Ursula Meyer-König

Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus Infections by Real-Time PCR

ABSTRACT The herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV) can cause life-threatening infections of the central nervous system and lead to severe infections in immunocompromised subjects and newborns. In these cases, rapid diagnosis is crucial. We developed three different real-time PCR assays based on TaqMan chemistry for the LightCycler instrument to detect HSV-1, HSV-2, and VZV. When the TaqMan assays were compared to our in-house nested PCR assays, the test systems had equal sensitivities of ≤10 plasmid copies per assay. When clinical samples were investigated by TaqMan PCR to detect HSV-1, HSV-2, and VZV DNA, 95, 100, and 96% of the samples determined to be positive by nested PCR, respectively, were positive by the real-time PCR assays. The specificities of all PCR assays were almost 100%. Furthermore, the TaqMan PCR assays could be performed within 2.5 h, whereas nested PCR results were available after 9 h. In addition to offering more rapid results, the TaqMan PCR assays appear to be less expensive than nested PCR assays due to less hands-on time. In summary, TaqMan PCR is an excellent alternative to conventional nested PCR assays for the rapid detection of HSV-1, HSV-2, and VZV in clinical samples.

Glycoprotein B genotype correlates with cell tropism in vivo of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) strains can be classified into four genotypes of the glycoprotein B (gB). In a previous study, the gB genotype 1 was found more frequently in bone marrow transplant recipients with nonfatal HCMV infection than in patients who died from HCMV disease [Fries et al. (1994): Journal of Infectious Diseases 169:769–774]. The distribution and cell tropism of different gB types in vivo were investigated. The gB type of HCMV was determined in blood or urine specimen from 76 organ and 47 bone marrow transplant recipients using PCR and restriction fragment length polymorphism (RFLP). The leukocyte populations (polymorphonuclear leukocytes, monocytes, T lymphocytes, non‐T lymphocytes) of 20 viremic patients were purified by a fluorescence‐activated cell sorter (FACS) and examined for HCMV infection by PCR. Sequence analysis of four randomly selected strains showed that gB types were similar to published sequences and no atypical gB types were found. Within the compartments blood and urine, the gB types were almost equally distributed, whereas the gB type 1, in contrast to gB types 2 and 3, did not infect T lymphocytes in vivo. These data show that the gB type correlates with viral tropism in vivo and thus provides further evidence that the gB variation may indeed influence the virulence of HCMV. J. Med. Virol. 54:75–81, 1998.

Human cytomegalovirus impairs dendritic cell function: a novel mechanism of human cytomegalovirus immune escape

Human cytomegalovirus (HCMV) employs multiple mechanisms to evade the immune system and succeeds to persist lifelong in the host. Human dendritic cells (DC) are the main antigen‐presenting cells and play the key role in inducing and maintaining immune responses. Here, we studied the interaction of HCMV with DC. We found that DC, irrespectively of their stage of maturation, were fully permissive for HCMV when endothelial cell‐adapted HCMV strains were applied. When fibroblast‐adapted strains were used, viral replication was abrogated at the level of immediate early (IE) and/or early (E) gene expression. Irrespective of the HCMV strain used, infection of DC prevented the signal delivery essential for T cell activation in a multistep manner. Furthermore, we observed an altered expression of adhesion molecules. This might contribute to an impairment of DC migration. Our data indicate that a soluble factor induced by IE and/or E genes is involved in these processes. The impairment of DC function upon HCMV infection may contribute to virus‐mediated immunosuppression and help the virus to establish persistence in the host.

Intragenic Variability of Human Cytomegalovirus Glycoprotein B in Clinical Strains

Human cytomegalovirus (HCMV) strains can be classified into four glycoprotein B (gB) genotypes, and there has been evidence of differences in viral virulence. In this study, intragenic variability of HCMV gB strains was analyzed. The gB gene was amplified by nested polymerase chain reaction using samples from immunosuppressed patients. The genotype of fragments corresponding to the cleavage site of gB was determined by restriction fragment analysis; fragments corresponding to the N- and C-termini (gBn and gBc) were sequenced and compared with published sequences. At the cleavage site, the four known genotypes were found. Typing revealed four major genotypes at the N-terminus and two at the C-terminus. In 22 of 44 strains, the gB type determined at the cleavage site was different from the gBn or gBc type (or either), indicating that intragenic variability within the gB gene occurs frequently.

Human cytomegalovirus glycoprotein B genotypes in renal transplant recipients

Based on sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) strains can be classified into four gB genotypes. In a previous study of bone marrow transplant recipients, infection with the gB type 1 correlated with a more favorable clinical outcome than infection with the gB types 2, 3, or 4. The gB type was determined in 60 renal transplant and in 47 bone marrow transplant recipients using PCR and restriction analysis. All HCMV variants in patient specimens could be assigned to one of the four previously described gB types. Two or more specimens obtained from 39 patients were analysed; in 31 of these patients the gB type was the same in all samples. The gB type did not correlate with the clinical outcome or the level of viremia in renal transplant recipients.

Glycoprotein B Genotype of Human Cytomegalovirus: Distribution in HIV-infected Patients

Glycoprotein B (gB) is involved in cell to cell transmission of human cytomegalovirus (HCMV) and may be a critical factor in tissue tropism and viral pathogenesis. We analyzed the distribution of the four known gB genotypes of HCMV in 99 HIV-positive patients. 29 patients had HCMV retinitis, and 70 patients had asymptomatic HCMV infection. DNA was isolated from blood, urine, and aqueous humor, and gB genotypes were determined by PCR and restriction analysis. Infections with gB type 1 were less frequent in patients with retinitis than in patients with asymptomatic HCMV infection (17% versus 37%; p = 0.05). Furthermore, the gB type was correlated with dissemination of infection. In patients with HCMV detected in only one compartment (blood or urine) the gB type 1 was found more frequently than in patients with HCMV detected in at least two compartments (p = 0.01). The data show that gB genotypes differ in their association with clinical disease, and indicate that the gB genotype may contribute to the course of HCMV infection.

Cytomegalovirus Pp65 Antigen-guided Preemptive Therapy With Ganciclovir In Solid Organ Transplant Recipients: A Prospective, Double-blind, Placebo-controlled Study

BACKGROUND The aim of this study was to evaluate pp65 antigen-guided antiviral therapy in preventing human cytomegalovirus (HCMV) infection in solid organ transplant recipients. METHODS Ten kidney and two liver transplant recipients with asymptomatic HCMV infection were randomized either for i.v. ganciclovir or placebo treatment in a prospective, double-blind study. All patients were positive by HCMV pp65 antigen test at levels >5 positive cells/2 x 10(5) investigated cells. RESULTS No cases of HCMV end-organ disease occurred. In contrast to patients on placebo (5/7), none of the patients on ganciclovir (0/5) developed HCMV-associated symptoms (P=0.01). However, because of the small number of patients, all three high-risk patients (donor seropositive, recipient seronegative) were randomized to placebo and all three developed symptoms. CONCLUSIONS Preemptive antiviral therapy guided by the pp65 antigen test seems to have a beneficial effect on preventing HCMV-associated symptoms in kidney and liver transplant recipients.

Infection of Blood and Bone Marrow Cells with the Human Cytomegalovirus in Vivo

The human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. Both, primary infection and reactivation of latent virus can cause disease. Peripheral blood leukocytes (PBL) most likely play an important role in viral persistence and dissemination of infection. However, an open question has been whether HCMV actively replicates in PBL in vivo and whether the progenitor cells in the bone marrow are also infected. Previous studies on this issue are controversial. Here we summarize data on the tropism of HCMV for mature leukocyte populations as well as bone marrow progenitor cells during HCMV viremia. All cell populations were highly purified by a fluorescence activated cell sorter (FACS) and analyzed by PCR for the presence of viral genomic DNA. Moreover, mature leukocyte populations were investigated for mRNA expression of regulatory and viral structural proteins. We could show, that HCMV DNA was detected most frequently in granulocytes and monocytes as well as in CD34+ progenitor cells of immunosuppressed patients. Viral mRNA expression was found in granulocytes, monocytes, and lymphocyte fractions. In contrast, no HCMV DNA was found in healthy, seropositive individuals.

Liver Failure due to Disseminated HSV-1 Infection in a Newborn Twin

Most cases of neonatal herpes simplex virus (HSV) infection result from contact with maternal genital tract secretions and are caused by infections with HSV type 2. We report on a fatal HSV-1 infection in a newborn twin presenting with liver failure. The infection was acquired by single contact with an aunt. The route of transmission was proven by PCR followed by restriction endonuclease fingerprinting and DNA sequencing. This report demonstrates that liver failure may be an early and single symptom in life-threatening neonatal HSV-1 infection.

Laboratory diagnosis of HCMV-related disease in renal transplant patients - pp65 antigen detection versus nested PCR.

BACKGROUND Sixty-five renal transplant (Tx) recipients were monitored for signs and symptoms of human cytomegalovirus (HCMV) infection. OBJECTIVES Different diagnostic markers were evaluated for early and correct diagnosis of HCMV disease. STUDY DESIGN Blood and urine samples were obtained in weekly intervals and the following markers were determined: (1) IgG and IgM antibodies in serum using immunofluorescence and ELISA tests; (2) viral shedding in urine by rapid centrifugation culture (RCC); (3) viral antigen (pp65) in peripheral blood leukocytes (PBL) by immunofluorescence and (4) viral DNA in PBL by nested PCR (NPCR). RESULTS Twenty-two patients remained free of HCMV infection, 18 patients developed clinical symptoms of HCMV disease, and 25 patients remained asymptomatic in spite of laboratory signs of HCMV infection. For the early detection of HCMV disease, the highest sensitivity was achieved using NPCR (100%) and pp65 antigen detection (94%). RCC and IgM serology were less sensitive (62% and 40% respectively). The differences of sensitivity were significant. Clinical specificity was 47% for NPCR, 79% for pp65 antigen detection, 66% for RCC, and 68% for IgM serology. CONCLUSION In contrast to NPCR, pp65 antigen detection was closely correlated with the appearance of clinical disease and proved to be a useful marker in the monitoring of antiviral therapy.