Detection of Heteroresistant Mycobacterium tuberculosis by Pyrosequencing

Engström, Arne; Hoffner, Sven; Juréen, Pontus

doi:10.1128/jcm.01761-13

Cited by 10 publications

(3 citation statements)

References 15 publications

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“…Similar findings have been reported previously in an HRM assay for pyrazinamide resistance detection (46). Heteroresistance has been identified previously in many studies using DNA sequencing (47,48). Mixed patterns have been reported in India at as high as 29 to 34% of isolates for codons 531, 526, and 516 (25,44).…”

Section: Discussionsupporting

confidence: 74%

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: 74%

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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“…There were two Rifampicin-resistant MTB populations from one MTB culture isolate with a different base sequence of the rpoB gene based on the Pyrosequencing. 18 Discrepancy could relate with different samples used. 16 This study used two samples from one subject who had not received MDR-TB drugs yet for the Xpert MTB/RIF assay and the Pyrosequencing tests.…”

Section: Resultsmentioning

confidence: 99%

Comparison of Interpretation between Pyrosequencing and Xpert MTB/RIF Assay in Multidrug-Resistant Tuberculosis

Choerunnisa

Rita

Tjandrawati

et al. 2020

mkb

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Indonesia is one of the countries with the highest multidrug-resistant tuberculosis cases in the world. Rapid molecular test using the Xpert MTB/RIF assay is one of the detection methods for MDR-TB. Early detection of MDR-TB is crucial for early initiation of treatment. However, Xpert MTB/RIF assay only detects the rpoB gene mutations associated with Rifampicin resistance. Recently, WHO recommends the use of Pyrosequencing, a DNA sequencing method that can detect not only the rpoB gene but also katG and/or inhA gene mutations associated with Isoniazid resistance. The aims of this study were to compare the interpretation between the two methods and to determine the differences in codon mutation position detection of the rpoB gene and mutation detection of the katG and/or inhA gene. This was a cross-sectional comparative observational study on patients ≥18 years old interpreted as RR-TB patients based on Xpert MTB/RIF assay results who had not received MDR-TB drugs at Dr. Hasan Sadikin General Hospital, Bandung, Indonesia. Results showed there were 40 Rifampicin-resistant TB subjects interpreted by Xpert MTB/RIF assay while Pyrosequencing interpreted 30 MDR-TB, 9 RR-TB and one Isoniazid-resistant TB subjects in January - February 2020. The detection of rpoB gene codon mutation position between Xpert MTB/RIF assay and Pyrosequencing methods was not significantly different (p=0.389). Pyrosequencing had detected 27 katG gene mutations, 3 inhA gene mutations, one katG and inhA gene mutation. To conclude, Pyrosequencing can be used for accurate detection of Rifampicin and Isoniazid resistance in MDR-TB.

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“…This is because heteroresistance represents various evolutionary stages of drug resistance during treatment 1 . Moreover, a small proportion of the heteroresistant population may remain undetectable using molecular diagnosis techniques due to the presence of any mutation in the targeted region 9 .…”

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confidence: 99%

Heteroresistance to rifampicin & isoniazid in clinical samples of patients with presumptive drug-resistant tuberculosis in Central India

Desikan¹,

Panwalkar²,

Punde³

et al. 2023

Indian Journal of Medical Research

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Background & objectives: A combination of resistant and susceptible Mycobacterium tuberculosis (MTB) isolated from clinical specimens is referred to as heteroresistance. Heteroresistance leads to difficulties in drug resistance testing and may adversely affect treatment outcomes. The present study estimated the proportion of heteroresistance among MTB in clinical samples of presumptive drug-resistant tuberculosis (TB) patients in Central India. Methods: A retrospective analysis of data generated from line probe assay (LPA) at a tertiary care hospital in Central India between January 2013 and December 2018 was carried out. A heteroresistant MTB in a sample was indicated by the presence of both wild-type and mutant-type patterns on an LPA strip. Results: Data analysis was carried out on interpretable 11,788 LPA results. Heteroresistance in MTB was detected in 637 (5.4%) samples. Of these, heteroresistance in MTB was detected in 413 (64.8%), 163 (25.5%) and 61 (9.5%) samples with respect to rpoB , katG and inhA genes, respectively. Interpretation & conclusions: Heteroresistance is considered a preliminary step in the development of drug resistance. Delayed or suboptimal anti-tubercular therapy in patients with heteroresistance of MTB may elicit full clinical resistance and negatively impact the National TB Elimination Programme. Further studies are, however, needed to determine the impact of heteroresistance on treatment outcomes in individual patients.

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Detection of Heteroresistant Mycobacterium tuberculosis by Pyrosequencing

Cited by 10 publications

References 15 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

Comparison of Interpretation between Pyrosequencing and Xpert MTB/RIF Assay in Multidrug-Resistant Tuberculosis

Heteroresistance to rifampicin & isoniazid in clinical samples of patients with presumptive drug-resistant tuberculosis in Central India

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