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. 2023 Aug 17;11(4):e0111423.
doi: 10.1128/spectrum.01114-23. Epub 2023 Jun 26.

Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing

Nabila Ismail #  1 , Anzaan Dippenaar #  1   2 , George Morgan  3 , Melanie Grobbelaar  1 , Felicia Wells  1 , Jessica Caffry  3 , Cristiana Morais  3 , Krzysztof Gizynski  3 , David McGurk  3 , Eduardo Boada  3 , Heather Murton  3 , Robin M Warren  1 , Annelies Van Rie  2
Affiliations

Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing

Nabila Ismail et al. Microbiol Spectr. .

Abstract

Mycobacterium tuberculosis whole-genome sequencing (WGS) is a powerful tool as it can provide data on population diversity, drug resistance, disease transmission, and mixed infections. Successful WGS is still reliant on high concentrations of DNA obtained through M. tuberculosis culture. Microfluidics technology plays a valuable role in single-cell research but has not yet been assessed as a bacterial enrichment strategy for culture-free WGS of M. tuberculosis. In a proof-of-principle study, we evaluated the use of Capture-XT, a microfluidic lab-on-chip cleanup and pathogen concentration platform to enrich M. tuberculosis bacilli from clinical sputum specimens for downstream DNA extraction and WGS. Three of the four (75%) samples processed by the microfluidics application passed the library preparation quality control, compared to only one of the four (25%) samples not enriched by the microfluidics M. tuberculosis capture application. WGS data were of sufficient quality, with mapping depth of ≥25× and 9 to 27% of reads mapping to the reference genome. These results suggest that microfluidics-based M. tuberculosis cell capture might be a promising method for M. tuberculosis enrichment in clinical sputum samples, which could facilitate culture-free M. tuberculosis WGS. IMPORTANCE Diagnosis of tuberculosis is effective using molecular methods; however, a comprehensive characterization of the resistance profile of Mycobacterium tuberculosis often requires culturing and phenotypic drug susceptibility testing or culturing followed by whole-genome sequencing (WGS). The phenotypic route can take anywhere from 1 to >3 months to result, by which point the patient may have acquired additional drug resistance. The WGS route is a very attractive option; however, culturing is the rate-limiting step. In this original article, we provide proof-of-principle evidence that microfluidics-based cell capture can be used on high-bacillary-load clinical samples for culture-free WGS.

Keywords: Mycobacterium tuberculosis; cell capture; culture-free sequencing; microfluidics; tuberculosis; whole-genome sequencing.

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Conflict of interest statement

The authors declare a conflict of interest. GM-employed by QuantuMDx Ltd JC-employed by QuantuMDx Ltd CM-employed by QuantuMDx Ltd KG-previously employed by QuantuMDx Ltd DM-employed by QuantuMDx Ltd EB-employed by QuantuMDx Ltd HM-previously employed by QuantuMDx Ltd

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