Individual NAT Research Articles

Trends in hepatitis E virus infection: Analyses of the long-term screening of blood donors in Hokkaido, Japan, 2005-2019.

After experiencing several cases of transfusion-transmitted hepatitis E (TT-HE) in Hokkaido, Northern Japan, hepatitis E virus (HEV) screening in blood donors, using a nucleic acid amplification test (NAT), was introduced in 2005. The frequency of HEV RNA-positive donations (2005-2019) was investigated, and the HEV RNA-positive specimens were phylogenetically analyzed. In August 2014, the 20-pooled NAT (20P-NAT) was replaced with an individual-NAT (ID-NAT) system. Until 2019, the frequency of HEV RNA-positive donors was 0.011% (289/2,638,685) with 20P-NAT and 0.043% (597/1,379,750) with ID-NAT, and no TT-HE cases were observed in Hokkaido. The prevalence among male, but not female donors, increased significantly between 2015 and 2019. Eighty-nine percent of HEV isolates from donors were genotype 3 and the remainder were genotype 4, and many clusters existed in each genotype. ALT levels at the time of donation were significantly higher in donors with genotype 4. Four subgenotypes, namely 3a (37%), 3b (41%), 3e (6%), and 4c (10%), comprised 94% of the total. During this period, the most identified subgenotype, 3a, transitioned to 3b. Majority of the HEV strains within the same clusters were detected in the same geographical region around the same period. Many of the human HEV isolates were shown to coexist closely with animal HEV isolates phylogenetically. In Hokkaido, multiple divergent HEV strains have been circulating, and small outbreaks of hepatitis E have occurred in the last 15 years. The results suggested that HEV NAT can contribute significantly in ensuring safety during blood transfusions.

Characteristic of HBV nucleic acid amplification testing yields from blood donors in China

BackgroundNucleic acid amplification testing (NAT) for blood screening has been previously performed in some countries to determine NAT yields. The current study sought to explore the non-discriminating reactive NAT yields using individual-NAT (ID-NAT) and characteristics of HBV NAT yields through a 10-year retrospective analysis in Zhejiang, China.MethodsBlood donations were analyzed using individual-NAT mode by the transcription-mediated amplification (TMA) method. Supplementary HBV serological tests were performed using chemiluminescent immunoassay, and HBV viral load assay was performed by real-time polymerase chain reaction. Follow-up studies were performed in partial donors with low HBV viral loads.ResultsNon-discriminating reactive NAT yields and HBV NAT yields varied in different years. The yields ranged from 853.73 per million to 2018.68 per million and 624.60 per million to 1669.50 per million, respectively. In the 476 NAT yields, 19 were probable window periods (WP), 33 probable occult hepatitis B virus infections (OBIs), 409 were confirmed OBIs and 15 were chronic HBV infections. ID-NAT results were categorized in four groups, and the findings showed that the levels of HBV DNA viral loads were different in the four different groups (χ2 = 275.02, p < 0.01). HBV viral load distribution was significantly different between anti-HBs positive and anti-HBc positive samples (χ2 = 49.429, p < 0.01). Notably, only 42.03% donors were NAT repeated positive in the 138 repeat donors’ follow up tests.ConclusionNAT screening of blood donations can reduce the risk of transfusion-transmitted HBV infections. Positive proportions of anti-HBs and anti-HBc are correlated with the HBV viral load level. However, low level of viral load donors pose risks in HBV NAT assays, and show fluctuating state for HBV viral load and leads to non-repeated NAT results during follow up studies.

Emerging infectious disease and transfusion-transmitted infection

Hepatitis B, hepatitis C and HIV infections are well-known infectious diseases caused by blood products, but recently there have been almost no reports. In 2011, hepatitis E virus (HEV) antibody test was covered by insurance in Japan, and certain numbers of transfusion-transmitted HEV were reported, and five cases were recognized in 2019. As the Japanese Red Cross has started to examine individual NAT of HEV for all blood donors since August 2020, and the number of transfusion-transmitted HEV will decrease. In addition, Trypanosoma cruzi antibody test for selective blood donors has been examined since August 2016, and the risk of Chagas disease infection by blood transfusion will be decreased. In the future, it is important for protecting against the transfusion-transmitted infection to inspect bacterial tests and know the information of emerging infectious disease occurring overseas. When the emerging infectious diseases such as DENV (dengue virus), WNV (West Nile virus), Zika (Zika virus) and CHIKV (chikungunya virus) occur in Japan, it is necessary to stimulate countermeasures and prepare what kind of tests should be performed for blood donors or criteria for blood donation.

Prevalence of antibody to Hepatitis B core antigen and Hepatitis B virus DNA in HBsAg negative healthy blood donors.

BackgroundHepatitis B virus is one of the most important blood born viruses. Although the sensitivity of screening tests has been considerably increased, transmission may still occur due to window period or occult hepatitis B infections (OBIs). This study was aimed at evaluating the prevalence of the anti-HBc and identifying the HBV DNA in HBsAg negative blood donors.MethodsThe Blood samples from 2031 HBsAg-negative blood donors were divided into three aliquots and tested for anti-HBc, anti-HBs and HBV DNA. Serologic screening including anti-HBc and anti-HBs was performed. As a confirmatory test, all positive results for anti-HBc were retested with another kit. Two positive results were considered for anti-HBc positivity. All HBsAg negative selected donations were tested by PCR assay on pooled specimens (five samples per pool), plasma samples found to be HBsAg negative but anti-HBc positive were selected for a single-unit specimen Real-Time assay.ResultsThe study population had a mean age of 33.25 ± 10.09 years were mainly composed of males (94.8 %). The seroprevalance rate was 4.9 % for Anti-HBc and 31.9 % for HBsAb. The majority (58.6 %) of Anti-HBc positive cases were regular blood donors with 42–49 years being the largest age group (41.4 %). Neither individual NAT nor pooled NAT test detected any HBV DNA.ConclusionHowever, Screening of anti-HBc Ab is proposed as a method to identify previous contact with HBV, but there is controversy in literature data regarding the cost-benefit of exclusion of positive anti-HBc Ab in blood donors. Our data does not suggest HBc-Ab test as a screening tool in the study setting.

Evaluation of the Procleix Ultrio Elite Assay and the Panther-System for Individual NAT Screening of Blood, Hematopoietic Stem Cell, Tissue and Organ Donors

Background: The performance of the multiplex Procleix Ultrio Elite assay as individual donor nucleic acid test (ID-NAT) for the detection of HIV-1, HIV-2, HCV, and HBV was evaluated in a retrospective, single center study. Methods: ID-NAT results of 21,181 blood donors, 984 tissue donors, 293 hematopoietic stem cell donors and 4 organ donors were reviewed in synopsis with results of serological screening and additional discriminatory and repetitive NAT in case of positive donors. Results: Specificity of the initial Procleix Ultrio Elite assay was 99.98% and after discriminatory testing 100.00%. Initially invalid results were observed in 75 of 21,181 blood donors (0.35%) but 16 of 984 tissue donors (1.62%, p < 0.001) which included non-heart-beating (‘cadaveric') donors. All these had valid negative ID-NAT results after repeated testing or testing of 1:5 diluted specimens in case of tissue donors. Occult hepatitis B (defined here as HBV DNAemia without HBsAg detection) was demonstrated by ID-NAT in two anti-HBc-positive tissue donors and suspected in two other tissue donors, where a definite diagnosis was not achieved due to the insufficient sample volumes available. Conclusion: The Procleix Ultrio Elite assay proved to be specific, robust and rapid. Therefore, routine ID-NAT may also be feasible for organ and granulocyte donors.

Virus de l’hépatite E, implications en transfusion sanguine

Hepatitis E virus (HEV) is a non-enveloped RNA virus transmitted by the fecal-oral route. Autochthonous hepatitis E occurring in developed countries is caused by genotypes 3 and 4 and is a zoonotic infection. Humans are infected mostly after ingestion of undercooked meat from infected animals. Most HEV 3 and 4 infections are clinically inapparent. However, genotype 3 (HEV 3) can lead to chronic hepatitis in immuno-compromised patients such as organ-transplant recipients and patients with haematological malignancies. In Europe, HEV 3 is implicated in transfusion-transmitted HEV infection. In France, as observed in several European countries, prevalence of HEV RNA and specific IgG antibodies are high indicating that viral circulation is important. The systematic HEV NAT screening of blood donations used for preparation of solvent detergent plasma indicate that 1 to 2218 donation is infected by HEV RNA. The need or implementation's impacts of safety measures to prevent HEV transmission by blood transfusion are under reflexion by French's health authorities. The HEV NAT screening is the only available tool of prevention. Alternative strategies are under investigation including individual or mini pool NAT testing all or part of blood donations.

31 MONOCYTE/MACROPHAGE PERIBILIARY RECRUITMENT BY PKHD1-DEFECTIVE CHOLANGIOCYTES INDUCES EXPRESSION OF αvβ6 INTEGRIN ON BILIARY CYSTS VIA TGF-β1 AND TNF-α IN CONGENITAL HEPATIC FIBROSIS

implementation of the current HBV-DNA screening assay, as well as a look-back of blood recipients. Methods: Sequence analysis revealed identical HBV sequences in donor and recipient. Back-up samples testing negative by NAT minipool at previous donations were individually tested by more sensitive assays (Roche Individual NAT, and home-made real-time PCR of S region). When available, pre and post-transfusion samples of recipients were tested for HBV markers. Sequence analyses were conducted by ABIPrism 3100. Results: 28 back-up samples (1–3 per patient) from 13 OBI carriers (2 anti-HBc only, 2 anti-HBs only, 8 anti HBs/antiHBc, 1 without any marker) were available. Overall, 62% tested positive with at least one of the two assays. Of them, 42% were positive by Individual NAT, and 58% by home-made protocol. Six donors gave at least one unit to 4 recipients, for whom both preand post-transfusion samples were available. One recipient acquired infection, as indicated by seroconversion and ALT elevation, two had pre-existing immunity to HBV, and one remained seronegative. Conclusions: A substantial proportion of donations containing HBVDNA are not identified by current NAT minipool assays. These donations can cause acute infections in recipients. Current approaches are highly ineffective, and alternative strategies based on more sensitive protocols or anti-HBc testing should be developed.

OC-6 Monocyte/macrophage peribiliary recruitment by Pkhd1-defective cholangiocytes induces expression of αvβ6 integrin on biliary cysts via TGFβ1 and TNFα in congenital hepatic fibrosis

implementation of the current HBV-DNA screening assay, as well as a look-back of blood recipients. Methods: Sequence analysis revealed identical HBV sequences in donor and recipient. Back-up samples testing negative by NAT minipool at previous donations were individually tested by more sensitive assays (Roche Individual NAT, and home-made real-time PCR of S region). When available, pre and post-transfusion samples of recipients were tested for HBV markers. Sequence analyses were conducted by ABIPrism 3100. Results: 28 back-up samples (1–3 per patient) from 13 OBI carriers (2 anti-HBc only, 2 anti-HBs only, 8 anti HBs/antiHBc, 1 without any marker) were available. Overall, 62% tested positive with at least one of the two assays. Of them, 42% were positive by Individual NAT, and 58% by home-made protocol. Six donors gave at least one unit to 4 recipients, for whom both preand post-transfusion samples were available. One recipient acquired infection, as indicated by seroconversion and ALT elevation, two had pre-existing immunity to HBV, and one remained seronegative. Conclusions: A substantial proportion of donations containing HBVDNA are not identified by current NAT minipool assays. These donations can cause acute infections in recipients. Current approaches are highly ineffective, and alternative strategies based on more sensitive protocols or anti-HBc testing should be developed.

Detection of West Nile virus RNA and antibody in frozen plasma components from a voluntary market withdrawal during the 2002 peak epidemic

The US West Nile virus (WNV) epidemic in the summer and fall of 2002 included the first documented cases of transfusion-transmitted WNV infection. In December 2002, the FDA supported a voluntary market withdrawal by the blood banking community of frozen blood components collected in WNV high-activity areas. At the time, the prevalence of viremia and serologic markers for WNV in the blood supply was undefined. In collaboration with America's Blood Centers, 1468 frozen plasma components (of approx. 60,000 frozen units voluntarily withdrawn from the market) were selectively retrieved from the peak epidemic regions and season (June 23, 2002-September 28, 2002). These units were unlinked, subaliquoted, and tested by WNV enzyme immunoassays (EIAs; Focus Technologies and Abbott Laboratories) and nucleic acid amplification tests (NATs; Gen-Probe Inc. and Roche Molecular Systems). Of the 1468 EIA results from Abbott and Focus, 7 were anti-immunoglobulin M (IgM)- and anti-immunoglobulin G (IgG)-reactive by both assays, 8 and 1 were IgM-only-reactive, and 8 and 23 were IgG-only-reactive, respectively. NAT by Gen-Probe and Roche Molecular Systems yielded one RNA-positive, antibody-negative unit containing approximately 440 RNA copies per mL. An additional 10-fold replicate NAT testing by Gen-Probe on 14 of 15 IgM-reactive specimens yielded 2 additional IgM- and IgG-reactive units with low-level viremia (i.e., 7/10 and 2/10 replicates tested reactive). The prevalence of acute (RNA-positive) and recent (IgM-seroreactive) WNV infections indicates that transfusion risk in high-risk areas could have been considerable and that voluntary market withdrawal of frozen components likely averted some WNV transfusion transmissions. The existence of very-low-level viremic units raises concerns, because WNV minipool NAT screening will miss such units and individual NAT may not completely correct this situation.