Prevalence of active HCV infection and genotypic distribution among the general population of district Mardan, Pakistan

Prevalência de infecção HCV ativa a distribuição genotípica entre a população geral do distrito de Mardan, Paquistão

N. Ullah M. A. Kakakhel Y. Bai L. Xi I. Khan B. S. Kalra T. Kumar H. Ahmad M. Shah L. Guanlan C. Zhang About the authors

Abstract

Hepatitis C virus (HCV) is the serious global public health burden of liver disease. Approximately 170 million people in the world are infected with (HCV). In Pakistan, where the disease has high occurrence rate. The present study envisages an up-to-date prevalence of HCV and genotypic distribution in the general population of Mardan District, Khyber Pakhtunkhwa (KP), Pakistan. The blood samples from 6,538 individuals including 3,263 males and 3,275 females were analyzed for hepatitis C surface antigen by Immuno-chromatographic test (ICT), Enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (PCR). It was found that 396 (12.13%) out of 3263 individuals contained antibodies in their blood against HCV, while among the different age groups, the highest incidences of HCV antibodies were found in the 31-40 age group (11.01%). The ICT positive samples were further screened by nested PCR to determine the existence of active HCV-RNA. It was identified that 7.11% (3263) of the total population (6538) tested was positive, among which the 461 (14.07%) females possessed antibodies in their blood against HCV. Our data showed total HCV infection in the investigated population was 5.78%. Higher percentage of HCV prevalence was detected in males than females in the age group 31-40 and 41-50. To compare the prevalence of HCV genotypes age-wise in male and female genotype 3a was found most prevalent genotype followed by 1a, 2a and 3b, respectively.

Keywords:
HCV; ELISA; PCR; prevalence; Mardan

Resumo

O vírus da hepatite C (HCV) é o grave problema de saúde pública das doenças hepáticas. Aproximadamente 170 milhões de pessoas no mundo estão infectadas com HCV; no Paquistão, a doença tem alto índice de ocorrência. O presente estudo prevê uma prevalência atualizada do HCV e distribuição genotípica na população geral do distrito de Mardan, Khyber Pakhtunkhwa (KP), Paquistão. As amostras de sangue de 6.538 indivíduos, incluindo 3.263 homens e 3.275 mulheres, foram analisadas para o antígeno de superfície da hepatite C por teste imunocromatográfico (ICT), ensaio imunoenzimático (ELISA) e reação em cadeia da polimerase de transcrição reversa (PCR). Verificou-se que 396 (12,13%) de 3.263 indivíduos continham anticorpos no sangue contra o HCV, enquanto entre as diferentes faixas etárias as maiores incidências de anticorpos anti-HCV foram encontradas na faixa etária de 31 a 40 anos (11,01%). As amostras positivas para ICT foram posteriormente rastreadas por nested PCR para determinar a existência de HCV-RNA ativo. Identificou-se que 7,11% (3.263) do total da população (6.538) testada foram positivos, dentre os quais 461 (14,07%) mulheres possuíam anticorpos no sangue contra o HCV. Nossos dados mostraram que a infecção total pelo HCV na população investigada foi de 5,78%. Maior porcentagem de prevalência de HCV foi detectada em homens do que em mulheres nas faixas etárias de 31-40 e 41-50. Para comparar a prevalência de genótipos de HCV com relação à idade no genótipo masculino e feminino 3a foi encontrado o genótipo mais prevalente seguido por 1a, 2a e 3b, respectivamente.

Palavras-chave:
HCV; ELISA; PCR; prevalência; Mardan

1. Introduction

HCV is a chronic infectious disease of the liver caused by a single strand RNA of Hepatitis C virus The virus belongs to the genus hepacivirus specifically affects the liver leading to morbidity and mortality (Bashir et al., 2017BASHIR, T., ASIM, M., AHSAN, M., ZAFAR, M.Z. and HUSSAIN, K., 2017. A case report: patient with the history of hepatitis C virus. Journal of Antivirals & Antiretrovirals, vol. 9, no. 2, pp. 55-57. http://dx.doi.org/10.4172/1948-5964.1000162.
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; Tariq et al., 2016TARIQ, S., BATOOL, Z., JAHANZEB, V., TARIQ, Q.-A. and DURRANI, S.H. 2016. Hepatitis C virus genotype distribution amongst HCV positive patients present ING at a private tertiary care hospital of Peshawar, Khyber Pakhtunkhwa. Journal of Rehman Medical Institute, vol. 2, no. 2, pp. 33-39.). Which were learnt about 15 years later after the discovery of HCV. According to the World health organization (WHO), HCV has infected almost 200 million individuals worldwide and resulted in 350,000 deaths (Tariq et al., 2016TARIQ, S., BATOOL, Z., JAHANZEB, V., TARIQ, Q.-A. and DURRANI, S.H. 2016. Hepatitis C virus genotype distribution amongst HCV positive patients present ING at a private tertiary care hospital of Peshawar, Khyber Pakhtunkhwa. Journal of Rehman Medical Institute, vol. 2, no. 2, pp. 33-39.). On comparison, the progression of HCV is more widespread in countries where HCV prevalence is low such as developing world of West of Europe, North America, and Australia, or intermediate in Japan, Italy, and Spain respectively (Afzal et al., 2016AFZAL, M.S., SHAH, Z.H. and AHMED, H., 2016. Recent HCV genotype changing pattern in the Khyber Pakhtunkhwa province of Pakistan; is it pointing out a forthcoming problem? The Brazilian Journal of Infectious Diseases, vol. 20, no. 3, pp. 312-313. http://dx.doi.org/10.1016/j.bjid.2015.12.011. PMid:26963150.
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; Ahmed et al., 2006AHMED, S.I., MAHMUD, M.R., KHAN, N.Y., NASEEMULLAH, M. and HANIF, M., 2006. Pegylated interferon and ribavirin in HCV genotype 3 detectable patients after 12 weeks of conventional interferon-ribavirin treatment. Pakistan Journal of Gastroenterology, vol. 20, pp. 58-62.). The major mood of HCV transmission occurred from person to person by blood transfusion, dental and surgical instruments, reused razors, illegal drugs abuses, ear, and nose piercing and tattooing with unsterilized needles, unhygienic or poor sexual practices (Qureshi et al., 2013QURESHI, H., MOHAMUD, B.K., ALAM, S.E., ARIF, A. and AHMED, W., 2013. Treatment of hepatitis B and C through national programme: an audit. JPMA. The Journal of the Pakistan Medical Association, vol. 63, no. 2, pp. 220-224. PMid:23894899.). Although, the signs and symptoms do not appear obviously. However, HCV infected individuals may suffer from lethal liver cancer/dysfunction and liver injury (Imran et al., 2013IMRAN, M., MANZOOR, S., ASHRAF, J., KHALID, M., TARIQ, M., KHALIQ, H.M. and AZAM, S., 2013. Role of viral and host factors in interferon based therapy of hepatitis C virus infection. Virology Journal, vol. 10, no. 1, pp. 299. http://dx.doi.org/10.1186/1743-422X-10-299. PMid:24079723.
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).

The general population of Pakistan are infected with HCV, indicating that with the second-highest HCV burden after Egypt (Averhoff et al., 2012AVERHOFF, F.M., GLASS, N. and HOLTZMAN, D., 2012. Global burden of hepatitis C: considerations for healthcare providers in the United States. Clinical Infectious Diseases, vol. 55, suppl. 1, pp. S10-S15. http://dx.doi.org/10.1093/cid/cis361. PMid:22715208.
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; Wedemeyer et al., 2014WEDEMEYER, H., DUBERG, A.S., BUTI, M., ROSENBERG, W.M., FRANKOVA, S., ESMAT, G., ÖRMECI, N., VAN VLIERBERGHE, H., GSCHWANTLER, M., AKARCA, U., ALEMAN, S., BALIK, I., BERG, T., BIHL, F., BILODEAU, M., BLASCO, A.J., BRANDÃO MELLO, C.E., BRUGGMANN, P., CALINAS, F., CALLEJA, J.L., CHEINQUER, H., CHRISTENSEN, P.B., CLAUSEN, M., COELHO, H.S., CORNBERG, M., CRAMP, M.E., DORE, G.J., DOSS, W., EL-SAYED, M.H., ERGÖR, G., ESTES, C., FALCONER, K., FÉLIX, J., FERRAZ, M.L., FERREIRA, P.R., GARCÍA-SAMANIEGO, J., GERSTOFT, J., GIRIA, J.A., GONÇALES JUNIOR, F.L., GUIMARÃES PESSÔA, M., HÉZODE, C., HINDMAN, S.J., HOFER, H., HUSA, P., IDILMAN, R., KÅBERG, M., KAITA, K.D., KAUTZ, A., KAYMAKOGLU, S., KRAJDEN, M., KRARUP, H., LALEMAN, W., LAVANCHY, D., LÁZARO, P., MARINHO, R.T., MAROTTA, P., MAUSS, S., MENDES CORREA, M.C., MORENO, C., MÜLLHAUPT, B., MYERS, R.P., NEMECEK, V., ØVREHUS, A.L., PARKES, J., PELTEKIAN, K.M., RAMJI, A., RAZAVI, H., REIS, N., ROBERTS, S.K., ROUDOT-THORAVAL, F., RYDER, S.D., SARMENTO-CASTRO, R., SARRAZIN, C., SEMELA, D., SHERMAN, M., SHIHA, G.E., SPERL, J., STÄRKEL, P., STAUBER, R.E., THOMPSON, A.J., URBANEK, P., VAN DAMME, P., VAN THIEL, I., VANDIJCK, D., VOGEL, W., WAKED, I., WEIS, N., WIEGAND, J., YOSRY, A., ZEKRY, A., NEGRO, F., SIEVERT, W. and GOWER, E., 2014. Strategies to manage hepatitis C virus (HCV) disease burden. Journal of Viral Hepatitis, vol. 21, suppl. 1, pp. 60-89. http://dx.doi.org/10.1111/jvh.12249. PMid:24713006.
http://dx.doi.org/10.1111/jvh.12249...
). The chronic HCV infection depends on several risk factors, encompassing the ethnic group, gender type, host genetic factors, age at the time of infection, immune response, and development of jaundice and viral genotype as well subtypes (Afzal et al., 2014AFZAL, M.S., AHMED, T. and ZAIDI, N.U., 2014. Comparison of HCV prevalence in pakistan and iran; an insight into future. Hepatitis Monthly, vol. 14, no. 1, pp. e11466. http://dx.doi.org/10.5812/hepatmon.11466. PMid:24497874.
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). Replication of Hepatitis C virus by mean of RNA-dependent RNA polymerase is error-prone resulting in the severe heterogenetic and different variant of HCV. As per estimation, 2-10 per nucleotide mutations occur per year (Attaullah et al., 2017ATTAULLAH, S., KHAN, S. and ZAHID, M., 2017. Heterogeneous distribution of HCV genotypes and growing menace of mixed HCV infection in blood recipients in Khyber Pakhtunkhwa. The Brazilian Journal of Infectious Diseases, vol. 21, no. 4, pp. 489-490. http://dx.doi.org/10.1016/j.bjid.2016.12.009. PMid:28282507.
http://dx.doi.org/10.1016/j.bjid.2016.12...
; Riaz et al., 2016RIAZ, S., BASHIR, M.F., HAIDER, S. and RAHID, N., 2016. Association of genotypes with viral load and biochemical markers in HCV-infected Sindhi patients. Brazilian Journal of Microbiology, vol. 47, no. 4, pp. 980-986. PMid:27528079.; Smith et al., 1997SMITH, D.B., YAP, P.L., SIMMONDS, P., LAWLOR, E., POWER, J., PATHIRANA, S. and DAVIDSON, F., 1997. The origin of hepatitis C virus genotypes. The Journal of General Virology, vol. 78, no. Pt 2, pp. 321-328. http://dx.doi.org/10.1099/0022-1317-78-2-321. PMid:9018053.
http://dx.doi.org/10.1099/0022-1317-78-2...
). Based on phylogenetic and sequencing analysis, the HCV genome is classified into 7 major and 67 known subtypes and further 20 provision subtypes. Among these genotypes 3 and 1 are world widely distributed (Messina et al., 2015MESSINA, J.P., HUMPHREYS, I., FLAXMAN, A., BROWN, A., COOKE, G.S., PYBUS, O.G. and BARNES, E., 2015. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology, vol. 61, no. 1, pp. 77-87. http://dx.doi.org/10.1002/hep.27259. PMid:25069599.
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). The identification of genotype is the fundamental measure for the sustainable virological response (SVR), for the selection of therapeutic stratagems and clinical treatment (Zein, 2000ZEIN, N.N., 2000. Clinical significance of hepatitis C virus genotypes. Clinical Microbiology Reviews, vol. 13, no. 2, pp. 223-235. http://dx.doi.org/10.1128/CMR.13.2.223. PMid:10755999.
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). On the ease of availability and low-cost income was an option for treatment of interferon (IFN), high SVR against specific genotypes in Pakistan. The pegylated IFN (PEG-IFN), ribavirin, or various treatment combinations were used (Afzal, 2017AFZAL, M.S., 2017. Hepatitis C virus and interferon-free antiviral therapeutics revolution: implications for Pakistan. Viral Immunology, vol. 30, no. 4, pp. 252-257. http://dx.doi.org/10.1089/vim.2016.0164. PMid:28118096.
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; Ahmed et al., 2006AHMED, S.I., MAHMUD, M.R., KHAN, N.Y., NASEEMULLAH, M. and HANIF, M., 2006. Pegylated interferon and ribavirin in HCV genotype 3 detectable patients after 12 weeks of conventional interferon-ribavirin treatment. Pakistan Journal of Gastroenterology, vol. 20, pp. 58-62.; Shah et al., 1997SHAH, H.A., JAFRI, W., MALIK, I., PRESCOTT, L. and SIMMONDS, P., 1997. Hepatitis C virus (HCV) genotypes and chronic liver disease in Pakistan. Journal of Gastroenterology and Hepatology, vol. 12, no. 11, pp. 758-761. http://dx.doi.org/10.1111/j.1440-1746.1997.tb00366.x. PMid:9430043.
http://dx.doi.org/10.1111/j.1440-1746.19...
; Yu et al., 2007YU, J., WANG, G., SUN, L., LI, X. and LI, S., 2007. Predictive value of rapid virological response and early virological response on sustained virological response in HCV patients treated with pegylated interferon α‐2a and ribavirin. Journal of Gastroenterology and Hepatology, vol. 22, no. 6, pp. 832-836. http://dx.doi.org/10.1111/j.1440-1746.2007.04904.x. PMid:17565637.
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).

The initial diagnosis of HCV can be achieve by mean of anti-HCV antibodies test or Enzyme-Linked Immunosorbent Assay (ELISA) (Khan et al., 2013KHAN, A., TAREEN, A.M., IKRAM, A., RAHMAN, H., WADOOD, A., QASIM, M. and KHAN, K., 2013. Prevalence of HCV among the young male blood donors of Quetta region of Balochistan, Pakistan. Virology Journal, vol. 10, no. 1, pp. 83. http://dx.doi.org/10.1186/1743-422X-10-83. PMid:23497435.
http://dx.doi.org/10.1186/1743-422X-10-8...
). But due to false accuracy results, PCR method holds an advantage over other techniques such as (ELISA), Recombinant Immunoblot assay (RIBA), Enzyme Immunoassay for the Qualitative Detection (EIA), ICT and so on, being a quick and reliable tool to diagnose, identify genotype, and quantify active HCV-RNA.

Previously, rare studies have been undertaken in different regions of Pakistan (Ahmad et al., 2007AHMAD, N., ASGHER, M., SHAFIQUE, M. and QURESHI, J.A., 2007. An evidence of high prevalence of Hepatitis C virus in Faisalabad, Pakistan. Saudi Medical Journal, vol. 28, no. 3, pp. 390-395. PMid:17334466.; Aslam and Aslam, 2001ASLAM, M. and ASLAM, J., 2001. Seroprevalence of the antibody to hepatitis C in select groups in the Punjab region of Pakistan. Journal of Clinical Gastroenterology, vol. 33, no. 5, pp. 407-411. http://dx.doi.org/10.1097/00004836-200111000-00013. PMid:11606859.
http://dx.doi.org/10.1097/00004836-20011...
; Kumar et al., 2020KUMAR, T., ZADA, S., IRFAN, M., BATOOL, H. and SAJJAD, W., 2020. Serological Prevalence of Hepatitis B Virus in Peshawar, Khyber Pakhtunkhwa, Pakistan. Pakistan Journal of Zoology, vol. 52, no. 2, pp. 525. http://dx.doi.org/10.17582/journal.pjz/20190213140254.
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, 2017KUMAR, T., AHMAD, N., HAYAT, M.K., GAO, B.-X., FAISAL, S., ILAHI, N., ALI, B., ZADA, S. and SAJJAD, W., 2017. Prevalence and genotypic distribution of hepatitis C virus in Peshawar KPK, Pakistan. Hayati Journal of Biosciences, vol. 24, no. 1, pp. 22-25. http://dx.doi.org/10.1016/j.hjb.2017.04.002.
http://dx.doi.org/10.1016/j.hjb.2017.04....
). Thus, urging needs to study the relationship between HCV and population-based on their route of entry, age, and gender with their overall HCV genotypes in Pakistan. Therefore, Mardan is a congested city of Khyber Pakhtunkhwa, Pakistan (Figure 1), was selected due to lack of information and knowledge about the HCV infection, limited health facilities, and other unfavorable environmental conditions. A survey-based prevalence, epidemiology, and genotypic distribution of HCV age base infection in the district Mardan can provides a solid foundation ailment strategy conferring to viral genotype.

Figure 1
Map of the Khyber Pakhtunkhwa province Pakistan. The district Mardan in encircled, where the present study was conducted for determining the prevalence of active HCV infection and genotypic distribution (Khan et al., 2011KHAN, M., HUSSAIN, F. and MUSHARAF, S., 2011. A fraction of fresh water algae of Kalpani stream and adjoining area of district Mardan, Pakistan. International Journal of Biosciences, vol. 1, pp. 2220-6655.): Genotypes of HCV.

2. Materials and Methods

2.1. Population sampling

This study was conducted in district Mardan a north-west city of Khyber Pakhtunkhwa province of Pakistan. Blood samples of 6,538 (Males: 3,263 [49.90%], females: 3275 [50.0%]) individuals were taken and stored at 4 °C for serum separation. All the individuals were categorized into different age groups 11-20, 21-30, 31-40, 41-50, 51-60, and 61-70-or above respectively. The population was selected based on visiting the concerned laboratories for diagnosis. The participants younger than 9-10 years were excluded. Data from three years (2017 to 2020), of each patient were taken. All the patient information was exclusively used for research purposes only. The study was approved by the Ethical Committee and collections center or laboratories with the collaboration of the School of Life Sciences, Lanzhou University P.R China, and the Virtual University of Pakistan.

2.2. Sera-diagnosis via ICT and ELISA

For anti-HCV antibodies test about 5-ml of the blood was collected in a disposable syringe from the radial vein of patients and stored in sterilized gel tubes. The serum was collected by coagulation of blood and stored at -20 °C until next use. Then sera were screened for the presence of anti HCV antibodies using a strip-based technology ICT commercially available from (ACON®, ACON Laboratories Inc., San Diego, CA 92121, USA) and ELISA from S.A. Barcelona Spain as recommended by the manufacturer instructions. The positive samples were further processed for molecular detection and genotypes. The Prevalence rate was determined as frequency based with the help of following Formula 1:

Prevalence a s % = 100 × Number of Positive Samples Number of Total Samples Measured (1)

2.3. HCV confirmation by PCR and genotyping

From anti-HCV antibodies positive samples, total RNA was isolated followed by (Kessler et al., 2002KESSLER, H.H., CLARICI, A.M.K., STELZL, E., MÜHLBAUER, G., DAGHOFER, E., SANTNER, B.I., MARTH, E. and STAUBER, R.E., 2002. Fully automated detection of hepatitis C virus RNA in serum and whole-blood samples. Clinical and Diagnostic Laboratory Immunology, vol. 9, no. 6, pp. 1385-1388. http://dx.doi.org/10.1128/cdli.9.6.1385-1388.2002. PMid:12414781.
http://dx.doi.org/10.1128/cdli.9.6.1385-...
), then cDNA was synthesized using gene-specific reverse primers by reverse transcription, using Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase at 37oC for 50 mins. The viral RNA was confirmed through PCR primers (Table 1). For the genotypic investigation, total RNA from the positive sample was run by (Ohno et al., 1997OHNO, O., MIZOKAMI, M., WU, R.-R., SALEH, M.G., OHBA, K., ORITO, E., MUKAIDE, M., WILLIAMS, R. and LAU, J.Y., 1997. New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a. Journal of Clinical Microbiology, vol. 35, no. 1, pp. 201-207. http://dx.doi.org/10.1128/jcm.35.1.201-207.1997. PMid:8968908.
http://dx.doi.org/10.1128/jcm.35.1.201-2...
). Briefly, reverse transcription PCR (RT-PCR) was performed for viral cDNA synthesis using core-gene specific reverse primers, and multiplex-PCR was performed. Nested PCR based two reaction mixtures containing two sets of primers (Table 2), were employed. The PCR-thermal cycler conditions were set 94 °C for 5 mins for pre-denaturation, followed by denaturation at 94 °C for the 30s (30 cycles). The primer annealing temperature was adjusted at 64 °C for the 30s; and with an extension of 72 °C for 30s, the final cooling temperature of 4 °C for 10 mins. The same conditions were applied for the nested PCR. Conversely, only primer annealing was carried out at 53 °C. At last, the amplified products of PCR were visualized on the gel documentation system after subjecting to electrophoresis on 2% agarose gel using with the DNA marker.

Table 1
Prevalence of active HCV among the different age groups of males as revealed by ICT, ELISA, and PCR tests.
Table 2
Prevalence of active HCV among the different age groups of females as revealed by ICT, ELISA, and PCR tests.

3. Results

3.1. Age-specific HCV frequency

A total of 6,538 participants including 3,263 males and 3,275 female blood samples were collected. All blood samples were tested for the presence of anti-HCV antibodies by using ICT and ELISA. Overall, tested samples were categorized into age groups that fall into seven different age categories 11 to 70 years or above. At first all the male samples were positive by ICT 15.96% and were further assessed by ELISA. The ELISA results revealed that out of the total number 12.13% were positive (Table 1). The nested PCR assay of the samples positive by either ICT or ELISA revealed that 232 of 7.11% males are with active HCV RNA in their blood (Table 1). To calculate the age-wise prevalence in males, our results shown that all the age groups were affected while the HCV prevalence fluctuates in different age groups. To find highest incidence rate of 11.01% was recorded from the age group of 31-40 years, followed by 7.58% in the age group of 41-50. However, the lower HCV incidence of 1.04% was perceived in the age group of 60-70 years. Contrariwise, no HCV incidence was noted from the age group of 70 or above years (Table 1).

Following the same approach, the age-wise prevalence in female gender was also assessed (Table 2). To identify highest incidence rate 7.10% was observed in the age group of 41-50 years, followed by 4.17% in the age group of 31-40. Similarly to detect lower incidence 1.55% was observed in the age group of 61-70 years (Table 2). These results revealed that different six age groups were affected significantly. Despite the fact, there were no PCR positive samples were detected in the age groups of 70 or above. Interestingly, it was observed that the age groups, the 41-50 year the HCV prevalence was higher in the all adults age group that is 21-40 years. However surprisingly, prevalence was highest in males from 31-40 and in females from 41-50 (Tables 1 and 2). We investigated that 11.01% of 99 males showed the higher positive PCR results in 31-40 age group for HCV (Table 1), while the prevalence in females was 7.10% (61) in the 41-50 age group (Table 2).

Taken together 6,538 contributors’ blood was tested for liver function through ICT and ELISA. For HCV antibodies ICT 6,538 results of 15.01% were positive. The serum samples which were assured affirmative using ICT were furthermore investigated by ELISA. Of which 11.39% obtained positive. The ELISA positive samples were furthermore sorted for RT-PCR analysis in which 5.78% samples were confirmed to be active infected of HCV and harbor HCV-RNA (Table 1). The overall data shows the highest prevalence of anti-HCV antibodies tested by ICT, ELISA, and HCV-RNA among the general population of Mardan, KPK Pakistan in males than females (Table 3). The typical gel photograph is presented (Figure 2).

Table 3
Overall distribution of the ICT, ELISA, and PCR based HCV results of males and females from Mardan, Khyber Pakhtunkhwa, Pakistan.
Figure 2
Representative gel photograph of HCV amplified products. Lane 1 represents negative control. HCV, Hepatitis C virus, lanes 2 to 8 are the typical positive samples for active HCV showing expecting 220 bp band of hepatitis C virus and lane, 8 is molecular marker.

3.2. Genotypes frequency between male and female

The prevalence of different genotypes from total HCV-RNA positive samples was further analyzed in the general population. Different HCV genotypes were detected by using allele-specific primers (Ohno et al., 1997OHNO, O., MIZOKAMI, M., WU, R.-R., SALEH, M.G., OHBA, K., ORITO, E., MUKAIDE, M., WILLIAMS, R. and LAU, J.Y., 1997. New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a. Journal of Clinical Microbiology, vol. 35, no. 1, pp. 201-207. http://dx.doi.org/10.1128/jcm.35.1.201-207.1997. PMid:8968908.
http://dx.doi.org/10.1128/jcm.35.1.201-2...
). In order, to compare the prevalence of HCV genotypes according to age in male patients were categorized into 7 age groups following the prevalence from (Table 1). HCV genotype 3a was detected 31.94% of 74 with anti-HCV positive individuals. Moreover, genotype 1a was observed 17.24% of 40, followed by genotype 2a of 9.48%, and genotype 3b was 9.05%, a typical identified genotypes gel photograph (Figure 3). Furthermore, it was observed 17.24% of 40 tested samples where no genotype PCR band was observed and affirmed as untypable by the present method employed. Correspondingly, also mixed infection of HCV genotypes was detected 15.06% of 35 (Table 4).

Figure 3
Typical Gels patterns of PCR products of different HCV genotypes identified in Mardan, KPK, Pakistan. Lane 1 = Molecular marker; Lane 2 = 208 bp HCV genotypes 1a; Lane 3 = untypable genotype; Lane 4 = 176 bp genotypes 3b; Lane 5 = 232 bp genotype 3a; Lane 6 = 190 bp genotype 2a; Lane 7 = negative control.
Table 4
Distribution of active HCV genotypes among the different age groups of males as revealed by PCR.

In the same way, to compare the age-wise prevalence of HCV genotypes in female patients also categorized into 7 age groups (Table 5). These results showed that genotype 3a was detected in female patients 38.35% of 56. Thus, these results showed that genotype 3a was significantly more abundant in female patients than males. These shreds of evidence showed that genotype 3a was dominant genotype circulating in general population of Mardan. Furthermore, genotype 1a (16.43% of 24), and genotype 2a (9.58% of 14) and genotype 3b (8.2% of 12) were found in female. Additionally, mixed infection of HCV genotypes 15.08% of 22 and untypable genotype 12.32% of 18 was observed in female of Mardan population. Besides these genotypes 5a and 6a were not detected in both HCV-RNA positive patients of general population of Mardan KPK.

Table 5
Distribution of active HCV genotypes among the different age groups of females as revealed by PCR.

3.3. Online data retrieval and its comparison

To elaborate our investigation a systematic review of the literature was conducted using online available tools, PubMed, PakMedinet, and World Wide Web portal (http://webfoundation.org/). Taken together 18 studies and average prevalence of HCV in terms of the individual province/ cities were compared with our present investigations (Table 6).

Table 6
Reviewed of literature demonstrating the prevalence of hepatitis C across the different locations of Pakistan from 1999 till now. Each technique of study with its population size and type has been mentioned.

Moreover, aiding funds to facilitate the healthcare system and better treatment facilities. Other preventive measures such as public awareness and vaccination programs are extreme need to fortify. The government and health authorities such as national institute of health (NIH) need to re-enforce the management and control strategies. This was an initial study and case-control investigation should be conducted in the future.

4. Discussion

The emerging infection and diseases pose a serious threat to human health. All these mostly occurred in underdeveloped countries like Pakistan due of limited resources. The new arising pathogens are characterized by a new model of pathogenicity (Khalil et al., 2017KHALIL, A.T., ALI, M., TANVEER, F., OVAIS, M., IDREES, M., SHINWARI, Z.K. and HOLLENBECK, J.E., 2017. Emerging viral infections in Pakistan: issues, concerns, and future prospects. Health Security, vol. 15, no. 3, pp. 268-281. http://dx.doi.org/10.1089/hs.2016.0072. PMid:28636447.
http://dx.doi.org/10.1089/hs.2016.0072...
). Hepatitis viral rate is alarming in Pakistan (Al Kanaani et al., 2018AL KANAANI, Z., MAHMUD, S., KOUYOUMJIAN, S.P. and ABU-RADDAD, L.J., 2018. The epidemiology of hepatitis C virus in Pakistan: systematic review and meta-analyses. Royal Society Open Science, vol. 5, no. 4, 180257. http://dx.doi.org/10.1098/rsos.180257. PMid:29765698.
http://dx.doi.org/10.1098/rsos.180257...
). The increasing HCV infections precisely depict the prevalence of HCV, and the biogeographic distribution of its genotypes. In this study, 6,538 HCV-suspects were randomly collected from the congested city of Mardan.

The prevalence of HCV has been observed by ICT 15.01%, ELISA 11.39% and PCR 5.78%, which comparatively much varies than many cities of Pakistan (Table 1-6), by comparing to Rawalpindi 2.45% (Masood et al., 2007MASOOD, R., SARDAR, M.A. and MALLHI, A.A., 2007. Seroprevalence of hepatitis B and C among the healthy blood donors at Fauji Foundation Hospital, Rawalpindi. Pakistan Journal of Medical Sciences, vol. 23, pp. 64-67.), Islamabad 5.31% (Idrees et al., 2008IDREES, M., LAL, A., NASEEM, M. and KHALID, M., 2008. High prevalence of hepatitis C virus infection in the largest province of Pakistan. Journal of Digestive Diseases, vol. 9, no. 2, pp. 95-103. http://dx.doi.org/10.1111/j.1751-2980.2008.00329.x. PMid:18419643.
http://dx.doi.org/10.1111/j.1751-2980.20...
), Buner 5% (Muhammad and Jan, 2005MUHAMMAD, N. and JAN, M.A., 2005. Frequency of hepatitis” C” in Buner, NWFP. Journal of the College of Physicians and Surgeons, vol. 15, no. 1, pp. 11-14. PMid:15670516.), Multan 4.06% (Idrees et al., 2008IDREES, M., LAL, A., NASEEM, M. and KHALID, M., 2008. High prevalence of hepatitis C virus infection in the largest province of Pakistan. Journal of Digestive Diseases, vol. 9, no. 2, pp. 95-103. http://dx.doi.org/10.1111/j.1751-2980.2008.00329.x. PMid:18419643.
http://dx.doi.org/10.1111/j.1751-2980.20...
), Northern areas of Pakistan 25.70% (Tariq et al., 1999TARIQ, W.U., HUSSAIN, A.B., KARAMAT, K.A., GHANI, E., HUSSAIN, T. and HUSSAIN, S., 1999. Demographic aspects of hepatitis C in northern Pakistan. JPMA. The Journal of the Pakistan Medical Association, vol. 49, no. 8, pp. 198-201. PMid:10641504.), and Faisalabad 20.89% (Ahmad et al., 2007AHMAD, N., ASGHER, M., SHAFIQUE, M. and QURESHI, J.A., 2007. An evidence of high prevalence of Hepatitis C virus in Faisalabad, Pakistan. Saudi Medical Journal, vol. 28, no. 3, pp. 390-395. PMid:17334466.). We have also found that the overall percentage was significantly higher in men than women over the last 3 years. This higher prevalence of HCV in male might be due the higher exposure in environment, but HCV is more prevalent among male than females of 2013 and 2017 years (Kumar et al., 2017KUMAR, T., AHMAD, N., HAYAT, M.K., GAO, B.-X., FAISAL, S., ILAHI, N., ALI, B., ZADA, S. and SAJJAD, W., 2017. Prevalence and genotypic distribution of hepatitis C virus in Peshawar KPK, Pakistan. Hayati Journal of Biosciences, vol. 24, no. 1, pp. 22-25. http://dx.doi.org/10.1016/j.hjb.2017.04.002.
http://dx.doi.org/10.1016/j.hjb.2017.04....
). Although, epidemiology of HCV exhibit variation from region to region or even within the same population (Wild and Hall, 2000WILD, C.P. and HALL, A.J., 2000. Primary prevention of hepatocellular carcinoma in developing countries. Mutat. Res. Mutation Research, vol. 462, no. 2-3, pp. 381-393. http://dx.doi.org/10.1016/S1383-5742(00)00027-2. PMid:10767647.
http://dx.doi.org/10.1016/S1383-5742(00)...
). Such differences of the prevalence of HCV might be associated with different lifestyles, age factors, poor literacy rate and health facilities, low socio-economic status, and lack of awareness (Shah et al., 1997SHAH, H.A., JAFRI, W., MALIK, I., PRESCOTT, L. and SIMMONDS, P., 1997. Hepatitis C virus (HCV) genotypes and chronic liver disease in Pakistan. Journal of Gastroenterology and Hepatology, vol. 12, no. 11, pp. 758-761. http://dx.doi.org/10.1111/j.1440-1746.1997.tb00366.x. PMid:9430043.
http://dx.doi.org/10.1111/j.1440-1746.19...
). Considering the age group distribution, the prevalence of HCV infection showed gradually increased among the middle age of 31-50 years. These may reflect that in 31–40 years age group, males have highest prevalence than females while in 41–50 years age group females were more affected than males. It will be interesting further to find out the factors associated in this gender specification.

Previously studies has shown that the severity, prognosis of disease and response to therapy may vary according to the genotypes (Antaki et al., 2013ANTAKI, N., BIBERT, S., KEBBEWAR, K., ASAAD, F., BAROUDI, O., ALIDEEB, S., HADAD, M., ABBOUD, D., SABAH, H., BOCHUD, P.-Y. and NEGRO, F., 2013. IL28B polymorphisms predict response to therapy among chronic hepatitis C patients with HCV genotype 4. Journal of Viral Hepatitis, vol. 20, no. 1, pp. 59-64. http://dx.doi.org/10.1111/j.1365-2893.2012.01621.x. PMid:23231085.
http://dx.doi.org/10.1111/j.1365-2893.20...
; Wyles and Gutierrez, 2014WYLES, D.L. and GUTIERREZ, J.A., 2014. Importance of HCV genotype 1 subtypes for drug resistance and response to therapy. Journal of Viral Hepatitis, vol. 21, no. 4, pp. 229-240. http://dx.doi.org/10.1111/jvh.12230. PMid:24597691.
http://dx.doi.org/10.1111/jvh.12230...
). Our data showed that HCV genotype 3a is the most prevalent in the Mardan population, which are corroborated with (Ahmad et al., 2010AHMAD, W., IJAZ, B., JAVED, F.T., JAHAN, S., SHAHID, I., KHAN, F.M. and HASSAN, S., 2010. HCV genotype distribution and possible transmission risks in Lahore, Pakistan. World Pakistan Journal of Gastroenterology, vol. 16, no. 34, pp. 4321-4328. http://dx.doi.org/10.3748/wjg.v16.i34.4321. PMid:20818816.
http://dx.doi.org/10.3748/wjg.v16.i34.43...
; Aziz et al., 2013AZIZ, H., RAZA, A., MURTAZA, S., WAHEED, Y., KHALID, A., IRFAN, J., SAMRA, Z. and ATHAR, M.A., 2013. Molecular epidemiology of hepatitis C virus genotypes in different geographical regions of Punjab Province in Pakistan and a phylogenetic analysis. International Journal of Infectious Diseases, vol. 17, no. 4, pp. e247-e253. http://dx.doi.org/10.1016/j.ijid.2012.09.017. PMid:23183233.
http://dx.doi.org/10.1016/j.ijid.2012.09...
; Khan et al., 2011bKHAN, S., ATTAULLAH, S., AYAZ, S., KHAN, S.N., SHAMS, S., ALI, I., BILAL, M. and SIRAJ, S., 2011b. Molecular epidemiology of HCV among health care workers of Khyber Pakhtunkhwa. Virology Journal, vol. 8, no. 1, pp. 105. http://dx.doi.org/10.1186/1743-422X-8-105. PMid:21385397.
http://dx.doi.org/10.1186/1743-422X-8-10...
). The frequency of genotype 1a was observed in male and female (17.24% and 16.43%) respectively. However, the frequency of the patients infected the method employed with unidentifiable or mix genotype in both genders. In most of previous studies reveal the detection of an unknown/undetermined HCV genotype (Ali et al., 2014ALI, S., AHMAD, A., KHAN, R.S., KHAN, S., HAMAYUN, M., KHAN, S.A., IQBAL, A., KHAN, A.A., WADOOD, A., UR RAHMAN, T. and BAIG, A.H., 2014. Genotyping of HCV RNA reveals that 3a is the most prevalent genotype in mardan, pakistan. Advances in Virology, vol. 2014, 606201. http://dx.doi.org/10.1155/2014/606201. PMid:24715902.
http://dx.doi.org/10.1155/2014/606201...
; Rauf et al., 2011RAUF, A., NADEEM, M.S., ALI, A., IQBAL, M., MUSTAFA, M., MUZAMMAL LATIF, M., LATIF, M.Z., AHMED, N. and SHAKOORI, A.R., 2011. Prevalence of hepatitis B and C in internally displaced persons of war against terrorism in Swat, Pakistan. European Journal of Public Health, vol. 21, no. 5, pp. 638-642. http://dx.doi.org/10.1093/eurpub/ckq084. PMid:20601693.
http://dx.doi.org/10.1093/eurpub/ckq084...
). These studies specified that identification of these unknown genotype(s), and exploration of its/their possible role for clinical investigations, diagnosis, sequencing and development of Hepatitis C, vaccine development and other prevention measures still remains challenging.

Genotype 4a is very rare in Pakistan and also absent in present study, nonetheless, a study on Lahore city in Pakistan identified 4a genotype (Ahmad et al., 2010AHMAD, W., IJAZ, B., JAVED, F.T., JAHAN, S., SHAHID, I., KHAN, F.M. and HASSAN, S., 2010. HCV genotype distribution and possible transmission risks in Lahore, Pakistan. World Pakistan Journal of Gastroenterology, vol. 16, no. 34, pp. 4321-4328. http://dx.doi.org/10.3748/wjg.v16.i34.4321. PMid:20818816.
http://dx.doi.org/10.3748/wjg.v16.i34.43...
), in the Middle East (Gower et al., 2014GOWER, E., ESTES, C., BLACH, S., RAZAVI-SHEARER, K. and RAZAVI, H., 2014. Global epidemiology and genotype distribution of the hepatitis C virus infection. Journal of Hepatology, vol. 61, no. 1, suppl., pp. S45-S57. http://dx.doi.org/10.1016/j.jhep.2014.07.027. PMid:25086286.
http://dx.doi.org/10.1016/j.jhep.2014.07...
). Genotypes 5a and 6a were also missing in present observations. These studies highlight a important clinical parameters (Ahmad et al., 2010AHMAD, W., IJAZ, B., JAVED, F.T., JAHAN, S., SHAHID, I., KHAN, F.M. and HASSAN, S., 2010. HCV genotype distribution and possible transmission risks in Lahore, Pakistan. World Pakistan Journal of Gastroenterology, vol. 16, no. 34, pp. 4321-4328. http://dx.doi.org/10.3748/wjg.v16.i34.4321. PMid:20818816.
http://dx.doi.org/10.3748/wjg.v16.i34.43...
). Moreover, HCV has six different genotypes, which have further subtypes. These genotypes confirmed 30-35% nucleotide differences in HCV genome (Smith et al., 2014SMITH, D.B., BUKH, J., KUIKEN, C., MUERHOFF, A.S., RICE, C.M., STAPLETON, J.T. and SIMMONDS, P., 2014. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology, vol. 59, no. 1, pp. 318-327. http://dx.doi.org/10.1002/hep.26744. PMid:24115039.
http://dx.doi.org/10.1002/hep.26744...
). However, it has been claimed that age is the main and important risk factor for HCV (Kumar et al., 2020KUMAR, T., ZADA, S., IRFAN, M., BATOOL, H. and SAJJAD, W., 2020. Serological Prevalence of Hepatitis B Virus in Peshawar, Khyber Pakhtunkhwa, Pakistan. Pakistan Journal of Zoology, vol. 52, no. 2, pp. 525. http://dx.doi.org/10.17582/journal.pjz/20190213140254.
http://dx.doi.org/10.17582/journal.pjz/2...
, 2017KUMAR, T., AHMAD, N., HAYAT, M.K., GAO, B.-X., FAISAL, S., ILAHI, N., ALI, B., ZADA, S. and SAJJAD, W., 2017. Prevalence and genotypic distribution of hepatitis C virus in Peshawar KPK, Pakistan. Hayati Journal of Biosciences, vol. 24, no. 1, pp. 22-25. http://dx.doi.org/10.1016/j.hjb.2017.04.002.
http://dx.doi.org/10.1016/j.hjb.2017.04....
; Mengal et al., 2012MENGAL, M.A., ABBAS, F., MENGAL, M.A., SHAFEE, M., BABAR, S. and ATIQUE, A., 2012. Passive surveillance of anti-hepatitis C virus antibodies in human subjects of four medical units of Balochistan, Pakistan. International Journal of Agriculture and Biology, vol. 14, no. 4, pp. 585-589.). Therefore, based our study different age groups containing HCV infection and even though comparing the different age groups, the adult male population was the most dominant in HCV prevalence at the age of 31-40 (11.01%) followed by females in the age group 41-50 (7.10%).The highest prevalence of HCV infection existed among middle age of male and old age female with 3a genotype. Therefore, most of the young age individuals remain ignorant about their status of HCV infection. Taken together age has been a main factor in HCV studies, with infection more predominant in older ages of population (Kumar et al., 2020KUMAR, T., ZADA, S., IRFAN, M., BATOOL, H. and SAJJAD, W., 2020. Serological Prevalence of Hepatitis B Virus in Peshawar, Khyber Pakhtunkhwa, Pakistan. Pakistan Journal of Zoology, vol. 52, no. 2, pp. 525. http://dx.doi.org/10.17582/journal.pjz/20190213140254.
http://dx.doi.org/10.17582/journal.pjz/2...
, 2017KUMAR, T., AHMAD, N., HAYAT, M.K., GAO, B.-X., FAISAL, S., ILAHI, N., ALI, B., ZADA, S. and SAJJAD, W., 2017. Prevalence and genotypic distribution of hepatitis C virus in Peshawar KPK, Pakistan. Hayati Journal of Biosciences, vol. 24, no. 1, pp. 22-25. http://dx.doi.org/10.1016/j.hjb.2017.04.002.
http://dx.doi.org/10.1016/j.hjb.2017.04....
; Mengal et al., 2012MENGAL, M.A., ABBAS, F., MENGAL, M.A., SHAFEE, M., BABAR, S. and ATIQUE, A., 2012. Passive surveillance of anti-hepatitis C virus antibodies in human subjects of four medical units of Balochistan, Pakistan. International Journal of Agriculture and Biology, vol. 14, no. 4, pp. 585-589.).

The Mardan city was taken as a second model city after Peshawar the capital province of Khyber Pakhtunkhwa, as entire districts have a mixed population (Kumar et al., 2020KUMAR, T., ZADA, S., IRFAN, M., BATOOL, H. and SAJJAD, W., 2020. Serological Prevalence of Hepatitis B Virus in Peshawar, Khyber Pakhtunkhwa, Pakistan. Pakistan Journal of Zoology, vol. 52, no. 2, pp. 525. http://dx.doi.org/10.17582/journal.pjz/20190213140254.
http://dx.doi.org/10.17582/journal.pjz/2...
). Among the Khyber Pakhtunkhwa districts Mardan has been facing basic health requisites problems and suffer from natural disasters including floods, population pressure and earthquakes. However, most of previous findings concern (Table 6), about ICT and ELISA or other non-PCR based (Ahmad et al., 2010AHMAD, W., IJAZ, B., JAVED, F.T., JAHAN, S., SHAHID, I., KHAN, F.M. and HASSAN, S., 2010. HCV genotype distribution and possible transmission risks in Lahore, Pakistan. World Pakistan Journal of Gastroenterology, vol. 16, no. 34, pp. 4321-4328. http://dx.doi.org/10.3748/wjg.v16.i34.4321. PMid:20818816.
http://dx.doi.org/10.3748/wjg.v16.i34.43...
). In our findings, RT-PCR depict the more exact picture of active HCV prevalence and genotyping distribution in different age groups of males and females.

Mardan is in the southwest of province Khyber Pakhtunkhwa, Pakistan. The proportion of the urban district is 20.2% whereas 79.8% is the rural proportion. A report conducted by Government of Pakistan, compared to urban areas HCV prevalence is more dominant in rural areas (Afridi et al., 2013AFRIDI, S.Q., ZAHID, M.N., SHABBIR, M.Z., HUSSAIN, Z., MUKHTAR, N., TIPU, M.Y., AKHTAR, F. and YAQUB, T., 2013. Prevalence of HCV genotypes in district Mardan. Virology Journal, vol. 10, no. 1, pp. 90. http://dx.doi.org/10.1186/1743-422X-10-90. PMid:23514695.
http://dx.doi.org/10.1186/1743-422X-10-9...
). Based on our prevalence frequency of different HCV genotyped reported so far lower in females 4.45% in comparison with males 7.11%. Our investigations suggest that the males community is acquiring HCV infection more rapidly due to maximum exposure to the external environment. On the other hand, barber shaving, Homosexuality may refer to males who have sex with males (MSM), or females who have sex with female (FSF) and use of drugs are common practices in these areas. The arguments are in line for higher occurrence of HCV among males, however in contrast females living house life. Overall, our data support the hypothesis of a homogeneous distribution of HCV infection between the male and female related to various outdoor factors. Similarly, HCV higher prevalence in males followed by females was observed when risk factors regarding hepatitis B and C (Khan et al., 2011aKHAN, S., ATTAULLAH, S., ALI, I., AYAZ, S., KHAN, S.N., SIRAJ, S. and KHAN, J., 2011a. Rising burden of Hepatitis C Virus in hemodialysis patients. Virology Journal, vol. 8, no. 1, pp. 438. http://dx.doi.org/10.1186/1743-422X-8-438. PMid:21920054.
http://dx.doi.org/10.1186/1743-422X-8-43...
). Furthermore, our finding indicates the dominant genotypes 3a and 1a, among the tested population. These results validate the findings of (Ahmad et al., 2010AHMAD, W., IJAZ, B., JAVED, F.T., JAHAN, S., SHAHID, I., KHAN, F.M. and HASSAN, S., 2010. HCV genotype distribution and possible transmission risks in Lahore, Pakistan. World Pakistan Journal of Gastroenterology, vol. 16, no. 34, pp. 4321-4328. http://dx.doi.org/10.3748/wjg.v16.i34.4321. PMid:20818816.
http://dx.doi.org/10.3748/wjg.v16.i34.43...
; Kumar et al., 2017KUMAR, T., AHMAD, N., HAYAT, M.K., GAO, B.-X., FAISAL, S., ILAHI, N., ALI, B., ZADA, S. and SAJJAD, W., 2017. Prevalence and genotypic distribution of hepatitis C virus in Peshawar KPK, Pakistan. Hayati Journal of Biosciences, vol. 24, no. 1, pp. 22-25. http://dx.doi.org/10.1016/j.hjb.2017.04.002.
http://dx.doi.org/10.1016/j.hjb.2017.04....
), reported that 59.9% and 16.5% of individuals were infected with 3a and 1a genotypes, both these being higher percentages than the other genotypes detected. Genotype 3a of 34.1% as the most prevalent genotype in district Swat, followed by 2a (8.1%), 3b (7%), 1a (5.4%) and mixed genotype 7.6% (Inamullah et al., 2011INAMULLAH, I., IDREES, M., AHMED, H., ALI, M., ALI, L. and AHMED, A., 2011. Hepatitis C virus genotypes circulating in district Swat of Khyber Pakhtoonkhaw, Pakistan. Virology Journal, vol. 8, pp. 16. PMid:21235746.). Another study of 49.5% prevalence genotype 3a described in district Swat (Ahmad et al., 2009AHMAD, A., AHMAD, B., ALI, A. and AHMAD, Y., 2009. Seroprevalence of HBsAg and anti-HCV in general healthy population of Swat district with frequency of different HCV Genotypes. Pakistan Journal of Medical Sciences, vol. 25, pp. 744-748.). To mitigate the risks associated with HCV infection, it is important to understand the interactions of pathogen-host-environment and monitor the molecular evolution and genomic surveillance.

Based on our visual investigation, most of the patients associated with low economic status and visited by other local medical practitioners. Lack of emergency medical services and initial help to victims are commonly practiced by untrained worker might be another major cause hepatitis C (Bukhari, 2013BUKHARI, M.M., 2013. Standard interferon and ribavirin response in patients with chronic hepatitis C. Rawalpindi Journal of Rawalpindi Medical College, vol. 17, pp. 297-299.). Use of no proper sterilization and contaminated equipment may represent another possible reason for high prevalence. Besides this sharing personal possession such as shaving razors, nail cutters, the needle used for ear and nose piercing, toothbrushes, and personal close or/and transgender sexual contact with an infected person (Qureshi et al., 2013QURESHI, H., MOHAMUD, B.K., ALAM, S.E., ARIF, A. and AHMED, W., 2013. Treatment of hepatitis B and C through national programme: an audit. JPMA. The Journal of the Pakistan Medical Association, vol. 63, no. 2, pp. 220-224. PMid:23894899.).

The results of our study showed prevalence and distribution of HCV genotypes varies with age. For example, genotype 3a was more common in male the ages 31-40, while it was more prevalent in female between ages 41-50. To our knowledge, it is the comprehensive report to evaluates the distribution of genotypes of HCV in Mardan, but it has some limitations, such as subtype with a relatively small sample size and a selection bias is possible given the use of the data from a multiple laboratory. But our study may be helpful for understanding the genotype distribution of HCV in Mardan and can provide important information about HCV prevalence among different ages and sexes. However, Khyber Pakhtunkhwa may not have much strength or capacity to mitigate these problems itself immediately. By so far, great push up efforts are required by the national/international community to reduce the existing threat.

5. Conclusion

In conclusion, HCV prevalence and genotype distribution has a certain relationship with different age and gender. We found that HCV 3a is a predominant genotype followed by 1a, 2a, and 3b in Mardan. Further studies are needed to collect bigger samples to estimate the different epidemiology of the HCV genotypes. As males are more frequently prone to high-risk than female also indicating that the infection is more proliferated between the adult group either males or females. To control HCV disease, there is a dire need to design a treatment achieving maximum control with minimal hostile effects. Major awareness events should be launched by Government and Semi government authorities or non-government organizations (NGOs), ample use of vaccine and other preventive measures to cease the outspread.

Acknowledgements

The authors express their sincere gratitude to the local Medical Centers and Hospitals of Mardan, Khyber Pakhtunkhwa, Pakistan, for granting this award for their research. Also extend esteemed thanks to all the concerned friends, doctor clinicians/patients for their cooperation as well as their support throughout this study. Authors are also very grateful for the Chinese Government Scholarship Council for the award of Doctoral studies.

The study was funded by Jiangsu Science and Technology Major Project (No. BA2016036), Lanzhou Science and Technology Funds (No. 2015-3-81), and Gansu Science and Technology Major Project (No. 17ZD2FA009).

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Publication Dates

  • Publication in this collection
    14 July 2021
  • Date of issue
    2023

History

  • Received
    26 Oct 2020
  • Accepted
    04 Dec 2020
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