Mandira Varma-Basil

Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis

ABSTRACT The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS6110 Clustering Rather than Ethambutol Resistance

ABSTRACT Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis Isolates from India and Mexico by a Molecular Beacon Assay

ABSTRACT We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Role of Mycoplasma pneumoniae infection in acute exacerbations of chronic obstructive pulmonary disease

Eighty per cent of the cases of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) have an infective aetiology, atypical bacteria including Mycoplasma pneumoniae accounting for 5-10 % of these. However, the importance of association of M. pneumoniae with episodes of AECOPD still remains doubtful. The present study was therefore undertaken to delineate the extent of involvement of M. pneumoniae in patients with AECOPD at a referral hospital in Delhi, India. Sputum samples and throat swabs from a total of 100 AECOPD patients attending the Clinical Research Center of Vallabhbhai Patel Chest Institute, Delhi, were collected during a 2-year period (January 2004-June 2006). The samples were investigated for the presence of aerobic bacterial pathogens and M. pneumoniae. Diagnosis of infection with M. pneumoniae was based on culture, serology, direct detection of M. pneumoniae specific antigen and PCR. Bacterial aetiology could be established in 16 of the 100 samples studied. Pseudomonas spp. were recovered from eight cases, Streptococcus pneumoniae from four and Klebsiella spp. from two cases. Acinetobacter sp. and Moraxella catarrhalis were isolated from one case each. Serological evidence of M. pneumoniae infection and/or detection of M. pneumoniae specific antigen were seen in 16 % of the cases. One case with definite evidence of M. pneumoniae infection also had coinfection with Pseudomonas spp. However, no direct evidence of M. pneumoniae infection was found in our study population as defined by culture isolation or PCR. In conclusion, although the serological prevalence of M. pneumoniae infection in our study population was significantly higher than in the control group, there was no direct evidence of it playing a role in AECOPD.

Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis

Purpose Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. Materials and Methods We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. Results Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. Conclusion These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.

Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR

We developed a novel indirect sandwich immuno-polymerase chain reaction (I-PCR) assay for the detection of mycobacterial antigen 85B (Ag85B, 30kDa, Rv1886c) in pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients. The amino-modified reporter DNA was covalently attached with the antidetection antibody through a heterobifunctional cross-linking agent succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. The detection limit of Ag85B by I-PCR was found to be 1 femtogram (fg)/mL, which was 10(6)-fold lower than an analogous enzyme-linked immunosorbent assay (ELISA). The sensitivities of 85% and 77% with I-PCR and 77.6% and 62.5% with ELISA were observed in smear-positive and smear-negative PTB patients, respectively, with high specificity. On the other hand, sensitivities of 84% and 63.7% with I-PCR and 68% and 47.5% with ELISA were observed in confirmed and clinically suspected EPTB cases, respectively, with high specificity.

Development and Clinical Evaluation of sdaA Loop-Mediated Isothermal Amplification Assay for Detection of Mycobacterium tuberculosis with an Approach To Prevent Carryover Contamination

ABSTRACT A rapid and sensitive loop-mediated isothermal amplification assay for the sdaA gene of Mycobacterium tuberculosis was developed using a dUTP-uracil-N-glycosylase (dUTP-UNG) strategy to prevent carryover contamination. Evaluation of the assay using clinical specimens (n = 648) showed high specificity (97.2%) and sensitivity (100%), demonstrating its potential as a diagnostic test for tuberculosis, especially in resource-limited settings.

Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis.

We were pleased to read the article entitled “The IL-17F sequence variant is associated with susceptibility to tuberculosis” in your reputed journal (Peng et al., 2013). The authors in order to address the role of IL17 gene polymorphisms in tuberculosis show that a variant in IL17F gene is associated with tuberculosis. The study uses a good sample size and very apt conclusions are drawn.Wewish to report here that independently we have also examined the role of rs2275913 and rs763780 single nucleotide polymorphisms of IL17A and IL17F genes respectively on the development of pulmonary (PTB) and lymph node tuberculosis (LNTB) in north Indians and correlated the serum IL-17A and IL-17F levels associated with them.We in our study did not find any association between the IL17F gene polymorphism and TB. To our knowledge, it is the first study to investigate the IL17 gene polymorphisms and their correlationwith serum IL-17 levels. This communication is no criticism of the reported work per se but ameans to report the different results we obtained in an unrelated ethnic population adding a different vantage point for the role of IL17 sequence variants in tuberculosis. The role of CD4 T cells and T helper (Th) 1 cytokines are critical and well characterized in the cell mediated response to Mycobacterium tuberculosis (M.tb), but it has emerged that the Th1 axis alone is not enough to counter the infection (Goldsack and Kirman, 2007) and there can be a mixed bag of other factors involved. IL-17 is a novel family of proinflammatory cytokines produced by Th17 cells in response to IL-23 (Kawaguchi et al., 2004). The IL-17 axis is very crucial for initial immune responses in tuberculosis (Khader and Cooper, 2008). To investigate the significance of the abovementioned genetic polymorphisms in the IL17 gene in human tuberculosis susceptibility, genomic DNA was isolated from 215 patients comprising of both PTB 88

Development of a Novel PCR Restriction Analysis of the hsp65 Gene as a Rapid Method To Screen for the Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in High-Burden Countries

ABSTRACT The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.