AST LEVELS WITH HEPATITIS B VIRUS DNA VIRAL LOAD

Objective:-The present study aimed to correlate the levels of ALT & AST with Hepatitis B Virus DNA Viral load by Real Time PCR in HBsAg Positive Patients Methods: Study conducted on 4927 patients in Meerut, India, which was performed in central research station labortory of Microbiology at netaji subhash Chandra Bose Medical College and Hospital Between November 2016 to April 2018 The sera were separated and screened for HBsAg,HBeAg by ELISA kit and DNA Viral load.PCR Positive Sample were analysed for ALT and AST Results: 245 positive for HBsAg. 55 (1.12%) were HBeAg positive. Out of 55 HBeAg cases, 35 were female and 92 female were negative for HBeAg, this was stastically significant (P< 0.039).In PCR, The results show that in 200 of patients were not detectable serum HBV DNA and 16(16%) were PCR positive, 12.18% were below 2000 IU/mL DNA levels, 1.7% were between > 2000 IU/mL to 20000 IU/mL HBV DNA levels and 2.1 % were >20000 IU/mL HBV DNA levels. ALT was significant (P<0.004) when correlate with DNA viral load. The correlation between AST & ALT was also significant (P< 0.025). Conclusions: The study showed a strong correlation between ALT and HBV DNA viral load.In lowincome countries, the management strategy, using HBsAg, HBeAg and ALT, seems adequate for the confirmation and diffrentiation of Hepatitis B virus inactive, active & chronic hepatitis B patients. Further studies are needed to standardize and improve the management of this group of patients


INTRODUCTION:
The human hepatitis B virus (HBV) is a smallenveloped DNA virus causing acute and chronic hepatitis.Despite the availability of a safe and effective vaccine, HBV infection still represents a major global health burden, with about 350-400 million people chronically infected worldwide and approximately 600,000 deaths per year due to HBV-associated liver pathologies [1].Between 15 and 40% of chronically infected individuals may develop severe liver disease and hepatocellular carcinoma (HCC), while the remaining become inactive carriers.[2].
HBV is present in blood, saliva, semen, vaginal secretions and menstrual blood of infected individuals and easily transmitted through contact with infected body fluids [3].Perinatal vertical transmission is the most common mode of transmission worldwide [4].In households of a chronically infected individual, HBV infection can occur via person-to-person, nonsexual contact [5].
During HBV disease progression, after seroconversion (HBeAg (+) to HBeAg (-), HBeAg consists of two clinical forms; one known as chronic inactive with low persistant aminotransferase levels and HBV DNA levels (≤ 100,000 copies/ml) and second with no HBeAg, high ALT and HBV DNA levels (≥ 100,000 copies/ml).In low-income countries like Pakistan, Nepal, India etc many patients refused to do PCR and liver biopsy procedure due to poverty and cost of these tests.Beside these challenges, the growing concern is the early detection of viral hepatic disease and liver damage.For this purpose, in routine laboratory tests, elevated alanine aminotransferase (ALT) levels are used as indicators of liver cell injury and as non-invasive diagnostic tests [6].Elevated AST levels are usually predominant in liver cirrhosis with increased ALT levels [7,8].During assessment of liver disease due to hepatitis, serum AST and ALT levels are most commonly used serum markers to detect acute and chronic hepatocytes cytotoxicity [9][10][11].Now a days, the main emphasis of workers is early detection of liver damage due to chronic HBV, however, there is always questions about the effectiveness of these test because of their low sensitivity [12,13].Several studies in Italy, China, Korea and Hong Kong showed that ALT levels higher than the normal limits are strongly associated with an increased risk of liver cirrhosis in HBV infected patients [14][15][16][17].Recent studies revealed that in patients with HBeAg (-), high ALT levels greater than 0.5x to the upper limit of normal (ULN) relate to advance fibrosis and ALT > 30 IU/L and 19 IU/L in male and female respectively, [9,10].After HBeAg seroclearance, two disease states, not necessarily static, are possible: Some patients remain in an inactive carrier (IC) state, defined by the European Association for the Study of the Liver (EASL) as fulfilling the following criteria: (1) HBeAg negativity; (2) anti-Hbe positivity; (3) persistently normal ALT (PNALT) levels (<40 IU/mL, with measurements at least every 3-4 months during 1 year); (4) serum HBV DNA levels <2000 IU/mL.At the Management of Hepatitis B workshop in 2000, an arbitrary level of 20,000 IU/mL was adopted as the serum HBV DNA cut-off level distinguishing active and inactive chronic hepatitis.[18] EASL acknowledges that there can be inactive HBV carriers with DNA levels between 2000 and 20,000 IU/mL.HBV DNA levels in HBeAg-negative chronic hepatitis B can fluctuate from undetectable to >2,000,000 IU/mL [19] some inactive carriers occasionally have HBV DNA levels between 2000 and 20,000 IU/mL, a single HBV DNA level between 2000 and 20,000 IU/mL appears to be a ''gray area'' which can correspond to both active CHB or inactive carriers.These studies indicates ALT level as a reliable serum marker leading to fact that HBV natural history can vary from one population to another [20].Thus, the aim of this study was to Correlatation of ALT & AST levels with Hepatitis B Virus DNA Viral load by Real Time PCR as the HBV is increasing in India.

Study background and subjects
This was conducted on 4927 patients.Blood sample were collected in clean, sterile, small test tube from suspected HBV infections and its sequelae patients from Meerut and prosses in central research center labortory of Microbiology at netaji subhash Chandra Bose Subharti Medical

Sample collection and processing
Five milliliter blood samples received in the Serology Section of Department of Microbiology from patients suspected of acute infectious hepatitis were analyzed.The sera were separated and screened for HBsAg by Hepa Card (J.Mitra & Co. Pvt.Ltd.New Delhi, India) and positive serum was stored in frozen (-20 °C) until tested for the viral markers.The positive serum samples for HBsAg by Hepa Card were tested again for HBsAg using commercially available ELISA kit (ERBA Transasia Bio-medicals Ltd.Daman, India).[21] Serum samples tested positive for HBsAg were tested for HBeAg (ELISA; Beijing Kewei Clinical Diagnostic Reagent Inc.

Beijing, China). [22]
DNA isolation from the serum samples was performed using the "QIAamp DNA Mini Kit" (Qiagen, Germany) following the manufacturer's recommendations.[23] The isolated DNA was amplified by Real Time PCR by using artus® HBV RG PCR Kit (Qiagen, Germany) following the manufacturer's recommendations [24] All PCR Positive Sample were analysed for ALT and AST using UV(IFCC) kinetic method by Randox Laboratories Ltd Kit (140 London Wall,London,UK) following the manufacturer's recommendations [25]

Statistical analysis
Obtained data were analyzed by using the SPSS software for windows version 16.The comparison of data in respect of age groups and gender were performed by Z-test and Karl pearson corelation test was used for correlatation of DNA viral load with ALT & AST.P < 0.05 was consider to be statistically significant.

Results
Of the 4927 serum sample, 2218 (45.01%) male and 2709 (54.98%) female were tested for HBsAg with the age range 21 -65.In this study, it was observed that 245 were positive and 4682 were negative.In 245 positive cases 118 were male and 127 were female.In 4682 negative cases 2100 were male and 2582 were female.Highest positive case were found in the age group of 21-30.[Table 1] All 245 HBsAg positive cases was tested for HBeAg and DNA Viral load.HBeAg positive were 55(1.12% ), 20 male were positive for HBeAg and 98 male were negative for HBeAg and Highest HBeAg positive male were found in the age group of 21-30.Out of 55 HBeAg casees, 35 were female and 92 female were negative for HBeAg, this was stastically significant (P< 0.039) by using Z test.[ In recent study by Kim et al. (2011) validate the performance of ALT and HBV DNA, and found that these markers may also used for discriminating patients with HBeAg (-) active carriers from inactive [31].Recent studies showed that for HBeAg (-) patients, low HBV DNA levels are associated with less liver damage although some studies were unable to observe such relationship [32,33].These findings suggest that HBV DNA load and ALT are most convenient techniques to predict active chronic HBV patients.

Ethical approval & Funding
Ethical approval for the study was taken from institutional research ethical committee.
No funding to declare.
College and Hospital Between November 2016 to April 2018.INCLUSION CRITERIA: Age between 21 -65 years with suspected HBV infections and its sequelae patients.Ability to provide written informed consent indicating awareness of the investigational nature of this study.EXCLUSION CRITERIA: If they were received any Immunization for HBV Co-infection:-HBV -HCV, HBV-HIV, HBV -HDV , Liver disease due to other viruses, Alcohols, Diabetics, Autoimmune disease, Immunomodulatory drugs (including systemic steroids, interferons, interleukins, or other cytokines)

Table 2 ]
In

Table 3 ]
All the 16 PCR positive patiants were analysed for AST & ALT to correlate with

Table 4 ]
Tables for, Correlatation of ALT & AST levels with Hepatitis B Virus DNA Viral load by Real Time PCR in HBsAg Positive Patients

Table 1 :
Distribution of Male and Female among HBsAg positive and HBsAg negative patients at different age group

Table 2 :
Distribution of HBeAg in Male and Female among HBsAg +Ve at different age group

Table 2 :
Distribution of HBV DNA viral load at different age group Note: 7 samples was cancel due to low volume/ spoilage of sample

Table 3 :
Corelation of AST & ALT with HBV DNA Viral Load 18% were below 2000 IU/mL HBV DNA levels, 1.7% were between > 2000 IU/mL to 20000 IU/mL HBV DNA levels and 2.1 % were >20000 IU/mL HBV DNA levels.In all the 16 PCR positive Patients, ALT was significant when correlate with DNA viral load but the corelation of AST with HBV DNA viral Load was Nonsignificant, leading to the conclusion that inflammation increases in patients with elevated