Accuracy of screening for HBV (NAT, HBSAg, anti-HBC) in blood donors from the Brazilian Amazon from 2105 to 2018

doi:10.21203/rs.3.rs-2448104/v1

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Research Article

Accuracy of screening for HBV (NAT, HBSAg, anti-HBC) in blood donors from the Brazilian Amazon from 2105 to 2018

https://doi.org/10.21203/rs.3.rs-2448104/v1

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Background

Hepatitis B (HBV) is an infectious disease of worldwide importance and of great interest to transfusion medicine. The Amazon region has areas of high endemicity, outlining a worrying scenario for local transfusion safety, and it is necessary to investigate its impact on public health and blood component stocks.

Objective

To investigate the accuracy of HBV screening (NAT, HBSAg, anti-HBC) in blood donors from the Hemocentro do Amazonas, Brazil, from 2105 to 2018.

Methods

Blood donors reactive in serological and molecular screening for HBV were tested for viral load detection using the qPCR method at the reference center for liver diseases in the State of Amazonas to verify the accuracy of the screening established in Brazilian blood centers.

Results

A total of 230,591 blood donations were analyzed, with detection of 3,932 inaptitudes by serological and molecular tests, of which 3,104 samples were reactive for some marker for HBV (78.94%). Among the results found, no detection of viral load was observed in 100% of donors reactive to HBsAg alone, while 100% of donors with the NAT-/HBsAg+/Anti-HBc + profile had a detectable viral load.

Conclusion

The positivity for some HBV marker in blood donors was the biggest reason for discarding blood bags in the period; Serological and molecular screening proved to be safe and reliable; The evasion of individuals from continuing the investigation at the referral center can impact the epidemiological control of hepatitis B.

hepatitis B virus

epidemiology

blood donors

blood transfusion

According to the World Health Organization (WHO), 257 million people are chronic carriers of the hepatitis B virus (HBV), equivalent to about 3.5% of the global population (1). Despite studies showing a progressive reduction in endemicity rates, the epidemiology of hepatitis B is not homogeneous in the Brazilian territory (2). High prevalence rates are described in populations with less urban complexity, as remnants and/or residents in isolated areas, including quilombolas and indigenous peoples, who have difficulties in accessing regular health services, presenting greater exposure and, therefore, greater risk of disease transmission (3).

Hepatitis B virus is related to a wide range of clinical manifestations, occurring in some cases in acute or chronic form or in most cases in the chronic phase which varies in severity from asymptomatic to oligosymptomatic severity, characterized from atypical flu-like symptoms to severe clinical conditions, such as cirrhosis and hepatocellular carcinoma (4, 5).

From the performance of the molecular test "nucleic acid test" for the detection of HBV (NAT HBV) in the screening of blood donors, a variable prevalence was found for carriers of viral DNA for HBV in asymptomatic donors negative for HBsAg, which characterized a new clinical form of infection, determined as occult infection by the hepatitis B virus - HBV (OBI) (2, 5).

Chronic hepatitis is asymptomatic in most cases, with clinical manifestations occurring only in advanced stages of the disease, with moderate to severe hepatic involvement, including fibrosis, cirrhosis, hepatocellular carcinoma, including involvement of other organs. Often, the diagnosis is made by chance, based on sporadic changes in routine evaluation tests or screening in blood banks, with viral hepatitis, mainly types B and C, appearing most often in the chronic phase of these diseases. (6).

Throughout Brazil, hepatitis B screening consists of performing HBSAg and anti-HBC tests. Anti-HBc IgM is a marker of the onset of infection, while antibodies specific for HBeAg and HBsAg indicate a favorable resolution of the infection (7, 8). Recent studies show that the assessment of the patient's viral load is the most informative marker of the evolution of liver disease caused by HBV (Fig. 1) (9–13).

Even with current and more accurate tests, the residual risk of transmission that HBV confers on blood stocks is still worrying, mainly due to very low viral loads presented by asymptomatic individuals with negative serology and asymptomatic individuals with viral DNA undetectable by molecular methods. qualitative, such as NAT HBV. Currently, after carrying out the tests for the detection of HBV in the screening of candidates for blood donation, a significant reduction in its transmission via blood transfusion was observed (14).

The Nucleic Acid Test (NAT) was introduced in blood donor screening in the late 1990s in Germany, Austria, and Japan (15). In Brazil, it became mandatory in the screening of blood donors as of 2014 (16). This test directly detects viral DNA from the highly preserved region of the S protein coding gene, using an automated platform through a mini-pool of six samples (MP-NAT) or individual samples (ID-NAT) for confirmation of viral presence, with sensitivity greater than 95%, thus conferring superiority over serological methods (15, 17). However, studies have demonstrated interference in the sensitivity of detection in diluted samples, mainly of occult infection with viral load at levels below 50 IU/mL, which can even make detection by NAT-ID difficult (4). In addition, NAT sensitivity can also be compromised by the technique used to extract the genetic material, as well as by variations in the HBV DNA sequence, mainly observed in the HBV/D genotype (17). One cannot deny the notoriety of NAT-HBV in reducing the seroconversion window to a maximum of 15 days, much smaller than the serological tests that can reach up to 82 days (18). However, studies demonstrate that the very low levels of viral DNA justify the substitution of MP-NAT HBV for ID-NAT, due to its high sensitivity (19–22. Another important molecular test is the Real Time Polymerase Chain Reaction (qPCR) used to confirm the presence of HBV DNA, considered the gold standard for the detection of low viral load and monitoring of individuals with chronic infection (20).

The states belonging to the western Brazilian Amazon (Acre, Rondônia, Roraima and Amazonas) are among the regions with the highest global prevalence of HBV. Indigenous populations living in the basins of the Amazon, Juruá, Purus and Madeira rivers, with high incidence rates, present approximately 55% of individuals reactive to Anti-HBc (Antibodies against the core antigen of hepatitis B virus) and 10% to HBsAg (Hepatitis B surface antigen) (23). Moresco et al. (2014), demonstrated a prevalence of 2.7% of occult hepatitis in blood donors in the interior of the state of Amazonas, with a non-reactive serological profile for HBsAg and a reagent for Anti-HBc using quantitative molecular tests (24). Currently, an ultrasensitive chemiluminescence assay has been validated to surpass molecular detection in some cases and to reduce the immunological window time to 14 days, although still in experimental studies (25).

Reactivity to Anti-HBc in isolation has been reported in different populations, ranging from 1–32%. Besides approximately 40% of individuals with this profile of results, this serological marker has low specificity and high rates of false-positives, requiring additional confirmatory tests such as molecular tests to retain the result (25–26). In contrast, several studies have shown that Anti-HBc is the only serological marker found in occult infection with NAT negative HBV, being considered an important complement to transfusion safety (20, 27–30).

NAT tests involve molecular techniques of real-time PCR (qPCR) and were developed for detection of viral nucleic acid in the period that precedes the systemic production of antibodies, that is, the initial phase of the infection or immunological window. With the Bio-Manguinhos NAT test, the immunological window was reduced to 10 to 12 days for HIV, HCV, HBV, increasing transfusion safety. The methodological flow follows the following steps: (a) Sample preparation in a mini pool of six or individual; (b) extraction of nucleic acid from the biological sample (plasma); (c) nucleic acid amplification; (d) detection of the nucleic acid by real-time PCR (31).

According to the Ministry of Health’s technical manual on hepatitis published in 2018, the NAT HBV test detects the presence of viral particles in the acute and chronic phases of hepatitis B, starting at 10 copies/mL of plasma or serum with a specificity above 99% (32–33)

Figure 2 summarizes the interpretation of serological and molecular markers for hepatitis B, including the Anti-HBs used to better understand the infectious scenario that can be found in the screening of blood donors, adapted from a recently performed multicenter study.

In the Standard Operating Procedure (SOP) of the Blood Cycle department of the HEMOAM Foundation, the flow of referrals to the FMT-HVD from donors who showed reactive results in screening for hepatitis B is depicted in Fig. 3.

In this context, the present study aimed to investigate the great relevance of the prevalence of hepatitis B in blood donors in the blood center of the State of Amazonas and its impact on the disposal of blood components, as well as on public health, investigating the importance of serological screening and molecular and protocol traceability for viral load detection in the HBV screening profiles defined for this purpose.

Blood transfusion in Brazil is safe, due to the clinical and laboratory screening established in the country for hepatitis B, as described above, and also largely due to the loyalty of donors with negative screening for HBV.

Currently, the National Network of Hematology and Hemotherapy Services (HEMORREDE), in endemic regions for hepatitis B, finds it difficult to provide blood components with negative HBV screening to serve the specific population of each location, with a substantial amount of blood bags donated and ruled out due to its reactivity to HBV, justifying specific research and projects that investigate the direct correlation between blood donor screening and viral load detection (2).

The present study was carried out on the information on the results of the HBV screening (HBsAg, NAT HBV and anti-HBc) in individuals who donated blood at the Hospital Foundation of Hematology and Hemotherapy of Amazonas – HEMOAM in the period from January 2015 to December 2018. From these data, we extracted the positive results of the screening test for HBV performed at HEMOAM and statistically analyzed the concomitance and accuracy of reactive profiles found in the same donor by comparing them with the serological and molecular screening for HBV of these donors, carried out at Fundação of Tropical Medicine Doutor Heitor Vieira Dourado - FMT-HVD, which is the reference center for liver diseases in the State of Amazonas.

It is important to state that, for this study, we considered only the first blood donation from donors, that is, repetitions of donations by the same donor were not considered.

Study design

Descriptive retrospective study that will use the database of donor registration of the blood center of Amazonas (Hemosys) and the electronic medical records system of the Fundação de Medicina Tropical Dr. Heitor of Vieira Dourado.

Population, sample and data collection

The first phase of the study corresponded to the collection of data from all donors who were reactive in at least one of the serological screening tests for hepatitis B, namely: HBsAg, anti-HBc and NAT HBV, from December 2014 to December de 2018. These donors corresponded to the study sample. To characterize this sample, information of epidemiological importance was obtained, such as: age, sex, socioeconomic level, education, type of donation and donor.

The second phase of data collection corresponded to the collection of information on the diagnostic investigation of donors from the first phase who were referred to the Fundação de Medicina Tropical do Amazonas (FMT/AM). Therefore, information was needed to allow the location of these individuals in the electronic medical record system of the FMT - HVD/AM, such as: full name, mother's name, date of birth, address and telephone number. Donors who agreed to participate in the second phase of the study were asked to sign an informed consent form. After the participants' consent, data from tests recorded in the FMT-HVD/AM medical record were collected, which provided information on the diagnostic confirmation of hepatitis B, such as: anti-HBe, anti-HBs, ALT, AST, viral load quantification.

Methods for detection of Hepatitis B

Tests performed at HEMOAM

The technical procedures were performed according to the specifications of the reagents and equipment manufacturers, using the commercial kits routinely adopted by HEMOAM:

To perform the HBsAg and Anti-HBc tests, the chemiluminescence immunoassay (CMIA) method was used using the ARCHITECT HBsAg Qualitative II® kits with a sensitivity of 99.52% and ARCHITECT Anti-HBc II® with a sensitivity of 99.10%, (35).

To carry out the molecular screening, the NAT HIV/HCB/HBV Kit, manufactured by Bio Manguinhos®, FIOCRUZ, Ministry of Health, was used, which is performed using the RT PCR method using a vacuum Silica membrane extraction method used in a minipool of 6 samples (MP NAT) and individual samples (ID NAT), with a detection limit of 300 IU/mL in MP NAT.

It is noteworthy that the reactive results in screening for HBV were confirmed by repeating the CMIA tests for HBsAg and Anti-HBc using the same methodology in duplicate, while the molecular test for DNA-HBV was performed on an individual sample.

Tests performed at FMT-HDV

The determination of serological markers for hepatitis B is performed using the automatic analyzer LIAISON XL (Diasorin Murex®) of chemiluminescence tests with solid phase technology using paramagnetized microparticles.

The tests were carried out: Qualitative determination of surface antigens (HBsAg); Quantitative determination of antibodies to surface antigens (anti-HBS); Qualitative determination of total antibodies against core antigens (anti-HBc); Quantitative determination of antibodies to antigens and (HBeAg); Qualitative determination of total antibodies against core antigens

The viral load detection protocol obtained in the FMT-HVD employs the quantitative methodology of qPCR using the ABBOTT Real Time HBV® KIT, according to the manufacturer's recommendations.

Serum or plasma HBV DNA can be quantified using nucleic acid amplification or signal amplification technologies (from abbot package insert). The Abbott RealTime HBV assay uses PCR technology along with real-time homogeneous fluorescence detection to quantify HBV DNA.

The assay is standardized according to the World Health Organization (WHO) International Standard for Hepatitis B Virus DNA (NIBSC Code 97/746) (11) and results are presented in International Units per mL (IU/mL) or copies/mL.

The concentration of HBV DNA in a sample or control is calculated from a memorized calibration curve or a calibration curve created by calibrators within a calibration or sample processing. The Abbott m2000rt instrument automatically reports results to the Abbott m2000rt workstation. Assay results can be displayed in IU/mL or in Log IU/mL. Assay results can also be reported in copies/mL or log copies/mL, using a mean conversion factor of 3.41 (1UI = 3.41 copies). Note: The assay is calibrated against the WHO International Standard for HBV. The conversion factor of 3.41 is based on an average conversion factor over the dynamic range of the assay. (Abbot kit insert)

The viral load detection protocol obtained at FMT-HVD employed the quantitative methodology of qPCR using the ABBOTT Real Time HBV® KIT, according to the manufacturer's recommendations.

Data analysis

Data obtained was entered into Microsoft Excel spread sheet for cleaning and exported to Statistical Analysis Software (SPSS Version 19) to descriptive analyses to frequencies. The prevalence of the HBsAg and anti-HBc serological markers and their respective 95% confidence intervals (95% CI) were determined.

A total of 230,591 blood donations were analyzed in the period, with 3,932 reactive for some serological/molecular marker, with the highest percentage of prevalence related to HBV (3,104 / 78.94%), that is, corresponding, on average, to 80% of discards during the period studied, followed by reactive screenings for syphilis (476 / 12.10%), HCV (163 / 4.14%), HIV I and II (80 / 2.03%), HTLV (57 / 1.44%) and Chagas Disease (52 / 1.32%).

It is important to highlight that all donors were able to undergo hematological screening, with the completion of hematocrit, as well as in clinical screening, with verification of blood pressure, temperature and application of a standardized clinical questionnaire throughout the national territory. Therefore, it was not possible to clinically detect the stage of HBV infection in donors, as they are asymptomatic, using only laboratory indicators. Regarding reactivity to HBV, considering the mandatory national protocols, in the screening routine performed at HEMOAM, the following percentages were detected: NAT HBV: 3.02%; HBsAg: 9.92% and Anti-HBc: 93.8%. It is worth mentioning that the anti-HBS test, used in the diagnosis of the stage of the disease in national reference centers, is not mandatory to be performed in Brazilian blood centers. However, in the national protocol, donors with reactive NAT HVB and HBsAg markers are referred to the state reference center for liver diseases, which in the city of Manaus is the FMT-HDV.

However, as we understand that the combinations between these markers present greater reflections on the clinical status between the donor and the HBV infection, we will present from that point on, the results considering the profiles of results found for hepatitis B, as shown in Table 1.

Among the results found, HBsAg and/or NAT HBV reactive individuals, with a profile for referral and obtaining viral load values, constituted the number of 314 (10.11%) samples from a total of 3,104 donors (Table 1), except for The 2,790 reagents were found only for Anti-HBc (profile 6), whose donors are not referred to a reference center as shown in Fig. 2 and according to the protocol used in HEMOAM.

As shown in Fig. 4, of 314 donors with a referral profile to the FMT-HVD referral center, 73 (23.2%) were found, but the qPCR result was found in only 51 (70%) donors who fulfilled the referral of HEMOAM and continued the investigation of the infection for HBV.

The Table 02 presents the records found and not found in the FMT-HDV according to the reactivity profiles for HBV. The results of the qPCR tests of the 55 donors are shown in Table 3. The results of laboratory profiles on HEMOAM and qPCR performed on FMT-HDV are shown in Table 04.

In this research, a 4-year survey was carried out in the HEMOAM database, which returned 230,591 blood donations with 3,932 (1.70%) retentions in serological and molecular screenings, presenting the highest prevalence of disposal associated with reactivity for HBV detection (3,104 / 78.94%). Among these, anti-HBc reacted in 2,913 (93.8%) donors, followed by HbsAg reactive in 308 (9.92%) and NAT HBV in 94 (3.02%), with one or more positive tests in the same donation. Retentions for HBV were followed by reactive screenings for syphilis (476/12.10%), HCV (163/4.14%), HIV I and II (80/2.03%), HTLV (57/1.44%) and Chagas Disease (52/1.32%).

It can be seen in the results of this study that of 3,104 reactive donors for some HBV marker, 314 (10%) presented a referral profile, according to the HEMOAM criteria for performing the HBV qPCR for viral load detection and, if confirmed, the beginning of treatment with the reference institute FMT-HDV. This is because 2,913 (93.8%) donors had only reactive anti-HBc alone, which did not include them in the referral criteria for viral load detection.

The significance of blood donors with isolated reactive anti-HBc covers a vast scientific discussion. This reaction profile opens the understanding that these donors may be at different stages of infection by HBV disease, for example: Third stage of acute infection, which can be proven with the presence of anti-HBc of IgM class and anti-HBe, or still in the phase of recovery of immunity with the presence of anti-HBS and/or anti-HBe, that is, there are doubts between recovery of immunity, false positive result or the identification of a chronic carrier. In Brazil, blood components with isolated reactive anti-HBC results are sent for disposal and the donor is considered definitively unfit. For these reasons, the investigation of anti-HBS and anti-HBe in blood centers area not performed, also considering the introduction of NAT HBV in 2014. These donors with isolated reactive anti-HBC are attended by the medical service of the Blood Center, where they are clinically evaluated, being instructed to perform anti-HBS and anti-HBe tests. In any case, even so, there is the possibility of non-investigation of these results by the donor, which may constitute a bias for the epidemiological control of hepatitis B in the state of Amazonas.

In Germany, a country of low endemicity with a global prevalence rate for anti-HBc in blood donors of 0.22%, Houareau and Offergeld (2019) (36) evaluated 31,562,556 blood donors in 10-year period, observing 68,741 with negative HBsAg/Anti-HBc positive results who were submitted to HBV NAT. Of these, 84 (0.12%) had detectable viral load, 47 of which were detectable only with ID-NAT, thereby demonstrating the importance of using Anti-HBc to minimize HBV transmission through blood donations. However, in countries with low endemicity, the exclusion of donors with this profile does not compromise the population's blood supply. The safety of blood over the blood supply should be noted. Considering the proportion of viral load detection observed in the Houareau et al. (2019) studies, in 2,913 donors reactive only for Anti-HBc, we would find approximately 3 donors with detectable viral load, justifying the discarding of bags only with the result of isolated anti-HBc.

In the period from 2015 to 2018, 314 donors, with NAT HBV and/or HBSAg positive results, were formally referred by HEMOAM to the FMT-HVD reference center. However, we verified, through an active search in that institution, that of the 314 referred donors, only 73 (23.2%) actually sought the reference service for the continuity of the investigation for HBV, which was confirmed by the attendance records found by our team at the referral center. Of these 73 donors, 22 also had reactive screening results for HIV and syphilis and attended the FMT-HVD and they even had the first screening of tests but did not return for the qPCR, which is the reason why we found viral load check results from just 51 donors. The records of 241 (76.7%) donors were not found at the referral center, that is, despite the referral, they did not attend and therefore did not continue the investigation of the positive results found in the serological and molecular screening for HBV performed at HEMOAM. We understand that such individuals, unfit for blood donation, did not follow the referral because they were still asymptomatic, despite the reactivity of NAT and HBSAg, compromising the epidemiological control of hepatitis B in the State of Amazonas.

From the transfusion point of view, donors with a positive laboratory marker in the screening for HBV (NAT-HBV, HBSAg and anti-HBC) are considered definitively unfit, as determined by Brazilian legislation.

Among the donors tested for qPCR, 01 individual who belonged to profile 1 in the HEMOAM screening (NAT HBV +, HBsAg + and anti-HBc +), presented qPCR -, HBsAg +, anti-HBc +, HBeAg - and anti-HBe +, probably characterizing a phase conversion of the infection. With the NAT HBV + in the HEMOAM, the donor could be in a phase of chronic infection with active replication and three months after the donation, when he looked for the FMT-HVD, he could already be in a phase of inactive chronic infection when the qPCR is became negative and the presence of the anti-HBe marker was detected (37).

Another 02 individuals belonged to profile 3 in the screening performed at HEMOAM (NAT HBV+, HBsAg - and anti-HBc +) at HEMOAM, presented in FMT-HVD, qPCR -, HBsAg -, anti-HBc +, HBsAg -, anti-HBe - and anti-HBS+, probably demonstrating a transition from a chronic replication phase to a recovery phase of immunity. We observed that these individuals sought FMT-HDV, also 3 months after blood donation (4, 37, 38).

According to Candotti et al (2018), a range between 0.09% and 0.29% of donations with an initially positive result may become non-reactive in discriminatory tests. The reasons for this discrepancy remain unclear, but reflect Poisson distribution statistics of HBV DNA levels around the assay readout (25).

The 17 donors who belonged to profile 7 (NAT HBV -, HBsAg + and anti-HBc -), presented in the FMT-HVD, the HBsAg - anti-HBC - and qPCR - tests, which may be associated with a reduction in the cutoff of the serological tests used in the Blood Center with the objective of increasing the safety margin of the test, since, in Brazil, the blood centers are not intended to diagnose communicable diseases, but rather to prevent a possibly infected bag from being transfused, obviously carrying out the confirmatory procedures after the disposal of the donation.

According to the Federal legislation in force in Brazil, when confirmed seroconversion occurs, that is, determined by confirmatory tests, in a blood donor who in previous donations had non-reactive serology for HBV infection, a retrospective process must be instituted, being It is the responsibility of the hemotherapy service to summon and advise the donor with the results of reagent tests, forwarding him to the state referral center for confirmation of the diagnosis or, in the case of confirmatory tests carried out by the hemotherapy service, to refer him to follow-up and treatment (16).

Dodd et al. (2018) questioned in their study the use of the HBsAg test in the screening of blood donors, because in 22,370,271 donations analyzed over a 5-year period in the United States, the authors found 2,987 reactive HBSAg, of which only 144 were reactive in NAT HBV with no other marker detected. They also observed that 47 of these 144 reactive samples had a recent vaccination history. After high-sensitivity serological and molecular assays, the authors concluded that the frequency of HBV in this outcome profile would have an infectious risk of 1 in 4.4 million donations, a strong argument for discontinuing the use of HBsAg assay in donor screening tests (25). Kiely et al. (2018) corroborate the study by Dodd et al. (2018), which describes as the main cause for the false-positive results of HBsAg the vaccination against influenza, rabies and HBV itself or even due to cross-reactions of epitopes of HBsAg and viral antibodies resulting from the adjacent immune response (39, 40).

As for the 51 individuals who had a detectable viral load at the referral center, 12 donors who had profile 5 of reactivity (NAT HBV -, HBsAg + and Anti-HBc +) stand out, as 100% of the samples showed qPCR + in the FMT- HVD with a log average of 2.23 and a range of 1.08–3.7. For this discussion, it is important to consider that the HBV NAT tests in HEMOAM are performed in minipools (MP) of 06 samples with a detection sensitivity of 300UI / mL, while the qPCR performed in the FMT-HDV uses individual samples (ID) with a much higher sensitivity target sequence of the tests, the difference in sensitivity between the minipool (MP) and individual (ID) tests, as well as the stages of the disease itself regarding serological and molecular detection, showing the effectiveness of the complementarity between the tests performed in screening (25). The dilution factor introduced by the pooling process reduces the sensitivity of the HBV NAT-MP and thus its ability to detect the low logs of HBV DNA seen in most donors with occult infection (25).

Comparing with profile 7 where all samples had reactivity only for HBsAg, with qPCR -, in the profile 5, the concomitance of reactivity between HBSAg + and Anti-HBc + proved to be an important indicator for the detection of viral load.

In highly endemic countries, such as China, an anti-HBc detection rate of 48% has been observed in donations with non-reactive MP HBV DNA which shows reactivity of 6 to 68% when performed in ID-NAT. Alternatively, most ID-NAT users have adopted a serology algorithm to discriminate true and false initial reactive results. Several repeat tests are performed to identify low viral load donations using the multiplex assay or a second independent or in-house commercial assay, preferably targeting a different region of the viral genome. This approach has its disadvantages, as it has a high cost and NAT assays have different levels of sensitivity. Even the most sensitive assays can fail to consistently detect extremely low levels of HBV DNA. Sensitivity to NAT can be enhanced by several non-exclusive changes to standard procedures, aimed to increase the number of HBV DNA templates in the amplification reaction. This can be achieved by purifying viral DNA from large volumes of plasma and/or concentrating viral particles with high-speed centrifugation. However, these approaches are not suitable for screening large-scale blood donations (25).

Still addressing the investigation of the results regarding screening for HBV in our study, we observed that of 314 donors referred to the reference center, only 73 (23.2%) records were found. This constitutes another bias for the control of hepatitis B, as 241 (76.7%) donors did not continue the investigation of the positive results found in the serological and molecular screenings carried out at the Blood Center.

Profile 4 (NAT HBV +, HBsAg - and Anti-HBc -, with only 1 donor, whose information was not found in the FMT-HVD, represented 0.03% of the positive results of the study, it is probably the only case that can be considered as occult hepatitis B among the 230,591 donors studied, with a frequency of 0.43%, which unfortunately was not proven because the donor did not attend the referral center for further investigation, this being a limit of this study to prove a single suspicion of occult hepatitis B. In contrast to Pondé (2013) who considers this profile a relatively common finding especially in blood banks when the contact with HBV has been too recent for the detection of other serological markers of infection, or still being explained by the occurrence of X-mutant virus, generally characterized by an elimination of 8 nucleotides between positions 1770 and 1777 and a point mutation from a Thymine to a Cytosine in the X-ORF region. This deletion results in a C-terminal X protein, truncated with 134 amino acids (aa) due to a loss of 23aa and the addition of 3aa, which causes a loss of its transcriptional activity. Deletion of 8 nucleotides from the central core promoter/enhancer II complex sequence may decrease the function of this transcriptional regulatory element, pro moving suppression of viral replication and synthesis of HBcAg and HBsAg. Consequently, donors infected with defective HBV X-mutant are negative for HBsAg and Anti-HBc despite the presence of HBV replication and detectable DNA (37).

In summary, the results found in this study are of great significance in relation to all tested donors, as it was possible to perceive the reliability of the combination of tests used in serological and molecular screening performed in Brazilian blood centers, with the verification of the impact of the different profiles found in blood donations and confirmation of results with viral load detection at a referral center. At the same time, we observed the need for an active search for all reactive donors who, for some reason, did not continue the investigation.

Based on the results found, we conclude that:

1. There was a high prevalence of positive HBV laboratory indicators in blood donors from the Amazon Blood Center, being the biggest reason for discarding blood bags due to infectious causes;

2. Serological and molecular screening (NAT-MP HBV, HBsAg and Anti-HBc) has been shown to be safe and reliable, allowing the detection of hepatitis B at different stages of the disease, including possible occult infections and HBV gene variants.

3. The need for effective traceability was verified regarding the investigation of all donors with reactive indicators for HBV, even isolated anti-HBC, because despite being unfit, they can be asymptomatic carriers, impacting public health and epidemiological control of hepatitis B.

4. We verified the importance of strengthening education policies aimed at obtaining voluntary, altruistic donations, focusing on the loyalty of blood donors and thus reducing retention percentages.

Ethics approval and consent to participate

This project was approved by the Research Ethics Committees of Amazonas Hematology and Hemotherapy Hospital Foundation, under CAAE: 17625119.0.0000.0009 and of the Tropical Medicine Foundation Dr. Heitor Vieira Dourado, co-participant center of the project, under CAAE: 17625119.0.3001.0005, according to the resolution 466/2012 of the National Health Council, which advocates the guidelines and Regulatory Norms for Research Involving Human Beings

We declare that all experiments were performed in accordance with the Nuremberg Code of 1947 and the Universal Declaration of Human Rights of 1948; the Declaration of Helsinki, adopted in 1964 and its versions of 1975, 1983, 1989, 1996 and 2000; the International Covenant on Economic, Social and Cultural Rights, 1966; the 1966 International Covenant on Civil and Political Rights; the 1997 Universal Declaration on the Human Genome and Human Rights; the 2003 International Declaration on Human Genetic Data; and the 2004 Universal Declaration on Bioethics and Human Rights;

Informed consent was obtained from all participants and from the institution whose databases were used in the research. In the first phase of the study, only information from the databases of the Hospital Foundation for Hematology and Hemotherapy and the Dr. Heitor Vieira Dourado and, therefore, we only need the consent form of both institutions. In the second phase, donors with positive results were called for confirmation and viral load testing. After this survey, donors were selected, who signed the free and informed consent form and thus agreed to participate in the second phase of the study.

Consent for publication

Not Applicable

Availability of Data and Materials

All our data are deposited in a public bank, in which my research group owns. Can make it available whenever requested upon application to my email [email protected].

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM). Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' Contributions

Rosa Cristina Caldas Belota performed the collection of samples and performed the practical and laboratorial parts of the entire project. It should be noted that this project was the dissertation for his Master's Degree of Rosa Cristina Caldas Belota.
Rosa Cristina Caldas Belota, Jean de Melo Silvaand Eduardo Luiz do Nascimento digitization of results in data analysis programs.
Cláudia Maria de Moura Abrahim, Márcia Costa Castilho and José Pereira Moura Neto wrote the manuscript with support from Sérgio Roberto Lopes Albuquerque.
Márcia Costa Castilho, José Pereira Moura Neto and Sérgio Roberto Lopes Albuquerque assisted in the development of the project and contributed to the final version of the manuscript.

Sérgio Roberto Lopes Albuquerque and José Pereira de Moura Neto conceived the study, was the idealizer of the project and advised Rosa Cristina Caldas Belota.

Acknowledgments

We especially to staff of the Department of Technology and Information of Fundação Hospitalar de Hematologia e Hemoterapia do Amazonas and of Fundação de Medicina Tropical Dr. Heitor Vieira Dourado for the technical support. Finally, the authors thank the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support.

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Table 1. Distribution for the prevalence of hepatitis B virus (HBV) infection in blood donors screening from Manaus-Amazonas of January 2015 to December 2018.

Infection Profile	Screening for HBV	n	%
1	NAT (+) / HBsAg (+) / Anti-HBc (+)	85	2.73
2	NAT (+) / HBsAg (+) / Anti-HBc (-)	3	0.1
3	NAT (+) / HBsAg (-) / Anti-HBc (+)	5	1.59
4	NAT (+) / HBsAg (-) / Anti-HBc (-)	1	0.03
5	NAT (-) / HBsAg (+) / Anti-HBc (+)	33	1.1
6	NAT (-) / HBsAg (-) / Anti-HBc (+)	2790	89.9
7	NAT (-) / HBsAg (+) / Anti-HBc (-)	187	6.02
Total		3104	100

NAT: nucleic acid testing

HbsAg: Hepatitis B surface antigen

Anti-HBc: Antibodies against the core antigen of hepatitis B virus

Table 2. Donor records found by HBV outcome profile.

Infection Status		Records found		Records not found		Total
		n	%	n	%	n	%
1	NAT (+) / HBsAg (+) /Anti-HBc (+)	20	23.5	65	76.5	85	100
2	NAT (+) / HBsAg (+) / Anti-HBc (-)	0	0	3	100	3	100
3	NAT (+) / HBsAg (-) / Anti-HBc (+)	2	40	3	60	5	100
4	NAT (+) / HBsAg (-) / Anti-HBc (-)	0	0	1	100	1	100
5	NAT (-) / HBsAg (+) / Anti-HBc (+)	12	36.4	21	63.60	33	100
7	NAT (-) / HBsAg (+) / Anti-HBc (-)	17	9,0	170	90,9	187	100
	Total	51	17.5	263	82.5	314	100

NAT: nucleic acid testing

HbsAg: Hepatitis B surface antigen

Anti-HBc: Antibodies against the core antigen of hepatitis B virus

Table 3. NAT, HBsAg and Anti-HBc status according to viral load detection.

HEMOAM HBV profile		Viral charge
		Detectable		No Detectable		Total
		n	%	n	%	n	%
1	NAT (+) / HBsAg (+) / Anti-HBc (+)	19	95.0	1	5.0	20	100
3	NAT (+) / HBsAg (-) / Anti-HBc (+)	0	0	2	100	2	100
5	NAT (-) / HBsAg (+)/Anti-HBc (+)	12	100	0	0	12	100
7	NAT (-) / HBsAg (+) /Anti-HBc (-)	0	0	17	100	17	100
	Total	31	64.8	20	35.2	51	100

NAT: nucleic acid testing

HbsAg: Hepatitis B surface antigen

Anti-HBc: Antibodies against the core antigen of hepatitis B virus

Table 4 – HBV profiles obtained in HEMOAM and results of qPCR performed in FMT-HDV

HBV profile (HEMOAM)		qPCR (FMT-HVD)
HBV profile (HEMOAM)		n	average log	Range
01	NAT (+) / HbsAg (+) / Anti-HBc (+)	19	2,76	1,43 – 4,22
05	NAT (-) / HbsAg (+) / Anti-HBc (+)	12	2,23	1,08 – 3,7

NAT: nucleic acid testing

HbsAg: Hepatitis B surface antigen

Anti-HBc: Antibodies against the core antigen of hepatitis B virus

No competing interests reported.

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Accuracy of screening for HBV (NAT, HBSAg, anti-HBC) in blood donors from the Brazilian Amazon from 2105 to 2018

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