Molecular drug resistance profiles of Mycobacterium tuberculosis from sputum specimens using ion semiconductor sequencing

Park, Joonhong; Jang, Woori; Kim, Myungshin; Kim, Yonggoo; Shin, So Youn; Park, Kuhn; Kim, Myung Sook; Shin, Soyoung

doi:10.1016/j.mimet.2017.12.003

Cited by 11 publications

(15 citation statements)

References 28 publications

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“…Similarly, we also noticed high percent of agreement between phenotypic and genotypic DST results for EMB (93.3%) with high sensitivity (100%) and specificity (88.2%). These results are concordant with some of the published studies [9,10], which showed large overlap in the estimated prevalence of EMB resistance by genetic sequencing and the estimated prevalence by phenotypic testing. However, several other studies observed discrepancies between the presence of common mutations at codon 306 of embB gene and phenotypic EMB resistance [5,8].…”

Section: Resultssupporting

confidence: 91%

“…However, the mechanisms of drug resistance are complex and not completely understood. Therefore, one of the main limitations of such molecular tests is that they evaluate only limited number of mutations linked with drug resistance in TB [9].…”

Section: Introductionmentioning

confidence: 99%

“…In recent years, significant and continued progress in next-generation sequencing (NGS) made this technology a promising clinical tool in comprehensive analysis of gene variants linked to drug resistance in TB [9]. Besides known mutations, NGS facilitates the discovery of novel variants in the entire coding regions of several genes previously implicated in MDR and/or XDR-TB resistance [9].…”

Section: Introductionmentioning

confidence: 99%

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Next-generation sequencing of drug resistant <i>Mycobacterium tuberculosis</i> clinical isolates in low-incidence countries

Содья

Toplak

Koren

et al. 2020

Russian Journal of Infection and Immunity

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Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.

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Section: Resultssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Next-generation sequencing of drug resistant <i>Mycobacterium tuberculosis</i> clinical isolates in low-incidence countries

Содья

Toplak

Koren

et al. 2020

Russian Journal of Infection and Immunity

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“…But targeted molecular assays are challenged by diverse mutations scattered across the pncA gene (561 bp plus promotor region) that is coding for the pyrazinamidase PncA, the drug activator ( Miotto et al, 2014 , Palomino and Martin, 2014 ). DNA sequencing approaches like whole genome sequencing (WGS) or amplicon sequencing can overcome this limitation ( Park et al, 2018 , Walker et al, 2017 ). Nevertheless, sequencing is expensive, technically demanding and yet not suitable as a point of care test ( Willby et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis

Havlicek

Dachsel

Slickers

et al. 2019

Diagnostic Microbiology and Infectious Disease

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Pyrazinamide (PZA) is a key antibiotic for the treatment of drug susceptible tuberculosis. PZA-resistance is mainly mediated by mutations in the pncA gene; however the current gold standard is a phenotypic drug susceptibility test requiring a well-adjusted pH-value for reliable results. Our melting curve assay detects a non-wild type genotype in selected pncA regions in at least 3750 gene copies/mL within 2.5 hours. The prototype assay was further evaluated by analyzing 271 Mycobacterium tuberculosis complex isolates from Swaziland originating from a previously published drug resistance survey and including 118 isolates with pncA mutations. Sensitivity was 83% (95% CI 75–89%) and specificity was 100% (95% CI 98–100%). Under consideration of further improvements with regard to the target range our melting curve assay has the potential as a rapid rule-in test for PZA susceptibility (wild type pncA ), however false resistant results (mutant pncA , but PZA susceptible) cannot be ruled out completely.

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“…There have been several reports using next-generation sequencing (NGS) technology to reveal drug resistance profiles in MTB [3–7]. Previous studies usually used only MTB that was resistant to one or more drugs, without including susceptible strains.…”

Section: Introductionmentioning

confidence: 99%

Application of next-generation sequencing to detect variants of drug-resistant Mycobacterium tuberculosis: genotype–phenotype correlation

Lee

et al. 2019

Ann Clin Microbiol Antimicrob

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BackgroundDrug resistance in Mycobacterium tuberculosis (MTB) is a major health issue worldwide. Recently, next-generation sequencing (NGS) technology has begun to be used to detect resistance genes of MTB. We aimed to assess the clinical usefulness of Ion S5 NGS TB research panel for detecting MTB resistance in Korean tuberculosis patients.MethodsMycobacterium tuberculosis with various drug resistance profiles including susceptible strains (N = 36) were isolated from clinical specimens. Nucleic acids were extracted from inactivated culture medium and underwent amplicon-based NGS to detect resistance variants in eight genes (gyrA, rpoB, pncA, katG, eis, rpsL, embB, and inhA). Data from previous studies using the same panel were merged to yield pooled sensitivity and specificity values for detecting drug resistance compared to phenotype-based methods.ResultsThe sequencing reactions were successful for all samples. A total of 24 variants were considered to be related to resistance, and 6 of them were novel. Agreement between the phenotypic and genotypic results was excellent for isoniazid, rifampicin, and ethambutol, and was poor for streptomycin, amikacin, and kanamycin. The negative predictive values were greater than 97% for all drug classes, while the positive predictive values varied (44% to 100%). There was a possibility that common mutations could not be detected owing to the low coverage.ConclusionsWe successfully applied NGS for genetic analysis of drug resistances in MTB, as well as for susceptible strains. We obtained lists of polymorphisms and possible polymorphisms, which could be used as a guide for future tests applying NGS in mycobacteriology laboratories. When analyzing the results of NGS, coverage analysis of each samples for each gene and benign polymorphisms not related to drug resistance should be considered.

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Molecular drug resistance profiles of Mycobacterium tuberculosis from sputum specimens using ion semiconductor sequencing

Cited by 11 publications

References 28 publications

Next-generation sequencing of drug resistant <i>Mycobacterium tuberculosis</i> clinical isolates in low-incidence countries

Next-generation sequencing of drug resistant <i>Mycobacterium tuberculosis</i> clinical isolates in low-incidence countries

Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis

Application of next-generation sequencing to detect variants of drug-resistant Mycobacterium tuberculosis: genotype–phenotype correlation

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