Molecular typing of methicillin-resistant Staphylococcus aureus: can PCR replace pulsed-field gel electrophoresis?

Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA). However, the method is time-consuming and expensive, and its discriminatory power may not be necessary in outbreak situations. We used a rapid multiplex PCR-based method with published primers and compared the results with those obtained by PFGE. A total of 75 clinical isolates were typed: 59 strains originated from our prospectively collected clinical strains and were epidemiologically unrelated; 16 strains came from an outbreak that was epidemiologically well defined in time and space. A primer mix of the spa gene, the coa gene, and the hypervariable region adjacent to mecA gene was used for multiplex PCR. Both PFGE and PCR clustered the 75 strains into 41 different genotypes. Concordance of the results was 100% for strains originating from the outbreak. Overall, both methods produced concordant results in 72% of cases. A total of 16% were clustered together by PFGE, but not by PCR and 12% were clustered together by PCR but not by PFGE, respectively. The turnaround time was only 8 h for PCR but 5 days for PFGE. This PCR-based method is excellent for rapid and inexpensive typing of MRSA in an outbreak setting, but the discriminatory power and reproducibility are still insufficient to replace PFGE in longitudinal studies in the endemic setting.     

Real-time PCR-based system for simultaneous quantification of human papillomavirus types associated with high risk of cervical cancer

We have previously shown that women with a high titer of human papillomavirus type 16 (HPV16) in cervical epithelial cells have an increased risk of developing cervical carcinoma in situ. In order to study the relationship between viral DNA amount and risk of cervical carcinoma for the HPV types most commonly found in cervical tumors, we developed a real-time PCR assay for the detection and quantification of HPV16, -18, -31, -33, -35, -39, -45, -52, -58, and -67. These HPV types are analyzed in two reaction tubes, allowing for independent quantification of three viral types, or groups of viral types, in each reaction. A separate reaction is used for estimating the number of a nuclear single-copy gene and is used to calculate the HPV copy number per genomic DNA equivalent in the sample. The system has a dynamic range from 10(2) to 10(7) HPV copies per assay and is applicable to both fresh clinical samples and DNA extracted from archival samples. Reconstitution experiments, made to mimic infections with several HPV types, shows that individual HPV types can be detected in a mixture as long as they represent 1 to 10% of the main type. The system was evaluated with respect to technical specificity and sensitivity, reproducibility, reagent stability, and sample preparation protocol and then used to analyze clinical samples. This homogeneous assay provides a fast and sensitive way for estimating the viral load of a series of the most frequent oncogenic HPV types in biopsies, as well as cervical smear samples.     

War Experiences and General Health Among People in Bosnia‐Herzegovina and Kosovo

War experiences may have an extensive impact on the health status of the exposed populations. This population‐based study aimed to examine the relationship between war experiences and self‐reported general health in representative sample surveys from Bosnia‐Herzegovina (n = 3,313) and Kosovo (n = 1,000). Data were collected with face‐to‐face interviews fielded in the winter of 2003–2004. Logistic regression analysis was used to compute unadjusted and adjusted odds ratios (ORs). The adjusted effects of the extensiveness of war experiences on poor health were positive in both countries, but they were statistically significant only for Bosnia‐Herzegovina: OR = 1.04, 95% CI [1.00, 1.08] for Bosnia‐Herzegovina and OR = 1.03, 95% CI [0.98, 1.09] for Kosovo. The strongest observed effect was found for Kosovo only: The extensiveness of war experiences was relatively strongly related to longstanding health problems, OR = 1.09, 95% CI [1.03, 1.15]. We found that war experiences may contribute to increased poorer health in the exposed populations; however, the effects 4–9 years after the war ended were modest. Hence, war experiences seemed to be more strongly related to war‐related distress and posttraumatic stress disorder than to self‐reported general health.