Dataset Information

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

ABSTRACT: Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

SUBMITTER: Votintseva AA

PROVIDER: S-EPMC5405248 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

Votintseva Antonina A AA Bradley Phelim P Pankhurst Louise L Del Ojo Elias Carlos C Loose Matthew M Nilgiriwala Kayzad K Chatterjee Anirvan A Smith E Grace EG Sanderson Nicolas N Walker Timothy M TM Morgan Marcus R MR Wyllie David H DH Walker A Sarah AS Peto Tim E A TEA Crook Derrick W DW Iqbal Zamin Z

Journal of clinical microbiology 20170308 5

Routine full characterization of <i>Mycobacterium tuberculosis</i> is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for <i>M. tuberculosis</i> WGS. We initially evaluated the method by using the Il ...[more]

PMID: 28275074

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Project description:BackgroundSputum smear microscopy is the most widely available diagnostic test for pulmonary tuberculosis in countries with a high burden of the disease. Improving its accuracy is crucial to achievement of case-detection targets established by the Millennium Development Goals. Unfortunately, many patients are unable to submit all of the specimens needed for examination or to return for treatment because standard sputum collection and reporting requires several clinic visits. To inform policy recommendations by a WHO-convened Expert Group, we aimed to assess the accuracy of sputum smear examination with strategies for obtaining sputum on 1 day compared with strategies for obtaining sputum over 2 days.MethodsWe did a systematic review and meta-analysis of research articles comparing the accuracy of front-loaded or same-day microscopy and standard sputum smear microscopy for diagnosis of culture-confirmed pulmonary tuberculosis. We searched Medline, Embase, Biosis, and Web of Science for articles published between Jan 1, 2005, and Feb 14, 2012. Two investigators identified eligible articles and extracted data for individual study sites. We generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when four or more studies were available.FindingsWe identified eight relevant studies from five articles enrolling 7771 patients with suspected tuberculosis in low-income countries. Compared with the standard approach of examination of two smears with Ziehl-Neelsen light microscopy over 2 days, examination of two smears taken on the same day had much the same sensitivity (64% [95% CI 60 to 69] for standard microscopy vs 63% [58 to 68] for same-day microscopy) and specificity (98% [97 to 99] vs 98% [97 to 99]). We noted similar results for studies employing light-emitting diode fluorescence microscopy and for studies examining three smears, whether they were compared with two-smear strategies or with one another.InterpretationSame-day sputum smear microscopy is as accurate as standard smear microscopy. Data from tuberculosis programmes are needed to document the changes required in the health system to successfully implement the strategy and understand its effects.FundingWHO and US National Institutes of Health.

Dataset Information

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

Publications

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

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