Dataset Information

Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant Mycobacterium tuberculosis from respiratory specimens.

ABSTRACT: An in-house-developed target amplicon sequencing by next-generation sequencing technology (TB-NGS) enables simultaneous detection of resistance-related mutations in Mycobacterium tuberculosis (MTB) against 8 anti-tuberculosis drug classes. In this multi-center study, we investigated the clinical utility of incorporating TB-NGS for rapid drug-resistant MTB detection in high endemic regions in southeast China. From January 2018 to November 2019, 4,047 respiratory specimens were available from patients suffering lower respiratory tract infections in Hong Kong and Guangzhou, among which 501 were TB-positive as detected by in-house IS6110-qPCR assay with diagnostic sensitivity and specificity of 97.9 and 99.2%, respectively. Preliminary resistance screening by GenoType MTBDRplus and MTBDRsl identified 25 drug-resistant specimens including 10 multidrug-resistant TB. TB-NGS was performed using MiSeq on all drug-resistant specimens alongside 67 pan-susceptible specimens, and demonstrated 100% concordance to phenotypic drug susceptibility test. All phenotypically resistant specimens with dominating resistance-related mutations exhibited a mutation frequency of over 60%. Three quasispecies were identified with mutation frequency of less than 35% among phenotypically susceptible specimens. They were well distinguished from phenotypically resistant cases and thus would not complicate TB-NGS results interpretations. This is the first large-scale study that explored the use of laboratory-developed NGS platforms for rapid TB diagnosis. By incorporating TB-NGS with our proposed diagnostic algorithm, the workflow would provide a user-friendly, cost-effective routine diagnostic solution for complicated TB cases with an average turnaround time of 6 working days. This is critical for timely management of drug resistant TB patients and expediting public health control on the emergence of drug-resistant TB.

SUBMITTER: Leung KS

PROVIDER: S-EPMC9505518 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant <i>Mycobacterium tuberculosis</i> from respiratory specimens.

Leung Kenneth Siu-Sing KS Tam Kingsley King-Gee KK Ng Timothy Ting-Leung TT Lao Hiu-Yin HY Shek Raymond Chiu-Man RC Ma Oliver Chiu Kit OCK Yu Shi-Hui SH Chen Jing-Xian JX Han Qi Q Siu Gilman Kit-Hang GK Yam Wing-Cheong WC

Frontiers in microbiology 20220909

An in-house-developed target amplicon sequencing by next-generation sequencing technology (TB-NGS) enables simultaneous detection of resistance-related mutations in <i>Mycobacterium tuberculosis</i> (MTB) against 8 anti-tuberculosis drug classes. In this multi-center study, we investigated the clinical utility of incorporating TB-NGS for rapid drug-resistant MTB detection in high endemic regions in southeast China. From January 2018 to November 2019, 4,047 respiratory specimens were available fr ...[more]

PMID: 36160212

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Project description:Next-generation sequencing (NGS) enables rapid identification of common and rare drug-resistant genetic variations from tuberculosis (TB) patients' sputum samples and MTB isolates. However, whether this technology is effective for formalin-fixed and paraffin-embedded (FFPE) tissues remains unclear. An amplicon-based targeted NGS sequencing panel was developed to predict susceptibility to 9 antituberculosis drugs, including 3 first-line drugs, by directly detecting FFPE tissues. A total of 178 tissue samples from TB patients who underwent phenotypic drug susceptibility test were retrospectively tested from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. Phenotypic drug susceptibility test results were used as the reference standard. We identified 22 high-quality mutations from 178 FFPE tissue samples, including 15 high+moderate+minimal confidence-level mutations associated with drug resistance (rpoB D435V, S450F/L; KatG S315T; inhA-fabG promoter c-15t; embB G406S, M306V; rpsL K43R, K88R, rrs a1401g, a514c; gyrA D94G/Y/A, A90V), 6 mutations not associated with resistance (rpoB D435Y, H445S, L430P, L452P; embB G406A/D), and one mutation site embB M306I defined as indeterminate. Compared to the phenotypic method, sensitivities (95% CI) for rifampicin, isoniazid, and ethambutol were 96% (79.65-99.90%), 93.55% (78.58-99.21%), and 71.43% (35.24-92.44%), respectively; while for second-line drugs, it varied from 23.53% (9.05-47.77%) for capreomycin to 86.84% (72.20-94.72%) for streptomycin. Specificities for all drugs were satisfactory (>94.51%). Therefore, important pathological FFPE tissue samples, despite partially degraded DNA, can be used as essential specimens for molecular diagnosis of drug resistant TB by amplicon-based targeted NGS technology. IMPORTANCE Amplicon-based targeted NGS technology focuses on a set of gene mutations of known or suspected associations with drug susceptibility in Mycobacterium tuberculosis (MTB). This method offers many benefits, such as low sequencing cost, easy customization, high throughput, shorter testing time and not culture dependent. Formalin-fixed and paraffin-embedded (FFPE) tissues are important pathological specimen in diagnosing tuberculous disease because they are noninfectious and provide excellent preservation of tissue morphology with low storage cost. However, the performance of amplicon-based targeted NGS method on FFPE samples has not been reported yet. Therefore, we evaluated the performance of this method using FFPE samples collected from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. We demonstrate that the amplicon-based targeted NGS method performs excellent on FFPE samples, and it can be applied to pathological diagnosis of drug resistant tuberculosis.

Project description:Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a practical approach for the rapid differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis.

Dataset Information

Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant Mycobacterium tuberculosis from respiratory specimens.

Publications

Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant <i>Mycobacterium tuberculosis</i> from respiratory specimens.

Similar Datasets

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