RESUMO
OBJECTIVES: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2â mg/L for lineage 1 compared with 0.5â mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8â mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS: In light of these findings, the provisional critical concentration of 1â mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.
Assuntos
Mycobacterium tuberculosis , Nitroimidazóis , Tuberculose , Antituberculosos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Tuberculose/microbiologiaRESUMO
Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the Bactec MGIT 960 system is unreliable, and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA-resistant Mycobacterium tuberculosis isolates. Sanger sequencing and/or whole-genome sequencing were performed to detect mutations in pncA, rpsA, panD, and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed using the Bactec MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA-resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in the pncA, rpsA, and panD genes. Of the 40 isolates, 16 (40%) were found to be susceptible using the reduced inoculum method (i.e., false resistance). No mutations were detected in two PZA-resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, East African Indian strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.
Assuntos
Mycobacterium tuberculosis , Pirazinamida , Amidoidrolases/genética , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Pirazinamida/farmacologiaRESUMO
Criteria defining bedaquiline resistance for tuberculosis have been proposed addressing an emerging concern. We evaluated bedaquiline phenotypic drug susceptibility testing (pDST) criteria using drug-resistant tuberculosis clinical isolates tested at five reference laboratories. Isolates were tested at the proposed bedaquiline MGIT960 and 7H11 agar proportion (AP) critical concentrations and also at higher dilutions. The epidemiological cutoff value for the broth microdilution (BMD) plates (frozen and dry) was investigated. Sanger sequencing was performed (atpE and Rv0678 genes) for any isolate testing resistant. The composite reference standard (CRS) defined susceptibility or resistance as is if all pDST methods agreed. If the pDST result was discordant, sequencing results were used for final classification. Geographically diverse and bedaquiline-unexposed isolates were tested (n = 495). The epidemiological cutoff value for BMD was confirmed to be 0.12 µg/ml. The majority of isolates were determined to be susceptible by all methods (467/495; 94.3%), and 28 were determined to be resistant by at least one method; 4 of these were determined to be resistant by all methods. Of the 28 resistant isolates, 12 harbored Rv0678 mutations exclusively. Isolates with insertions/deletions were more likely to be determined to be resistant by more than one method (5/7) compared to isolates with a single nucleotide polymorphism (1/5). Applying the CRS to 24 discordant pDST, BMD dry correctly detected most (15/24; 63%), followed by MGIT960 and BMD frozen (13/24; 61%) and lastly AP (12/24; 50%). Applying the CRS, the prevalence of bedaquiline resistance was 2.2% and ranged from 1.4 to 3.4%, depending on the method used. All methods performed well for bedaquiline susceptibility determination; however, resistance detected should be investigated by a second, alternative method.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Diarilquinolinas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical Mycobacterium tuberculosis isolates as susceptible/resistant to the drug. Three methods were used: Middlebrook 7H11 agar proportion (AP) assay, broth microdilution (BMD) assay, and mycobacterial growth indicator tube (MGIT) assay. Each of the five laboratories tested the 40-isolate (20 unique isolates, duplicated) EQA panel at three time points. The study validated the sensitivity and specificity of a bedaquiline MIC susceptibility breakpoint of 0.12 µg/ml for the BMD method and WHO interim CCs of 1 µg/ml for MGIT and 0.25 µg/ml for the 7H11 AP methods. Categorical agreements between observed and expected results and sensitivities/specificities for correctly identifying an isolate as susceptible/resistant were highest at the 0.25, 0.12, and 1 µg/ml bedaquiline concentrations for the AP method, BMD (frozen or dry plates), and MGIT960, respectively. At these concentrations, the very major error rates for erroneously categorizing an isolate as susceptible when it was resistant were the lowest and within CLSI guidelines. The most highly reproducible bedaquiline DST methods were MGIT960 and BMD using dry plates. These findings validate the use of standardized DST methodologies and interpretative criteria to facilitate routine phenotypic bedaquiline DST and to monitor the emergence of bedaquiline resistance.
Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Antituberculosos/farmacologia , Diarilquinolinas , Humanos , Testes de Sensibilidade Microbiana , Reprodutibilidade dos TestesRESUMO
We report on the first six cases of acquired resistance to bedaquiline in Pakistan. Seventy sequential isolates from 30 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing and by the detection of mutations in relevant genes. We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.
Assuntos
Antituberculosos/farmacologia , Clofazimina/farmacologia , Diarilquinolinas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Resistência Microbiana a Medicamentos/genética , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Paquistão , Estudos Retrospectivos , Tuberculose , Sequenciamento Completo do GenomaRESUMO
The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical Mycobacterium tuberculosis isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods (mirrored box, microscope, and Vizion digital viewing system), after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation.
Assuntos
Antituberculosos/farmacologia , Diarilquinolinas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Nitroimidazóis/farmacologia , Oxazóis/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose/tratamento farmacológico , Clofazimina/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Linezolida/farmacologia , Testes de Sensibilidade Microbiana/métodos , Reprodutibilidade dos TestesRESUMO
M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the MICs to be elucidated. The three participating laboratories each inoculated 38 96-well plates with 15 known M. tuberculosis strains (including the standard H37Rv reference strain) and, after 2 weeks' incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. The AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (>30000) of samples of M. tuberculosis from patients over the next few years.
Assuntos
Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana/instrumentação , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Automação Laboratorial , Testes Diagnósticos de Rotina , Farmacorresistência Bacteriana , Processamento de Imagem Assistida por Computador , Mycobacterium tuberculosis/crescimento & desenvolvimento , Reprodutibilidade dos Testes , SoftwareRESUMO
Low-level rifampin resistance associated with specific rpoB mutations (referred as "disputed") in Mycobacterium tuberculosis is easily missed by some phenotypic methods. To understand the mechanism by which some mutations are systematically missed by MGIT phenotypic testing, we performed an in silico analysis of their effect on the structural interaction between the RpoB protein and rifampin. We also characterized 24 representative clinical isolates by determining MICs on 7H10 agar and testing them by an extended MGIT protocol. We analyzed 2,097 line probe assays, and 156 (7.4%) cases showed a hybridization pattern referred to here as "no wild type + no mutation." Isolates harboring "disputed" mutations (L430P, D435Y, H445C/L/N/S, and L452P) tested susceptible in MGIT, with prevalence ranging from 15 to 57% (overall, 16 out of 55 isolates [29%]). Our in silico analysis did not highlight any difference between "disputed" and "undisputed" substitutions, indicating that all rpoB missense mutations affect the rifampin binding site. MIC testing showed that "undisputed" mutations are associated with higher MIC values (≥20 mg/liter) compared to "disputed" mutations (4 to >20 mg/liter). Whereas "undisputed" mutations didn't show any delay (Δ) in time to positivity of the test tube compared to the control tube on extended MGIT protocol, "disputed" mutations showed a mean Δ of 7.2 days (95% confidence interval [CI], 4.2 to 10.2 days; P < 0.05), providing evidence that mutations conferring low-level resistance are associated with a delay in growth on MGIT. Considering the proved relevance of L430P, D435Y, H445C/L/N, and L452P mutations in determining clinical resistance, genotypic drug susceptibility testing (DST) should be used to replace phenotypic results (MGIT) when such mutations are found.
Assuntos
Antibióticos Antituberculose/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Genótipo , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Rifampina/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Fatores de Tempo , Tuberculose/microbiologiaRESUMO
Two mycobacterial strains with close similarity to the Mycobacterium tuberculosis complex (MTBC) were isolated from cutaneous lesions of patients in the USA and Italy. At the phenotypic level, similarities to the MTBC included slow growth rate, rough morphotype of the unpigmented colonies and nearly identical high-performance liquid chromatography profiles of mycolic acids. In contrast to the MTBC, the strains were niacin- and nitrate-negative, and catalase-positive both at 68 °C and in semi-quantitative tests. The clinical isolates were more closely related to M. tuberculosis than to any other known mycobacterium and scored positive with commercial DNA probes (Hologic AccuProbe M. tuberculosis). Both average nucleotide identity and genome-to-genome distance suggested the strains are different from the MTBC. Therefore, given the distinguishing phenotypic and genomic-scale differences, we submit that the strains belong to a new species we have named Mycobacteriumdecipiens with type strain TBL 1200985T (=ATCC TSD-117T=DSM 105360T).
Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Filogenia , Tuberculose Cutânea/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Humanos , Itália , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Mycobacterium tuberculosis , Ácidos Micólicos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Estados UnidosRESUMO
BACKGROUND: Mycobacterium tuberculosis is responsible for high morbidity and mortality in immune-compromised hosts. CASE PRESENTATION: We present a rare case of cutaneous tuberculosis after orthotopic liver transplantation without involvement of any other organs. CONCLUSION: TB risk-factors assessment, careful LTBI screening and treatment according to national guidelines, as well as a reduction in missed opportunity for prevention are necessary to avoid MTB related disease in fragile patients.
Assuntos
Tuberculose Latente/diagnóstico , Transplante de Fígado/efeitos adversos , Tuberculose Cutânea/diagnóstico , Humanos , Tuberculose Latente/etiologia , Masculino , Pessoa de Meia-Idade , Mucosa/microbiologia , Mucosa/patologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Cutânea/etiologiaRESUMO
New therapeutics are urgently needed to fight tuberculosis and mycobacteria-related diseases that are a major health hazard especially in poor countries. Natural products have been the source of important antitubercular drugs in the past and still need to receive attention as a potent reservoir of chemical structures. Fifteen known and two new (+)-usnic acid (a benzofurandione formerly isolated from lichens) enamines and hydrazones are here described and tested against sensitive and multidrug-resistant strains of mycobacteria. Among several (+)-usnic acid conjugates, PS14 and PS18 showed potent activity against both susceptible and resistant Mycobacterium tuberculosis strains (MIC values of 1-32 and 2-32 mg/L, respectively) comparable with MIC of other antitubercular drugs already in use for tuberculosis treatment.
Assuntos
Antibacterianos/síntese química , Benzofuranos/síntese química , Desenho de Fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Benzofuranos/química , Benzofuranos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Mycobacterium tuberculosis/genéticaRESUMO
Four strains isolated in Iran from pulmonary specimens of unrelated patients are proposed as representative of a novel Mycobacterium species. Similarity, at the phenotypic level, with Mycobacterium kansasii is remarkable with the photochromogenic yellow pigmentation of the colonies being the salient feature. They differ, however, genotypically from this species and present unique sequences in 16S rRNA, hsp65 and rpoB genes. The average nucleotide identity and the genome-to-genome distance fully support the status of an independent species. The name proposed for this species is Mycobacterium persicum sp. nov. with AFPC-000227T (=DSM 104278T=CIP 111197T) as the type strain.
Assuntos
Pulmão/microbiologia , Mycobacterium/classificação , Filogenia , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Humanos , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Mycobacterium kansasii , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The characterization of five Iranian isolates, four from hospital haemodialysis water and one from the sputum of a patient, led to the detection of a novel mycobacterium species. The strains were characterized by mucoid colonies developing in 3-5 days at temperatures ranging from 25 to 37 °C. The biochemical test pattern was unremarkable while the HPLC profile of mycolic acids resembled that of Mycobacterium fortuitum. The sequences of three major housekeeping genes (16S rRNA, hsp65 and rpoB) were unique and differed from those of any other mycobacterium. Mycobacterium brisbanense, which is the species that shared the highest 16S rRNA gene sequence similarity (99.03â%), was distinct, as shown by the average nucleotide identity and by the genome to genome distance values (91.05 and 43.10â%, respectively). The strains are thus considered to represent a novel species of the genus Mycobacterium, for which the name Mycobacterium aquaticum sp. nov. is proposed. The type strain is RW6T (=DSM 104277T=CIP111198T).
Assuntos
Mycobacterium/classificação , Filogenia , Diálise Renal , Microbiologia da Água , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Genes Bacterianos , Humanos , Irã (Geográfico) , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Ácidos Micólicos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNAAssuntos
Tuberculose Extensivamente Resistente a Medicamentos , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/uso terapêutico , Surtos de Doenças , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/epidemiologia , Humanos , Itália/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologiaRESUMO
Identifying latently infected individuals is crucial for the elimination of tuberculosis (TB). We evaluated for the first time the performance of a new type of interferon-γ release assay, QuantiFERON-TB Plus (QFT-Plus), which includes an additional antigen tube (TB2), stimulating both CD4+ and CD8+ T-cells in contacts of TB patients.Contacts were screened for latent TB infection by tuberculin skin test, QFT-Plus and QuantiFERON-TB Gold in Tube (QFT-GIT).In 119 TB contacts, the overall agreement between QFT-Plus and QFT-GIT was high, with a Cohen's κ of 0.8. Discordant results were found in 12 subjects with negative QFT-GIT and positive QFT-Plus results. In analyses of markers of TB exposure and test results, the average time spent with the index case was the strongest risk factor for positivity in each of these tests. The difference in interferon-γ production between the two antigen tubes (TB2-TB1) was used as an estimate of CD8+ stimulation provided by the TB2. TB2-TB1 values >0.6â IU·mL-1 were significantly associated with proximity to the index case and European origin.QFT-Plus has a stronger association with surrogate measures of TB exposure than QFT-GIT in adults screened for latent TB infection. Interferon-γ response in the new antigen tube used an indirect estimate of specific CD8+ response correlates with increased Mycobacterium tuberculosis exposure, suggesting a possible role in identifying individuals with recent infection.
Assuntos
Busca de Comunicante/métodos , Testes de Liberação de Interferon-gama/métodos , Tuberculose Latente/diagnóstico , Adulto , Idoso , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Estudos Transversais , Feminino , Humanos , Incidência , Interferon gama , Itália , Tuberculose Latente/transmissão , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Mycobacterium tuberculosis , Teste Tuberculínico/métodos , Tuberculose Pulmonar/diagnósticoRESUMO
This study shows that the addition of a consensus 4-locus set of hypervariable mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) loci to the spoligotyping-24-locus MIRU-VNTR typing strategy is a well-standardized approach that can contribute to an improvement of the true cluster definition while retaining high typeability in non-Beijing strains.
Assuntos
Loci Gênicos , Repetições Minissatélites , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , Marcadores Genéticos , Genótipo , Humanos , Epidemiologia Molecular , Polimorfismo de Fragmento de Restrição , Kit de Reagentes para DiagnósticoRESUMO
OBJECTIVES: The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid. METHODS: MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation. RESULTS: We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88âââSTOP) and fbiA (Lys-250âââSTOP) have only been observed in strains resistant to delamanid. CONCLUSIONS: We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation.
Assuntos
Antituberculosos/farmacologia , Colorimetria/métodos , Indicadores e Reagentes/análise , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Nitroimidazóis/farmacologia , Oxazinas/análise , Oxazóis/farmacologia , Xantenos/análise , Automação Laboratorial/métodos , Genes Bacterianos , Polimorfismo Genético , Análise de Sequência de DNARESUMO
The name 'Mycobacterium alsiense', although reported in 2007, has not been validly published. Polyphasic characterization of three available strains of this species led us to the conclusion that they represent a distinct species within the genus Mycobacterium. The proposed novel species grows slowly and presents pale yellow-pigmented colonies. Differentiation from other mycobacteria is not feasible on the basis of biochemical and cultural features alone while genetic analysis, extended to eight housekeeping genes and one spacer region, reveals its clear distinction from all other mycobacteria. Mycobacterium asiaticum is the most closely related species on the basis of 16S rRNA gene sequences (similarity 99.3 %); the average nucleotide identity between the genomes of the two species is 80.72 %, clearly below the suggested cut-off (95-96 %). The name Mycobacterium alsense sp. nov. is proposed here for the novel species and replaces the name 'M. alsiense', ex Richter et al. 2007, given at the time of isolation of the first strain. The type strain is TB 1906T ( = DSM 45230T = CCUG 56586T).
Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Filogenia , Doenças Respiratórias/microbiologia , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ácidos Graxos/química , Humanos , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Ácidos Micólicos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. METHODS: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. RESULTS: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. CONCLUSIONS: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.
Assuntos
DNA Bacteriano/isolamento & purificação , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose Pulmonar/diagnóstico , Adulto , Feminino , Humanos , Masculino , Microscopia , Técnicas de Diagnóstico Molecular , Patologia Molecular , Sensibilidade e EspecificidadeRESUMO
Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.