Assessing the risk of CMV reactivation and reconstitution of antiviral immune response post bone marrow transplantation by the QuantiFERON-CMV-assay and real time PCR.

BACKGROUND: CMV reactivation is a major cause of severe complications in allogeneic hematopoietic stem cell transplant (HSCT) recipients. The risk of CMV reactivation depends on the serostatus (+/-) of the donor (D) and recipient (R). The reconstitution of CMV-specific T-cell responses after transplantation is crucial for the control of CMV reactivation. OBJECTIVES: The study aimed to determine the cellular immune status correlating with protection from high-level CMV viremia (>5000 copies/ml) and disease. STUDY DESIGN: We monitored CMV-specific cellular immune responses in 9 high-risk (D-/R+), 14 intermediate risk (D+/R+) and 3 low risk individuals (D+/R-), and 8 CMV negative controls (D-/R-). Interferon- γ (IFN-γ) levels as a marker for the CD8+ T-cell response were determined by the QuantiFERON-CMV-assay and compared to viral loads determined by PCR. RESULTS: Early CMV reactivation was detected in all high-risk and 13/14 intermediate risk individuals. High-level viremia was detected in 5/7 high and 7/14 intermediate risk patients. Reconstitution of the CMV-specific cellular immune response started from 3 months after transplantation and resulted in protection against CMV reactivation. Re-establishing of CMV-specific T-cell immune responses with IFN- γ levels >8.9 IU/ml is crucial for protection from high-level CMV viremia. CONCLUSIONS: Monitoring of HSCT-recipients with the QuantiFERON-CMV-assay might be of great benefit to optimize antiviral treatment.

Projecting effects of climate change on marine systems: is the mean all that matters?

Studies dealing with the effects of changing global temperatures on living organisms typically concentrate on annual mean temperatures. This, however, might not be the best approach in temperate systems with large seasonality where the mean annual temperature is actually not experienced very frequently. The mean annual temperature across a 50-year, daily time series of measurements at Helgoland Roads (54.2° N, 7.9° E) is 10.1°C while seasonal data are characterized by a clear, bimodal distribution; temperatures are around 6°C in winter and 15°C in summer with rapid transitions in spring and autumn. Across those 50 years, the temperature at which growth is maximal for each single bloom event for 115 phytoplankton species (more than 6000 estimates of optimal temperature) mirrors the bimodal distribution of the in situ temperatures. Moreover, independent laboratory data on temperature optima for growth of North Sea organisms yielded similar results: a deviance from the normal distribution, with a gap close to the mean annual temperature, and more optima either above or below this temperature. We conclude that organisms, particularly those that are short-lived, are either adapted to the prevailing winter or summer temperatures in temperate areas and that few species exist with thermal optima within the periods characterized by rapid spring warming and autumn cooling.

Instructive even after a decade: Complete results of initial virological diagnostics and re-evaluation of molecular data in the German rabies virus "outbreak" caused by transplantations.

In 2005, six patients in Germany received solid organs and both corneas from a donor with an unrecognized rabies infection. Initial virological diagnostics with the machinery available at the two national reference laboratories could quickly clarify the situation. Rabies virus antigen was detected in the organ donor's brain. In two of the three recipients with neurological alterations, intra vitam diagnosis was achieved by conventional RT-PCRs. Comparison of the phylogenetic relatedness of the different viral isolates proved transmission from the donor and, consequently, also established the diagnosis for the third patient. As indicated by the titre of neutralizing antibodies, the liver transplant recipient was protected from the lethal infection due to a vaccination against rabies virus, which he had received more than 15 years ago. All samples from the recipients of the corneas were invariably negative. Re-evaluation of the molecular data by real-time PCR did not lead to an improvement of intra vitam diagnosis but provided intriguing insights regarding the relative amounts of rabies virus RNA in different body fluids and peripheral organs. In saliva and skin, they were 250-200,000 times lower than in the infected patient's brains. Furthermore, in saliva samples taken serially from the same patient fluctuations by a factor of 160-500 were recorded. These findings highlight the problems of intra vitam diagnosis of rabies virus infections and make understandable why the virus can escape from all diagnostic attempts. Finally, in this context one should recall an almost trivial fact: Simple and appropriate postexposure prophylaxis could not only have saved the young organ donor's life but would also have prevented the whole transplantation-associated rabies "outbreak" in Germany.

Performance characteristics of the VERSANT hepatitis C virus RNA 1.0 (kPCR) assay.

HCV RNA assays are of central importance for virological diagnostics and for clinical planning and monitoring of an antiviral combination treatment of chronic HCV infections. The objective of the pre-market evaluation of the VERSANT HCV RNA 1.0 Assay (kPCR) was to collect analytical performance data for this new method of HCV RNA quantification and to compare them with the high standards that exist in this context. The assay exhibited a specificity of 100%. The mean intra- and inter-assay imprecision was 14.1% and 14.6%, respectively. The detection limit was determined to be 16IU/ml (95% confidence interval: 11.9-30.6IU/ml) and consequently corresponded to the manufacturer's claims (i.e. 15IU/ml). The test exhibited linearity for all HCV genotypes in a broad range from 15 to 10(8)IU HCV RNA/ml. Hence, the kPCR assay in general is well suitable for HCV RNA determinations in clinical practice. However, in a methodological comparison, a considerable under-quantification of the concentrations of HCV genotype 2 and 3 isolates was detected. Provided that the assay's manufacturer will quickly remedy this shortcoming, the VERSANT HCV RNA 1.0 (kPCR) can be called a completely reliable technique for HCV RNA quantification in routine virological diagnostics.

Dried blood spots--preparing and processing for use in immunoassays and in molecular techniques.

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

Clinical performance of the novel DiaSorin LIAISON(®) XL murex: HBsAg Quant, HCV-Ab, HIV-Ab/Ag assays.

BACKGROUND: The fully automated and closed LIAISON(®)XL platform was developed for reliable detection of infection markers like hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) antibodies (Ab) or human immunodeficiency virus (HIV)-Ag/Ab. To date, less is known about the diagnostic performance of this system in direct comparison to the common Abbott ARCHITECT(®) platform. OBJECTIVES: We compared the diagnostic performance and usability of the DiaSorin LIAISON(®)XL with the commonly used Abbott ARCHITECT(®) system. STUDY DESIGN: The qualitative performance of the above mentioned assays was compared in about 500 sera. Quantitative tests were performed for HBsAg-positive samples from patients under therapy (n=289) and in vitro expressed mutants (n=37). For HCV-Ab, a total number of 155 selected samples from patients chronically infected with different HCV genotypes were tested. RESULTS: The concordance between both systems was 99.4% for HBsAg, 98.81% for HCV-Ab, and 99.6% for HIV-Ab/Ag. The quantitative LIAISON(®)XL murex HBsAg assay detected all mutants in comparable amounts to the HBsAg wild type and yielded highly reliable HBsAg kinetics in patients treated with antiviral drugs. Dilution experiments using the 2nd International Standard for HBsAg (WHO) showed a high accuracy of this test. HCV-Ab from patients infected with genotypes 1-3 were equally detected in both systems. Interestingly, S/CO levels of HCV-Ab from patients infected with genotype 3 seem to be relatively low using both systems. CONCLUSIONS: The LIAISON(®)XL platform proved to be an excellent system for diagnostics of HBV, HCV, and HIV with equal performance compared to the ARCHITECT(®) system.

Detection of infections with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus by analyses of dried blood spots--performance characteristics of the ARCHITECT system and two commercial assays for nucleic acid amplification.

BACKGROUND: Nowadays, dried blood spots (DBS) are primarily used to obtain diagnostic access to risk collectives such as intravenous drug users, who are prone to infections with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Before DBS analyses can be used in this diagnostic context, however, a comprehensive evaluation of its performance characteristics must be conducted. To the best of our knowledge, the current study presents for the first time such essential data for the Abbott ARCHITECT system, which is currently the worldwide leading platform in this field of infection diagnostics. METHODS: The investigation comprised 1,762 paired serum/DBS samples and a total of 3,524 determinations with the Abbott ARCHITECT HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24-antigen/anti-HIV 1/2 assays as well as with the artus HBV LC PCR and VERSANT HCV RNA qualitative (TMA) tests. RESULTS: In the context of DBS testing, a specificity of 100% was recorded for the seven serological and molecular biological assays. The analytical sensitivity of HBsAg, anti-HBc, anti-HBs, anti-HCV, HIV-1-p24-antigen/anti-HIV 1/2, HBV DNA, and HCV RNA detections in DBS eluates was 98.6%, 97.1%, 97.5%, 97.8%, 100%, 93%, and 100%, respectively. DISCUSSION/CONCLUSIONS: The results obtained indicate that it is today possible to reliably detect HBsAg, anti-HBc, anti-HBs, anti-HCV and HIV-1-p24 antigen/anti-HIV 1/2 with state-of-the-art analytical systems such as the Abbott ARCHITECT in DBS eluates even when a comparatively high elution volume of 1,000 µl is used. They also provide evidence for the inherent analytical limits of DBS testing, which primarily concern the anti-HBc/anti-HBs system for individuals with HIV infections and nucleic acid tests with relatively low analytical sensitivity.

Composition of humic acid-degrading estuarine and marine bacterial communities.

We examined the bacterial decomposition of humic acids (HA) in two flow-through culture experiments, one inoculated by marine and one by estuarine bacterial communities. In both experiments, the cultures were fed with HA media of salinities of 28 and 14, close to their ambient and a distinctly different, foreign salinity. HA were decomposed to > 60% of the initial concentration within 70 days, and the foreign salinity yielded the highest decomposition. A detrended correspondence analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns showed that during incubation, the bacterial community composition underwent distinct changes. A phylogenetic analysis of DGGE bands excised and bacteria isolated at the end on HA as the sole carbon source showed that Alphaproteobacteria and Gammaproteobacteria largely dominated the communities in the marine flow-through cultures, whereas Gammaproteobacteria, Actinobacteria and Alphaproteobacteria dominated the estuarine communities. Eleven of 13 isolates obtained from both experiments were able to grow on HA as the sole carbon source, seven on phenol and three, affiliated to the Roseobacter clade, on various aromatic acids. The bacteria retrieved from the flow-through cultures were closely (96-99%) affiliated to organisms capable of degrading humic matter, aromatic and aliphatic compounds and also to other bacteria reported previously from the Wadden Sea and Weser estuary.