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

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.

Targeted-Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis Cultures Using Two Commercial Sequencing Platforms: Comparison of Analytical and Diagnostic Performance, Turnaround Time, and Cost.

BACKGROUND: The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics. METHODS: A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports. RESULTS: Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities. CONCLUSION: Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.

Diagnostic evaluation of an in-house developed single-tube, duplex, nested IS6110 real-time PCR assay for rapid pulmonary tuberculosis diagnosis.

OBJECTIVE: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis. METHODS: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain. RESULT: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57). CONCLUSION: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden.

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c (lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c (cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA, and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.