Rapid Genotypic Detection of rpoB and katG Gene Mutations in Mycobacterium tuberculosis Clinical Isolates from Northern India as Determined by MAS‐PCR

Gupta, Anamika; Prakash, Pradyot; Singh, Surya Kumar; Anupurba, Shampa

doi:10.1002/jcla.21558

Cited by 31 publications

(25 citation statements)

References 22 publications

(38 reference statements)

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“…Eight samples had both katG and inhA promoter mutations. Various Indian studies have reported katG mutations at codon 315 in the range of 80 to 89% (22,24,32) and inhA mutations in the range of 9% to 27% in INH-resistant strains (24,25,33). In our study, the INH monoresistance rate was ϳ21% (mutation in katG, inhA, or both), which was consistent with previous reports of 11 to 22% in India (29,34).…”

Section: Discussionsupporting

confidence: 91%

“…2B and C). These mutations have been reported previously at frequency estimates of 47 to 81% (codon 531), 7 to 45% (codon 526), and 5.6 to 22% (codon 516) in various studies in India (20)(21)(22)(23)(24)(25) and contribute to 70 to 95% of the total rpoB gene mutations leading to RIF resistance (22,26). We found that H526N was the most frequent mutation, followed by S531L (Fig.…”

Section: Discussionsupporting

confidence: 78%

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Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

et al. 2017

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Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INHinhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% ( value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR] 400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.KEYWORDS DNA sequencing, high-resolution melt curve analysis, tuberculosis, molecular diagnostics, multidrug resistance, sputum T uberculosis (TB) is one of the world's deadliest transmittable diseases (1). In 2015, worldwide, 10.4 million people were estimated to be infected with TB, with 1.4 million people dying from the disease. Drug-resistant TB is a menace for TB control worldwide, and 60% of reported TB patients estimated to have multidrug-resistant TB (MDR-TB) were not detected in 2015 (1). India holds the dubious distinction of harboring 27% of the global TB burden, with 2.5% of its new TB cases and 16% of previously treated cases having MDR-TB (1). Conventional Mycobacterium tuberculosis culturebased systems for first-line and second-line drug susceptibility testing (DST) are timeconsuming, while commercial liquid culture-based DST reduces turnaround times but

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 78%

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

et al. 2017

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“…In 2006 population genetics for isoniazid was studied throughout the globe, they revealed 49% mutation in katG gene in India isolates [24]. A recently Indian study, by Anamika et al found that the most common mutation in INH isolates is katG gene at codon 315 with the frequency 82.29% [25]. However, 30% of katG resistant clinical isolates of M. tb showed the frequency of KatG315 (Ser to Thr) in our study.…”

Section: Discussioncontrasting

confidence: 52%

Development of Sequence Based Molecular Diagnostic Test to Evaluate MDR and XDR in M. tuberculosis Patients from Western India

Potdar¹,

Thakur²

2013

AJIDM

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Globally, Tuberculosis is the most distress disease which remains as a major challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. The increasing knowledge in molecular biology techniques has improved our understanding of the molecular mechanisms of drug resistance for the major first and second-line anti-tubercular drugs. Thus, rapid and accurate diagnosis of Tuberculosis would improve patient care and limit its transmission. This study is aimed to identify mutations in drug resistance genes of first and second line drugs against Mycobacterium tuberculosis by using polymerase chain reaction (PCR) and DNA sequencing methods. This study involves sputum samples of 20 patients diagnosed to have pulmonary tuberculosis. The genomic DNA from clinical isolates was isolated using simple boiling method. PCR amplification and DNA sequencing were performed for katG, inhA, ndh, kasA, pncA, embB, rrs, rpsL, rpoB, gyrA, gyrB and thyA genes. Sequence analysis was done using Bioedit, a bioinformatics tool. The isolates of Mycobacterium tuberculosis from western part of India were analyzed by DNA sequencing revealed total 29 mutations in first line drugs. Out of them, 5(25%) in rpoB at codon 516 and 531, 6(30%) in katG at codon 315, 8(40%) in rpsL at codon 43 & 88, 3(15%) in embB at codon 306 and 7(35%) in pncA at codon 195.No mutations in inhA, ndh, kasA and rrs genes were observed in our study. In the second line drugs, 2(10%) at codon 90 & 100% mutations at codon 95 in gyrA gene, and no mutation in gyrB and thyA genes were observed.

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“…The proportion of mutation in rpoB 526 codon (30.0%) was lower than that in the study performed in India (45.22%) [29], but it was higher than the average prevalence in Asian countries in 2001 (11%) [30] and Latvia in 2012 (11.8%) [31]. The proportion of mutation in rpoB 526 codon was 14.9% in China [32].…”

Section: Discussioncontrasting

confidence: 77%

“…Moreover, mutation in codon 531 (10.0%) was lower compared to the results of other studies [33][34][35]. Yet, the proportion of mutations in codon 516 was higher than its prevalence in East Asia [30], Latvia [31], and India [29].…”

Section: Discussionmentioning

confidence: 66%

Prevalence and risk factors of drug-resistant tuberculosis in border provinces of Iran

Sahebi¹,

Ansarin²,

Farajnia³

et al. 2015

Postgraduate Medicine

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Rapid Genotypic Detection of rpoB and katG Gene Mutations in Mycobacterium tuberculosis Clinical Isolates from Northern India as Determined by MAS‐PCR

Abstract: MAS-PCR assays can be used for rapid detection of drug-resistant TB strains in routine diagnostic practice, enabling early administration of appropriate treatment regimens to the affected patients.

Cited by 31 publications

References 22 publications

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

Development of Sequence Based Molecular Diagnostic Test to Evaluate MDR and XDR in <i>M.</i> <i>t</i><i>uberculosis</i> Patients from Western India

Prevalence and risk factors of drug-resistant tuberculosis in border provinces of Iran

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