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Insgesamt geht man von ca. 200 Millionen chronischen Hepatilis-C-Virus (HCV) Trägern in der Welt aus. Der Hauptübertragungsweg der Hepatitis C ist seit der Einführung der Hepatitis C Testung im Blutspendewesen der i.v. Drogenabusus. Die Inzidenz von Neuinfektionen wird in Deutschland auf ca. 5.000/Jahr geschätzt, allerdings verlaufen die meisten akuten Infektionen unauffällig. Für das initiale Screening sind ELISA Tests zum Nachweis HCV spezifischer Antikörper am schnellsten und kostengünstigsten. Bei immungeschwächten Patienten können diese Tests allerdings aufgrund einer verzögerten oder fehlenden Immunantwort versagen. Falsch positive Resultate (insbesondere bei niedriger Reaktivität im Screening ELISA) können durch die Verwendung von rekombinanten Immunoblots verringert werden. In den letzten Jahren wurden Tests zum Nachweis des HCV Core Antigens entwickelt. Diese erwiesen sich als sehr sensitiv und vergleichbar mit der PCR für die Diagnose einer akuten HCV-Infektion. Zur Abklärung positiver oder unklarer serologischer Befunde oder zur Verlaufskontrolle der Viruslast chronisch infizierter Patienten sind Nukleinsäure Amplifikationstests (NAT) aufgrund ihrer höheren Sensitivität nach wie vor Mittel der Wahl. Die Entscheidung, welcher Patient behandelt werden sollte, ist von sehr vielen Faktoren abhängig. Diese sind das Alter des Patienten, der allgemeine Gesundheitszustand, das Risiko einer Zirrhose, Kontraindikation bzgl. der zu verwendenden Medikamente und die Wahrscheinlichkeit eines Therapieerfolgs (Viruslast, Genotyp). Es ist allgemein anerkannt, daß Patienten mit einer hohen Viruslast. (> 2 Million Kopien/ml) und der HCV-Genotyp l schlechter auf eine Therapie ansprechen.
Das erstmals Ende 2002 im Süden Chinas aufgetretene schwere akute respiratorische Syndrom (SARS) führte bis zum August 2003 zu insgesamt über 8000 Erkrankungen und über 700 Todesfällen. Eine von der Weltgesundheitsorganisation (WHO) ins Leben gerufene Kooperation verschiedener Laboratorien weltweit ermöglichte innerhalb von nur vier Wochen die Identifizierung des kausalen Agens, eines bislang unbekannten Coronavirus (vorläufig bezeichnet als SARS-assoziiertes Coronavirus oder SARS-CoV), welches die Koch’schen Postulate erfüllt. Der Erreger lässt sich (unter Hochsicherheitsbedingungen) gut in Zellkulturen vermehren, was weitere Studien zur Stabilität sowie die Entwicklung von antiviral wirksamen Substanzen und Impfstoffen erleichtert.
Obwohl schon rasch diagnostische Labortests, insbesondere zum Nachweis der viralen Nukleinsäure und virusspezifischer Antikörper, zur Verfügung standen, basiert die Falldefinition von SARS weiterhin auf klinisch-epidemiologischen Kriterien. In Hinblick auf die Gefahr eines (saisonalen) Wiederauftretens der Infektion müssen die verfügbaren Labormethoden dringend überprüft und weiter verbessert werden.
SARS ist ein gutes Beispiel dafür, wie schnell sich eine Infektionskrankheit über den internationalen Reiseverkehr ausbreiten kann, aber auch dafür, wie wichtig in einem solchen Falle eine gut koordinierte internationale Kooperation ist; durch Einsatz neuester, aber auch bewährter konventioneller Labormethoden und ständigen Austausch aktueller (Zwischen-)Ergebnisse sowie von Patientenproben und Reagenzien führte eine bisher einmalige Zusammenarbeit schnell zu einem Durchbruch. Dies lässt auf ähnliche Fortschritte beim Kampf gegen weitere neuartige Infektionserreger hoffen.
Herpes genitalis is caused mainly by herpes simplex virus type 2 (HSV-2) and to a lesser extent but with increasing frequency, by herpes simplex virus type 1 (HSV-1). Today, the diagnosis of genital herpes is based "on laboratory methods. Serology is useful to distinguish primary infection from latent infection and for seroepidemiological investigations. Newer type-specific antibody tests based on single recombinant or purified viral antigens have a higher sensitivity and specificity for detecting anti HSV-2 antibodies. The tests also allow the discrimination between HSV-1 or -2 specific antibodies. Since serology is not able to recognize reactivation, isolation in cell culture remains the standard. If cell culture is not available or optimal transport is not possible and rapid results are needed, direct antigen detection, or in selected cases, the highly sensitive and specific PCR should be used.
Testing for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by RT-PCR is a vital public health tool in the pandemic. Self-collected samples are increasingly used as an alternative to nasopharyngeal swabs. Several studies suggested that they are sufficiently sensitive to be a useful alternative. However, there are limited data directly comparing several different types of self-collected materials to determine which material is preferable. A total of 102 predominantly symptomatic adults with a confirmed SARS-CoV-2 infection self-collected native saliva, a tongue swab, a mid-turbinate nasal swab, saliva obtained by chewing a cotton pad and gargle lavage, within 48 h of initial diagnosis. Sample collection was unsupervised. Both native saliva and gargling with tap water had high diagnostic sensitivity of 92.8% and 89.1%, respectively. Nasal swabs had a sensitivity of 85.1%, which was not significantly inferior to saliva (p = 0.092), but 16.6% of participants reported they had difficult in self-collection of this sample. A tongue swab and saliva obtained by chewing a cotton pad had a significantly lower sensitivity of 74.2% and 70.2%, respectively. Diagnostic sensitivity was not related to the presence of clinical symptoms or to age. When comparing self-collected specimens from different material, saliva, gargle lavage or mid-turbinate nasal swabs may be considered for most symptomatic patients. However, complementary experiments are required to verify that differences in performance observed among the five sampling modes were not attributed to collection impairment.
Background: To minimize the risk of disease transmission in cornea transplantation, donor screening for blood-derived viral infections is mandatory. Ideally, pre-mortem blood samples are used, but based on availability, cadaveric blood samples of cornea donors may also be used. However, serological and nucleic acid amplification tests (NATs) need to be validated for the use of cadaveric specimens.
Methods: Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV) 1/2, and Treponema pallidum (syphilis)-specific serological and/or NAT assays were validated on different platforms (Abbott Alinity i, Alinity m, Roche Cobas 6800, and Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM)) using (un)spiked paired pre- and post-mortem cornea donor blood samples from the same individual (up to 23.83 h after death) of 28 individuals in accordance with the specifications of the German Federal Institute for Vaccines and Biomedicines (Paul-Ehrlich-Institut [PEI]). In addition, routinely HBV-, HCV- and HIV-PCR-negative tested post-mortem blood samples of 24 individuals were used to assess NAT specificity.
Results: For the majority of serological parameters on the Abbott Alinity i (HBsAg, anti-HBc, anti-HBs, anti-HCV, anti-HIV, anti-HTLV 1/2, and anti-Treponema pallidum), ratios of generated test results of (un)spiked paired pre- and post-mortem blood samples differed ≤25%, with an agreement of qualitative pre- and post-mortem test results ranging from 91.2 to 100%. For NAT parameters (HBV, HCV, and HIV) on the Cobas 6800, Alinity m, and CAP/CTM, no significant deviation in virus concentrations (factor >5) of spiked pre- and post-mortem blood samples could be observed. Ct-values of corresponding internal controls did also not differ significantly (>1.5 Ct-values). In addition, no false-positive test results were generated when specificity was assessed.
Conclusion: Overall, fluctuations of test results for serological and NAT parameters in pre- and post-mortem blood samples examined in this study, were only limited and within the range of what is also observed when routinely testing fresh patient specimens. We conclude that all examined assays are eligible for the screening of blood samples taken up to about 24 h after the occurrence of death.
Due to globally rising numbers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, resources for real-time reverse-transcription polymerase chain reaction (rRT-PCR)-based testing have been exhausted. In order to meet the demands of testing and reduce transmission, SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) are being considered. These tests are fast, inexpensive, and simple to use, but whether they detect potentially infectious cases has not been well studied. We evaluated three lateral flow assays (RIDA®QUICK SARS-CoV-2 Antigen (R-Biopharm), SARS-CoV-2 Rapid Antigen Test (Roche)), and NADAL® COVID-19 Ag Test (Nal von Minden GmbH, Regensburg, Germany) and one microfluidic immunofluorescence assay (SARS-CoV-2 Ag Test (LumiraDx GmbH, Cologne, Germany)) using 100 clinical samples. Diagnostic rRT-PCR and cell culture testing as a marker for infectivity were performed in parallel. The overall Ag-RDT sensitivity for rRT-PCR-positive samples ranged from 24.3% to 50%. However, for samples with a viral load of more than 6 log10 RNA copies/mL (22/100), typically seen in infectious individuals, Ag-RDT positivity was between 81.8% and 100%. Only 51.6% (33/64) of the rRT-PCR-positive samples were infectious in cell culture. In contrast, three Ag-RDTs demonstrated a more significant correlation with cell culture infectivity (61.8–82.4%). Our findings suggest that large-scale SARS-CoV-2 Ag-RDT-based testing can be considered for detecting potentially infective individuals and reducing the virus spread.
Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020
(2020)
Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.
Introduction: Reliable and cost-effective diagnostics for hepatitis E virus (HEV) infection are necessary. The aim of our study was to investigate which diagnostic test is most accurate to detect HEV infection in immunocompetent and immunosuppressed patients in a real world setting. Patients and Methods: We performed a retrospective analysis of 1165 patients tested for HEV antibodies and HEV PCR at the same time point. Clinical, laboratory and virological data were taken from patient charts. HEV IgA was measured in a subgroup of 185 patients. Results: HEV RNA was detectable in 61 patients (5.2%); most of them (n = 49, 80.3%/n = 43, 70.5%) were HEV IgM+ and IgG+; however, 12 patients (19.6%) were HEV RNA positive/HEV IgM negative and 17 patients (27.8%) were HEV RNA positive/HEV IgG negative. Ten HEV RNA positive patients (16.4%) had neither HEV IgG nor IgM antibodies. Importantly, all of them were immunosuppressed. HEV IgA testing was less sensitive than HEV IgM for HEV diagnosis. Conclusions: HEV infection can be overlooked in patients without HEV specific antibodies. Performing PCR is necessary to diagnose or exclude HEV infection in immunocompromised hosts. In immunocompetent patients, a screening based on HEV antibodies (IgG/IgM) is sufficient.
Determining the cell fate and the distribution of mesenchymal stromal/stem cells (MSCs) after transplantation are essential parts of characterizing the mechanisms of action and biosafety profile of stem cell therapy. Many recent studies have shown that MSCs migrate into injured tissues, but are only detectable at extremely low frequencies. We investigated the cell fate of MSCs after transplantation in an acute kidney injury (AKI) mouse model using in vivo bioluminescence imaging (BLI) and subsequent verification of cell migration using quantitative real-time polymerase chain reaction (qRT-PCR). The AKI was induced by a single injection of cisplatin (8 or 12 mg/kg). One day later, adipose-derived mesenchymal stromal/stem cells isolated from luciferase transgenic mice (Luc+-mASCs, 5 × 105) were intravenously transplanted. Migration kinetics of the cells was monitored using BLI on day 1, 3, and 6, and finally via quantitative real-time PCR at the endpoint on day 6. Using BLI, infused Luc+-mASCs could only be detected in the lungs, but not in the kidneys. In contrast, PCR endpoint analysis revealed that Luc-specific mRNA could be detected in injured renal tissue; compared to the control group, the induction was 2.2-fold higher for the 8 mg/kg cisplatin group (p < 0.05), respectively 6.1-fold for the 12 mg/kg cisplatin group (p < 0.001). In conclusion, our study demonstrated that Luc-based real-time PCR rather than BLI is likely to be a better tool for cell tracking after transplantation in models such as cisplatin-induced AKI.
Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.
Background: Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings.
Methods: For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers.
Results: Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77–0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring.
Conclusions: In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.