Antitubercular drug resistance in four healthcare facilities in North India.

Tuberculosis (TB) is a major public-health problem in India, having the highest number of incident and multidrug-resistant (MDR) TB cases. The study was carried out to appraise the prevalence of first-line anti-TB drug resistance in Mycobacterium tuberculosis (MTB) and its patterns among different types of TB patients from different settings in a province of North India. Of 3,704 clinical specimens, 345 (9.3%) were culture-positive, and drug-susceptibility testing was carried out for 301 MTB strains. A high level of primary and acquired drug resistance of MTB was observed in the region studied, with weighted mean of 10.5% and 28.08%, 12.81% and 29.72%, 17.12% and 29.94%, 11.97% and 27.84%, and 10.74% and 23.54% for rifampicin, isoniazid, streptomycin, ethambutol-resistant and MDR cases respectively. Drug resistance was significantly higher in pulmonary (p = 0.014) and acquired drug-resistant TB cases (p < 0.001). Any drug resistance (p = 0.002) and MDR TB were significantly (p = 0.009) associated with HIV-seropositive cases. An urgent plan is needed to continuously monitor the transmission trends of drug-resistant strains, especially MDR-TB strains, in the region.

Laboratory diagnosis of SARS-CoV-2 - A review of current methods.

At present the whole world is facing pandemic of the Coronavirus disease (COVID-19); caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has rapidly spreads across the world from its origin of Wuhan, China and affected millions people worldwide and make them to remain in their homes. The knowledge of available laboratory methods is essential for early and correct diagnosis of COVID-19 to identify new cases as well as monitoring treatment of confirmed cases. In this review we aim to provide the updated information about selection of specimens and availability of various diagnostic methods and their utility with current findings for the laboratory diagnosis of SARS-CoV-2 infection. This will guide the healthcare professionals and government organizations to make strategy for establishing diagnostic facilities for SARS-CoV-2 infections.

Role of spoligotyping and IS6110-RFLP in assessing genetic diversity of Mycobacterium tuberculosis in India.

In the present study, genetic diversity analysis of Mycobacterium tuberculosis isolated from patients attending a tertiary care hospital, North India, has been attempted. Eighty three isolates of M. tuberculosis were subjected to DNA fingerprinting using spoligotyping and IS6110-RFLP techniques. Spoligotype patterns showed that central Asian (32.5%), ill defined T (13.2%) and Beijing (10.8%) families were predominant in ongoing transmission of the bacterium. Two STs; ST26 (CAS_Delhi) and ST1 (Beijing) represented 36.1% of the total M. tuberculosis population in eastern Uttar Pradesh, North India. IS6110 RFLP analysis showed that isolates having low and zero copy number of the IS element were 15.6% and 19.2%, respectively. Out of the 47 isolates clustered by spoligotyping, 40 could be further differentiated as unique strains by IS6110-RFLP. Therefore, this study recommends that both the techniques be used simultaneously for DNA fingerprinting of M. tuberculosis in India.

Molecular typing of clinical Staphylococcus aureus isolates from northern India using coagulase gene PCR-RFLP.

Molecular typing of total 84 Staphylococcus aureus clinical isolates was performed using coagulase gene PCR. Out of 84 S. aureus strains total 33 different types of S. aureus strains were prevalent in this hospital and community. Types 2-7 and 9 were the most prevalent S. aureus strains accounting for more than 53% of total isolates. This technique is relatively inexpensive and is simple to perform and analyze.

Usefulness of IS6110-based restriction fragment length polymorphism analysis in fingerprinting of Mycobacterium tuberculosis isolates in North India.

OBJECTIVE/BACKGROUND: World Health Organization estimates that approximately one-third of the global community is infected with Mycobatcerium tuberculosis (MTB). Various molecular epidemiology methods were developed and found very promising for assessing the genetic diversity among MTB complex strain. The two major tools restriction fragment length polymorphism (RFLP) and spoligotyping were commonly used in various studies. Some Indian studies raise the question about the utility of IS6110-RFLP in India due to the presence of zero and low copy number of IS6110 element in MTB isolates. In this short study, we attempt to evaluate the usefulness of IS6110-RFLP in genotyping of MTB isolates in North India. METHODS: We conducted a short study involving 26 MTB isolates collected from Sawai Madhopur, Rajasthan, India. IS6110-RFLP analysis was performed as previously described method. In brief, the procedure involve MTB DNA digestion (PvuII restriction enzyme), electrophoresis on 1% agarose gel, transfer of DNA fragments on positively charged nylon membrane, hybridization with digoxigenin-labeled IS6110 probe, and detection by digoxigenin nucleic acid labeling and detection kit. RESULTS: IS6110-RFLP analysis of 26 MTB isolates showed presence of IS6110 element in varying range from 0 to 17 copies. Out of the 26 MTB isolates, two (7.8%), three (11.5%), and 21 (80.8%) showed zero, low, and multiple copy numbers, respectively. The isolates, which had IS6110 element, showed 22 different RFLP patterns. Two clusters of two isolates each were found, and 20 isolates showed unique RFLP pattern. The two clusters of isolates had 11 and 13 copy numbers of IS6110. CONCLUSION: In this study, we found that the majority of isolates were having multiple copy numbers of IS6110 element and showed a very diverse pattern. These results showed that the IS6110-RFLP analysis is still a promising genotyping method and has good discriminatory power to differentiate strains of MTB isolates in India.

Assessing genetic diversity of Mycobacterium tuberculosis by spoligotyping and IS6110-based restriction fragment length polymorphism in North India.

OBJECTIVE/BACKGROUND: Molecular epidemiology methods are very useful for differentiating between strains, assessing their diversity, and measuring the prevalence of the most circulating strain in an area. Various molecular typing methods using different molecular markers have been utilized worldwide, such as restriction fragment length polymorphism (RFLP), spoligotyping, Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeat (MIRU-VNTR), and Double repetitive element-PCR (DRE-PCR) typing, for simultaneous detection and epidemiologic typing of Mycobacterium tuberculosis. The present study is conducted to assess the genetic diversity of M. tuberculosis by IS6110-RFLP and spoligotyping in patients attending a tertiary care hospital in eastern Uttar Pradesh, North India. METHODS: A total of 83 representative isolates of M. tuberculosis were included in this study. These isolates were subjected to spoligotyping and IS6110-RFLP DNA fingerprinting techniques as described previously. RESULTS: The spoligotype patterns were compared with SpolDB4.0; patterns of 64 out of 83 M. tuberculosis isolates were matched with the available data, while 19 isolates were found to be orphan, that is, absent in the SpolDB4.0 database. The majority of the M. tuberculosis strains (56.5%) belong to central Asian (32.5%), ill defined T (13.2%), and Beijing (10.8%) families. On IS6110-RFLP analysis, in 19.2% (16/83) of these isolates, IS6110 element was not found (0 copy number strains). Further, 15.6% (13/83) isolates were found to be low-copy-number strains having less than six copies of IS6110 element, and the remaining 65.0% (54/83) were multiple-copy-number strains with six or more copies of the element. On comparing the results of spoligotyping and IS-6110-RFLP, a total of 47 isolates were clustered by spoligotyping; out of these isolates, 40 were found to be unique by IS6110-RFLP. CONCLUSION: Spoligotype analysis resulted in the grouping of a much larger number of isolates within apparently identical clusters compared with IS6110-RFLP typing, while IS6110-RFLP was not found to effectively distinguish between zero- and low-copy-number isolates. Therefore, we concluded that, in India, the use of both the techniques simultaneously for DNA fingerprinting of M. tuberculosis could be a better approach.

Primary and acquired drug resistance patterns of Mycobacterium tuberculosis isolates in India: a multicenter study.

Tuberculosis is the most prevalent infection worldwide. The emergence of drug-resistant Mycobacterium tuberculosis (M. tuberculosis) isolates emphasizes that it is necessary to monitor drug resistance of the organism against anti-tubercular drugs. We analyzed 327 M. tuberculosis isolates from patients who were cared for at three different health care centers, hereinafter known as study areas (SAs), in North India. Of the 327 total M. tuberculosis isolates, 255 were from a tertiary health care center (Varanasi, Uttar Pradesh [SA-1]), 48 were from a District tuberculosis center (Sawai Madhopur, Rajasthan [SA-2]), and 24 were from a different District tuberculosis center (Buxar, Bihar [SA-3]). Drug susceptibility testing against first-line antibiotics (viz. isoniazid, rifampicin, streptomycin, and ethambutol) was conducted for all the isolates using 1% proportional method. We found that the rates of acquired resistance were consistently higher than the rates of initial drug resistance. In new, untreated cases, a higher degree of MDR-TB was observed at SA-1 (13.3%) and SA-3 (25.0%), whereas it was observed in only 7.1% of the isolates at SA-2. In previously treated patients, MDR cases were found in 35.7% of the isolates from SA-1, 66.6% of the isolates from SA-2, and 43.8% of the isolates from SA-3. Resistance to a single drug was found at a much lower rate, ranging from 0.0 to 6.3% in new cases as well as previously treated cases. In conclusion, the primary resistance of M. tuberculosis is low, but acquired drug resistance is slightly higher in North India.

Molecular characterization of INH-resistant Mycobacterium tuberculosis isolates by PCR-RFLP and multiplex-PCR in North India.

In the present study, among 327 Mycobacterium tuberculosis (MTB) isolates collected from patients attending three different centres of North India, we attempted to find out the most common mutations occurring both at the Ser315 codon of katG and at the regulatory region of the mabA-inhA operon to evaluate their role for INH drug resistance in India. Out of 121 phenotypically INH-resistant MTB isolates, 88 (72.7%) were resistant to INH by genotypic methods viz., PCR-RFLP with MspI and SatI digestion and multiplex-PCR. PCR-RFLP results showed that 67 (55.4%) isolates had mutation in codon 315 of katG by SatI endonuclease. Among these, eight isolates that were found resistant by SatI PCR-RFLP were found to be sensitive by MspI PCR-RFLP. By multiplex-PCR we found 49 (40.5%), 21 (17.4%) and 10 (8.3%) isolates having AGC-->ACC substitution in katG only, mutation in inhA(C-15T) only and mutation in both respectively. Simultaneous use of both PCR-RFLP and multiplex-PCR can improve the detection rate of INH-resistant strains and may have an advantage over the liquid culture system of detecting drug resistance. These findings also enhanced our understanding about potential of resistance-related mutations in M. tuberculosis clinical isolates in India and could help in development and designing of molecular methods for revealing the drug susceptibility profiles of M. tuberculosis clinical isolates.