Anne Baasner

Role of Virus-Induced Myocardial Affections in Sudden Infant Death Syndrome: A Prospective Postmortem Study

The cause of sudden infant death syndrome (SIDS) is an unresolved problem of high relevance. Previous studies indicate a role of infections. In our prospective study, we investigated the frequency of virus-induced myocardial affections in SIDS. Postmortem samples from SIDS victims and control subjects were investigated prospectively. Pediatric cases of unnatural death served as controls. Samples were studied for enteroviruses, adenoviruses, parvovirus B19, and Epstein-Barr virus applying PCR. Immunohistochemical investigations for inflammatory cells, the necrosis marker C5b-9(m) complement complex, and the enteroviral capsid protein VP1 were performed. Overall, 62 SIDS victims were studied. As controls, 11 infants were enrolled. Enteroviruses were detected in 14 (22.5%), adenoviruses in 2 (3.2%), Epstein-Barr viruses in 3 (4.8%), and parvovirus B19 in 7 (11.2%) cases of SIDS. Control group samples were completely virus negative. Compared with controls, immunohistochemical investigations partially revealed a significant increase in the number of T lymphocytes in SIDS myocardial samples (p < 0.05). Furthermore, cases with elevated numbers of leukocytes and + necroses, and enteroviral VP1 macrophages, microfocal C5b-9(m) capsid protein within the myocardium were detected. Applying a comprehensive combination of molecular and immunohistochemical techniques, our results demonstrate a clearly higher prevalence of viral myocardial affections in SIDS. Our results emphasize the importance of PCR-based diagnosis of viral myocardial affections. We suggest preliminary criteria for cellular immunohistochemical diagnosis of viral myocardial affections derived from our findings. For future investigations in SIDS, we suggest a comprehensive approach that includes PCR and immunohistochemistry. Our results offer novel strategies for diagnosis of pediatric myocardial viral affections.

Polymorphic sites in human mitochondrial DNA control region sequences: population data and maternal inheritance

Sequence analysis of the mitochondrial DNA (mtDNA) control region is of central importance for forensic identity testing as well as for studies of human evolution. Here we report the sequencing data of the hypervariable regions I and II from 50 unrelated individuals from a western German population (Rhine area). In regions I and II, 52 and 26 sites of sequence polymorphism, respectively, were noted. Nucleotide substitution rather than insertion/deletion was the majority of variation. The distribution showed a large bias towards transitional changes than transversional changes. Furthermore we investigated uniparental inheritance in seven CEPH families each family with 7-9 maternal descendants. Most maternal relatives shared identical mtDNA sequences. Additionally sequences were compared for father:child pairs and as expected no evidence for paternal transmission of mtDNA was observed. The high variability of mtDNA control region sequences permits utility in forensic identity investigations. The data also indicate that the neomutation rate seems to be very low from one generation to the other.

Coxsackie B3 Myocarditis in 4 Cases of Suspected Sudden Infant Death Syndrome: Diagnosis by Immunohistochemical and Molecular-Pathologic Investigations

Immunohistochemical and molecular-pathologic techniques have improved the diagnosis of myocarditis as compared with conventional histologic staining methods done according to the Dallas criteria. Most investigations were carried out on adults, and only a few authors investigating childhood deaths applied these modern methods, used for diagnosing myocarditis. We report on four children under one year of age, who suddenly died without prodromal symptoms. Their deaths were attributed to SIDS (sudden infant death syndrome). Immunohistochemical (LCA, CD68, CD45R0, MHC-class-II-molecules, VP1-capsid-protein of enteroviruses) and molecular-pathologic (RT-PCR) investigations, however, suggested that death was caused by a coxsackie-B3-myocarditis. In the future, these methods should be used for investigating cases with suspicion of SIDS.

Fatal parvovirus B19 myocarditis in an 8-year-old boy.

A report is given on an 8-year-old boy who suddenly and unexpected died. Autopsy findings point to acute heart failure. Microscopic examination of the heart showed increased interstitial and perivasal fibrosis and myocarditis with macrophage infiltration. Polymerase chain reaction (PCR) analysis for parvovirus B19 was positive in heart samples and in the spleen. Immunostaining for parvoviral surface antigens was negative. Although the virus does not appear to have infected the cardiomyocytes, we speculate that myocarditis arose from immunological cross-reaction to epitopes shared between the virus and the myocardium.

PCR-Based Diagnosis of Enterovirus and Parvovirus B19 in Paraffin-Embedded Heart Tissue of Children with Suspected Sudden Infant Death Syndrome

The diagnosis of viral myocarditis remains difficult and generally depends on clinical and histologic criteria. Viral cultures and serology are often unrewarding with low yields. The purpose of this study was to analyze the usefulness of PCR in the rapid diagnosis of myocarditis in children. PCR was used to analyze 120 myocardial tissue samples from 60 cases of sudden infant death syndrome (SIDS) and 56 myocardial tissue samples from 36 cases with well-known causes of sudden death (11 children younger than 1 year and 25 children 1–10 years old). The myocardial tissue samples were evaluated for the presence of enteroviruses and parvovirus B19 using PCR primers designed to consensus and unique sequences of these viral genomes. Enteroviruses could be detected in 14 cases of SIDS, whereas the detection of enteroviral nucleic acid within the control group was negative. Seven cases with myocardial infection caused by parvovirus B19 were found in the SIDS group. The detection of parvoviruses in the control group of the 11 children younger than 1 year was negative, whereas 3 positive cases of parvoviruses could be detected in the control group of children from 1 to 10 years old. In the myocardial sample of one SIDS case, both enteroviruses and parvovirus B19 could be detected. Our results emphasize the importance of modern molecular biologic methods in cases of sudden infant death even when conventional histologic examination revealed no serious findings in heart muscle tissue.

Mitochondrial DNA in the central european population: Human identification with the help of the forensic mt-DNA D-Loop-Base Database

Sequencing of mtDNA is an advanced method for the individualisation of traces. Disadvantages of this method are expensive and time-consuming analysis and evaluation procedures as well as the necessary stock of population-genetic data which is still insufficient. Central European institutes of forensic medicine from Germany, Austria, and Switzerland have been working together since the beginning of 1998 to establish a mtDNA database. The aim is to build up a large stock of forensically established data and provide population-genetic data for frequency investigations, which will serve as a basis for expert opinions and scientific research. Good data quality is ensured by using original sequences only. Ring tests, which have been conducted to enhance analytical reliability, revealed a high correspondence rate of the analytical results obtained by the individual member institutes. Today 1410 sequences are available for comparison, of which 1285 sequences in the HV1 and HV2 regions cover the full ranges from 16051 to 16365 and from 73 to 340 (according to Anderson). The major part is formed by Central European sequences comprising 1256 data sets from Germany, Austria, and Switzerland. Today the database contains sequences from a total of 12 European, six African and three Asian countries including 100 sequences from Japan. This paper is aimed at discussing the individualisation potentials of mtDNA as well as the possibilities and limits of ethnic differentiation by means of pairwise sequence differences on the basis of the data stock available.

Immunohistochemical and molecular-pathological diagnosis of myocarditis in cases of suspected sudden infant death syndrome (SIDS) - A multicenter study

Virus-induced myocarditis is a common disease even in infants and children, but diagnosis can be difficult according to the Dallas-criteria, which have been criticised as being too unreliable. The diagnosis has been substantially improved due to immunohistochemical techniques for characterization and quantification of myocardial inflammatory reactions as well as molecular-pathological methods for viral genome detection. The published studies report on post-mortem samples from SIDS victims and controls which were prospectively investigated. Pediatric cases of unnatural deaths served as controls. The results demonstrate a clearly higher prevalence of viral myocardial infections in cases of suspected SIDS. Preliminary criteria for cellular immunohistochemical diagnosis of viral myocardial affections derived from these findings were suggested.

PCR-based diagnosis of adenovirus and Epstein-Barr virus in paraffin-embedded heart tissue

Abstract Immunohistochemical and molecular-pathological techniques have improved the diagnosis, but the incidence of virus-induced lethal myocarditis still remains unclear. Therefore, it is of great interest to investigate postmortem myocardial samples in cases of sudden infant death syndrome (SIDS). Adenoviruses and Epstein–Barr viruses are known as possible agents of myocarditis. Viral DNAs were specifically isolated and amplified from formaldehyde-fixed material. At autopsy, myocardial samples were taken from 62 SIDS cases from different regions and investigated with PCR. Adenoviral DNA was detected in 2/62 and Epstein–Barr viruses in 3/62 cases. Our results emphasize the importance of modern molecular-pathological methods in cases of sudden unexpected death.

PCR-based diagnosis of enterovirus and parvovirus B19 in paraffin-embedded heart tissue

Abstract Although immunohistochemical and molecular biological techniques have improved the diagnosis, the incidence of virus-induced lethal courses of myocarditis is still unclear. Therefore, it is desirable to investigate postmortem myocardial samples in cases of unknown cause of death. While enteroviruses are the most common agents of myocarditis, parvovirus B19 is also known to be highly cardiotropic. The enteroviral genome consists of a single-stranded RNA molecule. Parvovirus B19 is the only known human pathogen virus of the family Parvoviridae and consists of a linear single-stranded DNA molecule. In our investigation, RNA and DNA were specifically isolated and demonstrated from formalin-fixed material. Myocardial samples from 60 autopsy cases with unknown cause of death after autopsy were taken from different regions and investigated with a nested polymerase-chain-reaction (PCR). Enteroviruses could be detected in 14 cases. PCR revealed eight cases with myocardial infection due to parvovirus B19. In the myocardial sample of one case, both enteroviruses and parvovirus B19 were found. Our results emphasize the importance of modern molecular biological methods in cases of sudden death even when histological examination revealed no serious findings in heart muscle tissue.

Allele and Genotype Frequencies for the STR Locus D18S51 in a Western German Population

Genetic marker typing based on DNA amplification by the polymerase chain reaction (PCR) increasingly is being employed in forensic casework and for paternity testing. Allele frequencies were determined using PCR for 102 unrelated Germans (Rhine area) for the locus D18S51. Twelve alleles were observed, with frequencies ranging from 0.005 (allele 11) to 0.191 (allele 14). The observed heterozygosity was 0.867, and the power of discrimination was 0.968. There was no deviation from expectations under Hardy-Weinberg assumptions (P = 0.451).