Screening of HBV infection in multi-transfused previously vaccinated patients

Azza Aboelenin, Nermeen Eldesouky , Nehal Diaa , Fatma Abdel Wahab Abdel Maksoud* , Ilham Youssry

Kasr Al-Ainy Faculty of Medicine, Cairo University, Cairo, Egypt

Abstract Introduction: Blood and blood products transfusion is an essential therapy for many patients. However, it carries the risk of transmission of infection. The hepatitis B virus can be transmitted by blood transfusion. This highlights the importance of combined HBsAg and anti-HBc screening to eliminate blood-transmitted HBV. Aim of work: This study aimed to assess the prevalence of HBV among vaccinated multi-transfused patients in Egypt. Material and methods: The study was conducted on 805 multi-transfused patients who were followed up at the outpatient hematology clinic at Cairo University Children’s Hospital. Plasma blood samples were collected for an HBV rapid screening test, and serum samples were collected for serological and molecular analysis of positive cases. Results: The results of this study results of HBV screening using RDT revealed three positive cases out of 805. The first case showed HBsAg positive, HBsAb negative, HBcAb positive, and HBeAg positive, as well as a high viral load. The second case showed HBsAg positive, HBsAb negative, HBcAb positive, HBeAg negative, and an intermediate viral load. The third case showed HBsAg negative, HBsAb positive, HBcAb positive, HBeAg negative, with an undetectable titer. Conclusions: HBV infection persists among multi-transfused and previously vaccinated children. Therefore, the post-immunization immune status of these patients should be assessed, and strict hemovigilance should be followed. Keywords: HBV, hepatitis B virus, chemiluminescence, PCR Acta Haematol Pol 2024; 55, 6: 327–334

Introduction

Transfusion-related infections (TTIs) still represent a significant danger to patients worldwide. Any infection that spreads from person to person through parenteral blood or blood component delivery is referred to as a TTI [1]. Examples include Human T-cell Lymphotropic Virus (HTLV) I and II, syphilis, cytomegalovirus (CMV), malaria, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), human immunodeficiency virus (HIV), and West Nile virus [2, 3].

Hepatitis B is a potentially life-threatening infection caused by HBV. The high mortality rate from liver cancer and cirrhosis makes it a severe worldwide health concern [4]. Many people are asymptomatic during the initial infection. Symptoms may appear 1–6 months post-exposure [5]. The virus is transmitted vertically through perinatal transmission or horizontally by exposure to infectious blood or bodily fluids [6]. It is recommended that all blood be tested for hepatitis B before transfusion to prevent infection. The infection can be diagnosed 30–60 days after exposure [7]. The disease was first made preventable by vaccination in 1982 [8]. Vaccination is recommended by the World Health Organization on the first day of life if possible. Two or three more doses are required later to deliver the full effect [9]. In Egypt, mandatory vaccination against HBV was implemented in 1992 [10].

A safe and effective vaccine offers protection against hepatitis B. Preventing hepatitis B infection avoids complications, with a more than 95% success rate [11].

This study aimed to detect the prevalence of HBV infection in vaccinated and multi-transfused chronic hemolytic anemia pediatric patients attending the Cairo University Children’s Hospital hematology clinic.

Patients and methods

The study was conducted on 805 multi-transfused patients with chronic hemolytic anemia who attended the hematology clinic at Cairo University from August to December 2022. These patients received the HBV vaccine as part of the Egyptian government’s national vaccination program, as they were all born after 1992, the year in which the vaccine was made compulsory.

Patients’ ages ranged from 2 to 24 years with a mean of 13 ± 2 SD, 51.31% were male and 48.69% female. All included patients were screened for HBsAg using an immune-chromatographic rapid diagnostic test of pharmAtos Biodiagnostic laboratories. Of the 805 patients, there were 413 (51.31%) males and 392 (48.69%) females. Cases that revealed positive screening were further subjected to the following:

1. Serological study by Chemiluminescence Cobas 6000 for Hepatitis B surface Antigen (HBsAg), Hepatitis B surface Antibody (HBsAb), Hepatitis B core Antibodies (HBcAb), and Hepatitis B e Antigen (HBeAg);
2. PCR Real-time quantitative polymerase chain reaction (PCR);
3. Their families were tested by Chemiluminescence Cobas 6000 for HBsAg, HBcAb.

Sample collection

3 mL of venous blood was obtained from each patient on a sterile EDTA vacutainer for follow-up in the hematology clinic, and another 3 mL of blood was collected in a plane vacutainer tube.

Material and methods

Rapid test: The rapid diagnostic test (RDT) was selected for HBV screening because of its low cost and ability for point-of-care diagnosis. The test is an in vitro, visually read, qualitative immunoassay for detecting HBsAg in serum, plasma, or whole blood. It is rapid and easy to use.

Interpretation of RDT results

In positive results, as the serum is added, the antigen-antibody complex migrates towards the test zone (T), where it is captured by immobilized antibodies, forming a visible line. In negative results, the antigen is absent, and there is no visible line in the (T) zone. In an invalid result, the control line zone and the test line zone do not appear within 10 minutes.

Specificity and sensitivity

A total of 1,002 serum specimens were tested with this rapid one-step HBsAg test and compared to a reference kit (Chemiluminescence Cobas 6000). The results showed that 16 specimens were inconsistent with this rapid one-step test and the reference kit (positive accordance rate was 99.43%, negative accordance rate was 97.25%, and total accordance rate was 98.40%). Plasma specimens were tested and compared to serum specimens. The results showed a positive accordance rate of 100%, a negative accordance rate of 98.54%, and a total accordance rate of 99.01%.

Chemiluminescence immunoassay using a Cobas 6000 system Elecsyse 601 analyzer (Roche, USA) to assess HBsAg, Anti-HBs, HBeAg.

Real-time quantitative AmpliPrep/COBAS® TaqMan® system (Roche, USA)

The AmpliPrep/COBAS® TaqMan® HBV Test, version 2.0, is an in vitro nucleic acid amplification test (NAT) for quantifying HBV DNA in human plasma and serum. It uses the COBAS® AmpliPrep Instrument for automated specimen processing and the COBAS® TaqMan® Analyzer or COBAS® TaqMan® 48 Analyzer for automated amplification and detection.

Results

The screening for HBsAg by immune-chromatographic rapid diagnostic test revealed 802/805 (99.63%) negative cases. Only three cases, i.e. numbers 38, 91, and 442, were positive (0.37%), as depicted in Figure 1.

The only three positive cases were serologically investigated for HBsAg, HBs Ab, HBcAb, HBeAg and the PCR quantitative assay investigated for the viral load. Descriptive data for positive cases is set out in Table I, which shows the results of the serological assessment of HBsAg, HBsAb, HBcAb, HBeAg using chemiluminescence on the three positive cases.

The results of the PCR quantitative assay for the viral load of the three cases were as follows:

• Case 38 had a high titer of 17,000,000 IU/mL;
• Case 91 had an intermediate titer of 20,000 IU/mL;
• Case 442 had an undetectable titer of less than 10 IU/mL.

An ‘undetected’ result indicates that hepatitis B virus (HBV) DNA was not detected in the serum specimen. A result of less than 10 IU/mL indicates that HBV DNA was detected. However, the HBV DNA level present cannot be quantified accurately below this lower limit of quantification of this assay.

Family members of the three cases were serologically investigated. Table II sets out the results of the HBsAg and HBcAb tests performed on them.

Case 38 was an 11-year-old boy on regular blood transfusions, and we depended on the history of vaccination given by his mother. We revealed positive HBsAg, with a low level of HBsAb (2 mIU/mL) considered negative, positive HBcAb and positive HBeAg and a high titer viral load (17,000,000 IU/mL). This patient had chronic active hepatitis B infection with high infectivity and high viral load. The low level of HBsAb reflects his low immunity to HBV infection.

Case 91 was a 24-year-old female; she was on regular blood transfusions, her vaccination history was unknown, and she had positive HBsAg, with a low level of HBsAb (2 mIU/mL) considered negative, positive HBcAb and negative HBeAg with an intermediate titer of viral load (20,000 IU/ml). This patient had chronic hepatitis B infection with low infectivity and an intermediate viral load. The low level of HBsAb reflects her low immunity to HBV infection.

Case 442 was a 5-year-old boy. He was on regular blood transfusions, and we depended on the history of vaccination given by the mother, although there was no documentation of the doses received. He had negative HBsAg, an elevated level of HBsAb (20 mIU/mL) considered positive, positive HBcAb, and negative HBeAg with an undetectable titer of HBV of less than 10 IU/mL, so the patient was diagnosed as an occult hepatitis B patient.

Family members of the three cases showed negative results for all markers (HBsAg, HBcAb) except for the mother of case 38, who showed gray zone positivity for HBsAg, and her HBsAb was negative (2 mIU/mL). PCR of this mother showed an undetectable titer of less than 10 IU as shown in Figure 2. She gave no history of blood transfusions, and her vaccination history was undocumented.

Discussion

Hepatitis B is a potentially life-threatening liver infection caused by HBV. It is a major global health problem that can cause chronic infection and put people at high risk of death from cirrhosis and liver cancer.

Hepatitis B prevalence is highest in the WHO Western Pacific Region and the WHO African Region, where 6.2% and 6.1% of the adult population are infected, respectively. In the WHO Eastern Mediterranean Region, WHO South-East Asia Region, and WHO European Region, an estimated 3.3%, 2.0%, and 1.6% of the general population are infected, respectively. Additionally, 0.7% of people in the WHO Region of the Americas are infected [9].

According to WHO estimates, 1.5 million new cases of hepatitis B infection occur each year, and 296 million individuals worldwide were living with chronic illness in 2019. 820,000 deaths from hepatitis B are thought to have occurred in 2019, primarily from cirrhosis and hepatocellular cancer [12].

As of 2016, 27 million people (10.5% of those estimated to be living with hepatitis B) were aware of their infection, while 4.5 million (16.7%) of the people diagnosed were on treatment [13].

In 2015, Schweitzer et al. [14] reported the most robust estimates of the prevalence of chronic HBV by country and by region. Their findings highlighted both the substantial global burden of chronic HBV and the concentration of the epidemic in specific countries. This systematic review collated data from HBsAg seroprevalence studies including 109,415,627 individuals across 161 countries published between 1965 and 2013. The authors estimated that 3.61% of the global population lived with chronic HBV infection [14].

HBV prevalence by country showed significant worldwide variation, ranging from as low as 0.01% in Norway and the UK to more than 20% in countries such as South Sudan and Kiribati [15].

In Egypt, an estimated 3.3 million people are infected with HBV, which represents a moderate endemicity. The prevalence rate declined after the universal immunization program for infants was launched in 1992 [16]. A cross-sectional analysis of the prevalence of HBV in Egypt by Ismail et al. [17] in 2017 revealed a population prevalence rate of 1.4%. This study showed marked variations in the prevalence of infection nationwide, and within-household transmission appeared to play a powerful role in sustaining HBV infection rates in Egypt [17].

HBV also spreads by needle stick injury, tattooing, piercing, and exposure to infected blood and bodily fluids such as saliva and menstrual, vaginal, and seminal fluids. Sexual transmission of hepatitis B may occur, particularly in unvaccinated men who have sex with men and in heterosexual persons with multiple sex partners or contact with sex workers [18].

Perinatal or childhood infection has been associated with few or no symptoms but has a high risk of becoming chronic. A limited number of medications can be used to treat chronic hepatitis B in these age groups effectively; however, a safe and effective vaccine is available to prevent infection [19]. Follow-up of babies born to infected (HBsAg-positive) mothers involves four doses of vaccination: the first dose at birth, then 1, 2 and 12 months later, and/or a single dose of HBIG at birth [20].

The risk factors for HBV infection have been well-characterized in the literature in general. Even so, data specific to the Egyptian context is scarce, and multiple modes of transmission have been noted [21]. Previous work in Egypt has shown strong associations between distal risk factors such as literacy and HBV risk [17].

Proximate risk factors are better characterized and include intravenous drug use, which has been strongly associated with HBV risk status in studies in Egypt, tattooing and body piercing, barbershop use, and the presence of an infected family member [17, 22]. High rates of occult HBV infection reported in some studies from Egypt reflect the higher risk of iatrogenic exposure to which they were subject [23].

One of the major concerns in transfusion medicine centers on minimizing the risk for TTIs, including HBV [24]. Despite the use of effective risk reduction measures, the goal of zero residual risk remains out of reach [25]. Since HBV vaccination was introduced into the routine immunization for children in 1992, progress has been made in improving both the scale and coordination of public efforts to tackle HBV and HCV infection through the Egyptian National Strategy and the Plan of Action for the Prevention, Care, and Treatment of Viral Hepatitis.

Researchers have suggested that the residual risk for HBV transfusion transmission is “associated with the pre-seroconversion window period and occult HBV infection characterized by the absence of detectable HBsAg and extremely low levels of HBV DNA” [25]. Another study pointed out several factors that likely influence HBV transfusion transmission, including the volume of plasma transfused/viral load, the immune status of both recipient and donor concerning HBV, and the infecting strain of HBV [26].

Wang et al. [27] showed that HBV screening methods among blood donors differ between developed and developing countries. In developed countries like the US, blood donors are screened for HBsAg and anti-HBc. Persons positive for even one of them are disqualified based on evidence of ongoing or past infections. Combined HBsAg and anti-HBc screening virtually eliminates blood-transmitted HBV, with the rare exception of pre-HBsAg window-period donations eliminating most of the blood-transmitted HBV. In contrast, in developing countries endemic to HBV, adopting such a criterion would disqualify most volunteer blood donors. However, the advent of new viral detection technology, especially NAT, has concluded that c.10% of people with past HBV infection harbor viral DNA in their blood or blood cells [27].

The results of this HBV study screening revealed three positive cases. These initial results were further confirmed by testing the patients’ samples for various serological HBV markers using the chemiluminescence technique and quantitative viral load assay by real-time PCR.

Although the RDT is less specific than other immunological techniques (e.g. ELISA and chemiluminescence) and carries the risk of missing positive cases, this study was designed to screen as many patients as possible at the lowest cost. It is known that using more sensitive screening techniques for HBsAg (e.g. again ELISA and chemiluminescence), especially when combined with anti-HBcAb assay, can detect more positive cases with acute, chronic, or occult hepatitis B infection, or even resolved hepatitis B cases. Considering the specificity of the used assay and the results of confirmatory tests, the prevalence of HBV in the studied group was 0.37%.

It is worth mentioning that the discrepancy between positive HBsAg initial screening with RDT and negative chemiluminescence assay, in this case, comes from using another sample obtained five weeks later when the patient attended to receive another blood transfusion.

A prospective study was conducted at Mansoura University to estimate the real frequency of HBV and HCV among Egyptian β-thalassemic patients and to determine the infection-associated risk factors in this group. Fifty-eight (29.0%) were HBsAg positive, and 13 (6.5%) were anti-HBc positive by ELISA [28]. In another study conducted on transfusion-dependent thalassemic children attending the Pediatric Departments of both Sohag and Minia Universities in Upper Egypt from 2014-2015, the frequency of HBsAg positivity was 4.12% [29].

HBV screening in Cairo University Hospital’s blood bank is achieved by testing the donor samples for HBsAg using ELISA following the Egyptian national standards for blood banking. However, safer screening strategies are applied worldwide.

HBsAg tests are not sensitive enough during the window period (45–50 days for most sensitive assays) for HBV infection. Likewise, in the early convalescence phase of HBV infection acute phase, as well as in chronic HBV infections, very low levels of HBsAg are often present, which are not detected by the routinely used HBsAg assays [30]. Several reports on different HBV escape mutations, in which routine screening assays did not detect HBsAg [31].

Blood-borne transmission of HBV continues to occur despite the implementation of highly sensitive screening tests for HBsAg, suggesting that these assays are still not sensitive enough to prevent all infections [25]. Unlike HBsAg, anti-HBc may be present during the chronic carrier state and at the end of an acute resolving infection where HBsAg may be undetectable. HBV-positive donors who are analyzed when they present during these stages of the disease can often be identified by either HBV NAT or by testing for anti-HBc [30].The introduction of HBV NAT can overcome safety gaps left by HBsAg or anti-HBc testing in cases of occult HBV infection [32].

In this study, we depended on a mother’s history regarding HBV vaccination, and 2/3 positive cases had a low HBsAb titer. The Egyptian Ministry of Health has adopted a schedule for HBV immunization that involves three doses of yeast-recombinant hepatitis B vaccine administered to all infants at 2, 4, and 6 months, to coincide with other compulsory vaccines.

Optimal immunization is critical for all patients with thalassemia, especially transfused patients. Patients need to be immunized against hepatitis B, especially patients on chronic transfusions. Annual monitoring of titers and booster immunizations, when indicated, will ensure patients are well protected [33].

Salama et al. [34] conducted a study to assess the effectiveness of the HBV vaccination program among fully vaccinated children in Egypt. A total of 3,600 children aged from 9 months to 16 years who were fully vaccinated with the HBV vaccine during infancy were recruited. Sera were evaluated for HBsAg, anti-HBcab (total), and quantitative detection of hepatitis B surface antibody by ELISA. Samples positive for HBsAg or anti-HBcAb were subjected to quantitative HBV PCR. Sero-protection was detected among 2,059 children (57.2%) with mean titers of 75.4 ± 3.6 IU/L compared to 3.1 ± 2.1 IU/L among non-seroprotected children. HBsAg was positive in 0.11%, and breakthrough infection was found in 0.36% and 0.39%, depending on the positivity of anti-HBc and DNA detection, respectively. The prevalence of HBV infection was significantly higher among children aged ≥ 7 years (0.59%) compared to 0.07% among those aged under seven [34].In a cross-sectional study by El-Beshlawy et al. [35], 100 multi-transfused children were included, and screening for HBsAg, HBcAb by ELISA, only 12% of patients were either acutely or chronically infected with HBV. 46% were immune due to previous vaccination, and 39% were not protected from HBV infection. However, complete HBV vaccination in this study was achieved in 78/100 patients. The authors also found that among all multi-transfused children in the study, one in three of those who had completed their vaccinations were not protected [35].

El-Beshlawy et al. [35] showed HBsAg positivity had been 16.48% before the implementation of the national HBV vaccination. Establishing a vaccination program against HBV has had a notable impact on the prevalence of hepatitis B infection [16].

This decrease in the prevalence of hepatitis B infection reflects the efficacy of the vaccination program, and the effective screening of blood and blood products [36]. Despite the decline in the prevalence of HBsAg among Egyptian multi-transfused children over recent decades [37], it is still high compared to other countries, and this could be attributable to the high infection rates among those children despite vaccination against HBV, as they might not have responded adequately to vaccination or may not be fully vaccinated [34]. Post-vaccination infections may be a result of viruses with surface (S)-gene mutations in a region critical for antibody reactivity to the hepatitis B surface antigen (anti-HBs) [38].

Although blood is screened for HBV in blood banks in Egypt, transfusion-related HBV transmission may occur because screening in most blood banks is performed only for HBsAg, not for core antibodies [39, 40]. This has led to calls for the implementation of the anti-HBc test as a routine assay, which would undoubtedly eliminate possible HBV-infected blood units. Rejection of these units would decrease the risk of HBV transmission, particularly in immune-compromised recipients.

The results of this study together with those of Salama et al. [34] and El-Faramawy et al. [39], still reveal a gap regarding vaccination schedules that requires additional effort to ensure that the entire population has strictly completed their vaccinations, particularly in high-risk groups of patients.

In this study, the family members of the three cases were serologically investigated for HBsAg and HBcAb, and they all showed negative results except for the mother of case 38, who showed gray zone positivity for HBsAg while her HBsAb was negative (2 mIU/mL). Real-time PCR on her sample showed a titer of less than 10 IU. She gave no history of blood transfusions, and her vaccination history was unknown. Follow-up of this mother is needed, as she might have had a recent infection. Although the transmission mode is not yet apparent, the role played by household contact in active infections has been highlighted in a study by Talaat et al. [23].

In conclusion, to reduce the incidence of HBV in multi-transfused patients, we recommend active immunization against HBV for all patients on repeated blood transfusion therapy, plus strict follow-up and hemovigilance.

Article information and declarations

Data availability statement

Data that supports the findings of this study is available on request from the corresponding author.

Ethics statement

Informed consent from all individuals enrolled in the study was obtained.

Authors’ contributions

AA — idea for study, study design; NE — study design, manuscript revision; ND — practical part of screening, manuscript preparation; FAWAM — manuscript preparation; IY — clinical enrollment and follow-up of cases, manuscript revision.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Acknowledgements

Not applicable.

Conflict of interest

The authors declare no conflict of interest

Supplementary material

None.

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