Regulation of blood-screening in vitro diagnostics in sub-Saharan African countries remains a challenge.

According to the World Health Organization, blood must be screened for major transmitted infections before transfusion to prevent the possibility of passing an infection to the recipient. For accurate detection of infectious disease pathogens in the blood of donors, in-vitro diagnostic medical devices (IVDs) of high specificity and sensitivity should be used. In mature healthcare systems, the regulatory authorities authorize the usage of devices with the highest performance capabilities, which are also controlled through active market oversight. However, in Sub-Saharan African countries, the regulation of IVDs is often poorly developed. With the lack of stringent regulatory oversight, IVDs of poor quality can be put on the market and used for blood donor screening, which, ultimately, poses a great public health threat. The BloodTrain is a humanitarian project from the Germany Federal Ministry of Health that aims to help strengthen the regulatory authorities in Sub-Saharan partner countries. Here, we present the status of IVD regulation in the partner countries and the objectives that the BloodTrain project aims to achieve in the region toward regulating IVDs.

Performance of 20 rapid antigen detection tests to detect SARS-CoV-2 B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants using a clinical specimen panel from January 2022, Berlin, Germany.

BackgroundThere are conflicting reports on the performance of rapid antigen detection tests (RDT) in the detection of the SARS-CoV-2 Omicron (B.1.1.529) variant; however, these tests continue to be used frequently to detect potentially contagious individuals with high viral loads.AimThe aim of this study was to investigate comparative detection of the Delta (B.1.617.2) and Omicron variants by using a selection of 20 RDT and a limited panel of pooled combined oro- and nasopharyngeal clinical Delta and Omicron specimens.MethodsWe tested 20 CE-marked RDT for their performance to detect SARS-CoV-2 Delta and Omicron by using a panel of pooled clinical specimens collected in January 2022 in Berlin, Germany.ResultsWe observed equivalent detection performance for Delta and Omicron for most RDT, and sensitivity was widely in line with our previous pre-Delta/Omicron evaluation. Some variation for individual RDT was observed either for Delta vs Omicron detection, or when compared with the previous evaluation, which may be explained both by different panel sizes resulting in different data robustness and potential limitation of batch-to-batch consistency. Additional experiments with three RDT using non-pooled routine clinical samples confirmed comparable performance to detect Delta vs Omicron. Overall, RDT that were previously positively evaluated retained good performance also for Delta and Omicron variants.ConclusionOur findings suggest that currently available RDT are sufficient for the detection of SARS-CoV-2 Delta and Omicron variants.

Comparative sensitivity evaluation for 122 CE-marked rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021.

IntroductionNumerous CE-marked SARS-CoV-2 antigen rapid diagnostic tests (Ag RDT) are offered in Europe, several of them with unconfirmed quality claims.AimWe performed an independent head-to-head evaluation of the sensitivity of SARS-CoV-2 Ag RDT offered in Germany.MethodsWe addressed the sensitivity of 122 Ag RDT in direct comparison using a common evaluation panel comprised of 50 specimens. Minimum sensitivity of 75% for panel specimens with a PCR quantification cycle (Cq) ≤ 25 was used to identify Ag RDT eligible for reimbursement in the German healthcare system.ResultsThe sensitivity of different SARS-CoV-2 Ag RDT varied over a wide range. The sensitivity limit of 75% for panel members with Cq ≤ 25 was met by 96 of the 122 tests evaluated; 26 tests exhibited lower sensitivity, few of which failed completely. Some RDT exhibited high sensitivity, e.g. 97.5 % for Cq < 30.ConclusionsThis comparative evaluation succeeded in distinguishing less sensitive from better performing Ag RDT. Most of the evaluated Ag RDT appeared to be suitable for fast identification of acute infections associated with high viral loads. Market access of SARS-CoV-2 Ag RDT should be based on minimal requirements for sensitivity and specificity.

Establishment of a specimen panel for the decentralised technical evaluation of the sensitivity of 31 rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021.

IntroductionThe detection of SARS-CoV-2 with rapid diagnostic tests (RDT) has become an important tool to identify infected people and break infection chains. These RDT are usually based on antigen detection in a lateral flow approach.AimWe aimed to establish a comprehensive specimen panel for the decentralised technical evaluation of SARS-CoV-2 antigen rapid diagnostic tests.MethodsWhile for PCR diagnostics the validation of a PCR assay is well established, there is no common validation strategy for antigen tests, including RDT. In this proof-of-principle study we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from 1.1 × 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 RDT in up to six laboratories.ResultsOur results show that there is considerable variation in the detection limits and the clinical sensitivity of different RDT. We show that the best RDT can be applied to reliably identify infectious individuals who present with SARS-CoV-2 loads down to 106 genome copies per mL of specimen. For the identification of infected individuals with SARS-CoV-2 loads corresponding to less than 106 genome copies per mL, only three RDT showed a clinical sensitivity of more than 60%.ConclusionsSensitive RDT can be applied to identify infectious individuals with high viral loads but not to identify all infected individuals.

Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products.

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

WHO international standard for anti-rubella: learning from its application.

The WHO international standard for anti-rubella was first established in the 1960s when clinical diagnostics were in their infancy. Since the endorsement of the first international standard for anti-rubella IgG (RUBI-1-94), new rubella vaccines have been developed and global coverage of rubella vaccination has increased. Methods used to measure concentrations of anti-rubella IgG have also evolved to rapid, high-throughput binding assays, which have replaced often cumbersome and highly technical functional assays. During this timeframe, the protective concentration of antibody was set at 10 IU/mL by extrapolation of functional assay correlates; however, the subpopulation of antibodies within a polyclonal serum that confer protection remained undefined. Anti-rubella assays have variable formats, including antigens used, such that the same clinical sample tested on different assays can report different values with potentially devastating consequences, such as recommending to terminate pregnancy. WHO convened a meeting of experts in the rubella field to discuss the use of RUBI-1-94 and the potential future role of this international standard. The main conclusions of this meeting questioned the appropriateness of 10 IU/mL as the cutoff for protection and acknowledged the continuing role of RUBI-1-94 as a reference preparation to address analytical sensitivity and assay variation.

Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes.

For human hepatitis B virus eight distinct and two candidate genotypes are described. These genotypes differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. Comparative analysis of HBV genotypes revealed, with exception of HBV/G that shows impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established.

Hepatitis E viral loads in plasma pools for fractionation.

BACKGROUND: It is now recognized that blood donors may be silently infected with hepatitis E virus (HEV) and that plasma pools used in the manufacture of plasma-derived medicinal products may also contain detectable virus RNA. The occurrence of HEV-infected blood and plasma donors can vary considerably depending on local epidemiology. STUDY DESIGN AND METHODS: Manufacturing plasma pools from North America, Europe, the Middle East, and Asia were examined for the presence of HEV using transcription-mediated amplification of HEV RNA; confirmatory testing was performed using real-time reverse transcription polymerase chain reaction and sequencing. RESULTS: A total of 484 pools were tested. Asian pools were most frequently positive for HEV RNA and had higher viral loads, although none exceeding 300 IU/mL, and the sequenced strains (n = 5) clustered with Genotype 4, including one significantly divergent sequence. Only HEV Genotype 3 was identified in North American (n = 5) and European (n = 5) pools. There was no evidence of HEV in any pools tested from the Middle East. CONCLUSIONS: HEV was detected in manufacturing plasma pools from three different continents; viral loads were low-consistent with large pool sizes and moderate levels of HEV viremia at the individual donation level-but are nevertheless informative for risk assessment of plasma-derived medicinal products. Where sequencing was possible, analysis confirmed the presence of viruses consistent with locally circulating genotypes in the respective regions. The absence of HEV in Middle Eastern pools is consistent with the low prevalence of HEV in this region, likely due to low pork consumption.

Viral nucleic acids in human plasma pools.

BACKGROUND: The identification of viruses in human blood is required for epidemiologic surveillance and to detect potentially emerging threats to blood transfusion safety. STUDY DESIGN AND METHODS: Viral nucleic acids in plasma fractionation pools assembled from blood donors in the United States and Europe were analyzed by viral metagenomics. RESULTS: Anelloviruses were detected in each of the 10 plasma pools. Human pegivirus A (HPgV; GB virus type C) sequences were identified in eight of the 10 pools, more than 90% of which belong to Genotype 2. The recently described human HPgV2 in Flaviviridae was not detected. A small number of sequence reads of human papillomavirus were also detected in three pools. In one pool, two different gemycircularvirus genomes were identified and fully sequenced. The capsid protein of one gemycircularvirus shared 83% to 84% identity to those of genomes from human serum and sewage. The presence of the gemycircularvirus genomes in the plasma pool was independently confirmed and the viral concentration estimated by digital PCR at more than 10(6) copies/mL assuming their origin from single donors. CONCLUSION: Further research is required to elucidate whether gemycircularviruses can infect humans or are indicative of contamination occurring during phlebotomy, plasma pool processing, or ongoing donor fungal infections.

Standardization of NAT for Blood-Borne Pathogens.

Assays based on nucleic acid amplification technology (NAT) are increasingly used for screening of blood and for diagnosis or monitoring of patients. Both regulatory requirements for blood screening and international recommendations for the treatment of patients are based on common reference materials available globally for the standardization of NAT assays. World Health Organization International Standards (WHO ISs) and International Reference Panels (WHO IRPs) are primary reference materials. The characterization and manufacture of WHO reference materials as well as their evaluation is performed on behalf of the WHO by collaborating centers; their establishment is decided upon by the WHO Expert Committee on Biological Standardization (ECBS). The potency of the first WHO IS is defined by the 'international unit' (IU) which should be maintained upon replacement of the IS. The IU, unlike copy number or genome equivalent, is defined by the IS with a physical existence, is available worldwide, and allows traceability and comparability of results. The anticipated use of WHO ISs is the calibration of secondary standards or the validation of essential assay features, e.g. limit of detection.

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA.

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.

Sensitivity of hepatitis C virus core antigen and antibody combination assays in a global panel of window period samples.

BACKGROUND: Hepatitis C virus (HCV) antigen and antibody combination assays have been launched as a cost-effective alternative to nucleic acid testing (NAT) for reducing the antibody-negative window period (WP). Later, a HCV antigen chemiluminescence immunoassay (CLIA) became available. STUDY DESIGN AND METHODS: A panel composed of 337 HCV NAT-yield samples that were characterized for viral load (VL) and genotype was used to compare the sensitivity of two combination enzyme-linked immunosorbent assays (Monolisa, Bio-Rad; and Murex, formerly Abbott) and a HCV antigen CLIA (Abbott). Analytic sensitivity was compared with HCV RNA detection using Ultrio (Grifols) by testing serial dilutions of 10 genotype (gt)1 to gt4 samples. RESULTS: HCV antigen CLIA detected 92.4% of samples, whereas Monolisa and Murex detected 38.3 and 47.5%, respectively. In the HCV RNA VL range of 10(5) to 10(7) IU/mL, Monolisa and Murex detected 38% to 56% of gt1, 85% to 78% of gt2, and 21% to 37% of gt3. The overall geometric mean 50% limit of detection (range) of Ultrio on gt1 to gt4 dilution series was 3.5 (1.2-7.7) copies/mL, compared to 3.3 × 10(6) (4.4 × 10(5) -2.7 × 10(7) ), 3.4 × 10(6) (2.2 × 10(5) -4.2 × 10(7) ), and 2728 (415-7243) copies/mL for Monolisa, Murex, and HCV antigen CLIA, respectively. CONCLUSION: Analytical sensitivity of NAT was on average 1 million- and 780-fold higher than combination assays and HCV antigen CLIA, respectively. Relative sensitivities of combination assays differed for genotypes with Murex being more sensitive for gt1 and gt3 and Monolisa more sensitive for gt2. Although being less sensitive than NAT, combination assays could be considered in resource-limited settings since they detect 38% to 47% of seronegative WP donations.

Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany.

BACKGROUND: Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat). METHODS: Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems. RESULTS: Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening. CONCLUSION: HIV differs from other blood-borne viruses with regard to its fast evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1 variants.

How safe is safe: new human immunodeficiency virus Type 1 variants missed by nucleic acid testing.

BACKGROUND: Nucleic acid amplification techniques (NAT) in routine blood donor screening considerably reduce the diagnostic window phase period. Nevertheless, several reports of false-negative NAT results were published. Here, four cases of human immunodeficiency virus Type 1 (HIV-1) RNA-positive blood donations that escaped detection by NAT screening are described. STUDY DESIGN AND METHODS: A total of 2.7 million blood donations were screened for viral infections between January 2010 and October 2012 in our German Red Cross blood donation service. Four plasma specimens with false-negative NAT results were comparatively investigated with 12 CE-marked NAT assays. In two cases of putative HIV-1 variants the target region of the NAT assay was sequenced allowing comparison with the respective primers and probes. RESULTS: Most of the NAT assays used in routine blood donor screening with the 5'-long terminal repeat (LTR) as target region demonstrated deficiencies in detecting the viral variants and the low-viral-carrier donations. Sequence analysis revealed in one case a deletion of 56 nucleotides within the 5'-LTR preventing the binding of the probe accompanied by a neighbored insertion of another 52 nucleotides and several primer mismatches in another case. No false-negative results were obtained for these cases using dual-target assays. The viral load of the remaining two false-negative results was below the NAT's limit of detection. CONCLUSION: HIV-1 is characterized by a high mutation rate and rapid generation of new viral variants. By the use of one target region for HIV-1 NAT assays there is a certain risk of false-negative results. Employing HIV-1 multi- and dual-target assays in routine blood donor screening seems to be a reasonable possibility to minimize this problem.

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.

Blood screening nucleic acid amplification tests for human immunodeficiency virus Type 1 may require two different amplification targets.

BACKGROUND: Five cases of human immunodeficiency virus Type 1 (HIV-1) RNA-positive blood donations are described that escaped detection by three different CE-marked nucleic acid amplification technique (NAT) screening assays. These events were associated with two HIV-1 transmissions to recipients of blood components. The implicated NAT assays are monotarget assays and amplify in different viral genome regions (group-specific antigen or long terminal repeat). Investigations into the cause of the false-negative test results were initiated. STUDY DESIGN AND METHODS: Plasma specimens of the five NAT false-negative cases were comparatively investigated in 12 CE-marked HIV-1 NAT systems of differing design. The relative amplification efficiency for the HIV-1 variant was determined for each assay. Sequencing of the variants in the region targeted by each false-negative NAT assay allowed comparison with the respective primers and probes. RESULTS: Some of the NAT assays designed in a similar way to false-negative monotarget NATs also revealed deficiencies in detecting the viral variants. In each case sequencing of the assay target region in the variants demonstrated mismatches with primers and probes used by the assays. Some dual-target assays showed decreased amplification efficiency, but not false-negative results. CONCLUSION: HIV is characterized by its rapid evolution of new viral variants. The evolution of new sequences is unpredictable; NAT screening assays with a single target region appear to be more vulnerable to sequence variations than dual-target assays. Based on this experience with false-negative tests results by monotarget NAT assays, the Paul-Ehrlich-Institut is considering requesting dual-target NAT assays for HIV-1 blood donation screening in Germany.

Roadmap for harmonization of clinical laboratory measurement procedures.

Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions. Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.

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.

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 log(10) 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.