matrix-based-dna-extraction-for-targeted-next-generation-sequencing

Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS ...

Stellenbosch University

Green Point Tuberculosis Laboratory

 is the number one cause of hospital-acquired diarrhoea. Accurate diagnosis of  is of utmost importance as it guides patient management and infection control practices. Studies evaluating the performance of commercially available nucleic acid amplification tests (NAATs) versus algorithms are lacking in resource-limited settings.

Comparison of commercial assays and two-step approach to detect  in South Africa.

Nontuberculous mycobacteria (NTM) are a group of acid-fast mycobacteria other than  complex (MTBC) that cause pulmonary disease that is similar to the disease caused by MTBC. International guidelines for the diagnosis of pulmonary NTM disease are rigid and have remained unchanged for nearly 2 decades. In this opinion piece, we provide a new perspective on the traditional criteria by suggesting a diagnostic algorithm that incorporates direct molecular identification of NTM performed on raw sputum specimens (using Sanger or targeted deep sequencing approaches, among others) paired with traditional culture methods. Our approach ensures a more rapid diagnosis of pulmonary NTM disease, thus, facilitating timeous clinical diagnosis, and prompt treatment initiation, where indicated, and leverages recent advances in novel molecular techniques into routine NTM identification practice.

Incorporating direct molecular diagnostics in management algorithms for nontuberculous mycobacteria: Is it high time?

Misdiagnosis of non-tuberculous mycobacteria as tuberculous by the GeneXpert MTB/RIF Ultra: Fact or fiction?

Isolation of pure non-tuberculous mycobacteria culture from a mixed growth with conventional microbiology: Techniques not to be forgotten.

The GenoType CMdirect Ver 1.0 and GenoType Mycobacterium Common Mycobacteria Ver 2.0-line probe assays were utilized to identify nontuberculous mycobacteria (NTM) from decontaminated sputum in a GeneXpert MTB/RIF Ultra negative cohort. A preliminary NTM prevalence of 1.7 % was observed, with a 100 % specificity. The high specificity advocates the CMdirect as a possible rule-in test for NTM disease.

Identification of nontuberculous mycobacteria using the GenoType CMdirect and GenoType Mycobacterium CM line probe assays in GeneXpert MTB/RIF Ultra negative sputum: A South African prevalence study.

Nontuberculous mycobacteria (NTM) infections present a global health challenge. This study describes unidentified mycobacteria species from extrapulmonary sites, using advanced identification and sequencing techniques.

Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures.

Nontuberculous mycobacteria (NTM) are increasingly recognized opportunistic pathogens found ubiquitously in the environment. The presence of multiple NTM species at the site of disease complicates diagnosis and treatment.

Mixed nontuberculous mycobacteria in an immunocompromised patient with probable progressive multifocal leukoencephalopathy.

Xpert MTB/RIF Ultra (Ultra)-detected rifampicin-resistant tuberculosis (TB) is often programmatically confirmed using MTBDR. There are limited data on discordant results, including when re-tested using newer methods, like FluoroType MTBDR (FT-MTBDR) and targeted deep sequencing. MTBDR rifampicin-susceptible isolates from people with Ultra rifampicin-resistant sputum were identified from a South African programmatic laboratory. FT-MTBDR and single molecule-overlapping reads (SMOR; , , ) on isolate DNA were done (SMOR was used as a reference standard). Between 1 April 2021 and 30 September 2022, 8% (109/1347) of Ultra rifampicin-resistant specimens were MTBDR-susceptible. Of 89% (97/109) isolates with a sequenceable , SMOR resolved most in favor of Ultra (79% [77/97]). Sputum with lower mycobacterial load was associated with Ultra false-positive resistance (46% [11/24] of "very low" Ultra had false resistance vs 12% [9/73;  = 0.0004] of ≥"low"), as were Ultra heteroresistance calls (all wild-type probes, ≥1 mutant probe) (62% [23/37 vs 25% 15/60] for Ultra without heteroresistance calls;  = 0.0003). Of the 91% (88/97) of isolates successfully tested by FT-MTBDR, 55% (48/88) were FT-MTBDR rifampicin-resistant and 45% (40/88) susceptible, translating to 69% (47/68) sensitivity and 95% (19/20) specificity. In the 91% (99/109) of isolates with  and  sequenced, 62% (61/99) were SMOR isoniazid-susceptible. When Ultra and MTBDR rifampicin results are discordant, Ultra is more likely to be correct, and FT-MTBDR agrees more with Ultra than MTBDR; however, lower load and the Ultra heteroresistance probe pattern were risk factors for Ultra false rifampicin-resistant results. Most people with Ultra-MTBDR discordant resistance results were isoniazid-susceptible. These data have implications for drug-resistant TB diagnosis.

Xpert MTB/RIF Ultra-resistant and MTBDRsusceptible rifampicin results in people with tuberculosis: utility of FluoroType MTBDR and deep sequencing.

Mycobacterium Growth Indicator Tube (MGIT) 960 culture is critical for tuberculosis (TB) drug susceptibility testing (DST) but vulnerable to contamination. We evaluated the accuracy of Xpert MTB/XDR, a molecular DST for isoniazid, fluoroquinolone, amikacin, and ethionamide, on to-be-discarded contaminated growth.

Waste to Worth: A diagnostic accuracy of Xpert MTB/XDR on contaminated liquid cultures to salvage the detection of drug-resistant tuberculosis.

This study evaluated six DNA (Deoxyribonucleic acid) extraction methods for , focusing on cost, procedure time, yield, quality, DNA integrity, and suitability for amplicon-based sequencing. The extracted DNA was used in the GenoType Mycobacterium Common Mycobacterium (CM) line probe assay (LPA), which is routinely used for nontuberculous mycobacteria (NTM) identification in South Africa.

Article	Year
Comparison of commercial assays and two-step approach to detect in South Africa. African journal of laboratory medicine\| PubMed ID: 36263391	2022
Incorporating direct molecular diagnostics in management algorithms for nontuberculous mycobacteria: Is it high time? IJID regions\| PubMed ID: 38304760	2024
Misdiagnosis of non-tuberculous mycobacteria as tuberculous by the GeneXpert MTB/RIF Ultra: Fact or fiction? Southern African journal of infectious diseases\| PubMed ID: 38628427	2024
Isolation of pure non-tuberculous mycobacteria culture from a mixed growth with conventional microbiology: Techniques not to be forgotten. The Indian journal of tuberculosis\| PubMed ID: 39111929	2024
Identification of nontuberculous mycobacteria using the GenoType CMdirect and GenoType Mycobacterium CM line probe assays in GeneXpert MTB/RIF Ultra negative sputum: A South African prevalen... Diagnostic microbiology and infectious disease\| PubMed ID: 39293319	2024
Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures. IJID regions\| PubMed ID: 39483154	2024
Mixed nontuberculous mycobacteria in an immunocompromised patient with probable progressive multifocal leukoencephalopathy. IJID regions\| PubMed ID: 39810748	2025
Xpert MTB/RIF Ultra-resistant and MTBDRsusceptible rifampicin results in people with tuberculosis: utility of FluoroType MTBDR and deep sequencing. Antimicrobial agents and chemotherapy\| PubMed ID: 39918315	2025
Waste to Worth: A diagnostic accuracy of Xpert MTB/XDR on contaminated liquid cultures to salvage the detection of drug-resistant tuberculosis. Research square\| PubMed ID: 40297690	2025
DNA extraction: Implications for NTM identification and amplicon sequencing. Southern African journal of infectious diseases\| PubMed ID: 40469625	2025

Sarishna Singh

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