Ashrafali Mohamed Ismail

Antiviral Resistance Mutations and Genotype-Associated Amino Acid Substitutions in Treatment-Naïve Hepatitis B Virus-Infected Individuals from the Indian Subcontinent

Background/Aims: Antiviral resistance is a major challenge to the treatment currently available for hepatitis B virus (HBV). In this study, mutations that may affect the antiviral efficacy in treatment-naïve HBV-infected individuals were analyzed. Methods: Ninety-seven treatment-naïve HBV-infected individuals were included in this study. HBV reverse transcriptase (rt) domains were sequenced and nucleotide differences were compared to GenBank wild-type sequences. Furthermore, HBV genotypes, subgenotypes and subtypes were determined by analyzing surface gene sequences. Results: An adefovir-related rtI233V mutation was identified in 4 subjects. The rtS213T lamivudine and entecavir refractory mutant was presented in 3 individuals. Altogether, drug-related, atypical and novel HBVrt amino acid substitutions were seen in 73 positions. The HBV genotypes A, C, D and G were depicted in 15, 21, 60 and 1 individuals, respectively. There were 17 HBVrt amino acid substitutions that are associated with certain genotypes of HBV. Mutations in HBVrt corresponded to established surface gene mutations in 9 patients. Conclusion: This data shows that antiviral-resistant HBV strains do exist in treatment-naïve individuals in this region. Further studies are essential to characterize the role of HBVrt amino acid substitutions in response to anti-HBV therapy.

Performance Characteristics and Comparison of Abbott and artus Real-Time Systems for Hepatitis B Virus DNA Quantification

ABSTRACT Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log10 IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log10 IU/ml and limits of agreement of −0.91 to 1.11 log10 IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.

Association of HLA and TNF polymorphisms with the outcome of HBV infection in the South Indian population

The role of host genetic factors in the pathogenesis and outcome of hepatitis B virus (HBV) infection is not well known. We assessed the association of HLA and TNF (rs361525, rs1800629, rs1799724, rs1800630 and rs1799964) polymorphisms with HBV outcome in the South Indian population. Association of HLA polymorphism was analyzed in 90 individuals from each group, that is, spontaneous recovery (SR) and chronic-HBV (C-HBV) infection. The role of TNF polymorphisms was evaluated in 150 subjects with SR and 137 patients with C-HBV infection. After adjusting for age and sex, HLA-DRB1*07:01 was strongly associated with chronicity (corrected P-value (pc) <0.005, odds ratio (OR) 3.76, 95% confidence interval (CI) 1.84–7.68). The rs1800630 genotype was associated with HBV outcome in codominant (pc<0.01, OR=1.99, 95% CI 1.30–3.05) and dominant (pc<0.01, OR=2.28, 95% CI 1.35–3.84) analyzing models after adjusting for age and sex. Similarly, the rs1799964 genotype was associated with HBV outcome in codominant (pc=0.01, OR=1.57, 95% CI 1.09–2.27) and dominant (pc<0.01, OR=2.21, 95% CI 1.27–3.83) analyzing models. Haplotype analysis (rs1799964/rs1800630/rs1799724/rs1800629/rs361525) revealed that the CACGG haplotype was strongly associated with C-HBV infection (P=0.0004). Our study suggests that inheritance of HLA and TNF polymorphisms might explain the outcome of HBV infection in the South Indian population.

Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy

For hepatitis B virus (HBV)‐related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV‐vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non‐resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs ‘a’ determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence.

Recombination of the epsilon determinant and corneal tropism: Human adenovirus species D types 15, 29, 56, and 69

Viruses within human adenovirus species D (HAdV-D) infect epithelia at essentially every mucosal site. Hypervariable loops 1 and 2 of the hexon capsid protein contain epitopes that together form the epsilon determinant for serum neutralization. We report our analyses comparing HAdV-D15, 29, 56, and the recently identified type 69, each with highly similar hexons and the same serum neutralization profile, but otherwise disparate genomes. Of these, only HAdV-D type 56 is associated with epidemic keratoconjunctivitis (EKC), a severe infection of ocular surface epithelium and underlying corneal stroma. In the mouse adenovirus keratitis model, all four viruses induced inflammation. However, HAdV-D56 entry into human corneal epithelial cells and fibroblasts in vitro dramatically exceeded that of the other three viruses. We conclude that the hexon epsilon determinant is not a prime contributor to corneal tropism.

Molecular epidemiology and genetic characterization of hepatitis B virus in the Indian subcontinent.

BACKGROUND Hepatitis B virus (HBV) is a gradually evolving virus. The aim of this study was to characterize the distribution pattern of HBV genotypes and subgenotypes and HBsAg subtypes in chronic hepatitis B subjects from the Indian subcontinent. We also sought to investigate the genetic diversity of HBV genotypes and its influence on the therapeutic response. METHODS A total of 295 chronic hepatitis B subjects were studied. HBV genotypes and subgenotypes were determined using the generated HBV reverse transcriptase (rt) sequences. HBsAg subtypes were predicted using a newly developed automated program in Microsoft Visual Basic (VB6). Genetic diversity was characterized by calculating the mean genetic distance (d), the number of synonymous substitutions per synonymous site (dS), and the number of non-synonymous substitutions per non-synonymous site (dN). The virological response was measured by HBV DNA levels. RESULTS In southern India, the predominant HBV subgenotype/subtype was D2/ayw3 (79.1%). In eastern India, C1/adr (28.2%) was found to be the predominant subgenotype/subtype, followed by A1/adw2 (25.4%). In the north-eastern region, C2/adr, D2/ayw3, and D5/ayw3 were predominant and were each identified in 20.8% of subjects. In treatment-naïve subjects, the d, dS, and dN of genotype D sequences were higher compared to genotypes C and A. Additionally, the d, dS, and dN of HBV rt sequence were higher in subjects who subsequently showed a virological response to nucleos(t)ide analogues as compared to non-responders, irrespective of the genotypes tested (p=0.014 to p<0.0001). CONCLUSIONS We have described the distribution of HBV genotypes and subgenotypes and HBsAg subtypes in three major regions of the Indian subcontinent. HBV genetic diversity may play a pivotal role in the clinical outcome of chronic hepatitis B.

Low frequency of occult hepatitis B infection in anti-HBc seropositive blood donors: experience from a tertiary care centre in South India.

Dear Sir, Historically, transfusion-associated transmission was the major cause of hepatitis B virus (HBV) disease burden worldwide. Increased awareness of donor screening for hepatitis B surface antigen (HBsAg) has greatly reduced the rate of transmission of HBV. However, blood transfusion in the early acute phase of infection and HBV surface gene variants have justified the need for HBV core antibody (anti-HBc) screening and implementation of nucleic acid testing in donor populations. The residual risk of HBV transmission through blood transfusion is estimated to be 1 in 200,000 to 500,000 and is comparatively higher than that for hepatitis C virus (HCV) and human immunodeficiency virus (HIV)1. Occult infection could be one of the reasons for the relatively high risk of HBV infection. The clinical impact of occult HBV infection among recipients of blood and blood products is not completely known. The majority of anti-HBc positive individuals acquire HBV surface antibody (anti-HBs) through natural clearance of infection or as a result of HBV vaccination. However, an anti-HBs level that clearly precludes the circulation of HBV DNA and disease transmission has not yet been clearly identified. We attempted to address these issues by analysing the seroprevalence of HBV DNA in HBsAg negative, anti-HBc positive healthy blood donors with varying levels of anti-HBs antibody. Blood donors in our tertiary care hospital in south India were recruited in October 2008. Serum samples from these donors were tested for HBsAg, HIV and HCV antibody using Vitros ECI (Ortho-Clinical Diagnostics, Raritan, NJ, USA). A total of 1,300 replacement blood donors negative for these serological markers were investigated for the presence of anti-HBc antibody (Diasorin S.p.A. Saluggia, Italy). All the samples that were confirmed to be anti-HBc positive in Architect Anti-HBc II (Abbott, Weisbaden, Germany) were further tested for anti-HBs levels (AxSYM, Abbott, Weisbaden, Germany) and categorised into three groups according to these levels: anti-HBs negative, anti-HBs 100 mIU/mL. DNA was extracted from 200 μL of plasma using the QIAamp DNA blood MiniKit (Qiagen GmbH, Hilden, Germany) and HBV DNA was quantified using a CE-marked artus® HBV RG real-time polymerase chain reaction (PCR) (Qiagen GmbH, Hilden, Germany) in the Rotor-Gene 3,000 or 6,000 platform (Corbett Research, Mortlake, Australia). The assay targets the 134 bp region of the HBV core gene and the lower limit of detection (LLD) stated by the manufacturer is 20 IU/mL (system 1). Samples were further tested by another sensitive, FDA approved automated nucleic acid system (Abbott RealTime HBV, Weisbaden, Germany) targeting the surface region of the HBV genome. The LLD in this case is 10 IU/mL with a sample input of 500 μL (system 2). The study was approved by the Institutional Review Board of the hospital. The median age of the study donors was 32 (range, 14–65) years and most of the donor were male (89%). Of 1300 samples tested for anti-HBc, 217 (16.7%) were confirmed to be positive. When these anti-HBc positive blood donors were tested for anti-HBs, 86 (39.6%) were anti-HBs negative, 58 (26.7%) had an anti-HBs titre 100 mIU/mL. The available 184 samples from the anti-HBc seropositive blood donors were tested for HBV DNA and all were negative on testing in system 1 (artus® HBV RG PCR). When all these samples were further tested in the automated nucleic acid system 2 (Abbott RealTime HBV), two samples were found to be positive for HBV DNA with a viral load of <10 IU/mL (Figure 1). The overall prevalence of occult HBV in anti-HBc seropositive individuals was thus 1.1%. Figure 1 Flow Chart of the Study. Of the two HBV DNA positive donor samples, the anti-HBs titer of one sample was 63 mIU/mL and the other sample was anti-HBs negative. Previous reports have documented a prevalence of occult HBV of 7.5 to 30% in India2–5. In contrast, here we found a prevalence of 1.1% occult HBV in anti-HBc seropositive healthy blood donors. Such a low rate of occult HBV infection from a region of intermediate endemicity for HBV is noteworthy. The differences in rates of occult HBV seen in this population questions the uniformity of our screening practices. Though most studies have used sensitive nested PCR, HBsAg assays used in the diagnosis of occult HBV are highly varied (Table I). The difference in rates of occult HBV reported across the country may be due to the varying sensitivity of HBsAg assays used for donor screening. Donors may well be HBsAg positive when screened by more sensitive HBsAg assays, hence eliminating them from the category of donors with so-called occult infection. Furthermore, contamination in the nested PCR approach for nucleic acid detection is possible and replicate testing to discriminate true and false positive HBV DNA results is required. Table I Occult HBV infection in Indian anti-HBc seropositve blood donors. In a context of limited resources, anti-HBc can be an affordable marker for diagnosing occult HBV infection. However, there are reports of occult infection in anti-HBc seronegative individuals. There is, therefore, a residual risk of HBV transmission in transfusion settings that employ anti-HBc alone as a supplementary marker for occult HBV screening. Moreover, the use of anti-HBc screening relies on the geographic endemicity of HBV. Screening for anti-HBc and excluding about 20% of anti-HBc positive blood donations without knowing the HBV DNA status will result in a higher discard rate of blood units. Anti-HBs is the antibody protecting against HBV and might serve to neutralise the infectivity of these virions. Earlier studies suggested that blood units with >100 mIU/mL of anti-HBs antibody were safe for transfusion. The two HBV DNA positive donors in our study had anti-HBs titers 100 mIU/mL is not common and may not require further HBV DNA testing. However, different assays used for anti-HBs testing may also account for the variability of anti-HBs titres. Moreover, transfusions cannot be considered solely on anti-HBs levels as there are studies that have shown the presence of HBV DNA in individuals with >100 mIU/mL of anti-HBs antibody. Anti-HBs as a screening assay in blood donors does, therefore, need further evaluation as additional testing of anti-HBs in this setting will add to further cost and delay to the release of blood units. Our attempt to rule out false negative HBV DNA results by using a second real-time system with enhanced sensitivity picked up two samples with a viral load of <10 IU/mL. The estimated LLD (95% detection limit) of this assay as determined by the WHO International Standard for HBV was 1.43 IU/mL (unpublished data). This ensures the reliability of the HBV DNA status reported in this study. The presence of very low HBV DNA levels in blood donors necessitates the need for highly sensitive assays with a LLD of less than 10 IU/mL for the correct diagnosis of occult HBV infection. The lower prevalence of HBV DNA seen in anti-HBc seropositive donors provides indirect evidence that the preponderance of HBV DNA in anti-HBc seronegative donors will be much lower. Although we did not see high rates of occult infection in our setting, nucleic acid testing is still recommended as the residual risk of HBV transmission from healthy donors remains. Follow-up of our two HBV DNA positive blood donations could have helped to understand the clinical significance of these occult infections. In summary, the presence of HBV DNA in anti-HBc seropositive blood donations was low in our setting. The different rates of occult HBV infection reported across the country may be due to the varying sensitivity of HBsAg assays used for donor screening. The implementation of anti-HBc screening in donor populations is limited as excluding isolated anti-HBc blood donations will result in higher discard rates of blood units especially in higher endemicity regions. Nucleic acid testing cannot be exempted as there is a residual risk of HBV transmission in healthy blood donors. Anti-HBc and nucleic acid testing in transfusion settings can be minimised by the use of very sensitive HBsAg screening assays. Larger, multicentre studies are required to understand the actual burden of occult HBV infection in transfusion settings.

Impact of rtI233V mutation in hepatitis B virus polymerase protein and adefovir efficacy: Homology modeling and molecular docking studies

Adefovir is an adenosine analogue approved by the Food and Drug Administration for the treatment of chronic hepatitis B. Mutations occurring in the hepatitis B virus (HBV) reverse transcriptase (rt) domains are shown to confer resistance to antiviral drugs. The role of the rtI233V mutation and adefovir resistance remains contradictory. In this study, it was attempted to evaluate the impact of putative rtI233V substitution on adefovir action by homology modeling and docking studies. The HBVrt nucleotide sequence containing rtI233V mutation was obtained from the treatment-naive chronic hepatitis B subject. The three dimensional model of HBV polymerase/rt was constructed using the HIV-1rt template (PDB code: 1RTD A) and the model was evaluated by the Ramachandran plot. Autodock was employed to dock the HBV polymerase/rt and adefovir. The modelled structure showed the amino acid rtI233 to be located away from the drug interactory site. The substitution of isoleucine to valine did not appear to affect the catalytic sites of the protein. In addition, it does not alter the conformation of bent structure formed by residues 235 to 240 that stabilizes the binding of dNTPs. Therefore, it was predicted that rtI233V substitution may not independently affect the antiviral action of adefovir and incoming dNTP binding.

Association of mannose-binding lectin polymorphisms and HBV outcome in a South Indian population

Mannose binding lectin (MBL) is an important innate immune system pattern recognition molecule. The MBL gene polymorphisms are reported to play a crucial role in outcome of hepatitis B virus (HBV) infection. In this study, we ascertained the association of MBL genotypes with HBV outcome in a South Indian population. The MBL gene polymorphisms at codons 52, 54 and 57 of exon I, and promoter polymorphisms at −221 were typed by polymerase chain reaction‐sequence specific primer in spontaneously recovered and in chronic HBV group. The allele frequency of codon 52 ‘C’ was significantly higher in chronic HBV group than in the recovered group (98.5% vs. 93.6%; P = 0.003) and codon 52 ‘T’ was significantly higher in recovered group than in the chronic group (6.4% vs. 1.5%; P = 0.003). In multivariate analysis, after adjusting for age, sex and state of origin, codon 52 ‘CC’ and ‘CT’ genotypes were significantly associated with chronicity and recovery respectively [odds ratio (OR), 0.25; 95% confidence interval (CI), 0.08–0.80, P = 0.02] in co‐dominant analyzing models. This was re‐affirmed in analysis performed exclusively on Tamil Nadu subjects (OR, 0.23; 95% CI, 0.06–0.93, P = 0.039). The frequency of low/none haplotype (XY/O) was significantly higher in recovered group than in chronic group (15.6% vs 7.5%) and associated with spontaneous recovery (OR, 2.28; 95% CI, 1.04–4.99, P = 0.035). Our results provide preliminary evidence that inheritance of codon 52 genotypes and XY/O haplotype associated with low MBL level substantially determine the outcome of HBV infection in a sympatrically isolated South Indian population.

Genomic analysis of a large set of currently—and historically—important human adenovirus pathogens

Human adenoviruses (HAdVs) are uniquely important “model organisms” as they have been used to elucidate fundamental biological processes, are recognized as complex pathogens, and are used as remedies for human health. As pathogens, HAdVs may effect asymptomatic or mild and severe symptomatic disease upon their infection of respiratory, ocular, gastrointestinal, and genitourinary systems. High-resolution genomic data have enhanced the understanding of HAdV epidemiology, with recombination recognized as an important and major pathway in the molecular evolution and genesis of emergent HAdV pathogens. To support this view and to actualize an algorithm for identifying, characterizing, and typing novel HAdVs, we determined the DNA sequence of 95 isolates from archives containing historically important pathogens and collections housing currently circulating strains to be sequenced. Of the 85 samples that were completely sequenced, 18 novel recombinants within species HAdV-B and D were identified. Two HAdV-D genomes were found to contain novel penton base and fiber genes with significant divergence from known molecular types. In this data set, we found additional isolates of HAdV-D53 and HAdV-D58, two novel genotypes recognized recently using genomics. This supports the thesis that novel HAdV genotypes are not limited to “one-time” appearances of the prototype but are of importance in HAdV epidemiology. These data underscore the significance of lateral genomic transfer in HAdV evolution and reinforce the potential public health impact of novel genotypes of HAdVs emerging in the population.