Biomedical subjects

M Zerbini

Publications and source records attributed to M Zerbini.

At least 73 records · Page 4Linked to original sources

PubMed1993

Nested polymerase chain reaction assay for the detection of B19 parvovirus DNA in human immunodeficiency virus patients.

Persistent B19 parvovirus infection has been recognized in immunocompromised patients, often occurring with a low-titer viremia. In this study, nested polymerase chain reaction (PCR) for the detection of B19 parvovirus DNA was carried out on the sera of 49 human immunodeficiency virus (HIV)-1-seropositive patients, negative for the detection of B19 DNA at dot blot hybridization assay and with different values of serum anti-B19 IgM (27 patients proved positive and 22 negative). Of the 49 HIV-seropositive samples tested by nested PCR, seven were positive for the detection of B19 DNA. All seven belonged to the group of subjects seropositive for specific anti-B19 IgM. The study shows that, in the presence of specific B19 IgM, circulating virus may still be present but can be detected only by PCR. In that B19 infection can occur with low-titer viremia in immunocompromised patients, PCR may be the only method for virus detection.

Adult↗

PubMed1993

In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labelled probe.

An in situ hybridization assay using a digoxigenin-labelled probe was developed to detect B19 DNA in bone marrow erythroid elements of immunodeficient patients with hypoplastic anaemia. A 700 bp Bam HI-Hin dIII fragment of B19 DNA was used to construct the probe by incorporating deoxyuridine triphosphate labelled with digoxigenin. The in situ hybridized B19 DNA probe was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. Dark blue coloured inclusions at the enzyme site were detected in the nuclei of B19 infected erythroid cells at different stages of cell differentiation. Six out of the nine patients studied showed a positive reaction by in situ hybridization assay. The assay we developed proved highly specific and sensitive and it appears to be a suitable diagnostic test for investigating the possible role of B19 infection as a cause of haematopoietic disorders in immunocompromised hosts.

Anemia, Aplastic↗

PubMed1993

Microplate capture hybridization of amplified parvovirus B19 DNA fragment labelled with digoxigenin.

A capture hybridization technique in microplate has been developed for the identification of polymerase chain reaction (PCR) amplified B19 DNA fragment in clinical specimens. The amplified 104 bp B19 DNA fragment, located in the gene coding for structural proteins, was directly labelled during the amplification reaction by incorporation of digoxigenin-labelled dUTP. The amplified product was then captured by a probe immobilized on microplate wells. The capture hybridization reaction was visualized as an enzyme-linked immunosorbent assay using anti-digoxigenin Fab fragment labelled with peroxidase. Thirty-five serum samples were tested by our capture hybridization assay and the results were in accordance with the results obtained by Southern blot analysis of PCR amplified product. Our microplate capture hybridization assay showed a high sensitivity and reproducibility and appears to be a practical and reliable test for routine screening of B19 parvovirus DNA in clinical specimens.

Base Sequence↗

PubMed1993

Efficient parvovirus B19 DNA purification and molecular cloning.

A simple and efficient method of purification and molecular cloning of Parvovirus B19 DNA directly from small quantities of viremic sera was developed. Purified virions were lysed in annealing conditions, then viral DNA purification in double strand (ds) DNA form was achieved using an affinity DNA binding matrix. Affinity purification yielded a consistently high recovery of viral DNA. Using affinity purified ds viral DNA, we efficiently and stably cloned the complete coding internal unique sequence of B19 DNA. In our cloning strategy AatII and BamHI restriction endonuclease sites were exploited. This permitted cleavage of the 5.0 kbp AatII fragment in two AatII-BamHI fragments which could be efficiently cloned in a directional way in pUC18 plasmid vector. The availability of the two cloned AatII-BamHI fragments thus allowed the construction of a full length clone in a single ligation reaction.

Cloning, Molecular↗

PubMed1993

Symptomatic parvovirus B19 infection of one fetus in a twin pregnancy.

Twin pregnancies complicated by infection due to parvovirus B19 are uncommon. We report a case in which only one fetus developed a symptomatic infection, which presented at first as ascites and pleural effusion; later, meconium peritonitis developed. Hydrops spontaneously resolved, and at birth meconium peritonitis was successfully treated with surgery. However, even with the positive outcome of this pregnancy, a long-term follow-up is needed to exclude damage other than injury to the erythropoietic system.

Adult↗

PubMed1993

In situ hybridization at the ultrastructural level: localization of cytomegalovirus DNA using digoxigenin labelled probes.

We describe a procedure for detecting and localizing cytomegalovirus DNA sequences based on in situ hybridization at the ultrastructural level. A digoxigenin-labelled probe, identified with an anti-digoxigenin colloidal gold-labelled antiserum, was employed on infected cells embedded in a new acrylic resin (Bioacryl). The silver enhancement method on the same specimen was used to more easily reveal the reaction also at low magnification. The immunolocalization was characterized by high specificity with virtually no background staining and a good maintenance of submicroscopic cell features.

Acrylic Resins↗

PubMed1992

Different syndromes associated with B19 parvovirus viraemia in paediatric patients: report of four cases.

The broad spectrum of clinical manifestations associated with B19 parvovirus often allows the infection to go unrecognized. We tested for the presence of B19 parvovirus and the specific serological response in serum from hospitalized patients submitted for viral investigations without any specific indications for B19 parvovirus. We diagnosed human parvovirus B19 infection in four paediatric patients showing different clinical manifestations. The patients, aged between 5 and 8 years, were admitted to hospital for: (1) petechial rash; (2) mononucleosis-like syndrome; (3) neurological syndrome; and (4) respiratory disease (in an immunodeficient patient).

Child↗

PubMed1992

Detection of CMV DNA in clinical samples of AIDS patients by chemiluminescence hybridization.

A sensitive chemiluminescence dot-blot hybridization assay for the detection of CMV DNA in clinical samples of AIDS patients is described. In the chemiluminescence hybridization assay, digoxigenin-labelled CMV DNA probes were used and when hybridized they were detected by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. Adamantyl 1,2-dioxetane phenyl phosphate was used as the chemiluminescent substrate. The results were recorded by instant photographic films. The results obtained with the chemiluminescence hybridization assay were compared with the results obtained by hybridization with colourimetric detection. The chemiluminescent assay proved specific, sensitive and reliable and thus can be used as a valuable routine diagnostic test for the detection of CMV DNA in clinical samples.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Chemiluminescent assay for the detection of viral and plasmid DNA using digoxigenin-labeled probes.

A dot-blot hybridization immunoenzymatic assay with a chemiluminescent endpoint was developed for the rapid and sensitive detection of viral and plasmid DNAs. Digoxigenin-labeled probes were used to detect cytomegalovirus, parvovirus B19, and plasmid pBR328 DNAs. Hybridized probes were immunoenzymatically visualized by anti-digoxigenin Fab fragments labeled with alkaline phosphatase, and adamantyl 1,2-dioxetane phenyl phosphate was used as chemiluminescent substrate. Results were recorded by instant photographic films. The chemiluminescent hybridization assay was performed in about 8 hr and was able to detect as little as 50-10 fg of homologous target DNA.

Cytomegalovirus↗

PubMed1991

Chemiluminescence dot blot hybridization assay for detection of B19 parvovirus DNA in human sera.

A chemiluminescence dot blot hybridization assay was used for the detection of B19 parvovirus DNA in human sera by using digoxigenin-labeled probes. The probes were revealed immunoenzymatically by use of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The chemiluminescence signal was obtained by reacting the labeled probe-target complex with an enzyme-triggerable dioxetane substrate. The emitted photons were detected with instant photographic films. In the search for B19 parvovirus DNA, 2,808 serum samples were analyzed.

DNA Probes↗

PubMed1991

5-Azacytidine induction of a cellular heat shock protein and expression of the major immediate early protein of human cytomegalovirus in Vero cells.

Human cytomegalovirus can support an abortive infection in Vero cells and only some immediate early events are expressed in a low proportion of the cell population. It has been shown that cellular factors partially remove blocks in cells abortively or latently infected by HCMV, whereas 5-azacytidine is known to act on gene expression and cell differentiation. In this work we present evidence that the treatment of HCMV infected Vero cells with 5-azacytidine induces the expression of the major immediate early protein (68K) of Human cytomegalovirus by enhancing a 72K cellular heat shock protein.

Animals↗

PubMed1990

In situ detection of cytomegalovirus DNA in biopsies of AIDS patients using a hybrido-immunocytochemical assay.

An in situ hybrido-immunocytochemical assay, with a digoxigenin-labelled probe, was used to show the presence of cytomegalovirus DNA in both paraffin and frozen sections from tissue blocks of 5 AIDS patients. The hybridization probe was constructed by using two different DNA fragments of the repeated sequences of the CMV genome. The CMV DNA probe hybridized in situ was immunocytochemically visualized by anti-digoxigenin Fab fragments labelled with alkaline phosphatase. This hybridization procedure proved to be sensitive, specific, and provided good resolving power. Thus, it might effectively be employed in immunohistological and virological laboratories for the diagnosis of CMV infections in AIDS patients; indeed it might even be applied further in the virological context.

Acquired Immunodeficiency Syndrome↗

PubMed1990

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.

Adolescent↗

PubMed1990

Rapid screening for B19 parvovirus DNA in clinical specimens with a digoxigenin-labeled DNA hybridization probe.

A rapid dot blot hybridization assay for the detection of B19 parvovirus DNA in human sera was developed. Small portions of four serum samples were mixed, filtered onto a nylon membrane, and hybridized with a digoxigenin-labeled DNA probe; for each membrane, 380 serum samples could be tested. When a dot was positive by the hybridization assay, the four serum samples dotted together were separately tested to identify the sample positive for B19 DNA. A total of 10,150 serum samples submitted for viral serological and laboratory investigation with no specific requests for B19 testing were analyzed. Nine serum samples were positive for B19 DNA by dot blot hybridization assay, and the results were confirmed by electron microscopy. This method has proven to be reliable, economical in terms of time and costs, and useful for large-scale screening of clinical specimens, both for diagnostic work and for a source of antigen.

DNA Probes↗

PubMed1990

Rapid detection of cytomegalovirus DNA in urine samples with a dot blot hybridization immunoenzymatic assay.

A dot blot hybridization immunoenzymatic assay for the rapid detection of cytomegalovirus DNA in urine samples was developed by using a digoxigenin-labeled probe which was immunoenzymatically visualized by antidigoxigenin Fab fragments labeled with alkaline phosphatase. A total of 516 urine samples from different groups of subjects were analyzed, and the hybridization assay was able to yield results within 24 h. The results obtained were compared with results for detection of cytomegalovirus antigens in infected cell cultures.

Antigens, Viral↗

PubMed1989

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes.

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.

Cells, Cultured↗

PubMed1989

Immunological status to Epstein-Barr virus and cytomegalovirus in patients with genital condylomata.

Serological patterns against Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) were determined in patients with genital condylomata (GC). The Ig G antibody values to EB-induced virus capsidic antigens (VCA), early antigens (EA) and Ig M to VCA were significantly higher in the study group than in the controls. Moreover, the concomitant presence of EBV-Ig G anti-VCA greater than or equal to 1/320, EBV-Ig G anti-EA greater than or equal to 1/20 and EBV-Ig M anti VCA greater than or equal to 1/20 was observed in 13 serum samples of genital condylomata patients, while, in only 2 serum samples of the healthy controls, the same serological pattern was present. The distribution of antibody values to CMV-induced LA, EA and IEA showed a significantly increased prevalence in the study group in comparison with the controls: the concomitant presence of antibody with a titre greater than or equal to 1/320 for CMV-LA, greater than or equal to 1/20 for CMV-EA and greater than or equal to 1/20 for CMV-IEA was observed in 15 serum samples of GC patients and in only 3 serum samples of the control group. Our results suggest that the active or recent EBV and CMV infections we observed in genital condylomata patients may be a consequence of impaired immunity in these patients, but it does not exclude a possible role of EBV and CMV in perpetuating human papilloma virus-induced cell proliferation.

Adult↗