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1.
Emerg Infect Dis ; 28(7): 1431-1436, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35731170

RESUMO

We report the unusual genotypic characterization of a bacterium isolated from a clinical sample of a patient who grew up in Bangladesh and lives in the United States. Using whole-genome sequencing, we identified the bacterium as a member of the Mycobacterium tuberculosis complex (MTBC). Phylogenetic placement of this strain suggests a new MTBC genotype. Even though it had the same spoligotype as M. caprae strains, single-nucleotide polymorphism-based phylogenetic analysis placed the isolate as a sister lineage distinct from M. caprae, most closely related to 5 previously sequenced genomes isolated from primates and elephants in Asia. We propose a new animal-associated lineage, La4, within MTBC.


Assuntos
Mycobacterium tuberculosis , Animais , Bangladesh/epidemiologia , Genótipo , Humanos , Mycobacterium tuberculosis/genética , Filogenia , Sequenciamento Completo do Genoma
2.
J Clin Microbiol ; 59(4)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32999007

RESUMO

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multidrug-resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations that do not confer high levels of RIF resistance, as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF MICs compared to fully susceptible strains but remain phenotypically susceptible by mycobacterial growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here, we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1,779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 2.5-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 µg/ml in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT but were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.


Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , New York , Rifampina/farmacologia
3.
J Clin Microbiol ; 59(1)2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33055186

RESUMO

Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of Mycobacterium tuberculosis isolates for species identification, in silico spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases. The results were compared prospectively in real time to those obtained with our current clinically validated Illumina MiSeq sequencing assay for M. tuberculosis and phenotypic drug susceptibility testing results when available. Our assessment of 431 sequenced samples over a 32-week period demonstrates that, when using the proper quality controls and thresholds, the MinION can achieve levels of genotyping analysis and phenotypic resistance predictions comparable to those of the Illumina MiSeq at a very competitive cost per sample. Our results indicate that nanopore sequencing can be a suitable alternative to, or complement, currently used sequencing platforms in a clinical setting and has the potential to be widely adopted in public health laboratories in the near future.


Assuntos
Mycobacterium tuberculosis , Sequenciamento por Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Filogenia
4.
Artigo em Inglês | MEDLINE | ID: mdl-30962348

RESUMO

Using 894 phylogenetically diverse genomes of the Mycobacterium tuberculosis complex (MTBC), we simulated in silico the ability of the Hain Lifescience GenoType MTBC assay to differentiate the causative agents of tuberculosis. Here, we propose a revised interpretation of this assay to reflect its strengths (e.g., it can distinguish some strains of Mycobacterium canettii and variants of Mycobacterium bovis that are not intrinsically resistant to pyrazinamide) and limitations (e.g., Mycobacterium orygis cannot be differentiated from Mycobacterium africanum).


Assuntos
Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/classificação , Tuberculose/microbiologia , Técnicas de Genotipagem , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação
5.
PLoS Pathog ; 12(12): e1006111, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27973588

RESUMO

Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M. tuberculosis pulmonary infection and human disseminated M. bovis BCG infection. We found that new mutations accumulate during in vitro culture, but that in vivo, purifying selection against new mutations dominates, indicating that M. tuberculosis follows a dominant lineage model of evolution. Comparing bacterial populations passaged in T cell-deficient and immunocompetent mice, we found that the presence of T cells is associated with an increase in the diversity of the M. tuberculosis genome. Together, our findings put M. tuberculosis genetic evolution in a new perspective and clarify the impact of T cells on sequence diversity of M. tuberculosis.


Assuntos
Evolução Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Tuberculose/genética , Tuberculose/imunologia , Animais , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Epidemiologia Molecular
6.
Emerg Infect Dis ; 23(10): 1749-1751, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28930016

RESUMO

We report a case of lymphadenitis caused by Mycobacterium orygis in an immunocompetent person in Stony Brook, New York, USA. Initial real-time PCR assay failed to provide a final subspecies identification within the M. tuberculosis complex, but whole-genome sequencing characterized the isolate as M. orygis.


Assuntos
Genoma Bacteriano , Linfadenite/diagnóstico , Mycobacterium/genética , Idoso , Emigrantes e Imigrantes , Feminino , Humanos , Índia , Linfonodos/microbiologia , Linfonodos/patologia , Linfadenite/microbiologia , Linfadenite/patologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , New York , Filogenia , Sequenciamento Completo do Genoma
7.
J Clin Microbiol ; 55(6): 1871-1882, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28381603

RESUMO

Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterize Mycobacterium tuberculosis and other M. tuberculosis complex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.


Assuntos
Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Sequenciamento Completo do Genoma/métodos , Biologia Computacional/métodos , Humanos , New York , Estudos Prospectivos , Estudos Retrospectivos , Tuberculose/microbiologia
8.
BMC Infect Dis ; 16(1): 764, 2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27993132

RESUMO

BACKGROUND: The Xpert® MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. METHODS: An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. RESULTS: Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%-95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%-99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). CONCLUSIONS: The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.


Assuntos
Antibióticos Antituberculose , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/isolamento & purificação , Rifampina , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bioensaio , Estudos de Casos e Controles , DNA Bacteriano/análise , Países em Desenvolvimento , Reações Falso-Positivas , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Prevalência , Estudos Retrospectivos , Sensibilidade e Especificidade , Tuberculose/epidemiologia , Tuberculose/microbiologia , Estados Unidos/epidemiologia , Adulto Jovem
9.
N Engl J Med ; 367(11): 1020-4, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22913660

RESUMO

BACKGROUND: In January 2012, on the basis of an initial report from a dermatologist, we began to investigate an outbreak of tattoo-associated Mycobacterium chelonae skin and soft-tissue infections in Rochester, New York. The main goals were to identify the extent, cause, and form of transmission of the outbreak and to prevent further cases of infection. METHODS: We analyzed data from structured interviews with the patients, histopathological testing of skin-biopsy specimens, acid-fast bacilli smears, and microbial cultures and antimicrobial susceptibility testing. We also performed DNA sequencing, pulsed-field gel electrophoresis (PFGE), cultures of the ink and ingredients used in the preparation and packaging of the ink, assessment of source water and faucets at tattoo parlors, and investigation of the ink manufacturer. RESULTS: Between October and December 2011, a persistent, raised, erythematous rash in the tattoo area developed in 19 persons (13 men and 6 women) within 3 weeks after they received a tattoo from a single artist who used premixed gray ink; the highest occurrence of tattooing and rash onset was in November (accounting for 15 and 12 patients, respectively). The average age of the patients was 35 years (range, 18 to 48). Skin-biopsy specimens, obtained from 17 patients, showed abnormalities in all 17, with M. chelonae isolated from 14 and confirmed by means of DNA sequencing. PFGE analysis showed indistinguishable patterns in 11 clinical isolates and one of three unopened bottles of premixed ink. Eighteen of the 19 patients were treated with appropriate antibiotics, and their condition improved. CONCLUSIONS: The premixed ink was the common source of infection in this outbreak. These findings led to a recall by the manufacturer.


Assuntos
Cosméticos/efeitos adversos , Surtos de Doenças , Tinta , Infecções por Mycobacterium não Tuberculosas/etiologia , Mycobacterium chelonae/isolamento & purificação , Tatuagem/efeitos adversos , Feminino , Humanos , Masculino , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Mycobacterium chelonae/genética , New York/epidemiologia , Análise de Sequência de DNA , Pele/microbiologia , Pele/patologia
10.
Mol Cell Probes ; 29(6): 514-516, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26334290

RESUMO

We have developed a single tube TaqMan(®) real-time PCR assay that differentiates the full-length and truncated erm(41) gene to predict inducible resistance to clarithromycin in Mycobacterium abscessus. A study of 87 clinical isolates found this assay to be 90.8% concordant to conventional drug susceptibility testing results for the prediction of inducible clarithromycin drug resistance.


Assuntos
Proteínas de Bactérias/genética , Micobactérias não Tuberculosas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Claritromicina/farmacologia , Farmacorresistência Bacteriana , Humanos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/genética
11.
J Clin Microbiol ; 52(11): 3973-7, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25210065

RESUMO

Mycobacterium xenopi is an opportunistic mycobacterial pathogen of increasing clinical importance. Surveillance of M. xenopi is hampered by the absence of tools for genotyping and molecular epidemiology. In this study, we describe the development and evaluation of an effective multilocus sequence typing strategy for M. xenopi.


Assuntos
Tipagem de Sequências Multilocus/métodos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium xenopi/classificação , Mycobacterium xenopi/genética , Humanos , Epidemiologia Molecular/métodos , Infecções por Mycobacterium não Tuberculosas/epidemiologia
12.
Open Forum Infect Dis ; 11(8): ofae421, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39119477

RESUMO

Background: Isoniazid-resistant, rifampin-susceptible tuberculosis (Hr-TB) is associated with poor treatment outcomes and higher rates of acquisition of further drug resistance during treatment. Due to a lack of widespread diagnostics, Hr-TB is frequently undetected and its epidemiology is incompletely understood. Methods: We studied the molecular epidemiology of Hr-TB among all patients diagnosed with culture-positive pulmonary tuberculosis between January 1 and June 30, 2017, at an urban referral tuberculosis clinic in Port-au-Prince, Haiti. Demographic and clinical data were extracted from the electronic medical record. Archived diagnostic Mycobacterium tuberculosis isolates were tested for genotypic and phenotypic isoniazid resistance using the Genotype MTBDRplus assay (Hain, Nehren, Germany) and culture-based testing, respectively. All isoniazid-resistant isolates and a randomly selected subset of isoniazid-susceptible isolates underwent whole-genome sequencing to confirm the presence of mutations associated with isoniazid resistance, to validate use of Genotype MTBDRplus in this population, and to identify potential transmission links between isoniazid-resistant isolates. Results and Conclusions: Among 845 patients with culture-positive pulmonary tuberculosis in Haiti, 65 (7.7%) had Hr-TB based on the Genotype MTBDRplus molecular assay. Age < 20 years was significantly associated with Hr-TB (odds ratio, 2.39; 95% confidence interval, 1.14, 4.70; P = .015). Thirteen (20%) isoniazid-resistant isolates were found in 5 putative transmission clusters based on a single nucleotide polymorphism distance of ≤ 5. No patients in these transmission clusters were members of the same household. Adolescents are at higher risk for Hr-TB. Strains of isoniazid-resistant M tuberculosis are actively circulating in Haiti and transmission is likely occurring in community settings.

13.
Nat Microbiol ; 9(8): 2113-2127, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39090390

RESUMO

Several human-adapted Mycobacterium tuberculosis complex (Mtbc) lineages exhibit a restricted geographical distribution globally. These lineages are hypothesized to transmit more effectively among sympatric hosts, that is, those that share the same geographical area, though this is yet to be confirmed while controlling for exposure, social networks and disease risk after exposure. Using pathogen genomic and contact tracing data from 2,279 tuberculosis cases linked to 12,749 contacts from three low-incidence cities, we show that geographically restricted Mtbc lineages were less transmissible than lineages that have a widespread global distribution. Allopatric host-pathogen exposure, in which the restricted pathogen and host are from non-overlapping areas, had a 38% decrease in the odds of infection among contacts compared with sympatric exposures. We measure tenfold lower uptake of geographically restricted lineage 6 strains compared with widespread lineage 4 strains in allopatric macrophage infections. We conclude that Mtbc strain-human long-term coexistence has resulted in differential transmissibility of Mtbc lineages and that this differs by human population.


Assuntos
Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis , Simpatria , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/classificação , Tuberculose/transmissão , Tuberculose/microbiologia , Tuberculose/epidemiologia , Busca de Comunicante , Feminino , Adulto , Masculino , Macrófagos/microbiologia , Incidência , Filogenia
14.
J Clin Microbiol ; 51(7): 2232-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23658258

RESUMO

Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct "spacers" separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.


Assuntos
Magnetismo , Microesferas , Tipagem Molecular/métodos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Genótipo , Haiti , Humanos , Epidemiologia Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/epidemiologia
15.
Front Public Health ; 11: 1206056, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37457262

RESUMO

Mycobacterium tuberculosis complex (MTBC) infections are treated with combinations of antibiotics; however, these regimens are not as efficacious against multidrug and extensively drug resistant MTBC. Phenotypic (growth-based) drug susceptibility testing on slow growing bacteria like MTBC requires many weeks to months to complete, whereas sequencing-based approaches can predict drug resistance (DR) with reduced turnaround time. We sought to develop a multiplexed, targeted next generation sequencing (tNGS) assay that can predict DR and can be performed directly on clinical respiratory specimens. A multiplex PCR was designed to amplify a group of thirteen full-length genes and promoter regions with mutations known to be involved in resistance to first- and second-line MTBC drugs. Long-read amplicon libraries were sequenced with Oxford Nanopore Technologies platforms and high-confidence resistance mutations were identified in real-time using an in-house developed bioinformatics pipeline. Sensitivity, specificity, reproducibility, and accuracy of the tNGS assay was assessed as part of a clinical validation study. In total, tNGS was performed on 72 primary specimens and 55 MTBC-positive cultures and results were compared to clinical whole genome sequencing (WGS) performed on paired patient cultures. Complete or partial susceptibility profiles were generated from 82% of smear positive primary specimens and the resistance mutations identified by tNGS were 100% concordant with WGS. In addition to performing tNGS on primary clinical samples, this assay can be used to sequence MTBC cultures mixed with other mycobacterial species that would not yield WGS results. The assay can be effectively implemented in a clinical/diagnostic laboratory with a two to three day turnaround time and, even if batched weekly, tNGS results are available on average 15 days earlier than culture-derived WGS results. This study demonstrates that tNGS can reliably predict MTBC drug resistance directly from clinical specimens or cultures and provide critical information in a timely manner for the appropriate treatment of patients with DR tuberculosis.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Sequenciamento de Nucleotídeos em Larga Escala , Testes de Sensibilidade Microbiana , Reprodutibilidade dos Testes , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose/diagnóstico
16.
Tuberculosis (Edinb) ; 142: 102380, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37543009

RESUMO

Whole-genome sequencing (WGS) can predict drug resistance and antimicrobial susceptibility in Mycobacterium tuberculosis complex (MTBC) and has shown promise in partially replacing culture-based phenotypic drug susceptibility testing (pDST). We performed a two-year side by side study comparing the prediction of drug resistance and antimicrobial susceptibility by WGS molecular DST (mDST) to pDST to determine resistance at the critical concentration by Mycobacterial Growth Indicator Tube (MGIT) and agar proportion testing. Negative predictive values of WGS results were consistently high for the first-line drugs: rifampin (99.9%), isoniazid (99.0%), pyrazinamide (98.5%), and ethambutol (99.8%); the rates of resistance to these drugs, among strains in our population, are 2.9%, 10.4%, 46.3%, and 2.3%, respectively. WGS results were available an average 8 days earlier than first-line MGIT pDST. Based on these findings, we implemented a new testing algorithm with an updated WGS workflow in which strains predicted pan-susceptible were no longer tested by pDST. This algorithm was applied to 1177 isolates between October 2018 and September 2020, eliminating pDST for 66.6% of samples and reducing pDST for an additional 22.0%. This algorithm change resulted in faster turnaround times and decreased cost while maintaining comprehensive antimicrobial susceptibility profiles of all culture-positive MTBC cases in New York.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/genética , New York , Testes de Sensibilidade Microbiana , Isoniazida , Algoritmos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
17.
Rev Panam Salud Publica ; 31(3): 221-4, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22569696

RESUMO

OBJECTIVE: To determine the prevalence of multidrug-resistant tuberculosis (MDR-TB) among patients with new smear-positive pulmonary TB in Port-au-Prince, Haiti. METHODS: Sputum samples were cultured from 1 006 patients newly diagnosed with TB in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced. All isolates with rpoB mutations were sent to the New York State reference laboratory for conventional drug susceptibility testing (DST). All isolates were also tested with the GenoType MTBDRplus line-probe assay. RESULTS: Mycobacterium tuberculosis was isolated from 906 patients. Twenty-six (2.9%) of the isolates had missense mutations or deletions in rpoB and were resistant to rifampin by DST. All 26 were also resistant to isoniazid and classified as MDR-TB. Forty-six control isolates without rpoB mutations were found to be rifampin sensitive by DST. The GenoType MTBDRplus line-probe assay correctly identified 26 MDR-TB strains. It misclassified one pansusceptible isolate as rifampin resistant. CONCLUSIONS: This study shows an MDR-TB prevalence of 2.9% in newly diagnosed TB patients in Haiti and suggests that rpoB sequencing and hybridization assays are good screening tools for early detection of MDR-TB.


Assuntos
Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto , Antituberculosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Cidades/epidemiologia , Estudos Transversais , RNA Polimerases Dirigidas por DNA , Feminino , Haiti/epidemiologia , Humanos , Isoniazida/farmacologia , Masculino , Mycobacterium tuberculosis/genética , Prevalência , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/genética , Adulto Jovem
18.
Front Microbiol ; 13: 992610, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36299734

RESUMO

Nontuberculous mycobacteria (NTM) are environmental bacteria commonly found in soil and water in almost every part of the world. While usually non-pathogenic, they can cause acute respiratory and cutaneous infections under certain circumstances or in patients with underlying medical conditions. Contrary to members of the Mycobacterium tuberculosis complex, documented human-to-human transmissions of NTM have been rarely reported and most cases result from direct environmental exposure. Here we describe the identification of a new NTM species isolated from a hand laceration of a New York State patient after a fall. This new NTM forms rough, orange pigmented colonies and is naturally resistant to doxycycline and tobramycin. Whole genome analysis reveal no close relatives present in public databases, and our findings are in accordance with the recognition of a new taxonomic species of NTM. We propose the name Mycobacterium salfingeri sp. nov. for this new NTM representative. The type strain is 20-157661T (DSM = 113368T, BCCM = ITM 501207T).

19.
J Clin Microbiol ; 49(7): 2562-7, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21593269

RESUMO

Members of the Mycobacterium tuberculosis complex (MTBC) differ in virulence attributes, drug resistance patterns, and host preferences. The rapid differentiation of these species to determine zoonotic or human sources of tuberculosis disease or to direct treatment can benefit both public health and patient management. Commercially available assays cannot differentiate these species, and published assays have not been evaluated directly on clinical specimens. A real-time PCR assay for the differentiation of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, and M. canettii was developed. The presence or absence of regions of difference (RD) between the genomes of members of the MTBC allowed for the design of a single-tube five-plex real-time PCR assay to differentiate these species. This assay assesses the presence of RD1, RD4, RD9, RD12, and a region exterior to RD9 which is present in all MTBC members. To evaluate the performance of this assay, 192 clinical specimens positive for MTBC by real-time PCR were tested, resulting in a 94% correlation of the real-time PCR with the identification results obtained with cultured material. Additionally, 727 Bactec MGIT 960-positive cultures were tested, resulting in a 97% concordance between the methods. This real-time PCR is an inexpensive and rapid (2.5-h) method performed in a closed-format system and requiring minimal hands-on time that can be implemented in a clinical laboratory and used directly on clinical specimens.


Assuntos
Técnicas de Laboratório Clínico/métodos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Primers do DNA/genética , Humanos , Mycobacterium tuberculosis/genética
20.
J Clin Microbiol ; 48(4): 1182-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20107097

RESUMO

Our laboratory has developed a rapid, sensitive, and specific molecular approach for detection in clinical specimens, within 48 h of receipt, of both Mycobacterium tuberculosis complex (MTBC) DNA and mutations within the 81-bp core region of the rpoB gene that are associated with rifampin (RIF) resistance. This approach, which combines an initial real-time PCR with internal inhibition assessment and a pyrosequencing assay, was validated for direct use with clinical specimens. To assess the suitability of real-time PCR for use with respiratory, nonrespiratory, acid-fast bacillus (AFB)-positive and AFB-negative specimens, we evaluated specimens received in our laboratory between 11 October 2007 and 30 June 2009. With culture used as the "gold standard," the sensitivity, specificity, and positive and negative predictive values were determined for 1,316 specimens to be as follows: for respiratory specimens, 94.7%, 99.9%, 99.6%, and 98.6%, respectively; for nonrespiratory specimens, 88.5%, 100.0%, 100.0%, and 96.9%, respectively; for AFB-positive specimens, 99.6%, 100.0%, 100.0%, and 97.7%, respectively; and for AFB-negative specimens, 75.4%, 99.9%, 98.0%, and 98.4%, respectively. PCR inhibition was determined to be minimal in this assay, occurring in 0.2% of tests. The rpoB gene pyrosequencing assay was evaluated in a similar prospective study, in which 148 clinical specimens positive for MTBC DNA by real-time PCR were tested. The final results revealed that the results of direct testing of clinical specimens by the pyrosequencing assay were 98.6% concordant with the results of conventional testing for susceptibility to RIF in liquid culture and that our assay displayed adequate sensitivity for 96.6% of the clinical specimens tested. Used together, these assays provide reliable results that aid with the initial management of patients with suspected tuberculosis prior to the availability of the results for cultured material, and they also provide the ability to predict RIF resistance in Mycobacterium tuberculosis-positive specimens in as little as 48 h from the time of clinical specimen receipt.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , Rifampina/farmacologia , Análise de Sequência de DNA , RNA Polimerases Dirigidas por DNA , Humanos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/isolamento & purificação , Sensibilidade e Especificidade , Tuberculose/microbiologia
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