PREVALENCE OF DIFFERENT HEPATITIS B VIRUS GENOTYPES AND RISK FACTORS ASSOCIATED AMONG SELECTED YEMENI PATIENTS WITH CHRONIC HEPATITIS B INFECTION
Eman Mohammad Al-Shawkany1, Abdul-Al-Raoof Mohammad AlShawkany2, Saleh S Bahaj1,
Arwa Mohammed Othman1, Hassan A Al-Shamahy1, Azhar Azher Mohammed Al-Ankoshy3
1Medical Microbiology and Clinical Immunology Department, Faculty of Medicine and Health Sciences, Sana’a University.
2Molecular Genetics Department, College of Veterinary Medicine, Sana`a University, Yemen.
3Departement of physiology, Jabir Ibn Hayyan Medical University, Faculty of Medicine/Iraq.
Background and aims: Hepatitis B virus infection is a significant public health crisis global. Hepatitis B virus genotyping is an important tool in epidemiological studies to determine the category and extent of treatment and to predict the outcome of chronic infections, for instance hepatocellular carcinoma and cirrhosis. The study designed to determine the prevalence of hepatitis B virus genotypes among Yemeni patients with chronic hepatitis B (CHB) and to evaluate some of the associated risk factors.
Methods: Fifty patients (38 males, 12 females) with chronic hepatitis B from Al-Thawra Modern General Hospital, Al-Kuwait University Hospital, and AL-Gomhoria Hospital were included. HBV DNA was first detected by conventional PCR then HBV genotypes were determined using nested and multiplex PCR.
Results: Mixed HBV genotypes (A+B+C+D+E), (A+B+C+D+E+F), and (A+B+C+D) were found to be the most prevalent (60 %), it is followed by genotype D (16 %), genotype B (16%) and genotype A (8%), whereas C, E, and F genotype were not found individually among the study population. Blood transfusion was associated with mixed infection (χ2=13.06; p=0.005).
Conclusions: In assumption, this study demonstrates the general prevalence of hepatitis B virus genotypes among HBV-infected Yemeni hepatitis B patients who request medical consideration in a hospital. In mono-genotype HBV infection, genotype B and D were the most prevalent genotypes. In HBV mixed genotype infection, the A/B/C/D/E genotype was the most prevalent in the study area. In the future, based on genotype, clinical trials and treatment regimens must be individually assumed to efficiently manage chronic HBV infection. To this end, a prospective nationwide population study of HBV genotype spreading and clinical outcomes is suggested.
Keywords: chronic hepatitis B, HBV genotype, nested-PCR, prevalence, Yemen.
One of the global public health problem is Hepatitis B virus (HBV) infection. HBV is belongs to the Hepadnaviridae family with a partially double-stranded DNA1. It has been expected that about two billion people global have a proof of past or present infection with HBV and more than 358 million people have chronic lifelong infection and about 887 000 people die every year due to the outcomes of hepatitis B2,3. The endemicity of hepatitis B virus was expected in Yemen, where the prevalence of positive HBsAg in the general population and HCWs ranged from 8% to 20%, among infants, it was 4.1%, and up to 50% of health workers and populations usually had prior serological evidence of Hepatitis B virus infection in old reports. On the other hand, recent studies indicated that the rate of HBsAg, which ranges from 0.7-2% among the general population and to 4% among risk groups such as HCWs, as well as HBV decreased more among children4-11. Though HBV contains DNA genome, it replicates via an RNA intermediate and due to lacking of proofreading activity for spontaneous error of viral reverse transcriptase, nucleotide mutations of HBV genome lead to the occurrence of various genotypes and subtypes4. Currently, ten HBV genotypes (A-J) and twenty-four sub-genotypes (A1–A3, B1–B5, C1–C6, D1–D6 and F1–F4) are reported13. HBV genotypes show a distinct geographic and ethnic distribution. Genotype A is the most commonly distributed genotype in Europe, USA, Canada, Brazil, India, Central African countries, Tunisia and Benin14-16. Genotype B is predominant in Taiwan, Philippines, Japan, Hong Kong, China, Thailand, Indonesia, Vietnam, and USA15-18. Genotype C is prevalent in Australia, Melanesia, Micronesia, Polynesia, Indonesia, China, Hong Kong, Korea, Taiwan, Vietnam, Thailand, Japan, India, Solomon Islands, Brazil and USA. Genotype D is widespread in Mediterranean region, Spain, Czech Republic, Russia, Turkey, Albania, Afghanistan, South Asia, Middle East, Tunisia, Iran, Solomon Islands, Polynesia, Melanesia, Micronesia, Brazil and USA18-20. Genotype E is found endemically in Western Africa while genotype F is widely distributed in new world countries. Genotypes G has been reported from France, and Germany and North America. Genotype H is recorded from Central America, South America and Mexico while Genotype I was isolated in Vietnam and Laos21-23.
Different HBV genotypes are also related with dissimilar clinical phenotypes and prognosis. The rate of chronicity following acute genotypes A and D infection were reported to be high compared with genotypes B and C1, 14-26. Genotype C infection alone has been found to be associated with a significantly higher risk of cirrhosis and hepatocellular carcinoma than genotype B infection27, 28. In contrast, Genotype B infection has a slower progression to liver cirrhosis than genotype C29. In addition, the response to antiviral therapy, mainly to interferon, is related to HBV genotypes. Patients with genotype A have been reported to be more sensitive for treatment by interferon α as compared to those infected with genotype D30. HBV genotype B develops antiviral resistance more than genotype C31. This study aimed to determine the prevalence of different genotypes of hepatitis B virus among selected Yemeni patients with chronic hepatitis B (CHB) and to study the associated risk factors of contracting HBV infection.
SUBJECTS AND METHODS
Fifty patients with CHB were enrolled in this cross-sectional study. According to a random sampling descriptive study, the design and group effect was left equal to one, and the CHB population size equaled 5640 patients who were admitted to the main hospitals in Sana’a city32,33. Since the expected frequency of genotype A or D is 20%, and an acceptable margin of error for the prevalence of different HBV genotypes is 11% according to previous studies in region1,14-26; With a confidence level of 95% we need at least 50 randomly selected samples. The sample size was calculated using Epi Info version 6 (CDC). Patients from Al-Thawra General Modern Hospital, Kuwait University Hospital and Al-Jumhouri Hospital in Sana’a-Yemen from December 2016 to June 2017 were selected from the diagnostic patient lists by systematic random selection (all 10 in the list are from the hospital records).
Inclusion criteria: Both males and Females infected with HBV for more than six months. Their HBs Ag test was positive but their HBc IgM test was negative.
Exclusion patients: Excluded patients were acute hepatitis B (Anti-HBc IgM test was positive), have mixed infected with HBV and HCV, any patient treated with antiviral of HBV, and patients who had liver cirrhosis or hepatocellular carcinoma.
Demographic data were collected at the time of sample collection using a predesigned questionnaire. Also questionnaire included risk factors determinants of HBV infections. Then all statistical analyzes of the data were performed using the Statistical Package for Social Sciences (SPSS) version 24 and Excel 2007. Quantitative data were presented as means and stander divisions whereas nominal data was presented as numbers and percentages. Chi-square test was used for verifying existence of associations. P values≤0.05 were considered statistically significant.
Ethical approval was obtained from the Medical Research and Ethics Committee of the College of Medicine and Health Sciences, Sana’a University with reference number (11) on 14/08/2015. All data, including patient identification, was also kept confidential. A brief explanation of the purpose and importance of the study was given to each participant in order to obtain verbal consent and obtain signature to prevent misunderstanding.
Specimens collection: Five ml venous blood were collected from each patient by the first author. Laboratory work was carried out at National Center of Public Laboratories (NCPHL).Two ml of whole blood was collected in an EDTA tube for detection of HBV- DNA while three ml was put in a plain tube for detection of Hepatitis B surface Ag (HBsAg) and liver enzymes AST and ALT. Demographic data were collected at the time of sample collection using a predesigned questionnaire.
Detection of Hepatitis B surface antigen and anti-HBc-IgM: HBsAg and anti-HBc IgM in patient serum were detected by ELISA method (Closed system-Abbott diagnostic). Samples that were positive for HBsAg and anti-HBc IgM negative were enrolled in this study.
Estimation of the serum levels of alanine and aspartate transaminase: Serum level of AST and ALT were measured using Enzyme kinetics method (kit -AGAPPE, spectrophotometer-Bayer Diagnostic RA-50Clinical chemistry-Ireland).
Determination of HBV- DNA by conventional PCR:
a. Virus DNA Extraction and PCR amplification
DNA of HBV was extracted using AccuPrep® Genomic DNA Extraction Kit (Bioneer, Korea) in accordance with the manufacturer’s instructions. Extracted DNA was stored at -20°C for later analysis. HBV-DNA was then amplified using 1508 bp of P through S genes using universal primers, (FA2F) sense primer was reported by (S1-2) antisense primer that was described by Naito et al, 200134, shown in Table 1 using AccuPower® ProFi Taq PCR PreMix (bioneer Korea-Bio meter system, Germany)34. The PCR program was run for one cycle as: initial denaturation at 94°C for 5 minutes, 35 cycles consisted of denaturation at 94°C for 30 sec, annealing at 57°C for one minute followed by extension at 72°C for 1.5 minutes. The final extension was 72 °C for 5 minutes.
b. Determination of HBV genotypes
Genotyping system was based on nested PCR, using type specific primers for determination of six genotypes A through F of HBV, using (AccuPower® Gold Multiplex PCR PreMix from Bioneer-Bio metar system, Germany). The nested PCR primers were designed based on the conserved nature of the nucleotide sequences in regions of the P through S genes. The genotypes can be determined according to differences in the sizes of amplified DNA, in respective of the six HBV genotypes Table 2. Two nested PCRs were performed in different mixtures for each sample: mix 1 (sit 1) applied for identification of genotypes A, B, C with B2 sense universal primer and mix 2 (set 2) for genotypes D, E, F by B2R antisense universal primer. The nested PCR mixture made of 1μL aliquot of the first-round of PCR product in each of mix A and mix B, 1µl of each type specific primers (10 p mole) and 11µl ddH2O. The nested PCRs were amplified for 40 cycles with the following parameters: initial denaturation at 95°C for 10 minutes, 20 cycles of amplification at 94°C for 20 seconds, 58°C for 20 seconds, and 72°C for 30 seconds, and 20 cycles of 94°C for 20 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. Amplicon products were electrophoresed on a 3% agarose gel, stained with ethidium bromide, and evaluated under UV light.
The mean age of HBV patients was 32.64± 7.67 years. Most of CHB patients (24, 48%) were at age group 20-30 years old and only two (4%) CHB patients at age group >50 years old. Majority of CHB patients were males (38, 76 %) table (3). Distribution of HBV genotypes among study population is shown in table (4). Out of 50 patients, 30 patients (60%) had mixed genotypes, followed equally by genotype B and genotype D (8, 16%) and finally genotype A (4, 8%). Genotypes C, E, and F were not found alone but found in combinations with other genotypes. Mixed genotypes included A+B+C+D+E (20, 66.67%) followed by A+B+C+D+E+F genotypes (6, 20%) and finally genotypes A+B+C+D (4, 13.33%.). The association between HBV genotypes and certain risk factors is shown in table (5). The association between the blood transfusion and HBV genotypes was found to be statistically significant (χ2=13.06; p=0.005). However, the surgical and dental procedures had no association with HBV genotypes in study groups (χ 2= 3.96; p=0.27; χ 2=1.39; p=0.71, respectively).
HBV infection is a significant health problem in Yemen with intermediate to high endemicity of hepatitis B35. HBV genotypes have attracted more attention as they may influence disease progression and outcome of HBV-associated chronic liver disease, in addition to patient's response to antiviral treatments 36. Therefore, this study focuses on evaluating the prevalence of the HBV genotype in Yemen. This molecular genotyping of hepatitis B virus was the first of its kind in Yemen using a polymerase chain reaction (PCR)-based method, and no data on hepatitis B virus genotypes and mutations in hepatitis patients have been previously reported. However, there was a previous study in genotyping of HCV conducted in Yemen37. The small sample size may be an important limitation of this study, but we can justify this for two reasons: first by calculating the sample size using previous data from Yemen and the region, this calculation confirmed that 50 samples could be sufficient to achieve significant results similar to those that might be obtained of a larger sample size; Secondly, the cost of genetic testing was high for Sana'a University, which approved funds to conduct only 50 genetic tests.
The mean age of the studied group is 32.64±7.67 years, which means that the registered patients were born before the implementation of the national program for neonatal hepatitis B vaccination in Yemen. The majority of CHB patients were predominantly men (38, 76%) versus women (12, 24%). The tendency of hepatitis B infection to be more common in males than females may be because males are exposed to risk factors more frequently than females. Other studies from Yemen7-10, Saudi Arabia38, Bahrain20, Rwanda39 and Pakistan40 reported that hepatitis B infection is more prevalent among males than females. The current study revealed that the majority of Yemeni patients with CHB are infected with multiple HBV genotypes. CHB patients with mixed infection had four (A/B/C/D) to six (A/B/C/D/E/F) different HBV genotypes which might indicate co-infection or superinfection with different genotypes. The most common mixed genotypes were A+B+C+D+E while the least common mixed genotypes were A+B+C+D+E+F. Genotypes B, D and A were found to be mono-infection among CHB patients while Genotypes C, E, and F were only found in combinations with other genotypes. This result was similar to that described by Rashid and Saleh41, who found all Iraqi patients in their study had mixed infections42. However, our result differs from that reported from different countries around the world which reported that many patients are affected mainly by one genotype16,42-46. For example, a study from Saudi Arabia, the country bordering Yemen, found genotype D to be the most common genotype among Saudi patients with CHB47. It also differs from that reported in the UAE which reported that many Emirati patients with viral hepatitis are commonly infected with either genotype D or A48. Moreover, the Egyptian study revealed that all CHB patients had genotype D in which sub-D1 genotype was dominant49. With regard to risk factors, blood transfusion was found to be significantly associated with transmission of HBV genotypes in which patients may be exposed to co-infection or super-infection by transfusion of contaminated blood.
No surgical history or dental operation was found to be significantly associated with HBV genotypes infection. A blood transfusion may result in mixed infection in recipients if blood from donors who are carriers of hepatitis B virus are not tested or tested using low-sensitivity laboratory techniques8-10. Shortcoming of this study was the fairly small sample size. Only 50 patients were tested for HBV genotypes, which may not represent the accurate picture of HBV genotypes among HBV patients.
In conclusion, this study demonstrates the general prevalence of hepatitis B virus genotypes among HBV-infected Yemeni hepatitis B patients who seek medical attention in a hospital. In mono-genotype HBV infection, genotype B and D were the most prevalent genotypes. In HBV mixed genotype infection, the A/B/C/D/E genotype was the most prevalent in the study area. Clinical trials and treatment regimens should be hypothesized individually based on genotype to effectively manage chronic HBV infection in the future. To this end, a prospective national population study of HBV genotype distribution and clinical outcomes is recommended. Testing blood donors with highly sensitive tests is also essential to avoid cross-infection and severe infection with hepatitis B virus.
EMA, AMA, AMO, HAA, AA and SSB contributed equally to the design, implementation, statistical analysis and manuscript drafting. All authors read and approved the final manuscript.
Authors are grateful to all patients who participated in this study.
CONFLICT OF INTEREST
There is no conflict of interest with this research.