Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development.

The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA+ CD4+ Tmem cells from blood and CD69+ Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.

Algorithms for the Testing of Tissue Donors for Human Immunodeficiency Virus, Hepatitis B Virus, and Hepatitis C Virus.

BACKGROUND: Although transmission of pathogenic viruses through human tissue grafts is rare, it is still one of the most serious dreaded risks of transplantation. Therefore, in addition to the detailed medical and social history, a comprehensive serologic and molecular screening of the tissue donors for relevant viral markers for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) is necessary. In the case of reactive results in particular, clear decisions regarding follow-up testing and the criteria for tissue release must be made. METHODS: Based on the clinical relevance of the specific virus markers, the sensitivity of the serological and molecular biological methods used and the application of inactivation methods, algorithms for tissue release are suggested. RESULTS: Compliance with the preanalytical requirements and assessment of a possible hemodilution are mandatory requirements before testing the blood samples. While HIV testing follows defined algorithms, the procedures for HBV and HCV diagnostics are under discussion. Screening and decisions for HBV are often not as simple, e.g., due to cases of occult HBV infection, false-positive anti-HBc results, or early window period positive HBV NAT results. In the case of HCV diagnostics, modern therapies with direct-acting antivirals, which are often associated with successful treatment of the infection, should be included in the decision. CONCLUSION: In HBV and HCV testing, a high-sensitivity virus genome test should play a central role in diagnostics, especially in the case of equivocal serology, and it should be the basis for the decision to release the tissue. The proposed test algorithms and decisions are also based on current European recommendations and standards for safety and quality assurance in tissue and cell banking.

High Prevalence of Infectious Adeno-associated Virus (AAV) in Human Peripheral Blood Mononuclear Cells Indicative of T Lymphocytes as Sites of AAV Persistence.

Seroepidemiology shows that infections with adeno-associated virus (AAV) are widespread, but diverse AAV serotypes isolated from humans or nonhuman primates have so far not been proven to be causes of human disease. In view of the increasing success of AAV-derived vectors in human gene therapy, definition of the in vivo sites of wild-type AAV persistence and the clinical consequences of its reactivation is becoming increasingly urgent. Here, we identify the presumed cell type for AAV persistence in the human host by highly sensitive AAV PCRs developed for the full spectrum of human AAV serotypes. In genomic-DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV positive, predominantly AAV type 2 (AAV2) (77%), AAV5 (19%), and additional serotypes. Roughly 11% of the blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, 76% of whom were AAV positive. Of these, at least 45% displayed mixed infections. Follow-up of single blood donors over 2 years allowed repeated detection of the initial and/or additional AAV serotypes, suggestive of fluctuating, persistent infection. Leukocyte separation revealed that AAV resided in CD3+ T lymphocytes, perceived as the putative in vivo site of AAV persistence. Moreover, infectious AAVs of various serotypes could be rescued and propagated from numerous samples. The high prevalence and broad spectrum of human AAVs in leukocytes closely follow AAV seroepidemiology. Immunosuppression obviously enhances AAV replication in parallel with activation of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), reminiscent of herpesvirus-induced AAV activation. IMPORTANCE: Adeno-associated virus is viewed as apathogenic and replication defective, requiring coinfection with adenovirus or herpesvirus for productive infection. In vivo persistence of a defective virus requires latency in specialized cell types to escape the host immune response until viral spread becomes possible. Reactivation from latency can be induced by diverse stimuli, including infections, typically induced upon host immunosuppression. We show for the first time that infectious AAV is highly prevalent in human leukocytes, specifically T lymphocytes, and that AAV is strongly amplified upon immunosuppression, along with reactivation of latent human herpesviruses. In the absence of an animal model to study the AAV life cycle, our findings in the human host will advance the understanding of AAV latency, reactivation, and in vivo pathogenesis.

Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study.

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1ß, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

European Multicenter Study on Analytical Performance of Veris HIV-1 Assay.

The analytical performance of the Veris HIV-1 assay for use on the new, fully automated Beckman Coulter DxN Veris molecular diagnostics system was evaluated at 10 European virology laboratories. The precision, analytical sensitivity, performance with negative samples, linearity, and performance with HIV-1 groups/subtypes were evaluated. The precision for the 1-ml assay showed a standard deviation (SD) of 0.14 log10 copies/ml or less and a coefficient of variation (CV) of ≤6.1% for each level tested. The 0.175-ml assay showed an SD of 0.17 log10 copies/ml or less and a CV of ≤5.2% for each level tested. The analytical sensitivities determined by probit analysis were 19.3 copies/ml for the 1-ml assay and 126 copies/ml for the 0.175-ml assay. The performance with 1,357 negative samples demonstrated 99.2% with not detected results. Linearity using patient samples was shown from 1.54 to 6.93 log10 copies/ml. The assay performed well, detecting and showing linearity with all HIV-1 genotypes tested. The Veris HIV-1 assay demonstrated analytical performance comparable to that of currently marketed HIV-1 assays. (DxN Veris products are Conformité Européenne [CE]-marked in vitro diagnostic products. The DxN Veris product line has not been submitted to the U.S. FDA and is not available in the U.S. market. The DxN Veris molecular diagnostics system is also known as the Veris MDx molecular diagnostics system and the Veris MDx system.).

Prognostic factors for remission of and survival in acquired hemophilia A (AHA): results from the GTH-AH 01/2010 study.

Acquired hemophilia A (AHA) is caused by autoantibodies against factor VIII (FVIII). Immunosuppressive treatment (IST) results in remission of disease in 60% to 80% of patients over a period of days to months. IST is associated with frequent adverse events, including infections as a leading cause of death. Predictors of time to remission could help guide IST intensity but have not been established. We analyzed prognostic factors in 102 prospectively enrolled patients treated with a uniform IST protocol. Partial remission (PR; defined as no active bleeding, FVIII restored >50 IU/dL, hemostatic treatment stopped >24 hours) was achieved by 83% of patients after a median of 31 days (range 7-362). Patients with baseline FVIII <1 IU/dL achieved PR less often and later (77%, 43 days) than patients with ≥1 IU/dL (89%, 24 days). After adjustment for other baseline characteristics, low FVIII remained associated with a lower rate of PR (hazard ratio 0.52, 95% confidence interval 0.33-0.81, P < .01). In contrast, PR achieved on steroids alone within ≤21 days was more common in patients with FVIII ≥1 IU/dL and inhibitor concentration <20 BU/mL (odds ratio 11.2, P < .0001). Low FVIII was also associated with a lower rate of complete remission and decreased survival. In conclusion, presenting FVIII and inhibitor concentration are potentially useful to tailor IST in AHA.

Regular blood donation may help in the management of hypertension: an observational study on 292 blood donors.

BACKGROUND: Hypertension is one of the leading global risks for cardiovascular events worldwide. There is preliminary evidence that regular blood donation may be beneficial. STUDY DESIGN AND METHODS: Unselected blood donors were included in this observational study. Blood pressure (BP) was measured before and after blood donation, with participants donating between one and four occasions in a 1-year study period. RESULTS: In this study, 292 donors were enrolled. At baseline, 146 had elevated BP (> 140/90 mmHg). In hypertensives, after four blood donations, systolic and diastolic blood pressure (SBP and DBP, respectively) decreased from a mean of 155.9 ± 13.0 to 143.7 ± 15.0 mmHg and from 91.4 ± 9.2 to 84.5 ± 9.3 mmHg, respectively (each p < 0.001). There was a clear dose effect with decreasing BP by the increasing number of blood donations. After at least four blood donations, donors with Stage II hypertensive baseline values (≥ 160 mmHg SBP and/or ≥ 100 mmHg DBP) were found to have the most marked reduction in BP, with 17.1 mmHg (95% confidence interval [CI], -23.2 to -11.0; p < 0.0001) and 11.7 mmHg (95% CI, -17.1 to -6.1; p = 0.0006) for SBP and DBP, respectively. The decrease in BP was not significantly associated with changes of blood count or variables of iron metabolism. CONCLUSIONS: Regular blood donation is associated with pronounced decreases of BP in hypertensives. This beneficial effect of blood donation may open a new door regarding community health care and cost reduction in the treatment of hypertension.

Validation of Serological Testing for Anti-Treponema pallidum from Postmortem Blood on the Siemens-BEP(®)-III Automatic System.

BACKGROUND: Infectious disease marker testing is obligatory for the release of human tissue for transplantation. Most CE-marked tests are not validated for postmortem blood. In a previous study we have validated the testing for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc. Here, we present the validation of testing for antibodies against T. pallidum, which is the last marker obligatory for tissue release for transplantation. METHODS: 17 samples of postmortem sera and 10 samples of both pre-und postmortem sera were obtained from cornea donors and tested for anti-T. pallidum on the Siemens-BEP-III-System. These sera were spiked with anti-T. pallidum-positive standard sera in concentrations which give low- and high-positive results at the respective dilution. RESULTS: Two of the unspiked postmortem sera were false-positive most likely due to intense hemolysis (free hemoglobin > 50 mg/dl). Of the 25 negative postmortem sera, none of the spiked samples was false-negative after 0, 24 and 60 h. CONCLUSION: There is no indication that postmortem samples give false-negative or false-positive results with the test system and test kits used in cases of low hemolysis. The procedure described might serve as a model for validating other test kits on postmortem samples.

Recognition mechanisms of hemoglobin particles by monocytes - CD163 may just be one.

Hemoglobin-based oxygen carriers (HBOCs) as blood substitutes are one of the great hopes of modern transfusion and emergency medicine. After the major safety-relevant challenges of the last decades seem to be largely overcome, current developments have in common that they are affected by degradation and excretion at an early stage in test organisms. Several possible mechanisms that may be responsible for this are discussed in the literature. One of them is CD163, the receptor of the complex of haptoglobin (Hp) and hemoglobin (Hb). The receptor has been shown in various studies to have a direct affinity for Hb in the absence of Hp. Thus, it seems reasonable that CD163 could possibly also bind Hb within HBOCs and cause phagocytosis of the particles. In this work we investigated the role of CD163 in the uptake of our hemoglobin sub-micron particles (HbMPs) in monocytes and additionally screened for alternative ways of particle recognition by monocytes. In our experiments, blockade of CD163 by specific monoclonal antibodies proved to partly inhibit HbMP uptake by monocytes. It appears, however, that several other phagocytosis pathways for HbMPs might exist, independent of CD163 and also Hb.

Effects of phlebotomy-induced reduction of body iron stores on metabolic syndrome: results from a randomized clinical trial.

BACKGROUND: Metabolic syndrome (METS) is an increasingly prevalent but poorly understood clinical condition characterized by insulin resistance, glucose intolerance, dyslipidemia, hypertension, and obesity. Increased oxidative stress catalyzed by accumulation of iron in excess of physiologic requirements has been implicated in the pathogenesis of METS, but the relationships between cause and effect remain uncertain. We tested the hypothesis that phlebotomy-induced reduction of body iron stores would alter the clinical presentation of METS, using a randomized trial. METHODS: In a randomized, controlled, single-blind clinical trial, 64 patients with METS were randomly assigned to iron reduction by phlebotomy (n = 33) or to a control group (n = 31), which was offered phlebotomy at the end of the study (waiting-list design). The iron-reduction patients had 300 ml of blood removed at entry and between 250 and 500 ml removed after 4 weeks, depending on ferritin levels at study entry. Primary outcomes were change in systolic blood pressure (SBP) and insulin sensitivity as measured by Homeostatic Model Assessment (HOMA) index after 6 weeks. Secondary outcomes included HbA1c, plasma glucose, blood lipids, and heart rate (HR). RESULTS: SBP decreased from 148.5 ± 12.3 mmHg to 130.5 ± 11.8 mmHg in the phlebotomy group, and from 144.7 ± 14.4 mmHg to 143.8 ± 11.9 mmHg in the control group (difference -16.6 mmHg; 95% CI -20.7 to -12.5; P < 0.001). No significant effect on HOMA index was seen. With regard to secondary outcomes, blood glucose, HbA1c, low-density lipoprotein/high-density lipoprotein ratio, and HR were significantly decreased by phlebotomy. Changes in BP and HOMA index correlated with ferritin reduction. CONCLUSIONS: In patients with METS, phlebotomy, with consecutive reduction of body iron stores, lowered BP and resulted in improvements in markers of cardiovascular risk and glycemic control. Blood donation may have beneficial effects for blood donors with METS. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01328210 Please see related article: http://www.biomedcentral.com/1741-7015/10/53.

Virus NAT for HIV, HBV, and HCV in Post-Mortal Blood Specimens over 48 h after Death of Infected Patients - First Results.

OBJECTIVE: According to EU regulations (EU directive 2006/17/EC), blood specimens for virologic testing in the context of post-mortal tissue donation must be taken not later than 24 h post mortem. METHODS: To verify validity of NAT in blood specimens collected later, viral nucleic acid concentrations were monitored in blood samples of deceased persons infected with HIV (n = 7), HBV (n = 5), and HCV (n = 17) taken upon admission and at 12 h, 24 h, 36 h and 48 h post mortem. HIV and HCV RNA were quantified using Cobas TaqMan (Roche), HBV DNA was measured by in-house PCR. RESULTS: A more than 10-fold decrease of viral load in samples taken 36 h or 48 h post mortem was seen in one HIV-infected patient only. For all other patients tested the decrease of viral load in 36-hour or 48-hour post-mortal samples was less pronounced. Specimens of 3 HIV- and 2 HBV-infected patients taken 24 h post mortem or later were even found to have increased concentrations (>10-fold), possibly due to post-mortem liberation of virus from particular cells or tissues. CONCLUSION: Our preliminary data indicate that the time point of blood collection for HIV, HBV and HCV testing by PCR may be extended to 36 h or even 48 h post mortem and thus improve availability of tissue donations.

Validation of Virus NAT for HIV, HCV, HBV and HAV Using Post-Mortal Blood Samples.

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 false-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 40 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-Württemberg-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-Würtemberg-Hesse NAT system.

Bacterial safety study of the production process of hemoglobin-based oxygen carriers.

Hemoglobin microparticles (HbMP) produced with a three-step procedure, including coprecipitation of hemoglobin with manganese carbonate, protein cross-linking, and dissolution of the carbonate template were shown to be suitable for application as artificial oxygen carriers. First preclinical safety investigations delivered promising results. Bacterial safety plays a decisive role during the production of HbMP. Therefore, the bioburden and endotoxin content of the starting materials (especially hemoglobin) and the final particle suspension are intensively tested. However, some bacteria may not be detected by standard tests due to low concentration. The aim of this study was to investigate how these bacteria would behave in the fabrication process. Biocidal effects are known for glutaraldehyde and for ethylenediaminetetraacetic acid, chemicals that are used in the fabrication process of HbMP. It was shown that both chemicals prevent bacterial growth at the concentrations used during HbMP fabrication. In addition, the particle production was carried out with hemoglobin solutions spiked with Escherichia coli or Staphylococcus epidermidis. No living bacteria could be detected in the final particle suspensions. Therefore, we conclude that the HbMP fabrication procedure is safe in respect of bacterial contamination.

Adding a piece to the puzzle of Latin American blood donors and the potential risk of Trypanosoma cruzi transmission in Germany.

Introduction: Chagas disease (CD) is caused by the Trypanosoma cruzi (T. cruzi) infection and has become a global health concern due to population mobility, as well as non-vectorial transmission routes. Several countries outside Latin America (LA) have reported transfusion-associated transmission, but equivalent studies in Germany are lacking. This study aims to collect first data on the risk of transfusion associated transmission as well as LA blood donors originating from CD endemic countries in Germany. Materials and methods: A total of 305 blood donors who were assumed to be at risk for T. cruzi infection were retrospectively (267) as well as prospectively (38) selected at German blood donation sites in Bavaria and Berlin, and all retrospectively as well as 27 prospectively selected were serologically screened. Prospective study subjects additionally filled out a questionnaire. Results: All samples tested seronegative for T. cuzi specific antibodies. Prospectively enrolled study subjects all had high socio-economic status including good education. Knowledge regarding CD was limited but willingness to donate frequently was high. Blood donation rates from donors born in LA countries seem to increase from 2015. Discussion: Although no transfusion associated T. cruzi infection has been documented in Germany, it has likely already happened unnoticed, or will do in the near future. Performing risk-adapted serology-based blood donor screenings in Germany could avoid transfusion-associated transmission events as well as contribute to active case detection. Moreover, larger, and ongoing studies are needed to increase the evidence base as well as end the neglect of CD in Germany.

SARS-CoV-2 T Cell Response in Severe and Fatal COVID-19 in Primary Antibody Deficiency Patients Without Specific Humoral Immunity.

Morbidity and mortality of COVID-19 is increased in patients with inborn errors of immunity (IEI). Age and comorbidities and also impaired type I interferon immunity were identified as relevant risk factors. In patients with primary antibody deficiency (PAD) and lack of specific humoral immune response to SARS-CoV-2, clinical disease outcome is very heterogeneous. Despite extensive clinical reports, underlying immunological mechanisms are poorly characterized and levels of T cellular and innate immunity in severe cases remain to be determined. In the present study, we report clinical and immunological findings of 5 PAD patients with severe and fatal COVID-19 and undetectable specific humoral immune response to SARS-CoV-2. Reactive T cells to SARS-CoV-2 spike (S) and nucleocapsid (NCAP) peptide pools were analyzed comparatively by flow cytometry in PAD patients, convalescents and naïve healthy individuals. All examined PAD patients developed a robust T cell response. The presence of polyfunctional cytokine producing activated CD4+ T cells indicates a memory-like phenotype. An analysis of innate immune response revealed elevated CD169 (SIGLEC1) expression on monocytes, a surrogate marker for type I interferon response, and presence of type I interferon autoantibodies was excluded. SARS-CoV-2 RNA was detectable in peripheral blood in three severe COVID-19 patients with PAD. Viral clearance in blood was observed after treatment with COVID-19 convalescent plasma/monoclonal antibody administration. However, prolonged mucosal viral shedding was observed in all patients (median 67 days) with maximum duration of 127 days. PAD patients without specific humoral SARS-CoV-2 immunity may suffer from severe or fatal COVID-19 despite robust T cell and normal innate immune response. Intensified monitoring for long persistence of SARS-CoV-2 viral shedding and (prophylactic) convalescent plasma/specific IgG as beneficial treatment option in severe cases with RNAemia should be considered in seronegative PAD patients.

Plasma viscosity in giant cell arteritis.

BACKGROUND: Diagnosis of giant cell arteritis (GCA) is based on criteria of the American College of Rheumatology. However, not all GCA patients meet these criteria and treatment may be delayed in individual patients, leading to an increased risk of complications. METHODS: In an observational study, we investigated acute phase response markers in GCA and non-GCA patients matched for erythrocyte sedimentation rate and CRP levels. RESULTS: Plasma viscosity (PV) was significantly elevated in all GCA patients, but normal in non-GCA patients. CONCLUSIONS: Our data suggest that PV may reflect a more specific component of the acute inflammatory response in patients with GCA. Analysis of PV may significantly contribute to a reliable diagnosis early in the course of the disease, particularly in patients with suspected GCA that do not meet current diagnostic criteria.

Validation of the Serological Testing for Anti-HIV-1/2, Anti-HCV, HBsAg, and Anti-HBc from Post-mortem Blood on the Siemens-BEP-III Automatic System.

BACKGROUND: Some properties of blood are modified post mortem. These modifications might give false-negative or false-positive results in infectious disease testing. Most CE-marked test equipment for infectious serology testing is not validated for testing post-mortal blood. Validation, however, is obligatory, if the results are used for the release of tissues for transplantation. MATHODS: Samples of pre- and post-mortem sera were obtained from 20 cornea donors, and the results were compared for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc on the Siemens-BEP-III Automatic System. Negative post-mortem sera were spiked with standard sera (PEI anti-HCV IgG, PEI HBsAg ad 1000 standard, anti-HBc IgG (WHO) NIBSC 95/522, PEI anti-HIV-IV) in concentrations which give low- and high-positive results for the respective marker. RESULTS: All pre-mortem sera were negative for all markers. None of the post-mortem samples was false-positive. None of the spiked postmortem samples was false-negative. Technical errors occurred during the validation process but could be detected and eliminated. Serum samples should be centrifuged immediately after collection, and it must be taken into account that post-mortem serum could rarely lead to blockage of pipetting systems due to clotting phenomena. CONCLUSION: There is no indication that post-mortem samples give false-negative or false-positve results with the test system and test kits used. The procedure described might serve as a model for validating other test kits on post-mortem samples.

Improvement of an ultrasensitive human immunodeficiency virus type 1 real-time reverse transcriptase-polymerase chain reaction targeting the long terminal repeat region.

BACKGROUND: Human immunodeficiency virus Type 1 (HIV-1) assays applying nucleic acid testing (NAT) rely on HIV-1 sequence-specific primers and probes. Their hybridization can be limited or abolished by genetic polymorphisms occurring in the target sequence. STUDY DESIGN AND METHODS: Blood donations are routinely tested for HIV-1/2 antibodies and for HIV-1 RNA in our blood transfusion unit. Recently, HIV-1 RNA was undetectable with an established in-house real-time long terminal repeat (LTR) reverse transcriptase-polymerase chain reaction (RT-PCR) in two cases, whereas serologic assays were positive. The reason for this discrepancy was elucidated by sequencing of the NAT target region in the respective single donations. An improved primer was designed and tested on HIV-1 reference panels and blood donations to ensure reliable detection of HIV-1 RNA. RESULTS: Direct sequencing of the target region, isolated from samples of two unrelated HIV-positive blood donors, revealed one and four mismatches in the hybridization domain of the forward primer, respectively. Both viruses belong to HIV-1 Subtype B. LTR RT-PCR with an additional forward primer was suitable for all strains of HIV-1 tested with high sensitivity. CONCLUSIONS: Surveillance of HIV-1 genetic diversity is essentially required to continually evaluate its impact on performance of diagnostic and patient monitoring assays.

Validation of a hospital-laboratory workstation for immunohematologic methods.

BACKGROUND: The FREELYS Nano system (Diagast) is a manual workstation for ABO/D grouping, Rh phenotyping, K typing, and antibody screening (ABS) for immunoglobulin G (IgG) antibodies only and works with the erythrocyte-magnetized technology (EMT). The principle of EMT is based on magnetization of red blood cells and avoids centrifugation and washing steps. STUDY DESIGN AND METHODS: A total of 304 samples were tested with our routine blood bank methods, 100 samples for ABO/D grouping, 196 samples (100 at first evaluation, 96 at second evaluation) for Rh phenotyping and K typing (PK7200, Olympus), and 108 samples for ABS (DiaMed). All samples were tested in parallel with the FREELYS Nano. RESULTS: We found a 100% concordance between the observed (FREELYS Nano) and the expected (Olympus PK7200) results for ABO/D grouping in all 100 samples. For Rh phenotyping and K tests, in 24 of 100 samples false-positive reactions were observed in the first evaluation by the FREELYS Nano. After changing the test kit batch for Rh phenotyping by the manufacturer, a complete concordance in Rh phenotyping and K tests was observed in a second evaluation. For ABS, the FREELYS Nano showed in 4 of 108 samples (3.7%) false-negative reactions for IgG antibodies (two anti-K, one anti-E, one anti-C(w)), and one (0.9%) false-positive reaction. CONCLUSIONS: The FREELYS Nano is reliably suited to ABO/D grouping, Rh phenotyping, and K testing. The rate of false-negative reactions for IgG antibodies should be reduced.

Effect of CYSTUS052 and green tea on subjective symptoms in patients with infection of the upper respiratory tract.

Examples of medicinal herbs that have been perpetuated along several generations based simply on a folk tradition are Cistus and green tea. The principal active constituents of the genus Cistus and green tea are polyphenolic compounds. Polyphenols exhibit a wide range of antibacterial, antifungal and antiinflammatory effects. The present work aimed to investigate the clinical effect of a Cistus extract (CYSTUS052) in comparison with green tea on 300 patients with infections of the upper respiratory tract. Due to the lack of clinical study data on their efficacy in patients, this is a report of the findings of our study on the clinical efficacy of CYSTUS052 in patients with the upper respiratory tract infections (URTIs). This study observed a total of 300 patients (277 completers) treated with CYSTUS052 given in lozenges compared with treatment with an extract of green tea. The patients scored the subjective severity of target symptoms using a predefined scale. The score of subjective symptoms decreased over the course of treatment with CYSTUS052, whereas treatment with green tea resulted in a less significant decrease of symptoms. CYSTUS052 therefore proved to be an effective adjuvant for the treatment of respiratory infections.