteria to a particular host, animal or

Soon after the discovery of IS*6110* as a specific element in MTBC, its usefulness as diagnostic tool was explored. Subsequently, at the beginning of the nineties it was demonstrated that two strains isolated of different episodes of a patient had the same IS*6110*-RFLP pattern, in turn, a high degree of polymorphism was observed between strains isolated from different patients (Otal et al., 1991). The fact that IS*6110* varies in copy number and location in the bacterial genomes, along with its stability over time showed their usefulness in genotyping of the MTBC. This IS has been successfully used throughout the world for identifying and

TB is a major public health problem in humans affecting many countries and large numbers of people. There are many reasons to explain the global relevance of this disease, including poverty, the limited vaccine efficacy and the persistence of the pathogen itself. One crucial factor is the difficulty in diagnosis TB. Currently, the main impediment is the lack of adequately sensitive, specificity and rapid tests. Culture and smear microscopy are probably the most common tools used worldwide for confirming the identification of TB in clinical samples. But culture is time consuming and smear microscopy is not specific enough. This has led to its gradual replacement in the developed world by more sensible, specific and

human (Otal et al., 2008). Several of these features are being reviewed herein.

usefulness as target of PCR detection.

Fig. 1. Structural organization of IS*6110*

**3. The heads: Usefulness of IS***6110*

characterize members of this complex.

rapid methods, such as PCR.

**3.1 IS***6110* **in the detection of members of the MTB complex** 

part 3.1.2).

Because of the increased accessibility and convenience of PCR-based detection techniques, these are suitable to replace conventional culture methods. Since bacterial growth is not required, PCR can give results rapidly in as short a period as 1 day. Further PCR modifications, as nested–PCR or multiplex-PCR, can be used to improve results. Over the years, a significant improvement of PCR technologies has been achieved with the development of real-time PCR for the detection of target genes of *M. tuberculosis* in clinical specimens. The main advantages of real-time PCR are a shortened turnaround time, automation of the amplification and product detection and a decreased in the risk of crosscontamination (Espy et al., 2006).

#### **3.1.1 Advantages of IS***6110* **as target of the MTBC**

To obtain species-specific pathogen identification and detection in clinical samples, specific primers have been designed and tested using PCR-based methods, targeting different genomic sequences of *M. tuberculosis*. These have included IS*6110*, *hsp65*, TRC4 and *mpt40* (Bannalikar et al., 2006; Narayanan et al, 2001; Savekoul et al., 2006; Tumwasorn et al., 1996; Wei et al., 1999). Among these, the most widely investigated has been the IS*6110* being reported as a specific sequence of MTBC (Brisson-Noel et al. 1991; Eisenach, 1994; Sankar et al., 2011a). IS*6110* is an ideal target for PCR. IS*6110* is usually a multi-copy element and randomly distributed throughout the genome. The presence of multiple copies improves the sensitivity of the PCR amplification (Mathema et al., 2006; Sankar et al., 2011a).

Different oligonucleotides derived from that sequence have been successfully used to detect *M. tuberculosis* in all type of clinical specimens. Table 2 summarizes a list of the primers more frequently used in the literature. A problem found was that authors give different names to the same primers. The primers IS1 and IS2 (Eisenach et al., 1990) are the most frequently used, these oligonucleotides amplified a final product of 123 bp from 759 to 881 nucleotide position of IS*6110* (Table 2).

A search in the databases PubMed since 1991 using "IS*6110*" and "diagnostic" as keywords, allowed the identification of 138 papers that showed how IS*6110* could be a useful tool in diagnostic of TB. In 105 of these papers the diagnostic is based on PCR. Up to 5 of the 11 works published during the seven first months of 2011 applied the real time PCR technique in tuberculosis diagnostic using IS*6110* as target sequence.

In most of the cases the authors applied in-house PCR methods and compared results to other methods. Some authors concluded that IS*6110*-based PCR could be used routinely in clinical laboratories for rapid detection of *M. tuberculosis*, in sputum samples allowing early diagnosis and treatment (Ereqat et al., 2011). Evaluation of in-house PCR showed that variability in sensitivity and specificity is high (Cho et al., 2007).

The usefulness of IS*6110* in the detection and identification of MTBC in clinical samples has been demonstrated in many studies, either detecting IS*6110* as single target (Sankar et al., 2010a; Gupta et al., 2010; Inoue et al., 2011) or together to other specific targets (Sankar et al., 2010b; Leung et al., 2011). Multiplex PCR assay can be used for the simultaneous detection of other coinfections in clinical samples (Boondireke et al., 2010).

Additionally, in some cases, the location of IS*6110* specific to one strain can be used. PCR with primers targeting IS*6110* and the flanking region allowed identify and differentiate that

IS*6110* the Double-Edged Passenger 65

good approach could be a multiplex real-time PCR targeting IS*6110* and another target, as

As for DNA detection, another problem using IS*6110*-PCR is that it can detect non-viable mycobacteria for patients with earlier culture-positive specimens that had become culture

*M. bovis* strains usually contain one to five copies of IS*6110* (Otal et al., 2008), making the use of this IS less advantageous for the detection of this bacteria. The use of an immunomagnetic separation capture followed by PCR based on IS*6110* showed a detection threshold corresponding from 10 CFU in PBS to 1000 CFU for *M. bovis* in infected bovine fresh tissues, providing a sensitive, rapid and specific technique for the diagnosis of bovine tuberculosis

On the other hand, Sankar *et al*. analysed the sequence diversity of IS*6110* by using *in silico* approach. They found that IS*6110* insertion sequences harboured variations in its sequence and there are divergences within the copies of one strain. They collected a list of primers from those successfully used in the conventional PCR for the diagnosis of TB, but the reported data showed variation in the sensitivity and specificity for different regions of IS*6110*. All these data suggest that care must be taken when designing specific primers for IS*6110* detection. The authors recommended develop multiplex PCR assays targeting more

Indeed, the IS*6110* is still a favourite target sequence in the diagnosis of TB. Recently, a high sensitivity and specificity has been reported for the GeneXpert system, a real-time PCR assay that simultaneously detects both MTBC and rifampin resistance. However the accuracy of the Xpert MTB/RIF test for the detection of *M. tuberculosis* complex in paucibacillary samples was found to be lower than that of an in-house IS*6110* real time PCR

DNA fingerprinting of *M. tuberculosis,* based on the variability in both the number and the genomic position of IS*6110*, was standardised in 1993 to generate fingerprints, which permit comparison of the results obtained by different laboratories (van Embden et al., 1993). Such standardization has facilitated investigations into the international transmission of tuberculosis and has allowed to identify specific strains with unique properties such as high infectivity, virulence or drug resistance. Although other techniques based on this insertion sequence and other repetitive elements were described, IS*6110*-RFLP demonstrated the best discriminatory power and reproducibility and was accepted as the gold standard method for *M. tuberculosis* genotype (Kremer et al., 1999). Up to now it is the best-validated genotyping method, however, the requirement of growth culture and the poor discrimination found among the low copy number of IS*6110* strains (LCS), have led to search a better method, based on PCR, discriminative enough to be used on epidemiology. The application of IS*6110* as molecular tool has given a different global vision on TB. IS*6110*- RFLP has shown to be of great value in, among others: distinguish between recent transmission and reactivation, reinfection, mixed infections, studies of outbreaks, confirmation or rule out laboratory errors. It has also been useful to identify some strains

for example to use multiplex PCR using *hsp*65, protein B or MPB64 genes as targets.

negative following anti tuberculosis drug therapy (Causse et al., 2011).

than one region of the genome of *M. tuberculosis* (Sankar et al., 2011a).

routinely used since 2004 (Armand et al., 2011).

**3.2 Typing of members of the MTBC** 

(Garbaccio et al., 2010).


particular strain. This approach can be a useful tool in diagnosis and epidemiological studies.

Table 2. List of primers successfully used in IS*6110*-PCR for the detection of MTBC.

#### **3.1.2 Disadvantages of IS***6110* **as target of the MTBC**

However its wide applicability, targeting IS*6110* may not be by itself sensitive enough to diagnose 100% of the cases. Studies in India documented that 41% of *M. tuberculosis* isolates harboured a single copy of IS*6110* and 1% with no copy (Narayanan et al., 2002). In these situations the use of other targets for PCR in addition to IS*6110* for the detection of TB can be of help (Narayanan et al., 2001; Das et al., 1995; Chauhan et al., 2007; Kusum et al., 2011). A

particular strain. This approach can be a useful tool in diagnosis and epidemiological

GCCGGATCAGCGATCGT Real Time

TB1 / 104 - 123 GTGCGGATGGTGGCAGAGAT Nested PCR Boondireke

INS1 / 631-650 CGTGAGGGCATCGAGGTGGC PCR Hermans

ISI / 762-781 CCTGCGAGCGTAGGCGTCGG PCR Eisenach

CAAAGCCCGCAGGACCACGA Real Time

/ 367-392 CCGGCCAGCACGCTAATTAACGGTTC Nested PCR Cheng et al., 2004

However its wide applicability, targeting IS*6110* may not be by itself sensitive enough to diagnose 100% of the cases. Studies in India documented that 41% of *M. tuberculosis* isolates harboured a single copy of IS*6110* and 1% with no copy (Narayanan et al., 2002). In these situations the use of other targets for PCR in addition to IS*6110* for the detection of TB can be of help (Narayanan et al., 2001; Das et al., 1995; Chauhan et al., 2007; Kusum et al., 2011). A

Table 2. List of primers successfully used in IS*6110*-PCR for the detection of MTBC.

GCAAAGTGTGGCTAACCCTGAA

/1062 - 1077 CCGAGGCAGGCATCCA Real Time

CGTAGGCGTCGGTGACAAA

TGCCCAGGTCGACACATAGGTGA

CCACAGCCCGTCCCGCCGAT

/ 769- 746 TGTGGCCGGATCAGCGATCGTGGT

**3.1.2 Disadvantages of IS***6110* **as target of the MTBC** 

/ 455-472 CTGCACACAGCTGACCGA / 670-652 CGTTCGACGGTGCATCTG

TTCGACGGTGCATCTG

/1112 - 1132 GATCGTCTCGGCTAGTGCATT /1095 -1111 TCGGAAGCTCCTATGAC

TB4 / 387 -406 CCTGATGATCGGCGATGAAC TB2 /132 - 152 AGCACGATTCGGAGTGGGCA TB3 / 255 - 273 TCAGCGGATTCTTCGGTCG

IS2 / 854-883 CTCGTCCAGCGCCGCTTCGG

**Sequence (5´-3´) Method Reference** 

PCR

PCR

PCR

Leung et al., 2011

Lemaitre et al., 2004

et al., 2010

et al., 1990

et al., 1990

Inoue et al., 2011

studies.

**region** 

740

629

666

**Name / Target** 

MTB-F / 724-

MTB-R / 608 -

MTB-P / 651 -

INS2 / 856 -

TB130-F / 710

TB130-R / 817-

TB130-P / 742-

875


839

761

good approach could be a multiplex real-time PCR targeting IS*6110* and another target, as for example to use multiplex PCR using *hsp*65, protein B or MPB64 genes as targets.

As for DNA detection, another problem using IS*6110*-PCR is that it can detect non-viable mycobacteria for patients with earlier culture-positive specimens that had become culture negative following anti tuberculosis drug therapy (Causse et al., 2011).

*M. bovis* strains usually contain one to five copies of IS*6110* (Otal et al., 2008), making the use of this IS less advantageous for the detection of this bacteria. The use of an immunomagnetic separation capture followed by PCR based on IS*6110* showed a detection threshold corresponding from 10 CFU in PBS to 1000 CFU for *M. bovis* in infected bovine fresh tissues, providing a sensitive, rapid and specific technique for the diagnosis of bovine tuberculosis (Garbaccio et al., 2010).

On the other hand, Sankar *et al*. analysed the sequence diversity of IS*6110* by using *in silico* approach. They found that IS*6110* insertion sequences harboured variations in its sequence and there are divergences within the copies of one strain. They collected a list of primers from those successfully used in the conventional PCR for the diagnosis of TB, but the reported data showed variation in the sensitivity and specificity for different regions of IS*6110*. All these data suggest that care must be taken when designing specific primers for IS*6110* detection. The authors recommended develop multiplex PCR assays targeting more than one region of the genome of *M. tuberculosis* (Sankar et al., 2011a).

Indeed, the IS*6110* is still a favourite target sequence in the diagnosis of TB. Recently, a high sensitivity and specificity has been reported for the GeneXpert system, a real-time PCR assay that simultaneously detects both MTBC and rifampin resistance. However the accuracy of the Xpert MTB/RIF test for the detection of *M. tuberculosis* complex in paucibacillary samples was found to be lower than that of an in-house IS*6110* real time PCR routinely used since 2004 (Armand et al., 2011).

### **3.2 Typing of members of the MTBC**

DNA fingerprinting of *M. tuberculosis,* based on the variability in both the number and the genomic position of IS*6110*, was standardised in 1993 to generate fingerprints, which permit comparison of the results obtained by different laboratories (van Embden et al., 1993). Such standardization has facilitated investigations into the international transmission of tuberculosis and has allowed to identify specific strains with unique properties such as high infectivity, virulence or drug resistance. Although other techniques based on this insertion sequence and other repetitive elements were described, IS*6110*-RFLP demonstrated the best discriminatory power and reproducibility and was accepted as the gold standard method for *M. tuberculosis* genotype (Kremer et al., 1999). Up to now it is the best-validated genotyping method, however, the requirement of growth culture and the poor discrimination found among the low copy number of IS*6110* strains (LCS), have led to search a better method, based on PCR, discriminative enough to be used on epidemiology.

The application of IS*6110* as molecular tool has given a different global vision on TB. IS*6110*- RFLP has shown to be of great value in, among others: distinguish between recent transmission and reactivation, reinfection, mixed infections, studies of outbreaks, confirmation or rule out laboratory errors. It has also been useful to identify some strains

IS*6110* the Double-Edged Passenger 67

populations or by a combination of both (Barniol et al., 2009). One study was carried out to evaluate the origins of the resistant isolates in Finland, a country with a low incidence of TB. They have raised worries concerning the risk of disease in near-frontier contacts and they conclude that it is very probable that cases of MDR in Finland are mostly caught abroad

Several studies illustrate the situation in the highest TB incidence areas, such as two areas of India (Shanmugam et al., 2011; Purwar et al., 2011) or Uganda (Asiimwe et al., 2009). Other study that gives an overview of the distribution of genotypes of *M. tuberculosis* in Korea, found that drug resistance phenotypes were more strongly associated with Beijing family (see part 4.1.3). The Beijing genotype strains are also a major cause of TB (75% of MDR-TB) in the Aral Sea region, they are also strongly associated with drug resistance, independent of previous TB treatment and may be strongly contributing to the transmission of MDR-TB (Cox et al., 2005). In a population-based study carried out in rural China, the association between the Beijing family showed that a specific IS*6110*-RFLP and MIRU genotype 223325173533 were associated

The frequency and determinants of exogenous reinfection and of endogenous reactivation of TB in patients previously treated are poorly understood. The importance of reinfection as a cause for recurrence of TB is unclear and has potential public-health implications. Different studies have used IS*6110* genotyping to answer this question. The possibility of genotyping the isolates from initial and recurrent disease episodes allows to differentiate an episode of

At this respect, differences are shown depending on the incidences of TB and of the HIV status of the patients. In Spain, a country with a low incidence rate of TB, two studies on this issue were conducted. In the Gran Canaria Island, 2.4% of the cases had recurrent TB in a 5 years-period. Up to 44% of them corresponded to exogenous reinfection proved by IS*6110* genotypes (Caminero et al., 2001). In a second study conducted in Madrid extended twelve years, up to 3.1% of the patients had a second episode of TB. Only one recurrent case showed different genotypes, suggesting exogenous re-infection. Re-infection is possible among people in low-risk areas, but the rates are lower than those occurring in high-risk areas (Cacho et al., 2007). On the other hand, in countries with high incidence as India, most of the recurrences after successful treatment of TB are due to exogenous reinfection in HIVinfected persons, in contrast to endogenous reactivation in HIV-uninfected persons. Strategies for prevention and treatment of TB infection must take these findings into consideration (Narayanan et al., 2010). Conversely, one study carried out in Karinga Malawi, concluded that HIV increases the rate of recurrent TB by increasing the rate of reinfection disease (Crampin et al., 2010). Other authors reviewed different studies on recurrence and argued that, apart from extreme situations, the problem of recurrence due to

reinfection has few implications for TB-control programmes (Lambert et al., 2003).

A common dilemma of the different markers used for typing tuberculosis, including IS*6110*, is how to interpret the variability of the patterns. If two *M. tuberculosis* isolates from 2

with MDR and with increased transmissibility (Hu et al., 2011).

**3.2.2 Recurrent tuberculosis: Relapse or reinfection?** 

reinfection from that of relapse of TB.

**3.2.3 Limits of IS***6110* **as epidemiological tool** 

(Vasankari et al., 2011).

that may differ in transmission, suggesting that more virulent strains could show different pathogenesis and epidemiological characteristics. The establishment of Databases of the RFLP patterns has allowed to analyse the risk factors for tuberculosis and to detect the prevalent strains and/or the most transmitted strains, among the studied populations.

#### **3.2.1 Recent transmission & population studies**

The relatively higher rate of IS transposition on genomes compared to that of mutations in structural genes and other loci has elicited strong interest in the applications of ISs as genetic markers to study bacterial population genetics and phylogeny, especially for species with conserved genomes, as is the case of IS*6110* for *M. tuberculosis* (Fang et al., 2001). At the beginning of the nineties it was demonstrated the utility of IS*6110* in epidemiology (Otal et al., 1991). On the basis of IS*6110*-RFLP, recent transmission of TB has been associated to those patients whose isolates presented the same RFLP pattern or were included in a "cluster". The use of IS*6110*-RFLP analysis in population studies has considerably advanced our knowledge of the epidemiology of *M. tuberculosis*. Above all, large population studies have led to better understand how transmission occurs in the population. One study carried out in The Netherlands concluded that a short time span between the first two patients in a cluster was the strongest predictor for large cluster episodes (Kik et al., 2008). In this regard, after two population studies carried out in Zaragoza, Spain, along three years each, a change in patterns' transmission of TB was detected (López-Calleja et al., 2007). One susceptible strain designed as "MTZ" caused a susceptible outbreak involving more than one hundred inhabitants (18% of the TB cases). This kind of studies have made possible the detection and characterization of specific *M. tuberculosis* epidemic strains (Lopez-Calleja et al., 2009).

Recent studies indicate that multidrug-resistant *M. tuberculosis* has emerged in many countries for the past few years, without the concomitant development of health systems able to provide adequate treatment. MDR and XDR strains can be transmitted among the population (Bifani et al., 1996; Samper et al., 1997; Samper et al., 2005). It is known that the pattern of IS*6110*-RFLP does not usually change after acquisition of resistances of the strain, nevertheless, the complementary characterization of the genes conferring the resistance helps in contact tracing (Gavin et al., 2009). More recently, drug-resistance and molecular epidemiology of TB in the Murmansk region was investigated in a 2-year population-based surveillance of the civilian population. The study showed that MDR-TB strains were actively transmitted in the northern Russia (Mäkinen et al., 2011). In Ukraine, where increase of TB cases is maintained, the number of drug-resistant isolates was reported to be growing steadily, and transmission of drug-resistant isolates seems to contribute to the spread of resistant TB (Dymova et al., 2011). The MDR-TB genotyping databases allow the comparison of *M. tuberculosis* strains to improve the application of appropriate public health actions at a national level and, ideally, it should be extended across country borders (Bifani et al., 2001; Gavin et al., 2011; Ritacco et al., 2011).

The current population studies have been essential not only to gain a better understanding of how to implement effective TB control measures but also to analyse the importance of immigration. In Germany, the dynamics of TB transmission between TB high-prevalence immigrant and TB low-prevalence local populations confirm that there is no significant TB transmission from high to low-prevalence population. This could be probably due to the good performance of TB screening programmes, to low degree of mixing high to low

that may differ in transmission, suggesting that more virulent strains could show different pathogenesis and epidemiological characteristics. The establishment of Databases of the RFLP patterns has allowed to analyse the risk factors for tuberculosis and to detect the prevalent strains and/or the most transmitted strains, among the studied populations.

The relatively higher rate of IS transposition on genomes compared to that of mutations in structural genes and other loci has elicited strong interest in the applications of ISs as genetic markers to study bacterial population genetics and phylogeny, especially for species with conserved genomes, as is the case of IS*6110* for *M. tuberculosis* (Fang et al., 2001). At the beginning of the nineties it was demonstrated the utility of IS*6110* in epidemiology (Otal et al., 1991). On the basis of IS*6110*-RFLP, recent transmission of TB has been associated to those patients whose isolates presented the same RFLP pattern or were included in a "cluster". The use of IS*6110*-RFLP analysis in population studies has considerably advanced our knowledge of the epidemiology of *M. tuberculosis*. Above all, large population studies have led to better understand how transmission occurs in the population. One study carried out in The Netherlands concluded that a short time span between the first two patients in a cluster was the strongest predictor for large cluster episodes (Kik et al., 2008). In this regard, after two population studies carried out in Zaragoza, Spain, along three years each, a change in patterns' transmission of TB was detected (López-Calleja et al., 2007). One susceptible strain designed as "MTZ" caused a susceptible outbreak involving more than one hundred inhabitants (18% of the TB cases). This kind of studies have made possible the detection and characterization of specific *M. tuberculosis* epidemic strains (Lopez-Calleja et al., 2009).

Recent studies indicate that multidrug-resistant *M. tuberculosis* has emerged in many countries for the past few years, without the concomitant development of health systems able to provide adequate treatment. MDR and XDR strains can be transmitted among the population (Bifani et al., 1996; Samper et al., 1997; Samper et al., 2005). It is known that the pattern of IS*6110*-RFLP does not usually change after acquisition of resistances of the strain, nevertheless, the complementary characterization of the genes conferring the resistance helps in contact tracing (Gavin et al., 2009). More recently, drug-resistance and molecular epidemiology of TB in the Murmansk region was investigated in a 2-year population-based surveillance of the civilian population. The study showed that MDR-TB strains were actively transmitted in the northern Russia (Mäkinen et al., 2011). In Ukraine, where increase of TB cases is maintained, the number of drug-resistant isolates was reported to be growing steadily, and transmission of drug-resistant isolates seems to contribute to the spread of resistant TB (Dymova et al., 2011). The MDR-TB genotyping databases allow the comparison of *M. tuberculosis* strains to improve the application of appropriate public health actions at a national level and, ideally, it should be extended across country borders (Bifani et al., 2001;

The current population studies have been essential not only to gain a better understanding of how to implement effective TB control measures but also to analyse the importance of immigration. In Germany, the dynamics of TB transmission between TB high-prevalence immigrant and TB low-prevalence local populations confirm that there is no significant TB transmission from high to low-prevalence population. This could be probably due to the good performance of TB screening programmes, to low degree of mixing high to low

**3.2.1 Recent transmission & population studies** 

Gavin et al., 2011; Ritacco et al., 2011).

populations or by a combination of both (Barniol et al., 2009). One study was carried out to evaluate the origins of the resistant isolates in Finland, a country with a low incidence of TB. They have raised worries concerning the risk of disease in near-frontier contacts and they conclude that it is very probable that cases of MDR in Finland are mostly caught abroad (Vasankari et al., 2011).

Several studies illustrate the situation in the highest TB incidence areas, such as two areas of India (Shanmugam et al., 2011; Purwar et al., 2011) or Uganda (Asiimwe et al., 2009). Other study that gives an overview of the distribution of genotypes of *M. tuberculosis* in Korea, found that drug resistance phenotypes were more strongly associated with Beijing family (see part 4.1.3). The Beijing genotype strains are also a major cause of TB (75% of MDR-TB) in the Aral Sea region, they are also strongly associated with drug resistance, independent of previous TB treatment and may be strongly contributing to the transmission of MDR-TB (Cox et al., 2005). In a population-based study carried out in rural China, the association between the Beijing family showed that a specific IS*6110*-RFLP and MIRU genotype 223325173533 were associated with MDR and with increased transmissibility (Hu et al., 2011).
