Genetic diversity within diagnostic sputum samples is mirrored in the culture of Mycobacterium tuberculosis across different settings.

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.

Monitoring of First-line Drug Resistance Mutations Outside the Scope of Xpert MTB/RIF Ultra is Needed for Successful Control of DR-TB in Southern Mozambique.

Multidrug-resistant(MDR) tuberculosis in Southern Africa is of great concern, exacerbated by the spread of a clone harboring a mutation missed by Xpert Ultra. In Southern Mozambique, the presence of such mutation and rising cases of non-MDR isoniazid resistance highlights the need to ensure accurate detection of antimicrobial-resistance in the country.

Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes.

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis, identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study's main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.

Use of next generation sequencing technologies for the diagnosis and epidemiology of infectious diseases. / Uso de las tecnologías de secuenciación masiva para el diagnóstico y epidemiología de enfermedades infecciosas.

For the first time, next generation sequencing technologies provide access to genomic information at a price and scale that allow their implementation in routine clinical practice and epidemiology. While there are still many obstacles to their implementation, there are also multiple examples of their major advantages compared with previous methods. Their main advantage is that a single determination allows epidemiological information on the causative microorganism to be obtained simultaneously, as well as its resistance profile, although these advantages vary according to the pathogen under study. This review discusses several examples of the clinical and epidemiological use of next generation sequencing applied to complete genomes and microbiomes and reflects on its future in clinical practice.

Towards next-generation diagnostics for tuberculosis: identification of novel molecular targets by large-scale comparative genomics.

MOTIVATION: Tuberculosis (TB) remains one of the main causes of death worldwide. The long and cumbersome process of culturing Mycobacterium tuberculosis complex (MTBC) bacteria has encouraged the development of specific molecular tools for detecting the pathogen. Most of these tools aim to become novel TB diagnostics, and big efforts and resources are invested in their development, looking for the endorsement of the main public health agencies. Surprisingly, no study has been conducted where the vast amount of genomic data available is used to identify the best MTBC diagnostic markers. RESULTS: In this work, we used large-scale comparative genomics to identify 40 MTBC-specific loci. We assessed their genetic diversity and physiological features to select 30 that are good targets for diagnostic purposes. Some of these markers could be used to assess the physiological status of the bacilli. Remarkably, none of the most used MTBC markers is in our catalog. Illustrating the translational potential of our work, we develop a specific qPCR assay for quantification and identification of MTBC DNA. Our rational design of targeted molecular assays for TB could be used in many other fields of clinical and basic research. AVAILABILITY AND IMPLEMENTATION: The database of non-tuberculous mycobacteria assemblies can be accessed at: 10.5281/zenodo.3374377. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Development and application of affordable SNP typing approaches to genotype Mycobacterium tuberculosis complex strains in low and high burden countries.

The Mycobacterium tuberculosis complex (MTBC) comprises the species that causes tuberculosis (TB) which affects 10 million people every year. A robust classification of species, lineages, and sub-lineages is important to explore associations with drug resistance, epidemiological patterns or clinical outcomes. We present a rapid and easy-to-follow methodology to classify clinical TB samples into the main MTBC clades. Approaches are based on the identification of lineage and sub-lineage diagnostic SNP using a real-time PCR high resolution melting assay and classic Sanger sequencing from low-concentrated, low quality DNA. Thus, suitable for implementation in middle and low-income countries. Once we validated our molecular procedures, we characterized a total of 491 biological samples from human and cattle hosts, representing countries with different TB burden. Overall, we managed to genotype ~95% of all samples despite coming from unpurified and low-concentrated DNA. Our approach also allowed us to detect zoonotic cases in eight human samples from Nigeria. To conclude, the molecular techniques we have developed, are accurate, discriminative and reproducible. Furthermore, it costs less than other classic typing methods, resulting in an affordable alternative method in TB laboratories.