Infectivity of Hepatitis B Virus Surface Antigen-Positive Plasma With Undetectable HBV-DNA: Can HBsAg Screening Be Discontinued in Egyptian Blood Donors?

Hepatitis B Virus (HBV) infectivity data were reviewed and the 50% infectious dose (ID50) was reassessed in different HBsAg-positive infection stages enabling modelling of transfusion-transmitted (TT)-HBV infection risk if HBsAg donor screening was replaced by individual donation nucleic acid amplification technology (ID-NAT). Quantitative HBsAg and HBV-DNA assays were performed against international standards to compare the ratio between potential infectious HBV virions and subviral HBsAg particles in Egyptian HBsAg-positive blood donors as well as in Japanese chimpanzee samples of known infectivity. HBV-DNA load below the quantification limit of detection was estimated against a reference standard by replicate NAT testing (n = 25). Infectivity of chimpanzee samples collected during ramp-up and declining viremic phase were tested in a human liver chimeric mice (HLCM) model and compared with published infectivity data from different HBsAg-positive infection stages. Lowest estimates of ID50 in HBsAg-positive plasma were 3-6 HBV virions in chimpanzee studies. Infectivity decreased approximately 10-100-fold in the declining viremic phase using HLCM. In acute phase samples, HBV to HBsAg particle ratios varied between 1:102-104 but in HBsAg-positive blood donors this particle ratio reached 1:106-1012 when viral load was below 100 HBV-DNA copies/mL. Modelled TT-HBV risk of an HBsAg-positive/ID-NAT nonreactive blood transfusion was estimated at 5.5%-27% for components containing 20-200 mL of plasma when assuming an ID50 of 316 (point estimate between 100 and 1000) virions. It cannot be ensured that discontinuation of HBsAg donor screening and reliance on ID-NAT alone is safe.

Ultrasensitive PCR system for HBV DNA detection: Risk stratification for occult hepatitis B virus infection in English blood donors.

Occult hepatitis B (HBV) infection (OBI), characterized by low viral loads, accounts for much of the risk of HBV transfusion-transmitted infection. With anticore antibodies (anti-HBc) screening introduced in England, the imperative to identify OBI donors has increased. We aimed to develop an ultra-sensitive PCR system and investigate risk factors for HBV DNA presence in blood donations. Seven extraction methods and three PCR assays were compared. The optimal system was sought to determine HBV DNA presence in anti-HBc-positive donations. Predictors of DNA positivity were subsequently investigated. Extraction from 5 mL of plasma increased sample representation and resulted in HBV DNA detection in low viral load samples (~0.5 IU/mL). Screening of 487 763 donations in 2022 identified two OBI donors and 2042 anti-HBc-positive donors, 412 of the latter with anti-HBs < 100 mIU/mL. Testing of 134 anti-HBc-positive donations utilizing the 5 mL extraction method identified two further HBV DNA-positive donations. Higher anti-HBc titer and anti-HBs negativity were significant predictors of DNA detectability in anti-HBc-positive donations. An ultrasensitive PCR assay identified potentially infectious donations increasing HBV DNA detection in anti-HBc-positive donors from 0.5% to 1.9%. Anti-HBc titers may further complement the risk stratification for DNA positivity in anti-HBc screening and minimize unnecessary donor deferral.

Confirmation of HBV infection of HBsAg-negative NAT suspicious sample from blood donors / 中国输血杂志

【Objective】 To explore the HBV infection of initially reactive but discriminatory test non-reactive (NAT suspicious) samples of voluntary blood donors after PANTHER individual nucleic acid testing (ID-NAT) in Tianjin. 【Methods】 From January to August 2021, after routine testing and PANTHER ID-NAT, a total of 66 HBsAg-NAT reactive but discriminatory test non-reactive samples(referred to as NAT suspicious samples) were tested from 69 362 blood samples. Among which, 23 samples were selected by simple random sampling method and enriched by ultra-high speed centrifugation. HBV DNA was detected by supersensitive fluorescence quantification PCR (qPCR)and ID-NAT, and electrochemiluminescence was supplemented for two and half pairs of hepatitis B detection. 【Results】 Among 23 suspicious NAT samples, 14 were confirmed HBV DNA positive by serological and molecular biological tests, and the anti-HBc positive rate of HBV infected individuals was 92.8%. 92.8% (13/14) of the infected individuals were occult hepatitis B virus infection(OBI). A total of 10 samples were detected for viral load by qPCR, of which 5 were quantifiable, with viral load of (11~464) IU/mL and a median of 15.4 IU/mL. 【Conclusion】 60% of the NAT suspicious samples were detected as HBV DNA positive. Anti-HBc testing can exclude most OBI undetectable by NAT, and the sensitivity of NAT should be improved to ensure the safety of blood transfusion.

Infectivity of hepatitis B virus (HBV) surface antigen (HBsAg) positive plasma with undetectable HBV-DNA: Can HBsAg screening be discontinued in Egyptian blood donors?

HBV infectivity data were reviewed and the 50% infectious dose (ID50 ) was reassessed in different HBsAg positive infection stages enabling modelling of transfusion-transmitted (TT)-HBV infection risk if HBsAg donor screening was replaced by individual donation nucleic acid amplification technology (ID-NAT). Quantitative HBsAg and HBV-DNA assays were performed against international standards to compare the ratio between potential infectious HBV virions and subviral HBsAg particles in Egyptian HBsAg positive blood donors as well as in Japanese chimpanzee samples of known infectivity. HBV-DNA load below the quantification limit of detection was estimated against a reference standard by replicate NAT testing (n = 25). Infectivity of chimpanzee samples collected during ramp-up and declining viremic phase were tested in a human liver chimeric mice (HLCM) model and compared with published infectivity data from different HBsAg positive infection stages. Lowest estimates of ID50 in HBsAg positive plasma were 3-6 HBV virions in chimpanzee studies. Infectivity decreased approximately 10-100-fold in the declining viremic phase using HLCM. In acute-phase samples, HBV to HBsAg particle ratios varied between 1:102 -104 but in HBsAg positive blood donors this particle ratio reached 1:106 -1012 when viral load was below 100 HBV-DNA copies/ml. Modelled TT-HBV risk of an HBsAg positive/ID-NAT nonreactive blood transfusion was estimated at 9%-46% for components containing 20-200 ml of plasma assuming an ID50 of 316 (point estimate between 100 and 1000) virions. In the Egyptian setting, discontinuation of HBsAg donor screening and reliance on ID-NAT alone seems to be unsafe.

Prevalence of Occult Hepatitis B Virus Infection in Blood Donors with Negative ID-NAT in Switzerland.

Introduction: Screening of hepatitis B surface antigen (HBsAg) and individual-donation nucleic acid amplification testing (ID-NAT) of blood donors have become standard to detect hepatitis B virus (HBV) infection. However, there is still a residual risk of HBV transmission by blood components of donors suffering from occult HBV infection (OBI). Therefore, many countries implemented universal testing of anti-HBV core antigen (anti-HBc) antibodies in order to increase blood safety. In Switzerland, anti-HBc testing is not part of the routine blood donor-screening repertoire. Therefore, we sought to assess prevalence of donors with OBI in a Swiss blood donor collective. Methods: Blood donations were prospectively investigated for the presence of anti-HBc antibodies during two time periods (I: all donors, March 2017; II: first-time donors only, April 2017 until February 2018). Anti-HBc-positive findings were confirmed by an anti-HBc neutralization test. Discarded plasma samples of anti-HBc-confirmed positive donors were ultracentrifuged and subsequently retested by regular HBV-ID-NAT to search for traces of HBV. Results: During time period I, 78 (1.6%) individuals out of 4,923 donors were confirmed anti-HBc-positive. Sixty-nine (88%) anti-HBc-positive samples were available and processed by ultracentrifugation followed by repeat HBV-ID-NAT. Four samples (5.8%) were found positive for HBV DNA. Sixty-five (94.2%) samples remained HBV NAT-negative upon ultracentrifugation. During time period II, 56 (0.9%) donor samples out of 6,509 exhibited anti-HBc-confirmed positive. Fifty-five (98%) samples could be reassessed by HBV-ID-NAT upon ultracentrifugation. Three (5.5%) samples contained HBV DNA and 52 (94.5%) samples remained HBV NAT-negative. Conclusion: Overall, we detected 7 viremic OBI carriers among 11,432 blood donors, which tested negative for HBV by standard HBV-ID-NAT and HBsAg screening. In contrast, OBI carriers showed positive anti-HBc findings which could be confirmed in 83.8% of the cases. Thus, OBI might be missed by the current HBV screening process of Swiss blood donors. We suggest to review current HBV screening algorithm. Extended donor screening by anti-HBc testing may unmask OBI carriers and contribute to blood safety for the recipient of blood products.

Nucleic acid technology(NAT) testing for blood screening: A comparative study on individual donation and minipool NAT test / 中国输血杂志

【Objective】 To compare the detection performance of Cobas s201, a minipool(MP) nucleic acid test(NAT) system, and Panther, a individual donation(ID) NAT system, in blood donor screening. 【Methods】 NAT was conducted on 126 359 blood samples, and initially reactive (IR) samples were either discriminated or resolved by ID testing.The non-discriminated reactive (NDR) samples implicated in Panther sysytem were subjected to ID-NAT by Cobas s201. Some non-repeatable reactivet(NRR) and repeatable reactive (RR) samples implicated in Cobas s201 system were subjected to ID-NAT by Panther. 【Results】 61 MP-IR cases were implicated in a total of 85 128 samples that detected by Cobas 201, and 29(0.34‰) were RR after resolved by ID testing. 74(1.79‰)IR samples were implicated in 41 231 samples that detected by Panther, and 22 (29.73%) were DR-HBV after discriminatory test. Among the NDR 28 samples detected by Panther multiplex system, 7 were positive by Cobas s201 single sample (PP1) whereas non-reactive in simulated MPs of six by Cobas 201.In 28 RR samples resolved by Cobas 201, 24 positive and 4 negative samples were retested by Panther. Among the 11 samples presenting inconsistent retest results by Panther and Cobas 201, 10 were anti-HBc positive, carrying low viral load HBV. 【Conclusion】 The NAT-yield by Panther was significantly higher than that by Cobas s201. Some samples with negative discriminatory results were OBI, and it is necessary to further track and verify the unidentified samples. Cobas s201 is more suitable for a wide array of MP-NAT testing while Panther sample loading, which is flexible and easy to operate, is more suitable for ID-NAT with medium sample size.

Prevalence of Hepatitis B and C Viral Markers in Blood Donors Deferred from Donating Blood on the Basis of a History of Jaundice of Unknown Origin.

BACKGROUND AND AIMS: The aim of the present study was to know the prevalence of viral hepatitis markers among blood donors deferred on the basis of a history of jaundice of unknown origin and to predict the impact of this deferral on blood safety. METHODS: Observational study included 200 blood donors deferred on the basis of a history of jaundice of unknown origin and carried out serology and Individual Donation Nucleic Acid Testing (ID-NAT) for hepatitis B and hepatitis C. RESULTS: Five (2.5%) out of 200 blood donors deferred on the basis of history of jaundice were reactive on ELISA. Three out of five were reactive for HBsAg; whereas two for anti-HCV antibodies. Out of the 12 ID-NAT initial reactive, 10 samples discriminated on further testing; 4 for HBV, 5 for HCV and 1 was co-infection (HBV + HCV). The odds of being picked up as sero reactive on ELISA was 2.53 (95% CI; 1.04-6.19) and being picked up as ID-NAT yield was 13.08 (95% CI; 5.29-32.37) in donors deferred on a history of jaundice of unknown origin as compared to selected donors without a history of jaundice, with the difference in means achieving statistically significance at P = 0.03 and <0.001 respectively.The potential of deferral on a history of jaundice of unknown origin has a capacity to interdict 2-3 HBV and/or HCV reactive blood donors on serology and 5 HBV and/or HCV reactive blood donors on ID-NAT from entering the quarantine blood supply per 100 donors. CONCLUSION: The findings of the present study support that a deferral for a history of jaundice of unknown origin after attaining the age of 12 years in the present scenario of transfusion transmissible infectious disease screening in India.

NAT positivity in seronegative voluntary blood donors from western India.

BACKGROUND AND OBJECTIVES: Prevalence and composition of Hepatitis B, Hepatitis C and HIV-1, NAT positive but seronegative voluntary blood donors from western part of India is yet to be documented. MATERIAL AND METHODS: Over last 2 1/2 years all the seronegative voluntary blood donors were tested using 10 minipools on a semiautomated NAT testing platform. The positively tested donors were followed up for at least five months for development of seropositivity. RESULTS: 79532 seronegative donations were tested by 10 minipool (MP) NAT leading to 51 positive sample (44 Hep B, 5 HIV 1 and Hep C positive). All the HIV and Hep C NAT positive donors eventually developed seropositivity and out of 44 Hep B NAT positive donors, 31 developed seropositivity within six months of follow up, following counseling of the donors. This data translate into NAT yield of 1:1559 donors for all virus taken together. NAT yield for Hep B 1:1807 donors were much higher than HIV 1 in 1:15906 and HCV yield of 1:39761. Semiautomated minipool NAT testing system was found to be cost effective way for improving blood safety. INTERPRETATION AND CONCLUSION: Seronegative NAT yield in voluntary blood donors are quiet high in western part of India and in line with rest of the country is mainly due to Hepatitis B infection. Implementation of strict donor screening, Hep B vaccination of the population and sample mutation of NAT testing should be under taken on war footing.

Confirmation and follow up of initial "NAT yields": Prospective study from a tertiary healthcare center in India.

BACKGROUND: In India variable rate of "NAT yield" has been demonstrated in several published reports. This study was performed with the objective to know the rate of "true NAT yield" in blood donors by confirmation with supplementary tests and follow up of initial "NAT yield" donors. MATERIALS AND METHODS: A total of 48,441 blood donors were tested for HIV, HBV and HCV with enhanced chemiluminescence and ID-NAT. To know the true NAT yield status confirmation of NAT yield donors was done as with an array of serological tests, repeat ID-NAT and alternate NAT. RESULTS: The cumulative initial "NAT yield" rate was 1:4404 (11/48,441). Seven of 11 initial "NAT yields" were for hepatitis B whereas two each were in HIV and HCV. Among the 11 "NAT-yield" donors, eight donors were followed-up for confirmation. Out of these eight donors only 4 were found to be true HBV NAT yields. Out of four true NAT yields two were window period donations while the other two were occult hepatitis B infection with anti-HBcore total positive. CONCLUSION: Our findings suggest that all "initial NAT yields" may not be "true NAT yields". We would also like to suggest that to demonstrate the true "NAT yield" status supplementary tests and donor follow up are important to differentiate true NAT yields.

Hepatitis B core antibody testing in Indian blood donors: A double-edged sword!

BACKGROUND: Until lately, anti-HBc antibodies were considered an effective marker for occult Hepatitis B virus (HBV) infection and have served their role in improving blood safety. But, with the development of advanced tests for HBV DNA detection, the role of anti-HBc in this regard stands uncertain. MATERIALS AND METHODS: Anti-HBc and HBsAg ELISA and ID-NAT tests were run in parallel on donor blood samples between April 1, 2006 and December 31, 2010 at the Department of Transfusion Medicine, Indraprastha Apollo Hospitals, New Delhi. A positive ID-NAT was followed by Discriminatory NAT assay. RESULTS: A total of 94 247 samples were tested with a total core positivity rate of 10.22%. We identified nearly 9.17% of donors who were reactive for anti-HBc and negative for HBsAg and HBV DNA. These are the donors who are potentially non-infectious and may be returned to the donor pool. CONCLUSION: Although anti HBc testing has a definite role in improving blood safety, centers that have incorporated NAT testing may not derive any additional benefit by performing anti-HBc testing, especially in resource-limited countries like ours.