Ethical approval was obtained from the institutional review board (IRB) of the Pakistan Kidney and Liver Institute and Research Center (PKLI-RC; approval # PKLI-73). A copy of the consent form is provided as Extended data. Written consent was obtained from all the participants prior to interviews for the use of their epidemiological data for research and publication. Parental consent from minor participants was obtained for participants aged 16 years or below. It was signed by the parent or guardian who accompanied the patient on behalf of the minor. This consent form was provided in both the local language and in English. A copy of the minor consent form duly signed by the principle investigator of study was also provided to the parents of the minor participant. In cases where parents were not willing to participate in the study and did not sign the minor consent form, the patients were not included in the study.

Punjab is one of the most populated provinces of Pakistan, with a population of 110 million ¹⁸ , and has a high burden of hepatitis B and C. The PKLI-RC is state-of-the-art tertiary care center in Punjab’s provincial capital, Lahore. The hospital is currently providing clinical services for liver and kidney diseases, including kidney transplants. Given the burden of hepatitis B and C in the province, the PKLI-RC has established an outreach program (Hepatitis Prevention and Treatment Program), which has been providing free-of-charge, evidence-based hepatitis B and C preventive, screening, diagnostic, vaccination and treatment services to the population of Punjab since March 2017. The Hepatitis Prevention and Treatment Program’s (HPTP) services are being delivered through 24 Hepatitis Prevention and Treatment Clinics (HPTCs), which are based in Lahore and 23 other districts of the Punjab province. In addition to the PKLI-RC’s HPTP, two other similar government-funded hepatitis control programs are providing necessary care services to the population of Punjab. This cross-sectional study is based on the data of individuals who visited the HPTCs from March 2017 to August 2018 for screening, diagnosis and treatment of hepatitis.

All the visiting individuals were screened for HBV and HCV after their registration in the HPTCs’ Electronic Medical Record (EMR). Patients were included in the study after signing the consent form once the study had been explained to them by the investigator ¹⁹ . Patients who did not sign the consent form to participate in the study were screened and treated but they were not included in the study analysis. Patients were included irrespective of their age, gender, ethnicity and locality within Punjab Province. Patients were excluded from the study if they were unwilling to participate in the study or came from any other province of the country. Face-to-face interviews were conducted after voluntary informed consent was given. A total of 141,705 individuals were registered and screened in at all HPTCs and were then interviewed at the health center, the majority being carried out at the HPTC-Lahore clinic. After signing the consent form, patients were interviewed about their exposures to risk factors by trained interviewers and/or medical officers using a questionnaire ²⁰ . Each question was made clear to the patient in order to get the true answer according to the patient’s knowledge and memory. The criteria for inclusion in further analysis was no previous history of hepatitis. Of the 141,705 patients interviewed, only 12,427 met the criteria and only their data was used in the final analysis.

The predictor variables included in the analyses were: age (in years); gender (male/female/transgender); ever having had intravenous (IV) injections (and number of injections in the last five years), ever having had blood transfusions, ever having had dental procedures, being hospitalized within the last five years, ever having had surgery, having taken recreational drugs in the last five years, having had a circumcision performed by a barber (males only) , shaving by barbers, visiting a beauty parlor within the last five years, ever having had hijama therapy, having a body piercing in the last five years, having a tattoo in the last five years and ever having performed self-flagellation with sharp objects. The outcome variables were serostatus for hepatitis B (HBsAg) and hepatitis C (anti-HCV).

The data was extracted from a centralized electronic medical records (EMR) database in Excel format. An epidemiological questionnaire, also part of the EMR system, was used to collect the data on individuals’ demography and hepatitis-related risk factors. The questionnaire was piloted on 30 participants to obtain estimates about the expected response rates, data quality, validity and comprehensibility of the questionnaire. These participants were patients who visited these clinics routinely and were coming for a screening or checkup. They were provided with a consent form for their willingness to participate in the study. After signing the consent form, they were interviewed by medical officers using the pilot questionnaire. After reviewing the results of piloted questionnaire, a few variables were excluded from the final questionnaire (are you taking drugs; have you been bitten by any animal in last five years; are you taking cannabis) because these variables were found to be irrelevant and response rate for these variables were very low. The overall response rate was 96.66% (29/30) and the data quality was good in terms of dimensions such as ‘consistency’, ‘accuracy’, ‘completeness’, and ‘timeliness’. The internal consistency was measured by Cronbach’s alpha, with an alpha value of 0.87. The normality of data was assessed for the pilot survey to evaluate the fitness of data.

Questionnaires were completed by staff during face-to-face interviews, conducted in the local language by a trained interviewer. Interviewers were bachelor nurses or medical officers who had been trained by an epidemiologist to carry out interviews. The data was exported to Epi-Data for revisions such as data cleaning and data validation. Where >10% of all variables in any patient’s questionnaire were missing or not provided, the patient was excluded from analysis.

A 5ml blood sample was collected from each patient using disposable vacuum syringes by nurses at the health centers. Samples collected at the sentinel sites were transported daily to the central laboratory for laboratory diagnosis. Serum specimens from all samples were separated using standard protocols and stored at 20°C on the same day until tested for HBsAg or Anti-HCV in the central PKLI laboratory in Lahore following WHO standard procedures. HBsAg and anti-HCV analyses were performed, the details of which are given below.

A chemiluminescent micro particle immunoassay (CMIA) anti-HCV assay (ARCHITECT Anti-HCV assay, ABBOTT Diagnostics; Catalog no. 6L47) was conducted for the qualitative detection of antibodies against HCV in the patients’ serum and plasma. Recombinant HCV sample antigen-coated paramagnetic micro-particles and anti-human acridinium-labeled conjugates are fixed to create the reaction mixture and then incubated for 5 minutes at 25°C. Ancillary wash buffer was added to the mixture after washing, followed by another wash cycle and incubation for 5 minutes at 25°C. Pre-Trigger (Pre-Trigger Solution containing 1.32% w/v hydrogen peroxide) and Trigger solutions (containing 0.35N sodium hydroxide) are added to the mixture. The resulting chemiluminescent reaction was measured in relative-light-units (RLUs) using a luminometer. The presence of anti-HCV antibodies is determined by comparing the signals shown in the reaction to an active-calibration curve. If the signal in the specimen is greater or equal to the cutoff signal, the sample is then considered anti-HCV positive and vice versa. The ARCHITECT Anti-HCV assay calculates a result based on Sample RLU/Cutoff RLU (S/CO). The cutoff calculation is done such that; calibrator 1 Mean RLU Value × 0.074 = Cutoff RLU. Specimens with S/CO values <1.00 are considered nonreactive by the ARCHITECT Anti-HCV assay and need not be tested further. Specimens with S/CO values ≥ 1.00 are considered reactive by the ARCHITECT Anti-HCV assay. A two-step immunoassay for qualitative detection of HBsAg using CMIA technology was used. The ARCHITECT HBsAg assay (Catalog no. 6C36) utilized a four parameter logistic curve-fit (y-weighted) to generate a calibration curve. Specimens with concentration values < 0.05 IU/mL was considered nonreactive by the criteria of ARCHITECT HBsAg. Specimens with concentration values ≥ 0.05 IU/mL were considered reactive by the criteria of ARCHITECT HBsAg.

Data was analyzed using SPSS version 22 (IBM, NY, USA). Results are reported as percentages and odds ratios. Descriptive statistics of prevalence are reported in Table 1. To analyze the association of independent variables with the outcome variables, logistic regression was performed ²¹ . Variables with a significance level of <0.25 were retained in the final logistic model. Regression analysis of the data was conducted using multivariable logistic regression model. Goodness of fit of the model used was assessed using the Hosmer-Lemeshow test and the likelihood-ratio test. QGIS Version 3.2.2 ²² was used for geographical distribution and heat maps were developed based on coordinates data collected during interviews to present the seroprevalence of HBV and HCV at the district level.

Descriptive statistics were Figure 1 gives the overall prevalence of HBsAg and anti-HCV in the study population, which was 8.4% and 42.7%, respectively. Of those with HCV infection, seroprevalence of co-infection with HBV was recorded to be 4.2%. The seroprevalence data stratified by various categories is presented in Table 1. Briefly, the prevalence of co-infection was higher in males (5.7%) than females (3.2%). The transgender population had a significantly higher seroprevalence of HBsAg (11.8%) and anti-HCV (58.8%) compared to other gender groups. The chi-square test found a directly associated trend of increased HCV and co-infection was found with increasing age. Geographical heat maps for the seroprevalence of HBV and HCV are presented in Figure 2. Muzaffargarh district had the highest HBV seroprevalence at 26%, followed by Rajanpur (20.3%), Lodhran (10.6%), and Shujabad (10.3%). HCV seroprevalence was significantly higher (P value <0.05) in Shujabad district (66.4%), followed by Muzaffargarh (65.2%), Nankana Sahab (62.5%), and Lodhran (61.8%).

In the multivariable logistic model, age group (16–30 years) was found to be a significant (p-value <0.05) risk factor (OR=4.2) for HBV occurrence, followed by age groups 31–45 years (OR=3.4), and >61 group (OR=3.2). Males had 2.1-fold higher risk of HBV infection than females. The other risk factors found to be significantly associated with the occurrence of HBV included exposure to hijama therapy, circumcision performed by barbers, barber shaving, recreational drug use, tattooing, beauty parlor visits, IV injections and having >10 injections ( Table 2).

HCV occurrence was significantly associated with an increase in age of the patients. Belonging to the older age group (>61) was found to be a risk factor (OR=56.5; 95% C. I=40.6-78.6) for HCV infection. Exposure to hijama therapy was also recorded as a risk factor (OR=6.0). Females had a 1.3-fold higher risk of HCV than males. The other significant risk factors for HCV included circumcision by barbers, recreational drug usage, tattooing, piercing, blood transfusion, dental procedures, surgical procedures and prior hospitalization ( Table 3).

Age showed a significant association with HBV and HCV co-infection, as before. Patients in the >61 years age group had a 29.3-fold (95% C. I=6.9-124.6) higher risk of co-infection than the younger age groups. The other significant risk factors included barber shaving, self-flagellation, recreational drug use, circumcision by barbers, piercing, beauty parlor visits, history of blood transfusion and having >10 IV injections ( Table 4).

We have found age to be a significant risk factor associated with the occurrence of HBV ( Table 2). The younger age group (16–30 years) was at a 4.2-fold higher risk of HBV than the older age groups. The variation in the seroprevalence of HBV in different age groups could be due to the changing immune response of the body to infectious agents at certain ages, as also supported by the findings reported previously (Cheng et al., 2007). We also found males to be at a higher risk and barber shaving was significantly associated with HBV occurrence. Exposure to barber shaving, IV injections, and hijama therapy could be promoting the risk of HBV occurrence in males in our study population. Frequent exposure to barber shaving, surgical and dental procedures, blood donation and a higher number of injections being important risk factors for HCV infection has been reported elsewhere ¹⁰ . Our results are in line with the findings reported from other regions of the world ^{21, 25, 30} .

Hepatitis C viral infection has been reported to be an emerging epidemic in Pakistan ²⁵ . Here, an elevating trend of HCV seroprevalence was recorded with an increase in age ( Table 3). The highest seroprevalence of HCV was recorded in age group ≥61 years, followed by age group 46–60. This is likely due to a cumulative effect whereby the exposure to multiple risk factors increases with age. For instance, the number of child births, recreational drug use, exposure to barbers and numbers of injections all increase with age. These results are in line with the findings of 29. Hijama therapy was identified as a potential risk factor for HCV infection. This might be due to unhygienic environments where the procedure is carried out and the use of unsterilized instruments that are not properly autoclaved and are used on multiple individuals ²⁷ . Circumcision performed by barbers was also recorded as a risk factor in our study. This could be attributable to the use of contaminated instruments on multiple individuals ²⁶ . Other exposure variables found in this study have previously been reported, such as history of hospitalization, surgical procedures, recreational drug use, IV injections, tattooing, piercing, barbers shaving, beauty parlor visit, blood transfusion and self-flagellation with sharp objects ^{31, 32} .

The main strength of this study is that this involved 24 clinics based in various parts of the province and had a large sample size of 141,705 individuals, who were screened for HBV and HCV infections through ELISA. Data was collected through a centralized EMR system assuring the accuracy, completeness, consistency, reliability and repeatability of the data. Of the 141,705-study population, we only interviewed 12,427 participants for identification of risk factors associated with viral hepatitis. However, this sizeable number is still large enough to provide us reliable estimates ( Table 2– Table 4) on the role of various risk factors in the occurrence and spread of hepatitis in the local population. The limitations of the study are that all 141,705 individuals were not interviewed due to loss to follow up or low response levels in signing the consent form.

Figshare: Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multi-center cross-sectional study, 2017–18 (Extended Data File). https://doi.org/10.6084/m9.figshare.9891260.v1 ²³

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Figshare: Epidemiology of Viral Hepatitis B and C in Punjab, Pakistan: A Multicenter Cross-sectional Study, 2017–18. https://doi.org/10.6084/m9.figshare.9702845.v1 ²⁴

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Figshare: Questionnaire.docx for Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multi center cross-sectional study, 2017–18. https://doi.org/10.6084/m9.figshare.9993044.v1 ²⁰

Figshare: Epidemiology of viral hepatitis B and C in Punjab, Pakistan: a multicenter cross-sectional study, 2017–18 (Consent Form). https://doi.org/10.6084/m9.figshare.9730592.v2 ¹⁹

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).