Dataset Information

Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda.

ABSTRACT: Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance. A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%. Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda. Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country.

SUBMITTER: Ssengooba W

PROVIDER: S-EPMC4856735 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda.

Ssengooba Willy W Meehan Conor J CJ Lukoye Deus D Kasule George William GW Musisi Kenneth K Joloba Moses L ML Cobelens Frank G FG de Jong Bouke C BC

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 20160223

Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resista ...[more]

PMID: 26917365

Similar Datasets

Project description:Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance.To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods.The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites.Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance mutations to improve the design of tuberculosis control measures, such as diagnostics, and inform patient management.

Project description:Background: Tuberculosis disease, caused by Mycobacterium tuberculosis, is a major public health problem. The emergence of M. tuberculosis strains resistant to existing treatments threatens to derail control efforts. Resistance is mainly conferred by mutations in genes coding for drug targets or converting enzymes, but our knowledge of these mutations is incomplete. Whole genome sequencing (WGS) is an increasingly common approach to rapidly characterize isolates and identify mutations predicting antimicrobial resistance and thereby providing a diagnostic tool to assist clinical decision making. Methods: We applied machine learning approaches to 16,688 M. tuberculosis isolates that have undergone WGS and laboratory drug-susceptibility testing (DST) across 14 antituberculosis drugs, with 22.5% of samples being multidrug resistant and 2.1% being extensively drug resistant. We used non-parametric classification-tree and gradient-boosted-tree models to predict drug resistance and uncover any associated novel putative mutations. We fitted separate models for each drug, with and without "co-occurrent resistance" markers known to be causing resistance to drugs other than the one of interest. Predictive performance was measured using sensitivity, specificity, and the area under the receiver operating characteristic curve, assuming DST results as the gold standard. Results: The predictive performance was highest for resistance to first-line drugs, amikacin, kanamycin, ciprofloxacin, moxifloxacin, and multidrug-resistant tuberculosis (area under the receiver operating characteristic curve above 96%), and lowest for third-line drugs such as D-cycloserine and Para-aminosalisylic acid (area under the curve below 85%). The inclusion of co-occurrent resistance markers led to improved performance for some drugs and superior results when compared to similar models in other large-scale studies, which had smaller sample sizes. Overall, the gradient-boosted-tree models performed better than the classification-tree models. The mutation-rank analysis detected no new single nucleotide polymorphisms linked to drug resistance. Discordance between DST and genotypically inferred resistance may be explained by DST errors, novel rare mutations, hetero-resistance, and nongenomic drivers such as efflux-pump upregulation. Conclusion: Our work demonstrates the utility of machine learning as a flexible approach to drug resistance prediction that is able to accommodate a much larger number of predictors and to summarize their predictive ability, thus assisting clinical decision making and single nucleotide polymorphism detection in an era of increasing WGS data generation.

Project description:Background Tuberculosis is one of the deadliest disease caused by Mycobacterium tuberculosis. Its treatment still becomes a burden for many countries including Indonesia. Drug resistance is one of the problems in TB treatment. However, a development in the molecular field through Whole-genome sequencing (WGS) can be used as a solution in detecting mutations associated with TB- drugs. This investigation intended to implement this data for supporting the scientific community in deeply understanding any TB epidemiology and evolution in Papua along with detecting any mutations in genes associated with TB-Drugs. Result A whole-genome sequencing was performed on the random samples from TB Referral Laboratory in Papua utilizing MiSeq 600 cycle Reagent Kit (V3). Furthermore, TBProfiler was used for genome analysis, RAST Server was employed for annotation, while Gview server was applied for BLAST genome mapping and a Microscope server was implemented for Regions of Genomic Plasticity (RGP). The largest genome of M. tuberculosis obtained was at the size of 4,396,040 bp with subsystems number at 309 and the number of coding sequences at 4326. One sample (TB751) contained one RGP. The drug resistance analysis revealed that several mutations associated with TB-drug resistance existed. In details, mutations of rpoB gene which were identified as S450L, D435Y, H445Y, L430P, and Q432K had caused the reduced effectiveness of rifampicin; while the mutases in katG (S315T), kasA (312S), inhA (I21V), and Rv1482c-fabG1 (C-15 T) genes had contributed to the resistance in isoniazid. In streptomycin, the resistance was triggered by the mutations in rpsL (K43R) and rrs (A514C, A514T) genes, and, in Amikacin, its resistance was led by mutations in rrs (A514C) gene. Additionally, in Ethambutol and Pyrazinamide, their reduced effectiveness was provoked by embB gene mutases (M306L, M306V, D1024N) and pncA (W119R). Conclusions The results from whole-genome sequencing of TB clinical sample in Papua, Indonesia could contribute to the surveillance of TB-drug resistance. In the drug resistance profile, there were 15 Multi Drugs Resistance (MDR) samples. However, Extensively Drug-resistant (XDR) samples have not been found, but samples were resistant to only Amikacin, a second-line drug. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-08139-3.

Project description:BackgroundDiagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis.MethodsBetween Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance.FindingsWe characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7-93·7) and 98·4% specificity (98·1-98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes.InterpretationA broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early.FundingWellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.

Project description:Defining the precise relationship between resistance mutations and quantitative phenotypic drug susceptibility testing will increase the value of whole-genome sequencing (WGS) for predicting tuberculosis drug resistance. However, a large number of WGS data sets currently lack corresponding quantitative phenotypic data-the MICs. Using MYCOTBI plates, we determined the MICs to nine antituberculosis drugs for 154 clinical multidrug-resistant tuberculosis isolates from the Shenzhen Center for Chronic Disease Control in Shenzhen, China. Comparing MICs with predicted drug-resistance profiles inferred by WGS showed that WGS could predict the levels of resistance to isoniazid, rifampicin, streptomycin, fluoroquinolones, and aminoglycosides. We also found some mutations that may not be associated with drug resistance, such as EmbB D328G, mutations in the gid gene, and C-12T in the eis promoter. However, some strains carrying the same mutations showed different levels of resistance to the corresponding drugs. The MICs of different strains with the RpsL K88R, fabG1 C-15T mutations and some with mutations in embB and rpoB, had MICs to the corresponding drugs that varied by 8-fold or more. This variation is unexplained but could be influenced by the bacterial genetic background. Additionally, we found that 32.3% of rifampicin-resistant isolates were rifabutin-susceptible, particularly those with rpoB mutations H445D, H445L, H445S, D435V, D435F, L452P, S441Q, and S441V. Studying the influence of bacterial genetic background on the MIC and the relationship between rifampicin-resistant mutations and rifabutin resistance levels should improve the ability of WGS to guide the selection of medical treatment regimens. IMPORTANCE Whole-genome sequencing (WGS) has excellent potential in drug-resistance prediction. The MICs are essential indications of adding a particular antituberculosis drug dosage or changing the entire treatment regimen. However, the relationship between many known drug-resistant mutations and MICs is unclear, especially for rarer ones. The results showed that WGS could predict resistance levels to isoniazid, rifampicin, streptomycin, fluoroquinolones, and aminoglycosides. However, some mutations may not be associated with drug resistance, and some others may confer various MICs to strains carrying them. Also, 32.3% of rifampicin (RIF)-resistant strains were classified as sensitive to rifabutin (RFB), and some mutations in the rpoB gene may be associated with this phenotype. Our data on the MIC distribution of strains with some rarer mutations add to the accumulated data on the resistance level associated with such mutations to help guide further research and draw meaningful conclusions.

Dataset Information

Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda.

Publications

Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets