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+. RESULTS: Our study demonstrated good overall agreement between ART, HC2 and INNO-LiPA. In the group of the CIN3+ specimens HR-HPV was detected by ART in 93.07% (95% CI: 88.12-98.02), while HR-HPV detection rates with HC2 and INNO-LiPA were 91.09% (95% CI: 85.53-96.65) and 95.05% (95% CI: 90.82-99.28), respectively. The typing capability of ART for HPV16, HPV18 and a pool of twelve other HR-HPV types was investigated by comparison with INNO-LiPA demonstrating high overall assay concordance (89.81%; k 0.87). CONCLUSIONS: The Abbott RealTime assay showed similar clinical performance for detection of CIN3+ compared with HC2. The high level of automation and ability to identify HPV16, HPV18 and other HR-HPV make this assay a very attractive option for HR-HPV testing, potentially improving patient management by risk stratification of cytological abnormal populations.

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 40.9 mo) using colposcopy, PAP-smear, biopsy and HPV-testing with Hybrid Capture 2. RESULTS: Hysterectomy was performed as part of the primary management in 47 patients, who were excluded from the follow-up (FU) analysis. Out of 119 women closely followed-up, additional therapeutic procedures were performed in 69. At study conclusion, 7 patients (5.9%) showed persistent disease, while 8 (6.7%) had progressed to invasive adenocarcinoma (AC). Positive HR-HPV test was the only independent predictor of disease recurrence (adjusted OR=2.72; 95%CI 1.08-6.87), and together with free cone margins (OR=0.20; 95%CI 0.04-0.92), HR-HPV positivity was also the single most powerful predictor of disease progression to AC, with OR=3.74; 95%CI 1.84-7.61 (p=0.0001) in multivariate PA-GEE. CONCLUSIONS: These results suggest that testing HR-HPV positive at any time point during FU is the most significant independent predictor of progressive disease, while showing free margins in cone has a significant protective effect against progression to AC. Furthermore, because 4.3% women with persistent, recurrent or progressive disease experienced a late (5th and 6th FU) diagnosis of HG-CGIN or microinvasive AC, a close surveillance should be scheduled for at least three years in conservatively treated AIS patients.

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. Analysis of serological and virological maternal B19 markers of infection demonstrated that neither B19 IgM nor B19 DNA detected all maternal infections. IgM serology correctly diagnosed 94.1% of the B19 infections, while DNA testing correctly diagnosed 96.3%. The maximum sensitivity was achieved with the combined detection of both parameters. B19 vertical transmission was observed in 39% of the pregnancies, with an overall 10.2% rate of fetal deaths. The highest rates of congenital infections and B19-related fatal outcomes were observed when maternal infections occurred by the gestational week 20. B19 fetal hydrops occurred in 11.9% of the fetuses, and 28.6% resolved the hydrops with a normal neurodevelopment outcome at 1- to 5-year follow-up. In conclusion, maternal screening based on the concurrent analysis of B19 IgM and DNA should be encouraged to reliably diagnose maternal B19 infection and correctly manage pregnancies at risk.

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. The assay was applied to the analysis of 71 specimens, positive to HPV 16 as a sole infection, from women with abnormal cervical smears, precisely 31 low-grade squamous intraepithelial lesions and 40 high-grade lesions. Samples negative to both transcripts were found only in low-grade cervical lesions. Three different transcription profiles were found in the low- and high-grade lesions analyzed: in low-grade lesions samples positive only to E6*II and in high-grade lesions samples positive only to E6*I were detected. In low- and high-grade lesions, samples positive to both E6*I and E6*II were found. In the samples positive for both transcripts, the E6*I/E6*II ratio was higher than that in the majority of high-grade lesions and lower than that in all the low-grade lesions. Analyzing the transcription pattern in relation to E2/E6 ratio and to the DNA physical state, the presence of high values of E6*I was associated mainly with low values of E2/E6 ratio and of mixed DNA forms. The detection of HPV 16 E6*I/E6*II mRNAs may serve to identify transcription patterns indicative of cervical disease progression and help physicians to decide clinical management.

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. STUDY DESIGN: A high-throughput two-step LNA real time PCR assay was developed consisting of real time PCR reactions with fluorescent Locked Nucleic Acid (LNA) probes. The first step permits classification into three prognostic-risk groups of nine HR HPV genotypes (16, 18, 31, 33, 35, 45, 52, 56 and 58) most frequently found associated with cervical lesions in Europe. The second step allows us to genotype/quantify the HPV-DNA only when clinical, epidemiological or prophylactic aims exist. RESULTS: The specificity, repeatability, detection and quantitation limit, and linearity of the assay were evaluated and appear to be in agreement with guidelines for the validation of analytical procedures. The overall genotype concordance on cervical samples between our assay and INNOLiPA test was 94% (k 0.83) indicating good agreement. CONCLUSIONS: The two-step PCR assay can give much information relative to the predictive value of different HR HPV types and can quantify the genotype-specific viral load. In particular, its ability to detect and quantify nine HR HPV genotypes can help provide more efficient and successful patient care and may be useful for the monitoring of the efficacy of HPV vaccines.

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. This technique is not able to recognize mixed DNA forms (integrated plus episomal DNA); therefore, it detects only the presence of pure integrated DNA. Both E1 and E2 genes were detected in 84.6% and in 62.9% of low and high-grade lesions, respectively. The rate of samples with disrupted/deleted genes was significantly higher in high-grade cervical intraepithelial neoplasia than in low-grade cervical intraepithelial neoplasia (P<0.05). In high-grade cervical intraepithelial neoplasia the disruption/deletion pattern involved both E1 and E2 genes and E2 gene was always involved, while in the low grade cervical intraepithelial neoplasia only E1 gene was involved. In conclusion, in high-grade cervical lesions E2 gene seems a suitable target to identify HPV 16 DNA integration into cellular genome.

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. The data showed that B19 virus is able to bind and possibly enter HepG2 cells, but that viral genome replication or transcription is not supported and that viral proteins are not produced. As far as HepG2 cells can be considered a representative model system, any possible pathogenic role of B19 virus on the liver cannot be ascribed to infection or to a direct cytopathic effect on hepatocytes.

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. The analysis performed indicate that the synthesis of viral nucleic acids is correlated to the progression through the S phase of the cell cycle, that an extended pattern of transcriptional activity occurs throughout the course of infection, with a maximal rate of transcription preceding the onset of S-phase dependent replication of the viral genome, and that utilization of transcript processing signals is relatively constant throughout the course of infection. The information obtained led to the definition of a unified model of functional and expression profiling of parvovirus B19 genome.

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. A cut-off value was determined by using the receiver-operator characteristic analysis to distinguish between low-grade and high-grade CIN. Quantitative data showed that both p16 INK4A expression parameters increased with worsening grades of CIN and, when combined to obtain the p16 INK4A expression score, they showed a sharp discrimination among different lesions. The differences between the average p16 score of CIN1 versus CIN2 (0.57 versus 1.05), of CIN1 versus CIN3 (0.57 versus 1.31) and of CIN1 versus CIN2/3 (0.57 versus 1.20) were statistically significant. The quantitative evaluation of p16 INK4A expression by CL IHC assay could be therefore an interesting adjuvant method to distinguish different CIN grades and to predict the risk of progression of early CIN lesions.

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. The 6-month post-operative HPV status was correlated with the pre-operative HPV genotype and with the residual or recurrent disease within 24 months. It was observed that the residual or recurrent disease in women with persistence of HPV 16 and/or HPV 18 was higher (82.4%) than in women with persistence of at least one HR-HPV type of group 2 (HPV 31, 33, 35, 45, 52, and 58) (66.7%) and at least one type of group 3 (HPV 39, 51, 56, 59, 68, 26, 53, 66, 73, and 82) (14.3%). These data defined HR-HPV groups for the risk of progression of disease and suggested that the identification of persistent infection with different HR-HPV genotypes has the potential to improve the management of these patients.

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. PCR-ELISA is performed in 1 d and is very flexible, with the ability to process simultaneously up to 96 or 384 samples. This technique is potentially automatable and does not require expensive equipment, and thus can be fundamental in laboratories without access to a real-time PCR thermocycler. PCR-ELISA has mainly been used to detect infectious agents, including viruses, bacteria, protozoa and fungi. A PCR-ELISA protocol for the qualitative detection of papillomavirus genomes and simultaneous typing of different genotypes are detailed here as an example of the technique.

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. The target can be quantified in relation to a titration curve of different dilutions of the competitor. The competitor can alternatively be used at a unique optimal concentration to allow for standardized detection of the target sequence. PCR-ELISA can be performed in 1 d in laboratories without access to a real-time PCR thermocycler. This technique is applied in diagnostics to monitor the course of infections and drug efficacy. Competitive PCR-ELISA protocols for the quantitative and for the standardized detection of parvovirus B19 are detailed here as an example of the technique.

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. All the 72 LSIL were CINI, 83 of the 94 HSIL were CINII/III and 11 SCC (Squamous Cervical Carcinoma). Cytological specimens were analysed by two different SYBR Green Real-time PCR assays (RT-PCR). Specific primers for both E2 and E6 viral genes and GAPDH cellular gene were designed to determine viral load, physical status and E2/E6 ratio. RESULTS: The viral load was significantly higher in HSIL than in LSIL. In CINI episomal DNA was prevalent (72.2%), mixed forms (episomal and integrated) were 27.8%, suggestive of an early integration of viral DNA into cellular genome, no pure integrated forms were detected. However in CINII/III mixed DNA forms were prevalent (73.5%). In SCC pure integrated DNA was prevalent (81.8%) in absence of episomal forms. E2/E6 ratio decreased significantly from CINI to CINII/III and SCC with a linear trend. The logistic regression analysis showed that viral load higher than 1.38x10(6) genome copies per 300 ng of total DNA associated with E2/E6 ratio lower than 0.90 was highly significant in differentiating CINII/III versus CINI, while the only E2/E6 value lower than 0.17 was significant in differentiating SCC from CINI. CONCLUSIONS: Viral load higher than 1.38x10(6) genome copies per 300 ng of total DNA and E2/E6 ratio values allow HPV16 infected women with high grade cervical intraepithelial lesions to be recognized.

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. The assay was used to analyze the parvovirus B19 infection process in cell cultures and to quantify the amount of viral nucleic acids at different times after infection. The chemiluminescent ISH-PNA assay is characterized by high resolution providing a sharp localization of B19 nucleic acids within single cells, with higher sensitivity with respect to conventional colorimetric ISH detection. Thanks to the high detectability and wide linear range of chemiluminescence detection, an objective evaluation of the percentage of infected cells, which reached its maximum at 24 h after infection, following a B19 virus infectious cycle could be accurately evaluated. Chemiluminescence detection also allowed the quantitative analysis of viral nucleic acids at the single-cell level, showing a continuous increase of the content of viral nucleic acids in infected cells with time after infection. The developed chemiluminescent ISH-PNA assay could thus represent a potent tool for the assessment of viral infections and for the quantitative evaluation of the virus nucleic acid load of infected cells in virus studies and diagnostics.

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. This review provides an update on the different approaches currently available to detect, remove or inactivate B19 virus in order to enhance the safety margins of plasma products. Nucleic acid amplification techniques are the methods of choice for the detection of viruses, due to their high specificity and sensitivity. NAT assays are beneficial tools for the identification of contaminated mini-pools or plasma pools and the quantification of B19 contamination. They may also be valuable for testing the removal of B19 virus during manufacturing: since the virus may not be completely inactivated or removed by chemical or physical treatments, residual B19 contamination should always be checked. Solvent-detergent treatments fail to destroy B19 capsids because of the absence of a lipid-envelope, and heat treatments (pasteurization and dry-heat methods) cannot guarantee a complete viral inactivation because of the variable heat sensitivity 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. Bovine interferon-gamma cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) encoding for a putative 166 amino acid protein (22KDa) was cloned and expressed into baculovirus transfer vector pBlueBac 4.5/V5 His. This vector was co-transfected with Autografa californica multiple nuclear polyhedrosis virus (AcMNPV) DNA into Spodoptera frugiperda cells (Sf9) and the recombinant virus, named AcBoIFN-gamma, was then recovered. Recombinant BoIFN-gamma (rBoIFN-gamma His) was accumulated in the serum-free medium of AcBoIFN-gamma-infected cells. The nickel affinity spin column purified rBoIFN-gamma His was shown to be a glycosylated 20-22 KDa protein as confirmed by SDS-PAGE glycan determination and showed antiviral activity in vitro against the bovine viral diarrhoea-mucosal disease virus (BVD/MD). The production of this bioactive rBoIFN-gamma His will allow us to explore this cytokine as a potential vaccine adjuvant or therapeutic agent for bovine diseases.

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. All data were compared with data obtained with a standardized DNA-based ISH assay and confirmed by a PCR-ELISA. RESULTS: PNA-ISH detected B19 genome in a higher number of B19-infected UT-7/EpoS1 cells and with a more defined localization of viral nucleic acids than the standardized DNA-ISH assay. Moreover, PNA-ISH was able to detect B19 genome in all positive archival samples, whereas DNA-ISH failed in 5 samples. PNA-ISH detected more positive samples than DNA-ISH when consecutive specimens were analyzed, and a close agreement was found with PCR-ELISA results. CONCLUSIONS: The PNA-ISH assay had sensitivity and specificity comparable to a PCR assay and was more practical and quicker to perform than standard hybridization assays. The assay may be a suitable diagnostic test for the detection of viral nucleic acids in clinical specimens.