Apparently non-specific results found using a norovirus antigen immunoassay for fecal specimens from neonates.

Norovirus is increasingly recognized as a frequent cause of non-bacterial gastroenteritis. Despite a 10-fold increase in the number of cases reported following the availability of enzyme immunoassays, there are no reports yet from preterm neonates. We report on a sudden clustering of antigen-positive enzyme immuno assays results in a level III neonatal intensive care unit, involving 22 of 43 infants screened. Although antigen-positive samples were significantly associated with bloody stools (P<0.001) and gastric residues (P<0.02), norovirus infection could not be confirmed by reverse-transcriptase polymerase chain reaction or electron microscopy. We question the validity of the so called norovirus-specific antigen assays and warn against overreacting to positive enzyme immunoassays results without reverse-transcriptase polymerase chain reaction confirmation especially in the neonatal setting.

Visualization and microscopic quantification of HCMV-peptide-specific cytotoxic T lymphocytes using tetramer binding.

To monitor the frequencies of virus-specific cytotoxic T lymphocytes (CTLs), FACS analyses were performed detecting lymphocyte-specific surface molecules and tetramer binding, as marker for peptide-specificity. Aim of this investigation was to establish an alternative protocol for the quantification of virus-specific CTLs using tetramer binding and microscopic analyzing. The frequencies of HCMV-pp65-peptide-specific CTLs in the blood of eight different HLA-A*0201-positive, HCMV-IgG antibody-positive donors were analyzed with both methods. Using FACS analyses, a median of 0.8% and, using the microscopic analyses, a median of 3.0% was detected in the CD3+CD8+ cells. After enrichment of HCMV-pp65-peptide-specific CTLs using the interferon-gamma secretion assay followed by expansion in cell culture, a median of 90.6% using FACS analyses and a median of 87.1% using the microscopic analyses was detected. Thus, the staining protocol presented in this investigation is an alternative approach to detect and to quantify virus-specific CTLs in low as well as in high frequencies.

Cytomegalovirus infection after orthotopic liver transplantation is restricted by a pre-existing antiviral immune response of the recipient.

Infection of the liver by the human cytomegalovirus (HCMV) frequently occurs after orthotopic liver transplantation (OLT). However, the role of viral replication and the inflammatory reaction in the development of HCMV-associated liver dysfunction is unclear. To address this question in vivo, 84 liver biopsy specimens from 74 patients who received an orthotopic liver transplant were investigated by immunohistochemical detection of viral antigens and cell type specific marker proteins. The extent of viral replication was correlated with the HCMV antibody status of donor and recipient. HCMV immediate early antigens were found in 25 of 84 liver tissue sections investigated, hepatocytes being the predominant target cells. Bile duct epithelial cells, endothelial cells, mesenchymal cells and sinusoidal lining cells were also found susceptible to HCMV infection. The detection of viral capsid antigens, nuclear inclusions in infected cells and foci of infected cells were suggestive of permissive infection in these cells. In 25 HCMV-positive liver biopsy specimens, the median extent of HCMV infection was 0.33 (0.02-5.67) infected cells/mm(2) liver tissue. Primary infection of liver transplant recipients (D+/R-) was associated with a significantly higher extent of organ involvement as compared to reinfection or reactivation (D+/R+). In contrast, the extent of inflammatory infiltrates in areas of infected liver cells was higher in tissues of patients with pre-existing immunoreactivity (R+) compared to patients without pre-existing immunoreactivity (R-). In conclusion, these results favour the assumption that the immune response to HCMV is effective in restricting viral spread in the liver.

Human cytomegalovirus as a direct pathogen: correlation of multiorgan involvement and cell distribution with clinical and pathological findings in a case of congenital inclusion disease.

The human cytomegalovirus (HCMV), a member of the Herpesviridae, is the most frequent cause of congenital virus infections and a major cause of morbidity and mortality in immunocompromised patients. Due to the lack of an appropriate animal model, insight into the pathogenesis of HCMV infections originates primarily from in situ examination of HCMV-infected tissues. Although in immunocompromised adults such tests are complicated frequently by the presence of additional misleading pathogens, the absence of additional pathogens renders congenital inclusion disease the most suitable access for investigation of pathogenetic aspects of HCMV infections. Immunohistochemical examination of tissue sections from a boy with fatal congenital inclusion disease was undertaken to detect the extent of multiorgan and cell involvement. Adrenal gland, bone marrow, diencephalon, heart, kidney, liver, lung, pancreas, placenta, small bowel and spleen were included in this study. Detection of virus antigens from different phases of viral replication revealed that all investigated organs were infected by HCMV. Simultaneous detection of cell type specific marker molecules showed that a variety of cell types stained positive for HCMV antigens including endothelial cells, epithelial cells, smooth muscle cells, mesenchymal cells, hepatocytes, monocytes/macrophages and granulocytes. The lung, the pancreas, the kidneys and the liver were the major target organs with a high number of HCMV infected cells. This correlated with multiorgan failure as the cause of death and strongly indicates direct pathogenetic effects of HCMV.

Hepatocytes are permissive for human cytomegalovirus infection in human liver cell culture and In vivo.

The cytopathic potential of human cytomegalovirus (HCMV) in human liver cells was analyzed in cell culture and in tissue sections from patients with HCMV hepatitis. Liver cell cultures, consisting of hepatocytes, bile duct epithelial cells, and stromal cells were infected by various HCMV strains. Cytopathic effects, viral gene expression, and virus production were detected. Infected cell types were identified by immunocytochemical double labeling. Hepatocytes were the predominant target cells of HCMV infection in liver tissues and in cell culture. Late-stage infected cultured hepatocytes produced infectious progeny virus, and infectious virus was propagated from liver tissue specimens. HCMV infection in cultured liver cells closely resembled in vivo infection of the liver with regard to the target cell spectrum and the permissive course of infection. It is concluded that HCMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes.