Factors predicting the outcome of conservatively treated adenocarcinoma in situ of the uterine cervix: an analysis of 166 cases.

Objective: The present study assessed the clinical outcome of patients conservatively treated for cervical adenocarcinoma in situ (AIS) and their predictive factors using univariate and multivariate population averaged (PA) generalized estimating equation (GEE) model in a longitudinal setting.

Methods: A series of 166 consecutive women (mean age 39.8 yrs; range 23-63 yrs) underwent conservative treatment of AIS as the primary treatment and were followed-up (mean 40.

Comparison of Abbott RealTime High Risk HPV and Hybrid Capture 2 for the detection of high-risk HPV DNA in a referral population setting.

Background: The Abbott RealTime High Risk HPV assay (ART) is an automated multiplex real-time PCR test for detection of DNA from 14 high risk (HR) HPV types in cervical specimens and simultaneous distinction of HPV16 and HPV18 from other HR-HPV.

Objectives: To evaluate the performance of the ART assay in specimens referred for HPV testing to our laboratory (referral population) by comparison with historical data from HC2 and INNO-LiPA as well as histological status, if available.

Study Design: 412 cervical specimens were collected from women between 18 and 70 years of age: 301 previously tested by HC2 without clinical data and 111 previously tested by HC2 and INNO-LiPA with histological diagnosis of CIN3+.

Evidence of human bocavirus viremia in healthy blood donors.

Human bocavirus DNA was detected by means of a quantitative, real-time polymerase chain reaction at low levels in the 5.51% of sera obtained from healthy blood donors, suggesting that viral detection in blood is not necessarily associated with disease status.

Gestational and fetal outcomes in B19 maternal infection: a problem of diagnosis.

Parvovirus B19 infection during pregnancy is a potential hazard to the fetus because of the virus' ability to infect fetal erythroid precursor cells and fetal tissues. Fetal complications range from transitory fetal anemia and nonimmune fetal hydrops to miscarriage and intrauterine fetal death. In the present study, 72 pregnancies complicated by parvovirus B19 infection were followed up: fetal and neonatal specimens were investigated by serological and/or virological assays to detect fetal/congenital infection, and fetuses and neonates were clinically evaluated to monitor pregnancy outcomes following maternal infection.

Molecular analysis of HPV 16 E6I/E6II spliced mRNAs and correlation with the viral physical state and the grade of the cervical lesion.

The presence of HPV 16 E6*I/E6*II spliced transcripts, in cervical lesions of different grade, was analyzed to characterize the transcription pattern. The presence and amount of spliced transcripts were correlated with DNA viral markers such as E2/E6 ratio and physical state. The detection of HPV 16 E6*I/E6*II mRNAs was set up by an SYBR Green real-time reverse transcriptase PCR assay with an optimal dynamic range and sensitivity.

High-throughput two-step LNA real time PCR assay for the quantitative detection and genotyping of HPV prognostic-risk groups.

Background: Human papillomavirus (HPV) infection is a necessary event in the development of cervical carcinoma. High risk (HR) HPV genotypes, however, may progress differentially from low grade lesions to malignancy.

Objectives: The necessity to genotype and quantify HPV-DNA in cervical screening programs, in the follow up post-surgical treatments and in monitoring the effectiveness of HPV vaccination programs, requires access to economical, high-throughput and flexible molecular technologies.

Disruption of HPV 16 E1 and E2 genes in precancerous cervical lesions.

The presence of HPV 16 E1 and E2 genes was detected in cervical cytological samples using polymerase chain reaction assays. A total of 48 samples were analyzed from patients with HPV 16 infections associated with 13 low-grade cervical intraepithelial neoplasia and 35 high-grade cervical intraepithelial neoplasia. Disruption/deletion sites, within E1 and E2 genes, were detected using 6 primer pairs spanning the entire gene sequences.

HepG2 hepatocellular carcinoma cells are a non-permissive system for B19 virus infection.

Parvovirus B19 has been associated with liver dysfunction and has been considered a potential aetiological agent of fulminant hepatitis and hepatitis-associated aplastic anaemia. The possible effects of B19 virus infection on the liver have been investigated using HepG2 hepatocellular carcinoma cells as a model system, but the reported results are inconsistent. To investigate this relationship further, this study followed the course of B19 virus infection of HepG2 cells in terms of viral DNA, RNA and protein production by quantitative PCR, RT-PCR and immunofluorescence assays.

Functional analysis and quantitative determination of the expression profile of human parvovirus B19.

Comprehension of the pathogenetic potential of human parvovirus B19 requires the definition of the complete spectrum of cellular tropism and a functional analysis of the viral genome in infected cells. In this study, we carried out a systematic functional analysis of B19 virus genome in the course of infection of susceptible bone marrow mononuclear cells and myeloblastoid UT7/EpoS1 cells, in terms of dynamics of nucleic acid synthesis. A PCR array was designed and a comprehensive analysis was performed by quantitative PCR and RT-PCR, yielding extended information on the presence and abundance of the diverse classes of viral nucleic acids, on the temporal regulation of genome expression and on its relationship with the cell cycle.

Chemiluminescent quantitative immunohistochemical p16 INK4A localization as a marker for cervical intraepithelial neoplasias.

A quantitative evaluation of p16 INK4A overexpression together with its topographical localization in the epithelium of cervical biopsies from non-neoplastic lesions and cervical intraepithelial neoplasias (CIN 1, 2, and 3) was obtained by the development of an objective and sensitive immunohistochemical assay with chemiluminescent detection (CL IHC assay). The cervical biopsy samples were also checked for the presence of human papillomavirus nucleic acids. The quantitative evaluation of p16 INK4A expression was performed by combining 2 parameters: (1) intensity of the chemiluminescent-positive signal in the epithelium and (2) percentage of epithelium interested by the overexpression of p16 INK4A, to obtain a p16 INK4A expression score.

Meningoencephalitis with persistent parvovirus B19 infection in an apparently healthy woman.

A case of meningoencephalitis, associated with persistent parvovirus B19 infection, is described in a 36-year-old immunocompetent woman. Parvovirus B19 DNA was detected in samples of cerebrospinal fluid and serum; no parvovirus B19-specific clinical symptoms were seen, but neurological episodes were observed in the presence of parvovirus B19 infection and despite the onset of a specific immune response.

Correlation of high-risk human papillomavirus genotypes persistence and risk of residual or recurrent cervical disease after surgical treatment.

The evidence on genotype-specific risk in women infected with human papillomavirus (HPV) with normal cytology and the importance of the distinction of high-risk (HR)-HPV genotypes in the management of low-grade lesions suggest that the distinction of HR-HPV genotypes has the potential to improve the follow-up of patients treated for high-grade cervical lesions. The aims of this study were to define the persistence of the different HR-HPV in the follow-up of surgical treated women, to detect the changes of genotypes from the pre- to the post-operative status, and to evaluate whether genotype-specific persistence can predict the development of residual or recurrent disease during the follow-up. HR-HPV detection and genotyping was carried out by the Linear Array HPV Genotyping Test on cervical cytological samples from 72 women treated by surgery.

Competitive PCR-ELISA protocols for the quantitative and the standardized detection of viral genomes.

Competitive PCR-ELISA combines competitive PCR with an ELISA to allow quantitative detection of PCR products. It is based on the inclusion of an internal standard competitor molecule that is designed to differ from the target by a short sequence of nucleotides. Once such a competitor molecule has been designed and constructed, target and competitor sequences are concurrently PCR-amplified, before hybridization to two different specific probes and determination of their respective OD values by ELISA.

Qualitative PCR-ELISA protocol for the detection and typing of viral genomes.

PCR is an established technique providing rapid and highly productive amplification of specific DNA sequences. The demand for equally rapid, sensitive and objective methods to achieve detection of PCR products has led to the coupling of PCR with ELISA. PCR-ELISA involves direct incorporation of labeled nucleotides in amplicons during PCR-amplification, their hybridization to specific probes and hybrid capture-immunoassay in microtiter wells.

Viral DNA load, physical status and E2/E6 ratio as markers to grade HPV16 positive women for high-grade cervical lesions.

Objectives: Cervical intraepithelial neoplasias (CIN) associated with high-risk (HR) human papillomavirus infection, in addition to HR-HPV typing need other viral marker testing to distinguish a subset of lesions with clinical relevant infections. This study has evaluated the significance of viral markers, such as viral load, physical status and E2/E6 ratio, to stratify HPV16 infected women at a single point in time for grade of cervical lesions.

Methods: One hundred sixty-six cytological specimens were selected from women with low (n=72) and high (n=94) grade squamous intraepithelial lesions (SIL), and positive to HPV16.

PNA-based probe for quantitative chemiluminescent in situ hybridisation imaging of cellular parvovirus B19 replication kinetics.

To allow the ultrasensitive localization and the quantitative detection of parvovirus B19 nucleic acids in single infected cells at various times post-infection, a peptide nucleic acid (PNA)-based in situ hybridisation (ISH) assay with chemiluminescent detection has been developed. The assay is based on the use of a biotin-labelled PNA probe detected by a streptavidin-linked alkaline phosphatase and a chemiluminescent dioxetane phosphate derivative substrate. The luminescent signal was quantified and imaged with an ultrasensitive nitrogen-cooled CCD camera connected to an epifluorescence microscope.

Prevention of iatrogenic transmission of B19 infection: different approaches to detect, remove or inactivate virus contamination.

Parvovirus B19 is a frequent contaminant of human blood and plasma derivatives and iatrogenic transmission of B19 infection has been shown to occur through the administration of contaminated products. Manufacturing procedures, generally used for removal or inactivation of enveloped viruses (HIV, HCV and HBV) are not always effective in the elimination of B19 virus. A certain risk of contamination remains for some plasma derivatives due to the high-titer viral load in the starting blood donations and the extreme heat resistance and small size of the virus.

Expression of bioactive recombinant bovine interferon-gamma using baculovirus.

The precise role of bovine interferon-gamma (BoIFN-gamma) in disease and therapy is still poorly defined. Clearly it is involved in defence against parasites, bacteria, viruses and possibly tumor cells. This paper reports the expression of BoIFN-gamma in a baculovirus system to generate a fully functional recombinant protein.

Peptide nucleic acid-based in situ hybridization assay for detection of parvovirus B19 nucleic acids.

Background: Peptide nucleic acid (PNA) molecules are known to bind complementary nucleic acid sequences with a much stronger affinity and with more stable binding than DNA or RNA molecules. We chose parvovirus B19, which is diagnosed by detection of nucleic acids by in situ hybridization assay (ISH) and/or PCR, as an experimental model to develop an ISH assay that uses biotinylated PNA probes to detect viral genome in clinical specimens.

Methods: We first optimized the PNA-ISH assay on B19-infected and mock-infected UT-7/EpoS1 cells and then tested the assay on archival B19 specimens and on consecutive specimens.

HPV DNA patterns and disease implications in the follow-up of patients treated for HPV16 high-grade carcinoma in situ.

Twenty-five patients with high-grade cervical lesions associated with HPV16 infection were studied at the time of surgical treatment and followed up after conization. Before surgical treatment, the following parameters were analyzed: (1) physical status of HPV16 DNA, (2) viral load, (3) cytological presentation confirmed by histological diagnosis, and (4) colposcopy. At the time of conization, (5) margin presentation, and (6) cone biopsy were evaluated.

Parvovirus B19 genome as a single, two-state replicative and transcriptional unit.

The variation in the amount of parvovirus B19 DNA and different classes of RNA in permissive and non-permissive infected cells was analysed by means of quantitative real-time PCR and RT-PCR assays. In the permissive bone marrow mononuclear cells, UT7/Epo and KU812Ep6 cells, viral DNA usually increased within 48 hpi, rarely exceeding 2 Logs with respect to input DNA. Viral RNA was always present within 2-6 hpi, its increase paralleled that of viral DNA up to 36-48 hpi, and all the different classes of viral RNA were constantly represented in stable relative amounts throughout the infection cycle.

Recurrent erythema in patients with long-term parvovirus B19 infection.

We describe 3 patients with long-term parvovirus B19 infection (defined as detectable parvovirus B19 DNA load for >6 months after the onset of symptoms), which we monitored by serial testing for parvovirus B19 load and the presence of parvovirus B19-specific antibodies in blood. The patients showed recurrent erythema at intervals of several months.

High-throughput polymerase chain reaction chemiluminescent enzyme immunoassay for typing and quantifying human papillomavirus DNAs.

A miniaturized polymerase chain reaction (PCR) chemiluminescent enzyme immunoassay (CLEIA) based on a 384-well microtiter plate for detection and typing of oncogenic high- and low-risk human papillomavirus (HPV) in genital lesions is described. The assay relies on PCR consensus amplification, hybridization of the digoxigenin-labeled product by means of type-specific biotin-labeled oligoprobes immobilized on the streptavidin-coated wells of a 384-well microtiter plate, and quantification by means of a horseradish peroxidase-labeled antidigoxigenin antibody and chemiluminescence detection. The method provides semiquantitative information on the viral load, with a limit of detection of 10-50 DNA copies for HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 58, and 59 and high reproducibility (intraassay CV 7.