Marialuisa Zerbini

Presence of high-risk mucosal human papillomavirus genotypes in primary melanoma and in acquired dysplastic melanocytic naevi

Summaryu2002 Backgroundu2002 Some studies have shown that cutaneous and mucosal melanoma biopsy specimens harbour human papillomavirus (HPV), suggesting that this virus may play a role in development and progression of the tumour.

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.

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.

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.

Assessment of the presence of mucosal human papillomaviruses in malignant melanomas using combined fluorescent in situ hybridization and chemiluminescent immunohistochemistry

Backgroundu2002 The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high‐risk (HR) HPV genotypes in primary melanoma by PCR.

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

BACKGROUND AND OBJECTIVESnHigh-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.nnnSTUDY DESIGNnthe 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.nnnRESULTSnprotease 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.nnnCONCLUSIONSnHC-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.

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.

Persistent parvovirus b19 infection resulting in carpal tunnel syndrome

A woman showing chronic arthropathy after a parvovirus B19 infection was followed for 1 year with quantitative determination of parvoviral DNA, and specific immunoglobulin (Ig)M and IgG in blood. B19 arthopathy resulted in carpal tunnel syndrome, needing surgery, after 8 months of persistent B19 viraemia, when IgM was already cleared but IgG values were still high.nnCarpal tunnel syndrome (CTS) is the most common peripheral compression neuropathy, but many aspects of its aetiology are not at all clear. CTS is often termed as idiopathic but it has also been attributed to a variety of underlying disorders and processes, and sometimes CTS has been reported as secondary to infectious diseases of bacterial, mycotic and viral origin, including parvovirus B19. Parvovirus B19 in humans can cause acute infections but also persistent infections with continuous virus production both in immunocompetent and in immunosuppressed individuals.1 Parvovirus B19 is generally associated with erythema infectiosum, arthritis …

Human papillomavirus in melanoma: reply from authors

SIR, We are grateful to Dr Guerrini and colleagues for their interest in our study and we take this opportunity to clarify our opinions. We demonstrated the presence of high-risk mucosal human papillomavirus (HPV) genotypes from a considerable number of dysplastic naevi and primary melanomas using two different polymerase chain reaction (PCR)–enzyme-linked immunosorbent assay methods, concluding that the presence of the virus may be a cofactor in development and progression of these pathologies. We agree with other colleagues that our results need further evaluation. In particular, PCR is a highly sensitive technique, able to detect the presence of total DNA from tissue samples at a level of about one genome per cell. All previous studies aimed to detect HPV DNA in skin cancer using PCR procedures were in most cases unable to conclude that the DNA detected was entirely derived from cancer cells. In fact, PCR procedures are not suitable to ascertain if the HPV DNA detected is derived exclusively from cancer cells, but rather if its presence is due to a tumour surface contamination. On the other hand, different methods, including immunohistochemistry (IHC) and in situ hybridization (ISH), have been demonstrated as more specific, but less sensitive than PCR methods. On the basis of these considerations, we are submitting for publication a second part of our studies, concerning the evaluation of the presence of mucosal high-risk HPV in primary melanoma and its colocalization with a tumoral melanocytic marker in the same section, using a very sensitive method that combines an enzyme-amplified fluorescent ISH with a chemiluminescent IHC method for the detection of the tumoral melanocytic marker HMB-45. The antimelanoma monoclonal antibody HMB-45 used in the chemiluminescent IHC is widely used in diagnostic pathology owing to its great specificity and sensitivity in identifying pigmented tumours such as malignant melanoma, while normal melanocytes are unreactive.