Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

To elucidate the role of human herpesvirus (HHV)-6 and -7 (HHV-7) in pityriasis rosea (PR), we measured their DNA load in plasma, peripheral blood mononuclear cells (PBMC), and tissues using a calibrated quantitative real-time PCR assay. We also studied HHV-6-and HHV-7-specific antigens in skin by immunohistochemistry and anti-HHV-7 neutralizing activity using a syncytia-inhibition test. Plasma and PBMC were obtained from 31 PR patients (14 children, 17 adults), 12 patients with other dermatites, and 36 blood donors. Skin biopsies were obtained from 15 adults with PR and 12 with other dermatites. HHV-6 and HHV-7 DNA were detected in 17% and in 39% of PR plasmas, respectively, but in no controls. HHV-7 viremia was associated with a higher PBMC load and, in adults, with systemic symptoms. HHV-7, but not HHV-6, levels in PBMC were higher in PR patients than in controls. HHV-6 and HHV-7 antigens were found only in PR skin (17% and 67% of patients analyzed, respectively), indicating a productive infection. Syncytia-neutralizing antibodies were found in PR patients and controls, but their titers were lower in patients with HHV-7 viremia. These data confirm the causal association between PR and active HHV-7 or, to a lesser extent, HHV-6 infection.

Urinary tract infections (UTIs) are among the most frequent community-acquired infections worldwide. Escherichia coli is the most common UTI pathogen although underlying host factors such as patients' age and gender may influence prevalence of causative agents. In this study, 61 273 consecutive urine samples received over a 22-month period from outpatients clinics of an urban area of north Italy underwent microbiological culture with subsequent bacterial identification and antimicrobial susceptibility testing of positive samples. A total of 13 820 uropathogens were isolated and their prevalence analyzed according to patient's gender and age group. Overall Escherichia coli accounted for 67.6% of all isolates, followed by Klebsiella pneumoniae (8.8%), Enterococcus faecalis (6.3%), Proteus mirabilis (5.2%), and Pseudomonas aeruginosa (2.5%). Data stratification according to both age and gender showed E. coli isolation rates to be lower in both males aged ≥60 years (52.2%), E. faecalis and P. aeruginosa being more prevalent in this group (11.6% and 7.8%, resp.), as well as in those aged ≤14 years (51.3%) in whom P. mirabilis prevalence was found to be as high as 21.2%. Streptococcus agalactiae overall prevalence was found to be 2.3% although it was shown to occur most frequently in women aged between 15 and 59 years (4.1%). Susceptibility of E. coli to oral antimicrobial agents was demonstrated to be as follows: fosfomycin (72.9%), trimethoprim/sulfamethoxazole (72.9%), ciprofloxacin (76.8%), ampicillin (48.0%), and amoxicillin/clavulanate (77.5%). In conclusion, both patients' age and gender are significant factors in determining UTIs etiology; they can increase accuracy in defining the causative uropathogen as well as providing useful guidance to empiric treatment.

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Given empirical evidence that the relative accuracy of HPV-testing on self- vs clinician-samples is robust across clinical settings, the VALHUDES protocol offers a framework for validation of HPV assay/self-sample device combinations that can be translated to a primary screening setting.