The hepatitis C virus (HCV) is a single-stranded RNA virus. It is classified under the genus Hepacivirus and the family Flaviviridae. A lipid membrane is maintained by two or more envelope proteins (E). The envelope encases the nucleocapsid, composed of several copies of a tiny fundamental protein known as core or C, which contains the RNA genome.(1) A critical aspect of hepatitis C is its potential to result in chronic liver disease, such as cirrhosis, ultimately culminating in hepatocellular carcinoma (HCC).The primary mode of transmission for the hepatitis C virus is via intravenous exposure. Chronic HCV infection is quite probable; 80–100% of individuals who get an initial infection retain HCV.Up to 85% of individuals infected with the hepatitis C virus exhibit no signs of chronic hepatitis.Preliminary clinical findings indicate that hepatitis C infection is significantly associated with several symptoms that are not hepatic in nature. Type II diabetic mellitus (DM) has been identified as a potential indicator of HCV infection that is not associated with hepatic involvement.It is now evident that hepatitis C increases the likelihood of developing diabetes, particularly type 2 diabetes. Numerous studies have shown that individuals with diabetes, particularly those with type 2 diabetes mellitus, have a significant presence of anti-HCV antibodies.The liver plays a crucial role in this process and is fundamental to the development of insulin resistance. Individuals with liver failure often have difficulties in glucose metabolism, with around 20% of this population exhibiting overt diabetes.Type 2 diabetes (T2D) is a common complication of all liver diseases, irrespective of their etiology, particularly in late stages of the condition.Individuals may develop insulin resistance (IR) independently of HCV; nevertheless, substantial clinical and experimental data indicates that HCV contributes to its development. This is essential because insulin resistance not only accelerates the course of chronic hepatitis C but also alters the efficacy of antiviral therapy. (7). This research aims to evaluate the seroprevalence of HCV and its association with individuals suffering from type 2 diabetes mellitus.

Study Design and Setting

This study was conducted in Kirkuk City, Iraq, from December 2012 to May 2013, at Kirkuk General Hospital.

Participants

Peripheral venous blood samples (5 ml) were collected from 391 diabetic patients (ages 22-81) and 288 non-diabetic controls (ages 22-81) who attended Kirkuk General Hospital. Diabetes types were classified based on the history of treatment with oral hypoglycemic agents or insulin at the time of study inclusion. Patients over 40 years old and on oral hypoglycemic agents were categorized as having type 2 diabetes. The control group comprised non-diabetic individuals recruited concurrently from the same hospital. The Hepatitis C Virus (HCV) status of all participants, both diabetic and non-diabetic, was unknown at the time of enrollment and blood sample collection.

Laboratory Procedures

Blood serum was separated to detect HCV-specific antibodies using the Enzyme-Linked Immunosorbent Assay (ELISA) method (CTK Biotech Inc., USA), following the manufacturer's protocol. The optical density (OD) values were measured at 450 nm using an ELISA reader. Results were classified as seropositive or seronegative for HCV antibodies.

Following HCV antibody testing, liver function tests were conducted to study their association with HCV seropositivity. These included measurements of Alanine Aminotransferase (ALT) using an ALT biochemical kit (RANDOX, UK), Aspartate Aminotransferase (AST) using an AST biochemical kit (RANDOX, UK), and Alkaline Phosphatase using a Biomerieux kit (France).

Additionally, the study examined the relationship between HCV infection and the levels of Interleukin-10 (IL-10). Serum IL-10 levels were measured in all diabetic patients using a kit from Omnikine Co., USA.

Table 1 presents the distribution of HCV antibodies among diabetic and control groups, highlighting a significant difference in the prevalence of HCV infection between the two groups. Among the diabetic patients (n = 391), 26 individuals (6.65%) tested positive for HCV antibodies, while a vast majority, 365 individuals (93.35%), were negative. In contrast, the control group (n = 288) had a much lower prevalence, with only 1 individual (0.34%) testing positive for HCV antibodies and 287 individuals (99.66%) testing negative. The difference in HCV antibody prevalence between diabetic and control groups was statistically significant (P-value < 0.01), indicating a higher rate of HCV infection among diabetic patients compared to non-diabetic controls

Table 1: Distribution of HCV Antibodies in Diabetic and Control Groups

P-value: < 0.01

Table 2 shows the association of HCV seropositivity with sex among diabetic patients. Out of 146 male diabetic patients, 11 (7.53%) were seropositive for HCV antibodies. In comparison, among 245 female diabetic patients, 15 (6.12%) were seropositive. Although the seropositivity rate is slightly higher in males than in females, the difference is not statistically significant (P > 0.05). This indicates that there is no significant association between sex and the prevalence of HCV infection among diabetic patients in this study.

Table 2: Association of HCV Seropositivity with Sex in Diabetic Patients

Statistical Significance: P > 0.05

Table 3 presents the distribution of HCV seropositivity across different age groups among diabetic patients. The highest rate of seropositivity is observed in the 52-61 age group, with 11 out of 122 patients (9.01%) testing positive for HCV antibodies. This is followed by the 42-51 age group with a seropositivity rate of 7.24% (5 out of 69 patients) and the 62-71 age group with 7.14% (5 out of 70 patients). Lower rates are observed in the 32-41 (6.45%) and 72-81 (3.26%) age groups. No HCV seropositive cases were found in the youngest age group (22-31 years). Despite variations in seropositivity rates among age groups, the differences were not statistically significant (P > 0.05).

Table 3: Distribution of HCV Seropositivity by Age Groups in Diabetic Patients

Statistical Significance: P > 0.05

Table 4 demonstrates the frequency of anti-HCV antibodies among diabetic patients based on their exposure to surgical operations. Among the 26 HCV seropositive diabetic patients, a significantly higher proportion (65.38%, 17 out of 26) had a history of surgical operations compared to those who were unexposed (34.62%, 9 out of 26). This suggests a strong association between surgical operation exposure and HCV seropositivity among diabetic patients. The difference between the exposed and unexposed groups was statistically significant (P < 0.01), indicating that surgical procedures may be a significant risk factor for HCV infection in this population. This finding underscores the importance of strict infection control practices in surgical settings to minimize the risk of HCV transmission, especially among vulnerable diabetic patients.

Table 4: Frequency of Anti-HCV Antibodies Among Diabetic Patients Based on Surgical Operation Exposure

Statistical Significance: P < 0.01

For ALT levels, a higher percentage of HCV-positive diabetic patients (80.77%) showed elevated ALT levels compared to HCV-negative diabetic patients (59.18%) and non-diabetic patients (42.71%). In contrast, a smaller proportion of HCV-positive diabetic patients (19.23%) had normal ALT levels compared to HCV-negative diabetic patients (40.82%) and non-diabetic patients (57.29%). This indicates that HCV-positive diabetic patients are more likely to have elevated ALT levels, which may reflect liver damage or inflammation associated with HCV infection.

For AST levels, a similar pattern is observed. A larger proportion of HCV-positive diabetic patients (65.38%) had increased AST levels compared to HCV-negative diabetic patients (46.85%) and non-diabetic patients (42.36%). Conversely, 34.62% of HCV-positive diabetic patients had normal AST levels, compared to 53.15% of HCV-negative diabetic patients and 57.64% of non-diabetic patients. The elevated AST levels among HCV-positive diabetic patients further suggest liver involvement or injury.

For Alkaline Phosphatase levels, 69.23% of HCV-positive diabetic patients had increased levels, which is higher than both the HCV-negative diabetic group (51.00%) and the non-diabetic group (34.38%). Meanwhile, the proportion of patients with normal levels of Alkaline Phosphatase was lower in HCV-positive diabetic patients (30.77%) compared to HCV-negative diabetic patients (49.00%) and non-diabetic patients (65.62%). This trend implies that elevated Alkaline Phosphatase is more prevalent among HCV-infected diabetic patients.

Overall, the data indicates a notable association between HCV infection and elevated liver enzyme levels (ALT, AST, and Alkaline Phosphatase) among diabetic patients, highlighting potential liver dysfunction or damage in this subgroup.

Table 5: Relation of ALT, AST, and Alkaline Phosphatase Levels with Study Groups

Notes:

• ALT: Alanine Aminotransferase, Normal range: up to 12 U/L.

• AST: Aspartate Aminotransferase, Normal range: up to 12 U/L.

• Alkaline Phosphatase: Normal range: 21-92 U/L.

The data shows that HCV-positive diabetic patients have a significantly higher IL-10 level (98.5 ± 4.77 pg/ml) compared to HCV-negative diabetic patients (67.55 ± 4.11 pg/ml) and non-diabetic individuals (40.17 pg/ml), with a statistically significant difference (P = 0.008).

Table 6: Relation of HCV infection with IL-10 level in the study groups.

The hepatitis C virus attacks both the liver and extr hepatic organs. It may also coalesce with several other illnesses and ailments that are not associated with the liver. Several more hepatic disorders, including diabetes mellitus, have been recognized. (8) This research found that HCV infection was prevalent among diabetes individuals. Anti-HCV antibodies were detected and quantified using ELISA in both diabetes patients and healthy controls. Liver function assays and interleukin-10 were further used. The research indicated that 6.65% of diabetic patients were infected with HCV, as determined by the ELISA technique (refer to Table 1). This was a substantial disparity compared to the control group, in which just 0.34% of diabetes individuals had an HCV infection (P < 0.01).  The results of the present study align with findings from previous research conducted in other nations. A research conducted in Kuwait revealed that 7% of diabetics were infected with HCV, compared to just 1% of healthy controls. In Turkey, 7.5% of individuals with diabetes were infected with HCV, while just 0.1% of the control group was affected.In an Italian investigation, 7.6% of individuals with diabetes tested positive for the HCV virus in their bloodstream.In Taiwan, 6.8% of individuals with diabetes were identified as having HCV.The variations in HCV seroprevalence between earlier studies and this one may be attributable to differing demographic characteristics of the two groups. Variation across studies may arise due to disparate control sources, case definitions, sample sizes, and participant demographics. Numerous elucidations have been provided on the heightened prevalence of HCV infections in individuals with diabetes. This encompasses frequent parenteral injections, the presence of diabetes or other indicators of hepatitis C virus infection, and the increased likelihood of glucose tolerance issues in individuals with liver illnesses. It has been proposed that diabetes mellitus is more prevalent in individuals with hepatitis C virus-induced cirrhosis than in those with cirrhosis resulting from other etiologies.This research revealed that 7.53 percent of diabetes males and 6.12 percent of diabetic women tested positive for HCV. No substantial correlation existed between the two groups; nonetheless, 62.6 percent of all participants in this research were women (345 out of 391 diabetes patients).  Numerous research could not identify a significant correlation between men and women regarding HCV sickness.(14–16) Numerous additional research contradicted this one.  Abass et al. (17) and Al-Khazraji et al. (18) identified a significant correlation between sexual activity and HCV infection. The discrepancies may arise from variations in sample sizes, patient demographics, and timing of blood collection. Moreover, diabetes is a condition that affects both males and females. The diabetes patients in this research were categorized by gender, revealing a higher hospital visitation rate among women compared to males. This corroborates the previous assertion that the prevalence of diabetes is higher among women than males. Some diabetic individuals may have refrained from visiting the center due to the perception that it would require significant effort and be a waste of time.It is estimated that annually, 1.5 million more women than males succumb to diabetes mellitus, resulting in a total of 1.5 million fatalities from the illness.The research indicated that individuals aged 52 to 61 had the greatest prevalence of HCV seropositivity among diabetes patients. The subsequent age group, 42–51 years, accounted for 9.01% and 7.24%, respectively. The age range of 22 to 31 years did not include any HCV seropositive individuals. No significant correlation existed between age and HCV infection. A multitude of research reached identical outcomes. Mehta et al. (21) discovered that individuals aged 50 to 54 had the greatest prevalence of HCV infection. The increased incidence of positives seen in older individuals may be attributed to their more frequent exposure to intravenous interventions compared to younger individuals, hence increasing the likelihood of transmission of the infection.(22) The present investigation revealed that 65.38% of diabetic individuals who tested positive for HCV had previously undergone surgery. This result was statistically significant. Al-Mashhadani et al. (23) shown that healthcare professionals are at an increased risk of contracting hepatitis C after surgical procedures or blood transfusions. Research conducted by Habib et al. (24) revealed that 32.3% of individuals who had surgery or received sutures tested positive for HCV. The percentage was significantly elevated in dental procedures, reaching 62.2%.  The research examined the correlation between HCV infection and liver function assessments. The study revealed that 80.77% of HCV seropositive diabetic patients had elevated ALT levels, in contrast to 59.18% of HCV seronegative patients and 42.71% of non-diabetic patients, demonstrating a very significant correlation among these groups (Table 5). Furthermore, it was shown that 65.38% of HCV seropositive diabetes patients had elevated AST levels, demonstrating a very significant correlation (Table 6). Additionally, 69.23% of HCV seropositive diabetic patients presented with higher alkaline phosphatase levels; however, no significant link was seen between these variables. Table 7The present research concurred with Ali et al. (25), who reported elevated blood ALT levels in 55.5% of individuals without HCV. Mehta et al. (21) also discovered that individuals with diabetes mellitus had elevated levels of transaminases. Ni et al. (26) discovered that the blood ALT level was elevated in 73.7% of the positive cases, in contrast to 18.5% of the seronegative individuals.  The elevated liver enzyme levels in those who tested positive for HCV resulted in increased liver inflammation. Individuals with chronic hepatitis C have a diverse spectrum of ALT values, ranging from normal to consistently high. Research indicates that individuals with normal ALT levels have delayed cirrhosis development and a reduced incidence of the condition.However, some individuals with HCV infection remain asymptomatic, and their ALT values remain within the normal range. In such instances, the infection may be detected only by screening, such as an anti-HCV antibody test. In cases of hepatitis, the ALT level typically increases; however, in around 20 to 30 percent of HCV carriers, it remains within the normal range.Prior studies have shown that elevated ALT levels are associated with an increased risk of HCC in a community-based population. Repeated measurements of ALT throughout time to identify persistent anomalies may assist in assessing the risk of HCC.The cytokine interleukin-10 is crucial for regulating cellular immunological responses to HTC.Thirty percent of participants in this research who tested positive for HCV had elevated levels of IL-10. A substantial correlation existed between HCV infection and elevated levels of IL-10, as seen in Table 8. This was contrasted with a control group of 22.57% non-diabetic individuals and 24.93% HCV seronegative individuals. The results concurred with Paladino et al. (31), who discovered that individuals with an HCV infection had elevated levels of IL-10. Additionally, Liu (32) shown that blood IL-10 levels in chronic HCV patients are significantly elevated compared to those in healthy individuals.This result contradicted Bozkaya et al. (33), who observed that when all patients were analyzed collectively, the quantity of patients exhibiting elevated IL-10 levels was not significantly different from that of the control group. IL-10, an anti-inflammatory cytokine, is recognized for its protective role against liver damage induced by viruses, alcohol, and inflammation. Its primary function in the body seems to be the inhibition and regulation of inflammatory responses, as well as the modulation of immune cell differentiation and proliferation.(34) The diminished antiviral cellular immune responses seen in individuals with chronic HCV infection are purportedly attributable to elevated levels of IL-10.(35)

from the current study we concluded that there was a significant relation between HCV infection and type 2 diabetes, increased risk of HCV infection with increasing of age. Surgical procedures were very important to establish HCV infection in the society.  Level of IL-10 was elevated in diabetic patients infected with HCV.

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the Akitab University, College of Medical Technologies. Kirkuk Iraq

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