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1.
Clin Infect Dis ; 47(2): 168-75, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18532886

RESUMO

BACKGROUND: Understanding the epidemiology of human Mycobacterium bovis tuberculosis (TB) in the United States is imperative; this disease can be foodborne or airborne, and current US control strategies are focused on TB due to Mycobacterium tuberculosis and airborne transmission. The National TB Genotyping Service's work has allowed systematic identification of M. tuberculosis-complex isolates and enabled the first US-wide study of M. bovis TB. METHODS: Results of spacer oligonucleotide and mycobacterial interspersed repetitive units typing were linked to corresponding national surveillance data for TB cases reported for the period 2004-2005 and select cases for the period 1995-2003. We also used National TB Genotyping Service data to evaluate the traditional antituberculous drug resistance-based case definition of M. bovis TB. RESULTS: Isolates from 165 (1.4%) of 11,860 linked cases were identified as M. bovis. Patients who were not born in the United States, Hispanic patients, patients <15 years of age, patients reported to be HIV infected, and patients with extrapulmonary disease each had increased adjusted odds of having M. bovis versus M. tuberculosis TB. Most US-born, Hispanic patients with TB due to M. bovis (29 [90.6%] of 32) had extrapulmonary disease, and their overall median age was 9.5 years. The National TB Genotyping Service's data indicated that the pyrazinamide-based case definition's sensitivity was 82.5% (95% confidence interval; 75.3%-87.9%) and that data identified 14 errors in pyrazinamide-susceptibility testing or reporting. CONCLUSIONS: The prevalence of extrapulmonary disease in the young, US-born Hispanic population suggests recent transmission of M. bovis, possibly related to foodborne exposure. Because of its significantly different epidemiologic profile, compared with that of M. tuberculosis TB, we recommend routine surveillance of M. bovis TB. Routine surveillance and an improved understanding of M. bovis TB transmission dynamics would help direct the development of additional control measures.


Assuntos
Mycobacterium bovis/isolamento & purificação , Tuberculose/epidemiologia , Tuberculose/microbiologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Criança , Pré-Escolar , Laticínios/efeitos adversos , Laticínios/microbiologia , Farmacorresistência Bacteriana , Emigrantes e Imigrantes , Feminino , Genótipo , Infecções por HIV/epidemiologia , Hispânico ou Latino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Vigilância da População , Pirazinamida/farmacologia , Fatores de Risco , Tuberculose/etnologia , Tuberculose/transmissão , Estados Unidos/epidemiologia
2.
J Clin Microbiol ; 43(2): 688-95, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15695665

RESUMO

Genotyping of Mycobacterium tuberculosis isolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS6110 fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping. M. tuberculosis isolates (n = 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS6110 fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS6110 fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS6110 fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS6110 fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.


Assuntos
Técnicas de Tipagem Bacteriana , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/diagnóstico , Impressões Digitais de DNA/métodos , Elementos de DNA Transponíveis/genética , DNA Bacteriano/análise , Genótipo , Humanos , Sequências Repetitivas Dispersas/genética , Mycobacterium tuberculosis/isolamento & purificação , Oligonucleotídeos/análise , Tuberculose Pulmonar/microbiologia , Wisconsin
3.
J Clin Microbiol ; 42(1): 474-7, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14715809

RESUMO

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Mycobacterium tuberculosis/genética , Genótipo , Mycobacterium tuberculosis/classificação , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico
5.
Antimicrob Agents Chemother ; 47(12): 3799-805, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14638486

RESUMO

Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were > or =50 microg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were > or =100 microg/ml, and these isolates were also resistant to > or =8 microg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were > or =32 microg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to > or =50 microg of ETH per ml had such mutations.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias , Catalase , Etionamida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Infecções Pneumocócicas/microbiologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Frequência do Gene , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Especificidade da Espécie
6.
Biotechniques ; 35(4): 786-94, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14579744

RESUMO

We developed schemes for rapid identification of Mycobacterium species and strain typing using a microfluidic labchip instrument. A 439-bp region of the gene that codes for the 65-kDa heat shock protein (hsp65), which has sequence polymorphisms specific for most mycobacterial species, was examined using PCR-restriction analysis (PRA). We performed PRA in duplicate, using 2 strains each of 12 species, and observed that fragment sizes (bp) determined automatically by the instrument were consistently smaller than the correct sizes for each of the species as determined by sequence analysis (mean variance, < 7 bp). Mycobacterium tuberculosis isolates were typed with the labchip instrument using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, which determines the number of copies of repeated units at 12 loci in the genome based on product size after PCR amplification. Seven strains with one to six repeat copies at each locus were examined. Sizes were smaller by a mean of 13.47 bp compared with correct sizes predicted by sequence analysis, but could be used to correctly identify all strains types. Isolates of Mycobacterium chelonae and Mycobacterium abscessus were typed using randomly amplified polymorphic DNA (RAPD) electrophoresis, and patterns obtained using the labchip instrument were compared with multilocus enzyme electrophoresis (MEE) types. Patterns were distinct and reproducible for all strains except those with closely related MEE types. The labchip instrument is a versatile alternative for sizing mycobacterial DNA fragments.


Assuntos
Perfilação da Expressão Gênica/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Bacteriano/genética , Mycobacterium tuberculosis/classificação , Especificidade da Espécie
7.
J Clin Microbiol ; 41(6): 2683-5, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12791904

RESUMO

Spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) were evaluated for the ability to differentiate 64 Mycobacterium tuberculosis isolates from 10 IS6110-defined clusters. MIRU-VNTR performed slightly better than spoligotyping in reducing the number of clustered isolates and the sizes of the clusters. All epidemiologically related isolates remained clustered by MIRU-VNTR but not by spoligotyping.


Assuntos
Técnicas de Tipagem Bacteriana , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Elementos de DNA Transponíveis , DNA Bacteriano , Genótipo , Humanos , Repetições Minissatélites/genética , Mycobacterium tuberculosis/isolamento & purificação , Oligodesoxirribonucleotídeos/análise , Polimorfismo de Fragmento de Restrição , Sequências Repetitivas de Ácido Nucleico/genética , Tuberculose/microbiologia
9.
Emerg Infect Dis ; 8(11): 1192-6, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12453342

RESUMO

The National Tuberculosis Genotyping and Surveillance Network was established in 1996 to perform a 5-year, prospective study of the usefulness of genotyping Mycobacterium tuberculosis isolates to tuberculosis control programs. Seven sentinel sites identified all new cases of tuberculosis, collected information on patients and contacts, and obtained patient isolates. Seven genotyping laboratories performed DNA fingerprinting analysis by the international standard IS6110 method. BioImage Whole Band Analyzer software was used to analyze patterns, and distinct patterns were assigned unique designations. Isolates with six or fewer bands on IS6110 patterns were also spoligotyped. Patient data and genotyping designations were entered in a relational database and merged with selected variables from the national surveillance database. In two related databases, we compiled the results of routine contact investigations and the results of investigations of the relationships of patients who had isolates with matching genotypes. We describe the methods used in the study.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Centers for Disease Control and Prevention, U.S./organização & administração , Tuberculose/epidemiologia , Tuberculose/prevenção & controle , Controle de Doenças Transmissíveis/organização & administração , Bases de Dados Factuais , Genótipo , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Vigilância de Evento Sentinela , Tuberculose/microbiologia , Estados Unidos
10.
Emerg Infect Dis ; 8(11): 1197-209, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12453343

RESUMO

We conducted a population-based study to assess demographic and risk-factor correlates for the most frequently occurring Mycobacterium tuberculosis genotypes from tuberculosis (TB) patients. The study included all incident, culture-positive TB patients from seven sentinel surveillance sites in the United States from 1996 to 2000. M. tuberculosis isolates were genotyped by IS6110-based restriction fragment length polymorphism and spoligotyping. Genotyping was available for 90% of 11923 TB patients. Overall, 48% of cases had isolates that matched those from another patient, including 64% of U.S.-born and 35% of foreign-born patients. By logistic regression analysis, risk factors for clustering of genotypes were being male, U.S.-born, black, homeless, and infected with HIV; having pulmonary disease with cavitations on chest radiograph and a sputum smear with acid-fast bacilli; and excessive drug or alcohol use. Molecular characterization of TB isolates permitted risk correlates for clusters and specific genotypes to be described and provided information regarding cluster dynamics over time.


Assuntos
Mycobacterium tuberculosis/genética , Vigilância de Evento Sentinela , Tuberculose/epidemiologia , Tuberculose/microbiologia , Adolescente , Adulto , Idoso , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Criança , Pré-Escolar , Análise por Conglomerados , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Feminino , Genótipo , Humanos , Lactente , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Grupos Raciais , Fatores de Risco , Fatores de Tempo , Tuberculose/tratamento farmacológico , Estados Unidos
11.
Emerg Infect Dis ; 8(11): 1210-5, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12453344

RESUMO

Quality assessment exercises were conducted to evaluate the reproducibility of IS6110 DNA fingerprinting performed by eight laboratories in the National Tuberculosis Genotyping and Surveillance Network. Three panels, each with 8 to 16 isolates, were typed at all laboratories, resulting in 280 images. When the pattern obtained by the majority for each isolate was used as the standard, exact matches were obtained for 73% of patterns; 90% and 97% of patterns matched within one- and two-band differences, respectively. A second approach involved retyping of randomly selected isolates at the Centers for Disease Control and Prevention. Retyping was done for 8-19 isolates per laboratory (76 total). Paired images matched exactly for 54% of isolates and within one and two band differences, 78% and 93%, respectively. We evaluated reasons for mismatching. We also evaluated the reproducibility of spoligotyping using a test panel of 13 isolates; a discrepancy of 1 in 91 results was noted.


Assuntos
Técnicas de Tipagem Bacteriana/normas , Laboratórios/normas , Mycobacterium tuberculosis/genética , Centers for Disease Control and Prevention, U.S./normas , Impressões Digitais de DNA/normas , DNA Bacteriano/análise , Genótipo , Humanos , Laboratórios/organização & administração , Mycobacterium tuberculosis/classificação , Polimorfismo de Fragmento de Restrição , Reprodutibilidade dos Testes , Tuberculose/diagnóstico , Tuberculose/microbiologia , Tuberculose/prevenção & controle , Estados Unidos
12.
Emerg Infect Dis ; 8(11): 1224-9, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12453346

RESUMO

DNA fingerprinting was used to evaluate epidemiologically linked case pairs found during routine tuberculosis (TB) contact investigations in seven sentinel sites from 1996 to 2000. Transmission was confirmed when the DNA fingerprints of source and secondary cases matched. Of 538 case pairs identified, 156 (29%) did not have matching fingerprints. Case pairs from the same household were no more likely to have confirmed transmission than those linked elsewhere. Case pairs with unconfirmed transmission were more likely to include a smear-negative source case (odds ratio [OR] 2.0) or a foreign-born secondary case (OR 3.4) and less likely to include a secondary case <15 years old (OR 0.3). Our study suggests that contact investigations should focus not only on the household but also on all settings frequented by an index case. Foreign-born persons with TB may have been infected previously in high-prevalence countries; screening and preventive measures recommended by the Institute of Medicine could prevent TB reactivation in these cases.


Assuntos
Impressões Digitais de DNA , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Adolescente , Adulto , Idoso , Criança , Busca de Comunicante , DNA Bacteriano/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vigilância de Evento Sentinela , Tuberculose/transmissão , Estados Unidos/epidemiologia
13.
Emerg Infect Dis ; 8(11): 1294-302, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12453359

RESUMO

As part of the National Tuberculosis and Genotyping Surveillance Network, isolates obtained from all new cases of tuberculosis occurring in seven geographically separate surveillance sites from 1996 through 2000 were genotyped. A total of 10883 isolates were fingerprinted by the IS6110-restriction fragment length polymorphism method, yielding 6128 distinct patterns. Low-copy isolates (those with six or fewer bands) were also spoligotyped. The distribution of specific genotype clusters was examined. Databases were also examined for families of related genotypes. Analysis of IS6110 patterns showed 497 patterns related to the W-Beijing family; these patterns represent 946 (9%) of all isolates in the study. Six new sets of related fingerprint patterns were also proposed for isolates containing 6-15 copies of IS6110. These fingerprint sets contain up to 251 patterns and 414 isolates; together, they contain 21% of isolates in this copy number range. These sets of fingerprints may represent endemic strains distributed across the United States.


Assuntos
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Vigilância de Evento Sentinela , Tuberculose/epidemiologia , Tuberculose/microbiologia , Impressões Digitais de DNA , Bases de Dados Factuais , Genótipo , Humanos , Mycobacterium tuberculosis/classificação , Polimorfismo de Fragmento de Restrição , Estados Unidos/epidemiologia
14.
J Clin Microbiol ; 40(5): 1592-602, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11980927

RESUMO

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.


Assuntos
Elementos de DNA Transponíveis , Repetições Minissatélites , Mycobacterium tuberculosis/isolamento & purificação , Sequência de Bases , Impressões Digitais de DNA , Humanos , Mycobacterium bovis/classificação , Mycobacterium bovis/genética , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Filogenia , Polimorfismo de Fragmento de Restrição , Tuberculose/microbiologia
15.
J Clin Microbiol ; 40(5): 1651-5, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11980936

RESUMO

Mycobacterium tuberculosis complex isolates from cerebrospinal fluid of 67 meningitis patients were obtained from six fever hospitals in Egypt. One M. bovis and 66 M. tuberculosis isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis of oxyR. Among the M. tuberculosis isolates, 53 unique strain types (with 3 to 16 copies of IS6110) were found by RFLP analyses. Nine clusters (eight with two isolates each and one with six isolates) were also found. Thirty-six spoligotypes, including at least 10 that have been previously reported from other countries, were also observed. Forty-one (62.1%) of the isolates were in spoligotype clusters, and 22 (33%) of the isolates were in RFLP clusters. Fifty-one of the isolates were susceptible in vitro to all of the antituberculosis drugs tested, 11 were monoresistant to capreomycin, rifampin, isoniazid (INH), pyrazinamide, or streptomycin (STR), 4 were resistant to STR and INH, and 1 was resistant to STR, INH, and ethambutol.


Assuntos
Proteínas de Ligação a DNA , Meningites Bacterianas/líquido cefalorraquidiano , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Proteínas de Bactérias/genética , Egito , Hospitais Especializados , Humanos , Meningites Bacterianas/microbiologia , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Filogenia , Reação em Cadeia da Polimerase/métodos , Proteínas Repressoras/genética , Fatores de Transcrição/genética
16.
J Infect Dis ; 185(5): 641-9, 2002 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11865421

RESUMO

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.


Assuntos
Elementos de DNA Transponíveis/genética , Evolução Molecular , Dosagem de Genes , Mycobacterium tuberculosis/classificação , Tuberculose Pulmonar/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites/genética , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase , Tuberculose Pulmonar/microbiologia
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