Biomedical subjects

Frank T Hufert

Publications and source records attributed to Frank T Hufert.

9 recordsLinked to original sources

PubMed2006

Tick-borne encephalitis virus in Clethrionomys glareolus in the Czech Republic.

A total of 474 specimens from 157 rodents caught at the military training area of Boletice in the south of the Czech Republic from May to November 1999 were screened for TBEV by nested PCR. TBEV-specific RNA was amplified from lung, kidney, and spleen derived from one Clethrionomys glareolus in the first RT-PCR round. Sequence analysis revealed a 100% identity to the TBEV strain Neudoerfl. TBEV presence in the sample was confirmed by mouse brain passage of backup samples and cell culture. The results support the observation that hantaviruses and TBEV transmission can occur sympatrically in the same rodent population.

Animals↗

PubMed2006

Interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells.

Severe acute respiratory syndrome (SARS) of humans is caused by a novel coronavirus of zoonotic origin termed SARS-associated coronavirus (SARS-CoV). The virus induces severe injury of lung tissue, as well as lymphopenia and destruction of the architecture of lymphatic tissue by as-yet-unknown mechanisms. In this study, the interaction of SARS-CoV with dendritic cells (DCs), the key regulators of immune responses, was analysed. Monocyte-derived DCs were infected with SARS-CoV and analysed for viability, surface-marker expression and alpha interferon (IFN-alpha) induction. SARS-CoV infection was monitored by quantitative RT-PCR, immunofluorescence analysis and recovery experiments. SARS-CoV infected both immature and mature DCs, although replication efficiency was low. Immature DCs were activated by SARS-CoV infection and by UV-inactivated SARS-CoV. Infected DCs were still viable on day 6 post-infection, but major histocompatibility complex class I upregulation was missing, indicating that DC function was impaired. Additionally, SARS-CoV infection induced a delayed activation of IFN-alpha expression. Therefore, it is concluded that SARS-CoV has the ability to circumvent both the innate and the adaptive immune systems.

Antigens, Surface↗

PubMed2006

Diagnostic value of reverse transcription-PCR for detection of cytomegalovirus pp67 in samples from solid-organ transplant recipients.

We evaluated a highly sensitive quantitative real-time one-step reverse transcription-PCR (RT-PCR) for detection of human cytomegalovirus pp67 transcripts in monitoring of solid-organ transplant recipients. Results were compared with those of pp65 antigen testing and quantitative DNA-PCR. Due to a low clinical sensitivity, the pp67 RT-PCR was not able to replace these assays.

Cytomegalovirus↗

PubMed2004

Cytomegalovirus infection in organ-transplant recipients: diagnostic value of pp65 antigen test, qualitative polymerase chain reaction (PCR) and quantitative Taqman PCR.

BACKGROUND: The human cytomegalovirus (CMV) is a major cause of morbidity and mortality in transplant patients. In this study, we compared the diagnostic value of pp65 antigen test, qualitative nested polymerase chain reaction (PCR), and quantitative Taqman PCR in predicting the clinical outcome of CMV infection. METHODS: A total of 169 samples derived from 59 organ-transplant recipients (kidney n= 46, liver n= 11, kidney and pancreas n= 2) were analyzed. Peripheral blood leukocytes (PBL) were isolated using dextran gradient centrifugation, and 2 x 10 cells were analyzed for pp65 antigen by immunofluorescence. A crude DNA extract obtained from the same number of cells was used for qualitative nested PCR and quantitative Taqman PCR analysis. RESULTS.: The correlation coefficient of pp65 antigen test and Taqman PCR was R= 0.699 (P = 0.001). With cut-off values for pp65 antigen test set at greater than 10 positive nuclei per 2 x 10 PBL, sensitivity was 91%, and positive predictive value (PPV) was 70%. When the corresponding cut-off value for Taqman PCR was applied (>125000 genome copies per 2 x 10 PBL), a sensitivity of 83% and a PPV of 68% were found. Both assays allowed for the monitoring of successful antiviral therapy. Although qualitative nested PCR was highly sensitive (95%), it was less useful in predicting CMV disease (PPV 47%) and in therapy control. CONCLUSION: Our data show that pp65 antigen test and Taqman PCR are almost equivalent in the monitoring of CMV infection and disease when identical cell numbers are used for both assays.

Adult↗

PubMed2004

Rapid detection protocol for filoviruses.

BACKGROUND: The incidence of filovirus disease outbreaks has been increasing in recent years. Although there have been advances in the developments of diagnostics, field tests are rare. Apart from family members of infected patients, health care workers are at high risk of being infected during the initial phase of an outbreak. RT-PCR has been shown to be helpful in containing outbreaks. OBJECTIVES: To develop Taqman-RT-PCR for the detection of Ebola-Zaire virus (EBOV-Z), Ebola-Sudan virus (EBOV-S) and Marburg virus (MBGV). STUDY DESIGN: Quantitative Taqman-RT-PCRs for the detection of these viruses were developed and established on a portable Smartcycler TD. RESULTS AND CONCLUSIONS: All three assays were highly sensitive and specific. The mobility of the assay system may help to contain future outbreaks.

Base Sequence↗

PubMed2004

High-efficiency detection of severe acute respiratory syndrome virus genetic material.

A Taqman amplicon targeting the nucleocapsid gene of severe acute respiratory syndrome coronavirus (SARS-CoV) is 5 log(10) times more sensitive for SARS-CoV target RNA extracted from infected cells and 2.79 log(10) times more sensitive for RNA extracted from patient material of the index case in Frankfurt than an amplicon targeting the polymerase gene.

Bronchoalveolar Lavage Fluid↗

PubMed2003

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.

Adaptation, Physiological↗

PubMed2003

Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR.

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 <or=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.

DNA Primers↗

PubMed2003

Rapid detection of human pathogenic orthobunyaviruses.

Modern detection and identification tools can help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging diseases. We developed highly sensitive one-step TaqMan reverse transcription-PCR assays with sensitivities ranging from 10(4) to 10(1) molecules for 11 human pathogens of the orthobunyaviruses. We compared the performances of these assays on three currently available cyclers (ABI-PRISM 7700, LightCycler, and SmartCycler). The assay for Oropouche virus (OROV) was tested using sera collected from days 1 to 5 after onset of OROV disease and was found to be greatly superior to an established nested PCR system. A mean copy number of 1.31 x 10(7) OROV RNA/ml of serum was detected. Diagnostic RNA detection can be used as early as day 1 after onset of OROV disease. The use of a mobile SmartCycler and a hands-on time of less than 3 h could help to intensify outbreak surveillance and control, especially in field studies.

Animals↗