Molecular analysis of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates by multiple allele specific polymerase chain reaction in Puducherry, South India

Ravibalan, Thirumurugan; Kathirvel, M; Vallayyachari, Kommoju; Kesavan, Surendar; Samrot, Antony V.; Muthuraj, Muthusivaramapandian

doi:10.1016/j.jiph.2015.05.003

Cited by 36 publications

(21 citation statements)

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“…The rpoB HRM assay had 100% diagnostic accuracy in SSM-positive samples. RIF monoresistance was detected in 11 versus 12 samples by HRM and DNA sequencing, respectively, which was consistent with previous findings of ϳ9% (28) and as high as 15.5 to 16.5% in drug-naive samples (21,29).…”

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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“…treated PTB patients120714.814.8LJRecords from state RNTCP50Bhat S et al, 2010 [86]Mangalore2010Newly diagnosed PTB patients5082.010.06.06.032.04.04.0LJTertiary hospital51Kandi S et al, 2013 [87]Hyderabad2010–2011Prev. treated PTB patients8450.013.12.41.233.333.3LJTertiary hospital52Therese K L et al (b), 2012 [88]Chennai2011Combined PTB patients16645.22.40.63.612.16.6BACTEC MicroMGITTertiary hospital53Selvakumar N et al, 2015 [26]Tamil Nadu2011–2012Combined PTB patients193426.69.61.06.0013.21.81.793.96.10.2LJDesignated microscopycenters54Gaude G S et al, 2014 [19]Belgaum2011–2012Combined PTB patients6669.710.63.036.4LJTertiary hospital55Thirumurugan R et al, 2015 [89]Puducherry2011–2013Combined PTB patients12770.916.554.3LJOutpat...…”

Section: Resultsmentioning

confidence: 99%

Prevalence of drug-resistant pulmonary tuberculosis in India: systematic review and meta-analysis

et al. 2017

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BackgroundDrug-resistant pulmonary tuberculosis (DR-TB) is a significant public health issue that considerably deters the ongoing TB control efforts in India. The purpose of this review was to investigate the prevalence of DR-TB and understand the regional variation in resistance pattern across India from 1995 to 2015, based on a large body of published epidemiological studies.MethodsA systematic review of published studies reporting prevalence of DR-TB from biomedical databases (PubMed and IndMed) was conducted. Meta-analysis was performed using random effects model and the pooled prevalence estimate (95% confidence interval [CI]) of DR-TB, multidrug resistant (MDR-) TB, pre-extensively drug-resistant (pre-XDR) TB and XDR-TB were calculated across two study periods (decade 1: 1995 to 2005; decade 2: 2006 to 2015), countrywide and in different regions. Heterogeneity in this meta-analysis was assessed using I2 statistic.ResultsA total of 75 of 635 screened studies that fulfilled the inclusion criteria were selected. Over 40% of 45,076 isolates suspected for resistance to any first-line anti-TB drugs tested positive. Comparative analysis revealed a worsening trend in DR-TB between the two study decades (decade 1: 37.7% [95% CI = 29.0; 46.4], n = 25 vs decade 2: 46.1% [95% CI = 39.0; 53.2], n = 36). The pooled estimate of MDR-TB resistance was higher in previously treated patients (decade 1: 29.8% [95% CI = 20.7; 39.0], n = 13; decade 2: 35.8% [95% CI = 29.2; 42.4], n = 24) as compared with the newly diagnosed cases (decade 1: 4.1% [95% CI = 2.7; 5.6], n = 13; decade 2: 5.6% [95% CI = 3.8; 7.4], n = 17). Overall, studies from Western states of India reported highest prevalence of DR-TB (57.8% [95% CI = 37.4; 78.2], n = 6) and MDR-TB (39.9% [95% CI = 21.7; 58.0], n = 6) during decade 2. Prevalence of pre-XDR TB was 7.9% (95% CI = 4.4; 11.4, n = 5) with resistance to fluoroquinolone (66.3% [95% CI = 58.2; 74.4], n = 5) being the highest. The prevalence of XDR-TB was 1.9% (95% CI = 1.2; 2.6, n = 14) over the 20-year period.ConclusionThe alarming increase in the trend of anti-TB drug resistance in India warrants the need for a structured nationwide surveillance to assist the National TB Control Program in strengthening treatment strategies for improved outcomes.

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“…The main impetus was the timely detection of multidrug-resistant TB, [16][17][18][19] as RMP resistance is a surrogate for isoniazid resistance. 20,21 The key lesson learnt from the Xpert roll-out was that access to diagnosis is not the only indicator for measuring successful implementation; the rate of unsuccessful tests (which add no value to treatment) is also an important indicator of the effectiveness of Xpert implementation over time. Although Xpert was able to de- Unsuccessful Xpert results…”

Section: Public Health Actionmentioning

confidence: 99%

Unsuccessful Xpert^® MTB/RIF results: the Nigerian experience

Gidado

Nwokoye

Nwadike

et al. 2018

public health action

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Setting: Nigeria, a high tuberculosis (TB) burden country. Objective: To study the rate, distribution and causes of unsuccessful Xpert ® MTB/RIF test outcomes, with the aim of identifying key areas that need to be strengthened for optimal performance of the assay. Design: This was a retrospective analysis of data uploaded between January and December 2015 from Xpert facilities to the central server using GXAlert. Result: Of 52 219 test results uploaded from 176 Xpert machines, 22.5% were positive for Mycobacterium tuberculosis, 10.8% of which were rifampicin-resistant; 4.7% of the total number of results were invalid, 4.2% had error results and 2.1% no result outcomes. Technical errors were most frequent (69%); these were non-seasonal and occurred in all geopolitical regions and at all health facility levels. Temperature-related errors were more prevalent in the North-West Region, with peaks in April to June. Peak periods for temperature and machine malfunction errors coincided with the periods of low utilisation of the assay. Conclusion:The key challenge affecting performance was poor adherence to standard operating procedures. Periodic refresher training courses, regular supervision, preventive maintenance of Xpert machines and proper storage of cartridges are strategies that could improve Xpert performance.

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Molecular analysis of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates by multiple allele specific polymerase chain reaction in Puducherry, South India

Abstract: The simplicity and specificity of the MAS-PCR assay allows for easy implementation in clinical laboratories to detect rifampicin drug resistance in MDR-TB strains.

Cited by 36 publications

References 20 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

Prevalence of drug-resistant pulmonary tuberculosis in India: systematic review and meta-analysis

Unsuccessful Xpert^® MTB/RIF results: the Nigerian experience

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Molecular analysis of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates by multiple allele specific polymerase chain reaction in Puducherry, South India

Abstract: The simplicity and specificity of the MAS-PCR assay allows for easy implementation in clinical laboratories to detect rifampicin drug resistance in MDR-TB strains.

Cited by 36 publications

References 20 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

Prevalence of drug-resistant pulmonary tuberculosis in India: systematic review and meta-analysis

Unsuccessful Xpert® MTB/RIF results: the Nigerian experience

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Unsuccessful Xpert^® MTB/RIF results: the Nigerian experience