RESUMO
Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase ß-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.
Assuntos
Infecções por Corynebacterium/diagnóstico , Infecções por Corynebacterium/microbiologia , Corynebacterium diphtheriae/isolamento & purificação , Corynebacterium pseudotuberculosis/isolamento & purificação , Corynebacterium/genética , Corynebacterium/isolamento & purificação , Difteria/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas de Bactérias/genética , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/patogenicidade , Corynebacterium pseudotuberculosis/genética , Difteria/microbiologia , Toxina Diftérica/biossíntese , Toxina Diftérica/genética , Inglaterra , Humanos , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
The European Diphtheria Surveillance Network (EDSN) ensures the reliable epidemiological and microbiologic assessment of disease prevalence in the European Union. Here, we describe a survey of current diagnostic techniques for diphtheria surveillance conducted across the European Union and report the results from three external quality assessment (EQA) schemes performed between 2010 and 2014.
Assuntos
Técnicas Bacteriológicas/normas , Difteria/diagnóstico , Ensaio de Proficiência Laboratorial , Técnicas Bacteriológicas/métodos , União Europeia , Humanos , Garantia da Qualidade dos Cuidados de SaúdeRESUMO
Cutaneous diphtheria is endemic in tropical countries but unusual in the United Kingdom. Four cases occurred in the United Kingdom within 2 months in 2002. Because cutaneous diphtheria causes outbreaks of both cutaneous and pharyngeal forms, early diagnosis is essential for implementing control measures; high diphtheria vaccination coverage must also be maintained.
