Rapid genomic first- and second-line drug resistance prediction from clinical <i>Mycobacterium tuberculosis</i> specimens using Deeplex-MycTB

Feuerriegel, Silke; Kohl, Thomas A.; Utpatel, Christian; Andres, Sönke; Maurer, Florian P.; Heyckendorf, Jan; Jouet, Agathe; Badalato, Nelly; Foray, Lynda; Kamara, Rashidatu Fouad; Conteh, Osman S.; Supply, Philip; Niemann, Stefan

doi:10.1183/13993003.01796-2020

Cited by 57 publications

(42 citation statements)

References 17 publications

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“…Consistently, the similar mean read depths obtained on the specimens graded 1+, 2+ and 3+ from the DRC survey also suggest a limit of detection ≤1+ (corresponding to 5000–10 000 genomes·mL −1 of sample) with the DNA extraction conditions used, accounting also for the fact that the equivalent of about a tenth of the available 1–5 mL of the DRC samples was generally used for DNA extraction and testing. Further consistent with such analytical sensitivity, in a recent study using QIAamp DNA Mini kits for DNA extraction, complete Deeplex Myc-TB phenotypic predictions could be made from all 37 smear-positive (including five scanty) and two smear-negative samples, in a pilot series of 50 clinical specimens [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs

Jouet¹,

Gaudin²,

Badalato³

et al. 2020

Eur Respir J

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Conventional molecular tests for detecting Mycobacterium tuberculosis complex (MTBC) drug resistance on clinical samples cover a limited set of mutations. Whole genome sequencing (WGS) typically requires culture. Here, we evaluated the Deeplex Myc-TB targeted deep sequencing assay for prediction of resistance to 13 anti-tuberculous drugs/drug classes, directly applicable on sputum. With MTBC DNA tests, the limit of detection was 100–1000 genome copies for fixed resistance mutations. Deeplex Myc-TB captured in silico 97.1–99.3% of resistance phenotypes correctly predicted by WGS from 3651 MTBC genomes. On 429 isolates, the assay predicted 92.2% of 2369 first- and second-line phenotypes, with a sensitivity of 95.3% and specificity of 97.4%. Fifty-six of 69 (81.2%) residual discrepancies with phenotypic results involved pyrazinamide, ethambutol, and ethionamide, and low-level rifampicin- or isoniazid-resistance mutations, all notoriously prone to phenotypic testing variability. Only 2 of 91 (2.2%) resistance phenotypes undetected by Deeplex Myc-TB had known resistance-associated mutations by WGS analysis outside Deeplex Myc-TB targets. Phenotype predictions from Deeplex Myc-TB analysis directly on 109 sputa from a Djibouti survey matched those of MTBSeq/PhyResSE/Mykrobe, fed with WGS data from subsequent cultures, with a sensitivity of 93.5/98.5/93.1% and specificity of 98.5/97.2/95.3%. Most residual discordances involved gene deletions/indels and 3–12% heteroresistant calls undetected by WGS analysis, or natural pyrazinamide resistance of globally rare “M. canettii” strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14 902 of 19 422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free. Deeplex Myc-TB may enable fast, tailored tuberculosis treatment.

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Section: Discussionmentioning

confidence: 99%

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs

Jouet¹,

Gaudin²,

Badalato³

et al. 2020

Eur Respir J

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“…sputum) is difficult 3 . We recently demonstrated that targeted genome sequencing using the commercially available Deeplex ® -MycTB has a high sensitivity and specificity for first- and second-line drug resistance prediction, including BDQ/CFZ resistance mutations that have been found in patients 47,57,58 . In combination with our comprehensive set of mutations that confer resistance to BDQ/CFZ, Deeplex®-MycTB and other targeted sequencing approaches allow for rapid detection of BDQ/CFZ resistance from clinical samples in few days using small portable genome sequencers such as the MinION 59 .…”

Section: Discussionmentioning

confidence: 99%

Deciphering Bedaquiline and Clofazimine Resistance in Tuberculosis: An Evolutionary Medicine Approach

Sonnenkalb

Carter

Spitaleri

et al. 2021

Preprint

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Bedaquiline (BDQ) and clofazimine (CFZ) are core drugs for treatment of multidrug resistant tuberculosis (MDR-TB), however, our understanding of the resistance mechanisms for these drugs is sparse which is hampering rapid molecular diagnostics. To address this, we employed a unique approach using experimental evolution, protein modelling, genome sequencing, and minimum inhibitory concentration data (MIC) combined with genomes from a global strain collection of over 14,151 Mycobacterium tuberculosis complex isolates. Overall, 189 genomic variants causing elevated BDQ and/or CFZ MICs could be discerned, with 175 (95.1%) variants affecting the transcriptional repressor (Rv0678) of an efflux system (MmpS5-MmpL5). Structural modelling of Rv0678 suggests four major mechanisms that confer resistance: impairment of DNA binding, reduction in protein stability, disruption of protein dimerization, and reduction in affinity for its fatty acid ligand. These modelling and experimental techniques will improve personalized medicine in an impending drug resistant era.

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“…Genomic DNA of 30 randomly selected clinical Mtbc isolates was extracted at the Kyrgyz NRL using the CTAB protocol (SOP provided in the supplement materials), photometrically checked and separated into two identical 25 µl aliquots. NRL and RCB independently prepared libraries using the Nextera XT kit following the instructions of the manufacturer and using a modified Nextera protocol, respectively 9 , 18 . Both laboratories independently performed and bioinformatically analyzed WGS of the isolates on MiSeq and NextSeq 500 (Illumina, USA), respectively.…”

Section: Methodsmentioning

confidence: 99%

Implementation of whole genome sequencing for tuberculosis diagnostics in a low-middle income, high MDR-TB burden country

Vogel¹,

Utpatel

Corbett³

et al. 2021

Sci Rep

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Whole genome sequencing (WGS) is revolutionary for diagnostics of TB and its mutations associated with drug-resistances, but its uptake in low- and middle-income countries is hindered by concerns of implementation feasibility. Here, we provide a proof of concept for its successful implementation in such a setting. WGS was implemented in the Kyrgyz Republic. We estimated needs of up to 55 TB-WGS per week and chose the MiSeq platform (Illumina, USA) because of its capacity of up to 60 TB-WGS per week. The project’s timeline was completed in 93-weeks. Costs of large equipment and accompanying costs were 222,065 USD and 8462 USD, respectively. The first 174 WGS costed 277 USD per sequence, but this was skewed by training inefficiencies. Based on real prices and presuming optimal utilization of WGS capacities, WGS costs could drop to 167 and 141 USD per WGS using MiSeq Reagent Kits v2 (500-cycles) and v3 (600-cycles), respectively. Five trainings were required to prepare the staff for autonomous WGS which cost 48,250 USD. External assessment confirmed excellent performance of WGS by the Kyrgyz laboratory in an interlaboratory comparison of 30 M. tuberculosis genomes showing complete agreeance of results.

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Rapid genomic first- and second-line drug resistance prediction from clinical Mycobacterium tuberculosis specimens using Deeplex-MycTB

Abstract: Targeted next generation sequencing using the Deeplex-MycTB assay can rapidly generate comprehensive drug resistance profiles from Mycobacterium tuberculosis complex cultures and directly from tuberculosis patient samples to guide personalized treatment.

Cited by 57 publications

References 17 publications

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs

Deciphering Bedaquiline and Clofazimine Resistance in Tuberculosis: An Evolutionary Medicine Approach

Implementation of whole genome sequencing for tuberculosis diagnostics in a low-middle income, high MDR-TB burden country

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