Tino F. Schwarz

Immunoblot detection of antibodies to Toscana virus

Sera from patients with sandfly fever caused by Toscana virus (TOSV) infection were tested by immunoblot for specific antibody response to TOSV derived from infected Vero‐E6 cells. The 28 kDa TOSV nucleoprotein (N) was identified as the major immunodominant protein recognized by immunoblot. In sera of patients with acute TOSV infection, specific antibodies of the IgM, IgA, and IgG class were detected. Using sandfly fever virus, serotypes Sicilian (SFSV) and Naples (SFNV), as antigens for immunoblot, TOSV antibody‐positive sera cross‐reacted with the corresponding N proteins. These sera reacted for IgM and IgG by SFSV immunoblot, and for IgM by SFNV immunoblot. The diagnosis of sandfly fever may be confirmed by TOSV immunoblot.

Acute parvovirus B19 infection mimicking myelodysplastic syndrome of the bone marrow

SummaryA 36-year-old, previously healthy woman was referred to our institution with pancytopenia and splenomegaly for suspected acute leukemia. Bone marrow aspiration showed marked dysplastic changes, excess of blasts, and only spurious red blood cell precursors. Action was taken to prepare allogeneic bone marrow transplantation from an HLA identical sibling for myelodysplastic syndrome. Repeat cytological examination of the bone marrow revealed striking hyperplasia of the red cell line with presence of abnormal giant proerythroblasts. Acute parvovirus B19 infection was suspected and confirmed by detection of anti-B19 IgM and B19 DNA. The underlying disease for this transient aplastic crisis was a formerly unknown hereditary spherocytosis.

Productive infection of in vitro generated haemopoietic progenitor cells from normal human adult peripheral blood with parvovirus B19: studies by morphology, immunocytochemistry, flow-cytometry and DNA-hybridization.

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19‐infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3‐ and CD14‐poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhll‐3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19‐containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow‐cytometry, demonstrating a selective inhibition of GlyA‐positive cells following B19‐inoculation. Typical morphological changes were observed on cytocentrifuge‐spots, and typical giant‐cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19‐DNA increased by about x 100 after 72 h of culture.

Heat stability of parvovirus B19 : kinetics of inactivation

Heat inactivation of parvovirus B19 (B19) was studied in a culture of hematopoietic progenitor cells generated in vitro from peripheral human blood. After inoculating cell cultures with identical volumes of plasma (MII) containing B19 (B19-MII) heat-treated (60 degrees C) for various periods of time, a time-dependent inactivation of the input virus was determined by a decrease of viral DNA replication. No B19 DNA was detected after infection with B19-MII heat-treated for 20 min or more by Southern blot. Viral B19 protein production decreased time-dependently and was not detected after infection with samples treated for 12 min at 60 degrees C or more determined by the enzyme immunoassay. This study indicates that infectivity of B19 virus in plasma can be reduced in vitro by heat-treatment (60 degrees C). However, this does not mean that the heat treatment completely inactivated B19 virus.

Diagnosis of Human Parvovirus B19 Infections by Polymerase Chain Reaction

The polymerase chain reaction (PCR) was used for detecting parvovirus B19 DNA in clinical specimens. A pair of oligonucleotide primers spanning the PstI-fragment of the B19 virus genome was used for PCR, and a PCR product of 727 bp was amplified. B19 virus DNA was detected in all sera (n = 26) of individuals in the incubation period and acute phase of infection. PCR was useful for detecting viral B19 DNA in amniotic fluid and fetal blood of hydropic fetuses, confirming fetal B19 virus infection.

Comparison of Seven Commercial Tests for the Detection of Parvovirus B19-Specific IgM

Several enzyme immunoassays (EIA) for the detection of parvovirus B19 IgM (anti-B19 IgM) are now commercially available. In this study, seven commercial EIAs (Biotrin, DAKO, Viramed, Viratech, R-Biopharm, Mast) were compared with an in-house EIA (MvP-EIA) using native viral B19 particles and the reference IgM radioimmunoassay (MACRIA). A total of 88 sera were tested. Results agreed in 39/88 (44.3%) sera, whereas 47/88 (53.4%) were discrepant and 2/88 (2.3%) gave an equivocal result. Assay sensitivity ranged from 70.3 to 100% and specificity, from 75.9 to 100%. The best results were obtained with two EIAs (Biotrin, DAKO) using baculovirus-expressed B19 proteins as antigen. This study has shown that baculovirus-expressed B19 antibody tests are suitable tools for detecting anti-B19 IgM.

Prevalence of antibodies against hepatitis A virus, hepatitis B virus, and treponema pallidum in mauritius

A seroepidemiological study on the prevalence of antibodies against hepatitis A virus (HAV), hepatitis B virus (HBV) and Treponema pallidum was conducted in various groups of the population of the state of Mauritus (Islands of Mauritus and Rodrigues). 618 sera were tested. The overall prevalence of anti-HAV was 86.1% and yielded and age-dependent increase. Serological evidence for acute or chronic HBV infection was found in 3.8%; 4.5% were positive for anti-HBc alone, and in 12.6% past HBV infection was detected. No age- or sex-dependent increase in the prevalence of anti-HBc was found. There were differences in the anti-HBc prevalence among the various groups of population ranging from 5.9 (flight personnel) to 58.3% (prison inmates). Treponemal antibodies were detected in 6.0% and showed a fairly marked age-dependent increase. Our study suggests that vaccination programmes against HAV and HBV would be beneficial for the Mauritian population.

Das humane Parvovirus B19 und seine klinische Bedeutung

ZusammenfassungDas humane Parvovirus B19 (B19) verursacht das Erythema infectiosum (Ringelröteln) und aplastische Krisen. In der Schwangerschaft kann B19 auf den Feten übertragen werden, wodurch es zum Hydrops fetalis kommen kann. In seltenen Fällen werden B19assoziierte Thrombozytopenien oder Vaskulitiden beobachtet. Bei Immunsupprimierten kann B19 eine persistierende Infektion verursachen. Für die Diagnostik stehen heute verschiedene Methoden zum Virus- sowie zum Antikörpernachweis zur Verfügung. Mit Ausnahme der chronischen Verläufe, bei denen sich Immunglobulin oft als wirksam erweist, bedarf die B19-Infektion meist keiner Therapie.SummaryHuman parvovirus B19 causes erythema infectiosum (fifth disease) and aplastic crisis. In pregnancy, B19 may be transmitted on to the fetus resulting in hydrops fetalis. B19 is sometimes associated with thrombocytopenia and vasculitis. In immunocompromised patients, B19 may persist causing chronic infection. Several methods for detecting B19 virus or specific antibodies are now available. B19 infection does not usually require therapy. Efficacy of immunoglobulins in chronic B19 infection has been demonstrated.

Detection of parvovirus B19 specific antibodies and DNa in sera of hemophiliacs

important. PCR complements traditional serological and other detection methods by allowing discrimination between and detection of specific nucleic acid sequences. In viral hepatitis for example, PCR is used to distinguish between viremic and non-viremic infection, and to monitor viral replication in patients undergoing interferon therapy. However, PCR analysis of biological samples such as blood and other body fluids, is often unreliable due to the presence of amplification inhibitors. These inhibitors are often difficult to separate from the nucleic acids, requiring long and inconvenient purification procedures for their removal. Even then, amplification may be compromised. The authors have developed a fast, safe and easy protocol to efficiently purify viral nucleic acids from serum for reliable PCR. It is shown, that the sensitivity of the PCR strongly depends on template quality and that our new extraction method provides better quality nucleic acids than standard acid-phenol extractions. Data are presented demonstrating that RNA from as little as 0.01 gl HCV (Hepatitis C virus) positive serum can efficiently be amplified after purification by our procedure. The new procedure combines the handling advantages of spin-column technology with the ability of silica to specifically bind nucleic acids. After sample lysis in a special lysis buffer containing a chaotropic salt, samples are loaded onto a spin-column by microcentrifugation. Nucleic acids are selectively bound to a silica membrane, and contaminants are washed away by two brief washes. Purified nucleic acid is then eluted in water or buffer, ready for direct addition to the PCR reaction. Moreover, this new method requires less hands-on time, uses no organic solvents or alcohol precipitations and minimizes the danger of contamination by infectious agents. It is ideal for simultaneous handling of multiple samples, enabling the preparation of 24 samples within 30 min. So far the method has successfully been tested for samples as different as fresh, frozen or dried whole blood in the presence of all common anti-coagulants, plasma, serum, buffy coat, bone marrow, mucus, cell suspensions, urine and tissue.

Parvovirus B19 Infection of the Fetus: Histology and in Situ Hybridization

Fetal tissues from 16 spontaneous abortions, two terminations, and one perinatal death, 18 of which were associated with maternal human parvovirus B19 infection, were examined for B19 infection by histology and in situ hybridization using a digoxigenin-labeled B19-DNA probe. In 15 spontaneous abortions and one termination, erythroblasts with intranuclear inclusions (lantern cells) reacted with B19-DNA by in situ hybridization. No internal or external fetal malformations were observed. Because 13 (86.7%) spontaneous abortions with lantern cells occurred between the 20th and 28th weeks of gestation, it is postulated that B19 infection may be a particular threat to the fetus during this stage of gestation.