Correlation Between Serum Hyaluronic Acid Level and Liver Fibrosis in Patients W

Scholars Journal of Applied Medical Sciences Abbreviated Key Title: Sch J App Med Sci ISSN 2347-954X (Print) | ISSN 2320-6691 (Online) Hepatology Journal homepage: https://saspublisher.com/sjams/

“Correlation between Serum Hyaluronic Acid Level and Liver Fibrosis in Patients with Chronic Hepatitis B: A Study in Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh” Md. Al-Mamun Hossain1*, Mobin Khan2, Salimur Rahman3, Mamun-AI- Mahtab4, Md. Kamal5, Md. Kutub Uddin Mollick6

1Assistant Professor, (Hepatology), OSD, DGHS, Deputed Department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh 2Ex. Professor, Department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh 3Ex. Professor, Department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh 4Professor & Chairman, Department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh 5Ex. Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh 6Associate Professor, Department of Hepatology, Khulna Medical college Hospital, Khulna, Bangladesh

DOI: 10.21276/sjams.2019.7.9.22 | Received: 01.09.2019 | Accepted: 12.09.2019 | Published: 20.09.2019

*Corresponding author: Md. Al-Mamun Hossain

Abstract Original Research Article

Introduction: Bangladesh is an intermediate endemic country for hepatitis B, with a huge burden of CHB patients. Early diagnosis and treatment can reduced the adverse sequlae of CHB. The aim of the study was carried out to evaluate the serum hyaluronic acid level as a non-invasive marker of hepatic fibrosis in chronic hepatitis B. Objective: To find out Correlation between serum hyaluronic acid level and liver fibrosis in patients with chronic hepatitis B. Methods: Enrolled patients for study were divided into two groups, Group-1, 40 patients with chronic hepatitis B. Group -2, thirty age and sex matched patients without fiver disease was served as control. Study was done in the department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, during the period of July 2007 to June 2009. We included patients with chronic hepatitis B characterized by, HBsAg positive for more than 6 months, HBeAg positive or negative, serum HBV DNA in PCR (more than 105 copies/ml in HBeAg positive or more than 104 copies/ml in HBeAg negative case), serum ALT normal or elevated and excluded the patients who were decompensated cirrhosis, co-infection with hepatitis C virus, with history of alcoholism, with anti HBV therapy, drug induced hepatitis, sonologic evidence of NAFLD and patients with rheumatological disorder. 5 ml of peripheral blood was taken from each subject in tasting state in complete rest for measurement of serum hyaluronic acid by using the Corgenix HA Test Kit. Liver biopsy was done each patient of group-1 for complete histopathological examination including HAI and Knodell score. Results: Mean age of the healthy subjects was (26.6±6.37) years and of the patients was a (27.67±6.85) years. There were 33% patients were minimal necro inflammation (HAM-3), 50% were mild (HA! =4-8), 17% were moderate (HAI= 9-12). Among the patients stage -1 fibrosis was 56.7% and stage -3 was 43.3%. in our study, Serum HA in CHB was (Mean ± SD), 70.3 ± 42.7 ng/ml and control patients was (Mean ± SD), 18.3 ± 9.0 ng/ml. Mean level of HA in different grades of histological activity index are shown that HA level is higher in moderate HAI score than mild and minimal. We found there was positive correlation between hyaluronic acid and HAI score (P < 0.005). So this test can be done to detect and assess the severity of different grades of histological activity. The mean HA level in stage -1 fibrosis is 38.5 ng/ml and stage -3 fibrosis is 111.95 ng/ml. We found there was highly significant correlation between hyaluronic acid and fibrosis (P < 0.001). Conclusion: There was strong positive correlation between serum hyaluronic acid levels and degree of liver fibrosis Serum hyaluronic acid can be used for assessment of liver fibrosis in patients of chronic hepatitis B during diagnosis and follow up. So it is concluded that hyaluronic acid may be a useful marker of hepatic fibrosis as well as histological activity. Keywords: Hepatitis B virus, serum hyaluronic acid, liver fibrosis, chronic hepatitis B. Copyright @ 2019: This is an open-access article distributed under the terms of the Creative Commons Attribution license which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use (NonCommercial, or CC-BY-NC) provided the original author and source are credited.

NTRODUCTION million people worldwide have chronic HBV infection I [1]. Chronic Hepatitis B (CHB) may be defined as Hepatitis B virus (HBV) infection is a global chronic necro inflammatory disease of the liver caused public health problem. It is currently estimated that 400 by persistent infection with hepatitis B virus [2].

Al-Mamun Hossain et al., Sch J App Med Sci, Sep, 2019; 7(9): 3017-3021

Chronic Hepatitis B can be subdivided into HBeAg hepatitis B characterized by, HBsAg positive for more positive and HBeAg negative CHB. It causes a than 6 months, HBeAg positive or negative, serum spectrum of different diseases ranging from clinically HBV DMA in PCR (more than 105 copies/ml in HBeAg asymptomatic carrier state to the development of positive or more than 104 copies/ml in HBeAg negative cirrhosis and hepatocellular carcinoma (HCC)[3]. case), serum ALT normal or elevated. We were Chronic HBV infection is responsible for 500,000 to 1.2 excluded the patients who were decompensated million deaths every year, and is the 10th leading cause cirrhosis, co-infection with hepatitis C virus, with of death worldwide [4]. It is particularly important in history of alcoholism, with anti HBV therapy, drug the Asian pacific region where the prevalence is high induced hepatitis, sonologic evidence of NAFLD and [5]. In this part of the world, the majority of HBV patients with rheumatological disorder. For group- 1, infection is acquired perinatally or in early childhood. patients presenting with HBsAg-positive was evaluated HBV infection is also a common problem in our by thorough history and physical examination. country mainly affects the younger population and lead Suggestive patients were underwent serum ALT, to the development of cirrhosis and HCC at younger HBeAg, Anti HBe, HBV-DNA (PCR) to diagnose ages [6, 7]. Liver biopsy has been the gold standard to chronic hepatitis B and others investigations like Anti evaluate the histological stage of liver fibrosis and an HCV, USG of hepatobiliary system to fulfill exclusion integral part of management of chronic hepatitis B; but criteria. For group-2, patients without clinical, the procedure is invasive, costly and carries a definite biochemical, serological evidence of liver disease, such risk of occasional complications such as post procedure as patients of irritable bowel syndrome, peptic ulcer pain, haemorrhage and even death[8]. Hence, several disease was taken and evaluated by thorough history non-invasive serological markers have been reported to and physical examination. Suggestive patients were predict the presence of significant fibrosis or cirrhosis underwent serum ALT, HBsAg, AntiHCV, USG of in patients with CHB, among them AST/ALT ratio, hepatobiliary system to exclude liver disease. After AST to platelet ratio index (APRI), and age platelet having consent 5 ml of peripheral blood was be taken count index (API) are based on routine laboratory from each patient of group-1 and group-2 in fasting results [9, 10]. Recently several clinical studies have state in complete rest and send to Dept. of Microbiology attempted to identify serum markers like hyaluronic and Immunology, BSMMU for measurement of serum acid, procollagen-III, collagen-IV, matrix hyaluronic acid by using the Corgenix HA Test Kit. metalloproteinases, tissue inhibitor metalloproteinases Each patient of group-1 was admitted in hospital and and imaging techniques like fibroscan that correlate after informed written consent from the patient liver with the degree of cirrhosis or fibrosis and thus could be biopsy was done and the specimens sent to the used in conjunction or in place of a liver biopsy. department of pathology, BSMMU for complete Among these hyaluronic acid is particular interest [11, histopathological examination including HAI and 12]. Hyaluronic acid is an unbranched high molecular Knodell score without any clinical, biochemical, weight polysaccharide that is widely distributed in the serological and virological information of the patients. extracellular spaces. Part of the hyaluronic acid enters All collected data was analyzed by SPSS version 10. the general circulation via the lymphatic system and is Values were expressed either as mean + SD or in rapidly cleared and degraded mainly in the hepatic frequency or in percentages. Comparison between sinusoidal endothelial cells [13, 14]. A fibrotic liver groups was done by Chi-squire test, student T test and shows both relative and absolute increase in hyaluronic ANOVA test. Pearson test was used for correlation acid. Circulating HA measurement may be helpful in analysis and p value of <0.05 was considered to be differentiating non-cirrhotic from cirrhotic liver, for statistically significant. monitoring liver function and for evaluating the extent of liver fibrosis [15]. ESULTS R The age range of the healthy subjects was 18- OBJECTIVE 50 years and mean age was (26.6±6.37) years. The age To find out Correlation between serum range of the patients was 18-50 years and the mean age hyaluronic acid level and liver fibrosis in patients with was (27.67±6.85) years. The highest incidence of CHB chronic hepatitis B as well as control was found at 20-29 age groups. A male preponderance was observed in 73.3% the study ETHODS population (n=70). Their sex matched in both case and M control. Biochemical profile of CHB patients is shown Enrolled patients for study were divided into the range of ALT was 19-103 (46.7±22.38) U/L and two groups, Group-1, forty patients with chronic AST was 15-130 (44.30±26.65) U/L the range of hepatitis B aged 18 to 50 years. Group -2, thirty age and prothrombin time was 12-16 (13.97±1.25) sec, platelets sex matched patients without liver disease was served count range was (160-450) ×10 9/L. Among the patients as control. Study was done in the department of HBeAg positive was 23.3% and HBeAg negative was Hepatology, Bangabandhu Sheikh Mujib Medical 76.7% and Anti HBe positive was 70% and Anti HBe University (BSMMU), Dhaka, during the period of July negative was 30%. Mean level of HA in different 2007 to June 2009. We included patients with chronic grades of histological activity index are shown that HA © 2019 Scholars Journal of Applied Medical Sciences | Published by SAS Publishers, India 3018

Al-Mamun Hossain et al., Sch J App Med Sci, Sep, 2019; 7(9): 3017-3021 level is higher in moderate HAI score than mild and minimal. Table-3: HA Level in different stages of fibrosis Table-1: Correlation between noninvasive markets (n=70) and fibrosis (N=40) Stages of Hyaluronic Minimum Maximum Variables Mean±SD r-value p-value fibrosis acid Mean ± HA 70.34±42.75 0.866 0.000 SD ALT 40.85±18.73 0.208 0.271 0 18.31 ± 9.020 3.6 46.5 AST 44.3±26.65 0.154 0.416 1 38.53 ± 13.613 20.5 62.5 APRI 0.50±0.41 0.103 0.587 3 111.95 ± 56.2 174.2 PT 13.9±1.2 0.126 0.507 29.344 Platelets count 263.33±66.14 0.059 0.758 Total 44.33 ± 40.333 3.6 174.2 DNA (log) 5.99±2.3 -0.113 0.552

Table-2: Summaries of HA Level in both group of patients (N=40) Group HA Level CHB (ng/ml Control (ng/ml) Mean 70.3 18.3 Minimum 20.5 3.6 Maximum 174.2 46.5 Std. Deviation 42.7 9.0 No 40 30

Fig-2: Correlations of HA with stage of fibrosis

Fig-1: Correlations of HA with grades of HAI score

Table-4: Comparison of Hyaluronic acid between control and two groups of fibrosis patients (N=40) Group Other Group Mean Different of HA between groups (ng/ml) P value Fibrosis stage 0 (control) Fibrosis stage 1 -20.220 0.001 Fibrosis stage 3 -93.636 0.000 Fibrosis stage 1 Fibrosis stage 0 20.220 0.001 Fibrosis stage 3 -73.417 0.000 Data were analyzed using ANOVA (Hochberg test)

There were 33% patients wore minimal necro diagnosis and treatment can reduced the adverse inflammation (HAI=1-3), 50% were mild (HAI=4-8), 1 sequlae of CHB. The study was carried out to assess 7% were moderate (HAI= 9-12) and none were severe usefulness of hyaluronic acid for assessing liver fibrosis (HAI = 13-18). Among the patients stage -1 fibrosis in CHB patients. Serum hyaluronic acid estimation is a was 56.7% and stage -3 was 43.3%. No patients in stage cheap, simple-to-perform, minimally invasive, not -2 or stage-4. The mean HA level in stage -1 fibrosis widely available, needs baseline tests, transfusion was 38.5 ng/mi and stage -3 fibrosis was 1 11.95 ng/ml. facilities, trained personnel for biopsy, follow-ups and pathological support with competent pathologist. Many ISCUSSION patients are reluctant to undergo the invasive procedure. D The CHB patients were included without having any Bangladesh is an intermediate endemic condition like any rheumatological diseases, renal country for hepatitis B, with a huge burden of CHB diseases, antiviral drugs therapy that may cause changes patients, 7.2%-7.5% of population [16,17]. Farly in serum HA. The age range of the patients was 1 8-50 © 2019 Scholars Journal of Applied Medical Sciences | Published by SAS Publishers, India 3019

Al-Mamun Hossain et al., Sch J App Med Sci, Sep, 2019; 7(9): 3017-3021 years and the mean age was (27.67+6.85) years. The ONCLUSION highest incidence, 64% of CHB patients were found at C 20-29 age groups which were similar to previous study The findings obtained from this study suggest done among our people [18, 19]. In this study, among that serum hyaluronic acid is a useful noninvasive CHB patients male were 73.3% and female were market of liver fibrosis. There was strong positive 26.7%. This male preponderance 73.3% was observed correlation between serum hyaluronic acid levels and in the study population which was similar in previous degree of liver fibrosis under the study conditions. study done among Bangladeshi general population [5, Serum hyaluronic acid can be used for assessment of 19]. There were age and sex matched in both case and liver fibrosis in patients of chronic hepatitis B during control. Among the patients HBeAg positive was 23.3% diagnosis and follow up. So it is concluded that and HBeAg negative was 76.7% and Anti HBe positive hyaluronic acid may be a useful market of hepatic was 70% and Anti HBe negative was 30%. fibrosis as well as histological activity. Predominance of HBeAg negative CHB patients was also similar in previous study done in Bangladesh [6, REFERENCE 17, 18]. Among the patients stage-1 fibrosis was 56.7% 1. Alam SH, AHMAD N, Alam KH, Mustafa G, and stage-3 was 43.3%. No patient in stage 2 because MOBIN K. Correlation between hepatitis B viral the histopathological examination was done in Knodell DNA load and extent of liver pathology in patients scoring system in which stage 2 fibrosis was absent. As with chronic hepatitis B. cirrhosis was clinically excluded from the study, so 2. Anna SF, Lok TD, Brain J, McMahon. Chronic stage 4 was absent in this study, even after that hepatitis B, AASLD practices guideline histological stage-4 fibrosis would be possible but Hepatology. 2007; 45:507-539. incidentally this stage-4 fibrosis or cirrhosis was not 3. Zakim D, Boyer TD. Hepatitis B and D: In, A found in any patient. The correlation between fibrosis textbook of Liver Disease. 4th edition. Elsevier with HA, ALT, AST, APRI, PT, platelets count and Science. 2003; 959-974. DNA count were done. The P value found that none of 4. Sherlock S, Dooley J. Chronic hepatitis: general them significantly correlate with fibrosis except HA. In features, and autoimmune chronic disease. this study serum HBV DNA levels did not correlate ______Disease of The Liver and Biliary System. with the fibrosis which was similar in previous study [1, 2002; 11:321-3. 18, 19]. As a result hyaluronic acid is one of the best 5. Liaw YF, Leung N, Guan R, Lau GK, Merican I, noninvasive makers for hepatic fibrosis. Mean level of McCaughan G, Gane E, Kao JH, Omata M, Asian‐ HA in different grades of histological activity index are Pacific consensus update working party on chronic shown that HA level is higher in moderate HAI score hepatitis B. Asian‐Pacific consensus statement on that mild and minimal. We found there was positive the management of chronic hepatitis B: A 2005 correlation between hyaluronic acid and HAI score (r = update. Liver international. 2005 Jun;25(3):472-89. 0.496, P < 0.005) which was similar with previous 6. Alam S, Ahmad N, Mustafa G, Alam K, Khan M. study [15, 21]. So this test can be done to detect and Characteristics of treatment naive chronic hepatitis assess the severity of different grades of histological B in Bangladesh: Younger populations are more activity. In our study, Serum HA in CHB was (Mean ± affected; HBeAg-negatives are more advanced. SD), 18.3 ± 9.0 ng/ml, minimum HA level was 3.6 g/ml Saudi journal of gastroenterology: official journal and maximum 46.5 g/ml. Among CHB patients 40% of the Saudi Gastroenterology Association. 2008 had HA levels more than 75 ng/ml, 30% more than Jan;14(1):15. 100ng/ml. The mean HA level in stage-1 fibrosis is 38.5 7. Khan M, Ahmad N. Seroepidemiology of HBV and ng/ml and stage-3 fibrosis is 111.95 ng/ml which was HCV in Bangladesh. International Hepatology related so previous study. We found there was highly Communications. 1996 May 1;5(1):27-9. significant correlation between hyaluronic acid and 8. Bravo AA, Sheth SG, Chopra S. Liver biopsy. N fibrosis (P < 0.001) [15, 20-24]. Thus the results of our Engl J Med. 2001; 344:495-500. study were comparable with those of other study done 9. Kim BK, Kim SA, Park YN, Cheong JY, Kim HS, earlier in different countries. So serum HA levels can be Park JY, Cho SW, Han KH, Chon CY, Moon YM, used for predicting the degree of fibrosis and also Ahn SH. Noninvasive models to predict liver excluding significant fibrosis, which is very important cirrhosis in patients with chronic hepatitis B. Liver especially in countries with high numbers of hepatitis international. 2007 Sep;27(7):969-76. patients. This study had several limitations. Sample size 10. Wai CT, Greenson JK, Fontana RJ, Marrero JA, was small the number, we could not differentiate all Noninvasive models for predicting histology in fibrosis stages as Metavir scoring and Masson’s- patients with chronic hepatitis B. Liver Trichrome stain was not done. The ALT levels in our international. 2006; 26:666-672. study patients were below two times than normal. 11. Zeng MD, Lu LG, Mao YM, Qiu DK, Li JQ, Wan Further large scale cohort study is recommended to MB, Chen CW, Wang JY, Cai X, Gao CF, Zhou validate these results. XQ. Prediction of significant fibrosis in HBeAg‐ positive patients with chronic hepatitis B by a

Al-Mamun Hossain et al., Sch J App Med Sci, Sep, 2019; 7(9): 3017-3021

noninvasive model. Hepatology. 2005 B in Bangladesh. Hepatobiliary Pancreat Dis Int. Dec;42(6):1437-45. 2008; 7:379-382. 12. Alex Yui Hui, Joseph Jao Sung. Identification of 19. Mahtab MA, Rahman S, Khan M, Kamal MD, Chronic Hepatitis B Patients without Significant Mamun AA, Karim MF. Viral load speaks little Liver fibrosis by a Simple Nonivasive Predictive about toll on liver. Hepatobiliary Pancreat Dis Int. Model. AMJ Gastroenterology 2005; 100:616-623. 2007; 6:483-486. 13. Fraser JR, Laurent TC, Laurent UB. Hyaluronan: 20. Geramizadeh B, Janfeshan K, Saberfiroozi M. its nature, distribution, functions and turnover. Serum hyaluronic acid as a nonivasive market of Journal of internal medicine. 1997 Jul;242(1):27- hepatic fibrosis in chronic hepatitis B. Saudi J 33. Gastroenterol. 2008; 14:174-7. 14. Fraser JR, Laurent TC, Engström‐Laurent A, 21. Khan JA, Khan FA, Dilwas M, Ijaj A, Khan NA, Laurent UG. Elimination of hyaluronic acid from Mehmood T. Serum hyaluronic acid as a market of the blood stream in the human. Clinical and hepatic fibrosis. J Coll Physicians Surg Pak. 2007; experimental pharmacology and physiology. 1984 17:323-326. Feb;11(1):17-25. 22. Yilmaz Ş, Bayan K, Tüzün Y, Dursun M, Kaplan 15. Montazeri G, Estakhri A, Mohamadnejad M, Nouri A, Özmen Ş, Canoruç F, Akkuş Z. Replacement of N, Montazeri F, Mohammadkani A, Derakhshan hystological findings: serum hyaluronic acid for MH, Zamani F, Samiee S, Malekzadeh R. Serum fibrosis, high‐sensitive C‐reactive protein for hyaluronate as a non-invasive marker of hepatic necroinflamation in chronic viral hepatitis. fibrosis and inflammation in HBeAg-negative International journal of clinical practice. 2007 chronic hepatitis B. BMC gastroenterology. 2005 Mar;61(3):438-43. Dec;5(1):32. 23. Plevris JN, Haydon GH, Simpson KJ, Dawkes R, 16. Ahmad N, Khan M. Chronic viral hepatitis update Ludlum CA, Harrison DJ, Hayes PC. Serum on management. J Bangladesh Coll Phys Surg. hyaluronan--a non-invasive test for diagnosing 2002; 20:141-149. liver cirrhosis. European journal of 17. Mahtab MA, Rahman S, Khan M, Karim MF, gastroenterology & hepatology. 2000 Kamal MD. Comparison between HBeAg positive Oct;12(10):1121-7. and HBeAg negative Chronic Hepatitis B in young 24. Chen YP, Feng XR, Dal L, Zhang L, Hou JL. Non- Bangladeshi patients. Bangladesh J Hepatol. 2009; invasive diagnostic screening of hepatic fibrosis in 1:10-14 patients with chronic hepatitis B. Chinese Medical 18. Ahmad N, Alam S, Mustafa G, AdnanABM, Baig Journal. 2004; 11:1109-1112. RH, Khan M. e-antigen-negative chronic hepatitis