Nikita Panwalkar

Frequency of mutations in rifampicin and isoniazid resistant isolates of M. tuberculosis: an analysis from Central India.

BACKGROUND The spread of drug-resistant tuberculosis has challenged tuberculosis control strategies globally. The present study aims to analyze the frequency of mutations in rpoB, katG and inhA genes in strains of M. tuberculosis complex (MTBC) circulating in Central India. It is anticipated that the findings may provide a starting point to understand the evolutionary success of drug-resistant strains of MTBC in this region. METHODS Line probe assay was carried out on 720 consecutive sputum samples of MDR suspects from June 2012 to May 2013. Mutation frequencies in the rpoB, katG and inhA genes were analyzed. RESULTS Mutations were identified in 269 (37.6%) samples, as follows: 55 (7.6%) samples had mutations conferring resistance to only isoniazid, 84 (11.6%) had mutations conferring resistance to only rifampicin and 130 (18%) isolates had mutations conferring resistance to both isoniazid and rifampicin. The most frequent mutation in the rpoB gene was at codon S531L, seen in 141 (19.5%) isolates. The most frequent mutation in the katG gene was at codon S315T1, seen in 151 (20.9%) isolates; and in the inhA gene at codon C15T, seen in 21 (2.9%) isolates. Some unidentified mutations were also observed. CONCLUSION The patterns and the frequency of the mutations identified in this study indicate the most frequent mutations at S531L codon in the rpoB gene, S315T1 codon in the katG gene and C15T codon in the promoter region of the inhA gene. Controlling the emergence and spread of MDR TB requires an understanding of the evolution of these mutations.

Genetic diversity of Mycobacterium tuberculosis isolates from central India

Background & objectives: There is a paucity of data available on genetic biodiversity of Mycobacterium tuberculosis isolates from central India. The present study was carried out on isolates of M. tuberculosis cultured from diagnostic clinical samples of patients from Bhopal, central India, using spoligotyping as a method of molecular typing. Methods: DNA was extracted from 340 isolates of M. tuberculosis from culture, confirmed as M. tuberculosis by molecular and biochemical methods and subjected to spoligotyping. The results were compared with the international SITVIT2 database. Results: Sixty five different spoligo international type (SIT) patterns were observed. A total of 239 (70.3%) isolates could be clustered into 25 SITs. The Central Asian (CAS) and East African Indian (EAI) families were found to be the two major circulating families in this region. SIT26/CAS1_DEL was identified as the most predominant type, followed by SIT11/EAI3_IND and SIT288/CAS2. Forty (11.8%) unique (non-clustered) and 61 (17.9%) orphan isolates were identified in the study. There was no significant association of clustering with clinical and demographic characteristics of patients. Interpretation & conclusions: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis.

Line probe assay for detection of Mycobacterium tuberculosis complex: An experience from Central India

Background & objectives: Mycobacterium tuberculosis complex may sometimes not be detected in sputum samples of suspected multidrug-resistant tuberculosis (MDR-TB) patients by line probe assay (LPA) even though they are smear positive for acid-fast bacilli (AFB). This retrospective analysis was attempted to understand and document our experience with LPA for detection of M. tuberculosis complex and diagnosis of MDR-TB under programmatic conditions. Methods: One thousand two hundred and ninety four sputum samples of MDR-TB suspects that were smear positive for AFB, and received from February to November 2013, were tested by LPA for the presence of M. tuberculosis complex and resistance to isoniazid (INH) and rifampicin as per the diagnostic mandate of an accredited reference laboratory. As per the mandate, those samples that were negative for M. tuberculosis complex were cultured, and the growth again tested by LPA. A retrospective analysis of the results was carried out. Results: M. tuberculosis complex could be detected in 1217 (94.04%) but not in 77 (5.9%) of smear-positive sputum samples. Of the 1217 positive samples, 232 (19.1%) were MDR, 130 (10.6%) were rifampicin monoresistant and 101 (8.3%) were INH monoresistant. Seven hundred and fifty four (61.9%) strains were found to be pansensitive. Overall, 5.1 per cent of the sputum samples were negative for M. tuberculosis complex by LPA and culture. In at least 10 (0.77%) sputum samples smear positive for AFB, M. tuberculosis complex could not be identified by LPA though M. tuberculosis was present, as evidenced by culture positivity. Interpretation & conclusions: LPA is a robust technique for diagnosis of drug-resistant TB that has provided the basis for rapid and effective control of drug-resistant TB in India. While the reasons for concomitantly negative LPA and culture results of smear-positive sputum samples from MDR-TB suspects may be many, the possible presence of non-tubercular mycobacteria in these samples and the likelihood of inappropriate therapy in these patients cannot be ruled out. Addition of culture to the diagnostic algorithm may enhance the diagnostic yield.

Public health relevance of non-tuberculous mycobacteria among AFB positive sputa

BACKGROUND Sputum smear microscopy for acid fast bacilli (AFB) is used by most public health programmes to detect tuberculosis. While most AFB in countries endemic for tuberculosis are Mycobacterium tuberculosis (MTB), some may also be non-tuberculous mycobacteria (NTM). The inability to differentiate NTM from MTB by sputum smear microscopy may lead to erroneous diagnoses of tuberculosis, leading in turn to inappropriate therapy. METHODS This was a retrospective study of consecutive sputum samples received from November 2013 to March 2015 in the Department of Microbiology, Bhopal Memorial Hospital & Research Centre, Bhopal, India. Samples underwent smear microscopy, line probe assay (LPA) for MTB complex, culture, biochemical tests and LPA for NTM. RESULTS Of 4095 sputum samples, 2886 were AFB smear positive (70.5%). Of these, MTB complex was detected in 2611 (90.5%) samples by LPA. Of the remaining 275 samples, 47 grew AFB on culture. Nine strains belonged to the MTB complex. The remaining 38 (1.3%) were NTM, and could be speciated in 26 strains; 14 (53.8 %) were M. abscessus; 10 (38.4%) M. intracellulare, one (3.8%) M. kansasii and one (3.8%) M. fortuitum. The remaining 12 NTM could not be speciated. CONCLUSION NTM were present in at least 1.3% of all smear positive samples. It is important for public health programs to recognize the avoidable burden on logistics, infrastructure and finances caused by this. Detection and quantification of this burden would help design an appropriate strategy for optimal tuberculosis control.

An HIV1/2 point of care test on sputum for screening TB/HIV co-infection in Central India - Will it work?

OBJECTIVE To determine whether the OraQuick® HIV-1/2 Assay (OraSure Technologies, Inc., Bethlehem, PA, USA) in sputum is a valid tool for HIV surveillance among TB patients. METHODS A cross sectional study was carried out on sputa of patients diagnosed with tuberculosis. Sputa were tested for antibodies to HIV using OraQuick® HIV-1/2 Assay (OraSure Technologies, Inc., Bethlehem, PA, USA). The results were compared with results of serum ELISA. RESULTS Compared to serum ELISA, the OraQuick® HIV-1/2 Assay in sputum specimens reported 90% sensitivity (9/10) and 100% specificity (307/307), with a positive predictive value of 100% (95%CI: 66.37%-100.00%) and a negative predictive value of 99.68% (95%CI: 98.20%-99.99%). CONCLUSIONS This testing method may provide a useful strategy for conducting HIV surveillance in possible co-infected TB patients at peripheral centres. Since there is no investment on infrastructure, it may be possible for paramedical health professionals to carry out the test, particularly in areas with low HIV endemicity.

A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India

This study was carried out to identify predominant spoligotypes responsible for transmission and prevalence of tuberculosis in central India since there is no data available about the genetic biodiversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in this region. 35 strains of Mycobacterium tuberculosis were subjected to spoligotyping according to the standard protocol. A total of 25 strains out of the 35 (71.42%) could be grouped in to 6 clusters. The largest cluster comprised 8 isolates. Unique (Non-clustered) spoligotypes were seen in 10 isolates, Nine strains did not match the data base (Spol DB-4 data base). The results indicate that there may be a number of orphan strains unique to this geographical area. Further studies on a larger sample size derived from this area would help us delineate the epidemiology of Mycobacterium tuberculosis infection in this area.

Comparision of performance characteristics of automated PCR systems with culture for detection of MTB complex from clinical samples in Central India

Most published studies on performance characteristics of the COBAS AMPLICOR (CA) system and Real Time PCR (RT-PCR) for detection of Mycobacterium tuberculosis have been carried out in countries other than India.[1] Strains of M. tuberculosis are known to vary in their genomic structure between geographical regions. Performance characteristics of these tests have not been determined on locally prevalent strains in Central India.

Trends in spoligotype patterns of Mycobacterium tuberculosis strains in central India

This study aims to understand trends in spoligotype patterns of Mycobacterium tuberculosis (MTB) in Central India. Elucidation of these trends may provide baseline information to understand the transmission dynamics of strains of MTB in the region. Spoligotyping was carried out on 340 MTB strains isolated from clinical samples received from 2007 to 2011. The prevalence of ST26/CAS1_Del, ST11/EAI3_IND, ST288/CAS2, ST25/CAS1_Del and Beijing lineages showed waxing and waning trends. ST26/CAS1_Del and ST11/EAI3_IND lineages were consistently present and were predominant. Well-established lineages showed a consistent presence in the community. New orphan lineages appeared to be less capable of establishing themselves.

Spoligotype defined lineages of Mycobacterium tuberculosis and drug resistance: Merely a casual correlation?

Drug-resistant tuberculosis (TB) is a major challenge to TB control strategy worldwide. Analysis of genetic polymorphism among drug resistant Mycobacterium tuberculosis (MTB) strains may help provide some insight into the transmission dynamics of these strains. Spoligotyping is a widely used technique to identify genetic polymorphism, based on 43 known spacers interspersed between direct repeat regions. Considerable work has been done in various parts of the world using this technique to identify and analyse the polymorphic nature of MTB. Many studies have been carried out to determine the association of drug resistance with spoligotype defined lineages, and much data has been produced over the years. New information continues to be generated. This review aims to put together the findings of relevant studies in an attempt to understand the correlation of drug resistance with spoligotype defined lineages of MTB. This would help provide a perspective of the available data that can be used as a starting point to understand the molecular epidemiology of drug resistant TB.

Acute Monoarthritis of the Wrist Joint: Tuberculosis or Not?

Background Extrapulmonary tuberculosis (EPTB) is known to have many and varied presentations. However, isolated involvement of bone with tubercular infection is uncommon. The clinical features of such infections are known to mimic chronic pyogenic osteomyelitis, Brodie abscess, or tumors, but not acute monoarthritis. Case Description We describe here an unusual case of tuberculous osteomyelitis that mimicked features of acute monoarthritis of the wrist joint. Literature Review Extraspinal tuberculous osteomyelitis is rare and comprises only about 2 to 3% of all cases of osteoarticular tuberculosis, with the hip and knee joints being the most commonly involved. An extensive literature review did not show any published report of tuberculous osteomyelitis presenting as acute monoarthritis of the wrist joint. Clinical Relevance This case underlines the importance of making EPTB an important differential diagnosis even in cases with clinical features that are completely inconsistent with tubercular infections.