Hepatitis C virus (HCV) presents a significant global health concern, affecting 3.3% of the world’s population. The primary mode of HCV transmission is through blood and blood products. Patients with beta thalassemia, who rely on lifelong blood transfusions, are particularly vulnerable to HCV infections. This study aimed to assess the prevalence of hepatitis C virus infections among multitransfused thalassemic patients in the twin cities of Pakistan’s capital. The clinical research, involving the enrollment of 262 multitransfused beta thalassemic patients residing in the capital twin cities of Pakistan. The investigation encompassed the evaluation of hepatitis C virus presence, alanine aminotransferase (ALT) levels, serum creatinine, hepatomegaly, splenomegaly, and the occurrence of splenectomy. The overall prevalence of Hepatitis C virus infections was notably high at 55.73%. This was particularly pronounced among patients aged 20 years and older, with a 100% infection rate. In HCV-positive thalassemic patients, the average ALT level was observed to be 98 U/L, while average creatinine values stood at 0.39 mg/dL. Additionally, hepatomegaly was prevalent in 82.20% of HCV-positive thalassemic patients, featuring an average liver size increase of 4.33 cm. Splenomegaly was evident in 67.12% of HCV-positive thalassemic patients, with an average spleen size augmentation of 4.46 cm. Splenectomy was identified in 15.75% of cases. The incidence of HCV infections in the thalassemic population of Pakistan is alarmingly high. Furthermore, the risk of contracting HCV infections escalates with the advancing age of thalassemic patients. Elevated ALT levels and hepatomegaly were pervasive among the majority of HCV-positive thalassemic patients. Consequently, there is a compelling need for rigorous screening of blood products prior to transfusion to mitigate the future burden of HCV in Pakistan.
In 1975, Hepatitis C Virus (HCV) was initially identified as Non-A, Non-B viral hepatitis (NANBH), marking the discovery of a novel Flavi-like virus responsible for transfusion-associated hepatitis (Feinstone et al., 1975; Alter et al., 1975; Houghton, 2009). This virus is classified within the Flaviviridae family and is characterized by a single-stranded RNA structure with dimensions ranging from 55 to 65 nm). The World Health Organization (WHO) has aptly likened Hepatitis C Virus to a “viral time bomb,” acknowledging its alarming global prevalence, affecting approximately 3.3% of the world’s population, encompassing nearly 200 million individuals who have contracted HCV. Out of this afflicted population, 130 million endure the burden of chronic HCV infection, facing the peril of developing liver cirrhosis and/or liver cancer. An annual influx of 2.3-4.7 million individuals contracts new HCV infections, with a significant 70% transitioning into chronic hepatitis. WHO’s statistical analysis in 2008 revealed liver cirrhosis to be the 18th leading cause of mortality, a ranking expected to escalate to the 13th position by 2030 (WHO, 2008, 2009a, b). In this context, WHO has declared South East Asia as a high-risk area for HCV, with a prevalence rate of 2.15% (WHO, 2000). The evolution of liver cirrhosis transpires in 20% of HCV-infected patients within 10-20 years, while the emergence of liver cancer is typically observed 20-40 years post-infection (Smith, 2005; Ansar and Kooloobandi, 2002).
The primary modes of HCV transmission include blood transfusions, intravenous drug administration, needle stick injuries, perinatal transmission, hemophilia, hemodialysis, and organ transplants, collectively representing the most common risk factors for HCV transmission. Additionally, several less conventional risk factors for HCV transmission include intranasal cocaine use, body piercing, tattoos, shared shaving equipment, sexual activity, erosion of knuckles due to boxing, and transmission from healthcare workers to patients (Eduardo, 2006).
In Pakistan, HCV infection is progressively acknowledged as a significant healthcare concern. Numerous studies have unveiled HCV infection prevalence estimates among adults and children, falling within the range of 2.4-6.5% and 0.44-1.6%, respectively (Luby et al., 1997; Jafri, 2003; Aziz et al., 2007; Jafri and Subhan, 2008). The healthcare professional community in Pakistan faces an elevated risk of HCV infection, with a prevalence rate ranging from 4-5.6%, surpassing the global average by a factor of 20 (Aziz et al., 2002; Mujeeb et al., 1998). Pertinently, WHO reports an annual count of 1.2 to 1.5 million transfusions in Pakistan, though regrettably, the nation grapples with a 40% deficit in the availability of blood and blood components. Meeting a nation’s basic blood requirements necessitates a minimum of 1% of the population contributing as blood donors, a threshold far from realization, with an estimated average donation rate of just 0.37%. This figure rises marginally to 0.75% in developing and transitional countries, in stark contrast to the 3% donation rate prevalent in developed countries. Furthermore, low-income countries witness a striking tenfold disparity in the average number of blood donations per 1000 individuals when compared to their affluent counterparts. The inadequacy of blood supply often compels healthcare providers in low-income nations to rely on replacement or paid blood donations. In the context of Pakistan, this is reflected in the startling statistics, where 75% (and at times even 90% in northern regions) of blood donations stem from replacement donors, while professional donors and unpaid voluntary donors contribute 15% and 10% respectively (WHO, 2009a, b; PRCS, 2007; Mujeeb et al., 1998; NACP, 2012; Ahmad et al., 2007). Adequate precautionary measures should be taken for the prevention of viral diseases (Saeed et al., 2021a, b, c).
Notably, a study examining “well-selected blood donors” unveiled an overall HCV antibody frequency of 1.87%, with a differentiation between first-time donors at 1.83% and second-time donors at 2% (Ali et al., 2003). A WHO survey report from 2010, encompassing 173 countries, underscored that a mere 47% of the total 93 million blood donations in developing nations underwent rigorous screening under quality assurance procedures (WHO, 2010). The challenges associated with sustaining blood safety in Pakistan are multifaceted, encompassing the lack of centralized blood transfusion services, inadequately trained and educated staff, intermittent power supply, suboptimal efficiency in blood transfusion services, limited political support and commitment, insufficient resources and infrastructure, the utilization of low-sensitivity test kits with budget-friendly pricing, and the commercialization of blood screening setups (Cheraghali, 2011; Zanetti, 1999).
In the context of Pakistan, thalassemia is regarded as one of the most prevalent and life-threatening genetic disorders. Each year, the nation witnesses the birth of 5000-9000 children afflicted with β-thalassemia. This autosomal recessive disorder exhibits an approximate carrier rate of 5-7%, representing nearly 9.8 million carriers (Ahmed et al., 2002; Khan et al., 2000). The practice of consanguineous marriages, particularly the union of cousins and relatives, is more prevalent in Pakistan than in other North African Muslim countries, establishing a compelling link between such unions and the incidence of thalassemia. Thalassemia patients, on average, undergo 25 blood transfusions annually. This high frequency of blood transfusions underscores the potential development of transfusion-transmitted infections (TTIs). Previous research indicates HCV prevalence within the multi-transfused thalassemia population of Pakistan to range from 34.8% to 60%, a statistic that is notably higher than in other Asian nations (Akhtar et al., 1998; Akhtar and Moatter, 2004; Abdul-Mujeeb et al., 1997; Bhatti et al., 1995; Khokhar et al., 2005; McCaughan, 2000). Factors such as low educational standards, compromised health status, and the inadequate implementation of international blood safety measures are primarily responsible for the dissemination of TTIs, including HBV, HCV, and HIV, through multiple blood transfusions (Afzaal Memorial Thalassemia Foundation, 2024; Katsanos et al., 2005).
This article is dedicated to exploring the seroprevalence of Hepatitis C virus in thalassemia patients residing in the capital twin cities of Pakistan, namely Islamabad and Rawalpindi. The study meticulously assesses the relationships between HCV positivity and various study variables, subjecting these associations to rigorous statistical validation. The findings of this study yield compelling justifications for the urgent implementation of blood safety standards within diverse public health sectors, encompassing both rural and urban areas of Pakistan.
2.1. Study settings and data collection
A cross-sectional study, involving 262 multi-transfused β-thalassemia patients selected through random sampling from the regions of Islamabad, Rawalpindi, and its suburban areas. The study received approval from the National University of Sciences and Technology (NUST) and The International Center of Medical Sciences Research (ICMSR), Islamabad Pakistan. After obtaining informed written consent and demographic history data from each participant, a comprehensive physical and clinical examination was performed.
The clinical evaluation included the assessment of hepatomegaly, splenomegaly, and the presence of splenectomy using ultrasound techniques in adherence to standard operating procedures. Pre-transfusion hemoglobin counts, alanine aminotransferase (ALT) levels, and serum creatinine levels were determined using a spectrophotometer. Venous blood samples were collected using sterile syringes and placed in vacutainer (EDTA) tubes. All blood samples were subjected for in vitro diagnostic analysis.
The collected sera were divided into two aliquots and stored at -80 °C for the screening of HCV-positive samples. The initial screening for HCV-positive β-thalassemia patients was performed using a third-generation Anti-HCV antibody test, following the guidelines provided by Accurate Diagnostics USA. This test device demonstrated high reliability, with a 99% correlation with a leading commercial HCV EIA test kit. The test exhibited a relative sensitivity, specificity, and accuracy for HCV screening of >99.8% (with a confidence interval of 98.7-100.0%), 99.9% (99.8-100.0%), and 99.9% (99.7-100.0%), respectively. Confirmation of viral RNA was conducted through HCV qualitative PCR analysis.
Statistical analysis of study variables was carried out using the Statistical Package for Social Sciences (SPSS version 17). The χ2 test was employed to identify relationships between HCV positivity and study variables, and significance was assessed at the 0.05 level with a 95% confidence interval.
3.1- Prevalence of hepatitis C virus in beta-thalassemia patients
Out of the 262 beta-thalassemia patients enrolled in the study, 146 individuals tested positive for HCV, resulting in a prevalence rate of 55.73%. The prevalence of HCV among male participants (157) was 54.77%, while among female participants (105), it was 57.14%.
The prevalence of HCV varied by age group: In patients aged 1-10 years, HCV prevalence was 48.47%. Among patients aged 11-20 years, HCV prevalence was higher at 68.42%. For patients older than 20 years, the prevalence rate was 100%.
3.2. Hepatomegaly in HCV-infected beta-thalassemia patients
Hepatomegaly, an increase in liver size, was observed in 74.8% of all cases. Among patients with Hepatomegaly, HCV was detected in 61.20% of cases. In the broader cohort of HCV-infected beta-thalassemia patients, hepatomegaly was observed in 82.20% of cases. On average, the increase in liver size among all HCV-infected individuals was approximately 4.33 cm, with 4 cm being the most common increment observed. Figure 1 indicates the increase in liver size in HCV-infected thalassemia patients.
3.3. Splenomegaly in HCV-infected beta-thalassemia patients
In our study, we observed an increase in spleen size, medically termed Splenomegaly, in 64.9% of the cases. Among the subset of beta-thalassemia patients infected with HCV, splenomegaly was identified in 67.12% of subjects. Notably, splenectomy, the surgical removal of the spleen, was deemed necessary in 15.75% of these cases. Consequently, only 17.12% of all HCV-infected beta-thalassemia patients exhibited a normal spleen. On average, the increase in spleen size among the entire cohort of HCV patients measured 4.46 cm, with the majority displaying a more moderate increase of 2-3 cm in spleen size. Figure 2 shows splenomegaly among HCV-infected thalassemia patients.
3.4. Alanine transaminase levels and serum creatinine counts in HCV-infected beta-thalassemia patients
In both male and female populations, the established normal ranges for Alanine Transaminase (ALT) and serum creatinine are 10-40U/L, 10-32U/L, and 0.7-1.4mg/dl, 0.6-1.1mg/dl, respectively. For the entire cohort of HCV-positive beta-thalassemia patients, the average ALT level was notably elevated at 98U/L, contrasting with an average ALT level of 71.5U/L observed among HCV-negative individuals. Similarly, among all HCV-positive patients, the average serum creatinine level was measured at 0.39mg/dl, compared to the average serum creatinine count of 0.37mg/dl found in HCV-negative individuals. The statistical analysis for the relationships among study variables are shown in Table 1.
Table 1
Statistical Analysis for Relationship Studies among Different Study Variables.
| Relationship Between Variables | Pearson Chi-Square test value | P-value (Confidence level) |
|---|---|---|
| Relationship Between Hepatomegaly and HCV Positive Thalassemia Patients | 26.719 | 0.021 |
| Relationship Between Splenomegaly with HCV Positive Thalassemia Patients | 21.129 | 0.33 |
| Relationship Between HCV Positive Individuals and ALT Level | 1.79 E2 | 0 |
| Relationship Between HCV Positive Individuals and Creatinine Level | 16.65 | 0.002 |
Pakistan, a developing country grappling with a significant socioeconomic burden of infectious diseases, faces challenges related to anemia, communicable diseases, malnutrition, thalassemia, obstetrical emergencies, traumatic injuries, and road traffic accidents. Pakistan’s burden of viral infections is mounting day by day (Saeed et al., 2023a, b, c; 2024; Saeed and Piracha, 2023; Piracha and Saeed, 2023; Piracha et al., 2023, 2024; Uppal et al., 2024). The increasing demand for blood transfusions, often life-saving, has raised concerns about the quality of transfusion practices due to commercialization. Many blood transfusion services operate within hospital settings, relying heavily on replacement blood donors (often family or friends) and paid professional donors. However, this reliance on replacement donors poses safety concerns as they may donate blood under familial or societal pressure, potentially concealing health issues or high-risk behavior. Thalassemia patients, who require around twenty-five blood transfusions annually, face an increased risk of acquiring blood-borne pathogens due to this practice (Daily Times, 2011). HCV in Thalassemia patients of Pakistan is shown in Table 2.
Table 2 HCV in Thalassemia patients of Pakistan: a systemic review of available data.
| Author (Year) | Population Size | Population Type | Region | Prevalence |
|---|---|---|---|---|
| Bhatti et al. (1995) | 35 | Thalassemia Patients | Rawalpindi | 60% |
| Moatter et al. (1999) | 100 | Thalassemia Patients | Karachi | 35% |
| Muhammad et al. (2003) | 80 | Thalassemia Patients | Peshawar | 36.20% |
| Akhtar and Moatter (2004) | 256 | Thalassemia Patients | Karachi | 34.80% |
| Younas et al. (2004) | 75 | Thalassemia Patients | Islamabad | 42% |
| Burki et al. (2005) | 180 | Thalassemia Patients | Islamabad | 41.70% |
| Shah et al. (2005) | 250 | Thalassemia Patients | Peshawar | 57% |
| Hussain et al. (2008) | 180 | Thalassemia Patients | Islamabad & Peshawar | 41.70% |
In Pakistan, marriages represent the union of two families, where individuals prioritize the well-being of their loved ones. During times of need for blood, patients and their families predominantly rely on family-based replacement blood donors, rather than volunteer donors. Consanguineous marriages, where individuals marry close relatives, are common in both rural and urban settings (Afzal et al., 1994; Waheed et al., 2009) . Marital commitments are often difficult to alter due to concerns about potential family disputes, leading to the acceptance of consanguineous marriages to avoid social conflicts. The rejection of marriage proposals, particularly for women, is considered a significant taboo that can detrimentally affect their social lives and prospects for future marriages. Premarital screening, although encouraged by Islamic teachings, can face resistance due to religious misinterpretations, presenting challenges to its success. Furthermore, the financial burden of treating thalassemia patients, with costs ranging from 4,500 to 7,500 Pakistani Rupees, presents a significant challenge, particularly for families from uneducated and economically disadvantaged backgrounds.
Various epidemiological studies suggest a substantial prevalence of HCV infection among transfusion-dependent beta-thalassemia major patients. The prevalence of HCV in thalassemia patients varies worldwide, with Egypt having the highest rate at 22% of the population. In the United States, the Center for Disease Control and Prevention reports an anti-HCV prevalence of 1.8%, resulting in 8,000 to 10,000 deaths annually. In Europe and South East Asia, the prevalence of HCV ranges from 1.03% to 2.13%. In Pakistan, the percentage prevalence of HCV in different populations varies, with the highest rates observed in injecting drug users and multitransfused populations. Thalassemia patients are particularly at risk of acquiring Hepatitis C due to the administration of unscreened blood collected during the donor window period. The seroprevalence of HCV infection in thalassemia patients varies across different regions, with Iran, India, Italy, and Malaysia reporting rates of 63.8%, 16.7%, 47%, and 23.8%, respectively. In Pakistan, the prevalence of HCV in thalassemia patients ranges from 20.5% to 60%, with different studies providing insights into the percentage prevalence of HCV in various thalassemia populations in the country.
Previous studies suggest that over time, the prevalence of HCV in thalassemia patients has been on a gradual rise. Hussain et al. (2008) reported a seroprevalence of HCV in thalassemia patients from Islamabad and Peshawar at 41.7%. In contrast, our study conducted in the twin cities of Islamabad and Rawalpindi in Pakistan reveals a higher prevalence of HCV among thalassemia patients, at 55.73% (Hussain et al., 2008; Mukherjee and Dhawan, 2024; Khan et al., 2000). Furthermore, our research indicates that HCV prevalence is notably higher among females compared to males. A consistent upward trend in HCV prevalence is observed across different age groups of thalassemia patients. The data clearly illustrates that as thalassemia patients age, the likelihood of acquiring HCV significantly increases, with prevalence ranging from 48.47% in the 1-10 years age group to 100% in those over 20 years of age. This data underscores that as the age of multitransfused beta thalassemia patients advances, so does the risk of HCV transmission.
A substantial majority (75%) of the enrolled participants exhibited hepatomegaly, coinciding with an increased prevalence of viral hepatitis at 61.2%. Similarly, a majority of beta thalassemia patients (65%) presented with splenomegaly, accompanied by an elevated prevalence of HCV at 67.1%. Among HCV-infected beta thalassemia patients, the maximum recorded increase in liver and spleen size was 10cm and 12cm, respectively. Graphical analysis indicates that individuals with hepatomegaly of 1cm and 10cm were least prevalent, accounting for only 0.7% and 1.37%, respectively. Most individuals with hepatomegaly displayed an increase in liver size ranging from 3-4cm. However, among patient groups with significantly enlarged liver size, the proportion of individuals was considerably lower, indicating an elevated risk of mortality.
Similarly, the graphical analysis of HCV-infected beta thalassemia patients with splenomegaly revealed that individuals with spleen sizes of 1cm and 12cm were least common, each representing 2.04% of cases. The majority of patients with splenomegaly exhibited a 2-3cm increase in spleen size. However, among patient groups with significantly enlarged spleen size, the proportion of individuals was notably lower, suggesting an increased risk of death among these patients. This data further underscores that as liver and spleen size increases in HCV-infected beta thalassemia patients over time, the risk of mortality also rises. An elevated ALT level is indicative of liver disease or an unhealthy liver state. Notably, the ALT count in HCV-infected beta thalassemia patients was significantly higher compared to HCV-negative beta thalassemia patients. However, the serum creatinine levels were approximately similar in both HCV-positive and HCV-negative thalassemia patients. The relationships observed between HCV-positive thalassemia patients and hepatomegaly, ALT levels, and creatinine levels were found to be highly significant.
Hepatitis C Virus is rapidly spreading within the population of beta-thalassemia type major in Pakistan, primarily due to fragmented, disorganized, and decentralized hospital-based blood transfusion systems. Consanguineous marriages are prevalent in Pakistani society and are significantly associated with a higher prevalence of thalassemia. The majority of thalassemia patients exhibited elevated ALT and creatinine values, along with hepatomegaly and splenomegaly. The risk of HCV infection increases with the advancing age of thalassemia patients. Implementing proper blood screening procedures before transfusion is strongly recommended to alleviate the future burden of HCV in Pakistan.
We would like to acknowledge the efforts of Muhammad Ahmad from Nishtar Medical College, Multan Pakistan.
