Monica Musiani

Active or recent parvovirus B19 infection in children with Kawasaki disease

Because parvovirus B19 occasionally causes some of the features typical of Kawasaki disease, we investigated B19 involvement in 15 children with Kawasaki disease. Active or recent B19 infection, as shown by B19-DNAaemia, positive B19-specific IgM antibodies, or both, was diagnosed in 10 patients (67%). A high frequency of all major criteria for diagnosis of Kawasaki disease (60%), anaemia (60%), coronary aneurysms (30%), and arthropathy (30%) was found in children with B19-associated Kawasaki disease. Thus B19 may have a pathogenic role in the development of Kawasaki disease, with other predisposing factors.

Factors predicting human papillomavirus clearance in cervical intraepithelial neoplasia lesions treated by conization

OBJECTIVE The objective was to identify the factors, if any, that may predict long-term results of CIN treatment and HPV clearance/persistence after locally excisional therapy. METHODS A series of 252 women with CIN lesions treated by conization were subjected to sequential HPV detection by repeated PCR during the prospective posttreatment follow-up. Factors predicting viral clearance during the follow-up (10.26 months) were elaborated using univariate and multivariate statistical techniques applied on epidemiological, clinical and biological data of the lesions. RESULTS Sensitivity of the PAP test in detecting high-grade lesions was 93.9%, and specificity 27.3%. Odds ratio for having CIN 3/Stage IA1 squamous cervical cancer in the cone with HSIL PAP test was 5.69; 77.8 and 22.2% residual disease were found among PCR-positive and -negative cases, respectively. HPV DNA was negative in 74/252 (29.8%) samples at the first PCR. Multivariate logistic regression analysis showed that HPV 16 was an independent explanatory factor for high-grade CIN (P = 0.0001). HPV clearance increased to 63.5% at completion of the follow-up, corresponding to the monthly clearance rate of 5.27%. In Kaplan-Meier analysis, the highly significant (P = 0.0001) predictors of HPV clearance/persistence were age, lesion grade in the biopsy, lesion grade in the cone, volume of the cone, length of active sexual life, and involvement of endocervical margin (P = 0.0013). In chi-square tests, high-risk HPV type (P = 0.001) was such a predictor. In multivariate (Cox) model, the significant independent predictors of HPV clearance were involved endocervical margin (P = 0.001), lesion grade in the cone (P = 0.004), high-grade lesion in the colposcopic biopsy (P = 0.023), age (P = 0.029), and HSIL in PAP smear (P = 0.029). CONCLUSIONS These data suggest that posttreatment follow-up should include both the PAP test and HPV detection techniques for early detection of any patients at increased risk for disease recurrence and progression, because of persistent oncogenic HPV types.

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.

Chemiluminescent Low-Light Imaging of Biospecific Reactions on Macro- and Microsamples Using a Videocamera-Based Luminograph

The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 μm. Other applications, such as the alkaline phosphatase localization in 8 μm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigenin-labeled probes and alkaline phosphatase-labeled anti-digoxigenin antibody, using chemiluminescent substrate for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 μm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.

B19 virus genome diversity: epidemiological and clinical correlations

Genetic analysis of parvovirus B19 has been carried out mainly to establish a framework to track molecular epidemiology of the virus and to correlate sequence variability with different pathological and clinical manifestations of the virus. A good amount of information regarding B19 virus sequence variability is available, and presently there are about 400 sequence records deposited in the nucleotide database of NCBI. A few are almost complete genomic sequences, and these allow the construction of a global alignment framework. Many others are partial genomic sequences, limited to selected regions, and these allow comparison of a higher number of isolates from well-defined epidemiological settings and/or pathological conditions. Most studies showed that the genetic variability of B19 virus is low, that molecular epidemiology is possible only on a limited geographical and temporal setting, and that no clear correlations are present between genome sequence and distinctive pathological and clinical manifestations. More recently, several viral isolates have been identified that show remarkable sequence diversity with respect to reference sequences. The identification of variant isolates added to the knowledge of genetic diversity in this virus group and allowed the identification of three divergent genetic clusters, about 10% divergent from each other and still quite distinct from other parvoviruses, that can be thought of as different genotypes within the human erythrovirus group and that show clearly resolved phylogenetic relationship. These variant isolates pose interesting questions regarding the real extent of genetic variability in the human erythroviruses, the relevance of these viruses in terms of epidemiology and their possible implication in the pathogenesis of erythrovirus-related diseases.

Detection of B19 parvovirus infections by a dot-blot hybridization assay using a digoxigenin-labelled probe

A non-radioactive dot-blot hybridization assay for the detection of B19 parvovirus infections was developed using a digoxigenin-labelled probe both on nylon and nitrocellulose filters. A 700 bp BamHI HindIII fragment of B19 DNA was used to construct the probe. Probe labelling was carried out by incorporating deoxyuridine triphosphate labelled with digoxigenin. The dot-blot hybridization assay was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. The specificity and sensitivity of digoxigenin-labelled B19 DNA probe was compared with the results obtained with 32P-labelled B19 DNA probe. Out of the 504 serum samples tested, 3 samples were positive in all the hybridization assays performed and 494 were negative, 7 serum samples gave a weak positive reaction when Dig-B19 probe was used on nitrocellulose filters. The 77 pharyngeal swabs tested were negative in all the hybridization assays performed. Our hybridization assay showed a high sensitivity and reproducibility and it appears to be a rapid, practical and reliable test for routine screening of B19 parvovirus DNA in large numbers of clinical specimens.

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. J. Med. Virol. 80:1434–1440, 2008.

IgG immune response to B19 parvovirus VP1 and VP2 linear epitopes by immunoblot assay

Human B19 parvovirus recombinant capsid proteins VP1 and VP2 were expressed in E. coli and purified. Recombinant proteins were used to detect a specific IgG immune response against VP1 and VP2 linear epitopes by immunoblot assay. A total of 222 serum samples from 218 apparently immunocompetent subjects with different clinical conditions and laboratory evaluations with regards to B19 infection were analyzed. The sera had previously been tested for B19 DNA and for specific IgM and IgG against VP2 conformational antigens by ELISA assay. The data show that, during the active or very recent phase of infection, IgG anti‐VP1 linear epitopes appear in concomitance and with the same frequency as IgG anti‐VP2 conformational antigens. IgG against conformational VP2 antigens and against linear VP1 epitopes seem to persist for months or years in the majority of individuals. IgG against VP2 linear epitopes are generally present during the active or very recent phase of infection and during the convalescent phase, while they are present only in about 20% of subjects with signs of a past B19 infection. J. Med. Virol. 57:174–178, 1999.

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.

Intra-uterine parvovirus B19 infection and meconium peritonitis

B19 fetal infection has been associated with hydrops or fetal death. We report four cases of meconium peritonitis in hydropic fetuses with laboratory diagnosis of B19 infection.