Diagnosis of intrauterine parvovirus B19 infection at birth - Value of DNA detection in neonatal blood and dried blood spots.

BACKGROUND: Diagnosis of congenital viral infection at birth is generally attempted by direct detection of the virus by PCR in various neonatal materials. How to reliably diagnose intrauterine infection with parvovirus B19 (B19 V) at birth is unknown. OBJECTIVES: To evaluate the performance of B19 V DNA detection in cord blood (CB) or neonatal dried blood spots (DBS) in diagnosing fetal infection. STUDY DESIGN: Two cohorts of children diagnosed prenatally with an intrauterine B19 V infection were included in this study. CB samples of intrauterine B19 V infections that were sent to a reference laboratory for congenital infections in Stuttgart, Germany in the period 1995-2014 were tested in triplicate for B19 V DNA by quantitative PCR. DBS from children with intrauterine B19 V infection that underwent IUT at the LUMC, Leiden, the Netherlands in the period 2009-2014 were tested for B19 V DNA by quantitative B19 V PCR in triplicate. RESULTS: Fourteen of twenty (70 %) CB samples tested positive for B19 V DNA. The positivity rate was 40 % (4/10) in those with a prenatal diagnosis <20 weeks gestation. When intrauterine B19 V infection was diagnosed thereafter, 100 % (10/10) samples were B19 V DNA positive. Of the thirteen available DBS, twelve (92 %) tested positive. Viral load in CB and DBS corresponded inversely with time from fetal diagnosis to birth. CONCLUSION: B19 V DNA can be detected in neonatal blood samples of children following intrauterine B19 V infection, although the possibility of false-negatives, even in severe infections, should be considered. B19 V viral load at birth correlates with timing of infection.

High prevalence of human parvovirus B19 DNA in myocardial autopsy samples from subjects without myocarditis or dilative cardiomyopathy.

Human parvovirus B19 has been linked to a variety of cardiac diseases, as well as to erythema infectiosum, acute arthropathy, and fetal hydrops. A causal association between viral infection and cardiac disease was frequently postulated following the detection of B19 DNA by PCR in endomyocardial biopsy specimens. Since the lifelong persistence of B19 DNA in bone marrow, skin, synovia, tonsils, and liver was previously reported, the aim of our study was to investigate the possibility of asymptomatic B19 DNA persistence in heart tissue. Myocardial autopsy and postmortem blood samples were prospectively collected from 69 bodies sent to the Department of Forensic Medicine, Freiburg University Medical Center, for inquests. All study subjects were screened for B19-specific antibodies using a commercial enzyme immunoassay. Tissue samples were analyzed by real-time PCR for the presence of viral DNA. Since the presence of B19 genotype 2, known to have been circulating before 1960, would prove long-lasting persistence, the presence of the B19 genotype was retrospectively determined in seven of the study subjects by melting temperature analysis and sequencing of the PCR product. B19 DNA was found in myocardial samples from 46 of 48 seropositive and in none of 21 seronegative individuals. B19 genotype 1 was found in three patients born between 1950 and 1969. Genotype 2 was found in four patients born between 1927 and 1957. Our findings suggest lifelong persistence of B19 DNA in heart tissue. Thus, the detection of B19 DNA in myocardial biopsy specimens alone is not sufficient to postulate a relationship between B19 infection and cardiac disease.

A convenient rapid culture assay for the detection of enteroviruses in clinical samples: comparison with conventional cell culture and RT-PCR.

A convenient rapid culture assay (RCA) for the detection of enteroviruses was evaluated against RT-PCR using 576 stool and 102 cerebrospinal fluid (CSF) samples. One hundred and ninety stool samples were also tested by conventional cell culture (CCC). The RCA used immunoperoxidase staining of cell culture plates with a blend of monoclonal antibodies (mAbs) against enterovirus VP1 on the second and sixth days after inoculation. This blend was composed of 5D8/1 (Dako) and four 'in-house' mAbs. CCC was performed using fluorescence staining with the Enterovirus Screening Set (Chemicon International) for culture confirmation. Detection of enteroviruses by the RCA was more successful in colonic carcinoma (CaCo-2) and rhabdomyosarcoma (RD) cells than in human embryonic lung fibroblasts, HEp2 and A549 cells. The performance of CCC in RD cells was hindered by rapid cell degeneration and non-specific staining of cells during culture confirmation. The sensitivity of the RCA compared to RT-PCR in stool samples was found to be 71 % (115/161) on the second day and 87 % (140/161) on the sixth day. The sensitivity of the RCA in CSF samples was 38 % (22/58) after 2 days and 59 % (34/58) after 6 days. The specificity of the RCA was 100 %. All CCC-positive samples were positive by the RCA. CCC required 3-14 days for virus recovery. In conclusion, the RCA has the same sensitivity as CCC, significantly shortens the time required for the detection of enteroviruses, and prevents pitfalls associated with using RD cells for CCC. For diagnosis of aseptic meningitis in CSF samples, RT-PCR should be performed.