PubMed Health⌕ Search

Biomedical subjects

M Musiani

Publications and source records attributed to M Musiani.

At least 19 recordsLinked to original sources
PubMed2007

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

BACKGROUND: 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. OBJECTIVES: To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells. METHODS: We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis. RESULTS: The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60-80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity. CONCLUSIONS: The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.

Adult↗
PubMed2005

Presence and type of oncogenic human papillomavirus in classic and in differentiated vulvar intraepithelial neoplasia and keratinizing vulvar squamous cell carcinoma.

The presence and type of oncogenic papillomavirus (HPV) in classic warty/basaloid vulvar intraepithelial neoplasia and in differentiated vulvar intraepithelial neoplasia and keratinizing vulvar squamous cell carcinoma was investigated using three techniques, that is, histology, in situ hybridization, and PCR-ELISA. HPV typing was performed using in situ hybridization and PCR-ELISA. Differentiated vulvar intraepithelial neoplasia and invasive keratinizing vulvar squamous cell carcinoma proved completely negative for HPV by PCR-ELISA, in situ hybridization, and histologic examination, while in classic vulvar intraepithelial neoplasia, a HPV positivity of 66.1% was found. HPV 16 was the predominant type, with HPV 35, 33, and 52 types found rarely and sometimes together with HPV 16. PCR-ELISA proved to be the most suitable method to detect and type mucosal oncogenic HPVs. The absolute absence of HPV DNA in differentiated vulvar intraepithelial neoplasias and in keratinizing vulvar squamous cell carcinoma suggests a strong HPV-independent pathway of malignant progression to invasive carcinoma.

Adult↗
PubMed2005

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

BACKGROUND: 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. OBJECTIVES: To investigate the presence of HPV DNA and the prevalence of different high-risk mucosal HPV genotypes in primary melanoma (PM) and in acquired dysplastic melanocytic naevi (ADMN). METHODS: Fifty-one PMs from 18 men and 33 women (median age 55.5 years), 33 ADMN from 15 men and 18 women (median age 35.1 years) and 20 control skin samples from nine men and 11 women (median age 43.5 years) were studied. All diagnoses were made after histological analysis. HPV DNA analysis was made using two different polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) methods, namely MY-PCR and GP-PCR. RESULTS: Using GP-PCR, mucosal HPVs were detected in 14 PMs (27%; P = 0.0166) and eight ADMN (24%; P = 0.0367), while with MY-PCR, mucosal HPVs were found in 11 PMs (22%; P = 0.04) and five ADMN (15%; P not significant). All control skin samples were negative for mucosal HPVs with both DNA amplification procedures. CONCLUSIONS: Using our PCR-ELISA methods, the detection of mucosal high-risk HPV genotypes in 24% of precursor lesions (ADMN) and in 27% of PMs adds to the body of evidence indicating a colocalization of mucosal HPV and tumoral melanocytic pathologies.

Adolescent↗
PubMed2004

Antibody response to B19 parvovirus VP1 and VP2 linear epitopes in patients with haemophilic arthritis.

Parvovirus B19 infection occurs very frequently in patients with haemophilia on account of its transmission with plasma derivatives. In order to achieve a more defined serological pattern for the study of the role of B19 infection in haemophilic arthritis, 53 serum samples from 37 patients with haemophilic arthritis were investigated for the presence of IgG immune response against B19 VP2 and VP1 linear epitopes and VP2 conformational antigen compared to the serological reactivity against B19 NS1 and to the presence of B19 DNA in the synovial membranes. An IgG immune response against VP1 and VP2 linear epitopes was detected by immunoblot assay using recombinant proteins expressed in Escherichia coli. Specific IgG against VP2 and VP1 linear epitopes were present in 84.90 and 92.45% of haemophilic arthritis patients and in 28.0 and 64.0% of the controls (P<0.001) respectively. All 53 sera of the haemophiliacs (100%) and 66.0% of the controls (P<0.001) were IgG positive and IgM negative against VP2 structural epitopes. Specific IgG against VP2 linear epitopes, which are a serological marker of active or very recent B19 infection, proved to be significantly associated with the presence of anti-NS1 antibodies and with the presence of B19 DNA in synovial tissue in patients with haemophilic arthritis. In conclusion, in these patients the presence of B19 IgG anti-VP2 linear epitopes, in absence of IgM anti-VP2 structural antigens, can be a useful serological marker to diagnose active, recent or persistent B19 infection.

Adolescent↗
PubMed2004

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.

Antibodies, Viral↗
PubMed2004

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.

Biopsy↗
PubMed2004

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.

Adolescent↗
PubMed2003

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.

Antibodies, Viral↗
PubMed2002

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.

Capsid Proteins↗
PubMed2002

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.

Antibodies, Viral↗
PubMed2002

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.

Blood Donors↗
PubMed2001

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.

Capsid↗
PubMed2001

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.

Adolescent↗
PubMed2001

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.

Cervix Uteri↗
PubMed2001

A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples.

A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described, using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR, hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step, using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision, Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate, the EnVision system was able to detect 10 fg of B19 DNA, while with the chemiluminescent substrate the sensitivity increased by up to 2 fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe, which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision detection system was used to analyse 760 serum samples; the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory.

DNA Probes↗
PubMed2001

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.

Adult↗
PubMed2001

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.

Adult↗