RESUMO
The gold standards for the diagnosis of Clostridium difficile infections (CDIs) are the cytotoxicity assay and the toxigenic culture. However, both methods are time-consuming and the results are not available before 24-48 h. We developed and evaluated a multiplex in-house real-time polymerase chain reaction (PCR) assay for the simultaneous detection of toxigenic strains of C. difficile and the presumptive identification of the epidemic NAP1/027/BI strain from stools. Amplifications were performed using specific primers for tcdB and tcdC on an ABI Prism 7300 (Applied Biosystems). The detection of amplicons was done using TaqMan probes. The analytical sensitivity of the multiplex real-time PCR for detecting tcdB was estimated to 10 CFU/g of stools. This assay was assessed from 881 consecutive unformed stools from patients suspected of having CDI. The gold standard was the toxigenic culture for the diagnosis of CDI and PCR ribotyping for the identification of the NAP1/027/BI strain. The prevalence of positive toxigenic culture was 9.31%. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 86.59%, 97.43%, 78.02%, and 98.57%, respectively, for the real-time PCR and 70.73%, 100%, 100%, and 97.08%, respectively, for the cytotoxicity assay.
Assuntos
Técnicas Bacteriológicas/métodos , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , Primers do DNA/genética , Fezes/microbiologia , Humanos , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Proteínas Repressoras/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: To assess the impact of the incidental relocation of an intensive care unit (ICU) on the risk of colonizations/infections with Pseudomonas aeruginosa exhibiting OprD-mediated resistance to imipenem (PA-OprD). AIM: The primary aim was to compare the proportion of PA-OprD among P. aeruginosa samples before and after an incidental relocation of the ICU. The role of tap water as a route of contamination for colonization/infection of patients with PA-OprD was assessed as a secondary aim. METHODS: A single-centre, observational, before/after comparison study was conducted from October 2013 to October 2015. The ICU was relocated at the end of October 2014. All P. aeruginosa-positive samples isolated from patients hospitalized ≥48 h in the ICU were included. Tap water specimens were collected every three months in the ICU. PA-OprD strains isolated from patients and tap water were genotyped using pulse-field gel electrophoresis. FINDINGS: A total of 139 clinical specimens of P. aeruginosa and 19 tap water samples were analysed. The proportion of PA-OprD strains decreased significantly from 31% to 7.7% after the relocation of the ICU (P = 0.004). All PA-OprD clinical specimens had a distinct genotype. Surprisingly, tap water was colonized with a single PA-OprD strain during both periods, but this single clone has never been isolated from clinical specimens. CONCLUSION: Relocation of the ICU was associated with a marked decrease in P. aeruginosa strains resistant to imipenem. The polyclonal character of PA-OprD strains isolated from patients and the absence of tap-water-to-patient contamination highlight the complexity of the environmental impact on the endogenous colonization/infection with P. aeruginosa.