Frequency and Genotype Distribution of Hepatitis C Virus in Pakistan

Hepatitis C Virus (HCV) possesses as a major public health issue around the globe and causes serious liver-related diseases, resulting in high morbidity and mortality rate (Qamar et al., 2021). HCV is a blood-borne pathogen that belongs to the family Flavirividae and contains a positive sense, single-stranded RNA genome. HCV was identified in 1989 which progresses with two types of virological responses as an acute infection and sometimes due to viral persistence in host causes life-threatening chronic infections (Kamani et al., 2020). Untreated acute infections lead to liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC) and these pathological complications ultimately leads to liver failure (Kuna et al., 2019). According to the estimates of the World Health Organization (WHO) around 71 million people are living with this malady worldwide. Nearly; 400,000 people die each year as a result of liver cirrhosis and hepatocellular cancer. Around the world, 30% of HCV positive patients develop liver cirrhosis (Siddique et al., 2020).

In Pakistan, almost 6% of the population is infected with Hepatitis C viral disease which relays a huge disease burden (Haqqi et al., 2019; Qamar et al., 2021). Some of the major risk factors that contribute to disease burden in Pakistan are unsafe medical practice, un-screening of blood products, drug abuse, underreporting of infected regions, excessive and reuse of syringes, etc. (Ullah et al., 2021; Jamil et al., 2020). To eliminate the burden of HCV from Pakistan, the treatment and prevention of HCV must become a national priority.

Currently, about eight major genotypes of HCV have been identified with 87 subtypes in total (Rao et al., 2021). The genotypic distribution pattern of HCV varies in different geographical areas but according to some studies genotype 1 and 3 are the most prevalent genotypes (Rao et al., 2021). Due to this vast heterogeneity of HCV, it is very difficult to develop an effective vaccine against it but there are some potential treatments such as direct-acting antivirals (DAAs) therapies which may provide a possibility to eliminate HCV propagation (Duncan et al., 2020). Each genotype has its therapeutic regimen because each genotype’s response to treatment differs (Lin et al., 2021). The pattern of HCV heterogeneity varies in different geographical regions of Pakistan. In Pakistan, genotype 3a is reported to be the most prevalent genotype, followed by untypeable and coinfection of 3a/3b (Hussain et al., 2021).

HCV screening and diagnosis usually involves serological, molecular and genotypic assays. Qualitative and quantitative screening by PCR plays an important role in the diagnosis of HCV, followed by detection of anti HCV antibodies through ELISA and viral genotyping (Chevaliez, 2019). Early detection of HCV is very challenging because 60-70% of HCV infections are asymptomatic (Gomaa et al., 2020). For the proper diagnosis and treatment, careful detection of viral genotype is the main target. To meet WHO targets to eliminate hepatitis C by 2030 involves easy access to the availability of affordable point-of-care diagnostics and direct-acting antiviral treatment, as well as managing chronic hepatitis C sequelae, which are of critical considerations (Jamil et al., 2020).

Pakistan has the second-largest hepatitis C infection load globally (Rao et al., 2021; Jamil et al., 2020; ). The current research identifies not only the frequency of HCV infection in suspected patients but also determines the geographical pattern of HCV genotypes in different geographical regions of Pakistan.

Ethical approval was taken from the heads of biomedical labs.

A retrospective study was conducted including suspected individuals from Pakistan in order to find out the frequency and genotypes of HCV in suspected individuals.

Pakistan is a country that lies in South Asia with a population of around 225.2 million people. Pakistan is the world’s fifth-largest populous country and has an area of 881,913 square kilometers, making it the 33rd largest country in the world. Data included in our study was collected from biomedical labs located in Rawalpindi.

From April 2020 to April 2021, 4827 samples data were collected from the Accurate Lab, Alpha Lab, and Biotech Lab. During this period from respective biomedical labs, data on suspected individuals were obtained in which blood samples were collected and diagnostics tests were performed.

Suspected individuals to all risk groups in reach were included in this study. Sampling was done by collecting data on HCV from biomedical laboratories. Our data was stratified according to different age groups (<15 - 75 and above) and gender (male, female) that were tested for HCV.

Suspected individuals were screened for the detection of HCV by molecular techniques. The last four years of data were generated against HCV from 2018-2021. Blood samples were collected from suspected individuals. Samples were processed and stored for HCV PCR at −4°C. All serum samples were tested for anti-HCV by the Healgen kit.

The detection of the virus was done by molecular analyses. All the samples were diagnosed for anti HCV antibodies using real-time PCR by Healgen kit according to manufacturer’s protocol followed by genotyping of suspected patients. The viral genotype was determined for positive HCV RNA by amplifying and sequencing the HCV genome’s core region, as described previously (Kichatova et al., 2018).

IBM SPSS statistical version 24.0 was used to examine the data. Pearson chi square test was applied on different variables (year, gender, age groups and genotype with prevalence rate) with a significant level of p <0 .05.

The results show the frequency of HCV among suspected individuals in different years, age groups and gender in Pakistan. Out of 4827 samples, 2739 (56.7%) were tested negative and 43.2% were observed positive results. Out of 2739 samples, 854 were screened for the genotype of HCV.

Frequency of HCV varies in different regions of Pakistan. The overall HCV frequency in the suspected population was observed to be 43.2%. According to our findings, regional HCV frequency estimate was highest in Balochistan (94.9%) followed by Azad Kashmir (55.4%). KP showed a frequency rate of (40.5%). In Punjab, the recorded frequency rate was (39.5%) and in Sindh, it was recorded to be (34.2%). The frequency of HCV varied significantly among the provinces of Pakistan. A detailed summary of results is shown in Table I and Figure 1.

According to year-by-year data analysis, the frequency of HCV in both sexes varies. Table I shows the seroprevalence of suspected males and females from the year 2018-2021. According to our data, a significant difference was observed in in comparing the data year wise. A high frequency of HCV infection rate (94.4%) in both males and females were observed in 2018 and a subsequent drop in the infection rate was observed (Table II).

A small variation was observed between the frequencies of HCV in the gender-based analysis as shown in Table III. Out of 2685, 1167 (43.5%) females were positive and 1518 (56.5%) were tested negative. Total suspected male individuals were n=2142, out of which 921 (42.9%) were tested positive and 1221 (57.0%) were reported to be negative.

In the present study, participants were divided into six age groups ranging from a minimum of under 15 and a maximum of 76 years and older (Table IV). All individuals went through diagnostic methods such as serology, PCR and genotyping. Substantial differences were observed in different age groups as, the high infection rate was observed in the age group 61-75 that was (42.9%), followed by a slight difference in the age group 76 and older that was recorded as (42.5%), <15 years showed positivity rate of (40.0%). The middle age group from 16-30 showed an infection rate of (33.2%) slightly less than that found for the 31-45 age group to be (38.3%). However, the results were statistically significant.

From total pooled of suspected individuals, 1938 (43.2%) of individuals had an active HCV infection, and out of which 854 people responded to genotyping HCV.

According to our results Genotype, 3a was a profound genotype in our sample population (n=652, 76.3%). In our data interpretation, the next significantly increasing genotype was untypeable (n=150, 17.5%), followed by genotype 3b (n=8, 0.9%) and some traces of mix genotype 3a/3b was found (n=9, 1.05%). However, genotypes of n=35, 41% individuals were not detected as shown in the table. The most important finding of our study was that all areas of Pakistan were highly infected by genotype 3a in a suspected population sample. The frequency of HCV was non-significant amongst Pakistani provinces (Table V).

Table VI shows the association between age and gender regarding HCV frequency. In bivariant analysis, the age group with highest seroprevalence in females was found to be in 61­­­­­-75 years (44.9%) and 46-60 year (42.6%). However, in males the highest prevalence was found in age group 76 and older (46.8%) followed by age group 46-60 years (42.6%). Our study was statistically significant in terms of females, but not in terms of males.

Genotyping analysis in different age groups and gender resulted in distribution as genotype 3a was frequent in both gender and in all age groups. In males, all ages were most affected by genotype 3a with a frequency rate of 76.9%, followed by untypeable (14.6%). According to analysis, age group 46 - 60 showed high frequency of un-typeable to be 19.2 percent. However, age group 61-75 was reported by mix infection of 3a/3b with a frequency of 4.3 percent, followed by group 31-45 with 2.2%. The results were shown to be negligible by statistical analysis, and there was no correlation between categorical variables.

Whereas in female’s all age groups showed high frequency of genotype 3a (76.9%), followed by untypeable (11.3%), age group 61-75 showed high infection rate by untypeable genotype. Age group 46-60 showed 1.3% frequency of infection by genotype 3b and mix genotype 3a/3b. The results were statistically significant when compared to males. Detailed summary of results is shown in Table VII.

Hepatitis C viral infection is a major cause of mortality and morbidity around the world. It is a leading source of chronic liver failure. Globally, around 71 million people are expected to be infected with HCV according to a WHO report. HCV related liver failure cause around

350,000 deaths worldwide (Khaliq et al., 2018; Martínez et al., 2021). The prevalence of HCV and its geographical distribution of genotypes are critical for the management of the growing incidence of HCV. Due to the lack of a viable vaccine, HCV eradication requires thorough screening programs and other potential treatments. However, due to the high incidence of infection, risk behaviors, and inadequate linkage to care, reaching this target will be difficult, and efforts should be focused on vulnerable people (Brain et al., 2020).

Pakistan has the second-highest hepatitis C load in the world (Mahmud et al., 2019). In Pakistan, the HCV disease burden is quickly increasing, with over 6% (10 million) of the population is infected (Kumar et al., 2017). Pakistan must set diagnosis and treatment targets for hepatitis C elimination to meet the WHO’s ambitious goal. To meet this lofty target, 90% of all hepatitis C patients must be diagnosed promptly, and approximately 80% of all eligible patients must be treated with direct-acting antivirals (Afzal et al., 2015).

The prevalence of HCV varies from province to province as a result of topographical factors which are different in different regions. According to our analysis frequency of HCV in the suspected population of Pakistan was 43.2%. in this current study high frequency of HCV was found in Balochistan (94.95%), however, a previous study of 2017 shows similar results in which Balochistan had a high-frequency rate of 25.77% (Arshad and Ashfaq, 2017). Followed by Balochistan, Azad Kashmir showed a high infection rate of HCV (55.4%). A similar study (Mahmud et al., 2019) shows a prevalence rate of 5.8% in both Azad Kashmir and Balochistan. According to (Mahmud et al., 2019) high prevalence rate was observed in Sindh (7.0%) comparing to our study was (34.2%). Our study population of Khyber Pakhtunkhwa showed a prevalence rate of (40.5%) and Punjab (39.5%) comparing to the study of (Mahmud et al., 2019) Khyber Pakhtunkhwa showed (6.6%) and prevalence rate of Punjab at (5.6%) respectively. Differences in prevalence rate might be due to the differences in sample size.

Hepatitis C was more widespread until 2018 with a frequency rate of 94.4% in both males and females, after that a proportionate drop in HCV infection rate was observed. Overall frequency of HCV was observed to be 43.5 percent. The difference in frequency might be due to sample size. In comparison to a meta- analysis from Pakistan which included studies from 1995-2019 found 29.79%, 32.33% and 54.4 percent pooled prevalence of HCV (Shayan et al., 2021).

We found statistical association between age and HCV prevalence. According to the current study, the incidence varied by age, but only in a gender-specific way. In our population-based study sex-wise distribution of HCV was found greater in females (43.5%) as compared to males (42.9%). Similar observations were reported by previous studies (Kumar et al., 2017; Rana et al., 2020) and neighboring countries like India (Kar et al., 2019), China (Madewell et al., 2020). In contrast, some studies supported the evidence that it is more common in males than in females (Ahsan et al., 2019) because of the fact that males and females have varied levels of exposure to HCV risk factors.

The current study states that the old age group of people 46years and older showed a high frequency of HCV infection. Or analysis was supported by some previous studies (Ahsan et al., 2019; Rana et al., 2020; Qamar et al., 2021). The prevalence of instances in this age range may be because old age has implications such as a reduced immune system, making the individual more susceptible to infections (Ali et al., 2009). In contrast (Khurram et al., 2021; Qureshi et al., 2010) found a significant frequency of HCV among the age group between 20-49 years. Contributing elements in this regard, however, are still unknown because different age groups are subjected to many different risk factors that are linked with the frequency of HCV infection in Pakistan.

Ahsan et al. (2019) shows high prevalence in females with age group 51-60 years old and in males high frequency was found in age group 51-60 and 61-70 above. In neighboring country, China had high frequency in males of age group 10–49 and 80–100 years whereas, females of age group 0–9 and 50–79 years showed high infection rate (Niu et al., 2016). However, in contrast with some previous studies showed prevalence in middle age group of 20-40 years in both males and females (Ahmad et al., 2010).

Genotype 1 and 3 are most prevalent genotypes around the world (Shah et al., 2021). Overall, our analysis showed that the patients’ age and gender composition did not differ significantly (P > 0.05) according to genotype. Our results showed predominance of genotype 3a in both genders and in all age groups, however, genotype 1 was not included in our study. A study in Pakistan Punjab province supported the fact genotype 3a is more prevalent and affects all ages of both gender (Hussain et al., 2021; Yousaf et al., 2021). Studies from countries like India also support our findings (Barman et al., 2018; Sood et al., 2018), Turkey (Tüzüner et al., 2018). In contrast, according to (Bulut, 2020; Wu et al., 2021) genotype 3a and 1a was predominant in males and 1b was higher in females. A similar study showed high prevalence of genotype 1 in age group of 50 above patients and genotype 3 in younger patients (Petruzziello et al., 2019). The findings of this study contradicted those of a study conducted in Luxembourg in which genotype 3a was predominant in males and genotype 2 was more frequent in females (Roman et al., 2008).

In the present study, 856 HCV-suspected patients from different regions of Pakistan were genotyped. According to this research and earlier studies, HCV genotype 3a is the most common in Pakistan, followed by untypeable, 3b and mix genotype 3a/3b. Using data acquired and pooled from past publications about the HCV distribution pattern in Pakistan from 2010 to 2021, we were able to display the pattern of HCV genotype infections. Genotype 3a was seen to be more frequent in this study and prior investigations from Pakistan (Haqqi et al., 2019; Umer and Iqbal, 2016; Aziz et al., 2013). Geographical location is the most important factor influencing HCV genotype distribution. (Mahmud et al., 2020) observed that genotype 3a is most common in Afghanistan, Iran and Pakistan as compared to other countries and was associated with injecting drug abuse. However, studies from other neighboring countries show high prevalence of genotype 1 followed by genotype 3 (Bulut, 2020; Qu et al., 2021; Shah et al., 2021). The probability of all HCV genotypes was estimated for each province as shown in Table V.

According to our analysis, Azad Kashmir showed a significantly high prevalence of genotype 3a (71.4%) which is close to a previous study (Rauf et al., 2013) in which genotype 3a was the most prevalent form (50%) in females and (40%) in positive male patients. By a recent study (Haqqi et al., 2019), HCV genotype 3a is the most common in Azad Jammu and Kashmir (AJK), accounting for 59% of cases. Only 4% of people had an unidentified genotype.

The significant predominance of genotype 3a was observed in Balochistan with a frequency rate of 74.2% followed by an untypeable 21.7%. The findings of this study were close to a study conducted in 2019 which also shows the high infectivity of genotype 3a in the Balochi population. However, a 2009 report from Balochistan was included to provide a clearer overview of HCV genotype frequency distribution in Pakistan, which agrees with our study and stated that genotype 3a was the most profound genotype in the province (Afridi et al., 2009). Similarly, a study conducted in 2019 contradicts as it shows the high prevalence of genotype 1 (31.5%) in Balochistan (Mahmud et al., 2019).

The high frequency of genotype 3a was found in our study which is 82.2% followed by an untypeable 13.3% and a very small proportion of genotype 3b, 2.2% was found among the KP population. Previous investigations have found a substantially greater frequency of HCV 3a in both KP (Inamullah et al., 2011) and Pakistan (ranging from 70 to 90%) (Attaullah et al., 2011). However, a study demonstrates the high occurrence of genotype 1b (11.6%) after genotype 3a (45.50%) (Gul et al., 2016). Similarly, a recent study from 2020 also shows that genotype 3a is most prevalent in KP (Mumtaz et al., 2020). Our findings also corroborate with some previous studies (Ahmad et al., 2010; Kumar et al., 2017), who found that (59.9%) and (45.5%) of the participants were infected with genotype 3a.

According to our results genotype, 3a was the most frequent genotype in Punjab as compared to others, and all previous studies agree with our analysis (Haqqi et al., 2019). As indicated in table 4, our study indicates the high incidence of HCV genotype 3a (77.4%) followed by untypeable (15.3%) in suspected patients of Punjab, as study from 2020 and 2019 states the high infection rate by genotype 3a followed by genotype 1 in Punjab (Khan et al., 2020; Mahmud et al., 2019). Our study is consistent with a previous study of (Aziz et al., 2013) who found 88.1% of genotype 3a frequency in Punjab. Similar investigations were reported by Bostan et al. (.

There was no data on genotypes from Gilgit and Sindh Province. Because of the disparity in results, which could be attributable to a difference in sample size, Pakistan needs to do more comprehensive and synchronous genotyping in these areas.

Elimination of HCV is a global challenge. Liver diseases related to HCV imposes huge disease burden in developing countries with limited resources like Pakistan. Hepatitis prevention and control has been remained a low political precedence in Pakistan, with inadequate implementation of standardized health related policies devised by WHO and poor regulation of government-sponsored treatment regimens by deputed provincial health ministries. HCV elimination necessitates a strong political will to devise such strategies in order to eliminate hepatitis C virus from Pakistan and also requires funding from local government sectors and other commercial groups.

HCV the blood-borne virus imposes the second largest disease burden in Pakistan with a 6% of prevalence rate. The result of this retrospective study concludes that a significant number of suspected individuals (43.2%) are suffering from hepatitis C viral infection. In Pakistan, genotype 3a is the most prevalent genotype and has an infection rate of 76.3%, with only a small variation in the distribution pattern of genotypes. It is clear from the previous investigations that the lack of an effective vaccine has a significant role in the rise of HCV infection worldwide. Furthermore, unidentified genotypes are found in a significant percentage of the Pakistani population, and it is a significant problem for Pakistani researchers to discover the whole genome sequence(s) of these genotypes and enhance processes for the early detection of associated infection because if HCV illness is progressed, the treatment regimens employed to control or cure becomes less efficient. As a result, the focus must be on creating a powerful HCV vaccine that can drastically lower its prevalence to even though Pakistan has taken steps to combat viral hepatitis, there is still much more to be done to meet WHO’s global goal of eliminating HCV infection by 2030.

The authors thank the department of Biosciences, COMSATS University, Islamabad. This research did not receive any specific grant from funding agencies in the public, commercial, or not-to-profit sectors.