Simona Venturoli

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.

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.

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.

Human papillomavirus DNA testing by PCR-ELISA and hybrid capture II from a single cytological specimen: concordance and correlation with cytological results.

BACKGROUND AND OBJECTIVES A persistent infection by high-risk HPV is now considered as the major cause of cervical carcinoma. The use of a single cytological specimen for HPV DNA testing by two different molecular methods was analyzed and validated. STUDY DESIGN HPV DNA testing by PCR-ELISA and hybrid capture II HPV test (HC-II), was investigated on 317 cytological samples obtained from Italian women. Two hundred twenty-seven women were referred to virological lab for HPV DNA testing during cytological routine screening and 90 during a cytological and virological follow-up after a conization or hysterectomy. RESULTS Overall, the concordance between the two assays was high (K=0.87). Compared with PCR-ELISA, the HC-II showed a sensitivity of 91.7% and a specificity of 95.4%. Although the analytical sensitivity of the PCR-ELISA was higher, the performance of the two tests did not differ in recognizing HPV DNA positive patients with either low or high-grade squamous intraepithelial lesions (LSIL or HSIL). HPV DNA positivity was directly correlated with the severity of cytological diagnosis (P<0.005). CONCLUSIONS In view of the comparable results obtained with the two assays and of the ease of use, and higher throughput of HC-II, it seems advisable, with a single cytological specimen, to employ the HC-II test as a first-line assay, either for screening or diagnosis, and to perform reflex PCR on positive samples, if typing of prevalent high risk HPVs is needed.

Occurrence and Clinical Role of Active Parvovirus B19 Infection in Transplant Recipients

Abstract To evaluate the occurrence and clinical role of active parvovirus B19 infection in solid organ and bone marrow transplant recipients, 256 serum samples from 212 transplant patients were investigated retrospectively by competitive polymerase chain reaction. Sera were drawn during the transplantation period and up to 6 months after transplantation during a nonepidemic 1-year period. Three patients were found positive for B19 DNA; only one liver transplant patient had a clinically overt B19 infection. Overall, the rate of active parvovirus B19 infection in transplant subjects was low (1.42%), probably due to the high number of actively or passively immunized subjects among transplant recipients; this may also account for the asymptomatic infections observed.

Standardization of a PCR-ELISA in Serum Samples: Diagnosis of Active Parvovirus B19 Infection

To standardize a PCR assay for the detection of parvovirus B19 DNA in serum samples three different sample treatments were evaluated on the basis of the efficiency of recovery, reproducibility, convenience of sample handling, and presence of PCR inhibitors. Moreover, the presence of an internal standard competitor as the working reagent at one defined concentration in a competitive PCR‐ELISA has been suggested as a valid tool to standardize and validate the assay. The results indicated that serum sample treatment by rapid heating fulfilled the criteria for a routine practice in the diagnostic laboratory. Titration experiments carried out to define the optimal amount of the internal standard competitor to use in PCR‐ELISA showed that at 2 ×102 competitor copies, any amplification interferences between target and competitor sequences were avoided. The internal standard competitor in a competitive PCR‐ELISA allows the detection of false‐negative results due to PCR inhibitors in the samples or large amounts of target DNA. Heating treatment and competitive PCR‐ELISA for the detection of parvovirus B19 DNA were applied to the testing of 347 serum samples, which were submitted to the laboratory for B19 investigation. Of the 34 serum samples that were positive for B19 DNA, 15 were from adult patients and 19 from pediatric subjects. B19 infection was associated with haematological disorders, nonimmunological foetal hydrops, atypical rash, arthropathies, hepatic dysfunction, nonspecific symptoms, and congenital infections. J. Med. Virol. 59:239–244, 1999.

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.