Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC-US).

OBJECTIVE: The effect of triaging women with atypical squamous cells of undetermined significance (ASC-US) with human papillomavirus (HPV) DNA testing has been well documented. New tests detecting HPV E6/E7 mRNA are emerging, claiming to be more specific for detecting high-grade disease. We evaluated the clinical performance of two HPV tests: the Linear Array HPV genotyping test (LA) detecting HPV DNA from 37 oncogenic and non-oncogenic HPV types and the Aptima HPV assay detecting E6/E7 mRNA from 14 oncogenic HPV types. METHODS: We identified 369 consecutive PreservCyt samples diagnosed with ASC-US tested for HPV DNA using the LA test. The Aptima HPV test was performed on residual material in the same vial. Follow-up of 325 women was available. The gold standard used was histologically confirmed cervical intraepithelial neoplasia (CIN) grade 2+ or 3+. RESULTS: LA and Aptima HPV assays were positive in 44.3% and 31.7% of the cases, respectively. The concordance was 81.2%. The two tests had identical sensitivity for detecting CIN3+ [92.6% (95% CI, 75.7-99.1)] but the Aptima HPV assay showed a significantly better specificity of 73.8% (95% CI, 68.5-78.7) versus 60.1% (95% CI, 54.3-65.7) for LA for detecting CIN3+. When using CIN2+ as the gold standard the sensitivity for LA was higher than for the Aptima HPV assay [93.8% (95% CI, 82.8-98.7) versus 87.5% (95% CI, 74.8-95.3)], but the specificity was higher for the Aptima HPV assay: 78.0% (95% CI, 72.6-82.7) versus 64.3% (95% CI, 58.3-69.9). CONCLUSIONS: Both tests showed good and equal clinical sensitivities for detecting CIN3+, but the Aptima HPV assay had significantly higher specificity for detecting CIN2+ and CIN3+ in women aged 30 years or older with ASC-US.

Screening for methicillin-resistant Staphylococcus aureus in clinical swabs using a high-throughput real-time PCR-based method.

The presence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and the community is a serious problem. Accordingly, a comprehensive plan has been implemented in the County of Vejle, Denmark, to identify colonised and/or infected individuals and to control the spread of MRSA. Since 2005, all patients and healthcare personnel have been screened for MRSA colonisation, involving analysis of 300-400 samples daily. To deal with this number of samples, a PCR-based method customised for high-throughput analysis and a system for fast reporting of MRSA carrier status were developed. Swab samples were incubated overnight in a selective tryptone soya broth and were analysed by PCR the following day. Using this strategy, non-colonised individuals were identified within 24 h, while MRSA-positive samples were analysed further by traditional microbiological methods to determine the resistance pattern. This is a cost-effective approach, as the greatest expense in hospitals involves the isolation of patients of unknown MRSA status. The method was evaluated by testing 2194 clinical samples, with a sensitivity and specificity of 100% and 94%, respectively. The analytical sensitivity was 97%, with 161 of 166 different MRSA strains and isolates generating positive results according to PCR analysis. Using four control strains, the inter-assay variation was revealed to be a maximum of 2.6%, indicating good reproducibility.

Real-time polymerase chain reaction is a sensitive and precise method for quantifying transcription from reporter plasmids.

Sensitive methods are required for studying promoter activity of weakly expressed genes using reporter systems. In this study it is shown that the sensitivity of the traditional enzymatic assay for measuring LacZ activity is too low when studying the promoter activity of epiregulin, a member of the epidermal growth factor (EGF) system. Consequently, a new real-time polymerase chain reaction (PCR) method was developed to evaluate the expression of the reporter gene LacZ. This method can be used for monitoring promoter activity of weakly expressed genes. T24A cells with LacZ expression driven by the cytomegalovirus (CMV) promoter were used to compare real-time PCR and the traditional enzymatic detection system. The real-time PCR method had a more than 1000-fold higher sensitivity than the enzymatic assay. LacZ mRNA could be quantified from as few as two cells with an imprecision of 19%. The increased sensitivity enabled the measurement of LacZ mRNA transcription driven by the epiregulin promoter in stably transfected T24A bladder cancer cells. This allowed a reproducible quantification of a 3-fold transcriptional increase from the epiregulin promoter caused by serum stimulation of stably transfected T24A cells. Clearly, this new method is well suited to quantify relatively small changes in transcriptional activity from weakly expressed genes.

Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin.

Protein kinase CK2 is a pleiotropic serine/threonine kinase which has been shown to phosphorylate numerous substrates. Evidence is accumulating that CK2 may exist complexed to a variety of cellular proteins, e.g. p53, MDM2, and A-Raf. Here, we explored the effects of the chemotherapeutic drugs cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after cisplatin and carboplatin treatment, the RNA level of p53 dropped by 59% +/- 9% and 86% +/- 8% respectively, whereas the observed level of p53 protein rose to 7 and 10 times over the untreated control, respectively. Treatment with 33 microM cisplatin prompted apoptosis as early as 4 h after drug treatment. More than 50% apoptotic cells were seen after 6 h. We conclude that cisplatin and its second generation drug carboplatin act similarly i.e. both drugs cause a concomitant decrease in p53 mRNA and an increase in p53 protein level. After 4 h treatment with either of the two drugs, p53 levels reach a threshold which leads to the initiation of apoptosis.