Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing
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. Mi...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-08-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.01114-23 |
| _version_ | 1797741777172561920 |
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| author | Nabila Ismail Anzaan Dippenaar George Morgan Melanie Grobbelaar Felicia Wells Jessica Caffry Cristiana Morais Krzysztof Gizynski David McGurk Eduardo Boada Heather Murton Robin M. Warren Annelies Van Rie |
| author_facet | Nabila Ismail Anzaan Dippenaar George Morgan Melanie Grobbelaar Felicia Wells Jessica Caffry Cristiana Morais Krzysztof Gizynski David McGurk Eduardo Boada Heather Murton Robin M. Warren Annelies Van Rie |
| author_sort | Nabila Ismail |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T14:31:35Z |
| format | Article |
| id | doaj.art-98ab556599db40239b79132b4ae9e34a |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-12T14:31:35Z |
| publishDate | 2023-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-98ab556599db40239b79132b4ae9e34a2023-08-17T13:04:13ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-08-0111410.1128/spectrum.01114-23Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome SequencingNabila Ismail0Anzaan Dippenaar1George Morgan2Melanie Grobbelaar3Felicia Wells4Jessica Caffry5Cristiana Morais6Krzysztof Gizynski7David McGurk8Eduardo Boada9Heather Murton10Robin M. Warren11Annelies Van Rie12Division of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South AfricaDivision of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South AfricaQuantuMDx Ltd., Newcastle upon Tyne, United KingdomDivision of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South AfricaDivision of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South AfricaQuantuMDx Ltd., Newcastle upon Tyne, United KingdomQuantuMDx Ltd., Newcastle upon Tyne, United KingdomQuantuMDx Ltd., Newcastle upon Tyne, United KingdomQuantuMDx Ltd., Newcastle upon Tyne, United KingdomQuantuMDx Ltd., Newcastle upon Tyne, United KingdomQuantuMDx Ltd., Newcastle upon Tyne, United KingdomDivision of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South AfricaTuberculosis Omics Research Consortium, Family Medicine and Population Health, Institute of Global Health, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, BelgiumABSTRACT 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.https://journals.asm.org/doi/10.1128/spectrum.01114-23Mycobacterium tuberculosistuberculosiswhole-genome sequencingmicrofluidicsculture-free sequencingcell capture |
| spellingShingle | Nabila Ismail Anzaan Dippenaar George Morgan Melanie Grobbelaar Felicia Wells Jessica Caffry Cristiana Morais Krzysztof Gizynski David McGurk Eduardo Boada Heather Murton Robin M. Warren Annelies Van Rie Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing Microbiology Spectrum Mycobacterium tuberculosis tuberculosis whole-genome sequencing microfluidics culture-free sequencing cell capture |
| title | Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing |
| title_full | Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing |
| title_fullStr | Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing |
| title_full_unstemmed | Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing |
| title_short | Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing |
| title_sort | microfluidic capture of mycobacterium tuberculosis from clinical samples for culture free whole genome sequencing |
| topic | Mycobacterium tuberculosis tuberculosis whole-genome sequencing microfluidics culture-free sequencing cell capture |
| url | https://journals.asm.org/doi/10.1128/spectrum.01114-23 |
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