Christian Aepinus

Evaluation of Two Nested PCR Assays for Detection of Histoplasma capsulatum DNA in Human Tissue

ABSTRACT In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.

Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system.

In humans and experimental murine models enteroviruses, and in particular coxsackieviruses of group B (CVB), may induce chronic myocarditis associated with a persistent type of heart muscle infection. Persistent myocardial infection has been characterized by restricted viral replication and gene expression, which is capable of sustaining chronic inflammation. Altered replication and transcription of the virus, in addition to an immune response insufficient to recognize and clear infected cells entirely, are essential mechanisms for initiation and maintenance of persistent heart muscle infection. Viral cytotoxicity was found to be crucial for organ pathology both during acute and persistent infection, indicating that enterovirus myocarditis is a virus-induced rather than an immune-mediated disease. Notably, resistance to the development of persistent heart muscle infection is not linked to the H-2 haplotype of the host. In addition to persistently infected myocytes, detection of the replicative minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen, predominantly in splenic B lymphocytes, during the course of the disease. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac1+ macrophages, no enteroviral genomes were identified in CD8+ T cells. Detection of infected activated B lymphocytes both in heart tissue of CVB3-infected immunocompetent mice and syngenic SCID mice receiving splenocytes from CVB3-infected donors support the concept that B cell traffic may contribute to maintenance of chronic disease. Dissection of the diversity of viral and host-specific determinants in susceptible and resistant hosts will allow us to define the protective mechanisms that mediate resistance to the development of life-threatening acute and chronic enterovirus myocarditis.

Nested PCR Assays for Detection of Blastomyces dermatitidis DNA in Paraffin-Embedded Canine Tissue

ABSTRACT A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family Onygenaceae. We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue.

Disseminated fatal human cytomegalovirus disease after severe trauma.

Objective: Disseminated human cytomegalovirus (HCMV) disease is considered to be uncommon in critically ill but otherwise not immunosuppressed patients. We describe the case of a trauma victim who developed fatal HCMV disease that initially presented as pseudomembranous colitis and resulted in sudden cardiac death. Design: Case report of fatal HCMV disease in a previously healthy patient after multiple trauma. Setting: Surgical intensive care unit (ICU). Patient: A 63‐yr‐old male patient with multiple injuries. Interventions and Measurements: Under ICU treatment, symptoms of HCMV reactivation presenting as pseudomembranous colitis appeared 32 days after trauma. Detailed laboratory examinations for HCMV infection were performed, including complement fixation titer, immunoglobulin G and M, polymerase chain reaction, and virus isolation. Results: The intravital detection of HCMV DNA in serum, leukocytes, and a colonic biopsy specimen indicated HCMV reactivation. Postmortem examination findings, including positive viral cultures, showed severe disseminated HCMV disease with involvement of the colon and myocardium. Conclusions: The lack of specific clinical symptoms of HCMV disease and the delay until viral culture results are available make an exact and timely diagnosis of HCMV disease difficult. Its prevalence in critically ill but otherwise not immunosuppressed patients is currently unknown and possibly underestimated. Because severe illness or trauma can cause immunodysfunction and, thus, may contribute to an increased rate of HCMV disease, detailed studies are warranted to evaluate the real risk in the ICU setting.

Coxsackie B virus and its interaction with permissive host cells

BACKGROUND Observations in humans and the results of experiments on laboratory animals have provided evidence that coxsackieviruses of group B (CVB) are major etiologic agents of acute and chronic enterovirus myocarditis and various other virus-induced diseases. OBJECTIVE This minireview briefly summarizes the investigations to elucidate various molecular mechanisms for the induction and maintenance of persistent CVB infections. With regard to the recent findings that CVB may use several different receptor proteins, this article focuses on virus-host cell interactions and the potential impact of these interactions for enteroviral replication. STUDY DESIGN The interaction of CVB with specific cell surface proteins was analyzed in cultured cell lines and murine tissues at the level of virus attachment and virus internalization. As example for the interaction of CVB with intracellular proteins, the state of p21rasGTPase-activating protein (RasGAP) was investigated in mock-infected and CVB3-infected HeLa cells. RESULTS AND CONCLUSIONS The experiments to elucidate the virus receptor interactions revealed the necessity to differentiate between CVB attachment proteins and proteins involved in virus internalization. Since more than one protein may be required to initiate the uptake of CVB into permissive host cells, a model of the putative interaction of these proteins within a multimeric receptor complex is proposed. It is further tempting to speculate that the presence of multiple attachment proteins may influence the tissue tropism of CVB as well as pathogenicity.

Molecular pathogenesis of Epstein-Barr virus associated posttransplant lymphomas: new insights through latent membrane protein 1 fingerprinting.

Background. Fingerprint amino acid patterns within the carboxy terminus of the latent membrane protein (LMP1) oncoprotein of Epstein-Barr virus (EBV) allow individual strain identification at the molecular level. LMP1 is expressed in the tumor cells of EBV-associated posttransplant lymphomas (PTLs) and the LMP1 genome is also identified in lymphocytes of most donors of allogeneic bone marrow. Therefore, LMP1 genotyping in donor lymphocytes and PTL tumor cells, together with sex chromatin determination of tumor cells, allows to determine the origin of PTL tumor cells and the origin of individual EBV strains harboured by them. Methods. We traced the origin of aggressive PTLs occuring in six patients after allogeneic T cell-depleted stem cell transplantation (allo-SCT). DNA was extracted from donor lymphocytes and PTLs of recipients and amplified with LMP1-specific primers in each case. A comparative sequence analysis of the fingerprint LMP1 region identified in donor lymphocytes and lymphoma was performed. Results. One lymphoma of donor origin occurred after highly selected CD34+ PBSCT and contained the same LMP1 genotype as the donor lymphocytes. Three lymphomas of recipient origin had deletions within the carboxy terminus of LMP1, not identified in the donor strains. All lymphomas occurred in the setting of allo-SCT and had a rapid clinical course. Conclusions. These results show that highly selected CD34+ PBSCT does not protect against transfer of EBV positive founder cells of donor type PTL and that, after allo-SCT, recipient type PTLs are not uncommon. Outgrowth of recipient type lymphoma may be favoured by LMP1 deletion variant strains present in recipient lymphocytes.