Molecular epidemiology, phylogenetic analysis and genotype distribution of hepatitis B virus in Saudi Arabia: Predominance of genotype D1.

Despite the implementation of various vaccination programs, hepatitis B virus (HBV) poses a considerable health problem in Saudi Arabia. Insight on HBV evolutionary history in the region is limited. We performed a comprehensive epidemiological and phylogenetic reconstruction based on a large cohort of HBV infected patients. Three hundred and nineteen HBV-infected patients with different clinical manifestations, including inactive and active chronic carriers and patients with cirrhosis and hepatocellular carcinoma (HCC), were enrolled in this study. The full-length large S gene was amplified and sequenced. Phylogenetic analysis was performed to determine the genotype and subgenotypes of the isolates. Phylogenetic tree analysis revealed that genotype D is the most dominant genotype among patients. Moreover, this analysis identified two strains with genotype E isolated from active carriers. Detailed phylogenetic analyses confirmed the presence of four HBV D subgenotypes, D1 (93%, n = 296), D2 (0.02%, n = 5), D3 (0.003%, n = 1), and D4 (0.003%, n = 1). In addition, six genotype D strains were not assigned to any existing HBV D subgenotype. The large S gene of eight strains showed signatures of genotype recombination between the genotypes D and A and between D and E. Several strains harbored medically important point mutations at the protein level. Along with the dominance of the HBV genotype D, isolation of the E genotype and several recombinant strains from patients with Saudi Arabian origin is an essential result for decisions involving therapeutic measures for patients. Development of vaccines and detection of diagnostic escape mutations at antigenic epitopes on the HBsAg will be valuable to public health authorities. Furthermore, the diversity at the nucleotide and amino acid levels and different proportions of dN/dS at the PreS1, PreS2, and HBsAg reveal the selective pressure trend from inactive status towards advanced liver diseases.

Association between IL-37 gene polymorphisms and risk of HBV-related liver disease in a Saudi Arabian population.

Interleukin-37 (IL-37) has recently been recognized as a strong anti-inflammatory cytokine having anti-tumor activity against hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected patients. HCC is a typical inflammation-related cancer, and genetic variations within the IL-37 gene may be associated with the risk of HBV infection. Identification of the allelic patterns that genetically have a high disease risk is essential for the development of preventive diagnostics for HBV-mediated liver disease pathogenesis. In this study, we aimed to investigate the association between single nucleotide polymorphisms (SNPs) within the IL-37 gene and disease sequelae associated with HBV infection. We genotyped ten IL-37 SNPs in 1274 patients infected with HBV and 599 healthy controls from a Saudi Arabian population. Among the selected SNPs, two SNPs (rs2723175 and rs2708973) were strongly associated with HBV infection, and six SNPs (rs2723176, rs2723175, rs2723186, rs364030, rs28947200, rs4392270) were associated with HBV clearance, comparing healthy controls and HBV infected-patients respectively. A suggestive association of rs4849133 was identified with active HBV surface antigen (HBsAg) carrier and HBV-related liver disease progression. In conclusion, our findings suggest that variations at the IL-37 gene may be useful as genetic predictive risk factors for HBV infection and HBV-mediated liver disease progression in the Saudi Arabian population.

The Correlation Between Hepatitis B Virus Precore/Core Mutations and the Progression of Severe Liver Disease.

Viral mutations acquired during the course of chronic hepatitis B virus (HBV) infection are known to be associated with the progression and severity of HBV-related liver disease. This study of HBV-infected Saudi Arabian patients aimed to identify amino acid substitutions within the precore/core (preC/C) region of HBV, and investigate their impact on disease progression toward hepatocellular carcinoma (HCC). Patients were categorized according to the severity of their disease, and were divided into the following groups: inactive HBV carriers, active HBV carriers, liver cirrhosis patients, and HCC patients. Two precore mutations, W28* and G29D, and six core mutations, F24Y, E64D, E77Q, A80I/T/V, L116I, and E180A were significantly associated with the development of cirrhosis and HCC. Six of the seven significant core mutations that were identified in this study were located within immuno-active epitopes; E77Q, A80I/T/V, and L116I were located within B-cell epitopes, and F24Y, E64D, and V91S/T were located within T-cell epitopes. Multivariate risk analysis confirmed that the core mutations A80V and L116I were both independent predictors of HBV-associated liver disease progression. In conclusion, our data show that mutations within the preC/C region, particularly within the immuno-active epitopes, may contribute to the severity of liver disease in patients with chronic hepatitis. Furthermore, we have identified several distinct preC/C mutations within the study population that affect the clinical manifestation and progression of HBV-related disease. The specific identity of HBV mutations that are associated with severe disease varies between different ethnic populations, and so the specific preC/C mutations identified here will be useful for predicting clinical outcomes and identifying the HBV-infected patients within the Saudi population that are at high risk of developing HCC.

Phylogenetic and nucleotide sequence analysis of influenza A (H1N1) HA and NA genes of strains isolated from Saudi Arabia.

INTRODUCTION: In early 2009, a novel influenza A (H1N1) virus appeared in Mexico and rapidly disseminated worldwide. Little is known about the phylogeny and evolutionary dynamics of the H1N1 strain found in Saudi Arabia. METHODOLOGY: Nucleotide sequencing and bioinformatics analyses were used to study molecular variation between the virus isolates. RESULTS: In this report, 72 hemagglutinin (HA) and 45 neuraminidase (NA) H1N1 virus gene sequences, isolated in 2009 from various regions of Saudi Arabia, were analyzed. Genetic characterization indicated that viruses from two different clades, 6 and 7, were circulating in the region, with clade 7, the most widely circulating H1N1 clade globally in 2009, being predominant. Sequence analysis of the HA and NA genes revealed a high degree of sequence identity with the corresponding genes from viruses circulating in the South East Asia region and with the A/California/7/2009 strain. New mutations in the HA gene of pandemic H1N1 (pH1N1) viruses, that could alter viral fitness, were identified. Relaxed-clock and Bayesian Skyline Plot analyses, based on the isolates used in this study and closely related globally representative strains, indicated marginally higher substitution rates than the type strain (5.14×10-3 and 4.18×10-3 substitutions/nucleotide/year in the HA and NA genes, respectively). CONCLUSIONS: The Saudi isolates were antigenically homogeneous and closely related to the prototype vaccine strain A/California/7/2009. The antigenic site of the HA gene had acquired novel mutations in some isolates, making continued monitoring of these viruses vital for the identification of potentially highly virulent and drug resistant variants.

Characterization of H5N1 influenza A virus that caused the first highly pathogenic avian influenza outbreak in Saudi Arabia.

INTRODUCTION: Saudi Arabia (SA) experienced a highly pathogenic avian influenza (HPAI) H5N1 outbreak in domesticated birds in 2007. METHODOLOGY: Forty-three hemagglutinin (HA) and 41 neuraminidase (NA) genes of HPAI H5N1 viruses were sequenced and phylogenetic analyses of completely sequenced genes were performed to compare with other viral HA and NA gene sequences available in the public databases. RESULTS: Molecular characterization of the H5N1 viruses revealed two genetically distinct clades, 2.2.2 and 2.3.1, of H5N1 viruses circulating in the area. Amino acid sequence analysis of the HA gene indicated that the virus from 2.2.2 contained the sequence SPQGERRRK-R/G at the cleavage site, while the virus from 2.3.1 contained the sequence SPQRERRRK-R/G. Additionally, a few mutations with amino acid substitutions such as M226I at N-link glycosylation site were identified in two of these isolates. Amino acid sequence of the NA gene showed a 20-amino-acid deletion in the NA stalk region, required for enhanced virulence of influenza viruses and its adaptation from wild birds to domestic chickens. As close contact between humans and birds is unavoidable, there is a need for a thorough understanding of the virus epidemiology, factors affecting the spread of the virus, and molecular characterization such as phylogeny and substitution rates of H5N1 viruses circulating in the region. CONCLUSION: Two genetically distinct clades were found to be circulating in the country, which could likely result in recombination and emergence of more virulent viral strains. These findings could be helpful for the authorities devising control measures against these viruses.

Molecular epidemiology of human astrovirus infections in Saudi Arabia pediatric patients.

Earlier work Tayeb et al. [Tayeb et al. (2008): J Med Virol 80: 1919-1929] set out to study the epidemiology of diarrhea viruses in pediatric populations. The study addressed initially rotavirus, enteric adenovirus, and astrovirus but was later expanded to include norovirus (NoV). Viruses were sought in fecal specimens and characterized for genotype using molecular methods (PCR, RT-PCR, and RFLP) for the first time in KSA. The survey focused on three locations; Jeddah, Makkah, and Riyadh. During the Hajj, the chief population fluxes are via Jeddah to Makkah. One thousand samples were obtained from children (aged 6 years or less) presenting with diarrhea and thus representing community acquired rather than nosocomial infections. Rotavirus was identified in 6% of the samples followed by NoV accounted for 3.5%, astrovirus 1.9%, and adenovirus 1.4%. Rotavirus G9 was characterized for the first time in Saudi Arabia. Adenoviruses were confirmed and further typed using hexon-specific PCR and RFLP. These data were published by Tayeb et al. [Tayeb et al. (2008): J Med Virol 80: 1919-1929]. However, the nature of astrovirus identified was not investigated further. Therefore, more analysis details are appropriate for astrovirus in the Kingdom. As an extension of earlier work carried out on astrovirus serotype distribution in the Kingdom [Tayeb et al. (2008): J Med Virol 80: 1919-1929], a major objective of the project is to use molecular methods to determine the distribution of astrovirus genotype. Such data will help to provide valuable insights into genetic identities and possible sources of virus strains involved not only in pediatric gastroenteritis but also possible outbreaks in the community.

Quasispecies of genotype 4 of hepatitis C virus genomes in Saudi patients managed with interferon alfa and ribavirin therapy.

BACKGROUND AND OBJECTIVES: Many patients with hepatitis C virus (HCV) infection do not respond to antiviral treatment, possibly due to viral quasispecies. We aimed to investigate whether the quasispecies population could be used as a predictor of response to therapy in our patients. METHODS: The quasispecies of HCV genotype 4 (HCV-4) were studied in 25 naïve Saudi patients at zero, three, and six months following interferon alfa and ribavirin combination therapy. Hypervariable region 1 within the E2/NS1 gene of the virus was analyzed by the single-strand conformation polymorphism (SSCP) technique after amplification. RESULTS: Pretreatment DNA bands by SSCP (2-7 bands) were detected in all patients. In those who achieved a complete virological response within six months (viral load P=.53). Two of the four patients with pretreatment high viral load and the same or decreased composition of quasispecies bands responded to the therapy. CONCLUSION: Quasispecies in our studied patients cannot be used to predict responsiveness to treatment, but may offer an explanation for failure of most HCV-4 patients to respond to interferon alfa and ribavirin therapy.

Molecular detection of hepatitis B, hepatitis C, and torque teno viruses in drug users in Saudi Arabia.

Injecting drug users are at increased risk of infection with hepatitis viruses and blood-borne pathogens. The aim of this study was to examine HBV, HCV, HDV, and TTV infections in Saudi drug users (N = 344). Extraction of nucleic acid from serum, reverse-transcription, amplification of viral nucleic acids, and HBV and HCV genotyping were done using established techniques. Of the analyzed samples, 41 (12%) contained detectable HBV DNA, 131 (38%) contained detectable HCV RNA, and 174 (51%) had detectable TTV DNA. The predominant HBV genotype was found to be genotype D and the predominant HCV genotype was found to be genotype 1b. All the samples were negative for HDV. Twelve samples (3.5%) were found to contain mixed HBV and HCV genomes, 24 samples (7%) were found to contain mixed HBV and TTV genomes, 82 samples (24%) were found to contain mixed HCV and TTV genomes, and 9 samples (2.6%) were found to contain mixed HBV, HCV, and TTV genomes. Identification of various infections in drug users will help the control of these infections in this group as well as in the community.

Enteric viruses in pediatric diarrhea in Saudi Arabia.

Between September 1st, 2002 and August 31st, 2003, a panel of 1,000 stool samples was collected from patients presenting with diarrhea in the three major urban centers of Saudi Arabia; Riyadh, Mecca, and Jeddah. Each sample was tested for rotavirus, and astrovirus by ELISA, G and P type was determined for all rotaviruses. Adenoviruses were sought by hexon-specific PCR and identified by RFLP. A subset of 253 samples was also tested for norovirus by ELISA. Data were analyzed for seasonality of infection, patient nationality and likelihood of hospitalization. Although the overall incidence of rotavirus identification in acute diarrheal stool continued to decline, this was still the virus identified most commonly (6%). Norovirus accounted for 3.5%, astrovirus, 1.9% and adenovirus, 1.4%. Type G9 rotavirus was found to be present (and already common) in 2003, predating its first reported identification in the country in 2004. Most of the virus infections (and most of the G9 detections) occurred in April, the month following the occurrence of the Hajj in the study year. Although most viruses were spread equally in the population, rotaviruses were significantly more common in non-Saudis than in Saudi citizens. Overall the data are consistent with an increase in all virus infections following al Hajj and the potential introduction of novel strains (such as the G9 rotaviruses) by pilgrims. Hospitalization was significantly associated only with norovirus infections.

Human papilloma virus-16/18 cervical infection among women attending a family medical clinic in Riyadh.

BACKGROUND: Prevalence information is lacking on human papillomavirus types 16 and 18 (HPV-16/18) infection in cervical tissues of women residing in Riyadh, Saudi Arabia. In addition, there are no observations on progression to cervical intraepithelial neoplasia (CIN). SUBJECTS AND METHODS: Pap smear and HPV-16/18 DNA detection by PCR followed by Southern blotting was performed on 120 subjects (Saudi and other Arab nationals) during routine gynecological examination. Some HPV-positive subjects were followed for 4 years, by Pap smear every 6 months and by HPV DNA detection at the end of 4 years. RESULTS: Overall HPV-16/18 prevalence was 31.6%. HPV-16 prevalence alone was 13.3%, HPV-16 as a mixed infection with HPV-18 was 15%, and all HPV-16 was 28.3%. HPV-18 alone was 3.3%, HPV-18 as a mixed infection with HPV-16 was 15%, and all HPV-18 was 18.3%. Ten subjects had cervical abnormalities with the Pap smear test, six of whom were HPV-16/18 positive, 1 with HPV-16, 1 with HPV-18, and 4 with a mixed infection of HPV-16/18. Of all 23 HPV-16/18-positive subjects, either as individual or mixed infection, followed for 4 years, 7 showed abnormal cytology, 6 at initial examination and 1 during follow-up. Of these 7, 6 reverted to normal without treatment and 1 was treated and became normal after 3 years. None of the subjects progressed to CIN-III. CONCLUSION: A high prevalence of HPV-16/18 was found, but with a low rate of progression to CIN. A significant association with abnormal cytology was found only in patients with HPV-16/18 mixed infection.

Molecular epidemiology and genotyping of TT virus isolated from Saudi blood donors and hepatitis patients.

BACKGROUND: In Saudi Arabia, the epidemiology and clinical significance of Torque Teno virus (TTV) infection alone and in patients with hepatitis virus infections have not been determined in a single study. In this paper, we molecularly investigated the rate and genotypes of TTV infection among Saudi Arabian blood donors and patients with viral hepatitis. The effect of TTV coinfection on viral hepatitis was also examined. SUBJECTS AND METHODS: DNA was extracted from the sera of 200 healthy blood volunteers, 45 hepatitis B virus patients, 100 hepatitis C virus patients, 19 hepatitis G virus patients, and 56 non-A-G hepatitis patients. TTV DNA was amplified using primers derived from the ORF1 and 5'UTR regions. The alanine aminotransferase (ALT) level was determined for each specimen. Sequencing of ORF1 amplicons was carried out to investigate TTV genotypes. RESULTS: Using primers derived from ORF1 and 5'UTR, TTV DNA was detected in 5.5% and 50.5%, respectively, of healthy blood donors, in 2.2% and 88.8% in hepatitis B patients, in 2.0% and 70% of hepatitis C patients, in 15.8% and 100% of hepatitis G patients, in 5.4% and 12.5% of non-A-G hepatitis patients and in 4.8% and 56.4% overall. No detrimental effect of TTV coinfection in viral hepatitis patients was noted. An overall prevalence of 4.8% and 56.4% was established. Phylogenetic analysis indicated that the most common genotype of TTV among Saudis is 2c. CONCLUSION: The rate of TTV infection among Saudi Arabians seems to be lower than that stated in previous reports on Saudi Arabia and in some other countries. The virus does not seem to worsen the status of those who are suffering from viral hepatitis infection.

A comparative study on the application of 3 molecular methods in epidemiological typing of bacterial isolates using MRSA as a prototype.

OBJECTIVE: To evaluate and compare 3 widely used molecular techniques, namely, restriction endonuclease analysis of plasmid deoxyribonucleic acid (REAP), randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for their suitability and usefulness in the typing and fingerprinting of bacterial isolates. METHODS: Twenty-four epidemiologically unrelated methicillin-resistant Staphylococcus aureus (MRSA) isolates were used to evaluate the molecular typing methods (REAP, RAPD and PFGE). The study was conducted at the Research and Diagnostic Laboratories of King Faisal Specialist Hospital and Research Center from January 2002 through January 2003. RESULTS: Only 20.8% of all isolates studied were of the same genotypes by all 3 methods. Two major clusters of strains each representing 33% of the total number of isolates were identified by REAP analysis. Each of RAPD and PFGE however, identified one major cluster represented by 54% and 83% of the total number of isolates, all 3 typing methods, therefore, showed the clonal genetic relatedness among distant MRSA isolates. However inter-strain comparison of fingerprint data generated from each method revealed differences in clonal representation of the MRSA isolates. CONCLUSION: Although a variety of molecular assays are available for typing of bacterial species, there is no single standardized protocol for routine analysis. Reproducibility and interpretation of genotypic data are therefore, highly dependent on methodologies employed by the individual laboratory. Our findings illustrate the importance of using a combination of methods in typing schemes of bacterial isolates. In terms of reproducibility and typeability we found that PFGE is superior to REAP and RAPD and, therefore, more suitable for routine, standardized tracing of nosocomial bacterial isolates.