Transfusion Transmissible Infections in Blood Donors in the Province of Bié, Angola, during a 15-Year Follow-Up, Imply the Need for Pathogen Reduction Technologies

Transfusion transmissible infections (TTIs), caused by hepatitis B virus (HBV), human immunode-ficiency virus (HIV), hepatitis C virus (HCV), and syphilis, have a high global impact, especially in sub-Saharan Africa. We evaluated the trend of these infections over time in blood donors in Angola. A retrospective cross-sectional study was conducted among blood donors in Angola from 2005 to 2020. Additionally, frozen samples obtained from blood donors in 2007 were investigated to identify chronic HCV carriers and possible occult HBV infection (OBI). The overall prevalence of HBV, HCV, HIV, and syphilis was 8.5, 3, 2.1, and 4.4%, respectively, among 57,979 blood donors. HBV was predominant among male donors, while the remaining TTIs were predominant among women. Donors >50 years had a significantly high prevalence for all TTIs. Chronic HCV infection was ab-sent in 500 samples tested and OBI was present in 3%. Our results show the continued high prev-alence of TTIs among blood donors in Angola. Most infections showed a significantly low preva-lence in years with campaigns seeking voluntary blood donors, thus, reinforcing the importance of this type of donor to ensure safe blood. Africa, with a high prevalence of diverse pathogens, should consider cost-effective pathogen reduction technologies, once they are commercially accessible, to increase the availability of safe blood.


Introduction
Transfusion transmissible infections (TTIs) have a high global public health burden. An estimated 354 million individuals worldwide are chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV) [1], which caused 1.1 million deaths globally in 2019 [1]. This number has surpassed that of deaths caused by the human immunodeficiency virus (HIV), which for 2020 was estimated at 680,000 globally. HIV infects approximately 37.7 million people worldwide, with 67% (25.4 million) living in Africa [1,2]. For syphilis, an estimated 18 million people were infected in 2012, representing 0.5% of the global population. Updated data for 2020 estimated 7.1 million new cases of syphilis worldwide, which surpasses the total of 4.5 million cases for the other three TTIs combined [1]. a total number of blood donors registered for each year, with percentages related to the distribution for the study period; b age groups are expressed in years and frequency calculated only for known ages; c frequencies calculated exclusively for known gender; d number of blood donors tested for each TTI with percentages calculated using the total number 6of blood donors registered.

HCV
Among the 1500 blood donors registered in 2007, 500 serum samples were obtained for further testing, 412 were men and 88 women, with ages ranging from 18 to 64 years (average 29.3 ± 9.2). Positivity for anti-HCV was identified in 20 (4%) blood donors who had undetectable HCV RNA, and 4/20 (20%) were positive in the second anti-HCV assay. Ninety percent were men donors, and their ages ranged from 18 to 39 years. Malaria was detected in 14/20 (70%) of the anti-HCV positive samples (Supplementary Table S1).

HBV-OBI
From the 500 serum samples from 2007, 170 residual samples were identified, with known negative results for TTIs, and volumes greater than 2 mL. HBV DNA was detected in 10/170 (5.8%) samples, with viral loads ranging from <10-37,652,193 IU/mL. Viral loads of <10 IU/mL were retested to exclude possible carry-over contamination, yielding identical results. The quantitative serological assay detected HBsAg in 5/10 samples, with concentrations varying from 0.05 to >150 IU/mL, three of which were likewise positive in the second HBsAg RDT (VIKIA ® HBsAg/bioMérieux, Marcy-l'Étoile, France) assay. Anti-HBc was detected in 4/5 samples of HBsAg+/HBV DNA+. The remaining five (2.9%) samples were classified as possible OBI, as they were HBV DNA+/HBsAg-, with anti-HBc IgG positive in two samples (Table 4). Anti-HBc IgM testing was performed in all three samples, all of which were negative.

Blood Donors
The increase in blood donations from 2016 to 2020 was mainly due to the high demand for blood transfusions to treat falciparum malaria anaemia, the leading cause of death in Angola. This disease, accounting for blood transfusion needs, rose sharply in 2016 and increased gradually thereafter. From 2005 to 2008 and 2013 to 2015, a total of 9,643,228 malaria cases, with 21,595 deaths, were registered compared with 29,591,660 cases and 61,368 deaths registered between 2016 and 2020. A three-fold increase in malaria cases and deaths and over a two-fold increase in blood donations (16,985 versus 40,984) were observed for the period 2016-2020. In addition to the rise in cases of malaria in 2016, Angola experienced a yellow fever epidemic during the same period, contributing to the high toll of deaths, overwhelming the health system, and consequently the need for large volumes of blood supplies, according to J. Martins, coordinator of the National Malaria Program [18]. In 2020, despite the high number of malaria cases, a decrease in the number of transfusions was registered. This decrease was due to the change in treatment protocol in 2019, substituting quinine for artesunate as the first choice to treat severe cases of malaria, as the latter quickly clears circulating parasites. Thus, patients are less likely to have haematological complications that require blood transfusion [19,20].
During the period 1992-2002, the civil war in Angola devastated the country, especially in the central and southern regions of the province of Bié, where this study took place [21]. In 2005, when the study was initiated, fragility in data collection was noted in the blood bank records, with missing demographic data detected in 76.92% of the data.
Following that year, data registration improved with the following percentage of missing data: 2006 = 1.84%, 2007 = 6.20%, 2008 = 6.03%, 2013 = 0.00%, and 2014 = 3.64%. In the second semester of 2014, the National Blood Institute of Angola defined and implemented methodological guidelines, which improved data recording, especially after 2015, with no missing demographic data observed after this period (Decree Nº 281/14). Regarding missing laboratory results, before 2015, blood donor screening for HBV, HCV, HIV, and syphilis was not 100%, despite the WHO recommendation that all blood donors be tested for infectious diseases. One might consider missing testing data due to failure to register the test results. However, according to laboratory personnel, this was less likely to have occurred as the Hospital Geral do Bié s blood bank implemented a local testing algorithm to overcome diagnostic test shortages. HBV was the initial test for screening blood donors; those testing negative were further screened sequentially for HIV, HCV, VDRL, and malaria. If any test was reactive, further screening ceased.

HBV
Sub-Saharan Africa is endemic for HBV and has an estimated overall prevalence of over 8% in the general population [34]. The prevalence of hepatitis B in blood donors depends on the number of infected individuals with hepatitis B in the general population and the type of donor. Replacement donors are said to have a higher prevalence when compared to VNRBD; the latter is identified as providing the safest blood, as most are at low risk for infectious diseases [33]. Since replacement donors are the majority in Angola, the results could reflect the overall country seroprevalence. The prevalence of HBV in blood donors in Angola reported by Apata et al. [35]

HCV
HCV prevalence is estimated to be above 3.5% in North Africa and 1.5-3.5% in Sub-Saharan Africa [34,36]. In this study, HCV was 3%, which was within the estimated standards for the region. Low prevalence was found in other sub-Saharan African countries: 1% in Malawi [26], 0.9% in Namibia [38], and 0.3% in Eritrea [39], and the highest prevalence was reported in Burkina Faso and Ghana [23,30], ranging from 7 to 9.4%.
In this study, a gradual increase in the prevalence of HCV was noted over the years, which may reflect not only the use of improved sensitivities in the RDT, but the fact that the province of Bié was isolated from the country and the world from 1992 to 2002; however, with definitive peace, there were demining and improvements in inland routes, which made access to this province easier. Following these improvements, the flow of individuals from within the country and neighbouring countries intensified, which may have likewise contributed to the increased prevalence of anti-HCV in the period analysed.

HIV
HIV infection remains a global public health concern, especially in Africa, particularly in the western and central regions, where 21% of new HIV infections are reported globally [29]. Blood transfusion is responsible for an estimated 1% of new HIV infections [40], and screening of blood donors in this region is predominantly performed with RDT. The performance of the latest RDTs for HIV is equivalent to automated enzyme-linked immunosorbent assays, as shown in several studies evaluating rapid HIV tests for transfusion screening in Africa, which reported sensitivities of approximately 100%, supporting its use for screening blood donors [3]. In Angola, the HIV prevalence in the general population is 2% [2,41], and the blood donor prevalence during the study period was identical (2.1%). Similar results were reported in Malawi; (1.9%) [26]. Higher HIV seroprevalence has been reported in Mozambique (8.5%) and Equatorial Guinea (7.83%) [14] and is related to the high prevalence in the general population. The Hospital Geral do Bié s blood bank uses RDT for blood donor screening, but since December 2019, additional testing for HBV, HCV, and HIV has been performed using an automated immunofluorescence assay (MINI VIDAS ® /bioMérieux S.A., Marcy-l'Étoile, France) to lower residual risk. The present national screening algorithm requires that all blood candidates be screened first by a RDT, and those with negative results will be tested using the commercial automated MINI VIDAS ® /bioMérieux assay. The prevalence of HIV in 2020 was comparable to the general prevalence observed over the study period, which supports the high specificity and sensitivity attributed to RDT for HIV and the improved performance in the testing assays over the years [3].

Syphilis
Syphilis remains a severe public health problem globally, with an incidence of 0.5 cases per 1000 inhabitants and a high prevalence in the African region, which requires extended precautions related to blood safety [14,42]. In this study, the prevalence of syphilis in blood donors was 4.4%, similar to that reported in Burkina Faso (3.9%) [23]. Equatorial Guinea (21.5%) and Tanzania (12.7%) were registered with the highest prevalence in the SSA region [14], and the lowest prevalence was found in Ethiopia (1.1%) and Mali (0.01%) [29,40]. These variations in prevalence may be due to differences in sensitivity and specificity of screening tests, preventive measures, the effectiveness of the screening program, choice of donors, and accessibility to healthcare [14,42,43].
The varied prevalence observed in this study for the TTIs was either higher or within the WHO estimates for low-income countries [33]. While HIV values were mainly within the interquartile range ( Figure 5), HCV and syphilis prevalence were typically above these ranges. In contrast, HBV prevalence was 50% higher and 50% within the WHO range.
The seroprevalence variations observed along the years in this study for HBV, HCV, HIV and syphilis infections might be attributed to the different analytical sensitivities in the tests used during the study period (Supplementary Table S2). the interquartile range ( Figure 5), HCV and syphilis prevalence were typically above these ranges. In contrast, HBV prevalence was 50% higher and 50% within the WHO range.
The seroprevalence variations observed along the years in this study for HBV, HCV, HIV and syphilis infections might be attributed to the different analytical sensitivities in the tests used during the study period (Supplementary Table S2).

TTIs and Demographic Characteristics
The prevalence of HBV was higher in men, similar to that observed in other countries in the region, such as Gabon, Ethiopia, Mali, and South Africa [14,29,40,44]. This finding can be explained by the fact that men are more prone to progress to chronic HBV infection, with an expected difference in sex distribution for chronic HBV carriers [45,46]. In contrast, women blood donors had a higher prevalence of HCV than that found in Gabon, where prevalence was similar in both sexes [14]. In Ethiopia, however, the prevalence of HCV infection is higher in men [40]. Women had a higher prevalence of HIV blood donors than men as reported in Angola [2,41], and this may be partially explained by the characteristics of the genital mucosa of women, which is subject to a greater risk of contracting the infection [28]. Globally, the number of new cases of HIV registered in 2019-2020 was similar for both sexes [1,2]; however, when limited to the SSA region, women had a higher number of new HIV infections than men [1]. Similar to HCV and HIV, syphilis had a higher prevalence in women donors (5.8 versus 3.9%) as reported in Namibia [38], although most SSA countries have demonstrated a higher prevalence of syphilis among men donors; Ethiopia, Cameroon, Gabon, Tanzania, Eritrea, Kenya, and Nigeria [14,25,28,32,40,47,48].
The seroprevalence of TTIs is associated with age, as there is more exposure to infections over time, which consequently results in a higher prevalence in advanced ages [40]. Studies conducted in Ethiopia support this association, and blood donors over 50 years old, in this study, had the highest prevalence for all four infections: HBV, HCV, HIV, and syphilis [40]. Studies in Mauritania showed the highest prevalence in this age group for HBV, HCV, and HIV; Burkino Faso, Eritrea, and Gabon for HIV and syphilis; Mali for HIV and HCV [14,23,29,31,47]; and Tanzania for HCV and syphilis [28]. Furthermore, South Africa had the highest prevalence of HCV in blood donors over 60 years old [44], suggesting possible parenteral exposure in the past.
Besides the fact that age is associated with infection exposure, there are additional reasons to justify the elevated prevalence of syphilis with age. The non-treponemal tests used frequently in blood banks in the African region may cross-react with antibodies from other acute febrile and autoimmune diseases, resulting in false-positive results. Another factor is the treponemal antibody test that remains positive for life in 85% of patients after treatment. A positive treponemal antibody test does not differentiate active infection from the past [42]; therefore, this persistence of antibodies in the absence of infection elucidates the increase in syphilis prevalence with increasing age of blood donors [14].

HCV: Blood Donor Specimens
Of the 500 blood donor specimens collected in 2007, 4% tested positive for anti-HCV and lacked detectable virus, confirming a low prevalence of chronic HCV and translating as past infection or cross-reactivity. Spontaneous clearance of HCV occurs in at least 25% of infected individuals, and anti-HCV antibodies may be detectable in the absence of the virus [49]. Although most of the anti-HCV-positive samples from this group tested positive for malaria, speculations have been made regarding false-positive anti-HCV tests and other infectious diseases [50], but no study has confirmed this association. Most seroprevalence studies in blood banks use anti-HCV as the sole marker for HCV prevalence in SSA; however, Vermeulen and collaborator-based South African blood donors have reported molecular detection of HCV RNA, with an overall prevalence of 0.03, comparable to the WHO estimates [33,44].

HBV: Occult B Infection
The blood bank in Bié, Angola, had used the same screening policy for HBV for over 15 years, with RDT as their main screening assay for HBsAg until 2019, when the commercial automated immunoassay system MINIVIDAS®/bioMérieux was introduced.
In this study, HBV DNA testing was able to detect blood donors with HBV infection missed by the standard screening protocol of Angola. The highly sensitive quantitative commercial HBsAg assay was unable to detect HBV infection in 3% (5/170), identified as possible OBI, and was within the range of <1-87%, reported in other geographic regions, but lower than what has been reported in other SSA countries that are highly endemic for hepatitis B (Nigeria, 17%; Sudan, 38%) [51,52]. As second-time samples were not available, one cannot rule out the prospect of samples being collected during an acute phase window period, when HBsAg is not detected before its appearance, especially for the three samples that were negative for anti-HBc IgG and IgM. Therefore, residual risk remains an issue for HBV and other TTIs, despite the introduction of sensitive commercial assays. High-income countries that have implemented NAT to screen blood donors report residual risks for TTIs to be <1 per 1 million units of blood tested [53][54][55]. In SSA countries, where NAT testing is scarce, a different scenario is observed with unsettling high residual risks for these infections reported that vary from 67 to 250 per 1 million units tested [56]. Concerns remain with emerging pathogens not routinely tested in blood banks that jeopardise the quality of blood [13]. Pathogen inactivation through different PRT in plasma and platelet are already widespread in high-income countries. However, whole blood PRTs are not yet commercially available but are presently undergoing clinical trials [55,56]. Considering the high demand for whole blood and elevated prevalences of TTIs in SSA countries, the effective use of PRT would be an opportunity to increase safe blood availability. A PRT for whole blood could revolutionise blood safety in countries that lack NAT testing and additionally prevent the widespread of new emerging infections through blood transfusions.  (years 2005, 2006, 2007, 2008, 2013, 2014, and 2015) and after year 2016 (years 2016, 2017, 2018, 2019, and 2020). The year 2016 was selected because there was a sharp increase in blood collection following outbreaks of several infectious diseases with high demands for blood and blood products [57]. The prevalence of each TTI was obtained by dividing the number of reactive donors by the total number of blood donors tested for each year for each specific infection. When estimating prevalence according to sex or age category, missing data were excluded for calculation purposes.

Blood Specimens
Frozen serum samples from 500 consecutive previously consenting blood donors at the HGB between February and July 2007 were investigated for HCV using serological and molecular tests. Following these tests, residual samples with volumes greater than 2 mL were selected to explore possible OBI. As the study used one-time blood donorstored samples, OBI was identified only as possible and not confirmed cases. This is because HBsAg may be similarly absent during the pre-conversion window period of acute HBV infection. Samples collected in 2007 were centrifuged, serum aliquoted into two cryotubes, and stored in a freezer at −30 • C at the HGB until they were sent to Brazil. The samples were shipped on dry ice to the Viral Hepatitis Laboratory/Oswaldo Cruz Institute/Oswaldo Cruz Foundation/Brazil and immediately stored at -80 • C until thawed for serological or molecular tests for HCV and HBV.

Serological Marker Detection Using Rapid Diagnostic Tests (RDT) at the HGB
Blood donors were tested following the testing policy at the HGB that established HBV for initial screening; those testing negative were further screened sequentially for HIV, HCV, syphilis, and malaria. The testing was performed using different RDTs according to availability. HBsAg RDT was used for HBV detection, with HBsAg Determine ® (Abbott Japan Co., Ltd., Tokyo, Japan) as the principal commercial kit used throughout the study period. RDTs for antibody detection were used for HCV, HIV, and syphilis. Anti-HCV Spot ® (Genelabs Diagnostics Pty Ltd, Singapore) is a major screening RDT for HCV testing, and HIV 1/2 Determine ® (Abbott Japan Co., Ltda., Tokyo, Japan) and Uni-Gold™ HIV (Trinity Biotech Plc., Bray, Ireland) were the main tests used for HIV screening in blood donors. Syphilis test results, besides RDT, were also based on non-treponemal antibody tests, the rapid plasma reagin (RPR) assay, and the Venereal Disease Research Laboratory (VDRL) assay. A range of diagnostic tests was used for each of the TTIs and is listed in Table Supplement 2, according to availability.

HCV Testing
The 500 blood donor specimens were tested using a commercial enzyme-linked immunosorbent assay, HCV Ultra Hepanostika ® (bioMerieux S.A., Craponne, France), according to the manufacturer s instructions, and were repeated in duplicate and considered positive if all results were reactive. Reactive specimens for anti-HCV were investigated for HCV RNA using a commercial quantitative PCR assay (m2000rt Abbott RealTime HCV ® Abbott Molecular Inc., Des Plaines, IL, USA), and samples with undetectable HCV RNA were additionally tested for anti-HCV using a second assay (CHIRON RIBA HCV 3.0 Strip Immunoblot Assay/Chiron Corporation, Emeryville, CA, USA).

Malaria Testing
To identify possible false-positive anti-HCV results, additional screening for Plasmodium falciparum and Plasmodium vivax malaria were included and performed using an RDT, SD BIOLINE ® Malaria Ag Pf/Pan Standard Diagnostics, Inc., Seoul, Korea.

HBV Testing
To explore possible OBI, residual samples from 500 blood donors collected in 2007, with volumes greater than 2 mL and known negative results for HBV, HIV, HCV, and syphilis were selected to investigate the presence of HBV DNA using a commercial quantitative PCR assay (Abbott RealTime HBV ® Abbott Molecular Inc., Des Plaines, IL, USA). Samples with detectable HBV DNA were retested for HBsAg using a different commercial RDT (VIKIA ® HBsAg/bioMérieux, Rio de Janeiro, Brazil), and a quantitative HBsAg assay (Liaison ® XL HBsAg Quant/DiaSorin, Saluggia, Italy), in addition to the anti-HBc testing (IgG and IgM) performed with commercial assays (Liaison XL ® /DiaSorin S.p.A., Italy).

Statistical Analysis
We estimated prevalence as the proportion of individuals who had a positive result in the HBsAg, anti-HCV, anti-HIV, or syphilis tests. The data were stratified using the year of occurrence, sex, age, and disease. The 95% confidence intervals were calculated using the Clopper-Pearson method. Analyses were performed using the R Studio statistical software (version 1.4). Statistical significance corresponds to P-values less than 0.05.

Conclusions
During the study, we found significant improvements in (1) blood bank input recording; (2) screening coverage for HBV, HCV, HIV, and syphilis; and (3) updated screening algorithms that included assays with higher analytical sensitivities. The decrease in TTI prevalence noted during VNRBD campaigns reinforces the importance of this type of donor to ensure safe blood. HBV has the highest burden of TTI in Angola and needs to be addressed with care, as it is a vaccine-preventable disease. Adult vaccination for HBV and treatment needs to be implemented by Angola s public health system to reduce new infections, which may be missed in the window periods by the present blood donor screening algorithm. Considering the high demand for whole blood and elevated prevalences of TTIs and emerging pathogens in SSA countries, the effective use of PRT in whole blood, once commercially available, will be an opportunity to increase safe blood availability.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/pathogens10121633/s1, Table S1: Characteristics of the 500 blood donor samples collected in 2007, and those positive for anti-HCV, Table S2 List of rapid tests used during the study period.  Informed Consent Statement: Written informed consent was obtained from all subjects to publish this paper, which had blood withdrawn in 2007. Patient consent was waived for the retrospective study that used blood donor records to obtain demographic information and screening test results.
Data Availability Statement: Data reported in this study are available upon request. Datasets obtained from the Hospital Geral do Bié s blood bank are not publicly disclosed as they contain confidential information, and thus in accordance with national regulations, must remain private.