Parvovirus B19 in pregnancy: possible consequences of vertical transmission.

OBJECTIVE: The aim was to determine the outcome of pregnancies complicated by maternal Parvovirus B19 (B19) infection. METHOD: Among 175 pregnant women referred to our clinic because of suspicion of a B19 infection, 63 with confirmed laboratory diagnosis of acute/recent B19 infection were followed up by ultrasound and Doppler measurement of the middle cerebral artery peak systolic velocity. RESULTS: The vertical transmission rate was 31.7% (20/63). Of the 20 infected, 8 had hydrops, 1 had signs suggestive of meconium peritonitis and 1 had an isolated hydrothorax. Three fetuses presenting with hydrops were treated with intrauterine blood transfusion. Two of them died while the last showed resolution of anemia. Among the five untreated hydropic fetuses, one presented with mild signs that resolved spontaneously, two died at 16 and 17 weeks of gestation and two had also cardiomegaly and the parents opted for elective termination of pregnancy. All the anemic fetuses had middle cerebral artery peak systolic velocity values more than 1.8 multiples of the median. No stillbirth occurred. CONCLUSIONS: The outcome of uncomplicated cases with B19 infection is good. In the presence of hydrops prognosis was very poor. It seems therefore logical to attempt to pick up this ominous signs early.

Parvovirus b19 DNA is commonly harboured in human skin.

BACKGROUND: Parvovirus B19 is the aetiological agent of erythema infectiosum. The presence of B19 DNA in lesional skin of other cutaneous manifestations has frequently been reported although there is disagreement on the role of the B19 virus in tissues. OBJECTIVES: To investigate the presence of B19 DNA (1) in skin lesions of patients with a described B19-related disease, (2) in skin lesions of B19-unrelated diseases and (3) in healthy skin. METHODS: A total of 121 skin samples were examined for the presence of B19 DNA by PCR assays and peptide-nucleic-acid-based in situ hybridisation techniques. RESULTS: B19 DNA was detected in 11/38 (28.9%) pityriasis lichenoides, 8/30 (26.7%) melanocytic naevi, 5/29 (17.2%) primary melanomas and 6/24 (25.0%) healthy skin biopsies. A difference in B19 DNA prevalence was observed in specimens grouped according to age, irrespective of pathologies. CONCLUSIONS: B19 DNA can be found in skin tissues of patients with pityriasis lichenoides as well as in lesions not related to B19 infection and in healthy controls. B19 DNA can be detected in skin of young subjects in a significantly high rate compared to adults, suggesting that viral persistence may be the usual outcome after primary infection.

Diagnosis of fetal parvovirus B19 infection: value of virological assays in fetal specimens.

OBJECTIVE: The purpose of our work was to examine the most reliable laboratory diagnosis of fetal parvovirus B19 infection in hydropic fetuses by evaluating the most appropriate clinical sample and laboratory test. DESIGN: B19 DNA detection in fetal samples and serological signs of B19 infection in the respective mothers. Samples collected between January 2000 and July 2008. SETTING: Microbiology, University of Bologna, Bologna, Italy. SAMPLES: One hundred thirty-five fetal samples (58 fetal cord blood and 77 amniotic fluid samples) and 109 serum samples collected from 109 pregnant women. METHODS: Validated and certified in situ hybridisation assay (ISH) and polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) were performed on fetal samples to detect B19 DNA. B19-specific antibodies were investigated in maternal serum samples by a commercial enzyme immunoassay. MAIN OUTCOME MEASURES: Parvovirus B19 DNA detection in fetal specimens was analysed in relation to maternal serological signs of infection. RESULTS: Parvovirus B19 DNA was detected in 22.41% of fetal cord blood and 36.36% of amniotic fluid samples. A statistically significant difference was found between DNA detection by ISH (23.70%) and PCR-ELISA (14.81%) (P= 0.004). Only 11.76% of fetuses with virological diagnosis of B19 infection were from women with serological signs of acute/recent B19 infection. CONCLUSIONS: Diagnosis of fetal parvovirus B19 infection cannot always rely on maternal serological investigations but rather on the virological analysis of fetal samples. Both fetal cord blood and amniotic fluid samples are suitable for diagnosis, but the detection of B19 DNA in the cells of amniotic fluid samples by ISH proved to be the most reliable diagnostic system.

Seroprevalence of IgG against conformational and linear capsid antigens of parvovirus B19 in Italian blood donors.

Serum samples from 446 Italian blood donors between 18 and 65 years of age were analysed for the presence of IgG against parvovirus B19 capsid proteins VP1 and VP2 including conformational and linear epitopes. The overall prevalence of IgG against parvovirus B19 capsid proteins VP1 and VP2 against at least one antigen type was 79.1 %. No significant difference was found between men and women. In the 18-27 years age group, 77.0 % of the population had experienced infection with the virus, reaching 88.5 % in the 48-57 years age group. The overall prevalence of IgG was 78.0 % against conformational VP1 + VP2 antigens, 74.9 % against conformational VP2, 70.9 % against linear VP1 and 23.3 % against linear VP2 in the analysis of the IgG response against different conformational and linear epitopes of VP1 and VP2. Although IgG against conformational VP1+VP2, conformational VP2 and linear VP1 was present in more than 60 % of subjects in all age groups, IgG against VP2 linear antigens was present in only 32% of subjects in the 18-27 years age group and then decreased to 20.5 % in the 28-37 years age group. A different trend was noted when IgG positivity against linear and conformational epitopes was analysed separately in men and women. A significant increase was found in seroprevalence of IgG against VP2 conformational antigens with increasing age in males and a significant decrease in seroprevalence of IgG against VP2 linear antigens in women with increasing age.

Molecular testing for detection of in vitro infectivity of plasma pools contaminated with B19 virus.

B19 virus can be transmitted by contaminated blood or blood products. Recent observations, in healthy volunteers, suggest that active B19 infection can follow the administration of plasma pools with a concentration > or =10(7) genome equivalents/ml (geq/ml) of B19 DNA. However, patients receiving batches with levels of virus DNA lower than 10(4) geq/ml do not show any evidence of transmission of the virus. The aim of the study was to show, by in vitro assays, a threshold of viral load in B19 contaminated plasma pools over which the infection can be transmitted. Twenty plasma pools, each containing 960 single donations, were tested to correlate the viral load and the level of antibodies anti-B19 with the in vitro infectivity and expression of B19 virus. All the plasma pools, titrated for B19 viral load by competitive PCR, were inoculated into KU812Ep6 erythroid human cell line. Five of the nine contaminated plasma pools, with a B19 DNA concentration > or =3.60 x 10(6) geq/ml, were able to infect KU812Ep6 cells. In vitro infectivity was shown in KU812Ep6 cells at 24 h post-infection by in situ hybridisation and amplification assays for viral DNA and RNAs. Plasma pools with a viral load in the range of 6.00 x 10(3)-8.96 x 10(4) geq/ml did not show infectivity when inoculated into KU812Ep6 cells. Medium-high titres of IgG antibodies anti-B19 were detectable in all the plasma pools and the neutralising activity associated with specific IgG anti-B19 may explain the lack of infectivity of plasma pools contaminated with a low viral load. In conclusion, in situ hybridisation and amplification assays for viral DNA and RNAs in KU812Ep6 cells inoculated with plasma pools can be valid assays to test for the presence of infectious virus in the production of B19-safe material.

Efficient treatment of paraffin-embedded cervical tissue for HPV DNA testing by HC-II and PCR assays.

BACKGROUND AND OBJECTIVES: High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. STUDY DESIGN: the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. RESULTS: protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance (K=0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. CONCLUSIONS: HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

Detection of parvovirus B19 IgG: choice of antigens and serological tests.

Serum samples were analysed for the presence of (a) IgG against VP1+VP2 using recombinant native conformational antigens by ELISA test (b) IgG against VP2 using recombinant native conformational antigens by ELISA test and (c) IgG against VP1 and against VP2 using denatured linear antigens by Western blot. Out of the 446 samples examined, 353 were positive for specific B19 IgG and out of these, 98.6 % proved positive in the ELISA assay using conformational VP1+VP2 antigens, 94.6% proved positive in the ELISA assay using conformational VP2 antigens, 89.5% were positive at the Western blot assay using denatured linear VP1 and VP2 antigens, with all proving positive for linear VP1 and only 29.5% out of the positive samples proving positive for linear VP2. Since all samples positive by Western blot proved positive by ELISA, our data show that recombinant capsids obtained either with VP1+VP2 or with VP2 alone, used in ELISA, are very useful for detecting the immune response against both conformational and native linear epitopes of B19 structural proteins although some sera may have antibodies directed exclusively against VP1+VP2 antigens and few may have antibodies directed exclusively against VP2 antigens alone.

Relevance of B19 markers in serum samples for a diagnosis of parvovirus B19-correlated diseases.

In order to evaluate the optimal and essential diagnostic test(s) for a correct diagnosis of B19 diseases, 344 consecutive serum samples were tested from 344 patients with clinical suspicion of B19 infection during an epidemic period (early Spring-Autumn 2000). Sera were tested for B19 DNA by a standardized competitive polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) and dot-blot hybridization and for specific IgM and IgG by ELISA. Of 344 patients examined, 125 were positive for markers of B19-associated disease: 49 had both B19 DNA and IgM, 50 had B19 DNA without IgM, and 26 had IgM without B19 DNA. After examination of the different patterns of B19 markers as diagnostic tools for B19 infection, IgM determination detected only 60% of B19-documented infections. IgM tests were nevertheless fundamental, as they were the unique diagnostic marker in 20.8% of documented infections (26 of 125 patients), in the diagnosis of recent, but still symptomatic infections when B19 DNA was no longer detectable. The determination of B19 DNA with PCR permitted detection of 79.2% of infections and therefore represented an essential test. PCR was fundamental for the diagnosis of B19 disease, as the unique diagnostic marker in 32% of documented infections (50 of 125 patients), both in acute infections at the onset of symptoms before the appearance of immunological response, and during the course of persistent B19 infections in which IgM had cleared. The contemporaneous determination of B19 DNA by PCR and specific IgM appears to be the most appropriate diagnostic protocol for the correct laboratory diagnosis of B19 infection.

Analysis of B19 virus contamination in plasma pools for manufacturing, by using a competitive polymerase chain reaction assay.

BACKGROUND AND OBJECTIVES: The presence of B19 virus in blood poses a risk of transmission of the virus via blood or blood products. Screening processes for manufacturing should be aimed at achieving production plasma pools with B19 virus contamination levels below 104 genome equivalents/ml (geq/ml) in order to prevent transmission of infection through plasma derivatives. MATERIALS AND METHODS: The suitability of a competitor plasmid as an internal analytical standard for the detection of B19 virus in plasma pools was assessed by using a competitive polymerase chain reaction (PCR) assay. Seventy-five plasma pools, each consisting of 960 single donations, were analysed for B19 virus contamination following a lysis treatment. RESULTS: The amount of competitor plasmid in the competitive PCR assay established, with good accuracy, a threshold value for discrimination of the viral load in plasma pools. Analysis of samples from plasma pools showed that 12% of pools were contaminated with B19 virus at levels above the set threshold value. CONCLUSIONS: The competitive PCR assay developed proved to be effective for discrimination of the B19 virus contamination level in screening of plasma pools for manufacturing.

Evaluation of commercial kits for the detection and typing of human papillomavirus in cervical swabs.

Human papillomaviruses (HPV) detection by MY consensus primers amplification within the L1 region and typing of prevalent genital HPVs by reference and commercial sets of probes was compared by PCR-ELISA systems. The specificity of commercial probes used in the L1 HPV Geno-Kit with respect to the reference probes proved to be 100%, with an overall agreement of 97.6%. The discordant results concerned only the detection of HPV 16, both as single genotype present in the sample and as multiple infections. The analytical sensitivity of the commercial probe for HPV 16 proved to be slightly less sensitive than the reference probe in the hybridisation conditions of the PCR-ELISA system. The L1 PCR-ELISA reference system was evaluated further in comparison with commercial E6/E7 consensus PCR and microplate hybridisation by typing kit. Amplified products of both the L1 and E6/E7 consensus regions were also analysed by agarose gel electrophoresis. An overall concordance of 95.2% was found. On account of its specificity and sensitivity the E6/E7 commercial system proved to be particularly useful for diagnostic laboratory, as it detects only the prevalent high risk genotypes. The agarose gel detection can therefore be used as screening test, thus reducing the costs, while the E6 E7 HPV Geno-Kit High Risk can be used when specific typing is required.

Diagnostic procedures in B19 infection.

In immunologic normal hosts, both children and adults, B19 can cause acute, generally self-limiting diseases. The infection leads to a viremia that can be present, at high titre, for about one week, then the onset of a specific immune response controls the infection. B19 infection in pregnancy can be associated with non-immunologic foetal hydrops or foetal death. In immunocompromised hosts, B19 can persist over several months and sometimes years. Persistent or recurrent B19 infections can be associated with chronic clinical manifestations or with transient clinical syndromes, generally related to the recrudescence of viral replication. Since the infection has been associated with a wide variety of clinical manifestations and some clinical features of B19 infection, such as anemia, artropathy and rash, can be common to other pathogens, a laboratory diagnosis of B19 infection is required. A diagnostic protocol must consider both the type of pathology and the type of patient. In immunocompetent individuals serological and virological testing is complementary, while in immunocompromised patients viral detection is the diagnosis of choice. Viral detection methods are generally based, nowadays, on the direct detection of B19 genome in clinical specimens. B19 DNA is mainly detected by hybridizations assays and by the most sensitive PCR assays. Serological diagnosis of B19 infection is generally achieved by detection of IgM and IgG antibodies to the B19 structural proteins VP1 and VP2. IgM detection is most often performed by capture assays, both in EIA and RIA formats, IgG are mainly detected by indirect EIA and immunofluorescence tests.

Detection of adeno-associated virus DNA in female genital samples by PCR-ELISA.

Adeno-associated viruses (AAV) are human parvoviruses that require helper function for their replication. Several studies have demonstrated that AAV DNA sequences can be found in the female genital tract but the incidence of infection seems very variable. A PCR-ELISA method detecting AAV DNA was developed for combining the specificity and the sensitivity of conventional PCR with an objective interpretation of the results. In the PCR-ELISA, a defined number of cells from cervical specimens were digested and amplified with concomitant digoxigenin labeling. Digoxigenin-labeled amplified products hybridized to a specific biotinylated probe were captured in streptavidin-coated microtiter wells by a biotin-streptavidin binding and were visualized by colorimetric immunoenzymatic reaction. PCR-ELISA was carried out in 110 cervical cytological specimens of women with or without the concomitant detectable presence of papillomavirus (HPV) DNA and the results were compared with those obtained by conventional PCR followed by dot blot hybridization. When compared to conventional PCR considered as reference standard, PCR-ELISA was found to be 98% sensitive and 96% specific. Out of the total 110 samples examined, 52.7% were positive for AAV DNA by both techniques, demonstrating a high prevalence of AAV infection in the uterine cervix. When analyzing samples with or without the presence of HPV DNA, 63.2 % of the samples were positive for HPV DNA and 41.5% of the samples were negative for HPV proved positive for AAV DNA by both PCR-ELISA and conventional PCR. Hence, PCR-ELISA, which can be completed in 1 day, proved to be a reliable method for an objective detection of AAV DNA in clinical samples. The present study showed a frequent infection of the cervical epithelium with AAV both in the presence and absence of HPV infection.

Identification of an immunodominant peptide in the parvovirus B19 VP1 unique region able to elicit a long-lasting immune response in humans.

The immune response against parvovirus B19 is mainly directed against the two structural proteins, VP1 and VP2. The amino terminal half of the VP1 unique region has been shown to elicit a dominant immune response in humans, more effective than other linear epitopes and also it has been seen to contain significant neutralizing linear epitopes. Three overlapping recombinant peptides corresponding to amino acids 2-40 (VP1-A), amino acids 32-71 (VP1-B), and amino acids 60-100 (VP1-C) of the VP1 unique region were produced by a procaryotic expression system. These peptides were used as antigens in a Western blot assay to detect specific immunoglobulin G (IgG) in serum samples from blood donors of different age groups with documented signs of a past B19 infection. Fragment VP1-C appeared significantly immunodominant over the other peptides, reacting with specific IgG in 86% of serum samples. The fragment VP1-C corresponds to a sequence with a known neutralizing activity and seems able to elicit a long-lasting immune response because specific IgG were present in blood donors of all age groups. VP1-C would therefore appear to be an attractive candidate as a component of a subunit vaccine.

Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA.

AIMS: To investigate the distribution and viral load of the most prevalent high risk human papillomavirus (HPV) types 16, 18, 31, 33, and 45 and low risk HPV types 6 and 11 in a variety of cervical lesions. METHODS: One hundred and seventy six cytological specimens from women with different cervical lesions were investigated. For an accurate standardisation of the sample, cervical cells were counted and a volume of the cell suspension processed by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). Semiquantitative determinations were achieved in relation to an external reference titration curve. RESULTS: HPV DNA was detected in 60.2% of the samples. HPV-16 was the prevalent genotype (57.6%), followed by HPV-33, HPV-31, HPV-6, HPV-18, and HPV-45. HPV-11 was not detected. HPV-16 showed a pronounced increase in prevalence with the evolution of cervical disease. Semiquantitative evaluation of the results showed that only HPV-16 DNA could reach very high values (> 1000 genome copies/cell) and a very high HPV-16 load correlated with the severity of cervical disease. CONCLUSIONS: Only HPV-16 load appears to be associated with the severity of cervical disease.

Differential IgM response to conformational and linear epitopes of parvovirus B19 VP1 and VP2 structural proteins.

The IgM immune response against conformational and linear epitopes of B19 structural proteins VP1 and VP2 was examined in serum samples with a suspect B19 infection to determine the most suitable antigen for use in IgM detection and also to evaluate a possible relationship between the course of B19 infection and the presence of epitope type-specific IgM. The detection of IgM against conformational epitopes was performed by ELISA using undenatured VP1 and VP2 antigens whereas the detection of IgM against linear epitopes was performed by Western blot assays using denatured VP1 and VP2. IgM immune response against VP1 conformational epitopes appeared dominant, being detected in all serum samples positive for specific IgM, whereas IgM against VP2 linear antigen were found less frequently, being identified in less than half of the B19 IgM positive sera. In the examination of the course of infection, IgM against VP1 conformational epitopes appeared in the active phase of B19 infection at the same time and with the same frequency as IgM anti VP2 conformational epitopes and anti linear VP1 epitopes. IgM against VP1 conformational epitopes were seen to be long-lasting because in the recent phase of infection they were still present when other specific IgM were absent. During the active phase of B19 infection, IgM against VP2 linear epitopes were less frequently found than other specific IgM and in the recent phase they underwent a rapid temporal diminution. The data demonstrate that a sensitive B19 IgM test needs to be performed in diagnostic laboratories by ELISA using conformational B19 antigens; Western blot assays can be used only as confirmatory tests using VP1 linear antigens.

Scanning force microscopy study on a single-stranded DNA: the genome of parvovirus B19.

The genome of parvovirus B19 is a 5600-base-long single-stranded DNA molecule with peculiar sequence symmetries. Both complementary forms of this single-stranded DNA are contained in distinct virions and they hybridize intermolecularly to double-stranded DNA if extracted from the capsids with traditional methods, thus losing some of their native structural features. A scanning force microscopy analysis of these double-stranded DNA molecules after thermal denaturation and renaturation gave us the chance to study the possible states that this DNA can assume in both its single-stranded and double-stranded forms. A novel but still poorly reproducible in situ lysis experiment that we have conducted on single virions with the scanning force microscope made it possible to image the totally unpaired state that the single-stranded DNA molecule most likely assumes inside the viral particle. Structural considerations on single molecules offer the opportunity for the formulation of plausible hypotheses on the interaction between the DNA and the viral structural proteins that could prove important for the DNA packaging in the capsid and, possibly, the viral infection mechanisms.

Different patterns of restriction to B19 parvovirus replication in human blast cell lines.

B19 parvovirus can replicate in erythroid progenitor cells and in a small number of human blast cell lines. To better understand and analyze the B19 virus replicative cycle, we performed and compared the infection of bone marrow cells and of different blast cell lines with erythroblastoid and megakaryoblastoid phenotypic characteristics (UT-7, TF-1, M-07, and B1647). Following in vitro infection, B19-specific nucleic acids were characterized with regard to the genome-replicative intermediates, the transcription pattern, and the localization of virus-specific nucleic acids inside infected cells. While all cell lines tested proved to be susceptible to B19 virus infection, two different patterns of restriction to replication of B19 virus were observed. In the first restriction pattern, observed in UT-7 cells, the single-stranded viral DNA was converted to double-stranded replicative intermediates, identical to those found in bone marrow cells, and a full set of viral transcripts were observed. However, replication and transcription were restricted to a small subset of cells, and production of capsid proteins was not detected. In the second restriction pattern, observed in TF-1, M-07, and B1647 cells, the single-stranded viral DNA was not converted to double-stranded replicative intermediates.

Tyramide signal amplification of biotinylated probe in dot-blot hybridization assay for the detection of parvovirus B19 DNA in serum samples.

Highly sensitive assay systems are necessary for large-scale virological screenings. We evaluated the use of tyramide signal amplification (TSA) for biotinylated probe in dot-blot hybridization assay to detect B19 DNA in serum samples. The probe was constructed by PCR and directly labeled with biotin during amplification reaction. The sensitivity of the dot-blot hybridization assay with TSA detection method was evaluated in comparison with a hybridization assay using the direct detection of biotinylated probe by streptavidin-biotin-alkaline phosphatase substrate. The TSA detection was able to detect 1 pg of B19 DNA and proved to be 10-50 times more sensitive than the hybridization assay with the direct detection of biotinylated probe. The analysis of 720 serum samples by TSA detection of biotinylated probe showed that the assay may be a valid diagnostic tool in routine testing of B19 DNA in serum samples.

Immunoreactivity against linear epitopes of parvovirus B19 structural proteins. Immunodominance of the amino-terminal half of the unique region of VP1.

Three peptides corresponding to the 2-100 amino acids of VP1 unique sequence (VP1-F1), to the 99-227 amino acids of VP1 unique sequence (VP1-F2) and to the 237-781 amino acids of VP1 protein common to VP2 (VP1-F3 = VP2) were produced by prokaryotic expression. The three peptides, which span the entire VP1 structural protein of parvovirus B19 and also the entire VP2 protein, were used to evaluate the immunoreactivity against linear epitopes of these fragments in a large number of serum samples taken in different clinical situations with regards to B19 infection and in some commercial preparations of aspecific immunoglobulins. The data demonstrated that the specific VP1-F1 fragment, corresponding to the amino-terminal half of the VP1 unique region, is immunodominant and can elicit a long lasting immune response in comparison with VP1-F2 and VP1-F3 = VP2. Data regarding the presence of specific IgG to the three fragments in commercial preparations of immunoglobulins demonstrated that the dominant immune response was also against VP1-F1 linear epitopes while IgG against VP1-F2 and IgG against VP1-F3 = VP2 could be found only in high concentrations of Ig preparations. The reported data can be useful as a basis for the development of a B19 recombinant vaccine.