Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Acknowledgments Some of the figures (Figures 4–6 and Supplementary Material S1) were generated using BioRender and draw.io, respectively. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Wellcome Foundation (grant #222941/Z/21/Z), the South African Medical Research Council, American Association of Zoo Veterinarians Wild Animal Health Fund [S005651 and S007355], the National Research Foundation South African Research Chair Initiative [grant #86949], and MHM was supported by Wellcome Trust (grant #216634/Z/19/Z). AGL is supported by the EDCTP TESA III network (CSA2020NoE-3104).Peer reviewedPublisher PD

Evaluation of whole- genome sequence data analysis approaches for short- and long- read sequencing of Mycobacterium tuberculosis

Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB) isolates can be used to get an accurate diagnosis, to guide clinical decision making, to control tuberculosis (TB) and for outbreak investigations. We evaluated the performance of long-read (LR) and/or short-read (SR) sequencing for anti-TB drug-resistance prediction using the TBProfiler and Mykrobe tools, the fraction of genome recovery, assembly accuracies and the robustness of two typing approaches based on core-genome SNP (cgSNP) typing and core-genome multi-locus sequence typing (cgMLST). Most of the discrepancies between phenotypic drug-susceptibility testing (DST) and drug-resistance prediction were observed for the first-line drugs rifampicin, isoniazid, pyrazinamide and ethambutol, mainly with LR sequence data. Resistance prediction to second-line drugs made by both TBProfiler and Mykrobe tools with SR- and LR-sequence data were in complete agreement with phenotypic DST except for one isolate. The SR assemblies were more accurate than the LR assemblies, having significantly (P<0.05) fewer indels and mismatches per 100 kbp. However, the hybrid and LR assemblies had slightly higher genome fractions. For LR assemblies, Canu followed by Racon, and Medaka polishing was the most accurate approach. The cgSNP approach, based on either reads or assemblies, was more robust than the cgMLST approach, especially for LR sequence data. In conclusion, anti-TB drug-resistance prediction, particularly with only LR sequence data, remains challenging, especially for first-line drugs. In addition, SR assemblies appear more accurate than LR ones, and reproducible phylogeny can be achieved using cgSNP approaches

Aquatic Hemiptera in Southwest Cameroon: Biodiversity of Potential Reservoirs of Mycobacterium ulcerans and multiple wolbachia sequence types revealed by metagenomics

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a neglected tropical disease associated with freshwater habitats. A variety of limnic organisms harbor this pathogen, including aquatic bugs (Hemiptera: Heteroptera), which have been hypothesized to be epidemiologically important reservoirs. Aquatic Hemiptera exhibit high levels of diversity in the tropics, but species identification remains challenging. In this study, we collected aquatic bugs from emerging foci of BU in the Southwest Region of Cameroon, which were identified using morphological and molecular methods. The bugs were screened for mycobacterial DNA and a selection of 20 mycobacteria-positive specimens from the families Gerridae and Veliidae were subjected to next-generation sequencing. Only one individual revealed putative M. ulcerans DNA, but all specimens contained sequences from the widespread alphaproteobacterial symbiont, Wolbachia. Phylogenetic analysis placed the Wolbachia sequences into supergroups A, B, and F. Circularized mitogenomes were obtained for seven gerrids and two veliids, the first from these families for the African continent. This study suggests that aquatic Hemiptera may have a minor role (if any) in the spread of BU in Southwest Cameroon. Our metagenomic analysis provides new insights into the incursion of Wolbachia into aquatic environments and generated valuable resources to aid molecular taxonomic studies of aquatic Hemiptera. © 2019 by the authors

Development and application of nanopore sequencing based methods for rapid, culture-free diagnosis of tuberculosis

Tuberculosis (TB) is a condition of global health concern with an estimated 1/3 of the human population infected. A growing percentage of these infections also demonstrate resistance to antibiotics, increasing morbidity and mortality in affected populations. The gold standard for drug susceptibility testing (DST), microbial culture, is very slow (weeks-months) and can’t provide the necessary information within a clinically useful timeframe. Culture DST also requires specialist equipment that is not broadly available, therefore drug-resistant TB (DR-TB) is underdiagnosed globally. These limitations mean there is an urgent need for the development and uptake of new, rapid, DST technologies. Existing molecular technologies such as Xpert MTB/RIF offer rapid TB diagnosis but are only capable of detecting Rifampicin resistance due to limitations in PCR multiplexing technology. Comparatively, the GenoType MTBDRplus and MTBDRsl assays provide broader DST testing capability but are far from comprehensive for detecting all important drug-resistance associated mutations. Targeted next-generation sequencing (tNGS) has the potential to rapidly diagnose TB and determine drug-resistance by amplification of known mutation loci. We developed a tNGS assay for DST covering 13 anti-tuberculous drugs using known SNPs (~200) in 16 Mycobacterium tuberculosis genes. Genotypic and phenotypic test performance were assessed during a blinded study of 392 contrived samples provided by the Foundation for Innovative New Diagnostics (FIND). This tNGS assay was found to have an overall genotypic sensitivity of 95% and specificity of 99% when compared to Illumina. The phenotypic sensitivity was 95%-97% and specificity was 96%-100% across all targeted drugs. Clinical metagenomics has the potential to diagnose TB, perform DST, and provide epidemiological information directly from sputum in a single assay. We developed a metagenomic sequencing based TB test and evaluated it on spiked sputum samples from collaborators at the Norfolk and Norwich University Hospital (NNUH). Analysis showed commensal bacteria were present in high numbers, accounting for the majority of reads, thereby reducing analytical sensitivity. Attempts to design a commensal depletion protocol proved unsuccessful and metagenomic development was halted. In conclusion, two approaches for rapid DST and TB diagnosis were designed and tested using contrived clinical samples. The tNGS method showed excellent potential for clinical use and is undergoing continued evaluation by FIND and the WHO under their Seq&Treat program. Continued development of the method has led to reductions in assay complexity, cost and turnaround time and use of the new WHO mutation list and simplified analysis tool will aid implementation of the test in the future

Using genomics to understand the origin and dispersion of multidrug and extensively drug resistant tuberculosis in Portugal.

Portugal is a low incidence country for tuberculosis (TB) disease. Now figuring among TB low incidence countries, it has since the 1990s reported multidrug resistant and extensively drug resistant (XDR) TB cases, driven predominantly by two strain-types: Lisboa3 and Q1. This study describes the largest characterization of the evolutionary trajectory of M/XDR-TB strains in Portugal, spanning a time-period of two decades. By combining whole-genome sequencing and phenotypic susceptibility data for 207 isolates, we report the geospatial patterns of drug resistant TB, particularly the dispersion of Lisboa3 and Q1 clades, which underly 64.2% and 94.0% of all MDR-TB and XDR-TB isolates, respectively. Genomic-based similarity and a phylogenetic analysis revealed multiple clusters (n = 16) reflecting ongoing and uncontrolled recent transmission of M/XDR-TB, predominantly associated with the Lisboa3 and Q1 clades. These clades are now thought to be evolving in a polycentric mode across multiple geographical districts. The inferred evolutionary history is compatible with MDR- and XDR-TB originating in Portugal in the 70's and 80's, respectively, but with subsequent multiple emergence events of MDR and XDR-TB particularly involving the Lisboa3 clade. A SNP barcode was defined for Lisboa3 and Q1 and comparison with a phylogeny of global strain-types (n = 28 385) revealed the presence of Lisboa3 and Q1 strains in Europe, South America and Africa. In summary, Portugal displays an unusual and unique epidemiological setting shaped by >40 years of uncontrolled circulation of two main phylogenetic clades, leading to a sympatric evolutionary trajectory towards XDR-TB with the potential for global reach

Genomic analyses of Mycobacterium tuberculosis from human lung resections reveal a high frequency of polyclonal infections

Polyclonal infections occur when at least two unrelated strains of the same pathogen are detected in an individual. This has been linked to worse clinical outcomes in tuberculosis, as undetected strains with different antibiotic resistance profiles can lead to treatment failure. Here, we examine the amount of polyclonal infections in sputum and surgical resections from patients with tuberculosis in the country of Georgia. For this purpose, we sequence and analyse the genomes of Mycobacterium tuberculosis isolated from the samples, acquired through an observational clinical study (NCT02715271). Access to the lung enhanced the detection of multiple strains (40% of surgery cases) as opposed to just using a sputum sample (0-5% in the general population). We show that polyclonal infections often involve genetically distant strains and can be associated with reversion of the patient's drug susceptibility profile over time. In addition, we find different patterns of genetic diversity within lesions and across patients, including mutational signatures known to be associated with oxidative damage; this suggests that reactive oxygen species may be acting as a selective pressure in the granuloma environment. Our results support the idea that the magnitude of polyclonal infections in high-burden tuberculosis settings is underestimated when only testing sputum samples

Improving diagnostic approaches in extensively and multidrug resistant tuberculosis: new diagnostic tools and repurposing drugs against X/MDRTB

Tuberculosis remains a major killer worldwide. Accurate, fast, and accessible diagnostics for both the disease and drug resistance remain challenges for tuberculosis (TB) control. In four different translational projects linked by the fact they dealt with laboratory diagnosis of drug resistance, I evaluated repurposed compounds and diagnosis of tuberculosis using resources already available in most microbiology laboratories in high-income countries and rapidly expanding to low and middle-income countries. Our aim was to utilise resources already available in most microbiology laboratories in high-income countries wherever possible with the belief that these systems would subsequently expand to low and middle-income countries. In Chapter 1, I found that the MGIT instrument missed true rifampicin resistance associated with specific rpoB mutations. This has now been overcome by the introduction of whole genome sequencing. In Chapter 2, carbapenems were tested against M. tuberculosis using different methodologies, showing that their role in the treatment of tuberculosis remains unclear. Testing in a more physiological model of the granuloma showed similar unclear results but confirmed the excellent activity of clofazimine, a drug that until recently could not be reliably tested in vitro. In Chapter 3, a MALDI-ToF MS lipidomic approach was used to identify M. tuberculosis and NTMs at species and some sub-species level. This approach could be easily introduced in UK microbiology laboratories today. In Chapter 4, urine was explored as a sample for the diagnosis of pulmonary tuberculosis using a lipidomic-MALDI-ToF MS approach and a PCR-based approach aiming to detect M. tuberculosis trans-renal DNA. Over the period of my PhD, I was part of the seminal projects that produced the evidence to introduce WGS for susceptibility testing and which recommended stopping routine phenotypic testing for these drugs. I was involved in the provision of phenotypic data, validation of comparative UK data, writing the standard operating procedure for DST and training technical staff to set up and read commercially available microtiter plates for slow growing mycobacteria. This included MGIT-based phenotypic test methodology which was used to pair with genotypic results. For first line drugs results were very good. Implementation of whole genome sequencing in the UK for the diagnosis, susceptibility testing and surveillance promised to overcome some of the issues mentioned above. However, 7 years later, a reliable phenotypic drug susceptibility testing methodology is desperately needed for both new compounds and to inform on those drugs for which genomic information is unclear or sparse. New understanding of pharmacokinetics-pharmacodynamics should also be incorporated in these models.Open Acces