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Andreas Nitsche

Publications and source records attributed to Andreas Nitsche.

25 records · Page 2Linked to original sources
PubMed2003

Detection of human polyomaviruses in urine from bone marrow transplant patients: comparison of electron microscopy with PCR.

BACKGROUND: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. METHODS: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. RESULTS: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 10(6) genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <10(3) GE/mL and 100% for urine samples containing 10(9) GE/mL. CONCLUSIONS: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.

BK Virus↗
PubMed2003

Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.

Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).

Animals↗
PubMed2003

Interleukin-3 promotes proliferation and differentiation of human hematopoietic stem cells but reduces their repopulation potential in NOD/SCID mice.

In the present study we explored systematically the influence of human interleukin-3 (IL-3) on the cord blood (CB) cell-derived production of human hematopoietic cells in the bone marrow, blood, and spleen of chimeric nonobese/severe combined immunodeficient mice ((NOD/SCID) mice. CB mononuclear cells and MACS-enriched CB CD34(+) cells were injected into irradiated NOD/SCID mice. The mice were additionally transplanted with a stably transfected rat fibroblast cell line expressing the human IL-3 gene (Rat-IL-3) constitutively, or with the nontransfected rat fibroblast cell line as a control (Rat-1). Rat-IL-3 mice displayed a higher engraftment of human hematopoietic cells in bone marrow, spleen, and peripheral blood compared with mice with Rat-1 cotransplantation. When we transplanted their total bone marrow cell population into secondary mice, surprisingly, mice transplanted with bone marrow cells from Rat-1 mice displayed a higher proportion of human hematopoietic cells compared with Rat-IL-3 mice. As expected, bone marrow cultures (BMCs) from Rat-IL-3 mice contained a higher proportion of human cells than Rat-1 bone marrow cells. However, when BMCs were passaged to new flasks, we observed a higher proportion of human cells in BMCs from Rat-1 mice compared with BMCs from Rat-IL-3 mice. IL-3 promotes the proliferation and differentiation of hematopoietic stem cells in chimeric bone marrow. In addition, IL-3 may play a role in the depletion of hematopoietic stem cells in chimeric bone marrow. In the absence of IL-3, the hematopoietic stem cells may remain in a quiescent state and proliferation can be induced by stimuli, including secondary transplantation or cell passage.

Animals↗
PubMed2002

Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture.

BACKGROUND: Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV. RESULTS: The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant. CONCLUSIONS: This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).

DNA, Viral↗
PubMed2002

Real-time PCR in virology.

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

DNA, Viral↗