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Rapid and sensitive method for the detection of B19 virus DNA using the polymerase chain reaction with nested primers.

The sensitivity of detection of B19 virus DNA in clinical specimens was evaluated by comparing the results of single PCR and nested PCR assays, with or without subsequent Southern blot hybridization to a radiolabelled B19 DNA probe. The inhibitory activity of human serum components on polymerase reaction was also determined. The sensitivity of B19 virus DNA detection decreased by a factor of 10(7) in the presence of 10% serum in the single PCR reaction mixture, and of 10(3) for nested PCR. When nested PCR products were analysed by Southern blot hybridization to a B19 radioactive DNA probe, the sensitivity of the assay increased to such a level of B19 DNA detection that the reaction was no longer influenced by the presence of serum inhibitors in the original sample. Less than ten B19 genome copies could thus be detected in a 10-microliters sample. A panel of 38 clinical samples, originating from patients with possibility of B19 virus infection, were assayed by this method. Only one sample was found to be positive after single PCR, whereas seven samples (including the former) gave a positive signal after nested PCR. The specificity of the nested PCR products was controlled by hybridization to the B19 DNA probe and DNA sequencing. No discrepancy in the results was observed between nested PCR alone and nested PCR followed by Southern blot analysis.

Adult↗

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↗

Antibody capture haemadherence tests for parvovirus B19-specific IgM and IgG.

In the absence of serum inhibitor human parvovirus B19 agglutinates primate red cells. Agglutination of baboon erythrocytes by inhibitor free baculovirus expressed VP1/VP2 proteins of B19 virus was enhanced by low pH. To avoid interference from inhibitor(s) present in serum specimens microtitre tests with an antibody capture configuration were designed and optimised. Antigen binding was demonstrated by the adherence of baboon erythrocytes. These tests (MACHAT, GACHAT) detected IgM or IgG anti-B19 in 1032 serum specimens with an overall sensitivity of 99%, specificity of 98.2% and positive predictive value of 96.8% with reference to the radioimmunoassays currently in diagnostic use. MACHAT and GACHAT are straightforward, and well suited to large scale research and seroepidemiological investigations. They are without the risks from infectious antigen and radiation associated with existing radioimmunoassays and allow the phenomenon of B19 virus haemagglutination to be put to practical advantage.

Adolescent↗

Genetic diversity in the non-structural gene of parvovirus B19 detected by single-stranded conformational polymorphism assay (SSCP) and partial nucleotide sequencing.

A homologous region in the parvovirus B19 non-structural gene (B19 nt 1399-1682) was examined in 50 samples from patients with a wide variety of B19-related disease from various countries by PCR amplification, single-stranded conformational polymorphism (SSCP) assay and nucleotide sequencing. Five SSCP types were confirmed by nucleotide sequence analysis. Of a total of 6 mutations, all were silent. Types 3 and 4 accounted for 92% of strains. There was no correlation between genome type and either clinical illness or patient age. However, there was a correlation between SSCP type and country of origin. Type 3 strains predominated in Japan (18/26) and the UK (6/8), whereas type 4 predominated in the USA (9/12). Notably, type 3 strains also predominated among females (14/18), whereas there were approximately equal numbers of strain types 3 (7/17) and 4 (8/17) among males; an observation which remains unexplained. Within the Japanese group, although type 3 strains predominated overall, strains isolated from 1981 to 1987 consisted of types 1 (2/15), 2 (1/15), 3 (8/15), and 4 (4/15), whereas strains isolated from 1990 to 1994 consisted almost entirely of type 3 (10/11).

Adolescent↗

Automated detection of digoxigenin-labelled B19 parvovirus amplicons by a capture hybridization assay.

An automated method to identify B19 amplicons, directly labelled with digoxigenin during amplification reaction was developed. The labelled amplicons were hybridized with a biotinylated B19 oligo-probe and captured on commercially available test tubes coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and the colourimetric reaction automatically evaluated as an immunoenzymatic assay. Fifty serum samples were tested by the assay and the results were in accordance with those obtained by Southern blot analysis of amplified products. Due to the high sensitivity, specificity and reproducibility shown, the assay seems to be a practical and reliable test for the diagnosis of B19 infection and can be easily adapted to identify any digoxigenin-labelled amplified product of viral genomes.

Bacterial Proteins↗

Evaluation of 4 commercial test kits for parvovirus B19-specific IgM.

Four commercial test kits for parvovirus B19 IgM were evaluated by testing 491 sera assembled into 7 panels. The serum panels were designed to assess sensitivity and specificity of the commercial assays and to reflect the various clinical settings in which acute B19 infection forms part of the differential diagnosis. A mu-capture radioimmunoassay (MACRIA) was used as the reference test. With respect to MACRIA, the commercial B19 IgM assays showed an overall sensitivity of 70.1-84.1% and specificity of 92.2 to 97.4%. Assay performance varied in different clinical situations. In sera from adults with acute B19 arthropathy, all 4 assays were 100% sensitive, but in children with fifth disease, the sensitivity ranged from 44.1 to 88.6%. The sensitivity of all 4 assays was also low when testing samples collected more than 6 weeks after onset of symptoms and in women with B19-associated embryopathy. Specificity was greater than 97% in healthy blood donors, but varied from 70.9 to 83.3% in patients acutely infected with other viruses, including rubella. Although the IgM test kits here evaluated may be usefully introduced for B19 diagnosis in certain settings, knowledge of their limitations will be important when results have been interpreted.

Acute Disease↗

Petechial glove and sock syndrome caused by parvovirus B19.

The petechial glove and sock syndrome is a recently described febrile dermatosis characterized by acral pruritus, edema, pain, petechiae, and an enanthem of petechiae and erosions; these features suggest a viral origin. We report a typical case in a 36-year-old woman. IgM antibodies to human parvovirus B19 (PVB19) were present, and acute and convalescent IgG antibodies demonstrated seroconversion, which suggested recent infection with PVB19. Results of tests for other viral and bacterial agents were negative. These results strongly implicate PVB19 as an etiologic agent in the petechial glove and sock syndrome.

Acute Disease↗

Parvovirus B19 outbreak in a developmental center.

Of 49 employees serving developmentally disabled persons, 13 (27%) were found to have parvovirus B19 infection on the basis of serum positivity for parvovirus B19 IgM by enzyme-linked immunosorbent assay. Two employees had severe peripheral symmetric polyarthropathy lasting longer than 6 months. Infection control measures are described.

Adult↗

Viruses and bone marrow failure.

Many agents are associated with bone marrow failure, including toxins, inherited metabolic defects, ionizing radiation, and viral infection. In most cases, the etiologic agent is unknown. Many of these unclassified cases have symptomatic, immunologic, or epidemiologic similarities to viral infections. Viruses from different taxonomic families have been implicated in bone marrow failure syndromes, and they appear to cause hematosuppression by a variety of mechanisms. Some of the viruses involved in relatively well characterized suppressive interactions will be reviewed, including parovovirus B19, dengue, hepatitis viruses, Epstein-Barr virus, cytomegalovirus and the human immunodeficiency virus.

Bone Marrow Diseases↗

Parvovirus B19 infection and hematopoiesis.

Parvovirus B19, the only known human pathogenic parvovirus, is highly tropic to human bone marrow and replicates only in erythroid progenitor cells. The basis of this erythroid tropism is the tissue distribution of the B19 cellular receptor, globoside (blood group P antigen). In individuals with underlying hemolytic disorders, infection with parvovirus B19 is the primary cause of transient aplastic crisis (TAC). In immunocompromised patients, persistent B19 infection may develop that manifests as pure red cell aplasia and chronic anemia. B19 infection in utero can result in fetal death, hydrops fetalis, or congenital anemia. Diagnosis is based on examination of the bone marrow and B19 virological studies. Treatment of persistent infection with immunoglobulin leads to a rapid marked resolution of the anemia.

Animals↗

Temporal evolution of risk estimates for presumed human teratogens.

We present preliminary data assessing a previously untried method of deriving estimates of risk from case reports on presumed human teratogens. We postulated that we could take advantage of biases inherent to case reports in order to generate one or more families of temporal curves that could be used to estimate the "true" risk of teratogenic exposure. Using this method (which we refer to as the "case-cumulative method") we found that two agents (parvovirus B19 and isotretinoin) demonstrated a logarithmic decrease in the estimated risk over time, as intuitively expected, while trimethadione and the coumarin derivatives showed a more complex pattern over time. Analysis of estimated risks quoted by reviews and large studies for these four agents showed large variability from estimate to estimate and no discernible temporal pattern. With further analysis of other agents, the case-cumulative method might eventually prove to be useful in teratogen counseling.

Abnormalities, Drug-Induced↗

Sudden cardiac death in a 5-year-old girl associated with parvovirus B19 infection.

We report on a 5-year-old girl who suddenly collapsed and died while dancing at a family party. Histological examination of the heart including the cardiac conduction system revealed lymphocytic infiltrations of the sinu-atrial node and perivascular infiltration in the atrio-ventricular region. Additionally, foci of mononuclear infiltrates were observed in the myocardium. Consequently, myocarditis was diagnosed as cause of death. The child also had lymphocytic conjunctivis, parotitis and tracheitis. Evaluation of infections by means of nested polymerase chain reaction revealed parvovirus B19 DNA (PVB19) in tissue samples of the trachea.

Child, Preschool↗

Presence of perineuritis in a case of papular purpuric gloves and socks syndrome associated with mononeuritis multiplex attributable to B19 parvovirus.

A previously healthy 16-year-old boy developed papular purpuric gloves and socks syndrome concomitant with mononeuritis multiplex attributable to acute infection with parvovirus B19. A cutaneous biopsy showed lymphocytic lichenoid vasculitis and perineuritis. We discuss the relevance of these pathologic findings, and we suggest a correlation with the patient's neurologic condition. This case illustrates the difficulties in differentiating dysesthesia accompanying cutaneous manifestations of exanthem from neurologic disease on the basis of the clinical appearance alone.

Adolescent↗

Parvovirus B19 in pregnancy.

Pregnant women who report possible exposure to a viral illness can present a clinical predicament for obstetric providers. Although some viruses are benign in pregnancy, others can have serious consequences. Parvovirus B19, the causative agent of fifth disease, is one of the more serious of the common viruses. Understanding the pathophysiology and appropriate testing for parvovirus will help providers better counsel their patients.

Adult↗