Tuberculosis (TB) management in endemic regions often grapples with resource constraints, including the scarcity of Mycobacterium tuberculosis (M.tb) culture laboratories. The emergence of M.tb strains with complex drug resistance profiles necessitates rapid and comprehensive drug susceptibility testing (DST) to guide patient treatment. However, traditional phenotypic DST (pDST) for M.tb is costly and time-consuming. In this study, we retrospectively enrolled 829 participants from six specialized TB treatment hospitals in China from September 2022 to July 2023. The diagnostic performance of TBseq test, a targeted-nanopore sequencing assay, was compared head-to-head with M.tb culture, acid-fast bacillus smear, quantitative polymerase chain reaction, and Xpert MTB/RIF Ultra by using clinical diagnosis as the reference standard. Subsequently, pDST for seven anti-TB drugs (rifampicin, isoniazid, ethambutol, streptomycin, levofloxacin, amikacin, and capreomycin) was performed. The resistance predictions provided by the TBseq test were compared with pDST results, which were used as a reference standard. The performance estimates of TBseq test were quantified through sensitivity, specificity, positive predictive value, negative predictive value, and the area under the receiver operating characteristic curve (AUC), providing a comprehensive assessment of its diagnostic accuracy. We found that TBseq test demonstrated significantly superior diagnostic performance for TB compared to other methods, achieving a sensitivity of 90.9% (95% CI: 88.9%-93.0%), specificity of 93.0% (95% CI: 97.2%-99.5%), and an AUC of 0.92 (95% CI: 0.876-0.963). TBseq test also exhibited robust predictive capabilities for drug resistance to the seven anti-TB drugs, with sensitivity and specificity consistently above 90% for all drugs. The AUC values ranged from 0.919 to 0.998, indicative of high diagnostic accuracy in forecasting drug resistance.IMPORTANCEOur results show that TBseq test offers excellent identification performance for tuberculosis (TB), significantly outperforming Mycobacterium tuberculosis (M.tb) culture, acid-fast bacillus (AFB) smear, qPCR, and Xpert MTB/RIF. Its diagnostic accuracy for anti-TB drug resistance is also superior, with sensitivity and specificity above 90% for all drugs tested. This method can be integrated into routine clinical diagnostic workflows, enabling early diagnosis and reporting of drug resistance simultaneously.

Pathogen sequencing is an important tool for disease surveillance and demonstrated its high value during the COVID-19 pandemic. Viral sequencing during the pandemic allowed us to track disease spread, quickly identify new variants, and guide the development of vaccines. Tiled amplicon sequencing, in which a panel of primers is used for multiplex amplification of fragments across an entire genome, was the cornerstone of SARS-CoV-2 sequencing. The speed, reliability, and cost-effectiveness of this method led to its implementation in academic and public health laboratories across the world and adaptation to a broad range of viral pathogens. However, similar methods are not available for larger bacterial genomes, for which whole-genome sequencing typically requires in vitro culture. This increases costs, error rates and turnaround times. The need to culture poses particular problems for medically important bacteria such as Mycobacterium tuberculosis, which are slow to grow and challenging to culture. As a proof of concept, we developed two novel whole-genome amplicon panels for M. tuberculosis and Streptococcus pneumoniae. Applying our amplicon panels to clinical samples, we show the ability to classify pathogen subgroups and to reliably identify markers of drug resistance without culturing. Development of this work in clinical settings has the potential to dramatically reduce the time of diagnosis of drug resistance for multiple drugs in parallel, enabling earlier intervention for high priority pathogens.

Culturing and genomic sequencing of Mycobacterium tuberculosis (MTB) from tuberculosis (TB) cases is the basis for many research and clinical applications. The alternative, culture-free sequencing from diagnostic samples, is promising but poses challenges to obtain and analyse the MTB genome. Paradoxically, culture is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. To unravel this paradox we generate high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics analysis. Careful downstream comparisons reveal sources of sputum-culture incongruences due to false positive/negative variation associated with factors like low input MTB DNA or variable genomic depths. After accounting for these factors, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants' frequency (0.98). The combined analysis from five different settings and more than 100 available samples shows that our results can be extrapolated to different TB epidemic scenarios, demonstrating that for the cases tested culture accurately mirrors clinical samples.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potent tool for detecting drug resistance in tuberculosis (TB); however, concerns about its reliability have been raised. In this study, we assessed the reliability of MassARRAY (Sequenom, Inc.), which is a MALDI-TOF MS-based method, by comparing it to the well-established GeneXpert assay (Cepheid) as a reference method.

A retrospective study was conducted using laboratory data retrieved from Henan Chest Hospital (Zhengzhou, China). To ensure a rigorous evaluation, we adopted a comprehensive assessment approach by integrating multiple outcomes of the Xpert assay across various specimen types.

Among the 170 enrolled TB cases, MassARRAY demonstrated significantly higher sensitivity (85.88%, 146 of 170) compared to the Xpert assay (76.62%, 118 of 154) in TB diagnosis (p < 0.05). The concordance in detecting rifampicin resistance between MassARRAY and the combined outcomes of the Xpert assay was 90%, while it was 97.37% (37 of 38) among smear-positive cases and 89.06% (57 of 64) among culture-positive cases. When compared to the phenotypic susceptibility outcomes of the 12 included drugs, consistency rates of 81.8 to 93.9% were obtained, with 87.9% for multiple drug resistance (MDR) identification.

MassARRAY demonstrates high reliability in detecting rifampicin resistance, and these findings may offer a reasonable basis for extrapolation to other drugs included in the test panel.

Pulmonary tuberculosis (PTB) and lung cancer (LC) have similar clinical symptoms and atypical imaging findings, which are easily misdiagnosed. There is an urgent need for a noninvasive and accurate biomarker to distinguish LC from PTB.

A total of 694 subjects were enrolled and divided into discovery set (n = 122), identification set (n = 214), and validation set (n = 358). Metabolites were identified by multivariate and univariate analyses. Receiver operating characteristic curve were used to evaluate the diagnostic efficacy of biomarkers.

Seven metabolites were identified and validated. Phenylalanylphenylalanine for distinguishing LC from PTB yielded an area under the curve of 0.89, sensitivity of 71%, and specificity of 92%. It also showed good diagnostic abilities in discovery set and identification set. Compared with that in healthy volunteers (median [interquartile range], 1.57 [1.01, 2.34] μg/mL), it was elevated in LC (4.76 [2.74, 7.08] μg/mL; ratio of median, [ROM] = 3.03, P < .01) and reduced in PTB (1.06 [0.51, 2.09] μg/mL; ROM = 0.68, P < .05).

The metabolomic profile of LC and PTB was described and a key biomarker identified. We produced a rapid and noninvasive method to supplement existing clinical diagnostic examinations for distinguishing LC from PTB.

Within-host Mycobacterium tuberculosis (Mtb) diversity may detect antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here, we compared three sample pre-processing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Mtb-positive NALC-NaOH-treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I, or benzonase digestion. Levels of contaminating host DNA and target Mtb DNA were assessed by quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. The pre-treatment sample has a high host-to-target ratio of DNA (6,168 ± 1,638 host copies/ng to 212.3 ± 59.4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3,422 ± 2,162 host copies/ng to 11,721 ± 7,096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb target for all three pre-processing methods. Comparing the results of the benzonase sample sequenced both with and without enrichment, the percent of sequencing reads mapping to the Mtb increased to 90.95% from 1.18%. We demonstrate a low limit of detection for a new custom dsDNA Mtb target enrichment panel that has a favorable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis, M. africanum Lineages 5/6 and M. bovis to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by M. tuberculosis, while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections.

Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients' disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. IMPORTANCE We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences.

Tuberculosis (TB), an airborne infectious disease caused by Mycobacterium tuberculosis complex (MTBC), remains a global health problem. West Africa has a unique epidemiology of TB that is characterized by medium- to high-prevalence. Moreover, the geographical restriction of M. africanum to the sub-region makes West Africa have an extra burden to deal with a two-in-one pathogen. The region is also burdened with low case detection, late reporting, poor treatment adherence leading to development of drug resistance and relapse. Sporadic studies conducted within the subregion report higher burden of drug resistant TB (DRTB) than previously thought. The need for more sensitive and robust tools for routine surveillance as well as to understand the mechanisms of DRTB and transmission dynamics for the design of effective control tools, cannot be overemphasized. The advancement in molecular biology tools including traditional fingerprinting and next generation sequencing (NGS) technologies offer reliable tools for genomic epidemiology. Genomic epidemiology provides in-depth insight of the nature of pathogens, circulating strains and their spread as well as prompt detection of the emergence of new strains. It also offers the opportunity to monitor treatment and evaluate interventions. Furthermore, genomic epidemiology can be used to understand potential emergence and spread of drug resistant strains and resistance mechanisms allowing the design of simple but rapid tools. In this review, we will describe the local epidemiology of MTBC, highlight past and current investigations toward understanding their biology and spread as well as discuss the relevance of genomic epidemiology studies to TB control in West Africa.