Genotyping Mycobacterium tuberculosis isolates with few copies of IS6110: Value of additional genetic markers.

PURPOSE: IS6110 restriction fragment length polymorphism (RFLP) analysis is widely used for molecular epidemiological studies of tuberculosis. Role of spoligotyping and Fluorescent Amplified Fragment Length Polymorphism (FAFLP) was studied in low-copy number IS6110 strains of Mycobacterium tuberculosis complex (Mtbc). METHODS: The study isolates included 70 strains of Mtbc collected from different regions of India. IS6110 restriction fragment, spoligotyping and FAFLP were performed for genotypic analysis. RESULTS: A single copy of IS6110 was found in 30% of isolates with 90.5% of them harboring characteristic 1.5-Kb IS6110 restriction fragment.IS6110RFLP identified 51 different types, FAFLP 41 types, and spoligotyping 31 types. Combination of all three techniques identified 67 different types.IS6110 RFLP analysis was found sensitive for genotyping isolates with more than one copy of IS6110 (Hunter Gaston Discriminatory Index (HGDI-1) while, neither spoligotyping (HGI-0.89) nor FAFLP (HGDI-0.92) or their combinations were as good. The discriminatory power of spoligotyping (HGDI- 0.89) in isolates with a single copy of IS6110 was higher than IS6110-RFLP.Clustering was reduced to 67% using spoligotyping and to 38% with FAFLP. CONCLUSION: Combination of FAFLP and Spoligotyping may prove to be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of IS6110 element.

Genetic biodiversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in India.

Spoligotyping was performed on 540 Mycobacterium tuberculosis isolates in order to evaluate the genetic biodiversity of tubercle bacilli in India. One hundred and forty seven patterns were unique and 393 were grouped in 48 clusters. Comparison with an international spoligotype database showed that the most predominant clades among tuberculosis (TB) isolates were Central Asian (CAS) and East-African Indian (EAI) with shared-types (ST) ST26 and ST11 alone being responsible for 34% of all TB cases. Twenty one (3.8%) isolates belonged to the Beijing genotype. Marked variations were observed among circulating strains, STs belonging to CAS family predominated in the North, whereas the EAI family was more common in the Southern India. TB in India is predominantly caused by strains belonging to the principal genetic group 1 (PGG1), suggesting that most of the TB burden in India may be traced to ancestral clones of the tubercle bacilli. This study gives an insight into the global M. tuberculosis genetic biodiversity in India, the predominant spoligotypes and their impact on disease transmission.

rpoB gene sequencing and spoligotyping of multidrug-resistant Mycobacterium tuberculosis isolates from India.

Multi drug-resistant Mycobacterium tuberculosis (MDR TB) has been well studied in outbreaks in settings of low endemicity in developed countries. However, the characteristics of MDR TB in the community with high endemicity such as India have not been well investigated. Mutations in the 81-bp rifampicin resistance-determining region of the rpoB gene were analyzed by DNA sequencing of 187 M. tuberculosis clinical isolates (149 resistant and 38 sensitive) from different parts of India. 146-Point mutations and two insertions were found in 146 of 149 resistant isolates in seven codons. The most common mutations were in codons 531 (59%), 526 (22%), and 516 (11.5%). Mutations were not found in three (2%) of the resistant isolates. N-terminal sequencing in these isolates showed no mutation at codon V176. None of the drug-susceptible isolates showed any mutation in the 437-bp rpoB gene segment sequenced. Genotypic analysis revealed a total of 80 different spoligotypes. A unique pattern was found in 65 (43.6%) isolates, whereas 84 (56.4%) were in 15 clusters. Comparison with an international spoligotype database showed ST26, Delhi type (18.1%), ST1, Beijing type (9.4%), and ST11 (5.4%), as the most common. The majority of isolates in the Beijing genotype (13/14) were associated with mutation 531TTG and similar drug-resistance patterns while other major clusters showed that the nature and frequency of occurrence of mutations in the rpoB gene were independent of spoligopatterns.

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis by in-house, reverse line blot assay.

Drug resistance in tuberculosis is a significant problem in countries endemic for tuberculosis. A sensitive, specific, and high-throughput reverse line blot assay (RLBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. DNA sequencing done for 72 resistant isolates from Delhi, for baseline data, showed mutations within the rpoB core region in all RIF-resistant strains. The RLBA includes oligonucleotide probes specific for wild-type and mutant sequences, allowing sensitive detection of both genotypes in a single assay. The assay based on reverse hybridization principle simultaneously detects 13 different mutations affecting 6 independent codons, including the most prevalent mutations at positions 531 and 526. Application of the method to a panel of 292 MDR TB isolates and susceptible strains from 5 different cities in India showed 98% concordance with the sequencing results. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in Mycobacterium tuberculosis.

Incidence and risk factors for extensively drug-resistant tuberculosis in Delhi region.

BACKGROUND: India with a major burden of multidrug-resistant tuberculosis (MDR-TB) does not have national level data on this hazardous disease. Since 2006, emergence of extensively drug-resistant TB (XDR-TB) is considered a serious threat to global TB control. This study highlights the demographic and clinical risk factors associated with XDR-TB in Delhi. METHODS: The study was conducted during April 2007 to May 2010. Six hundred eleven MDR-TB suspects were enrolled from four tertiary care hospitals, treating TB patients in Delhi and the demographic details recorded. Sputum samples were cultured using rapid, automated liquid culture system (MGIT 960). Drug susceptibility testing (DST) for Rifampicin (RIF) and Isoniazid (INH) was performed for all positive M. tuberculosis (M.tb) cultures. All MDR-TB isolates were tested for sensitivity to second-line drugs [Amikacin (AMK), Capreomycin (CAP), Ofloxacin (OFX), Ethionamide (ETA)]. RESULTS/FINDINGS: Of 611, 483 patients were infected with MDR M. tuberculosis (M.tb) strains. Eighteen MDR-TB isolates (3.7%) were XDR M.tb strains. Family history of TB (p 0.045), socioeconomic status (p 0.013), concomitant illness (p 0.001) and previous intake of 2(nd) line injectable drugs (p 0.001) were significantly associated with occurrence of XDR-TB. Only two of the patients enrolled were HIV seropositive, but had a negative culture for M. tuberculosis. 56/483 isolates were pre-XDR M. tuberculosis, though the occurrence of pre-XDR-TB did not show any significant demographical associations. CONCLUSIONS: The actual incidence and prevalence rate of XDR-TB in India is not available, although some scattered data is available. This study raises a concern about existence of XDR-TB in India, though small, signaling a need to strengthen the TB control program for early diagnosis of both tuberculosis and drug resistance in order to break the chains of transmission.

Characterization of predominant Mycobacterium tuberculosis strains from different subpopulations of India.

The predominant strains from India belong to Central-Asian (CAS) and the East-African-Indian (EAI) clade of Mycobacterium tuberculosis. The two clades have also been shown to be geographically partitioned. The study of such strains may help to understand the characteristics that make M. tuberculosis an effective pathogen and its overrepresentation in certain populations. M. tuberculosis isolates characterized by spoligotyping under a population based tuberculosis study covering different regions from the North and South India were further analyzed by restriction fragment length polymorphism (RFLP) and by deletion analysis of M. tuberculosis specific deletion region 1 (TbD1). The genetic relationship of the two clades inferred using different genetic markers showed good correlation. In the North where the CAS clade predominates the isolates are characterized by presence of high IS6110 copy number and absence of TbD1 region whereas in the South where the EAI clade predominates the isolates are characterized by low copy number of IS6110 and presence of TbD1 region. The ancestral EAI strains were found to be less often associated with drug resistance or young age as compared to the CAS clade. The study highlights that the EAI lineage is well established in India and that CAS may be emerging or more recently introduced to India. The results depict a distinction in the lineage of strains from the North versus South India indicating a need to study if the pathogen has adapted to specific human populations.

Predominant tuberculosis spoligotypes, Delhi, India.

One hundred five Mycobacterium tuberculosis clinical isolates from the Delhi area were typed by spoligotyping; 45 patterns were identified. Comparison with an international spoligotype database showed type 26, Delhi type (22%), type 54 (12%), and type 1, Beijing type (8%), as the most common. Eighteen spoligotypes did not match any existing database pattern.