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 ...

University of California, San Francisco

Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampin-resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods and explored the occurrence of pyrazinamide heteroresistance. We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the  gene, whole-genome sequencing (WGS), and phenotypic drug susceptibility testing. We calculated the diagnostic accuracy of the different methods and investigated the prevalence and clinical impact of  heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Köser classification and expert rules. We observed 100% agreement across genotypic methods for detection of  fixed mutations; only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% confidence interval [CI], 93.8 to 99.3%) and specificity of 100% (95% CI, 100 to 100%), both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI, 94.6 to 99.6%) and specificity of 97.8% (95% CI, 95.7 to 99.9%), and TDS had sensitivity of 98.8% (95% CI, 97.2 to 100%) and specificity of 97.8% (95% CI, 95.7 to 99.9%). We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mycobacterium tuberculosis isolates was lower than that identified for other first-line drugs.

Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis.

 (Mtb) persists in lung myeloid cells during chronic infection. However, the mechanisms allowing Mtb to evade elimination are not fully understood. Here, we determined that in chronic phase, CD11c monocyte-derived lung cells termed MNC1 (mononuclear cell subset 1), harbor more live Mtb than alveolar macrophages (AM), neutrophils, and less permissive CD11c MNC2. Transcriptomic and functional studies of sorted cells revealed that the lysosome biogenesis pathway is underexpressed in MNC1, which have less lysosome content, acidification, and proteolytic activity than AM, and less nuclear TFEB, a master regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in MNC1. Instead, Mtb recruits MNC1 and MNC2 to the lungs for its spread from AM to these cells via its ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome function of primary macrophages and MNC1 and MNC2 in vivo, improving control of Mtb infection. Our results indicate that Mtb exploits lysosome-poor monocyte-derived cells for in vivo persistence, suggesting a potential target for host-directed tuberculosis therapy.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Bedaquiline is a life-saving tuberculosis drug undergoing global scale-up. People at risk of weak tuberculosis drug regimens are a priority for novel drug access despite the potential source of Mycobacterium tuberculosis-resistant strains. We aimed to characterise bedaquiline resistance in individuals who had sustained culture positivity during bedaquiline-based treatment.

Bedaquiline resistance in patients with drug-resistant tuberculosis in Cape Town, South Africa: a retrospective longitudinal cohort study.

Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers.

Article	Year
Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis. Journal of clinical microbiology\| PubMed ID: 34757831	2022
Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection. Research square\| PubMed ID: 37398178	2023
Bedaquiline resistance in patients with drug-resistant tuberculosis in Cape Town, South Africa: a retrospective longitudinal cohort study. The Lancet. Microbe\| PubMed ID: 37931638	2023
primerJinn: a tool for rationally designing multiplex PCR primer sets for amplicon sequencing and performing in silico PCR. BMC bioinformatics\| PubMed ID: 38082220	2023

Jason Limberis

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