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Detection of occult hepatitis B virus infection among blood donors in Sudan

Mahmoud, Ola Abd El Kadera; Ghazal, Abeer Abd El Rahima; Metwally, Dalia El Sayeda; Elnour, Ahmed Mohamedb; Yousif, Ghanim Eltahir Ahmedc

The Journal Of The Egyptian Public Health Association: April 2013 - Volume 88 - Issue 1 - p 14–18
doi: 10.1097/01.EPX.0000427065.73965.c8
Original articles
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Background Sudan is classified among countries with a high hepatitis B surface antigen (HBsAg) endemicity of more than 8%. Cross-sectional studies have showed a marked increase in the prevalence of occult hepatitis B infection (OBI) in patients with cirrhosis or hepatocellular carcinoma. In terms of OBI infectivity by transfusion, it is largely unknown whether residual risk estimates translate into true rates of infection.

Aim The current study aimed to determine the frequency of OBI among blood donors in Sudan.

Materials and methods This study was carried out during the period between 2011 and 2012. It included 100 HBsAg-negative blood donors who attended the Central Blood Bank in Sudan. Sera collected from all donors were tested for HBsAg, antibodies against hepatitis B core antigen (anti-HBc), antibodies against hepatitis Be antigen (anti-HBe), and antibodies against hepatitis B surface antigen (anti-HBs) by enzyme-linked immunosorbant assay. Anti-HBc-positive patients were tested for hepatitis B virus (HBV)-DNA.

Results The anti-HBc was detected in 42% of the blood donors, among whom 90.5% were positive for HBV-DNA. Two main profiles have been detected, namely, the presence of the three genes (S, C, and X genes) together in 35.7% of the blood donors or the presence of the X gene in addition to the core gene.

Conclusion and recommendations With the use of HBsAg as the sole detection marker for HBV, there is a danger of HBV transmission through blood transfusion. Anti-HBc testing should be added to the routine blood donor screening test if occult hepatitis B is to be diagnosed.

aDepartment of Microbiology Medical Research Institute, University of Alexandria, Alexandria, Egypt

bDepartment of Haematology, Faculty of Medicine, University of Elimam Elmahdi, Kosti

cMedical Laboratory Science, University of Khartoum, Khartoum, Sudan

Correspondence to Dalia El Sayed Metwally, PhD, Department of Microbiology, Medical Research Institute, University of Alexandria, Alexandria, Egypt Tel: +01111365264; e-mail: dr.dalia.ragab@hotmail.com

Received November 29, 2012

Accepted January 20, 2013

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Introduction

Hepatitis B virus (HBV) infection is a major public health problem worldwide. Of the two billion individuals who have been infected worldwide, more than 240 million are chronic carriers of HBV 1. Approximately 15–40% of infected patients will develop cirrhosis, liver failure, or hepatocellular carcinoma (HCC) 2.

Implementation of hepatitis B surface antigen (HBsAg) in routine screening of blood donors in the early 1970s has considerably enhanced the safety of transfusion. The incidence of transfusion-transmitted hepatitis B has been reduced steadily over the last four decades. However, it was found that HBV transmission by blood components negative for HBsAg can still occur 3. HBV transmission remains the most frequent transfusion-transmitted viral infection. Thus, the term occult hepatitis B virus infection (OBI) was introduced. OBI is defined as the presence of HBV-DNA in blood (and/or) liver tissue with no detectable HBsAg, with or without antibodies against hepatitis B core antigen (anti-HBc) or antibodies against hepatitis B surface antigen (anti-HBs) 4.

Sudan is classified among the countries with a high HBV seroprevalence. Exposure to the virus varies from 47 to 78%, with a prevalence of HBsAg ranging from 6.8% in central Sudan to 26% in southern Sudan 5,6.

The prevalence of OBI is unclear and depends in part on the sensitivity of the HBsAg and DNA assays used as well as the prevalence of HBV infection in the study population. OBI varies considerably between different geographical regions. Studies have shown that the prevalence of OBI is closely related to the endemicity of HBV infection. Patients from countries highly endemic for HBV are more likely to develop OBIs 7.

OBI has been reported in 0.1–2.4% of HBsAg-negative, anti-HBc-positive (±anti-HBs) blood donors in western countries, where only 5% of the population has previous exposure to HBV, and in up to 6% of a similar cohort of donors who reside in endemic areas, where 70–90% of the population has been exposed to HBV 8,9.

The aim of this study was to determine the frequency of OBI among blood donors in Sudan.

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Materials and methods

A cross-sectional study was carried out during the period between 2011 and 2012. It included 100 consecutive HBsAg-negative samples collected from blood donors who attended the Central Blood Bank in Sudan.

Blood donors were subjected to a detailed questionnaire. Donors with risk factors for viral hepatitis were excluded from blood donation. The sample size was calculated using the NCSS 2004 and PASS 2000 program. A minimum sample size of 92 from a population of 120 produces a 95% confidence interval equal to the sample proportion ±0.03000 when the estimated proportion is 0.10000 10.

All relevant information was collected from each donor (after obtaining full consent) including personal data as (age, sex, etc) as well as health data (history of previous infection with viral hepatitis or HIV, previous surgical interference, and dentistry). The study was carried out after receiving the approval of the Ethics Committee in the Medical Research Institute.

Sera collected from all donors were tested for HBsAg by enzyme-linked immunosorbant assay, anti-HBc, antibodies against hepatitis Be antigen (anti-HBe), anti-HBs (Abott Murex Diagnostic Division, Kent, UK), and determination of liver enzymes: aspartate aminotransferase(AST), alanine aminotransferase (ALT), and total serum bilirubin 9,11.

HBsAg-negative blood donors were tested for the anti-HBc and anti-HBc-positive patients were tested for HBV-DNA.

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HBV-DNA extraction

DNA was extracted from 200 μl of serum using the QIAamp viral DNA mini kit (Qiagen Inc., Valencia, California, USA) following the manufacturer’s instructions.

Amplification of S and C and X genes by SYBR green real-time PCR (Applied Biosystems Inc., Foster city, California, USA) using specific primers 11.

Table

Table

The amplification reaction was performed as follows.

0.3 μl (30 pmol) of HB surface or core, or X gene sense primers, 0.3 μl (30 pmol) of HB S or C or X gene antisense primers, 12.5 μl SYBR green universal PCR master mix two-fold (Applied Biosystems), 10 μl of Qiagen extracted DNA, and H2O were added to bring the reaction to a final volume of 25 μl.

Thermal profile: AmpliTaq activation for 95°C for 10 min, followed by 40 cycles of PCR amplification, including denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by melting curve analysis to determine the purity and specificity of the amplification product. The melting curve analysis profile was 95°C for 1 min, 50°C for 30 s, and 95°C for 15 s.

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Determination of the HBV viral load for anti-HBc-positive cases using the Taqman probe technique (Artus-Biotech, Hamburg, Germany)

HBV-DNA was amplified using the Real Artus HBV TM RG PCR Assay (Artus-Biotech). Fifteen microliters TaqMan universal PCR master mix two-fold (Artus HBV RG PCR) were added to 10 μl of Qiagen extracted DNA to bring the reaction to a final volume of 25 μl. Real-time PCR was performed using the Mx3000P (Stratagene, La Jolla, California, USA) real-time PCR system. The reaction was carried out according to the following thermal profile: AmpliTaq gold activation at 95°C for 10 min, followed by 40 cycles of two PCR-step amplification, denaturation at 95°C for 15 s, followed by annealing and extension at 60°C for 1 min with end-point fluorescence detection.

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Results

The majority of blood donors included in the current study (74%) were between 25 and 39 years of age, and all donors were men because usually women do not donate blood in Sudan.

The biochemical, serological, and C, X, S gene markers for the 100 blood donors are listed in Table 1. Among the 100 blood donors included in this study, 42% were anti-HBc positive and 8% were anti-HBe positive. Among the 42 anti-HBc-positive blood donors, two main profiles could be detected, namely, the presence of the three genes together in 35.7% of the blood donors or the presence of the X gene in addition to the core gene in 35.7%. There was no statistically significant relation between the serological and gene profile of HBV in the study group (P=0.650) (Table 1).

Table 1

Table 1

Table 2

Table 2

Figure 1 and Table 2 show the distribution of C, S, and X genes among the 42 anti-HBc-positive blood donors.

Figure 1

Figure 1

All the anti-HBc-positive blood donors tested for viral load quantification using the Tagman probe technique (Artus-Biotech) were negative for HBV-DNA.

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Discussion

OBI is a worldwide diffused entity, although its distribution may reflect the general prevalence of the HBV in the various geographic areas and in the various populations 12. Carriers of occult infection may be a source of HBV transmission in the case of blood transfusion 13.

Among the 100 blood donors included in this study, 42% were anti-HBc positive. There are two main interpretations of the anti-HBc-only serostatus. The first interpretation is that after many years of HBV chronic carriage with nonproductive infection, the level of HBsAg in the circulation becomes too low to be detected. In a variable proportion of these samples, anti-HBe is detected, but with the titer of anti-HBe being lower than anti-HBc, there comes a point when the former is no longer detectable, leaving the latter as the only serological marker of infection. The second interpretation takes into consideration the relatively brief persistence of anti-HBs in individuals who have recovered from HBV. Over time, anti-HBs in immune individuals becomes no longer detectable and only anti-HBc remains 14,15.

The possibility of persistent HBV infection in anti-HBc-positive individuals has been supported by the fact that traces of HBV are often detectable in the blood for many years after clinical recovery from acute hepatitis 16.

Detection of HBV-DNA from serum or liver samples is considered the gold standard for the diagnosis of OBI. Experts have recommended the use of highly sensitive nested PCR or real-time PCR assays that can detect fewer than 10 copies of HBV-DNA for the diagnosis of OBI. In addition, testing for multiple targets on the HBV genome increases the HBV-DNA detection rates in patients with OBI 15,17.

Because viral DNA levels in OBI are very low, the identification of OBI is strongly dependent on both the sensitivity and the specificity of the assay 18.

Moreover, the technical procedures used so far have differed considerably from one study to another in terms of both specificity and sensitivity and, as a consequence, the results obtained have frequently been contradictory 12,19. The current technologies used for DNA detection are nested PCR, real-time PCR, and transcription mediated amplification. Primers must be specific for different HBV genomic regions and complementary to highly conserved (genotype shared) nucleotide sequences 9,20.

We used a commercial kit (Artus-Biotech) for the quantitative detection of the HBV-DNA viral load using the Taqman probe technique, but HBV-DNA could not be detected using this method. This may be attributed to the small region amplified by the kit (134 bp) with the possibility of the presence of mutations in the primers or probe sequences. Occult HBV strain populations harbor a genetic heterogeneity in viral regions (Pre-S/S, Pre-Core/Core; X, Polymerase) and regulatory elements (Core promoter, Enhancer I and II) potentially involved in viral replication and/or gene expression 18–21.

Our results are not in agreement with those of Allice et al. 22, who reported that HBV Cobas TaqMan real-time PCR seems completely appropriate for exploration of occult hepatitis B because of its high sensitivity and reliability: quantification with an internal control present in each sample, detecting the presence of an inhibitory, automation, and anticontamination system.

However, several studies 23–25 have reported that the use of the Cobas TaqMan HBV PCR assay to screen OBI has not provided information similar to that reported using nested PCR.

The 42 anti-HBc-positive blood donors were tested for HBV genes (S, C, and X) using SYBR green real-time PCR. The core gene was detected in 38 (90.5%) out of 42 blood donors. The X gene could be detected in 30 (71.4%) and the S gene in 42.8% of the donors. HBV-DNA could thereby be detected in 38 (90.5%) out of the 42 anti-HBc-positive blood donors.

The prevalence of serological markers in HBsAg-negative blood donors in different regions of the world was studied. The prevalence in North America showed that HBV-DNA was detected in 0.1–1.05% of those who were HBsAg negative and anti-HBc positive with or without anti-HBs and that HBV-DNA was detected in 2.03–2.8% in the anti-HBc-only category (no anti-HBs) [30–34].

The studies of prevalence in Europe have shown that HBV-DNA was detected in 0–1.59% of those who were HBsAg negative and anti-HBc positive (with or without anti-HBs) 31.

Studies of prevalence in the Middle East and Asia have shown that HBV-DNA was detected in 1.09–3% of those who were HBsAg negative and anti-HBc positive (with or without anti-HBs) and that HBV-DNA was detected in 8.1% in the anti-HBc-only category (no anti-HBs) 32–34.

The higher prevalence of OBI among Sudanese blood donors tested in this study could be attributed to the high prevalence of anti-HBc positivity (42%) and to the real-time SYBR green PCR technique targeting more than one gene.

Increasing attention has recently been focused on the impact of viral load on the evolution of chronic liver disease. The serum viral load correlates with the risk of progression to cirrhosis, and high HBV viremia may favor the development of HCC through a sustained inflammatory activity 21,35.

Occult HBV maintains several of the oncogenic mechanisms of HBV such as the capacity to be integrated in the host’s genome and production of transforming proteins. Therefore, it is conceivable that occult HBV increases the risk for development of HCC. Integration of HBV-DNA could also induce carcinogenesis through transactivation of other oncogenes. Both HBx protein and the truncated pre-S/S protein are potent transactivators and are commonly found in HCC tissue 21,22,35,36. In the present study, the X gene has been be detected in 71.4% of the 42 anti-HBc-positive blood donors.

The usefulness of monitoring liver enzymes and HBV-DNA levels in monitoring OBI has been reported 23,37. This was not the case in the present study as the majority of ALT, AST were in the normal range. Only two blood donors showed slightly elevated levels of AST and one showed elevated ALT.

Our data confirm the possibility of post-transfusion HBV infection as the prevalence of the anti-HBc among our blood donors was 42%, with the presence of HBV-DNA in 38 (90.5%) of these donors.

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Conclusion and recommendations

With the use of HBsAg as the sole detection marker for HBV, there is a danger of HBV transmission through blood transfusion. Anti-HBc testing should be added to the routine blood donor screening test if OBI is to be diagnosed.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

blood donors; occult hepatitis B virus; S, C, and X genes

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