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Investigation of Prevalence of HBV DNA in the Individuals of Negative HBs Ag in Yazd, Iran

  • Jamshid Ayatollahi
  • Vajihe Kargar ShoorokiEmail author
  • Masoud Doosti
  • Seyed Hossein ShahcheraghiEmail author
  • Faeze Sadat Heidari
  • Sodabe Hemati
  • Zohre Akhoundimeybodi
Medicine
Part of the following topical collections:
  1. Topical Collection on Medicine

Abstract

Occult hepatitis B infection is characterized by negative hepatitis B surface antigen (HBs Ag) and also detectable hepatitis B virus (HBV) DNA. Spontaneous HBs Ag-negative is a rare occurrence in chronic hepatitis B patients. This study aimed to determine the prevalence of HBV DNA in patients that surface antigen hepatitis B virus had lost. During this study, 3320 patients with chronic hepatitis B were followed, of which 1750 patients were men and 1570 patients women. They were HBs Ag-negative patients and AST and ALT levels were normal in them. To isolate HBV DNA, real-time PCR technique was performed, while ELISA technique was used to detect HBs Ag. Data collected and analyzed by SPSS software. Of the 3320 patients with chronic hepatitis B virus, 96 patients—including 62 men (64.5%) and 34 women (35.4%)—had no HBV surface antigen and a significant relationship was observed between HBs Ag clearance process and gender (p value = 0.05). The prevalence of HBV DNA was reported in 29.2% of cases. It is worth noting that between HBV DNA positive and using antiviral drugs, a significant relationship has been observed in patients (p = 0.0001). Our results showed a significant relationship between existence of HBV DNA and drug consumption.

Keywords

Occult hepatitis B HBs Ag-negative HBV DNA ELISA Real-time PCR Antiviral drugs 

Introduction

Hepatitis B virus (HBV) infects almost 2 billion people in different parts from the world, causing nearly 600,000 mortalities yearly because of acute and chronic consequences [1]. The prevalence of occult hepatitis B infection (OBI) is different depending on variable prevalence of infection in societies where the relevant study has been performed, on different samples (liver specimen or serum) to identify HBV DNA, and on different investigations that have been used in the research [2]. In the Middle East, some countries recorded large infection rates for HBV (Oman and Yemen) while some other such as Iran and Pakistan perceived the low rates for it [3, 4, 5].

Blood transfusion increases the risk of infection with pathogens especially HBV [5]. The danger of transfusion-transmitted HBV infection has been decreased by testing all blood donors for HBV surface antigen (HBs Ag) since 1970 [6]. Presently, HBs Ag screening test is a method utilized to decrease transmitted HBV by blood transfusion [6]. It reduces the viral infection rate to almost 7 to 32 per million blood units [6, 7].

The researchers believe that in endemic parts where the occurrence of OBI is a main problem, a better method such as virus nucleic acid tests is needed to screen the blood and blood products used for transfusion [6, 7, 8, 9]. OBI is detected by the presence of HBV DNA in the serum of HBs Ag-negative people with or without HBc Ab positivity [10]. When using available ELISA tests, there is still a potential risk for transmission of infection in the phase of OBI through blood transfusion. Identifying anti-HBc in serum indicates that the donor has been exposed to HBV in the past [11, 12, 14]. If HBs Ag is negative and anti-HBc is positive, some conditions may prevail: (a) remote infection with loss of measurable HBs Ag (occult HBV infection); (b) nonspecific, cross-reacting antibody; (c) the period when HBs Ag has disappeared; and (d) passive transfer of anti-HBc [11, 12, 13, 15, 16, 17].

In this study, HBc Ab was not investigated.

The aim of the present study was to determine the frequency of HBV DNA in the absence of HBs Ag.

Materials and Methods

This cross-sectional study was performed among patients with chronic hepatitis B—their HBs Ag had been negative—who in previous years and in 2016 referred to the infectious diseases part of Yazd medical sciences university and private laboratories of the city. Of the 3320 patients with chronic hepatitis B virus, 96 patients had no surface antigen HBV.

Using the sample size formula (N) and by taking 95% confidence level and according to the prevalence of DNA rate among negative HBs Ag donors between 1 and 3%, the minimum sample size was determined as 96.
$$ N=\frac{\left(z1-\frac{a}{z}+z1-\beta \right)2\times \left[p1\left(1-p1\right)+p2\left(1-p2\right)\right]}{\left(p1-p2\right)2} $$

Demographic data was included during the course of the disease, education level, age, sex, and disease. Qualitative variables were compared with chi-square and T tests. For this study, blood samples were taken from 96 cases who already chronically were with positive HBs Ag but at the moment, HBs Ag is not determinable. A questionnaire containing information such as clinical stage, antiviral therapy, age, and gender was used to collect patient information. It also contains demographic information for each patient, disease condition, radiographic data, and clinical trials.

Extraction of DNA

The High Pure Nucleic Acid Viral Kit (Roche Germany company) was used to extract virus DNA on plasma samples of patients negative HBs Ag in this study. Briefly, 200 μl binding buffer and 50 μl proteinase K with 4 μl poly A were poured in a 1.5-ml tube. Then, 200 μl plasma was added to the top mix. The samples were incubated at 72 °C for 10 min, and then 100 μl binding buffer was added. Centrifuging was performed in 8000 rpm for 10 min. Four hundred fifty microliter wash buffer was added and centrifuged the same as previous. Finally, elution buffer was added and centrifuged. The final extraction was placed at 4 °C for real-time PCR.

Real-time PCR

Real-time PCR test was performed using GeneProof HBV RG PCR Kit with catalog number (HBV / ISEX / 025) for proliferation of a 134-bp region of the genome of hepatitis B.

Before the start, the patient plasma sample and reaction components were brought to environment temperature. For each patient sample, four micro tubes (0.2 ml) for Rotor-Gene, respectively positive and negative control reactions for real-time PCR and positive and negative controls for extraction were placed.

Thirty microliter Master Mix and 2 μl internal controls were added to the tubes. Then, 20-μl patient sample and control was added to them. The sequence of forward primer was 5′-TAT GTT TCC CTC CTG CTG CT-3′ and the sequence of reverse primer was 5′-CCC CCA ACT CCC AAT TCT AT-3′.

ELISA Test

The method used for separating HBs Ag was Direct Sandwich ELISA using ELISA kit related to DiaSorin Company and was performed on samples of patients who during this time lost HBs Ag.

The presence or absence of anti-HBs was determined by comparing patient samples with the cut-off value. If absorbance value of the patient sample was greater than or equal to the cut-off value, the patient was considered reactive. If absorbance value of the patient sample was less than the cut-off value, the patient was considered non-reactive.

Data Analysis

The data will be entered into SPSS vs.22 software. Qualitative data was reported as percentages. T test was used for quantitative information. To investigate the relationship between the prevalence rate of DNA and clinical and demographic characteristics, Spearman’s correlation was used. The significance level was considered less than 0.05.

Results

Of the 3320 patients, 96 cases were HBs Ag negative. Patients who had negative HBs Ag test in two consecutive tests with time space of at least 6 months were considered as true negative and enrolled in the study. In this study, 96 patients with chronic hepatitis B were HBs Ag negative that included 62 men (64.5%) and 34 women (35.4%). The prevalence rate of HBs Ag negative in patients with chronic hepatitis B in between 3320 cases was 2.8%. It was found to be significantly related between HBs Ag negative and gender (p = 0.05) (Table 1).
Table 1

Frequency of HBs Ag negative in patients with chronic hepatitis B based on gender

HBs Ag

HBs Ag negative N (%)

Chronic hepatitis B N (%)

p value

Gender

   

Male

62 (64.5)

1750 (52.7)

0.05

Female

34 (35.4)

1570 (47.2)

Total

96 (100)

3320 (100)

The mean age of patients with chronic HBV was 37.1 ± 10.7, and age-related subjects were divided into four groups: the first group was 20–30 years, the second group was 31–40 years, the third group was 41–50 years, and the fourth group was older than 50 years.

The prevalence of HBs Ag among the age groups showed that there was no significant relationship between HBs Ag negative and age (p = 0.12). It is worth noting that there was not any patient with less than 18 years of age in this study.

Patients with chronic hepatitis B were divided based on consumption of drug into two categories—users and non-users of antiviral medicines. There was a significant relationship between negative HBs Ag and age (p = 0.04) (Table 2).
Table 2

Frequency of HBs Ag negative in patients with chronic hepatitis B based on consumption of drug

Patients

HBs Ag negative N (%)

Chronic hepatitis B N (%)

p value

Antiviral drug

   

Consumption of drug

61 (63.5)

1765 (53.1)

0.04

Non-consumption of drug

35 (36.5)

1555 (46.8)

Total

96 (100)

3320 (100)

There was no significant relationship between existence of HBV DNA and gender (p = 0.7). The average load of HBV in all positive samples was 5.7 ± 2.5 × 104 copies/ml. The lowest loading rate was 2345 copies/ml and the highest loading rate was 87,340 copies/ml.

The nucleic acid of HBV DNA was detected in 28 patients whose frequency distribution based on age groups has been shown in Table 3. There was no significant relationship between existence of HBV DNA and age of the patient (p = 0.81).
Table 3

Frequency of HBV DNA based on age groups

Age group (years)

HBV DNA negative N (%)

HBV DNA positive N (%)

p value

20–30

8 (11.76)

3 (10.71)

0.81

31–40

22 (32.35)

7 (25)

41–50

27 (39.7)

12 (42.85)

> 50

11 (16.17)

6 (21.42)

Total

68 (100)

28 (100)

The nucleic acid of HBV DNA was detected in 28 patients whose frequency distribution based on drug use has been indicated in Table 4. It shows a significant relationship between existence of HBV DNA and drug use (p = 0.0001).
Table 4

Frequency of HBV DNA based on consumption of drug

HBV DNA

HBV DNA negative N (%)

HBV DNA positive N (%)

p value

Antiviral drug

   

Consumption of drug

58 (85.2)

3 (10.7)

0.0001

Non-consumption of drug

10 (14.7)

25 (89.2)

Total

68 (100)

28 (100)

The results of standard samples in different dilutions to determine the load of the virus are shown in Fig. 1.
Fig. 1

Proliferation of standard samples in different dilutions from left to right (10−1–10−5) relatively

The results of testing different positive samples are shown in Fig. 2.
Fig. 2

Proliferation of positive samples

In this study, the samples with Ct value more than 35 were investigated as negative and samples with Ct lower than 35 were considered positive.

Discussion

The prevalence rate of HBs Ag negative in patients with chronic hepatitis B was 2.8% in the present study. It also indicated a significant relationship between HBs Ag negative and gender. It also showed a significant relationship between existence of HBV DNA and drug consumption. There was no significant relationship between existence of HBV DNA and age of the patient. Finally, there was a significant relationship between negative HBs Ag and age.

A study was done to detect the presence of HBV DNA among healthy blood donors that were HBs Ag negative. There were 64 women and 936 men in the study cases. A positive relationship was observed between HBc Ab positivity and age of the donors and also with HBV DNA positivity [18].

Another study was performed to evaluate infection markers with HBV and the potential importance of anti-HBc investigating blood donors to identify infection. The results showed that all blood samples were HBs Ag negative of which 43 (8%) were anti-HBc positive. From those which were positive for anti-HBc, five samples were also positive for HBV DNA [13].

A study was done to identify whether routine anti-HBc testing of blood donations could be an agent for preventing some transmitted HBV infections by blood transfusion. HBV DNA was identified in 4/14 (28.5%) of HBs Ag negative and anti-HBc positive samples. Also, anti-HBs antibody was determined in 2/4 (50%) of HBV DNA-positive samples [19]. In the present study, the prevalence rate of HBs Ag negative in patients with chronic hepatitis B was 2.8%.

Another study investigated the prevalence of OBI in a population from the Brazilian Amazon area, investigation of genotypes and S gene mutations. One hundred eighty-one patients with negative serology for HBs Ag and anti-HBs and negative anti-HBc were present in the mentioned study. HBV DNA was detected in 14.36% of cases. Genotyping showed the A type in 88.46% of HBV DNA-positive patients, with A1 subgenotype that was the most prevalent with 78.26% [20].

Conclusion

It should be noted that in individuals who have been positive for chronic HBs Ag and have recently been negative, this negative result may be due to the low titer of HBs Ag, and if PCR was performed for these patients, it may be positive.

Notes

Acknowledgments

The authors want to thank the staff of Infectious Diseases Research Center of Shahid Sadoughi University of Medical Sciences, Yazd.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Allain JP. Occult hepatitis B virus infection: implication in transfusion. Vox Sang. 2004;86(2):83–91.CrossRefGoogle Scholar
  2. 2.
    Hu KQ. Occult hepatitis B virus infection and its clinical implications. J Viral Hepatitis. 2002;9(4):243–57.CrossRefGoogle Scholar
  3. 3.
    Torbenson M, Thomas DL. Occult hepatitis B infection. Lancet Infectious Dis. 2002;2(8):479–86.CrossRefGoogle Scholar
  4. 4.
    Cacciola I, Pollicino T, Squandrito G, Cerenzia G, Villari D, de Franchis R, et al. Quantification of intrahepatic hepatitis B virus (HBV) DNA in patients with chronic HBV infection. Hepatol. 2000;31(2):507–12.CrossRefGoogle Scholar
  5. 5.
    Nordbo SA, Skaug K, Holter E, Waage A, Brinch L. Reactivation of hepatitis B virus infection in an anti-HBc and anti-HBs positive patient after allogeneic bone marrow transplantation. Europ J Hematol. 2000;65(1):86–7.CrossRefGoogle Scholar
  6. 6.
    Hennig H, Puchta L, Luhm J, Schlenke P, Gocrg S. Frequency and load of hepatitis B virus DNA in firsttime blood donors with antibodies to hepatitis B core antigen. Blood. 2002;100(7):2637–41.CrossRefGoogle Scholar
  7. 7.
    Allain JP. Occult hepatitis B virus infection. Transfus Clin Biol. 2004;11(1):18–25.CrossRefGoogle Scholar
  8. 8.
    Alavian SM, Fallahian F, Lankarani KB. The changing epidemiology of viral hepatitis B in Iran. The J Gastrointestin Liver Dis. 2007;16(4):403–6.PubMedGoogle Scholar
  9. 9.
    Datta S, Banerjee A, Chandra PK, Chakraborty S, Basu SK, Chakravarty R. Detection of a premature stop codon in the surface gene of hepatitis B virus from an HBs Ag and antiHBc negative blood donor. J Clinical Virol. 2007;40(3):255–8.CrossRefGoogle Scholar
  10. 10.
    Aguiar JI, Aguiar E, Paniago A, Cunha R, Galvao L, Daher R. Prevalence of antibodies to hepatitis B core antigen in blood donors in the middle west region of Brazil. Memorias do Instituto Oswaldo Cruz. 2001;96(2):185–7.CrossRefGoogle Scholar
  11. 11.
    Delavari M, Shahabinejhad N, Renzaho A, Zahedi M, Owhadi AR. Frequency of anti-HBc & HBV DNA detection in blood donors of Kerman province, Iran. J Blood Disorders Transfusion. 2011;2(1):1–4.Google Scholar
  12. 12.
    Behbahani BA, Mafi-Nejad A, Tabei SZ, Lankarani KB, Rashidi M. Indication of anti-HBc antibody screening and HBV -DNA detection in diagnosing latent hepatitis B virus infection. The Iran J Med Sci. 2005;30:28–33.Google Scholar
  13. 13.
    Pourazar A, Salehi M, Jafarzadeh A, Kazemi-Arababadi M, Oreizi F, Shariatinezhad K. Detection of HBV DNA in HBsAg negative normal blood donors. Iran J Immun. 2005;2:172–6.Google Scholar
  14. 14.
    Findik D, Arslan U, Baykan M. Determination of hepatitis B virus DNA incidence, viral load, and mutations in blood donors with HBs Ag and anti-HBs-negative serology and antibodies to hepatitis B core antigen. The Europ J Int Med. 2007;18(8):571–5.CrossRefGoogle Scholar
  15. 15.
    Seo DH, Whang DH, Song EY, Kim HS, Park Q. Prevalence of antibodies to hepatitis B core antigen and occult hepatitis B virus infections in Korean blood donors. Transfusion. 2011;51(8):1840–6.CrossRefGoogle Scholar
  16. 16.
    Bhatti FA, Ullah Z, Salamat N, Ayub M, Ghani E. Anti-hepatits B core antigen testing, viral markers, and occult hepatitis B virus infection in Pakistani blood donors: implications for transfusion practice. Transfusion. 2007;47(1):74–9.CrossRefGoogle Scholar
  17. 17.
    Saraswat S, Banerjee K, Chaudhury N, Mahant T, Khandekar P, Gupta RK. Post-transfusion hepatitis type B following multiple transfusions of HBsAg-negative blood. J of Hepatol. 1996;25(5):639–43.CrossRefGoogle Scholar
  18. 18.
    Vaezjalali M, Rashidpour S, Rezaee H, Hajibeigi B, Zeidi M, Gachkar L. Hepatitis B viral DNA among HBs antigen negative healthy blood donors. Hepatitis Month. 2013;13(3):6590.Google Scholar
  19. 19.
    Jafarzadeh A, Kazemi Arababadi M, Mirzaee M, Pourazar AA. Occult hepatitis B virus infection among blood donors with antibodies to hepatitis B core antigen. Acta Med Iran. 2008;46(1):27–32.Google Scholar
  20. 20.
    de Castro Sant’ Anna C, de Almeida MKC, Ferreira P, de Oliveira RG, Ferreira Baraúna AR, Costa Gonçalvez E, et al. Prevalence of occult hepatitis B in a population from the Brazilian Amazon region. J Med Virol. 2018;90(6):1063–70.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Jamshid Ayatollahi
    • 1
  • Vajihe Kargar Shooroki
    • 2
    Email author
  • Masoud Doosti
    • 1
  • Seyed Hossein Shahcheraghi
    • 1
    • 3
    Email author
  • Faeze Sadat Heidari
    • 2
  • Sodabe Hemati
    • 2
  • Zohre Akhoundimeybodi
    • 4
  1. 1.Infectious Diseases Research Center, Shahid Sadoughi HospitalShahid Sadoughi University of Medical SciencesYazdIran
  2. 2.Resident of Infectious and Tropical DiseasesShahid Sadoughi University of Medical SciencesYazdIran
  3. 3.Department of Modern Sciences & Technologies, Faculty of MedicineMashhad University of Medical SciencesMashhadIran
  4. 4.Shahid Sadoughi University of Medical SciencesYazdIran

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