Dataset Information

Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

ABSTRACT:

Background

Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs.

Methods

We isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene.

Results

We tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases.

Conclusion

This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.

SUBMITTER: Rowneki M

PROVIDER: S-EPMC7209238 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

Rowneki Mazhgan M Aronson Naomi N Du Peicheng P Sachs Paige P Blakemore Robert R Chakravorty Soumitesh S Levy Shawn S Jones Angela L AL Trivedi Geetika G Chebore Sheilla S Addo Dennis D Byarugaba Denis K DK Njobvu Panganani Dalisani PD Wabwire-Mangen Frederick F Erima Bernard B Ramos Eric S ES Evans Carlton A CA Hale Braden B Mancuso James D JD Alland David D

PloS one 20200508 5

<h4>Background</h4>Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs.<h4>Methods</h4>We isolated MTB DNA by boiling smear content in a C ...[more]

PMID: 32384098

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Project description:BackgroundEthiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations.MethodsWe enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs.ResultDNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%-95.8%) for isoniazid, 95.5% (95% CI, 91.2%-99.8%) for rifampicin, 98.6% (95% CI, 95.9-100%) for ethambutol, 91.3% (95% CI, 84.6-98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%-67.1%) for streptomycin.ConclusionWe detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future.

Project description:BackgroundExtrapulmonary tuberculosis (EPTB) refers to a form of Tuberculosis (TB) where the infection occurs outside the lungs. Despite EPTB being a devastating disease of public health concern, it is frequently overlooked as a public health problem. This study aimed to investigate genetic diversity, identify drug-resistance mutations, and trace ongoing transmission chains.MethodsA cross-sectional study was undertaken on individuals with EPTB in western Ethiopia. In this study, whole-genome sequencing (WGS) was employed to analyze Mycobacterium tuberculosis (MTB) samples obtained from EPTB patients. Out of the 96 genomes initially sequenced, 89 met the required quality standards for genetic diversity, and drug-resistant mutations analysis. The data were processed using robust bioinformatics tools.ResultsOur analysis reveals that the majority (87.64%) of the isolates can be attributed to Lineage-4 (L4), with L4.6.3 and L4.2.2.2 emerging as the predominant sub-lineages, constituting 34.62% and 26.92%, respectively. The overall clustering rate and recent transmission index (RTI) were 30 and 17.24%, respectively. Notably, 7.87% of the isolates demonstrated resistance to at least one anti-TB drug, although multi-drug resistance (MDR) was observed in only 1.12% of the isolates.ConclusionsThe genetic diversity of MTBC strains in western Ethiopia was found to have low inter-lineage diversity, with L4 predominating and exhibiting high intra-lineage diversity. The notably high clustering rate in the region implies a pressing need for enhanced TB infection control measures to effectively disrupt the transmission chain. It's noteworthy that 68.75% of resistance-conferring mutations went undetected by both GeneXpert MTB/RIF and the line probe assay (LPA) in western Ethiopia. The identification of resistance mutations undetected by both GeneXpert and LPA, along with the detection of mixed infections through WGS, emphasizes the value of adopting WGS as a high-resolution approach for TB diagnosis and molecular epidemiological surveillance.

Project description:Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Dataset Information

Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

Background

Methods

Results

Conclusion

Publications

Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

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