Detection of Anti-HBc (Total) and HBA-DNA in Hepatitis B Virus Surface Antigen Negative Blood Donors

Hepatitis B virus (HBV) infection remains a critical global public health concern. The introduction of hepatitis B surface antigen (HBsAg) testing in routine blood donor screening during the early 1970s significantly improved transfusion safety. Despite this progress, cases of HBV transmission from blood components testing negative for HBsAg have been documented. Research indicates that some donors, while negative for HBsAg but positive for anti-HBc, can still replicate the hepatitis B virus. These donors may carry and sustain HBV-DNA in their liver and bloodstream, representing a latent source of HBV transmission. This cross-sectional study involved 300 HBsAg negative individuals who visited the Transfusion Medicine Department at Chittagong Medical College Hospital and the Shandhani Chittagong Medical College unit for blood donation between July 2016 and June 2017. Of these participants, 58.66% were voluntary donors, while 41.33% were replacement donors. The study revealed that the majority of participants were aged 21–30 years, with a mean age of 27.09 years. Males accounted for 83% of the donors, whereas females comprised 17%. The findings showed that 49 donors (16.3%) tested positive for anti-HBc (total) among the 300 HBsAg-negative donors. Anti-HBc (total) positivity was more prevalent among replacement donors (9.31%) compared to voluntary donors (7%), with the difference being statistically significant (P < 0.05). Among the anti-HBc (total) positive donors, 21 (42.9%) were HBV-DNA positive, while 32 (65.3%) were positive for anti-HBs. Risk behaviors such as a history of jaundice, prior surgery, blood transfusions, and a family history of hepatitis B virus infection were notable among these donors.

In conclusion, the study underscores that relying solely on HBsAg for donor screening leaves a substantial number of HBV-infected donors unde- tected. Incorporating anti-HBc testing into donor screening protocols could substantially decrease HBV-infected donations, thereby reducing HBV transmission rates.

Introduction

Hepatitis B virus (HBV) is a widespread infectious agent with a significant global presence. Approximately 75% of the global population resides in regions with high infection rates. It is estimated that over 2 billion individuals have been or are currently infected with HBV, resulting in more than 350 million chronic carriers worldwide. The burden of infection is particularly pronounced in Asia, which accounts for over 75% of the global chronic cases [1]. The highest prevalence rates are reported in the WHO Western Pacific Region (6.2% of adults) and the WHO African Region (6.1%). The WHO Eastern Mediterranean Region, South-East Asia Region, and European Region show infection rates of 3.3%, 2.0%, and 1.6%, respectively. In the WHO Region of the Americas, the prevalence is 0.7% [2]. In South Asia, including Bangladesh, HBV infection is considered intermediate endemic, with prevalence rates ranging from 1.5% to 5.5% [3], [4]. Blood transfusion is an indispensable component of modern medical care. However, ensuring the safety of blood for transfusion remains a challenge. The World Health Organization (WHO) reports that approximately 92 million blood donations are collected globally each year, and these donations are screened for HBV, HCV, HIV (types 1 and 2), treponemal antibodies, and malaria antigens [5]. Despite advancements, the transmission of HBV via donated blood continues to occur. Since the 1970s, the screening of blood donors for hepatitis B surface antigen (HBsAg) has significantly mitigated the risk of transfusion-associated HBV infection. However, HBsAg-negative blood samples have occasionally been implicated in post-transfusion hepatitis cases [6]. The introduction of volunteer-based, regular blood donation programs combined with comprehensive medical screening and mandatory testing for HBsAg and anti-HBc has further reduced transfusion-associated HBV. Between 1981 and 1988, cases of transfusion-related hepatitis B reported to the American Red Cross decreased by approximately 50% [1]. Nonetheless, numerous studies have detected HBV DNA in the liver and blood of HBsAg-negative but anti-HBc-positive individuals. These findings suggest that recovery from acute hepatitis B may not result in complete viral clearance. Instead, the virus persists at minimal levels under immune control. This latent HBV infection has been observed even among individuals without liver disease, such as healthy blood donors or those with normal liver function tests [7], [8]. It has been established that blood donations from individuals who are HBsAg-negative but anti-HBc-positive can still transmit hepatitis B. Polymerase chain reaction (PCR) testing has confirmed that some HBsAg-negative, anti-HBc-positive donors continue to replicate HBV [9], [10]. Screening blood donors for anti-HBc antibodies has been suggested as a potential strategy to further reduce HBV transmission risks [11]. Previous studies have also demonstrated the feasibility of routine PCR screening for HBV in blood banks [12]. In Bangladesh, the current diagnostic protocol for detecting HBV in donated blood relies exclusively on HBsAg screening. However, evidence from various regions highlights the detection of HBV DNA in HBsAg-negative individuals, raising concerns about the risk of transfusion-transmitted HBV from latent carriers. The prevalence of latent HBV infection varies globally and correlates with the overall HBV endemicity of the region. For instance, studies in India have reported latent HBV infection rates ranging from 2.1% in Kolkata to 20.87% in New Delhi [13]–[15]. In Bangladesh, approximately 14% of healthy blood donors have been found to carry anti-HBc antibodies, and 30.7% of anti-HBc-positive individuals were reported to have latent HBV infection [16], [17]. The present study aims to determine the prevalence of anti-HBc antibodies among HBsAg-negative blood donors and assess the frequency of HBV DNA in anti-HBc-positive samples. The study was conducted at the Transfusion Medicine Department of Chittagong Medical College Hospital and the Shandhani Chittagong Medical College Unit.

Materials and Methods

The study was carried out in the Department of Microbiology of Chittagong Medical College and Center for Specialized Care and Research (CSCR), Chittagong, during the period of July 2016 to June 2017. It was designed as an observational cross section study. The study population consisted of 300 HBsAg negative blood donors who were donated blood in Transfusion Medicine Department of Chittagong Medical College Hospital and Shandhani, Chittagong Medical College Unit.

Selection Criteria

Samples which were negative for HBsAg and also for Treponemal antibody, anti-HCV, anti-HIV and malarial antigen were included in the study. Blood donors who were not fit for blood donations were excluded from the study.

Data Collection Tool

Donors visiting the Transfusion Medicine Department at Chittagong Medical College Hospital and the Shandhani Unit of Chittagong Medical College were informed about the study’s purpose and objectives. Only individuals who agreed to participate were included in the study, after providing written informed consent. A comprehensive pre-donation questionnaire was administered to gather socio-demographic details, such as age, gender, and occupation. The questionnaire also included inquiries about medical history, including jaundice, previous surgeries, history of blood transfusion, and family history of HBV infection. All donor histories and laboratory findings were meticulously documented in a pre-designed data sheet for each participant.

Sample Collection, Processing, and Preservation

Blood samples (5 mL) were collected under strict aseptic conditions using a 5 mL disposable syringe and transferred to sterile test tubes. The blood was allowed to clot, and serum was separated through centrifugation at 3000 rpm. Following serum separation, an immunochromatographic test (ICT) was conducted to detect HBsAg. For donors testing negative for HBsAg, further analysis for Anti-HBc (total) using ELISA was performed. Additionally, Anti-HBs testing via ELISA was carried out for donors positive for Anti-HBc (total). Remaining serum samples were stored in vials at −70 °C for subsequent polymerase chain reaction (PCR) analysis.

Laboratory Procedure

The presence of Hepatitis B virus surface antigen (HBsAg) was detected using the ICT method.

Test Principle

The One-Step HBsAg Test Device (Serum/Plasma) is a qualitative lateral flow immunoassay for the detection of HBsAg in serum or plasma. In the test line area of the assay, a membrane was pre-coated with anti-HBsAg antibodies. During the test, the serum or plasma sample reacts with the particles coated with anti-HBsAg antibodies. The mixture migrates up the membrane by capillary action through chromatography and reacts with the anti-HBsAg antibodies on the membrane to form a colored line. The presence of this colored line in the test area indicates a positive result, and its absence indicates a negative result. Serving as a control for the procedure, the control line area always displays a colored line, indicating that the correct sample volume has been added and the membrane is wetted.

Test Principle (PCR)

This diagnostic kit uses magnetic bead technology to extract HBV DNA from human serum. This real-time quantitative fluorescent PCR technology test uses a pair of specific primers that target the conserved sequence of HBV PCR and are accompanied by a specific fluorescent probe (the TaqMan probe is at the 5′ end of the other component marked with FAM). The PCR mix is used to quantitatively detect HBV DNA by changing the fluorescent signal. The PCR detection system uses positive internal control (the 5′ end of the TaqMan HBV internal control probe is labeled HEX) to monitor the presence of PCR inhibitors in the test sample by detecting whether the internal control is normal, thereby avoiding false negative results. This diagnostic kit employs the UNG enzyme + dUTP contamination prevention system. If samples are pretreated at 50 °C before PCR amplification, the UNG enzyme should be able to sufficiently degrade potentially undesirable PCR by-products to avoid false positive results. In the PCR detection system, the ROX reference dye was used to check sample loading variations and batch-to-batch differences. This makes it easier for the instrument to automatically analyze the relationship between the reported fluorescence and the internal reference fluorescence (ROX), allowing for more accurate quantification.

Processing of Specimens

A sufficient number of sterile 1.5 mL centrifuge tubes were prepared and appropriately labeled with identifiers for negative control, positive control, quantitative references A-D, and test specimens. Into each tube, 300 μL of HBV extraction solution 1 was added. Subsequently, 200 μL of each specimen, negative control, positive control, and quantitative references A-D was added to their respective tubes, and the lids were secured. Then, 100 μL of HBV extraction solution 2 was added to each tube, and the contents were thoroughly mixed using a vortex for 10 seconds. The tubes were kept at room temperature for 10 minutes before being centrifuged immediately. Following centrifugation, the tubes were placed on a magnetic bead separator for 3 minutes, after which the supernatant was carefully pipetted out.

Next, 600 μL of HBV extraction solution 3 and 200 μL of HBV extraction solution 4 were added to each tube, and the contents were mixed by vortexing for 5 seconds. The tubes were centrifuged again, and the magnetic bead separator was used as before. After 3 minutes, the pipette tip was positioned at the bottom of each tube to gently remove and discard the liquid. The tubes were held for 1 minute to allow any residual liquid to settle, which was then removed carefully from the bottom of the tubes.

Data Analysis

Experimental results were systematically documented and analyzed using SPSS software version 20.0. Qualitative data were represented as mean ± standard deviation (SD), while quantitative data were presented as numbers and percentages.

Action solution 4 into each tube. Vortex them for 5 seconds. Centrifuged them instantaneously and placed them again on the magnetic bead separator. 2.6 After 3 minutes, we placed the pipette tip at the bottom of the centrifuge tube and slowly pipetted and discarded all the liquids from the bottom. Hold it for 1 minute, then pipetted and discarded the residual liquids in the tube bottom.

Data analysis: The results of the experiments were recorded systematically, and statistical analysis was done by using SPSS software version 20.0. Qualitative data was expressed as mean ± SD while quantitative data was expressed as number and percentage.

Results

The present study was conducted among 300 HBsAg negative subjects visiting the Transfusion Medicine Department of Chittagong Medical College Hospital and Shandhani Chittagong Medical College Unit selected for blood donation during the period from July 2016 to June 2017.

Table I shows age distribution of study subjects where most were in the age group 21–30 years (57%). Mean age of the blood donors was 27.08 ± 7.452 years.

Table I. Age Distribution of the Study Subjects (n = 300)

Age interval	Frequency	Percent
<20 years	55	18.3
21–30	171	57.0
31–40	54	18.0
41–50	17	5.7
51–60	3	1.0
Total	300	100

Table II shows Anti HBc total positivity were more in male 42 (14%) than female 7 (2.33%). Among the 300 donors, male 249 (83%) were more than female 51 (17%). Male female ratio was 4.88:1.

Table II. Gender Distribution of Study Subjects (n = 300)

AntiHBc (total)	Sex		Total
	Male	Female
Positive	42 (14)	7 (2.33)	49 (16.33)
Negative	207 (69)	44 (14.66)	251 (83.66)
Total	249 (83)	51 (17)	300 (100)

Table III shows 49 (16.3%) donors were Anti HBc (total) positive among 300 HBsAg negative subjects.

Table III. Detection of Anti HBc (Total) Among HBsAg Negative Subjects (n = 300)

AntiHBc (total)	Frequency	Percent
Positive	49	16.3
Negative	251	83.7
Total	300	100

Table IV shows a comparison of type of donors and AntiHBc (total) positivity by Chi-square test. Anti HBc (total) positivity is more in replacement donors 28 (9.31%) than voluntary donors 21 (7%). There was statistically significant difference between type of donors and AntiHBc (total) positivity (P < 0.05).

Table IV. Relation of AntiHBc (Total) with Type of Donors (n = 300)

Type of donors	AntiHBc (total)		Total
	Positive	Negative
Replacement	28 (9.31)	96 (32)	124 (41.33)
Voluntary	21 (7)	155 (51.66)	176 (58.66)
Total	49 (16.33)	251 (83.66)	300 (100)

Table V shows that 21 (42.9%) donors were HBV-DNA positive among 49 Anti HBc (total) positive subjects.

Table V. Detection of HBV DNA by PCR Among AntiHBc (Total) Positive Subjects (n = 49)

HBV-DNA (PCR)	Frequency	Percent
Detected	21	42.9
Not detected	28	57.1
Total	49	100

Table VI shows among 21 HBV-DNA positive donors, DNA load were 10³–10⁵ copies/ml in 10 (47.61%) subjects and above 10⁵ copies/ml in 11 (52.39%) subjects.

Table VI. Quantitative HBV DNA Results Among the HBV DNA Positive Blood Donors (n = 21)

HBV DNA	Frequency	Percent
103–105 copies/ml	10	47.61
>105 copies/ml	11	52.39
Total	21	100

Table VII shows 32 (65.3%) were anti HBs positive among Anti HBc positive subjects.

Table VII. Detection of AntiHBs Among Anti HBc (Total) positive subjects (n = 49)

AntiHBs	Frequency	Percent
Positive	32	65.3
Negative	17	34.7
Total	49	100

Tables VIII and IX show some risk factors associated with AntiHBc positivity such as H/O jaundice, H/O blood transfusion, H/O surgery, Family H/O HBV infection. There was a significant association between the anti-HBc positivity and H/O jaundice (p < 0.05). Among 300 HBsAg negative blood donors 49 (16.33%) were antiHBc (total) positive. Among AntiHBc positive donors HBV-DNA was detected in 21 (42.85%) cases and 15 (30.61%) were also positive for AntiHBs.

Table VIII. Overall Demographic, Serological and Virological Characteristics among HBsAg (−ve) Blood Donors (n = 300)

Features	Total
HBsAg (negative)	300
Age years (mean ± SD)	27.08 ± 7.452
Male/Female	249/51
Male: Female	4.88:1
AntiHBc (Positive %)	49/300 (16.33%)
HBV-DNA (Positive %)	21/49 (42.9%)
AntiHBs (Positive %)	32/49 (65.3%)

Table IX. Risk Factors Associated with AntiHBc Positivity (n = 300)

Variable	Anti HBc (total)		Chi square value	P value
	Positive	Negative
H/O jaundice
Yes	26 (53.1)	71 (28.3)	11.501	0.001
No	23 (46.9)	180 (71.7)
H/O transfusion
Yes	2 (4.1)	4 (1.6)	0.928	0.385
No	47 (95.9)	247 (98.4)
H/0 surgery
Yes	9 (18.4)	33 (15.1)	0.926	0.335
No	40 (81.6)	218 (86.9)
F/H HBV infection
Yes	6 (12.6)	13 (5.6)	3.450	0.063
No	43 (87.4)	238 (94.4)

Discussion

Hepatitis B virus (HBV) is recognized as one of the most prevalent viruses transmitted through blood transfusions and organ transplants, often resulting in significant morbidity and mortality. Consequently, it is essential to screen all blood donors for viral infections [16]. With the advent of reliable serological methods for screening blood donors, cases of post-transfusion hepatitis have become rare. However, concerns regarding the safety of blood transfusions persist due to the potential for HBV infection among donors. In Bangladesh, HBsAg testing is currently used as the standard approach for detecting HBV infection in blood donors [17]. The diagnosis of HBV infection traditionally relies on the presence of HBsAg in the bloodstream [18]. Nevertheless, HBsAg screening alone does not entirely eliminate the risk of HBV transmission through transfusions, as the absence of this marker does not rule out the presence of HBV DNA. Consequently, many developed nations have revised their blood transfusion protocols to incorporate anti-HBc testing. Additionally, nucleic acid testing (NAT) has been introduced in several countries to further mitigate the risk of transfusion-associated hepatitis [19], [20].

In this study, 300 HBsAg-negative individuals were screened for Anti-HBc (total) using the ELISA method. Samples testing positive for Anti-HBc (total) underwent further analysis for HBV DNA using real-time PCR and Anti-HBs using ELISA. The study revealed a male predominance, with a male-to-female ratio of 4.88:1. Similarly, studies conducted by Islam et al. [16] and Lavanya et al. [21] in Bangladesh and India reported male-to-female ratios of 15.7:1 and 11.5:1, respectively. Additionally, this study found that 57% of donors were aged 21–30 years, aligning with similar research in Bangladesh, which reported that 60% of donors belonged to this age group [16]. In this study, the mean age of participants was found to be 27.08 ± 7.452 years, with an age range spanning from 18 to 60 years. A similar study reported the mean age of blood donors as 27.8 ± 6.4 years, ranging from 18 to 45 years [16], which aligns closely with the findings of the present study. In contrast, a higher mean age of 38 ± 11 years was reported among 1,000 donors in Iran [22]. This discrepancy could be attributed to geographical, racial, ethnic, and lifestyle differences in the study populations.

For HBsAg-negative blood donors, the current study observed an anti-HBc positivity rate of 16.33%. The prevalence was 7% among voluntary donors and 9.31% among replacement donors. Similar findings were reported in studies conducted in Dhaka by Jahan et al. [20] and Islam et al. [16], where the prevalence of anti-HBc was 20.6% and 14%, respectively. A study from India reported an overall prevalence of 8%, with 6% among voluntary donors and 9% among replacement donors [23]. Research from the Department of Transfusion Medicine at PGIMER, Chandigarh, found an anti-HBc prevalence of 8.4%, with 6.9% in voluntary donors and 10.4% in replacement donors [13]. A study from New Delhi noted that 18.9% of donors were reactive for anti-HBc [24], while research from West Bengal, Eastern India, reported a positivity rate of 18.3% among voluntary donors [15]. In Iran, the prevalence of anti-HBc among voluntary donors was 6.55% [9], and in Pakistan, where all donors were replacement donors, the rate was reported at 17.28%. Comparatively, the prevalence of anti-HBc in Europe and North America is significantly lower, with 0.63% in the UK [25] and 1.5% in Germany [7].

In this study, anti-HBc seroprevalence was lower among voluntary donors compared to replacement donors (7.0% vs. 9.31%), consistent with findings from other studies. The prevalence of HBV-DNA in HBsAg-negative samples also varies depending on regional infection rates. Reported rates of HBV-DNA in anti-HBc-positive donors include 0% in the UK [26], 3.7% in the USA [27], and 29.07% in Iran [28]. Other studies have reported HBV-DNA detection rates of 26.6% in Italy [32], 30% in India [14], 40% in Iran [23], and 90.5% in Egypt [6]. Similar findings were reported by Jahan et al. [20] and Rahman et al. [17] in Bangladesh, who observed HBV-DNA positivity rates of 8.5% and 30.7%, respectively. Studies have reported varying rates of HBV-DNA detection in blood donors, reflecting differences in occult HBV prevalence. These variations can be attributed to factors such as the endemicity of HBV infection, geographical differences, the sensitivity of diagnostic methods for detecting HBV DNA and other markers, sample size, and the characteristics of the study population.

In the present study, 32 (65.30%) of the 49 anti-HBc-positive donations were also positive, suggesting potential protection against HBV. However, 15 (30.61%) of these anti-HBs-positive samples were HBV-DNA positive. Similarly, an Indian study reported HBV-DNA positivity in 27.1% of the 164 anti-HBc-positive group and 30.0% of the 40 anti-HBs-positive group [14], aligning with our findings. Another study from Iran found anti-HBs antibodies in 50% of HBV-DNA-positive samples [29]. In Bangladesh, research revealed that 33.33% of anti-HBs-positive donors had detectable HBV DNA [17]. In this study, HBV DNA was detected in individuals positive for anti-HBc, with or without anti-HBs.

The presence of HBV DNA in anti-HBs and anti-HBc-positive individuals may indicate recovery from a prior HBV infection, with the virus persisting at low levels [30]. We also examined risk factors associated with anti-HBc positivity, including histories of jaundice, blood transfusion, surgery, and family history of HBV infection. A significant association was found between anti-HBc prevalence and a history of jaundice. A related study in India identified significant associations between anti-HBc prevalence and factors such as blood transfusion, surgery, ear piercing, family history of jaundice, tattoos, and beauty salon visits [21]. These findings highlight how geographical, racial, ethnic, and lifestyle differences among participants may influence risk factors.

Occult HBV infection (OBI) is increasingly recognized as a contributor to chronic liver disease and hepatocellular carcinoma (HCC) [31]. OBI also poses a risk for HBV transmission via blood transfusions and organ transplants, as evidenced by the high prevalence of occult HBV among blood donors in Chittagong, Bangladesh. This raises serious concerns about transfusion safety, as HBV-contaminated blood may be transmitted despite HBsAg screening. Our findings indicate that OBI is an emerging threat to transfusion safety in this community. To minimize HBV transmission risk, it is ideal to select HBsAg-negative individuals who are either anti-HBc negative or anti-HBc positive/anti-HBs positive/HBV DNA negative as regular blood donors. While this approach reduces transfusion-related HBV infections, it increases the cost of testing. Some countries have implemented routine anti-HBc screening for blood donors, reducing the risk of post-transfusion HBV infection [18], [32]. Incorporating anti-HBc testing into donor screening in resource-limited settings, such as Bangladesh, may similarly lower the number of HBV-infected donations. Although this could lead to the rejection of some donations, it would significantly enhance transfusion safety and reduce the risk of HBV-related complications.

Conclusion

HBV-DNA was found in HBsAg negative and AntiHBc (total) positive blood donors which indicates, these donors are potential source of HBV infection. So, only HBsAg screening test is not sufficient for safe blood transfusion.

Recommendations

Further large-scale multicentre study involving significant number of blood donors is recommended to determine whether antiHBc (total) and/or HBV-DNA by PCR should be done in addition to HBsAg to eliminate transfusion transmitted HBV infection.

Limitations

Due to high costs, HBV-DNA by PCR could not be tested in HBsAg negative and AntiHBc (total) negative blood donors. AntiHBcIgM could not be tested in HBsAg negative Blood donors due to lack of resources. Last, this study is a single centre study with limited time and resources.