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1.
Pediatr Infect Dis J ; 32(9): 942-5, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23587980

RESUMO

BACKGROUND: United States national surveillance data show that the use of culture for pertussis diagnostics has sharply declined, whereas polymerase chain reaction (PCR) is now the most common testing method. PCR testing for pertussis is rapid and sensitive, but the lack of standardization and variable specificity is concerning. METHODS: A web-based survey containing 12 questions was sent to public health, commercial and hospital-based US laboratories performing clinical diagnostics to determine the pertussis diagnostics used. An extensive real-time PCR (RT-PCR) questionnaire accompanied a proficiency panel assessing the types of extraction methods, RT-PCR methods and current quality control in place at the laboratories. The proficiency panel of 12 specimens containing Bordetella pertussis at various concentrations and negative controls was created to detect cross-contamination and assess the lower limit of detection. RESULTS: One hundred twenty-three (35%) of 355 respondents from the web-based survey performed diagnostic tests for the presence of B. pertussis. Eighty-three (71%) labs reported performing culture, whereas 67 (54%) labs used PCR. All 41 laboratories that consented to participate in the proficiency exercise used the IS481 RT-PCR target; however, a variety of extraction and RT-PCR methods were employed. The laboratories correctly identified 92% of the B. pertussis specimens, and 5% of the laboratories (1.8% of the panel specimens) reported at least 1 false-positive. CONCLUSIONS: The small percentage of false-positives suggests that adequate procedures are in place to prevent cross-contamination. Differing extraction and PCR methods as well as variable analytic sensitivity emphasize the necessity for an external well-defined quality control program and interlaboratory pertussis PCR harmonization.


Assuntos
Bordetella pertussis/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Coleta de Dados , Testes Diagnósticos de Rotina/métodos , Ensaio de Proficiência Laboratorial , Coqueluche/diagnóstico , Técnicas de Laboratório Clínico/normas , Testes Diagnósticos de Rotina/normas , Humanos , Controle de Qualidade , Estados Unidos
2.
Emerg Infect Dis ; 18(8): 1248-55, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22841154

RESUMO

Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935-2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970-1990 (94%) but declined to ≈ 70% after 1991 and has remained constant. During 2006-2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population.


Assuntos
Bordetella pertussis/genética , Bordetella pertussis/isolamento & purificação , Variação Genética , Coqueluche/microbiologia , Alelos , Antígenos de Bactérias/genética , Bordetella pertussis/classificação , Bordetella pertussis/imunologia , Notificação de Doenças/estatística & dados numéricos , Proteínas de Fímbrias/genética , Humanos , Repetições Minissatélites/genética , Tipagem de Sequências Multilocus , Toxina Pertussis/genética , Vacina contra Coqueluche/administração & dosagem , Vacina contra Coqueluche/imunologia , Estados Unidos/epidemiologia , Vacinação/estatística & dados numéricos , Fatores de Virulência de Bordetella/genética , Coqueluche/epidemiologia , Coqueluche/prevenção & controle
3.
J Clin Microbiol ; 49(12): 4059-66, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21940464

RESUMO

A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.


Assuntos
Técnicas Bacteriológicas/métodos , Infecções por Bordetella/diagnóstico , Bordetella bronchiseptica/isolamento & purificação , Bordetella parapertussis/isolamento & purificação , Bordetella pertussis/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Bordetella bronchiseptica/classificação , Bordetella bronchiseptica/genética , Bordetella parapertussis/classificação , Bordetella parapertussis/genética , Bordetella pertussis/classificação , Bordetella pertussis/genética , Primers do DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Toxina Pertussis/genética , Subunidades Proteicas/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA
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