PREVALENCE OF HEPATITIS C IN DIABETIC PATIENTS: A PROSPECTIVE STUDY

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1 Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 3 pp. 771ñ775, 2016 ISSN Polish Pharmaceutical Society PREVALENCE OF HEPATITIS C IN DIABETIC PATIENTS: A PROSPECTIVE STUDY NOSHEEN KANWAL 1, BUSHRA NASIR 1, MUHAMMAD ASAD ABRAR 1, IRAM KAUKAB 1, AHMAD NAWAZ 2, NAVEED NISAR 1 and GHULAM MURTAZA 2 * 1 Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan 2 Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, Pakistan Abstract: There is a strong evidence of the relationship between diabetes and hepatitis C however, there are certain gaps in the literature. Therefore, this study was carried out to determine the prevalence of hepatitis C in diabetic patients and risk factors associated with it, to evaluate the presence of possible relationship between hepatitis C and diabetes. Serological testing for anti HCV antibody was carried out on a sample of 100 diabetic patients visiting the diabetic clinic Nishtar Medical College and Hospital Multan. An anti HCV antibody test was carried out on HCV ELISA 3.0 (third generation) kit, locally purchased. Data about demographic information and history of risk factors for HCV was collected from diabetic patients using a structured questionnaire as an experimental tool, after taking informed consent. Data of about 100 non diabetic subjects (volunteer blood donors) was taken from the blood bank of that hospital. Prevalence rate of HCV infection among diabetic patients was recorded 19% and in the control group (non-diabetics) was 3%. Prevalence of HCV infection is higher in type 2 diabetic patients as compared to type 1 diabetic patients (84% vs. 16%). Diabetic patients between age group years of age has high prevalence rates (47%) as compared to healthy individuals. Female diabetic patients have higher seropositivity (74%) as compared to male diabetic patients (26%). High prevalence of HCV infection has been reported among diabetic patients with duration of disease = 11 years (47%). Most of the patients were married (95%) and from urban locality (89%) and almost all were poor (99%). HCV positive diabetic patients have also history of blood transfusion (16%), hospital admissions (84%), major surgical procedure (63%), family history of hepatitis C (16%), razor sharing among males (16%) and comb sharing (79%). There was not any I/V drug addict (or history of I/V drug addiction), and tattooing, nose/ear piercing from contaminated needle and toothbrush sharing have not been seen among the participants of research.the results showed that in the present study the prevalence of HCV infection is six times higher in diabetic patients as compared to non-diabetic subjects (control group). Keywords: hepatitis C, anti HCV antibody, diabetic patients, prospective study, risk factors, structured questionnaire, serological testing, seropositivity, blood transfusion Diabetes mellitus is one of the most common, non-communicable diseases worldwide. It is the main cause of heart diseases, stroke, kidney failure, lower limb amputations (non traumatic) and new cause of blindness in adults and seventh main cause of deaths in the United States (1). The prevalence of diabetes is rapidly increasing especially in children and young adults. The number of type 2 diabetic patients is increasing worldwide continuously. Approximately, 80% of the diabetic people live in middle and low income countries of the world. Most of the diabetic patients were between 4-60 years of age. About 183 million diabetic patients were undiagnosed; 4.6 million deaths were caused by diabetes in Every year about children develop type 1 diabetes (2). Today, hepatitis C is the most common blood born disease worldwide (3). Since the discovery of hepatitis C in 1989, it is the major cause of chronic liver disease and will be the substantial cause of mortality in the future. The complexity and uncertainty of hepatitis C relate to its geographical distribution and determination of its associated risk factors that accelerate progression of disease. In developed countries, it is the main cause of liver transplantation, and is the most common chronic blood born infection in the United States. The distribution of hepatitis C is quite variable geographically. The highest prevalence rates of hepatitis C have been reported in the countries of Africa and Asia. Lower prevalence rates have been recorded in the industrialized countries of North and Western Europe, North America and Australia (4-7). * Corresponding author: gmdogar356@gmail.com; phone: ; fax:

2 772 NOSHEEN KANWAL et al. Hepatitis C is both acute as well as chronic liver disease, most of the morbidity is associated with the development of chronic infection after initial acquisition. Determination of onset of HCV infection is difficult as most of the infections are asymptomatic initially (4). The principal objective of this research is to elucidate relationship between diabetes mellitus and hepatitis C. Whether diabetes is a risk factor for HCV infection or not, taking into consideration the significance of other risk factors for hepatitis C in diabetic patients. METHODOLOGY For this descriptive cross-sectional study, nonprobability purposive sampling technique was used. Sample size comprised of 100 diabetic patients as well as 100 healthy subjects (control group). Inclusion criteria comprised of both type 1 and 2 diabetic patients of any age, of either sex. Emergency cases, dialysis patients, transplant recipients and patients who didnít give consent for participation were excluded from study. Sample collection procedure A detailed, structured questionnaire was developed to record demographic and clinical history of patients. Hundred diabetic patients who visited the diabetic clinic Nishtar Hospital Multan were interviewed. Informed consent of every participant was taken before sampling, ensuring them the confidentiality and describing them the objectives of the study. Patients were interviewed for demographic information, duration of diabetes, mode of therapy, hospital admission, intravenous drug addiction, family history of hepatitis C, nose or ear piercing, tattooing, tooth brush sharing, razor sharing, comb sharing and blood glucose level. Sample collection was carried out by using sterile disposable syringes (Becton Dickinson, Pakistan). Approximately 3 ml of peripheral blood of every participant (diabetic patient) was obtained via veinopuncture technique and labeled accordingly. Then, the samples were transferred to the laboratory for analysis. Blood samples of 100 healthy volunteers (control group) visiting blood bank of Nishtar Hospital, Multan were also taken and tested for anti- HCV antibodies. Table 1. Percentages of various parameters. No. of obs. Variables Percentage 1 Type 1 diabetes 15% 2 Type 2 diabetes 85% 3 Male 35% 4 Female 65% 5 Married 95% 6 Unmarried 5% 7 Urban 91% 8 Rural 9% 9 Blood transfusion 17% 10 Hospital admission 66% 11 Major surgery 48% 12 Family history of HCV inf. 21% 13 Toothbrush sharing 0% 14 Comb sharing 79% 15 Razor sharing 8% 16 I/V drug addiction 0% 17 Tattooing 0% 18 Nose/Ear piercing from market 0% 19 HCV positive (diabetic) 19% 20 HCV positive male (diabetic) 26% 21 HCV positive female (diabetic) 74% 22 HCV positive (control group) 3%

3 Prevalence of hepatitis C in diabetic patients: a prospective study 773 Table 2. Distribution of HCV pattern in diabetic patients. No. of Anti HCV antibody Anti HCV antibody Variables observation (positive) (negative) Type of diabetes 1 Type 1 16% 84% Type 2 15% 85% Age group (years) 35 16% 17% % 25% % 41% > 55 5% 17% Gender 3 Male 26% 37% Female 74% 63% Marital status 4 Married 95% 95% Unmarried 5% 5% Locality 5 Urban 89% 91% Rural 11% 9% Duration of diabetes (years) % 45% % 28% 11 47% 27% 7 Blood transfusion 16% 17% 8 Hospital admission 84% 62% 9 Major surgery 63% 45% 10 Family history of HCV 16% 22% 11 Comb sharing 79% 79% 12 Razor sharing 16% 6% Test methodology After collection, blood samples (3 ml) were labeled and transferred to the laboratory for analysis. Each sample was centrifuged at 4000 rpm for 3-5 min. After centrifugation serum was separated from each specimen. These sera were tested immediately in 10 µl of negative control 3 wells, positive control 2 wells and remaining wells for specimens (total 96 microwells). For remaining 9 samples another the same kit was used. Hundred microliters of sample diluent was added to each well through micropipette (Analysis Technologies, USA). Microplate was covered with adhesive sheet and mixed well on vibrating mixer (Vortex Genie, USA). Then, wells were incubated at 37 O C for 30 min. Then, the plate was taken out and washed with 350 µl of diluted washing solution for 5 times (10 s soak time for each wash) and all liquid was aspirated from each well. Subsequently, 100 µl of enzyme conjugate was pipetted in each well. Microwells plate was covered with adhesive sheet and incubated for 30 min at 37 O C. Washing was repeated 5 times with 350 µl of diluted washing solution (10 s soak time for each wash) and all liquid was aspirated from each well and gently mixed the TMB substrate A and B in the ratio of 1 : 1 and 100 µl of the mixed substrate solution was added to each well. Wells were incubated for 10 min at room temperature. Hundred microliters of stop solution was then added to each well and absorbance was measured in the microplate reader (ELISA Reader Dia 710 Diamate, UK) at 450 nm with reference wavelength at 620 nm. A cut off value > 1 was considered reactive (7). Statistics Data analysis was carried out and descriptive analysis was performed. Results were expressed as percentages and mean. The binary logistic regression model was applied, which has given odds ratio (OR), 95% confidence interval (CI) and p-value (> 0.05 was considered significant statistically). Goodness-of-fit test was applied to determine that the applied regression model is the best fit.

4 774 NOSHEEN KANWAL et al. RESULTS AND DISCUSSION The study population comprised of 100 diabetic patients and 100 healthy subjects recruited from the blood bank. The mean age of diabetic patients was ± years and the mean duration of diabetes was 8.27 ± 5.24 years. Higher prevalence of HCV infection was estimated in females (74%) than males (26%). Most of the HCV positive patients were married (95%) and from urban locality (89%). Prevalence of HCV infection increased 47% with the increase in the duration of diabetes as maximum numbers of positive cases were found in the group of patients having 11 years of duration of diabetes. In HCV positive cases 84% patients have history of hospital admission and 16% have a history of blood transfusion, 63% have history of major surgery and 16% have a family history of hepatitis C. About 79% HCV positive patients share comb with others and 16% male patients share razors. Percentage of HCV positive patients in different age groups is given in Table 2 for age group 1 ( 35 yrs), age group 2 (36-45 yrs), age group 3 (46-55 Table 3. Binary logistic regression (Y versus X 1, X 2,...X 15 ). 95% CI Predictor Coefficient SE coefficient Z P Odds ratio Upper Lower Constant X X X X X X * X X X X X X Note 1: Y = Hepatitis C. 1. Coefficient of X 1 (type of diabetes) is 0.098, it means the prevalence of hepatitis C increases by from type 1 diabetes to type 2 diabetes. 2. Coefficient of X 2 (gender) is 0.899, which means the prevalence of HCV is more in females than males. 3. Coefficient of X 3 (age) is 0.016, which means the prevalence of hepatitis C in diabetics is increased by with every unit increase in age. 4. Coefficient of X 4 (marital status) is 1.48, which means the prevalence of hepatitis C is 1.48 greater in married than unmarried. 5. Coefficient of X 5 (residential status) is 0.244, which means the prevalence of hepatitis C is increased by from rural areas to urban areas. 6. Coefficient of X 6 (socioeconomic status) is , which means the prevalence of hepatitis C decreases by from poor to mediocre. 7. Coefficient of X 7 (duration of disease) is 0.043, which means the prevalence of hepatitis C is increased by by unit increases (year) in duration of disease. 8. Coefficient of X 8 (blood transfusion) is -0.57, which means the prevalence of hepatitis C is decreased by 0.57 in patients having no blood transfusion. 9. Coefficient of X 10 (hospital admission) is 1.699, which means the prevalence of hepatitis C is increased by by increasing hospital admissions. 10. Coefficient of X 11 (major surgical procedure) is 0.189, which means the prevalence of hepatitis C is increased by in patients having major surgical procedure during their lives. 11. Coefficient of X 12 (family history of HCV) is , which means the prevalence of hepatitis C decreased by in patients having no family history of hepatitis C compared to patients having a family history of hepatitis C. 12. Coefficient of X 15 (razor sharing) is 2.79, which means the prevalence of hepatitis C is increased by 2.79 by razor sharing in male diabetic patients. Note 2: In column of Odds ratios, the following items have been interpreted: 1. X 1 = type of diabetes OR = 1.10, which means type 2 diabetic patients have 10% more risk to develop hepatitis C than type 1 diabetic patients. 2. X 2 = gender OR = 2.46, which means female diabetic patients have 46% more risk to develop hepatitis C than man diabetic patients. 3. X 3 = age OR = 1.02, which means the risk of developing hepatitis C is increased by 2% with every unit (1 year) increases in age. 4. X 4 = marital status OR = 4.40, which means risk of developing hepatitis C is 40% more in married than unmarried. 5. X 5 = residential status OR = 1.28, which means risk of developing hepatitis C is 20% more in urban population than that of rural population. 6. X 7 = duration of disease OR = 1.04, which means risk of developing hepatitis C is increased by 4% by every unit (1 year) increase in age. 7. X 8 = blood transfusion OR = 0.56, which means the risk of the development of hepatitis C is less likely in those patients with no history of blood transfusion. 8. X 10 = hospital admission O.R = 5.47, which means risk of the development of hepatitis C is increased 5.47 times with hospital admissions. 9. X 11 = major surgical procedure OR = 1.21, which means risk of developing hepatitis C is increased by 21% in patients with history of major surgical procedures. 10. X 12 = family history of HCV OR = 0.53, which means the risk of the development of hepatitis C is increased by 53% in patients having a family history of hepatitis C. 11. X 15 = razor sharing OR = 16.39, which means the risk of the development of hepatitis C increased times by razor sharing in male diabetic patient.

5 Prevalence of hepatitis C in diabetic patients: a prospective study 775 yrs) and age group 4 ( 55 yrs). Moreover, the number of HCV positive patients increased with the increase in the duration of disease and their percentage is given for groups of duration of disease (group 1 (1-5 years), group 2 (6-10 years) and group 3 (11 and more years)). In Table 3, X 15 was significant as its p-value was less than 0.05, it means that razor sharing was significant and all other factors were non-significant. Additionally, the goodness of fit tests of the logistic regression model was applied and it was observed that all the three tests, i.e., Pearson, Deviance and Hosmer-Lemeshow have p-values greater than 0.05 (0.282, 0.551, 0.467, respectively), which shows that the model is a good fit. In this study, it was found that the prevalence of hepatitis C infection in type 2 diabetic patients was higher as compared to control group of healthy blood donors (19% vs. 3%). The results of this study are in agreement with the results of some studies conducted earlier in Pakistan as well as in other countries (6-9). This higher prevalence of hepatitis C in diabetic patients may be the complication of diabetes therapy (10). Furthermore, the prevalence of hepatitis C infection is higher in type 2 than type 1 diabetic patient (84% vs. 16%). Thus, the study establishes type 2 diabetes mellitus (D.M.) as a risk factor for hepatitis C, this study is in agreement with another study conducted earlier in Pakistan (7). Sixteen percent prevalence of HCV infection in type 1 diabetic patients reveals that there could be an association of type 1 D.M. upon the development of hepatitis C infection (8). It may be due to insulin therapy, which may increase the risk of acquiring viral hepatitis due to exposure of the needle (9). It was observed that older patients have higher incidence of HCV infection as compared to those in younger age groups (9). This high seropositivity in the older age group may be due to more parenteral exposures and more chances of transmission of infection as compared to younger age group. It was observed in this study that the risk of developing hepatitis C in diabetic patients increased with the increase in the duration of disease (4). This may be due to complication of diabetes therapy (2). The study also reveals that females have higher seropositivity as compared to males (74% vs. 26%) This result is in agreement with another study conducted earlier in Nigeria (9). It was also evident from this study that certain factors as age, gender, marital status, residential status, history of hospital admission, blood transfusion and major surgery, duration of diabetes, comb sharing, razor sharing are important factors for the development of hepatitis C in diabetic patients. CONCLUSION According to the findings of this study it is concluded that there is a strong association between diabetes mellitus and hepatitis C but it is necessary to evaluate further whether D.M. is a risk factor for hepatitis C or vice versa. It is also clear from this study that certain factors, including female gender, older age, marriage, urban locality, history of blood transfusion, longer duration of diabetes, hospitalization, major surgical procedure, comb sharing, razor sharing increase the risk of developing HCV infection. Anti- HCV antibody and liver function tests should be done immediately after the diagnosis of diabetes and should be repeated periodically during anti-diabetic drug therapy. Special care and attention is required for parenteral therapy of diabetic patients to minimize the risk of HCV infection. It is suggested that this study can be further expanded to monitor liver function tests during anti-diabetic drug therapy to elucidate the development of liver disease which could be due to drug therapy or due to increasing duration of diabetes. Fourth generation ELISA test or PCR test should be done to detect HCV RNA in diabetic patients, which are more sensitive and reliable than 3 rd generation ELISA test for the detection of anti-hcv antibodies. REFERENCES 1. National Diabetes Information Clearing House (NDIC) [diabetes.niddk.nih.gov]. 2. IDF Diabetes ATLAS, 5 th edn., International Diabetes Federation, Brussels Bruce R., Bacon M.D.: Am. J. Managed Care, 46, 927 (2011). 4. Shephard C.C., Findli L., Alter M.J.: Lancet Infect. Dis. 5, 558 (2005). 5. Lakshmi V., Reddy A.K., Dakshinamurty K.V.: Indian J. Med. Microbiol. 25, 140 (2007). 6. Jadoon N.: Virol. J. 7, 304 (2010). 7. Okan V., Araz M., Akhraran S.: Int. J. Clin. Prac. 56, 175 (2002). 8. Ndako J.A., Echeonwn G.O., Shidali N.N.: Virol. J. 6, 98 (2009). 9. Gavin N., Levinthal M.D.: Clin. Diabet. 17, 219 (2009). 10. Chen L.K., Tsai, Y.C., Lee S.D.: Diabetes Med. J. 3, 340 (2005). Received: