Prevalence of genotype D in chronic liver disease ...

Original Article

Prevalence of genotype D in chronic liver disease patients with occult HBV infection in northern region of India Meher Rizvi*, Mohd Azam*, Asfia Sultan*, Indu Shukla, Abida Malik, Masihur Reman Ajmal1, Fatima Khan, Hiba Sami *Departments of Microbiology and1 Medicine, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University (AMU), Aligarh, Uttar Pradesh, India

*These authors contributed equally to this work. Address for correspondence: Dr. Asfia Sultan, Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University (AMU), Aligarh, Uttar Pradesh, India. E-mail: [email protected]

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Background: Etiology of nearly 30% cases of chronic viral hepatitis remains undetected. Occult HBV infection (OBI) has emerged as an important clinical entity in this scenario. Apart from prevalence and clinical outcome of OBI patients genotype was determined in northern region of India. Materials and Methods: A total of 847 patients with chronic liver disease (CLD) were screened for common viral etiologies and others serological markers of HBV. Amplification of surface, precore and polymerase genes of HBV was performed in patients negative for other etiologies. Genotyping and sequencing of the precore region was performed for OBI cases. Results: Twenty-nine (7.61%) cases of OBI were identifiedof which 9 had chronic liver disease (CHD), 11 liver cirrhosis (LC) and 9 hepatocellular carcinoma (HCC). Majority of OBI cases were detected by amplification of surface gene 26 (89.6%), followed by pre-core gene 12 (41.3%). Their liver functions tests were significantly deranged in comparison to overt HBV cases. IgG anti HBc was present in 8 (27.6%) OBI cases. Mutation was observed in 8 (32%) in pre-core region at nt. 1896 of overt HBV cases. Genotype D was the predominant genotype. In conclusion: OBI in our study was characterized by predominance of genotype D and more severe clinical and biochemical profile in comparison to overt HBV. IgG anti HBc positivity could be utilized as a marker of OBI. We recommend use of sensitive nested PCR for diagnosis of OBI, amplifying at least surface and precore gene. KEY WORDS: CLD, gene amplification, HBV, OBI, surface gene

INTRODUCTION Chronic viral hepatitis is a major global health problem. Chronic HBV and HCV infection account for majority of chronic hepatits cases. However approximately 30% still remain undetected. OBI is being considered as a possible agent of cryptogenic hepatitis. OBI is defined as serologically undetectable hepatitis B surface antigen (HBsAg-ve), despite the presence of circulating HBV DNA.[1] The clinical implications of occult HBV infection are diverse. First of all, OBI poses potential risk of HBV transmission through blood transfusion, hemodialysis, and organ transplantation.[2,3] Second, it may serve as a cause of cryptogenic liver disease, contributing to acute exacerbation of chronic hepatitis B, or even fulminant hepatitis. Thirdly, it may be associated with development of hepatocellular carcinoma. Fourth, it may affect disease progression and treatment response of chronic hepatitis C. Implementation of HBV DNA screening has the potential to significantly reduce the ‘‘window period” (WP) and to reveal OBI.[4] The availability of PCR assays for HBV DNA allows the detection of 102 copies/mL compared with 106 copies/mL using hybridization INDIAN JOURNAL

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assays. Sensitive HBV DNA amplification assay is the gold standard assay for detection of OBI.[5] OBI varies significantly between different geographical regions. [2] Studies have shown that the prevalence of occult HBV infection is closely related to the endemicity of HBV infection.[6] Prevalence rates for OBI ranging from less than 1% to as high as 87% have been reported from different parts of the world.[7] Many studies have focussed on prevalence of OBI in healthy blood donors. Indian studies have shown 18.9% prevalence of OBI in healthy blood donors.[8] Data on OBI in south-east Asian countries in CLD patients in particular are sparse. We assessed the prevalence, clinical outcome and genotype of OBI in CLD patients negative for A-E viral hepatitis. MATERIAL AND METHODS The study was conducted over a period of two and a half years from August 2008 to July 2011 in the Departments of Microbiology and Medicine, J.N. Medical

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Rizvi, et al.: Occult HBV infection in chronic liver disease patients

College, A.M.U., Aligarh. Eight hundred and forty seven consecutive patients with hepatitis attending the Hepatitis clinic or admitted in the Medicine wards of Jawaharlal Nehru Medical College were screened for viral hepatitis. All patients underwent complete physical examination and detailed clinical history was elicited from them. The study was approved by the institutional ethical committee of J.N. Medical College, AMU, Aligarh. A written informed consent was obtained from each patient.

Detection of OBI Anti-HBcIgG positive and all cases of unknown etiology were subjected to amplification for HBV surface, pre-core and polymerase genes. Liver biopsies were not performed.

Exclusion criteria Patients with autoimmune hepatitis, alcoholic hepatitis, and drugs induced hepatitis, patients giving history of recent infection, surgery, trauma within the preceding 2 months, renal insufficiency or with other acute or chronic inflammatory diseases were excluded from the study. None of the participants had received any antiviral or immunosuppressive therapy before or during the course of the study.

Amplification of surface and pre-core gene of HBV Surface gene Outer PCR was performed using 2× Master Mix (Fermentas) with forward primer HBMF1; 5′-YCCTGCTGGTGGCTCCAGTTC-3′ and reverse primer HBMR2; 5′-AAGCCANACARTGGGGGAAAGC-3′ while nested PCR was performed using HBMF2; 5′-GTCTAGACTCGTGGTGGACTTCTCTC-3′ and HBMR2 primers (OPERON Biotechnologies, Cologne, Germany). The cycling condition for both first and second round of PCR were as follows: 96°C for 2 min for initial denaturation followed by 25 cycles of 94°C for 15 sec., 60°C for 45 s and 72°C for 45 s followed by a final extension at 72°C for 7 min on thermocycler (LABNICS, USA). Nested PCR amplified 485 bp fragments.

Collection of specimen Venous blood samples (5-10 ml) were obtained after taking informed consent from hepatitis patients. Serum was separated by centrifugation and stored at −40°C until further study. Routine investigations Serum amino alanine transaminase (ALT), serum aspartate amino transferase (AST) and alkaline phosphatase (ALP), bilirubin (direct and indirect) total bilirubin, albumin, globulin, creatinine and international normalized ratio for prothrombin time were performed. Specific investigations like ultrasonographic examination of liver, upper GI endoscopy and liver biopsy were performed wherever feasible. Primary screening 2347 patients were screened for HAV, HBV, HCV, HEV and HIV by commercially available ELISA kits according to the manufacturer’s instruction using the following kits: Third generation HBsAg and anti-HCV(J. Mitra& Co. Pvt. Ltd., India), fourth generation anti-HIV (J. Mitra & Co. Pvt. Ltd., India), antiHAV IgM and anti HEV IgM (DRG International, Inc., USA). ELISA was performed as per manufacturer’s instructions. Eight hundred forty-seven patients were diagnosed with chronic liver disease (415 chronic active hepatitis, 238 liver cirrhosis and 194 hepatocellular carcinoma). Of these, 385 had overt HBV infection, 73 (9.7%) were positive for HCV and 18 (3.5%) were HIV positive. A large number, 371 cases still remained unidentified. Secondary screening The unidentified cases as well as chronic HBV positive patients were screened for other HBV serological markers (anti HBcIgM, anti HBc total, HBeAg, anti HBe) by ELISA (DRG International, Inc., USA, Bio-Rad, France and Orgenics, Israel). ELISA was performed as per manufacturer’s instructions. 538

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Extraction of HBV DNA HBV DNA of all probable occult and 50 HBV positive cases were extracted from the serum samples using the standard phenolchloroform -isoamylalcohol method.[9]

Pre-core gene PCR was performed using 2× Master Mix (Fermentas) with forward primer 5′-TA (C/T) TAG GAG GCT GTA GGC AT-3′ and reverse primer5′-AGA ATA GCT TGCCTG AGT GC-3′ (OPERON Biotechnologies, Cologne, Germany). The cycling condition were as follows: 94°C for 3 min for initial denaturation followed by 39 cycles of 94°C for 30 s, 58°C for 50 s and 72°C for 50 s followed by a final extension at 72°C for 5 min to amplify a 321-bp fragment. Polymerase gene Outer PCR was performed using 2 × Master Mix (Fermentas) with forward primer (Outer PCR) HB1; 5′-ATCGTGGATGTGTCTGCGG-3′ and reverse primer HB2; 5′-GGCAACGGGGTAAAGGTTCA-3′ while nested PCR was performed using HB3; 5′-CATCTTCTTGTTGGTTCTTTCTG-3′ and HB4; 5′-TTAGGGTTTAAATGTATACCC-3′ (OPERON Biotechnologies, Cologne, Germany). The cycling condition for both outer and inner PCR were as follows: 96°C for 2 min for initial denaturation followed by 35 cycles of 94°C for 15 s, 58°C for 45 s and 72°C for 45 s followed by a final extension at 72°C for 7 min. Nested PCR amplified 415-bp fragment. The amplified products were electrophoresed on a 1.5% agarose gel stained with ethidium bromide and evaluated by gel doc (Bio Rad, USA). DNA sequencing of amplified products of pre-core gene was done by Macrogen, Inc. South Korea using commercially available Big Dye® termination kit. Twenty five HBeAg negative overt HBV andallOBI cases were sequenced.



Genotyping Genotyping was performed as described previously by Sami, et al.[10] Statistical analysis Statistical analysis was performed with the IBM SPSS Statistics 19. Results were expressed as means ± standard deviation or as percentages. Means were compared between groups by using the t-test, ANOVA (one way analysis of variance) and frequency distributions were compared by using the chi-square test. RESULTS Occult hepatitis B virus infection Twenty-nine (7.61%) cases of OBI with 95% confidence interval (CI) being +/- 2.61were detected. Among these, 18 (62%) were males while 11 (38%) were females. Mean age of these patients was 53 ± 6 years. A slight decline in the age of patients with occult HBV infection was observed.

patients polymerase gene was amplified. Nine cases were detected by both surface and pre-core primer sets while in 17 cases only surface gene was amplified and in 3 cases only pre-core gene was amplified. Clinical profile Distribution of overt and occult HBV in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma are given in Table 1. A larger percentage of OBI patients presented with HCC and LC as compared to patients with overt HBV infection. Among the 29 OBI patients, 9 (31%) had chronic active hepatitis (CH), 11(37.9%) had liver cirrhosis (LC) and 9 (31%) had hepatocellular carcinoma (HCC). Among 385 patients with overt HBV infection, 89 (23.1%) had CH, 75 (19.4%) had LC, 53 (13.7%) had HCC while 168 (43.6%) patients went into remission.

Majority of the OBI cases 26 (89.6%) were identified by amplification of surface gene [Figure 1] while 12 (41.3%) cases were identified using pre-core primers [Figure 2]. In 2 (6.8%)

Biochemical profile Total bilirubin levels were more deranged in OBI patients as compared to overt HBV cases. There was 6- to 8-fold rise in liver enzyme levels in patients with OBI as shown in Table 1.

Figure 1: Amplifica on of 485 bp product of surface gene in lane 3 and 4 obtained by nested PCR

Figure 2: Amplifica on of 321 bp product of precore gene in lane 1, 2 and 3 obtained by reverse and forward primer PCR

Table 1: Comparison of clinical profile of pa ents with Overt vs. Occult HBV Pa ent Profile No. of pa ents Mean age Gender: M/F HBsAg HBcAbIgM HBcAb (Total) HBe Ag HBeAb Total bilirubin mg/dl Direct bilirubin mg/dl ALT IU/L AST IU/L Prothrombin % of standard

CHB (n = 415) Overt HBV % OBI % n=89 (21) n=9 (2.1) 51 44 51/38 5/4 89 (100) 0 (0) 0 (0) 0 (0) 89 (100) 4 (44.4) 39 (43.5) 0 (0) 41 (46.7) 0 (0) 34 45 17 22 60 272 169 276 83 82

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LC (n = 238) Overt HBV % OBI % n=75 (31.5) n=11(4.6) 57 52 39/36 7/4 100 0 (0) Not done 0 (0) 75 (100) 2 (18.2) 21 (28) 0 (0) 47 (62.6) 0 (0) 61 67 32 39 117 167 67 95 55 63

HCC (n = 194) Overt HBV % OBI % n=53 (26.7) n=9(4.6) 65 63 41/12 6/3 100 0 (0) Not done 0 (0) 53 (100) 2 (22.2) 19 (35.8) 0 (0) 30 (56.6) 0 (0) 39 75 21 41 118 294 89 115 77 89


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Serological profile of patients Anti HBc was present in all chronic HBV patients. OBI patients were negative for IgM anti HBc, anti HBe, HBe Ag whileIgG anti HBc were present in 25.96% cases. Among the overt HBV group HBeAg was present in 35.79% of patients while anti HBe was present in 55.33% patients as seen in Table 1. Genotyping and DNA sequencing of pre-core gene region of HBV Genotype D was detected in 24(86%) of OBI cases. Precore mutation was observed in 8 cases at nt. 1896 of overt HBV cases. No mutations were observed in occult cases [Figure3]. DISCUSSION OBI is a challenging clinical entity. [5] It is recognized by two main characteristics: Absence of HBsAg, and low viral replication. HBV contains four open reading frames: The S gene, coding for the envelope proteins; the core gene, coding for the core and precore proteins; the P gene, coding for DNA polymerase; and the X gene, coding for a transcriptional transactivator.[11] Recent observations have suggested that the lack of HBsAg in OBI may be due to rearrangements in the HBV genome that interfere with gene expression or lead to the production of an antigenically modified S protein. [12] We undertook this study to assess the prevalence of OBI, its clinical implications and genotyping in CLD patients from Aligarh region of North India. HBV DNA was detected in 29 (7.61%) cases of HBV, HCV and HIV negative CLD individuals. The prevalence of OBI in our study was 7.6% which is in consonance with the intermediate level of endemicity of HBV in India. Alavian, et al. 2012 reported 5-55% prevalence of OBI among chronic HBV carriers.[13] Studies outside India have reported OBI prevalence of as high as 22% to as low as 1.9% in chronic hepatitis patients.[14,15]

Figure 3: Muta on in precore gene of HBV DNA at nt 1896 is observed 540

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In our study, HBV DNA was more frequently identified in patients with cirrhosis while it was identified more often in chronic hepatitis in other studies.[16] Our findings also suggest that OBI patients had higher incidence of LC (39.2%) and HCC (31%). A limitation of the study is that liver biopsies were not performed, as the occult status was determined much later. On analysing the prevalence of OBI in different chronic liver disorders, OBI was observed in 4.6% of LC and HCC cases each. Chung, et al. found HBV DNA in 28% of HBsAg-negative cirrhotic patients.[17] Shiota 2000, reported the detection of OBI in 69% of HCC patients which was much higher than in our study. However, their sample size was low.[18] Hasegawa, et al. 2005 reported a low correlation in which only one out of seven HCC cases were found to have OBI.[19] Occult HBV and its potential oncogenicity are traditionally considered a consequence of the capacity of the virus to be integrated into the host genome, although free episomal HBV genomes may persist in the liver cells during occult infection.[20] However, further genetic studies are needed to understand why patients progressed to LC and HCC despite the low viral load. Host responses may also play some role in progression of disease which have to be analyzed in future studies. The authors are analysing the role of Th1 and Th2 cytokines in the immunopathogenesis of OBI (unpublished data). In this study total bilirubin levels and PT INR values were significantly deranged in OBI patients as compared to overt HBV cases. Flares of hepatic transaminases has been reported to occur more frequently in patients with OBI than in patients who tested HBV-DNA negative.[21] In our study 27.6% of OBI cases were positive for anti HBc antibodies which was higher than in similar studies from Pakistan and Egypt who reported 17.2% and 21.47% positivity, respectively.[22,23] Studies in India have also reported varied prevalence of anti HBc ranging from 10.82% to 18.9%.[8,24] Majority of OBI cases in our study were detected by amplification of surface gene (89%), followed by 41.37% by pre-core gene and 6.89% by amplification of polymerase gene. Thus OBI need not necessarily be due to mutation in HBsAg. Amplification of surface gene alone would have resulted in missing 11% cases. Sensitivity of OBI detection increased when combined amplification of surface gene and pre-core gene was done. Polymerase gene amplification did not further increase the sensitivity. A surprisingly high mutation rate of 32% was observed in the precore region at nt. 1896 of HBeAg negative overt HBV cases. Other studies have also reported higher frequencies of precore mutations in chronic HBV which correlated with severe clinical outcome such as HCC.[25] It is probable that multiple precore or core mutations are required to increase viral virulence.[26] These mutations may alter the core protein, decreasing immune recognition by cytotoxic T cells which is a key mode of viral clearance, hence contributing to immune escape. Strangely no mutation was observed in occult cases. This suggests that OBI in our study were not due to mutations in the precore region. Low viral load may be the reason



for development of OBI in our study. The relatively more severe clinical progression of disease in OBI in this study may be due to a greater derangement in host immune response. Several host immune responses such as apoptosis, cytolytic and non cytolytic T cell responses have been linked to modulation of HBV replication and HBV protein synthesis in OBI.[27] Genotype D was the predominant genotype in OBI cases. This mirrors the predominance of genotype D in this region.[10] Moreover genotype D has been reported to be more virulent than genotype A.[28] However, further research is needed to shed light on the role of both HBV genotype D and the host immune response in the increased virulence of virus in OBI. More basic research may answer these questions. CONCLUSION

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We recommend use of sensitive nested PCR for diagnosis of OBI which can detect as low as 104 copies/ml of HBV DNA. It was observed that testing for multiple targets on the HBV genome increases the sensitivity for detection of OBI. In our study, a poorer clinical profile of OBI cases was observed in relation to overt HBV cases. However, specific guidelines for the management of OBI are yet to evolve. Further work is needed to clarify the clinical significance of OBI, its pathogenic consequences and progression to cirrhosis and hepatocellular carcinoma. ACKNOWLEDGEMENT

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This work was supported by grants from Department of Science and Technology (DST), Ministry of Science and Technology, India.

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How to cite this article: Rizvi M, Azam M, Sultan A, Shukla I, Malik A, Ajmal MR, et al. Prevalence of genotype D in chronic liver disease patients with occult HBV infection in northern region of India. Indian J Pathol Microbiol 2014;57:537-41. Source of Support: This work was funded by grants from Department of Science and Technology (DST), Ministry of Science and Technology, India. Conflict of Interest: None declared.


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