No Hepatitis Delta Virus Seropositivity among Blood Donors with Overt and Occult Hepatitis B Infection in Dalian, Liaoning Province, China.

Hepatitis delta virus (HDV) is an obligate satellite of hepatitis B virus (HBV). Dual HDV/HBV infection is associated with down-regulated HBV replication and fast progression to severe liver disease. Although HDV is transmissible through exposure to infected blood, data about HDV infection in blood donors remain scarce. Between 2011 and 2021, 869,633 donations were collected from prequalified donors in Dalian, China. In total, 1060 (0.12%) were confirmed HBsAg and/or HBV DNA-reactive. Subsequently, anti-HDV IgG was tested in 2175 donations, including 65 that tested HBsAg+ pre donation, 1017 confirmed HBV-positive (507 HBsAg+/HBV DNA+, 33 HBsAg+/DNA-, 477 HBsAg-/DNA+ (451 occult (OBI) and 26 acute infections)), 327 viral DNA non-repeated-reactive, 397 anti-HBc-only, and 369 anti-HBs-only. Two (0.09%) samples tested anti-HDV IgG weakly reactive but were unconfirmed by IgM and IgG repeat testing with alternative assays, suggesting an initial false reactivity. In addition, HDV testing in a subgroup of confirmed OBI donors, comprising 451 donors from Dalian and 126 archived samples of OBI donors from around the world, showed only one non-Chinese donor to be repeatedly anti-HDV-reactive, suggesting that HDV/HBV coinfection does not play a significant role in the genesis of OBI. The overall data suggested an extremely low prevalence of HDV infection among blood donors in Liaoning province, Northeast China.

Monitoring viral genomic sequences in transfusion-transmitted viruses.

BACKGROUND AND OBJECTIVES: Monitoring genomic sequences of blood-borne viruses infecting blood donors enables blood operators to undertake molecular epidemiology, confirm transfusion transmission and assess and characterize molecular and serological screening assays. The purpose of the study was to determine how blood operators globally value viral diversity surveillance and to assess its impact. MATERIALS AND METHODS: An electronic questionnaire was developed and circulated to members of the International Society of Blood Transfusion-transmitted infectious diseases working party. Responses were compiled and complete data sets were analysed. RESULTS: Ninety-seven percent of respondents agreed that monitoring viral genomic sequences was important to blood operators and the transfusion community. However, only 47% of respondents are currently doing this monitoring. The main limitations reported were a lack of financial resources and expertise. Sequencing techniques, primarily next-generation sequencing and also Sanger sequencing, were considered most appropriate, with the preferred option for testing being regional or national reference centres. Respondents agreed that engagement with public health authorities needs to be enhanced. CONCLUSION: Monitoring genomic sequences of blood-borne viruses is widely considered important by the transfusion community because of its direct applications for transfusion safety, and beyond for public health in general. Therefore, there is a need to strengthen collaboration between blood operators and public health authorities. While national and regional reference centres may be the most appropriate structure for such testing, international collaborations should not be overlooked. Overcoming financial barriers will be an important hurdle for many.

Occult Hepatitis B Virus Infection and Liver Fibrosis in Chinese Patients.

BACKGROUND: The impact of hepatitis B surface antigen (HBsAg)-negative/hepatitis B virus (HBV) DNA-positive occult HBV infection (OBI) on the severity of liver fibrosis remains unclear. METHODS: A total of 1772 patients negative for HBsAg but positive for antibody to hepatitis B core antigen (HBcAg), stratified by the presence or absence of OBI, were selected for long-term carriage leading to elevation of ≥2 of 4 liver fibrosis indexes-hyaluronic acid (HA), laminin, type III procollagen peptide (PCIII), and type IV collagen (CIV)-at testing in a Chinese hospital. Patients were tested for serum viral load, HBV markers, and histopathological changes in liver biopsy specimens. RESULTS: OBI was identified in 148 patients with liver fibrosis (8.4%), who had significantly higher levels of HA, laminin, PCIII, and CIV than 1624 fibrotic patients without OBI (P < .05). In 36 patients with OBI who underwent liver biopsy, significant correlations were observed between OBI viral load and serum HA levels (P = .01), PCIII levels (P = .01), and pathological histological activity index (HAI) scores (P < .001), respectively; HAI scores and PCIII levels (P = .04); HBcAg immunohistochemical scores and HA levels (P < .001); and HBcAg immunohistochemical scores and PCIII levels (P = .03). Positive fluorescent in situ hybridization results were significantly more frequent in patients with OBIs (80.6% vs 37.5% in those without OBIs). Among patients with OBIs, HBcAg was detected in the liver tissue in 52.8% and HBsAg in 5.6%. CONCLUSIONS: OBI status appears to be associated with liver fibrosis severity.

Insights on 21 Years of HBV Surveillance in Blood Donors in France.

Hepatitis B virus (HBV) infection is the most frequent viral infection found in blood donors (BDs) in France. We analyzed the epidemiological and sero-molecular data on HBV infection gathered over the past two decades by the French haemovigilance surveillance network, blood screening laboratories, and the national reference center for transfusion infectious risks (NRC). Between 2000 and 2020, 6149 of the 58,160,984 donations (1.06/10,000) tested HBV positive, 98% of them from first-time blood donors (FTBDs). In addition, 2212 (0.0071%) of the 30,977,753 donations screened for HBV DNA tested DNA positive, of which 25 (1.1%) were positive only for this marker. HBV prevalence decreased by 2.8-fold and the residual risk for transfusion-transmitted HBV infection decreased 13-fold and was divided by 13. The major risk factor for HBV infection was the origin of donors (endemic country, 66.5%), followed by parenteral exposure (10.7%). In the whole HBV-positive BD population, genotype D was predominant (41.8%), followed by genotypes A (26.2%) and E (20.4%), reflecting the geographical origin of donors. The low and decreasing prevalence and incidence of HBV infection in French BDs, coupled with a screening strategy using three HBV markers (HBsAg, anti-HBc and DNA), ensures a high level of blood safety, further reinforced by the implementation of pathogen-reduction measures.

Re-Entry Evaluation of Chinese Blood Donors with Unconfirmed Hepatitis B Screening Results.

The hepatitis B virus (HBV) remains a high priority for Chinese blood banks due to the high prevalence of infection. HBV blood safety has been significantly improved by the implementation of highly sensitive and specific serological and molecular HBV screening assays. The multiplication of viral markers tested and the ever-increasing analytical sensitivity of the tests can make the interpretation of the results difficult. False-positive or indeterminate results may lead to permanent donor deferrals and conflicts between donors and blood banks. To avoid blood shortages, blood services aim to limit unnecessary donor losses by developing procedures for the re-entry of donors temporarily deferred due to an unconfirmed HBV reactivity. The development of such procedures based on donor follow-up and HBV confirmation remains limited. A review of the scarce data available revealed considerable heterogeneity in testing methods and re-entry algorithms, limited validation studies, and a lack of accurate assessment of the residual infectious risk potentially associated with donor re-entry. In conclusion, systematic and widely validated confirmatory testing and prolonged follow-up are essential for safe re-entry of temporary deferred donors. Standardization of HBV testing methods and the establishment of dedicated expert laboratories are needed because of the complexity of HBV infection in blood donors.

Anti-HBc-nonreactive occult hepatitis B infections with HBV genotypes B and C in vaccinated immunocompetent adults.

Absence of anti-HBc reactivity with detectable anti-HBs was observed in blood donors with occult hepatitis B virus (HBV) infection (OBI). The prevalence and mechanisms underlying this uncommon condition were investigated over time in Chinese blood donors with OBI. Isolated anti-HBs OBI status was identified from 466,911 donors from Dalian, China, and monitored in follow-up (range: 2.6-84.3 months). HBV vaccination status was documented, and infecting viral strains were characterized. Of 451 confirmed OBIs (1:1035), 43 (9.5%; 1:10,858) had isolated anti-HBs as only serological marker. Isolated anti-HBs OBIs differed from anti-HBc-reactive OBIs by significantly younger age (median 24 years), higher HBV DNA (median: 20 IU/ml) and anti-HBs (median 60.5 IU/L) levels, paucity of mutations in HBV Core and S proteins, and high vaccination rate (72%). Vaccinated isolated anti-HBs OBIs (n = 31) differed from unvaccinated (n = 11) by significantly younger age (22 vs 38 years), higher anti-HBs level at index (48% vs 9% with anti-HBs >100 IU/L) and higher frequency of anti-HBs immune response (44% vs 20%). Of 15 vaccinated and 5 unvaccinated OBIs follow-up, 65% (8 vaccinated and 5 unvaccinated) became HBV DNA negative suggesting aborted recent infection, while 35% (7 vaccinated) had low persistent viraemia 2 to 65 months post index. In conclusion, isolated anti-HBs OBI in Chinese blood donors appears associated with young, vaccinated, adults exposed to HBV who predominantly develop low level aborted infection revealed by transient HBV DNA and immune anti-HBs response. However, a subset of individuals still experienced low but persistent viral replication whose clinical outcome remains uncertain.

Effectiveness of the HCV blood screening strategy through eighteen years of surveillance of HCV infection in blood donors in France.

BACKGROUND: The question of maintaining blood screening based on both Hepatitis C virus (HCV) infection antibodies (Ab) and Nucleic Acid Testing (NAT) has been raised in several countries. The French blood donor surveillance database was used to address this issue. MATERIALS AND METHODS: In France, HCV-NAT was implemented in mini pools (MP) in 2001 and in individual testing (ID) in 2010. HCV-positive donations are further investigated including detection of RNA with an alternative polymerase chain reaction assay: Amplicor HCV v2.0 (Roche; LOD95 50 IU/mL) from 2001 to 2006 and CobasTaqMan (CTM) HCV 2.0 assay (Roche; LOD95 9.3 IU/mL) since 2007. RESULTS: From 2001 to 2018, 3,058/48.8 million donations were confirmed HCV positive: 64.4% were Ab+/NAT+, 35.1% Ab+/NAT- and 0.5% Ab-/NAT+. From 2001 to 2018, the NAT yield decreased from 0.65 per million donations to 0, and NAT+ donations dropped from 77% to 46% of the total of HCV donations. 2,491/3,058 were further tested for HCV-RNA: 1,032 (816 NAT+, 216 NAT-) with Amplicor and 1,459 (897 NAT+, 562 NAT-) with CTM. Four (3 MP and 1 ID-NAT, 0.5%) of the 778 NAT negative donations had low viral loads. DISCUSSION: The decline in HCV-NAT yield cases raises the question of the relevance of NAT. Conversely, the increase in Ab+/NAT-donors, suggesting a growing number of resolved infections, argue for Ab discontinuation. In our experience, at least 0.5% of Ab+/NAT-donations had low RNA level when retested. Although the risk of viral transmission by such donations is probably low, the uncertainty associated with their infectivity goes against the removal of Ab in blood screening in our country.

Alternative hepatitis B virus DNA confirmatory algorithm identified occult hepatitis B virus infection in Chinese blood donors with non-discriminatory nucleic acid testing.

BACKGROUND: Multiplex viral nucleic acid testing (NAT) and a discriminatory testing algorithm have been used to detect viral infections in blood donors. Non-discriminated reactive (NDR) results may arise from low hepatitis B virus (HBV) DNA levels and are challenging for donor management by blood services. The aim of this study was to evaluate the performance and feasibility of alternative viral particle concentration methods to confirm and to characterise HBV infection status in NDR donors from Dalian, China, in order to improve routine donor management according to the potential residual risk estimate. MATERIALS AND METHODS: Individual donations were tested with ULTRIO Plus, and discriminated when reactive. Virions were concentrated from 12 and 6 mL plasma samples by ultracentrifugation (UC) and polyethylene glycol (PEG) precipitation, respectively. HBV DNA was detected with four nested polymerase chain reactions (95% limit of detection: 5-25 IU/mL). Amplified products were sequenced for definitive confirmation. Anti-HBc and anti-HBs were tested. RESULTS: Of 77,556 donors, 79 (0.1%) were NAT NDR. After viral particle concentration by UC and PEG precipitation, HBV DNA was detected in 46 (58.2%) and 34 (43.0%) NDR donors, respectively, including 61.7% of samples that were repeatedly non-reactive with multiple NAT testing. Anti-HBc and anti-HBs (median titre: 37 mIU/mL) were detected in 87.3% and 46.8% of NDR donors, respectively. Sequencing confirmed HBV DNA in 65.8% of NDR donors, of whom 96.2% were occult HBV carriers with rare mutations in S and core proteins. DISCUSSION: A HBV DNA confirmatory procedure with limited technical constraints was implemented successfully. The majority of NDR donors had occult HBV infections with extremely low viral DNA levels, which may constitute a potential residual threat for blood safety. Only a minority of anti-HBc+ NDR donors had anti-HBs levels high enough to consider their reinstatement as donors. The data support the permanent deferral of NDR donors to ensure maximum blood safety in areas of high HBV endemicity.

Viruses Special Issue "Transfusion-Transmitted Viral Infections".

Blood transfusion is a life-saving treatment that requires patients to have access to safe and adequate blood products that are available in a timely manner [...].

Mass SARS-CoV-2 molecular and serological screening of medical staff and patients in Hangzhou, China: no evidence of RNA detection, low seroprevalence, and limited exposure risk in the hospital setting.

BACKGROUND: To assess and limit the SARS-CoV-2 exposure risk from symptomless individuals in the hospital setting, molecular and serological screening of staff and patients attending a tertiary hospital in China was conducted. METHODS: SARS-CoV-2 RNA was tested by quantitative RT-PCR. Anti-SARS-CoV-2 IgM and IgG were screened initially with two lateral flow immunoassays (LFIs) and further confirmed with three chemiluminescence immunoassays (CLIAs). The assay performance was assessed using archived samples from 32 confirmed COVID-19 cases and 80 healthy individuals. RESULTS: Between April 24 and May 8, 2020, 16,043 subjects (7,392 medical staff, 4,714 inpatients, 1,209 chaperones, 1,705 outpatients, and 1,023 fever clinic patients) were screened. No subject tested positive for viral RNA. Seventy-three (0.46%) tested positive for IgM or IgG on the initial LFI screening, of whom 63 were investigated with CLIAs: 2 (0.01%) were confirmed as seroreactive and 18 (0.11%) were indeterminate. Unconfirmed seroreactivity was significantly more frequent in fever clinic patients. The CLIAs showed similar (95.0-100%) IgM or IgG specificity but higher IgG sensitivity (93.75-96.88% vs. 31.25-81.25%) than the LFIs. The confirmed seropositive cases included a previously discharged COVID-19 patient and an undiagnosed symptomless patient showing detectable IgM and IgG over 35 days of follow-up. No transmission was evidenced within the corresponding family cluster. CONCLUSIONS: Low SARS-CoV-2 prevalence and limited exposure risk were observed. Seroprevalence varied between 0.012% and 0.12% according to the testing algorithm and the confirmation criteria used, indicating that quality standards for serological tests are needed. Protective immunity in asymptomatic COVID-19 patients who recovered needs to be investigated further, but the associated risk of transmission appeared limited.

Development and clinical validation of loop-mediated isothermal amplification (LAMP) assay to diagnose high HBV DNA levels in resource-limited settings.

OBJECTIVE: A massive scale-up of testing and treatment is indicated to globally eliminate hepatitis B virus (HBV) infection. However, access to a polymerase chain reaction (PCR), a key test to quantify HBV DNA levels and determine treatment eligibility, is limited in resource-limited countries. We have developed and evaluated the loop-mediated isothermal amplification (LAMP) assay to diagnose clinically important HBV DNA thresholds defined by the WHO (≥20 000 and ≥ 200 000 IU/mL). METHODS: Pan-genotypic primer sets were designed on conserved HBV gene regions. Accuracy of LAMP to identify highly viraemic patients was evaluated in 400 and 550 HBV-infected people in France and Senegal, respectively. RESULTS: Our primers successfully detected eight major HBV genotypes/sub-genotypes (A1/2/3/B/C/D/E/F) with a detection limit ranging between 40 and 400 IU/mL. In France, the area under the receiver operating characteristic curve (AUROC), sensitivity and specificity of bead-based extraction and real-time turbidimetric LAMP were 0.95 (95% CI 0.93-0.97), 91.1% and 86.0%, respectively, to diagnose HBV DNA ≥20 000 IU/mL; and 0.98 (0.97-0.99), 98.0% and 94.6% for ≥200 000 IU/mL. The performance did not vary by viral genotypes. In Senegal, using a field-adapted method (reagent-free boil-and-spin extraction and inexpensive end-point fluorescence detection), the AUROC, sensitivity and specificity were 0.95 (0.93-0.97), 98.7% and 91.5%, respectively, to diagnose HBV DNA ≥200 000 IU/mL. The assay was not adapted to discriminate low-level viraemia. DISCUSSION: We have developed a simple, rapid (60 min), and inexpensive (US$8/assay) alternative to PCR to diagnose high viraemia ≥200 000 IU/mL. HBV-LAMP may contribute to eliminating HBV mother-to-child transmission by identifying high-risk pregnant women eligible for antiviral prophylaxis in resource-limited countries.

Role of core protein mutations in the development of occult HBV infection.

BACKGROUND & AIMS: Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. METHODS: An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo. RESULTS: A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. CONCLUSIONS: W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. LAY SUMMARY: Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively in vitro and in vivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.

A Comparison of Cell Activation, Exhaustion, and Expression of HIV Coreceptors and Restriction Factors in HIV-1- and HIV-2-Infected Nonprogressors.

Human immunodeficiency viruses induce rare attenuated diseases due either to HIV-1 in the exceptional long-term nonprogressors (LTNPs) or to HIV-2 in West Africa. To better understand characteristics of these two disease types we performed a multiplex comparative analysis of cell activation, exhaustion, and expression of coreceptors and restriction factors in CD4 T cells susceptible to harbor those viruses. We analyzed by flow cytometry the expression of HLA-DR, PD1, CCR5, CXCR6, SAMHD1, Blimp-1, and TRIM5α on CD4 T cell subsets from 10 HIV-1+ LTNPs and 14 HIV-2+ (12 nonprogressors and 2 progressors) of the ANRS CO-15 and CO-5 cohorts, respectively, and 12 HIV- healthy donors (HD). The V3 loop of the HIV-1 envelope from 6 HIV-1+ LTNPs was sequenced to determine the CXCR6-binding capacity. Proportions of HLA-DR+ and PD1+ cells were higher in memory CD4 T subsets from HIV-1 LTNPs compared with HIV-2 and HD. Similar findings were observed for CCR5+ cells although limited to central-memory CD4 T cell (TCM) and follicular helper T cell subsets, whereas all major subsets from HIV-1 LTNPs contained less CXCR6+ cells compared with HIV-2. All six V3 loop sequences from HIV-1 LTNPs contained a proline at position 326. Proportions of SAMHD1+ cells were higher in all resting CD4 T subsets from HIV-1 LTNPs compared with the other groups, whereas Blimp-1+ and Trim5α+ cells did not differ. The CD4 T cell subsets from HIV-1 LTNPs differ from those of HIV-2-infected subjects by higher levels of activation, exhaustion, and SAMHD1 expression that can reflect the distinct patterns of host/virus relationships.

The contribution of more sensitive hepatitis B surface antigen assays to detecting and monitoring hepatitis B infection.

BACKGROUND: Hepatitis B surface antigen (HBsAg) remains the main viral marker for screening and monitoring hepatitis B virus (HBV) infection. The quantification limit of most current HBsAg assays is around 0.05 IU/mL. The Lumipulse-G-HBsAg-Quant assay (Fujirebio) claims to obtain a tenfold improvement in sensitivity. This study aimed to assess the performance of this assay in detecting low HBsAg levels in clinical samples. METHODS: Three panels of stored frozen samples were selected on the basis of HBV-DNA and HBsAg values obtained previously with routine techniques. Panels 1 (n=13) and 2 (n=52) consisted of DNA-positive/HBsAg-negative samples from individuals in the window period and with occult HBV infection respectively. Panel 3 comprised 23 samples with low or discrepant HBsAg screening results. All these samples were tested retrospectively with the DiaSorin and Fujirebio HBsAg assays. RESULTS: Sixteen out of 65 samples (25 %), initially screened HBsAg negative, were reactive only with the Fujirebio assay (median value= 0.015 IU/mL; IQR= 0.012): three (23 %) samples from panel 1 and 13 (25 %) from panel 2. Thirteen of these 16 (81 %) had HBsAg values below 0.03 IU/mL with the DiaSorin assay. In panel 3, 22 (96 %) samples were quantified successfully with the Fujirebio assay (median: 0.32 IU/mL; IQR: 1.20) and 19 (83 %) with the DiaSorin assay (median: 0.31 IU/mL; IQR: 0.65). Concentrations obtained with the two assays showed good correlations (r=0.893, Spearman). CONCLUSIONS: HBsAg assays with enhanced analytical sensitivity could improve HBV serological profile interpretation with possible consequences on clinical management of infected patients, and on blood transfusion safety.

Follow-up program for blood donors with unconfirmed screening results reveals a high false-positive rate in Dalian, China.

BACKGROUND: Chinese blood donors with unconfirmed serological and/or molecular screening results are deferred permanently. This study investigated the implementation and performance of a follow-up program aiming to improve the notification and management of deferred donors in Dalian, China. STUDY DESIGN AND METHODS: From January 2013 to February 2018, 411,216 donations were tested for HBsAg, anti-HCV, anti-HIV/HIV antigen, and antibodies to Treponema pallidum. HBV, HCV, and HIV nucleic acid testing (NAT) was performed in mini-pools of six or in individual donations (IDs). Reactive donations were evaluated further with alternative serological assays and ID-NAT re-testing. A follow-up procedure was developed to evaluate a subset of deferred donors that were either potential NAT yield cases, serology non-reactive and NAT non-repeated reactive (NRR), or serology NRR irrespective of NAT result. RESULTS: Serological and molecular routine, plus supplemental testing, identified HBV, HCV, HIV, and TP in 503 (0.12%), 392 (0.09%), 156 (0.04%), and 2041 (0.49%) donations, respectively. Overall, 683 of 4156 (16.4%) eligible donors and 205 donors deferred prior 2013 participated in the program. They included 664 serology NRR and 224 NAT yield cases, and 58.8% repeat donors. All markers combined, follow-up documented false reactivity, primary acute infections, and OBI in 61.9% (550/888), 3.3% (29/888), and 12.8% (114/888) of these donors, respectively. Isolated anti-HBc or anti-HBs reactivity was observed in 22% of cases. CONCLUSION: Follow-up testing refined infection status in 78.0% (693/888) of deferred donors with unconfirmed screening results. This high false-positive rate encouraged to reevaluate the current screening strategies and to consider donor reentry.

Metagenomic analysis of potential pathogens from blood donors in Guangzhou, China.

OBJECTIVES: This study aimed to identify the emerging/reemerging pathogens in blood donation samples. BACKGROUND: A metagenomic analysis has previously been used to look for pathogens but in this study, the relationship with aminotransferase (ALT) is described. METHODS/MATERIALS: Excluding samples reactive to hepatitis B virus, hepatitis C virus, human immunodeficiency syndrome virus or syphilis and plasma samples were stratified into three groups of ALT levels (IU/L): A ≤ 50, B 51 to 69 and C ≥ 70, respectively. Each group was mixed in a pool of 100 samples, from which DNA and cDNA libraries were established for next generation sequencing and analysis. Pathogens of interest were identified by immunoassays, nested-polymerase chain reaction, phylogenetic analysis and pathogen detection in follow-up donors. RESULTS: Several new or reemerging transfusion-transmitted pathogens were identified; Streptococcus suis, Babesia species and Toxoplasma gondii were found in the three ALT groups, Epstein-Barr virus (EBV) only in group C. Ten S. suis nucleic acid positive samples were detected, all closely phylogenetically related to reference strains. A donor in group A carried both S. suis genome and specific IgM in follow-up samples. This strain was identified as nontoxic S. suis. Five samples contained a short fragment of Babesia species SpeI-AvaI gene, while T. gondii was identified in 20 samples as a short fragment of 18S rDNA gene. In group C, two samples contained EBV genome. CONCLUSIONS: Blood donations that contained S. suis, Babesia species and T. gondii sequences might represent potential transfusion risks. EBV, a potential cause of elevated ALT, was detected. Metagenomic analysis might be a useful technology for monitoring blood safety.

High rate of hepatitis C virus and human immunodeficiency virus false-positive results in serologic screening in sub-Saharan Africa: adverse impact on the blood supply.

BACKGROUND: False positivity in blood screening may cause unnecessary deferral of healthy donors and exacerbate blood shortages. An international multicenter study was conducted to estimate the frequency of HCV and HIV false seropositivity in seven African countries (Burundi, Cameroon, Democratic Republic of Congo, Madagascar, Mali, Mauritania, and Niger). STUDY DESIGN AND METHODS: Blood donations were tested for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) with rapid detection tests (RDTs), third-generation enzyme immunoassays (EIAs), or fourth-generation EIAs. HCV (456/16,613 [2.74%]) and HIV (249/16,675 [1.49%]) reactive samples were then confirmed with antigen/antibody assays, immunoblots, and nucleic acid testing. Partial viral sequences were analyzed when possible. RESULTS: The HCV reactivity rate with RDTs was significantly lower than with EIAs (0.55% vs. 3.52%; p < 0.0001). The HIV reactivity rate with RDTs was lower than with third-generation EIAs (1.02% vs. 2.38%; p < 0.0001) but similar to a fourth-generation assay (1.09%). Only 16.0% (57/357) and 21.5% (38/177) of HCV and HIV initial reactive samples, respectively, were repeatedly reactive. HCV and HIV infections were confirmed in 13.2% and 13.7%, respectively, of repeated reactive donations. The predominant HCV genotype 2 and 4 strains in West and Central Africa showed high genetic variability. HIV-1 subtype CRF02_AG was most prevalent. CONCLUSION: High rates (>80%) of unconfirmed anti-HCV and anti-HIV reactivity observed in several sub-Saharan countries highlights the need for better testing and confirmatory strategies for donors screening in Africa. Without confirmatory testing, HCV and HIV prevalence in African blood donors has probably been overestimated.

Occurrence of occult hepatitis B virus infection associated with envelope protein mutations according to anti-HBs carriage in blood donors.

OBJECTIVES: Occult hepatitis B virus infection (OBI) carries a risk of hepatitis B virus (HBV) transmission and hepatocellular carcinoma. As previous studies have had a limited sample size, the characteristics of OBI with genotype B and C (OBIB and OBIC) mutations relating to hepatitis B surface antibody (anti-HBs) elicited by vaccination or a limited host immune response to HBV have not been fully explored. METHODS: In this study, the occurrence of OBIB or OBIC strains associated with envelope protein (pre-S/S) amino acid substitutions obtained from 99 blood donors stratified according to anti-HBs carriage were characterized extensively. RESULTS: According to the presence of anti-HBs within each genotype, the number and frequency of substitution sites specific for anti-HBs(-) OBIB were higher than those specific for anti-HBs(+) OBIB strains (67 vs 31; 117 vs 41), but the reverse pattern was found in OBIC strains (3 vs 24; 3 vs 26). Mutations pre-s1T68I and sQ129R/L were found uniquely in 15-25% of anti-HBs(+) OBIB carriers and mutation pre-s1A54E was found preferentially in anti-HBs(+) OBIC, while 17 substitutions were found preferentially in 11-38% of anti-HBs(-) OBIB strains. In the major hydrophilic region (MHR) region, mutations sS167 in OBIB, sT118 in OBIC, and sA166 in both genotypes were possibly immune-induced escape mutation sites. CONCLUSIONS: Several mutations in pre-S/S of OBI appeared to be associated with carrier anti-HBs pressure, which might be risk factors for potential reactivation of viruses under anti-HBs selection in OBI carriers.

Occult hepatitis B infection among blood donors from Yaoundé, Cameroon.

BACKGROUND: In Cameroon, the prevention of hepatitis B virus (HBV) transmission by blood transfusion is still only based on hepatitis B surface antigen (HBsAg) screening. However, occult HBV infection (OBI) characterised by the absence of detectable HBsAg and low level of viral DNA remains a potential threat for blood safety. The prevalence of OBI was investigated in blood donors from Yaoundé to provide evidence-based recommendations to improve HBV blood safety. MATERIAL AND METHODS: Blood donations from August 1st, 2016 to March 31st, 2017 were routinely screened for HBV, human immunodeficiency virus (HIV), and hepatitis C virus (HCV) infections (Murex HBsAg Version 3, Murex HIV Ag/Ab Combination, and Murex HCV Ag/Ab Combination [DiaSorin]). Additional HBV investigations were performed, including hepatitis B core antibody ([HBc] Monolisa Anti-HBc PLUS; BIO-RAD) and HBV DNA tested in minipools of two samples using the quantitative Cobas Taqman HBV assay (Roche; LoQ: 6 IU/mL) and HBV DNA genotyping by sequencing. RESULTS: Of 1,162 donations analysed, 91 (7.8%) were reactive for HBsAg. All of them were also anti-HBc positive. Among the 1,071 HBsAg negative samples, 522 (48.7%) were reactive for anti-HBc. Six (0.56% of all donations) samples fulfilled the consensus definition of OBI and showed low HBV DNA loads (all <6 IU/mL). Following nested polymerase chain reaction amplifications, HBV DNA sequences were obtained for 4 of these samples (1 nearly whole genome [3123 nt], 2 Pre-S/S regions [1,356 nt], and 1 S region [445 nt]). Phylogenetic analysis identified genotype E in all samples. DISCUSSION: Around 1 in 100 Cameroonian blood donors screened who resulted HBsAg negative and anti-HBc positive carried occult HBV infection. HBsAg alone for screening prospective donors is not sufficient to eliminate the risk of HBV transfusion transmission in Cameroon, and because anti-HBc screening does not seem to be feasible without compromising blood supply, implementation of HBV nucleic acid testing could be considered when possible.