RESUMEN
Analysis of genome sequencing data from >100,000 genomes of Mycobacterium tuberculosis complex using TB-Annotator software revealed a previously unknown lineage, proposed name L10, in central Africa. Phylogenetic reconstruction suggests L10 could represent a missing link in the evolutionary and geographic migration histories of M. africanum.
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Evolución Biológica , Mycobacterium , Filogenia , Mycobacterium/genética , Programas Informáticos , África Central/epidemiologíaRESUMEN
BACKGROUND: Pulmonary tuberculosis (PTB) diagnosis relies on sputum examination, a challenge in sputum-scarce patients. Alternative non-invasive sampling methods such as face mask sampling (FMS) have been proposed. OBJECTIVE: To evaluate the value of FMS for PTB diagnosis by assessing its agreement with sputum samples processed by GeneXpert MTB/RIF (Ultra)(Xpert) testing, and describe FMS sensitivity and specificity. METHODS: This was a prospective study conducted at the Carrière TB clinic in Guinea. Presumptive TB patients willing to participate were asked to wear a surgical mask containing a polyvinyl alcohol (PVA) strip for thirty minutes. Subsequently, two spot sputum samples were collected, of which one was processed by microscopy on site and the other by Xpert in Guinea's National Reference Laboratory of Mycobacteriology (LNRM). The first 30 FMS were processed at the Supranational Reference Laboratory in Antwerp, Belgium, and the following 118 FMS in the LNRM. RESULTS: One hundred fifty patients participated, of whom 148 had valid results for both mask and sputum. Sputum smear microscopy was positive for 47 (31.8%) patients while sputum-Xpert detected MTB in 54 (36.5%) patients. Among the 54 patients testing sputum-Xpert positive, 26 (48.1%) yielded a positive FMS-Xpert result, while four sputum-Xpert negative patients tested positive for FMS and 90 patients were Xpert-negative for both sputum and mask samples, suggesting a moderate level of agreement (k-value of 0.47). The overall mask sensitivity was 48.1%, with 95.7% specificity. CONCLUSION: In our setting, Xpert testing on FMS did not yield a high level of agreement to sputum sample.
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Tuberculosis Pulmonar , Tuberculosis , Humanos , Esputo , Guinea , Máscaras , Estudios Prospectivos , Tuberculosis Pulmonar/diagnósticoRESUMEN
Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates. A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5'-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome. Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.
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Farmacorresistencia Bacteriana Múltiple , Mycobacterium tuberculosis , Amicacina/farmacología , Antituberculosos/farmacología , Capreomicina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Marcadores Genéticos , Estudio de Asociación del Genoma Completo , Kanamicina/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genéticaRESUMEN
Conventional molecular tests for detecting Mycobacterium tuberculosis complex (MTBC) drug resistance on clinical samples cover a limited set of mutations. Whole-genome sequencing (WGS) typically requires culture.Here, we evaluated the Deeplex Myc-TB targeted deep-sequencing assay for prediction of resistance to 13 anti-tuberculous drugs/drug classes, directly applicable on sputum.With MTBC DNA tests, the limit of detection was 100-1000 genome copies for fixed resistance mutations. Deeplex Myc-TB captured in silico 97.1-99.3% of resistance phenotypes correctly predicted by WGS from 3651 MTBC genomes. On 429 isolates, the assay predicted 92.2% of 2369 first- and second-line phenotypes, with a sensitivity of 95.3% and a specificity of 97.4%. 56 out of 69 (81.2%) residual discrepancies with phenotypic results involved pyrazinamide, ethambutol and ethionamide, and low-level rifampicin or isoniazid resistance mutations, all notoriously prone to phenotypic testing variability. Only two out of 91 (2.2%) resistance phenotypes undetected by Deeplex Myc-TB had known resistance-associated mutations by WGS analysis outside Deeplex Myc-TB targets. Phenotype predictions from Deeplex Myc-TB analysis directly on 109 sputa from a Djibouti survey matched those of MTBSeq/PhyResSE/Mykrobe, fed with WGS data from subsequent cultures, with a sensitivity of 93.5/98.5/93.1% and a specificity of 98.5/97.2/95.3%, respectively. Most residual discordances involved gene deletions/indels and 3-12% heteroresistant calls undetected by WGS analysis or natural pyrazinamide resistance of globally rare "Mycobacterium canettii" strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14â902 out of 19â422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free.Deeplex Myc-TB may enable fast, tailored tuberculosis treatment.
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Mycobacterium tuberculosis , Preparaciones Farmacéuticas , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
The definition of a genus has wide-ranging implications both in terms of binomial species names and also evolutionary relationships. In recent years, the definition of the genus Mycobacterium has been debated due to the proposed split of this genus into five new genera (Mycolicibacterium, Mycolicibacter, Mycolicibacillus, Mycobacteroides and an emended Mycobacterium). Since this group of species contains many important obligate and opportunistic pathogens, it is important that any renaming of species does not cause confusion in clinical treatment as outlined by the nomen periculosum rule (56a) of the Prokaryotic Code. In this study, we evaluated the proposed and original genus boundaries for the mycobacteria, to determine if the split into five genera was warranted. By combining multiple approaches for defining genus boundaries (16S rRNA gene similarity, amino acid identity index, average nucleotide identity, alignment fraction and percentage of conserved proteins) we show that the original genus Mycobacterium is strongly supported over the proposed five-way split. Thus, we propose that the original genus label be reapplied to all species within this group, with the proposed five genera potentially used as sub-genus complex names.
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Ácidos Grasos , Mycobacterium , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Mycobacterium/genética , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The diagnosis of tuberculosis (TB) using smear microscopy has been based on testing two specimens: one spot and one early morning sputa. Recently, the World Health Organization (WHO) has recommended to replace, whenever possible, microscopy with GeneXpert® MTB/RIF performed on a single specimen. However, as the bacterial load is higher in early morning specimens than in spot specimens, one could expect lower sensitivity of GeneXpert® MTB/RIF performed only on spot specimens. In this study, we compared results of GeneXpert® MTB/RIF on spot specimens versus early morning specimens, under programmatic conditions in Cotonou, Benin. METHODS: From June to September 2018, all sputa received from presumptive TB patients at the Supranational Reference Laboratory for Tuberculosis of Cotonou were included in the study. From each patient, two specimens were collected (one spot and one early morning) and GeneXpert® MTB/RIF was performed on both specimens. RESULTS: In total, 886 participants were included in the study, of whom 737 provided both sputa and 149 (16.8%) gave only the spot specimen. For the 737 participants who provided both sputa, GeneXpert® MTB/RIF was positive for both specimens in 152 participants; for three participants GeneXpert® MTB/RIF was positive on spot specimen but negative on morning specimen while for another three, the test was positive on morning specimen but negative on spot specimen. The overall percentage of agreement was excellent (99.2%) with a positive and negative percent agreement greater than 98%. CONCLUSION: For TB diagnosis under programmatic conditions in Cotonou, GeneXpert® MTB/RIF in spot specimens gave similar results with the test in morning specimens. Performing GeneXpert® MTB/RIF in both specimens did not significantly increase the number of cases detected. To avoid losing patients from the diagnostic cascade, it is preferable to test sputa produced at the time of the first visit at the health center.
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Mycobacterium tuberculosis , Benin , Farmacorresistencia Bacteriana , Humanos , Mycobacterium tuberculosis/genética , Rifampin , Sensibilidad y Especificidad , EsputoAsunto(s)
Antituberculosos , Diarilquinolinas , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Estándares de Referencia , Diarilquinolinas/uso terapéutico , Diarilquinolinas/farmacología , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient's sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI (n = 125, 94%) than with ETOH (n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage (n = 10, 41.7%) than with CPC (n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI (n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle (CT ) values significantly decreased compared to those prestorage with ETOH (difference, -1.1; 95% CI, -1.6 to -0.6; P = 0.0001) but not with CPC (P = 0.915) or OMNI (P = 0.33). For one patient's ETOH- and CPC-stored specimens with a CT of <10, Xpert gave results of rifampin false resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen. In conclusion, 28-day storage of sputum in OMNI, CPC, or ETOH at ambient temperature does not impact short-fragment PCR (Xpert), including for low smear grades. However, for long-fragment PCR, ETOH yielded a lower PCR positivity for low smear grades, while the performance of OMNI and CPC was excellent for all smear grades. (The study has been registered at ClinicalTrials.gov under registration number NCT02744469.).
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Mycobacterium tuberculosis/aislamiento & purificación , Manejo de Especímenes/métodos , Esputo/microbiología , Tuberculosis/diagnóstico , Cetilpiridinio/química , Etanol/química , Humanos , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Factores de TiempoRESUMEN
Rifampin heteroresistance-where rifampin-resistant and -susceptible tuberculosis (TB) bacilli coexist-may result in failed standard TB treatment and potential spread of rifampin-resistant strains. The detection of rifampin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment and may improve rifampin-resistant TB control. The limit of detection (LOD) of rifampin heteroresistance for phenotypic drug susceptibility testing by the proportion method is 1% and, yet, is insufficiently documented for RDTs. We, therefore, aimed to determine, for the four RDTs (XpertMTB/RIF, XpertMTB/RIF Ultra, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII), the LOD per probe and mutation, validated by CFU counting and targeted deep sequencing (Deeplex-MycTB). We selected one rifampin-susceptible and four rifampin-resistant strains, with mutations D435V, H445D, H445Y, and S450L, respectively, mixed them in various proportions in triplicate, tested them with each RDT, and determined the LODs per mutation type. Deeplex-MycTB revealed concordant proportions of the minority resistant variants in the mixtures. The Deeplex-MycTB-validated LODs ranged from 20% to 80% for XpertMTB/RIF, 20% to 70% for Xpert Ultra, 5% to 10% for GenoTypeMTBDRplusv2.0, and 1% to 10% for GenoscholarNTM+MDRTBII for the different mutations. Deeplex-MycTB, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII provide explicit information on rifampin heteroresistance for the most frequently detected mutations. Classic Xpert and Ultra report rifampin heteroresistance as rifampin resistance, while Ultra may denote rifampin heteroresistance through "mixed patterns" of wild-type and mutant melt probe, melt peak temperatures. Overall, our findings inform end users that the threshold for reporting resistance in the case of rifampin heteroresistance is the highest for Classic Xpert and Ultra to resolve phenotypic and genotypic discordant rifampin-resistant TB results.
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Antibióticos Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Técnicas de Diagnóstico Molecular/normas , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Proteínas Bacterianas/genética , Genotipo , Humanos , Límite de Detección , Pruebas de Sensibilidad Microbiana , Técnicas de Diagnóstico Molecular/métodos , Mutación , Mycobacterium tuberculosis/genética , Juego de Reactivos para Diagnóstico/normas , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.
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Genoma Bacteriano , Genotipo , Enfermedades Pulmonares/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium kansasii/genética , Brasil , Gráficos por Computador , ADN Bacteriano , Genómica , Humanos , Mycobacterium kansasii/aislamiento & purificación , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.
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Antituberculosos/farmacología , Interpretación Estadística de Datos , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Fenotipo , Análisis de Secuencia de ADN , Revisiones Sistemáticas como Asunto , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Objectives: Resistance-associated variants (RAVs) in Rv0678 , a regulator of the MmpS5-MmpL5 efflux pump, have been shown to lead to increased MICs of bedaquiline (2- to 8- fold) and clofazimine (2- to 4-fold). The prevalence of these Rv0678 RAVs in clinical isolates and their impact on treatment outcomes are important factors to take into account in bedaquiline treatment guidelines. Methods: Baseline isolates from two bedaquiline MDR-TB clinical trials were sequenced for Rv0678 RAVs and corresponding bedaquiline MICs were determined on 7H11 agar. Rv0678 RAVs were also investigated in non-MDR-TB sequences of a population-based cohort. Results: Rv0678 RAVs were identified in 23/347 (6.3%) of MDR-TB baseline isolates. Surprisingly, bedaquiline MICs for these isolates were high (> 0.24â mg/L, n = 8), normal (0.03-0.24 mg/L, n = 11) or low (< 0.03â mg/L, n = 4). A variant at position -11 in the intergenic region mmpS5 - Rv0678 was identified in 39 isolates (11.3%) and appeared to increase the susceptibility to bedaquiline. In non-MDR-TB isolates, the frequency of Rv0678 RAVs was lower (6/852 or 0.7%). Competition experiments suggested that rifampicin was not the drug selecting for Rv0678 RAVs. Conclusions: RAVs in Rv0678 occur more frequently in MDR-TB patients than previously anticipated, are not associated with prior use of bedaquiline or clofazimine, and in the majority of cases do not lead to bedaquiline MICs above the provisional breakpoint (0.24â mg/L). Their origin remains unknown. Given the variety of RAVs in Rv0678 and their variable effects on the MIC, only phenotypic drug-susceptibility methods can currently be used to assess bedaquiline susceptibility.
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Antituberculosos/farmacología , Clofazimina/farmacología , Diarilquinolinas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antiinflamatorios/farmacología , Antibióticos Antituberculosos/uso terapéutico , Antituberculosos/uso terapéutico , Ensayos Clínicos como Asunto , Clofazimina/uso terapéutico , Diarilquinolinas/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Prevalencia , Rifampin/uso terapéutico , Análisis de Secuencia de ADN , Tuberculosis Resistente a Múltiples Medicamentos/epidemiologíaRESUMEN
RATIONALE: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. OBJECTIVES: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. METHODS: We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. MEASUREMENTS AND MAIN RESULTS: The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. CONCLUSIONS: These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.
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Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Técnicas de Diagnóstico Molecular , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Genes Bacterianos/efectos de los fármacos , Genes Bacterianos/genética , Humanos , Mutación/efectos de los fármacos , Mutación/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Análisis de Secuencia de ADN , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Ethambutol (EMB) resistance can evolve through a multistep process, and mutations in the ubiA (Rv3806c) gene appear to be responsible for high-level EMB resistance in Mycobacterium tuberculosis We evaluated the prevalence of ubiA and embB (Rv3795) mutations in EMB-resistant strains originating from Africa and South Korea. No differences in embB mutation frequencies were observed between strains from both origins. However, ubiA mutations were present in 45.5% ± 6.5% of the African EMB-resistant isolates but in only 9.5% ± 1.5% of the South Korean EMB-resistant isolates. The ubiA mutations associated with EMB resistance were localized to regions encoding the transmembrane domains of the protein, whereas the embB mutations were localized to regions encoding the extramembrane domains. Larger studies are needed to investigate the causes of increased ubiA mutations as a pathway to high-level EMB resistance in African countries, such as extended EMB usage during tuberculosis treatment.
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Antituberculosos/farmacología , Etambutol/farmacología , Mutación/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Pruebas de Sensibilidad MicrobianaAsunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Antituberculosos/farmacología , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genéticaRESUMEN
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Proteínas Bacterianas/química , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano , Modelos Moleculares , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Estudio de Asociación del Genoma Completo , Humanos , Isoniazida/uso terapéutico , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Conformación Proteica , Análisis de Secuencia de ADN , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis Resistente a Múltiples Medicamentos/metabolismoRESUMEN
BACKGROUND: Molecular resistance testing fails to explain all fluoroquinolone resistance, with a continued need for a suitable rapid phenotypic drug susceptibility testing method. OBJECTIVE: To evaluate the optimal method for phenotypic fluoroquinolone susceptibility testing. METHODS: Using Löwenstein-Jensen medium, Middlebrook 7H11 agar, BACTEC-MGIT 960 and the resazurin microtitre plate assay, we determined susceptibility to fluoroquinolones in Mycobacterium tuberculosis and investigated cross-resistance between ofloxacin, levofloxacin, moxifloxacin and gatifloxacin. We compared MICs of all four fluoroquinolones for 91 strains on Löwenstein-Jensen (as the gold standard) with their MICs in resazurin plates, and with ofloxacin susceptibility at a single concentration in MGIT and on 7H11 agar, in addition to sequencing of the gyrAB genes. RESULTS AND CONCLUSIONS: Applying a cut-off of 2 mg/L ofloxacin, 1 mg/L levofloxacin and 0.5 mg/L moxifloxacin and gatifloxacin in all methods, some discordance between solid medium and MGIT methods was observed, yet this tended to be explained by MICs around the cut-off. The high discordance between Löwenstein-Jensen (LJ) and resazurin plates suggests that the currently applied cut-offs for all fluoroquinolones in the resazurin method should decrease and minor changes in colour (from blue to purple) be considered as meaningful. High-level resistance in all assays to all drugs correlated well with the presence of gyrA mutations, in support of recent findings that fluoroquinolone resistance should be tested at different concentrations, as patients with lower levels of resistance may continue to benefit from high-dose fluoroquinolone-based therapy.
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Antituberculosos/farmacología , Fluoroquinolonas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Medios de Cultivo/química , Girasa de ADN/genética , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Análisis de Secuencia de ADNRESUMEN
Mycobacterium avium represents a health concern for both humans and pigs. The characterisation of its subspecies is an important step improving the understanding of the epidemiology and the control of this pathogen. Ninety-two human M. avium strains were selected for a retrospective study. Subspecies determination by rpoB sequencing and IS1245/IS901 analysis showed that 98.9% of Belgian human M. avium strains belong to the subspecies hominissuis (MAH). Some of these MAH strains present particular IS1245/IS901 profiles (absence of IS1245 and false IS901 detection provoked by the presence of ISMav6). In addition, 54 MAH strains isolated from submandibular lymph nodes of Belgian pigs with lymphadenitis were included in this study. Genotyping of human and porcine isolates was performed using multispacer sequence typing (MST). In total, 49 different MST types were identified among pig (n = 11) and human (n = 43) MA isolates, with only five shared by both hosts. Among these MST types, 34 were newly identified. Our findings demonstrate the extensive genetic diversity among MAH isolates. Some genotypes were more prevalent in human or pigs but no correlation was observed between MST type and place of residence or the farm of origin for human and porcine isolates respectively, suggesting an environmental source of infection.
Asunto(s)
Mycobacterium avium/genética , Mycobacterium avium/aislamiento & purificación , Enfermedades de los Porcinos/microbiología , Tuberculosis/microbiología , Tuberculosis/veterinaria , Animales , Bélgica , Variación Genética , Genotipo , Humanos , Repeticiones de Minisatélite , Mycobacterium avium/clasificación , Filogenia , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Estudios Retrospectivos , Análisis de Secuencia de ADN , Porcinos , Tuberculosis/diagnósticoRESUMEN
Sputum samples from new tuberculosis (TB) cases were collected over 2 years as part of a prospective study in the northeastern part of Lima, Peru. To measure the contribution of recent transmission to the high rates of multidrug resistance (MDR) in this area, Mycobacterium tuberculosis complex (MTBc) isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and 15-locus mycobacterial interspersed repetitive-unit (MIRU-15)-variable-number tandem repeat (VNTR) analysis. MDR was found in 6.8% of 844 isolates, of which 593 (70.3%) were identified as belonging to a known MTBc lineage, whereas 198 isolates (23.5%) could not be assigned to these lineages and 12 (1.4%) represented mixed infections. Lineage 4 accounted for 54.9% (n = 463) of the isolates, most of which belonged to the Haarlem family (n = 279). MIRU-15 analysis grouped 551/791 isolates (69.7%) in 102 clusters, with sizes ranging from 2 to 46 strains. The overall high clustering rate suggests a high level of recent transmission in this population, especially among younger patients (odds ratio [OR], 1.6; P = 0.01). Haarlem strains were more prone to cluster, compared to the other families taken together (OR, 2.0; P < 0.0001), while Beijing (OR, 0.6; P = 0.006) and LAM (OR, 0.7; P = 0.07) strains clustered less. Whereas streptomycin-resistant strains were more commonly found in clusters (OR, 1.8; P = 0.03), clustering rates did not differ between MDR and non-MDR strains (OR, 1.8; P = 0.1). Furthermore, only 16/51 MDR strains clustered with other MDR strains, suggesting that patients with primary MDR infections acquired the infections mostly from index cases outside the study population, such as retreated cases.