Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Aluminium in plasma and tissues after intramuscular injection of adjuvanted human vaccines in rats.

Aluminium (Al) toxicokinetics after intramuscular (IM) injection of Al-adjuvanted vaccines is unknown. Since animal data are required for modeling and extrapolation, a rat study was conducted measuring Al in plasma and tissues after IM injection of either plain Al-hydroxide (pAH) or Al-phosphate (pAP) adjuvant (Al dose 1.25 mg), single human doses of three Al-adjuvanted vaccines (V1, V2, and V3; Al doses 0.5-0.82 mg), or vehicle (saline). A significant increase in Al plasma levels compared to controls was observed after pAP (AUC(0-80 d), mean ± SD: 2424 ± 496 vs. 1744 ± 508 µg/L*d). Percentage of Al dose released from injected muscle until day 80 was higher after pAP (66.9%) and AP-adjuvanted V3 (85.5%) than after pAH and AH-adjuvanted V1 (0 and 22.3%, resp.). Estimated absolute Al release was highest for pAP (836.8 µg per rat). Al concentration in humerus bone was increased in all groups, again strongest in the pAP group [3.35 ± 0.39 vs. 0.05 ± 0.06 µg/g wet weight (ww)]. Extrapolated amounts in whole skeleton corresponded to 5-12% of the released Al dose. Very low brain Al concentrations were observed in all groups (adjuvant group means 0.14-0.29 µg/g ww; control 0.13 ± 0.04 µg/g ww). The results demonstrate systemically available Al from marketed vaccines in rats being mainly detectable in bone. Al release appears to be faster from AP- than AH-adjuvants. Dose scaling to human adults suggests that increase of Al in plasma and tissues after single vaccinations will be indistinguishable from baseline levels.

Development of a World Health Organization International Reference Panel for different genotypes of hepatitis E virus for nucleic acid amplification testing.

BACKGROUND: Globally, hepatitis E virus (HEV) is a major cause of acute viral hepatitis. Epidemiology and clinical presentation of hepatitis E vary greatly by location and are affected by the HEV genotype. Nucleic acid amplification technique (NAT)-based assays are important for the detection of acute HEV infection as well for monitoring chronic cases of hepatitis E. OBJECTIVES: The aim of the study was to evaluate a panel of samples containing different genotypes of HEV for use in nucleic NAT-based assays. STUDY DESIGN: The panel of samples comprises eleven different members including HEV genotype 1a (2 strains), 1e, 2a, 3b, 3c, 3e, 3f, 4c, 4g as well as a human isolate related to rabbit HEV. Each laboratory assayed the panel members directly against the 1st World Health Organization (WHO) International Standard (IS) for HEV RNA (6329/10) which is based upon a genotype 3 a strain. RESULTS: The samples for evaluation were distributed to 24 laboratories from 14 different countries and assayed on three separate days. Of these, 23 participating laboratories returned a total of 32 sets of data; 17 from quantitative assays and 15 from qualitative assays. The assays used consisted of a mixture of in-house developed and commercially available assays. The results showed that all samples were detected consistently by the majority of participants, although in some cases, some samples were detected less efficiently. CONCLUSIONS: Based on the results of the collaborative study the panel (code number 8578/13) was established as the "1st International Reference Panel (IRP) for all HEV genotypes for NAT-based assays" by the WHO Expert Committee on Biological Standardization. This IRP will be important for assay validation and ensuring adequate detection of different genotypes and clinically important sub-genotypes of HEV.

Aluminium toxicokinetics after intramuscular, subcutaneous, and intravenous injection of Al citrate solution in rats.

Knowledge of dose linearity, plasma clearance, rate and extent of subcutaneous (SC) and intramuscular (IM) absorption of soluble aluminium (Al) citrate is considered a prerequisite for evaluation of toxicokinetic data obtained from SC or IM administration of Al adjuvants in medicinal products. Therefore, total Al plasma kinetics was investigated after SC, IM, and IV administration of single Al doses (36 and 360 µg/kg IM or SC; 30 and 300 µg/kg IV) given as citrate solution in rats. Control groups receiving vehicle (saline) were run in parallel to monitor background plasma Al levels over time resulting from dietary intake. Evaluation of Al plasma profiles was done by both non-compartmental analysis of baseline-corrected data and simultaneous model fitting to the raw data using a population kinetics approach. High and dose-independent total plasma clearance (6.6 mL/min/kg) was observed after IV administration corresponding to 60-82% of normal rat GFR. This supports the previous assumptions that parenterally administered Al citrate is more rapidly cleared from plasma than other Al species (e.g., chloride or lactate). Furthermore, plasma exposure of Al (Cmax and AUC0-inf) increased dose-proportionally at all administration routes. Fast and complete absorption of Al was observed at each dose level after both SC and IM administration (bioavailability estimates: 88 and 110%). Estimates for the first-order absorption rate constant ka correspond to absorption half-lives of 36 min (SC) and ≤ 13 min (IM). There was no increase in tissue Al content (whole bone and brain) after 36 µg/kg IM compared to control rats.

Harmonization of nucleic acid testing for Zika virus: development of the 1st World Health Organization International Standard.

BACKGROUND: With the ongoing public health emergency due to Zika virus (ZIKV), nucleic acid testing (NAT) is essential for clinical diagnosis and screening of blood donors. However, NAT for ZIKV has not been standardized, and this study was performed to establish a World Health Organization (WHO) International Standard (IS) for ZIKV RNA; WHO ISs have been used to improve detection and quantification of blood-borne viruses. STUDY DESIGN AND METHODS: The candidate IS (cIS), code number 11468/16, was prepared by heat inactivation and lyophilization of a ZIKV strain isolated from a patient in French Polynesia in 2013. The cIS was evaluated together with other reference materials, including both Asian and African ZIKV lineages as well as a panel of clinical samples and in vitro-transcribed RNAs. The samples for evaluation were distributed to 24 laboratories from 11 countries. The assays used consisted of a mixture of in-house developed and commercial assays (available or in development). RESULTS: The potencies of the standards were determined by quantitative and qualitative assays. In total, 37 sets of data were analyzed: 19 from quantitative assays and 18 from qualitative assays. Data demonstrated wide variations in reported potencies of the cIS and the other study samples. CONCLUSIONS: Assay variability was substantially reduced when ZIKV RNA concentrations from the biological reference materials and clinical samples were expressed relative to the cIS. Thus, the WHO has established 11468/16 as the 1st IS for ZIKV RNA, with a unitage of 50,000,000 IU/mL.

Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity.

LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.

World Health Organization International Standard to harmonize assays for detection of hepatitis E virus RNA.

Nucleic acid amplification technique-based assays are a primary method for the detection of acute hepatitis E virus (HEV) infection, but assay sensitivity can vary widely. To improve interlaboratory results for the detection and quantification of HEV RNA, a candidate World Health Organization (WHO) International Standard (IS) strain was evaluated in a collaborative study involving 23 laboratories from 10 countries. The IS, code number 6329/10, was formulated by using a genotype 3a HEV strain from a blood donation, diluted in pooled human plasma and lyophilized. A Japanese national standard, representing a genotype 3b HEV strain, was prepared and evaluated in parallel. The potencies of the standards were determined by qualitative and quantitative assays. Assay variability was substantially reduced when HEV RNA concentrations were expressed relative to the IS. Thus, WHO has established 6329/10 as the IS for HEV RNA, with a unitage of 250,000 International Units per milliliter.

Transfusion-Transmitted Bacterial Infections - Haemovigilance Data of German Blood Establishments (1997-2010).

SUMMARY: METHODS: In order to evaluate the benefit of risk minimisation measures, reporting rates of transfusion-transmitted bacterial infections (TTBI) were calculated on the basis of annual reports and distributed blood components. Following the implementation of risk minimisation measures in 2003 and 2008, a comparison of pre- and post-implementation periods was performed. RESULTS: During a period of 14 years, 90 cases of TTBI were confirmed, 34 were caused by red blood cell (RBC) concentrates, 5 by fresh frozen plasma, and 51 by platelet concentrates (PCs). The overall reporting frequency was 1 TTBI in 1.91 million RBC units; 1 TTBI in 0.094 million PC units, and 1 TTBI-associated fatality in 0.57 million PC units. From 2001-2004 the reporting rate was 13.7 per million PC units; 2005-2008, after the implementation of pre-donation sampling; it was 10.8 per million PC units (p > 0.5). After limitation of the shelf life (2008), the reporting rate decreased to 4.49 per million PC units (p = 0.12), and one case of related fatality was reported. Agents with low pathogenicity were reported in 14 of 41 immunosuppressed patients (34%) but only in 1 of 13 patients with non-haematological/oncological diseases. CONCLUSION: TTBI and associated fatalities could be gradually reduced by the risk minimisation measures, but further strategies such as implementation of sensitive screening tests or pathogen-reducing approaches should be discussed.

Liver cancer protease activity profiles support therapeutic options with matrix metalloproteinase-activatable oncolytic measles virus.

Primary and secondary cancers of the liver are a significant health problem with limited treatment options. We sought here to develop an oncolytic measles virus (MV) preferentially activated in liver tumor tissue, thus reducing infection and destruction of healthy tissue. We documented that in primary tumor tissue, urokinase-type plasminogen activator and especially matrix metalloproteinase-2 (MMP-2) are significantly more active than in adjacent nontumorous tissue. We then generated variants of the MV fusion protein by inserting different MMP substrate motifs at the protease cleavage site and identified the motif PQGLYA as the most efficient cleavage site as determined by syncytia formation on protease-positive tumor cells. The corresponding MMP-activatable oncolytic MV-MMPA1 virus was rescued and shown to be strongly restricted on primary human hepatocytes and healthy human liver tissue, while remaining as effective as the parental MV in the tumor tissue sections. Our findings underline the clinical potency of the MMP activation concept as a strategy to generate safer oncolytic viruses for the treatment of primary and secondary cancers of the liver.

Assessment of component-resolved in vitro diagnosis of celeriac allergy.

BACKGROUND: Previous studies have demonstrated insufficient sensitivity of commercially available celeriac extract reagents in the diagnosis of celeriac allergy. OBJECTIVE: We sought to assess the diagnostic performance of specific IgE determination based on recombinant and purified natural celeriac allergens in comparison with an extract-based assay and to investigate interference by IgE to cross-reactive carbohydrate determinants and its biologic activity. METHODS: Twenty-four subjects with a positive double-blind, placebo-controlled food challenge result to celeriac; 20 atopic control subjects with birch pollen allergy who tolerated celeriac; and 20 nonatopic subjects were enrolled. IgE binding was investigated for celeriac allergens (rApi g 1.01, rApi g 4, and nApi g 5), extract reagents (celeriac, birch, mugwort, and timothy grass pollen), birch pollen allergens (rBet v 1 and rBet v 2), and cross-reactive carbohydrate determinants by means of ImmunoCAP analysis. Biologic activity of allergens was determined based on basophil mediator release. RESULTS: Component-resolved ImmunoCAP analysis considerably increased the sensitivity to detect celeriac-specific IgE by 20%. Sensitization to carbohydrate structures was detected in 38% of patients with celeriac allergy, and there was an excellent correlation between sensitization to the glycoprotein Api g 5 and isolated glycan. Positive results among atopic control subjects were mainly caused by protein allergens, whereas the effect of carbohydrate epitopes was marginal. The ability of allergens to induce mediator release decreased in the order Bet v 1 > Api g 1 > Api g 5, confirming the low biologic activity of IgE to carbohydrate epitopes. CONCLUSION: Component-resolved diagnosis allowed an increase in diagnostic sensitivity from 67% to 88% compared with extract-based diagnosis. Sensitization to Api g 5 was attributable to its glycan moieties but did not interfere with diagnostic specificity.

Analysis of 5' junctions of human LINE-1 and Alu retrotransposons suggests an alternative model for 5'-end attachment requiring microhomology-mediated end-joining.

Insertion of the human non-LTR retrotransposon LINE-1 (L1) into chromosomal DNA is thought to be initiated by a mechanism called target-primed reverse transcription (TPRT). This mechanism readily accounts for the attachment of the 3'-end of an L1 copy to the genomic target, but the subsequent integration steps leading to the attachment of the 5'-end to the chromosomal DNA are still cause for speculation. By applying bioinformatics to analyze the 5' junctions of recent L1 insertions in the human genome, we provide evidence that L1 uses at least two distinct mechanisms to link the 5'-end of the nascent L1 copy to its genomic target. While 5'-truncated L1 elements show a statistically significant preference for short patches of overlapping nucleotides between their target site and the point of truncation, full-length insertions display no distinct bias for such microhomologies at their 5'-ends. In a second genome-wide approach, we analyzed Alu elements to examine whether these nonautonomous retrotransposons, which are thought to be mobilized through L1 proteins, show similar characteristics. We found that Alu elements appear to be predominantly integrated via a pathway not involving overlapping nucleotides. The results indicate that a cellular nonhomologous DNA end-joining pathway may resolve intermediates from incomplete L1 retrotransposition events and thus lead to 5' truncations.