Johannes Blümel

Occurrence of hepatitis E virus RNA in plasma donations from Sweden, Germany and the United States.

Dear Editor, We have investigated 165 010 plasma donations from Germany, Sweden and the United States for the presence of hepatitis E virus (HEV) RNA in plasma mini-pools of up to 96 donations using a proprietary internally controlled realtime RT-PCR assay; the 95% cut-off of the assay is 250 IU ⁄ ml as determined by dilution of the WHO International Standard for HEV RNA [1]. From Europe, 95 835 Swedish and 18 100 German donations were screened, of these, 12 Swedish and four German donations were positive for HEV RNA. Allowing for actual mini-pool size, the rate of HEV-positive donations was 1:7986 and 1:4525 for the Swedish and German donors, respectively. In contrast, no HEV-positive donations were identified in 51 075 donations from the United States. Whenever mini-pools were positive for HEV RNA, individual positive donations were resolved and excluded from pharmaceutical production; 12 of the samples were characterized by molecular and serological analysis (Table 1). Analysis of the HEV strains revealed genotype 3 in all cases. Genotyping was performed by amplification of the ORF2 ⁄ 3 region of the HEV genome using the OneStep RT-PCR kit (Qiagen GmbH, Hilden, Germany) and the forward primer 5¢-GGGTGGAATGAATAA CATGT and reverse primer 5¢-AGGGGTTGGTTGGATGAA or 5¢-GGGGCGCTGGGMCTGGTCACGCCAAG. Amplification products were sequenced directly; all sequenced strains were distinct from each other (accession numbers JN995562-JN995573). The closest RNA sequence matches from BLAST searches were between an HEV strain from a wild boar (accession number FJ705359) and samples 6 and 7 obtained from German donors. Viral loads varied between 3Æ2–5Æ7 log10 IU ⁄ ml HEV RNA and are in a similar range reported for Japanese blood donors [2]. Anti-HEV IgM and anti-HEV IgG in the individual donations were determined using EIAs from Wantai (Wantai, Beijing, China). The majority of samples were window-period donations. Only one viraemic sample was positive for IgM, another for IgG (confirmed by repeat testing using different kit lots). Three samples were initially reactive; however, repeat testing using different kit batches gave negative results. Such

Standardization of Hepatitis E Virus (HEV) Nucleic Acid Amplification Technique-Based Assays: an Initial Study To Evaluate a Panel of HEV Strains and Investigate Laboratory Performance

ABSTRACT The performance of hepatitis E virus (HEV) RNA nucleic acid amplification (NAT)-based assays has been investigated using a panel of HEV-containing plasma samples. The panel comprised 22 HEV-positive plasma samples representing 10-fold serial dilutions of HEV genotypes 3a, 3b, 3f, and 4c obtained from blood donors. Two negative-control plasma samples were included. All samples were blinded. The plasma samples were prepared as liquid/frozen materials and distributed to participants on dry ice. Laboratories were requested to test the panel using their routine HEV assays and to score samples as either positive or negative and could optionally return data in copies/ml for HEV RNA. Twenty laboratories from 10 different countries participated in the study. Data were returned by all participating laboratories; 10 laboratories returned quantitative data. All assays except one were developed in-house using conventional or real-time reverse transcriptase PCR (RT-PCR) methodologies. There was a 100- to 1,000-fold difference in sensitivity between the majority of assays, independent of the virus strain. Although the quantitative data were limited, for the samples in the range of ∼6 to 4 log10 copies/ml, the standard deviations of the geometric means of the samples ranged between 0.38 and 1.09. Except for one equivocal result, HEV RNA was not detected in the negative samples. The variability of assay sensitivity highlights the need for the standardization of HEV RNA NAT assays.

Parvovirus B19 transmission by heat‐treated clotting factor concentrates

BACKGROUND: Human parvovirus B19 (B19) DNA can be frequently detected in plasma‐derived coagulation factor concentrates. The production of some clotting factor products includes heat treatment steps for virus inactivation, but the effectiveness of such steps for B19 inactivation is unclear. Moreover, detailed transmission case reports including DNA sequence analysis and quantification of B19 DNA from contaminated heat‐treated blood components have not been provided so far. Therefore, the correlation between B19 DNA in blood components and infectivity remains unclear.

Seroprevalence and incidence of hepatitis E virus infection in German blood donors

Hepatitis E virus (HEV) is transmissible by transfusion. More data are needed about seroprevalence, incidence, and viremia in blood donors for the assessment of risk of transfusion‐transmitted (TT)‐HEV infections.

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.

Parvovirus B19 DNA in plasma pools and plasma derivatives.

Human parvovirus B19 (B19) has been transmitted by various plasma‐derived medicinal products. The aim of this study was to determine the frequency and the level of B19 DNA contamination in plasma pools destined for fractionation and in a broad range of plasma derivatives. In addition, removal of B19 DNA by the manufacturing process was investigated in cases where corresponding samples from plasma pool and product were available.

Widespread distribution of hepatitis E virus in plasma fractionation pools

Dear Editor, We have investigated plasma fractionation pools, used in the manufacture of plasma-derived medicinal products, for HEV RNA and anti-HEV IgG. Plasma pools were obtained from Europe, North America, the Middle East and Asia. Approximately 10% of pools were positive for HEV RNA, showing a widespread geographic distribution (Table 1). Positive pools originated from plasma sourced in North America (n = 1), Western Europe (n = 2) and Eastern Europe (n = 1). No positive samples were identified in the pools from the Middle East. Of the Asian pools, four were positive for HEV RNA. None of the positive pools exceeded a load of >1000 copies ⁄ ml HEV RNA. The low viral loads found in pools may explain why Mordow et al. [1] did not detect HEV RNA in any final preparations of plasma-derived coagulation factors, which undergo further processing steps after initial cryoprecipitation. Another possible factor is the sensitivity of HEV RNA NAT assays that have been shown to vary widely between laboratories [2] and is not currently standardized. HEV viraemia in blood donors can exceed 7 log10 HEV RNA copies ⁄ ml [3]; therefore, dependent on pool size and virus reduction steps used during manufacture, it is possible that in HEV RNA might still be detectable in some plasma derivatives. Phylogenetic analysis of the HEV strains identified in the pools (GenBank, accession numbers JN257704– JN257711) revealed that genotype 3 viruses were found in Europe and North America, whilst the Asian pools contained genotype 4 viruses that are more common in the region. All sequences were distinct from one other. The two genotypes identified in pools are found both in humans as well as in animal such as swine, with likely zoonotic transmission in some cases. The presence of anti-HEV IgG in pools was determined using enzyme immunoassays from MP Biomedicals (MP Biomedicals Asia Pacific, Singapore) and Axiom (Axiom Diagnostic, Bürstadt, Germany). Only Asian pools were found to have anti-HEV IgG levels greater than the cut-off of the MP Biomedicals assay, and results were confirmed using the Axiom kit, reflecting the seroprevalence of anti-HEV in the region. Whilst anti-HEV is detectable in some pools, how this correlates to neutralization of potential virus infectivity remains unknown. In a recent study, it was possible to propagate infectious HEV in culture using viraemic serum containing anti-HEV [4]. There are no reports of HEV transmission by pooled plasma, although HEV has been transmitted by transfusion. HEV can be removed by nanofiltration (<20 nm), and it may be inactivated by heat treatment, the effectiveness of which is dependent upon parameters for inactivation and composition of the matrix [5]. However, where anti-HEV levels are low, solvent ⁄ detergent-treated plasma, with no effective reduction steps against non-enveloped viruses such as HEV, could present a risk for transmission, and it may be prudent to test such plasma pools for HEV RNA.

Different susceptibility of B19 virus and mice minute virus to low pH treatment.

BACKGROUND:  Parvoviridae are small nonenveloped viruses that are known to be highly resistant to physico‐chemical treatments. Because low pH is frequently applied to process intermediates or final products, the impact of such conditions on the human erythrovirus B19 (B19V) and the mouse parvovirus (mice minute virus, MMV) was assessed, which is often used as a model for B19V. Owing to the lack of a suitable cultivation and/or detection system for B19V no such data exist so far.

Analysis of porcine circovirus type 1 detected in Rotarix vaccine.

A metagenomic analysis of live human vaccines has recently demonstrated the presence of porcine circovirus type 1 (PCV1) DNA in the paediatric vaccine Rotarix used in the prevention of acute gastroenteritis. Using real-time PCR for PCV1, titres of PCV1 DNA in several batches of Rotarix were found to be in the order of 6-7 log(10) copies per dose. Pre-treatment of the reconstituted vaccine with the nuclease Benzonase, followed by extraction of nucleic acid and quantification of PCV1 DNA by real-time PCR, revealed that there was no loss of PCV1 DNA titre compared to untreated controls, suggesting that the porcine viral DNA was present in the vaccine in an encapsidated form. PCV1 permissive PS cells, human HEK293 and Vero cells, used for vaccine production, were infected with Rotarix or PCV1, respectively, and subjected to immune fluorescence and RT-PCR. Viral genomes were present in Rotarix-incubated as well as PCV1-infected cells, while viral transcription was seen only in PCV1-infected cells. Similarly, PCV1-specific protein expression was observed in PCV1-infected cells, but not in cells treated with Rotarix. Passaging of the supernatant indicated productive infection in PCV1-infected PS cells, but not in HEK293 and Vero cells or in any cell line incubated with Rotarix. PCV1 DNA present in Rotarix was protected from Benzonase digestion; however, PCV1 was not recognized in immune electron microscopy and unable to infect PS, HEK293 or Vero cells, suggesting that the high amount of PCV1 DNA present in Rotarix does not reflect a corresponding proportion of biologically active virus particles.

Tissue donation and virus safety: more nucleic acid amplification testing is needed

A. Pruss, G. Caspari, D.H. Krüger, J. Blümel, C.M. Nübling, L. Gürtler, W.H. Gerlich. Tissue donation and virus safety: more nucleic acid amplification testing is needed.
Transpl Infect Dis 2010: 12: 375–386. All rights reserved