First Insights into the Phylogenetic Diversity of Mycobacterium tuberculosis in Nepal

Background Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. Methods and Findings We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. Conclusions We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.


Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a global health threat with an estimated nine million incident cases and 440,000 multidrug-resistant TB cases worldwide [1]. The incidence of TB was 163 per 100,000 population in 2010, and multidrug resistance (MDR) occurred in 2.9% of new cases and 11.7% of previously treated cases based on the most recent drug resistance survey in 2006 [2,3]. In Nepal, the National Tuberculosis Control Programme adopted Directly Observed Short Course therapy (DOTS) in 1995.
There are several genotyping techniques to define the genetic diversity of M. tuberculosis [7,27,28]. Spoligotyping is a widely used genotyping technique [29,30]. It is based on the repetitive DNA region known as the Direct Repeat (DR) locus in M. tuberculosis [28]. This region is characterized by series of direct repeats interspersed by short unique regions called ''spacers''. However, these spacers exhibit a high rate of change, and convergent evolution can lead to identical genetic character states in phylogenetically unrelated strains [5,31]. By contrast, genomic deletions and single nucleotide polymorphism (SNPs) evolve more slowly. Recent advances in comparative genomics have led to the development of more robust markers to study the genetic diversity [7,[32][33][34][35][36], and are therefore ideal for determining phylogenetic lineages and sub-lineages [11].
Nepal lies between two high TB burden countries, India and China which together account for one third of the world's TB cases [37]. To date, there are no data on the phylogenetic diversity of M. tuberculosis in Nepal. The aims of the study were to describe the main M. tuberculosis lineages and spoligotypes circulating in Nepal, and to explore possible associations with clinical and epidemiological characteristics.

Ethics Statement
This study was approved by the Nepal Health Research Council, Nepal and the Ethics Committee of the Canton of Basel (EKBB), Switzerland. All study participants provided written informed consent. After diagnosis, the TB cases were referred to DOTS centers for treatment as provided by the Nepal Government's National TB Control Program.

Study Setting
The study was based on a convenience sample of TB patients mainly representing populations from Kathmandu and the surrounding area. TB suspects who reported symptoms of TB including cough for more than two weeks, chest pain, night sweat and fever were recruited at the German Nepal Tuberculosis Project (GENETUP), Kathmandu, Nepal. Patients already undergoing DOTS therapy were also enrolled, if found smearpositive during follow-up visits. GENETUP is a national reference laboratory, technically and financially supported by ''Kuratorium Tuberkulose in der Welt e. V.'' (Gauting, Germany), and is the main referral center for culture and drug susceptibility testing to diagnose MDR and extensively drug-resistant TB.

Study Population and Data Collection
We included a total of 261 culture-confirmed TB cases diagnosed between August 2009 and August 2010. We collected socio-demographic and clinical data including previous TB episodes, treatment history, HIV, and BCG vaccination status. The information was collected by physicians and trained medical and nursing staff. A new case of TB was defined as a patient who had not taken anti-TB drugs for at least one month according to WHO guidelines [38]. A previously treated case was defined as a patient who received TB treatment for one month or more. BCG vaccination status was defined based on the presence or absence of a BCG scar.

Culture, DNA Extraction and Identification of M. tuberculosis Complex
Sputum samples were cultured on Löwenstein Jensen (LJ) growth medium following standard microbiological laboratory procedures. The DNA was extracted by re-suspension of MTBC colonies in 500 ml of sterile distilled water, heat killed at 90uC for one hour, and centrifuged. The supernatants were preserved at 4uC until further use. MTBC strains were identified by multiplex polymerase chain reaction (PCR) by targeting the rpoB gene region. We used the forward primers K-0155 (59-TCCTCGAT-GACGCCGCTTTCT-39) and K-0209 (59-AYATCGAC-CACTTCGGYAACC-39), and the reverse primer K-0156 . PCR conditions were as follows: initial denaturation step for 5 minutes at 96uC, 35 amplification cycles of 96uC for 40 secs (denaturation), 62uC for 30 secs (annealing), 72uC for 1 min (extension), and a final extension cycle of 7 minutes at 72uC. The amplicons were separated by electrophoresis on a 2% agarose gel. The PCR yielded a 849 bp amplicon in M. tuberculosis isolates, compared to a 1539 bp amplicon in non-tuberculous mycobacteria. All M. tuberculosis isolates were stored in glycerol medium at -70 o C.

Determination of the Main M. tuberculosis Lineages
We determined the main phylogenetic lineages of M. tuberculosis by real-time PCR using fluorescence-labeled probes (Taqman, Applied Biosystems, USA) targeting lineage-specific SNPs as previously described [4,32,36].

Spoligotyping
Spoligotyping was performed according to the manufacturer's instructions, using commercially available kits from Isogen Bioscience BV (Maarssen, The Netherlands) [30,39]. Spoligotyping patterns were defined according to the definitions in the SITVITWEB database (http://www.pasteur-guadeloupe.fr:8081/ SITVIT_ONLINE/) accessed on October 22, 2012. The SITVITWEB global database has documented 7,105 spoligotyping patterns from a global collection of 53,816 strains [40]. All patterns that could not be assigned were considered orphan spoligotypes.

Molecular Drug Resistance Testing
As phenotypic drug susceptibility testing results were not available for all strains, we used molecular methods to detect drug resistance in our study. Molecular drug resistance testing was performed on all strains by direct sequencing of the hotspot regions of the target genes for rifampicin (rpoB), isoniazid (inhA promoter region and katG), and streptomycin (rpsL). MDR strains were then further sequenced and analyzed for ethambutol (embB), fluoroquinolones (gyrA) and aminoglycoside (rrs) resistance by sequencing of the relevant gene segments. For the rpoB region, we used an in-house PCR assay with primer pair K-0155 and K-0209 as described above. For all other target genes, PCR primers and PCR conditions were adapted from previously published studies [41][42][43].The sequences were analyzed with M. tuberculosis H37Rv as reference sequence using the Staden software package [44,45], and compared to the publicly available web-based database (http://www. tbdreamdb.com/) [46]. Any drug resistance was defined as resistance to isoniazid, rifampicin, streptomycin, ethambutol, fluoroquinolones, and/or aminoglycosides. MDR was defined as resistance to at least isoniazid and rifampicin.

Statistical Analyses
We used Chi-square test to test the statistical significance of differences between groups in binary variables, and the Kruskal Wallis rank test for continuous variables. Logistic regression models were used to compare patient characteristics associated with Lineage 2 (includes the Beijing genotype) compared to all other lineages (Lineages 1, 3 and 4), adjusted for age, sex, treatment history, BCG vaccination status, and any drug resistance. All statistical analyses were performed in STATA 10.1 (Stata Corp., College Station, TX, USA).

Mycobacterium tuberculosis Genotyping and Lineage Assignment
We analyzed a total of 261 M. tuberculosis isolates (one from each patient). The SNP-typing results showed the presence of four different M. tuberculosis lineages ( Table 1). The most  India or Tibet (LAM9, H3, T2-T3, T1, XI, H1, and H3) according to the SITVITWEB database. Among the 30 (11.5%) Lineage 1 strains, only 18 (60.0%) matched the SITs of the East African Indian (EAI) family. Only two SIT types SIT 138 (EAI5; n = 10), and SIT 11 (EAI3_IND; n = 4) were represented by more than one strain. However, SIT 1734 (EAI1_SOM) present as a single isolate in our dataset was not reported before from the Indian sub-continent according to the SITVITWEB database.
When comparing SNP typing with the spoligotyping results, we found one case of ''pseudo-Beijing'' spoligotpype as previously reported (Table 1) [31].

Association between M. tuberculosis Lineages and Patient Characteristics
We observed that the proportion of female sex was different across the four main M. tuberculosis lineages. Lineage 2 isolates were more common among females (41.7%), compared to other lineages (range 13.3% to 27.4%, overall p = 0.016, Table 2). Moreover, any drug resistance was more frequently detected in Lineage 2 isolates (31.0%) than in any other lineages (range 13.2% to 14.6%, overall p = 0.011). Other patient characteristics such as age, previous treatment history, or BCG vaccination were not significantly associated with any of the four lineages (Table 2).
Because Lineage 2 (includes Beijing genotype) has been previously associated with particular characteristics   [17,19,21,47], and because Lineage 2 was the second most common lineage in our sample, we tested whether these characteristics were also associated with Lineage 2 in our setting by comparing our Lineage 2 isolates to the other lineages combined (

Discussion
We analyzed 261 M. tuberculosis isolates from Nepal using SNP typing and spoligotyping. We found that four main phylogenetic lineages of M. tuberculosis were present in Nepal. Lineage 2 (East-Asian Lineage, includes the Beijing genotype) and Lineage 3 (CAS/Delhi) were the most frequent, while Lineage 1 (Indo-Oceanic Lineage) and Lineage 4 were less prevalent. Spoligotyping revealed a large genetic diversity with the predominant spoligotyping families being Beijing and CAS/Delhi, and nearly 14% of spoligotyping patterns previously unreported.
Because Nepal is geographically located between India and Tibet (China), we expected to observe similar M. tuberculosis genotypes in Nepal as in these neighboring countries. Indeed, Lineage 3 (corresponds to Delhi/CAS spoligotype), which was the most common M. tuberculosis genotype in our sample, was previously shown to be predominant in Northern India [48][49][50]. Similarly, Lineage 2 (includes Beijing), which was the second most common genotype in our study has been reported as the most frequent among TB cases from China (including Tibet) [22,[51][52][53][54][55]. The prevalence of the Beijing genotype of 32.2% in our study is in the range of the prevalence reported from other Asian countries, ranging from 17% in Malaysia to 72% in Japan [56]. Lineage 1 which is association with South-Indian region, Bangladesh and the Philippines was also present in our study sample [4].
We observed a discrepancy between SNP typing and spoligotyping results. Spoligotyping is based on the highly variable DR locus, and convergent evolution may therefore lead to homoplasy in spoligotyping patterns [5]. We found a strain with a Beijing spoligotype, which was assigned to Lineage 3 (includes CAS genotype) rather than to Lineage 2 (includes Beijing) based on alternative molecular markers. We have previously published this phenomenon as ''Pseudo-Beijing'' [31]. In Asian countries with a high prevalence of Beijing spoligotypes, it is likely that this phenomenon may be observed in other settings.
We found that Lineage 2 was associated with female sex, which is in line with a previous study from Vietnam [57]. In contrast to other studies [57,58] however, we found no evidence for an association between Lineage 2 and age. Our observation may be explained by bacterial factors or genetic host factors. Young and middle-aged women may be more likely to progress from infection to disease than men [59,60]. Alternatively, our results may be influenced by recruitment of more young females than young males into our study. Indeed, females were younger than males in our study population. Overall, our study population showed a male-to-female ratio of 2.3:1 which is similar to the global estimate of 1.9:1 reported by WHO [2], and may reflect differences in access to health care [61,62]. Furthermore, sex differences in TB case notification rates among males and females have been noted before in other settings [63,64].
Lineage 2 was also associated with any drug resistance. This is consistent with previous studies from different settings [21,23]. The reasons for this association remain unknown [20], but the strain genetic background of Beijing strains [65] and their interactions with the human immune system may play a role [21]. Alternatively, this association might reflect higher relapse rates in patients infected with Beijing strains [66]. Indeed, in our study, Lineage 2 included more patients that were previously treated but this association was not statistically significant. Finally, previous studies hypothesized that Beijing strain may escape the protective immunity of BCG vaccination [21], but we found no evidence for such an association between Lineage 2 and BCG immunization. BCG immunization has been introduced in Nepal more than 30 years ago, with an estimated immunization coverage of 96% in 2009 [67]. However, larger studies may be required for a more complete understanding of the association between previous BCG vaccination and particular M. tuberculosis genotypes.
Our study has several limitations. First, the study was not population-based as patients were recruited only at GENETUP (Kathmandu), and patients diagnosed at other microscopy centers during the study period could not be included. Second, patients coming from more remote areas outside of Kathmandu might be more likely to be referred as drug resistance suspects. Therefore, this may have artificially increased the proportion of drug-resistant strains in our sample. Third, although our study covered samples from forty different districts of Nepal including those bordering with India and Tibet, half of patients were from the Kathmandu area. Therefore, the study results mainly reflect the genetic diversity of the strains from the patients who visited GENETUP.
In conclusion, we found a high diversity of M. tuberculosis genotypes in Nepal with representation of all four main M. tuberculosis lineages, and showed that Lineage 2 (includes Beijing genotype) was associated with female sex and any drug resistance. This study fills the gap on the map of the genetic population structure of M. tuberculosis in the Asian region by providing a first insight into the phylogenetic lineages of M. tuberculosis circulating in Nepal.