Reliability of CE-marked NATs for HIV-1 subtype C detection and quantitation.

We compared seven CE-marked HIV-1 RNA nucleic acid amplification technology (NAT) based assays for their detection efficiency and quantitation concordance in regard to HIV-1 subtype C. We used 398 plasma samples from South African repeat blood donors identified as HIV positive at occasion of routine screening NAT performed mainly during the years 2010-2013, with most plasma samples reflecting recent HIV-1 infections. All HIV-1 subtype C specimens were detected, independent of mono- or dual-target assay design. In the same time period new variants of HIV-1 subtype B had been identified which were missed by some mono-target assays, a finding which was not corroborated for subtype C in our study. A high level of concordance of HIV-1 subtype C quantitation was determined for the HIV-1 NATs, showing successful standardization in this diagnostic field.

Standardization of Nucleic Acid Tests: the Approach of the World Health Organization.

The first World Health Organization (WHO) international standards (ISs) for nucleic acid amplification techniques were established two decades ago, with the initial focus on blood screening for three major viral targets, i.e., hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1. These reference materials have subsequently found utility in the diagnosis and monitoring of a wide range of infectious diseases in clinical microbiology laboratories worldwide. WHO collaborating centers develop ISs and coordinate international studies for their evaluation. The WHO Expert Committee on Biological Standardization is responsible for the endorsement of new standardization projects and the establishment of new and replacement ISs. Potencies of ISs are defined in international units (IU); the reporting in IU for assays calibrated with an IS (or secondary standards traceable to the IS) facilitates comparability of results for different assays and determination of assay parameters such as analytical sensitivities.

Establishment of the Ph. Eur. Hepatitis A virus RNA for NAT testing BRP batch 1.

Detection of viral contamination in plasma donations is critical to prevent transmission of infectious diseases. The European Pharmacopoeia (Ph. Eur.) monograph 1646 'Human plasma (pooled and treated for virus inactivation)', requires that plasma pools used for the manufacture of this product be tested, among others, for the presence of hepatitis A virus RNA by nucleic acid testing (NAT) using a positive control containing 100 International Units (IU) of hepatitis A virus (HAV) RNA per mL. To this end, the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) organised an international collaborative study under the aegis of the Biological Standardisation Programme, for the establishment of the 1st Biological Reference Preparation (BRP) for HAV RNA for NAT testing. A freeze-dried candidate material was thus prepared and calibrated against the WHO 2nd International Standard for HAV for NAT (00/562) in a study in which thirteen European and North American laboratories including Official Medicines Control Laboratories (OMCLs), manufacturers of plasma-derived products, producers of in vitro diagnostic kits and a blood transfusion centre participated. Based on the outcome of the study, an HAV RNA content of 40 000 IU/vial (corresponding approximately to 4.6 log10 IU/vial) was assigned to the BRP, which was adopted by the Ph. Eur. Commission in March 2016 as Ph. Eur. hepatitis A virus RNA for NAT testing BRP batch 1.

[Legal basis and practice of examination of critical in vitro diagnostic devices]. / Rechtsgrundlagen und Praxis bei der Prüfung von kritischen In-vitro-Diagnostika.

In vitro diagnostic devices (IVD) are categorized into different risk classes depending on the potential consequences of false test results in transfusion medicine or on the individual patient. Test systems of higher risk may be assessed and examined by a third party in addition to the manufacturer's evaluation. The preapproval examination of essential performance features can assure minimum quality features prior to marketing of the IVDs. By batch testing the variation between different batches of an IVD is determined. Comparative testing in a re-evaluation scheme can define the current state of the art. The present European IVD directive stipulates batch testing for high-risk IVDs while the draft version of the new European IVD regulation also foresees independent product testing performed by European reference laboratories.

[The ways of harmonization of clinical laboratory measurement techniques].

The results of implementation of different clinical laboratory techniques are to be equal in clinically significant limits to be optimally applied in diagnostics of diseases and treatment of patients. When the results of laboratory tests are not standardized and harmonized for the very same clinical assay the results can be expressed by unmatched numbers. Unfortunately, in some handbooks the values are presented based on the results of application of specific laboratory techniques without considering possibility or likelihood of differences between various techniques. When this is a case, accumulation of data of diferent clinical research studies and working out of clinical handbooks on this basis will be inconsistent. Inadequate understanding of issue that the results of laboratory tests are not standardized and harmonized can lead to incorrect clinical, financial, managerial or technical decisions. The standardization of clinical laboratory techniques was applied to many measurands related to primary referent techniques (standard specimen of pure substance) or/and developed referent measurement techniques. However, harmonization of clinical laboratory techniques for those measurands which are not related any developed measurement techniques is quite problematic due to inadequate determination of measurand, its inadequate analytical specificity, insufficient attention to commutability of referent materials and poor systematic approach to harmonization. To overcome these issues an infrastructure is to be developed to support systematic approach to identification and prioritization of measurands which are to be harmonized on the basis of clinical importance and technical applicability. The management of technical implementation harmonization process for specific measurands.

Performance of hepatitis B surface antigen tests with the first WHO international hepatitis B virus genotype reference panel.

BACKGROUND: Standardization of hepatitis B surface antigen (HBsAg) tests is indispensable for consistent quality and comparability. Ideally, the assays should detect all known hepatitis B virus (HBV) genotypes equally well. OBJECTIVE: Development of an HBV genotype reference panel for HBsAg assays representing the most prevalent HBV subgenotypes to address commutability and traceability of the heat-inactivated 2nd WHO International Standard (IS) for HBsAg in relation to native HBsAg and to HBV genotypes. STUDY DESIGN: An HBV panel of 15 non-inactivated lyophilized specimens representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H was evaluated in parallel to the IS by 15 laboratories using 19 different HBsAg tests and tree unitages. The virus content of the samples was reduced by ultracentrifugation and dilution to <2×10(4) IU HBV DNA/mL. RESULTS: Twenty-two qualitative and 6 quantitative data sets were evaluated. Overall, the results demonstrated consistent detection of HBV genotypes by the majority of tests with a mean potency variability relative to the IS of 36%. Some assays showed significant genotype-dependent differences in analytical sensitivity. Some tests were more sensitive with the IS, others less. On average, one IU HBsAg corresponded to 0.88±0.20 ng HBsAg protein. CONCLUSIONS: The panel was accepted by the WHO as the "1st International Reference Panel for HBV genotypes for HBsAg-based assays". The panel is a helpful complementation to the IS to validate HBV genotype specific analytical test sensitivities.

First WHO International Reference Panel containing hepatitis B virus genotypes A-G for assays of the viral DNA.

BACKGROUND: WHO International Standards (IS) are provided for the calibration and validation of diagnostic and screening assays, e.g. for hepatitis B virus (HBV). HBV forms numerous subgenotypes and the current IS for HBV DNA reflects subgenotype A2. OBJECTIVE: A reference panel with the most prevalent subgenotypes should facilitate evaluation of genotype-specific detection efficiencies. STUDY DESIGN: 215 HBV positive plasma samples collected worldwide were characterized for HBV markers and sequenced. Fifteen subgenotype A1, A2, B2, B4, C2, D1, D3, E, F2 and G samples were selected for the panel. The lyophilized samples were tested in parallel with the IS in an international collaborative study with 16 laboratories using 13 different nucleic acid amplification techniques (NATs). RESULTS: Eight of 13 NAT had a HBV DNA detection efficiency which was independent of the genotype and consistent with the IS, while with five assays, certain deviations were noted, particularly with genotype F which was under quantitated or even missed by three assays. The panel was accepted by the WHO as the "1st WHO International Reference Panel for HBV Genotypes for HBV NAT-Based Assays". CONCLUSIONS: The evaluation of HBV DNA assays should include many different genotypes. The WHO Reference Panel is universally available for manufacturers of HBV DNA assays, diagnostic laboratories and control authorities to facilitate standardized validation of HBV genotype specific detection efficiency of both diagnostic (quantitative and qualitative) and screening NAT assays.

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

In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT in donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT for HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT for HCV should still be performed. If the NAT screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood bank laboratories in performing virological tests.

The harmonization of the regulation of blood products: a European perspective.

The development of blood products as medicines initially took place on the national level in various countries, which resulted in considerable diversity of mechanisms and stringency of regulatory oversight. The scenario changed dramatically with the catastrophic experience that severe virus infections had been transmitted by blood products world-wide. Blood products, which had been regulated differently in the member states, became subject to the European pharmaceutical legislation in 1989. A specialized directive regulating the blood transfusion sector and the collection of plasma for fractionation was enacted in 2002. The European Community, particularly the Commission and the European Medicines Agency, is continuously refining the requirements, providing detailed technical and scientific guidance. In addition, institutions of the Council of Europe play an important role in the transfusion sector, the elaboration of the European Pharmacopoeia prescriptions, and the co-ordination of Official Medicines Control Laboratory or Laboratories batch release. However, further and sustained efforts towards international harmonization are needed. There are already important mechanisms in place, such as the International Conference on Harmonization initiative, which is producing internationally recognized guidelines on central issues. Another important achievement is the common technical document format, which enables the use of uniform applications for marketing authorization. However, there is still room for progress, for example, questions regarding regulatory requirements for licensing of in vitro diagnostic devices, or mutual recognition of inspections. The World Health Organization continues to play an important role in harmonization, both substantially by the production of high-level guidance documents or the establishment of physical international standard preparations, and in a more general sense by providing a platform for international collaboration. A very important aspect is the transparency of the creation and refinement of regulatory requirements. It is currently the rule that draft legal texts, monographs and guidelines are published for a consultation period before adoption. Effort and attention are required to keep track of the developments. However, in the era of modern electronic communication tools, the necessary information can be found on websites and comments can easily be submitted. Networking and exchange of information will continue to be crucial for development and maintenance of sound and balanced regulatory requirements.

First case of hepatitis C virus transmission by a red blood cell concentrate after introduction of nucleic acid amplification technique screening in Germany: a comparative study with various assays.

BACKGROUND AND OBJECTIVES: Pooled nucleic acid amplification techniques (NAT) and donor screening for anti-hepatitis C virus (HCV) have reduced the diagnostic window period of HCV infection in the blood donor population from about 12 to 1 or 2 weeks. During that time, HCV RNA is hardly detectable by pooled or individual donation NAT. Here we describe a case of transfusion-acquired HCV infection from an extremely low-titre donation. After a repeat donor tested positive for HCV, a look-back procedure was initiated. A recipient of a red cell concentrate from the previous donation was identified and found to be infected with HCV as well. We compared several commercial NAT systems for their ability to detect the viraemic plasma. MATERIALS AND METHODS: Molecular analyses of HCV in donor and recipient samples were performed. The HCV-transmitting plasma was tested using different commercially available qualitative and quantitative NAT assays. RESULTS: HCV transmission was verified by molecular analyses and was assigned to genotype 2b. NAT with various commercial HCV assays detected the infection erratically in individual donations. However, the detection rate was not directly related to the claimed sensitivity of some HCV NATs. CONCLUSIONS: HCV transmission can be caused by donations that escape NAT detection even when tested in an individual donation. Comparison of different assays led to results that did not necessarily reflect the expected sensitivities. The need for standard materials representing further HCV genotypes is discussed.

Molecular epidemiology of West Nile Virus in humans.

After the introduction of West Nile Virus into the United States of America in 1999 followed by annual WNV epidemics during the mosquito seasons and spreading of the virus from the East (New York; 1999) to the West of the U.S. (California; 2003/2004) there appeared the question of whether a similar scenario could happen in Europe, too. To be able to answer this question the German Ministry of Health decided to investigate the prevalence and incidence of WNV infections in German blood donors. First a test algorithm was established taking into account the high level of cross-reactivity between different flavivirus infections in serological test systems. AntiWNV-suspicious specimens were further investigated for their neutralisation capacity and by an antiWNV confirmation assay developed in-house. As a preliminary result of our studies a very low prevalence of WNV infections in healthy German blood donors was measured. Development of highly specific test systems is necessary for accurate and reliable differential diagnosis of flavivirus infections.

Anti-HBc screening of blood donors: a comparison of nine anti-HBc tests.

BACKGROUND AND OBJECTIVES: Since voluntary introduction of hepatitis B virus (HBV) minipool nucleic acid amplification technology (NAT) at the German Red Cross, the expected residual risk of a transfusion-associated HBV infection has been estimated to be 1 : 500,000 - about 10 times higher than for human immunodeficiency virus (HIV) or hepatitis C virus (HCV) infection. Donors demonstrating chronic positivity for antibody to hepatitis B core antigen (anti-HBc), negativity for hepatitis B surface antigen (HBsAg) and polymerase chain reaction (PCR)-negative with a low virus load are a major cause of this increased risk. MATERIALS AND METHODS: Ten-thousand blood donors from our blood-donation centre were screened for anti-HBc using the current PRISM HBc and the new PRISM HBcore assay to evaluate the diagnostic sensitivity and specificity of these tests. PRISM HBc- or PRISM HBcore-reactive samples were further analysed using seven additional tests for anti-HBc, two tests for antibody to hepatitis B surface antigen (anti-HBs), one test for antibody to hepatitis B envelope antigen (anti-HBe) and three HBV NAT assays. RESULTS: From a total of 10,000 donors, nine and 14 samples were reactive only in the PRISM HBc and the PRISM HBcore, respectively, whereas 165 samples were reactive in both anti-HBc assays. Further analysis of these 188 anti-HBc-reactive specimens in a total of nine different anti-HBc assays revealed concordant results for 162 (86.2%) specimens. Sample cut-off values for anti-HBc were significantly (P < 0.01) lower for anti-HBc-only reactive samples compared with specimens that were also reactive for anti-HBs or anti-HBe. CONCLUSIONS: Both PRISM anti-HBc assays revealed that approximately 1.8% of non-prescreened blood donors from Germany were reactive for anti-HBc. Although sensitivity was comparable between both assays, specificity was increased significantly with the PRISM HBcore. High anti-HBc sample cut-off values were indicative for reactivity in other HBV parameters and for concordant results in the nine different anti-HBc assays. Look-back investigations are necessary to estimate the infection risk both of anti-HBc-only positive and of anti-HBc/anti-HBs-positive blood transfusions.

Collaborative study for establishment of a European Pharmacopoeia Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150 per cent of the estimated potency was determined. For the quantitative assay the semi-weighted combination of the 5 potency estimates lead to a combined potency of 5.83 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.04 log( 10) which corresponds to 91 to 110 per cent of the estimated potency. The semi-weighted combination of the results from both types of assay lead to a final potency estimate of 5.80 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.05 log( 10) which corresponds to 86 to 117 per cent of the estimated potency. A threshold control suitable for quantitative testing of B19 DNA and reflecting the requirements of the European Pharmacopoeia monograph Human anti-D immunoglobulin should contain 104 IU/ml of B19 DNA. Taking into account the assigned titre of 5.80 log( 10) IU/ml (630.957 IU/ml), a dilution of 10(-1.8) (1/63) of the candidate BRP should yield a positive response. This concentration was easily detected by all participating laboratories in all tests. The candidate BRP thus appeared to be suitable for the intended purpose. The candidate BRP was adopted as the European Pharmacopoeia BRP by the European Pharmacopoeia Commission at its session in June 2003 and is available from the European Directorate for the Quality of Medicines (Catalogue Number: Y0000285).

Causality assessment of suspected virus transmission by human plasma products.

BACKGROUND: Causality assessment of reports on suspected virus transmission is crucial for early detection of infectious plasma products. Commonly used algorithms, such as the WHO criteria, do not meet the specific requirements for causality assessment of suspected virus transmission. STUDY DESIGN AND METHODS: A special algorithm, based on nucleic acid amplification and gene sequencing technology, effectiveness of validated virus-inactivation methods, empirical data concerning the safety record of the product, and information on batch-related infection clusters, was developed. The algorithm is focused on laboratory test results or otherwise standardized data, with few clinical data being required. To facilitate practical application, the algorithm has been converted into a graphical decision tree. RESULTS: The feasibility of the algorithm is shown by causality assessment of sample cases. Three cases are presented with the details of each case used in the 12-question checklist. The answers provided by the checklist led to the causality classification. CONCLUSION: The algorithm is a tool for evaluating reports of suspected virus transmission in a standardized manner. It thus has the potential to improve early signal detection in pharmacovigilance of plasma products by confirmation or exclusion of suspected infectivity in most cases.

Hepatitis C virus transmission through quarantine fresh-frozen plasma.

In 1994, quarantine fresh-frozen plasma (Q-FFP) was introduced in Germany in order to reduce the risk of HIV and HCV transmission. In 1998, an acute HCV infection of a patient was reported to us. The look-back revealed that this patient had received two Q-FFP from a donor who had seroconverted for HCV in the meantime. Recipients of further plasma donations from this donor were identified. Back-up specimens of these donations were investigated in several laboratories. A total of 25 additional HCV-PCR positive plasma units had been transfused to 12 further patients. HCV infections were diagnosed in seven of these recipients, three patients had already been deceased. One of the remaining two recipients was already HCV positive prior to transfusion, in the other patient, no HCV infection was detectable. This patient had received three units of an "early" plasma donation , which was tested negative by PCR in one laboratory, but positive in the other. The subsequent, clinically infectious donation had the same discrepant PCR results. Thus, eight cases of HCV transmission were revealed and classified as "certain" with regard to causality, also due to an identical HCV genotype, i.e. 3e. Some of these infections would have been prevented by application of a different anti-HCV assay. The assay used in the respective plasmapheresis station was in-sensitive in this individual case for more than 400 days after the first PCR positive donation. This caused the release of the above mentioned infectious units. Upon re-testing the backups, three of four other anti-HCV assays revealed a positive result already 104 days after the first PCR-positive donation. The donor had increased ALAT levels (> 23 IU/L) at nine of 28 donations, two of these were higher than 2.5 times the upper normal limit, and two were higher than 68 IU/L, which is the cut-off value for male blood donors in Germany. The results of these (look-back) studies arouse several queries, i.e. differences in the diagnostic sensitivity between current anti-HCV and PCR tests, the accuracy of risk-estimates (especially when based on hemovigilance studies for Q-FFP), the value of ALAT testing, and currently practised release algorithms for Q-FFP.

A new cluster of hepatitis A infection in hemophiliacs traced to a contaminated plasma pool.

Recently, several clusters of hepatitis A have been observed among hemophiliacs linked to factor VIII concentrates treated for virus inactivation solely with the solvent/detergent (S/D) method, a procedure that does not affect nonenveloped viruses such as the hepatitis A virus (HAV). A new outbreak of hepatitis A in six hemophiliacs treated with the same lot of a factor VIII preparation occurred recently in Germany. The objective of the study was to clarify whether these diseases were caused by the administration of the S/D-treated plasma product, rather than a community-acquired infection. Polymerase chain reactions designed to detect HAV nucleic acid have been carried out in the implicated factor VIII lots, in the corresponding plasma pools, and in serum samples of four out of six infected individuals. The nucleic acid sequences were determined in samples that resulted in positive amplification products. HAV sequences were found in one of the two plasma pools used for manufacture of the incriminated product, in the incriminated lot itself, and in all recipient sera tested so far, although the latter were collected up to 7 weeks after the onset of jaundice. The sequences obtained were completely identical, revealing a unique HAV strain of genotype IA. This study provides conclusive evidence that hepatitis A can be transmitted by factor VIII concentrates treated solely by the S/D procedure for virus inactivation. This inactivation method is not effective against nonenveloped viruses. Since a number of hepatitis A transmission episodes have been described with such preparations during the past 10 years, their continued use seems to be questionable unless additional virus removal or inactivation steps are introduced to prevent the transmission of nonenveloped viruses. Molecular approaches again proved to be reliable tools for elucidating the chain of virus transmission.

Validation of HCV-NAT assays and experience with NAT application for blood screening in Germany.

Validation of HCV-NAT assays is an important prerequisite for the use of NAT for screening plasma or blood donations. The main NAT features to be validated are specificity, detection limit and robustness. Preliminary experience in Germany obtained with different methodical and logistic approaches shows the feasibility of HCV-NAT as a screening test for blood donations.