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Prevalence of SARS-CoV-2 antibodies is an essential indicator to guide measures. Few population-based estimates are available in Germany. We determine seroprevalence allowing comparison between regions, time points, socio-demographic and health-related factors.MuSPAD is a sequential multi-local seroprevalence study. We randomly recruited adults in five counties with differing cumulative SARS-CoV-2 incidence July 2020 - February 2021. Serostatus was determined using Spike S1-specific IgG ELISA. We determined county-wise proportions of seropositivity. We assessed underestimation of infections, county and age specific infection fatality risks, and association of seropositivity with demographic, socioeconomic and health factors.We found seroprevalence of 2.4 % (95%CI: 1.8-3.1%) for Reutlingen in June 2020 (stage 1) which increased to 2.9% (95%CI: 2.1-3.8%) in October (stage 2), Freiburg stage 1 1.5% (95% CI: 1.1-2.1%) vs. 2.5% (95%CI: 1.8-3.4%), Aachen stage 1 2.3% (95% CI: 1.7-3.1%) vs. 5.4% (95%CI: 4.4-6.6%), Osnabrück 1.3% (95% CI: 1.0-1.9%) and Magdeburg in Nov/Dec 2020. 2.4% (95%CI 1.9-3.1%). Number needed to quarantine to have one infected person quarantined was 8.2. The surveillance detection ratio (SDR) between number of infections based on our results and number reported to health authorities ranged from 2.5-4.5. Participants aged 80+ had lower SDR. Infection fatality estimates ranged from 0.2-2.4%. Lower education was associated with higher, smoking with lower seropositivity.Seroprevalence remained low until December 2020 with high underdetection. The second wave from November 2020 to February 2021 resulted in additional 2-5% of the population being infected. Detected age specific differences of SDR should be taken into account in modelling and forecasting COVID-19 morbidity.HighlightsEvidence before this studySeroepidemiological surveys on SARS-CoV-2 are a useful tool to track the transmission during the epidemic. We searched PubMed/the pre-print server medRxiv and included web-based reports from German health organizations using the keywords “seroprevalence”, “SARS-CoV-2”, “Germany” and similar other English and German terms in the period from January 1st, 2020 until March 2021. We identified 30 published studies in Germany which mostly report low SARS-CoV-2 seroprevalence (Added value of this studyWe provide the first comprehensive, high-precision multi-region population-based SARS-CoV-2 seroprevalence study with representative sampling following the WHO protocol in Germany. By measuring SARS-CoV-2 IgG, we explore immunity at regional and national level over time. We also assess risk factors and sample each region twice, which permits to monitor seroprevalence progression throughout the epidemic in different exemplary German regions.Implications of all the available evidenceOur results show low seroprevalence (

Background RNA viruses are associated with a high frequency of mutations because of the missing proofreading function of polymerases, such as reverse transcriptase. Between 2007 and 2010, six blood donations with false-negative nucleic acid technology (NAT) results were reported in Germany. Therefore, NAT screening in two viral genome regions was introduced by our blood donation service in 2010 on a voluntary basis and became mandatory in Germany since the beginning of 2015. Study design and methods Blood donor screening was done using, in parallel, the German Red Cross (GRC) HIV-1 CE long terminate repeats (LTR) PCR kit and the GRC HIV-1 gag CE PCR kit. In total, 7 million blood donations were screened during the study period from 2010 to 2014 with the GRC dual-target human immunodeficiency virus 1 (HIV-1) NAT system. Additionally, three suspicious specimens were analyzed by four monotargeted NAT assays and by five dual-target NAT assays. Results Three of 7 million donations tested negative using the 5'LTR-polymerase chain reaction, but they were positive if amplification was performed in the gag region. HIV antibodies were detected in all three donations. Nucleic acid sequence analysis identified a deletion of 22 bases within the 5'LTR probe binding region. Three different ltr-based monotargeted assays missed two donations, except for a low-reactive result obtained by one of the assays. In total, the detection rates for HIV-1-positive donations were 37.5% (3/8) for monotargeted assays and 100% (10/10) for dual-target assays. Conclusion The current data demonstrate that dual-target NAT systems reduce the risk of false-negative HIV-1 NAT screening results.

Background Human parvovirus B19 (B19V) infection and damage of circulating angiogenic cells (CAC) results in dysfunctional endogenous vascular repair (DEVR) with secondary end-organ damage. Trafficking of CAC is regulated by SDF-1α and the respective receptor CXCR4. We thus tested the hypothesis of a deregulated CXCR4/SDF-1α axis in symptomatic B19V-cardiomyopathy. Methods CAC were infected in vitro with B19V and transfected with B19V-components. Read-out were: CXCR4-expression and migratory capacity at increasing doses of SDF-1α. In 31 patients with chronic B19V-cardiomyopathy compared to 20 controls read-outs were from blood: migratory capacity, CXCR4 expression on CAC, serum SDF-1α; from cardiac biopsies: SDF-1α mRNA, HIF-1α mRNA, microvascular density, resident cardiac stem cells (CSC), transcardiac gradients of CAC. Results In vitro B19V-infected CAC showed up-regulation of surface CXCR4 with increased migratory capacity further enhanced by elevated SDF-1α concentrations. Overexpression of the B19V capsid protein VP2 was associated with this effect. Chronic B19V-cardiomyopathy patients showed increased numbers of ischaemia mobilised CAC but DEVR as well as diminished numbers of CAC after transcardiac passage. Cardiac microvascular density and CSC were significantly reduced in B19V-cardiomyopathy. Conclusions We thus conclude that B19V infection has a direct VP2-mediated negative impact on trafficking of CAC in the presence of impaired cardiac regeneration.

RNA viruses are associated with a high frequency of mutations because of the missing proofreading function of polymerases, such as reverse transcriptase. Between 2007 and 2010, six blood donations with false-negative nucleic acid technology (NAT) results were reported in Germany. Therefore, NAT screening in two viral genome regions was introduced by our blood donation service in 2010 on a voluntary basis and became mandatory in Germany since the beginning of 2015.Blood donor screening was done using, in parallel, the German Red Cross (GRC) HIV-1 CE long terminate repeats (LTR) PCR kit and the GRC HIV-1 gag CE PCR kit. In total, 7 million blood donations were screened during the study period from 2010 to 2014 with the GRC dual-target human immunodeficiency virus 1 (HIV-1) NAT system. Additionally, three suspicious specimens were analyzed by four monotargeted NAT assays and by five dual-target NAT assays.Three of 7 million donations tested negative using the 5'LTR-polymerase chain reaction, but they were positive if amplification was performed in the gag region. HIV antibodies were detected in all three donations. Nucleic acid sequence analysis identified a deletion of 22 bases within the 5'LTR probe binding region. Three different ltr-based monotargeted assays missed two donations, except for a low-reactive result obtained by one of the assays. In total, the detection rates for HIV-1-positive donations were 37.5% (3/8) for monotargeted assays and 100% (10/10) for dual-target assays.The current data demonstrate that dual-target NAT systems reduce the risk of false-negative HIV-1 NAT screening results.

Background Occult hepatitis B virus (HBV) infection (OBI) is identified in 1:1000 to 1:50,000 European blood donations. This study intended to determine the infectivity of blood products from OBI donors. Study Design and Methods Recipients of previous donations from OBI donors were investigated through lookback (systematic retrieval of recipients) or traceback (triggered by clinical cases). Serologic and genomic studies were undertaken on consenting donors and recipients. Multiple variables potentially affecting infectivity were examined. Results A total of 45 of 105 (42.9%) donor-recipients pairs carried antibodies to HBV core (anti-HBc) as evidence of previous HBV infection. Subtracting 15% of anti-HBc population background, the adjusted transmission rate was 28%. Anti-HBc prevalence increased to 28 of 44 (63.8%) in unvaccinated recipients receiving anti-HBs–negative OBI blood products. In contrast, four of 26 (15.4%) recipients of anti-HBs–positive products were anti-HBc positive. Transmission with anti-HBs–negative products depended on volume of plasma transfused (85%-100% with 200 mL of fresh frozen plasma [FFP], 51% with 50 mL in platelet concentrates [PCs], and 24% with 20 mL in red blood cells [RBCs], p

BACKGROUND: In February 2007, a 63-year-old man underwent surgery. Retrospective testing with nucleic acid testing (NAT) showed that the patient was human immunodeficiency virus Type 1 (HIV-1) positive 10 days after transfusion. The transfusion-transmitted infection had been identified by a donor-related lookback started in April 2007 after anti-HIV seroconversion.METHODS: Sequence analysis was performed in the gag-pol region as well as in the V3 loop env region. Archived plasma from the transmitting donation was investigated for the individual-donation NAT with the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Roche CAP/CTM HIV-1 test) and for HIV antigen/antibody combination testing (Abbott Architect). Additional testing was done on the donor's follow-up sample and on the recipient's sample. RESULTS: The Roche CAP/CTM HIV-1 test failed to detect viral RNA by minipool NAT in the index donation (April 2007) as well as in the donation that caused the infection (January 2007). Phylogenetic analysis showed a very high genetic similarity among viral sequences from both donor and recipient, proving the HIV-1 transmission by sequence data. CONCLUSION: This case represents the first documented HIV-1 transmission by transfusion of red blood cells after mandatory introduction of HIV-1 NAT for blood screening in Germany. Low viral load and mismatches in the primer/probe region might explain the detection failure of the NAT screening assay. A certain risk remains that new virus variants contain mutations at positions critical for amplification or detection of viral genomes. An option to reduce the risk of a detection failure by NAT is the simultaneous use of several conserved regions as amplification targets.

In this study we have investigated whether streptolysin O contributes to the virulence of group A streptococci. For this purpose we generated M-negative and SLO-negative mutants by insertion mutagenesis into the chromosome of an M type 1 strain. The inactivation of M1 protein expression was achieved by the construction of the integrative plasmid pSFABS, which contains the internal fragment abs of the emm1 gene. Integration of pSFABS by homologous recombination into the chromosome of strain 38 541 resulted in the generation of mutant EMM1. Inactivation of slo with plasmid pFWSLOD resulted in two different mutant forms. The homologous recombination with plasmid pFWSLOD carrying the two slo fragments slo1 (899 base pairs in the 5' region) and slo2 (709 base pairs in the downstream part) resulted in mutants SLO3, SLO4 and SLO17. In SLO17 a double crossover event took place with insertion of the spectinomycin resistance gene aad9 between the slo fragments 1 and 2. In mutants SLO3 and SLO4 the homologous recombination with the same plasmid led to the integration of the whole plasmid construct into the chromosome of strain 38 541. Both forms of mutation failed to express SLO. In mutant SLO4 additionally M1 protein expression was significantly decreased. The mutants EMM1 (M − , SLO + ) and SLO4 (M decreased, SLO − ) showed a reduced binding to collagen-coated surfaces. In contrast the mutants SLO3 and SLO17 (both M + , SLO − ) and the wild-type strain 38 541 (M + , SLO + ) showed an affinity to collagen similar to purified M1 protein. All mutants were less virulent for chicken embryos compared to the wild-type strain after infection by intravenous injection as well as by application onto the chorioallantoic membrane. The results show that besides M protein SLO can also influence virulence of group A streptococci. Moreover, it became obvious that streptococci need more than one tool to fully develop their infectious potential.

The M and M-like proteins of Streptococcus pyogenes are fibrous cell surface proteins. They have multiple binding sites for several human proteins and are composed of the C-terminal anchor domain, the alpha-helical coiled-coil domain, and the N-terminal non-coiled-coil domain. The coiled-coil domain of the M1 protein consists of repeat units called B, C, and D and a spacer unit S between B and C. Recombinant fragments A-B-S-C-D, A-B-S, B-S-C, S-C, S-C-D, C-D, and C of the coiled-coil domain were studied by analyzing their secondary structures and binding affinities to human serum albumin (HSA). As shown by circular dichroism, all fragments are in an alpha-helical conformation. C-D and S-C-D form coiled coils at room temperature and bind below 37 degrees C with high affinity to HSA. C-D and S-C-D unfold in two steps with Tm values of approximately 31 and approximately 65 degrees C; complex formation with HSA increases the unfolding temperatures. B-S-C has a lower alpha-helical content, a less pronounced coiled-coil conformation, and a reduced thermal stability, binds HSA weaker, and is only slightly stabilized by HSA binding in comparison to C-D and S-C-D. C and S-C are less stable than the other fragments and are not organized as coiled coils showing some features of alpha-helical single strands only below 20 degrees C, and binding of HSA was not observed. The results indicate that the formation of coiled-coil structures, supported by flanking D regions and, to a lesser extent also B regions, is essential for the binding of C repeat units to HSA.

Summary Objective: Commercial available NAT systems are usually not validated for screening of post-mortem blood samples. NAT testing might be challenging due to inhibitory substances in the cadaveric blood sample that cause falsely negative test results. Validation studies have to be performed to show the performance characteristics of the NAT assays for testing cadaveric blood. Methods: A set of 32 post-mortem serum and plasma samples from cornea donors and 20 control samples from blood donors, serologically and NAT negative for all investigated parameters, were spiked with defined concentrations of WHO reference material and tested for HIV-1, HCV, HBV, and HAV by NAT using DRK Baden-Wurttemberg-Hesse CE PCR kits. Analytical sensitivity, analytical specificity and reproducibility/precision were validated and compared with each other in both groups of samples. Results: The analytical sensitivity was 100% for control and post-mortem specimens when spiked with virus standards at concentrations of 3 × level of detection (LOD). Invalid results did not occur. The analytical specificity rate for all assays was 100%. Intra-assay variation was analyzed as a function of sample material and sampling time post mortem. Values of % coefficient of variation (%CV) were comparable for serum and plasma but slightly higher for post-mortem samples especially for those samples collected more than 24 h post mortem. Conclusion: Based on the presented validation, postmortem donor samples can be tested with the automated DRK Baden-Wurtemberg-Hesse NAT system.

Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMerieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT.

BACKGROUND: The risk of transfusion-transmitted human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections is predominantly attributable to donations given during the early stage of infection when diagnostic tests may fail. In 1997, nucleic acid amplification technique (NAT)-testing was introduced at the German Red Cross (GRC) blood donor services to reduce this diagnostic window period (WP). STUDY DESIGN AND METHODS: A total of 31,524,571 blood donations collected from 1997 through 2005 were screened by minipool NAT, predominantly with pool sizes of 96 donations. These donations cover approximately 80 percent of all the blood collected in Germany during that period. Based on these data, the WP risk in the GRC blood donor population was estimated by using a state-of-the-art mathematic model. RESULTS: During the observation period, 23 HCV, 7 HIV-1, and 43 HBV NAT-only–positive donations were detected. On the basis of these data and estimated pre-NAT infectious WPs, the residual risk per unit transfused was estimated at 1 in 10.88 million for HCV (95% confidence interval [CI], 7.51-19.72 million), 1 in 4.30 million for HIV-1 (95% CI, 2.39-21.37 million), and 1 in 360,000 for HBV (95% CI, 0.19-3.36 million). Based on observed cases of breakthrough infections, the risk of transfusion-related infections may be even lower. CONCLUSION: The risk of a blood recipient becoming infected with HCV, HIV-1, or HBV has reached an extremely low level. Introduction of individual donation testing for HCV and HIV-1 would have a marginal effect on interception of WP donations.

The emm3-gene and the emm78-gene encoding the M-protein type 3 (OF-) resp. type 78 (OF+) of Streptococcus pyogenes were completely sequenced (AC-No.: X80168) and the recombinant proteins expressed in E. coli. Concerning the type 3 the cell surface antigen as well as the isolated protein revealed a pronounced binding affinity towards the human plasma proteins human serum albumin (HSA) and fibrinogen (FBG). In comparison type 78 showed a reduced HSA-binding affinity. Several lines of evidence have led to the assumption that plasma protein binding is a general phenomenon and plays a significant role for survival and even expansion of pathogenic streptococcal strains having successfully invaded human patients (2).

M protein of Streptococcus pyogenes has been postulated as the major virulence factor of group A streptococci (GAS) based on its association with a phagocytosis-resistant phenotype (4). The antiphagocytic properties against human granulocytes of M proteins have been demonstrated by using defined isogeneic mutants lacking expression of M protein (1, 6). However, other proteins M-related proteins like Mrp, have also been described to contribute to streptococcal resistance against phagocytic attack (7). The genes encoding the proteins of the M-family are located in the vir-regulon and were regulated by the multigene regulator gene mga (3). But, hyaluronic acid capsule (HA), chemically completely different from streptococcal M proteins, also interferes in the phagocytic defense against streptococci (10) and it could be shown that both, M protein and HA, contribute to the virulence of one streptococcal strain (2, 9). In this study we investigated whether the chicken embryo model is worth to measure changes in virulence of streptococcal strains after isogeneic inactivation of genes encoding different M- and M-like proteins but also of genes encoding additional potential pathogenicity factors like streptococcal proteinase (SPEB), or the oligopeptide permease oppD.

Spinal cord chimeras were produced by replacing a small fragment of neural tube of a 2-day-old White Leghorn chicken embryo with a similar fragment from a Japanese quail embryo. The embryo mortality w