Simultaneous detection and ribotyping of Clostridioides difficile, and toxin gene detection directly on fecal samples.

BACKGROUND: Clostridioides difficile is the most common cause of nosocomial diarrhea. Ribotyping of cultured strains by a PCR-based test is used to study potential transmission between patients. We aimed to develop a rapid test that can be applied directly on fecal samples for simultaneous detection and ribotyping of C. difficile, as well as detection of toxin genes. METHODS: We developed a highly specific and sensitive primer set for simultaneous detection and ribotyping of C. difficile directly on total fecal DNA. Toxin genes were detected with primers adapted from Persson et al. (Clin Microbiol Infect 14(11):1057-1064). Our study set comprised 130 fecal samples: 65 samples with positive qPCR for C. difficile toxin A/B genes and 65 C. difficile qPCR negative samples. PCR products were analyzed by capillary gel electrophoresis. RESULTS: Ribosomal DNA fragment peak profiles and toxin genes were detected in all 65 C. difficile positive fecal samples and in none of the 65 C. difficile negative samples. The 65 samples were assigned to 27 ribotypes by the Dutch reference laboratory. Our peak profiles corresponded to these ribotypes, except for two samples. During a C. difficile outbreak, patients were correctly allocated to the outbreak-cluster based on the results of direct fecal ribotyping, before C. difficile isolates were cultured and conventionally typed. CONCLUSION: C. difficile ribotyping directly on fecal DNA is feasible, with sensitivity and specificity comparable to that of diagnostic toxin gene qPCR and with ribotype assignment similar to that obtained by conventional typing on DNA from cultured isolates. This supports simultaneous diagnosis and typing to recognize an outbreak.

Prevalence of plasmid-mediated AmpC in Enterobacteriaceae isolated from humans and from retail meat in Zagazig, Egypt.

Background: The objective of this study was to determine the prevalence of plasmid-mediated AmpC (pAmpC) among Enterobacteriaceae isolated from humans and from retail meat in Egypt. Methods: Enterobacteriaceae were isolated from patients with suspected bloodstream infection, human fecal samples, retail chicken meat samples and retail sheep meat samples. All group I Enterobacteriaceae were analyzed for presence of pAmpC genes by PCR. Antibiotic susceptibility testing was performed in all pAmpC positive isolates, followed by phenotypic and genotypic ESBL and carbapenemase testing on indication. Results: The prevalence of pAmpC among group I Enterobacteriaceae isolated from 225 patients with bloodstream infection was 5.6% [95%CI 2.2-13.4]. Among 100 patients with community-onset gastroenteritis the prevalence in fecal samples was 4.8% [95%CI 2.1-10.7]. The prevalence among 112 chicken carcasses and 100 sheep meat samples was 2.4% [95%CI 0.7-8.4] and 1.1% [95%CI 0.2-5.7], respectively. In half of the AmpC positive isolates we detected an ESBL gene and 2 isolates harbored a carbapenemase gene. In five isolates there was resistance to at least three important alternative antibiotic drugs. Conclusions: We consider the prevalence of pAmpC in Egypt, as found in our study, moderately low. To follow future trends in prevalence of pAmpC worldwide, a standardized screening algorithm for the detection of pAmpC is needed.

Clinical correlates of herpes simplex virus type 1 loads in the lower respiratory tract of critically ill patients.

BACKGROUND: The significance of isolation of herpes simplex virus (HSV) type 1 from the lower respiratory tract in critically ill patients on mechanical ventilation is still unclear. In the current study, we used polymerase chain reaction techniques to quantify HSV-1 to further evaluate its role. OBJECTIVES: The hypothesis was that high loads reflect invasive pulmonary disease related to prolonged mechanical ventilation and increased mortality, as opposed to shedding from the upper respiratory tract, which leads to lower viral loads. STUDY DESIGN: We prospectively studied 77 consecutive patients admitted to the intensive care unit and analyzed 136 tracheal aspirates or bronchoalveolar lavage fluids, taken when clinically indicated in the diagnostic workup of fever, radiologic pulmonary infiltrates, progressive respiratory insufficiency or combinations. Samples were cultured for bacteria and yeasts according to routine microbiological methods and HSV-1 loads were determined by real time quantitative PCR. Viral loads were expressed per number of cells recovered. RESULTS: HSV-1 load was directly related to the simplified acute physiology score II (rs=0.47, P=0.04) when the first specimen taken proved positive for HSV-1. HSV-1 positivity concurred with Candida spp. colonization. Patients with and without a HSV-1 load did not differ with respect to pulmonary and systemic courses and vital outcomes. CONCLUSIONS: The data suggest that HSV-1 in the lower respiratory tract originates from shedding in the upper respiratory tract in about 30% of critically ill patients, following immune suppression and reactivation, without invasively infecting the lung. No attributable mortality was observed.

Two years' prospective collection of molecular and epidemiological data shows limited spread of hepatitis A virus outside risk groups in Amsterdam, 2000-2002.

We performed a viral sequencing study on samples representing all reported primary cases of acute hepatitis A virus (HAV) infection reported for 2 years in Amsterdam. Two regions of HAV RNA were amplified, sequenced, and used for phylogenetic analysis. Of 156 cases, strains of 104 isolates (66.6%) clustered into 3 genotypes: 1A, 1B, and 3. Two separate transmission circles occurred, without mutual interrelation. In genotype 1A, 4 clusters occurred in men having sex with men (MSM), and the fifth cluster was related to a virus from Morocco. In genotype 1B, 6 small clusters were directly related to the Moroccan virus. In genotype 3, strains were related to a virus from Pakistan. Our analysis indicates that, to stop transmission of HAV in Amsterdam, the entire MSM population and travelers to countries where HAV is endemic, especially children, should be vaccinated. Prevention strategies need not include the vaccination of all children living in Amsterdam.

RAPD typing of Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens and Pseudomonas aeruginosa isolates using standardized reagents.

OBJECTIVE: To perform quality assessment of standardized random amplified polymorphic DNA (RAPD) analysis for epidemiologic typing of Klebsiella pneumoniae, K. oxytoca, Serratia marcescens and Pseudomonas aeruginosa. METHODS: Thirty K. pneumoniae, 15 K. oxytoca, 30 S. marcescens and 33 P. aeruginosa epidemiologically unrelated isolates and four collections of clinically related isolates of each species were included in the study. RAPD analysis was performed using Ready-To-Go RAPD Analysis beads with primer ERIC-1R and Ready-To-Go primer 2 for K. pneumoniae and K. oxytoca, primer set ERIC-2/1026 and Ready-To-Go primer 2 for S. marcescens, and primers D-10514 and D-14306 for P. aeruginosa. RESULTS: All epidemiologically unrelated K. pneumoniae and K. oxytoca isolates were distinguished. Twenty-nine types were distinguished among the 30 unrelated S. marcescens isolates and 32 types among the 33 unrelated P. aeruginosa isolates. Indistinguishable banding patterns were obtained in repeated analyses of two isolates and from 11 serial subcultures of three isolates of each species included in the study. The RAPD data from the clinically related isolates correlated with the epidemiologic origin of the isolates. CONCLUSIONS: The use of Ready-To-Go RAPD Analysis beads resulted in reproducible and stable banding patterns with a high discriminatory capacity, and the RAPD typing results corresponded with the epidemiologic origin of the isolates.