The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important, since (i) the oncogenic potential among the high-risk HPV genotypes varies in the pathogenesis of cervical cancer, (ii) monitoring multivalent HPV vaccines is essential to investigate the efficiency of the vaccines, and (iii) genotyping is crucial in epidemiologic studies evaluating HPV infections worldwide. Various genotyping assays have been developed to meet this demand. Comparison of different studies that use various HPV genotyping tests is possible only after a performance assessment of the different assays. In the present study, the SPF 10 LiPA version 1 and the recently launched Roche Linear Array HPV genotyping assays are compared. A total of 573 liquid-based cytology samples were tested for the presence of HPV by a DNA enzyme immunoassay; 210 were found to be positive for HPV DNA and were evaluated using both genotyping assays (163 with normal cytology, 22 with atypical squamous cells of undetermined significance, 20 with mild/ moderate dysplasia, and 5 with severe dysplasia). Comparison analysis was limited to the HPV genotype probes common to both assays. Of the 160 samples used for comparison analysis, 129 (80.6%) showed absolute agreement between the assays (concordant), 18 (11.2%) showed correspondence for some but not all genotypes detected on both strips (compatible), and the remaining 13 (8.2%) samples did not show any similarity between the tests (discordant). The overall intertest comparison agreement for all individually detectable genotypes was considered very good ( value, 0.79). The genotyping assays were therefore highly comparable and reproducible.Molecular and epidemiologic studies have shown that a persistent infection with high-risk human papillomavirus (HPV) is the most important risk factor for both cervical cancer and its precursors (9,11,29,33). Approximately 40 different HPV types can infect the mucosa of the anogenital tract. Based on their carcinogenicities, these anogenital HPV types have been subdivided into low-risk HPV (lr-HPV) types, probable highrisk HPV (hr-HPV) types, and hr-HPV types (27), although some controversy remains regarding the probable high-risk genotypes (30). Almost all squamous cell cervical cancers worldwide harbor hr-HPV types (36). Moreover, high-risk HPV DNA can be detected in 74% of the premalignant lowgrade cervical intraepithelial neoplasia (CIN) lesions and approximately 84% of the high-grade CIN lesions (25). Consequently, the efficacy of population-based screening programs solely using cervical cytology could benefit from adding hr-HPV testing (32). Accordingly, many ongoing international research projects assess the feasibility of introducing hr-HPV tests in available routine screening.For these screening purposes, several tests have been developed in order to distinguish high-risk HPV infections from no HPV infection. Among these are the signal amplification method Hybrid Capture II (hc2) (Digene Corp., Gaithersburg, Maryland) and the ...

The purpose of this study was to detect and genotype 16 different human papilloma virus (HPV) types simultaneously using a short fragment polymerase chain reaction (SPF) hybridization line probe assay (LiPA). 152 women who were referred to the gynecologist because of abnormal cervical smear underwent colposcopic examination and repeat cervical smear. In addition, the cervical scrapes were analyzed for the presence of HPV by a novel general HPV polymerase chain reaction assay followed by a single reaction genotyping assay allowing for a simultaneous detection and identification of 16 different HPV types. HPV DNA was detected in 38% of normal follow-up cervical scrapes, 51% of scrapes with atypical squamous cells of undetermined significance, 78% of scrapes with mild dysplasia (low grade squamous intraepithelial lesions), 86% of scrapes with moderate dysplasia (high grade squamous intraepithelial lesions), and in 88% of scrapes with severe dysplasia and carcinoma in situ. One case of invasive squamous cell carcinoma was positive for HPV 16. Overall, a single HPV type was detected in 56% of HPV positive scrapes, with HPV 16 being the most common and accounting for 45% of all single infections. Forty-four percent of the positive scrapes contained multiple HPV types, of which double infections prevailed. Follow-up results proved the reproducibility and reliability of SPF HPV LiPA. In conclusion, we have used and evaluated the SPF-HPV-LiPA system for the detection and genotyping of HPV infections. The combined detection-typing method proved to be sensitive, specific, simple, and fast, making mass screening of cervical scrapes accessible for routine practice and facilitating individual patient management.

The pathogenesis of coxsackie B virus (CVB) infections is generally studied in mice by intraperitoneal (i.p.) injection, whereas the gastrointestinal tract is the natural porte d'entrée in humans. The present study was undertaken to compare systematically the influence of infection route on morbidity and pathology. Swiss Albino mice were infected with CVB3 (Nancy) at different doses (5 x 10(3), 5 x 10(5), 5 x 10(7), 5 x 10(9) TCID50), given either i.p. or orally. Virus could be isolated from several organs (heart, spleen and pancreas), indicating systemic infection, irrespective of the infection route. Virus titres were 1-2 logs higher after i.p. infection, but kinetics were largely independent of infection route. Organs became negative for virus isolation after 21 days, with the exception of spleen tissue, which remained positive for up to 49 days. Thereafter, virus was detected only by immunohistochemistry and PCR up to 98 days post-infection (oral route). Histopathology showed mild inflammation and necrosis in heart tissue of all mice during the acute phase, with repair at later stages. Strikingly, pancreatic lesions were confined to the exocrine pancreas and observed only after i.p. infection. Under all experimental conditions, the pancreatic islets were spared. In contrast, immunohistochemistry showed the presence of viral VP1, protein 3A and alpha interferon (IFN-alpha) in exocrine as well as endocrine pancreas of all mice, irrespective of route and dose of infection. It is concluded that infection via the oral route protects the pancreas from damage, but not from infection, a process in which IFN-alpha is not the only factor involved.

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Previous studies have shown that enteroviral RNA can be detected in blood at the onset of type 1 diabetes (T1D). The infection may play a role in triggering T1D and genetic host factors may contribute to this process. We investigated (1) whether enterovirus is present at the onset of T1D in peripheral blood mononuclear cells (PBMC), plasma, throat, or stool, and (2) whether enteroviral presence is linked with HLA-DR type and/or polymorphisms in melanoma differentiation-associated gene 5 (MDA5) and 2'-5' oligoadenylate synthetase 1 (OAS1), factors of antiviral immunity. To this end, PBMC, plasma, throat, and stool samples from 10 T1D patients and 20 unrelated controls were tested for the presence of enteroviruses (RT-PCR), for HLA-DR type, and polymorphisms in MDA5 and OAS1. Enterovirus RNA was detected in PBMC of 4/10 T1D patients, but none of 20 controls. Plasma was positive in 2/10 T1D patients and none of 20 controls, suggesting that enteroviruses found at the onset of T1D are mainly present in PBMC. All throat samples from positive T1D patients were virus-negative and only 1 fecal sample was positive. The negative results for all throat and most stool samples argues against acute infection. Enterovirus presence was linked with HLA-DR4, but not with polymorphisms in MDA5 or OAS1.

Pneumocystis jiroveci pneumonia (PCP) is an opportunistic infection affecting immunocompromised patients. While conventional diagnosis of PCP by microscopy is cumbersome, the use of PCR to diagnose PCP has great potential. Nevertheless, inter-laboratory validation and standardization of PCR assays is lacking. The aim of this study was to evaluate the inter-laboratory agreement of three independently developed real-time PCR assays for the detection of P. jiroveci in bronchoalveolar lavage fluid samples. Therefore, 124 samples were collected in three tertiary care laboratories (Leiden University Medical Center, Maastricht Infection Center and Radboud University Nijmegen Medical Centre) and were tested by both microscopy and real-time PCR. Of 41 samples positive for P. jiroveci by microscopy, 40 were positive in all three PCR assays. The remaining sample was positive in a single assay only. Out of 83 microscopy-negative samples, 69 were negative in all three PCR assays. The other 14 samples were found positive, either in all three assays (n=5), in two (n=2) or in one of the assays (n=7). The data demonstrate high inter-laboratory agreement among real-time PCR assays for the detection of P. jiroveci.

BackgroundA new virus called the Merkel Cell Polyomavirus (MCPyV) has recently been found in Merkel Cell Carcinoma (MCC). MCC is a rare aggressive small cell neuroendocrine carcinoma primarily derived from the skin, morphologically indistinguishable from small cell lung carcinoma (SCLC). So far the actual presence of the virus in MCC tumour cells on a morphological level has not been demonstrated, and the presence of MCPyV in other small cell neuroendocrine carcinomas has not been studied yet.Methodology/Principal FindingsWe investigated MCC tissue samples from five patients and SCLCs from ten patients for the presence of MCPyV-DNA by PCR and sequencing. Electron microscopy was used to search ultrastructurally for morphological presence of the virus in MCPyV-DNA positive samples. MCPyV was detected in two out of five primary MCCs. In one MCC patient MCPyV-DNA was detected in the primary tumour as well as in the metastasis, strongly suggesting integration of MCPyV in the cellular DNA of the tumour in this patient. In the primary MCC of another patient viral particles in tumour cell nuclei and cytoplasm were identified by electron microscopy, indicating active viral replication in the tumour cells. In none of the SCLCs MCPyV-DNA was detected.Conclusions/SignificanceOur results strongly suggest that MCPyV is an oncogenic polyomavirus in humans, and is potentially causally related to the development of MCC but not to the morphological similar SCLC.