Seroprevalence and risk factors of hepatitis B virus among blood donors in district Charsadda Khyber Pakhtunkhwa Pakistan

Hepatitis B Virus (HBV) is a serious worldwide public health issue both in underdeveloped and developed countries. About two billion individuals were infected by HBV globally, of which 400 million were chronic HBV carrier. The aim of this retrospective study was to investigate the seroprevalence and some possible risk factors of hepatitis B virus infection among male blood donors in district Charsadda, Khyber Pakhtunkhwa Pakistan. A total of 460 male blood donors with age range 15-55 years were screened for HBsAg by Immunochromatographic technique (ICT) and 3 generation enzyme-linked immunosorbent assay (ELISA). Out of 460 samples, 11(2.39%) were found positive for HBsAg by ICT, and 13(2.82%) were HBsAg positive by ELISA. The mean seroprevalence of HBV from both ICT and ELISA was 2.60%. The highest seroprevalence (46.15%) was observed among the donor groups with age range of 21-30 years followed by (30.76%) among 15-20 years, (15.38%) among 31-40 years and the lowest seroprevalence (7.69%) among 41-55 years. Blood transfusion (30.76%) was the most apparent influencing risk factor for HBV followed by dental treatment (23.07%), Sexual partner positive for HBV infection (15.38%), surgery and shave by the barbers (7.69%) for each and unknown reason (15.38%).

. Some important ways for the transmission of HBV are the utilization of unsterilized medical instruments, blood transfusion, sharing of individual items, offering needles to drug addicts, barber risk, reuse of contaminated needles and syringes without sterilization for therapeutic injections, vertical transmission , furthermore by unsafe sexual interaction with HBV infected patients [9-12].The prevalence of HBV infection among the developing countries of Asia, the Pacific Islands and Africa is very high as compared to developed countries like Australia, Western Europe, and the USA, where the prevalence of HBV is very low [13].In Russia, Japan, and Eastern Europe about 2-8% of their population are infected with HBV.It is estimated by world health organization (WHO), that about 0.6 million deaths of the people occur annually due to HBV infection.As per 2010 report of WHO, it was observed that 0.12 billion individuals were infected with HBV in China followed by India with 0.04 billion and Indonesia with twelve million infected individuals [14].In Pakistan, HBV is also a prominent public health issue, and its rate of infection is rising rapidly [15].

Study design and sample collection
This retrospective study was conducted at the Department of Pathology, Hayatabad Medical Complex (HMC), Peshawar, from 3 rd September 2015 to 30 th August 2016.A total of 460 blood samples were collected from volunteer blood donors of district Charsadda and tested for hepatitis B surface antigen (HBsAg).Risk factors were evaluated carefully, and detailed medical history of each blood donor was recorded on structured questioner (Performa) and consent form.All the blood donors included in the study were male with age range 15-55 years.About 5 mL blood sample was collected from each blood donor with a disposable syringe under aseptic conditions and allowed to clot.Serum was separated from each blood sample in a centrifuge machine at 4000 rpm for 5 minutes and was transferred to sterilized test tubes for further qualitative and quantitative screening tests.The initial qualitative tests were carried out by chromatographic immunoassay (Acon, USA) for the detection of HBsAg in blood serum.All the positive and negative samples on ICT were further screened on 3 rd Generation ELISA Technique (EASE BN-96 TMB, Taiwan) to evaluate the specificity and sensitivity of ICT and ELISA assays.

Detection of hepatitis B surface antigen (HBsAg) by ICT
We used ICT strips (Acon, USA) according to maker's instructions for the detection of HBsAg.The test strip was removed from the foil pouch and was kept on a clean, dry surface.Then 100 μL serum sample was dispensed in the strip.After 15 minutes, the results were interpreted according to the appearance of color bands.The control was also run to check the validity of the strip.Both purplish red test bands and purplish red control band appeared on the membrane of the strip which showed a positive result.One red line appears in the layer of the strip in the control region (C).The appearance of no red line in the test area indicated a negative result [19].Detection of hepatitis B surface antigen (HBsAg) by ELISA All the positive and negative samples on ICT were further screened on third generation ELISA (EASE BN-96 TMB, Taiwan) according to maker's instructions.Three wells covered with anti-HBs were taken and placed in a holder.50 μL of the positive control, negative control and specimens were added to their respective wells.Then 50 μL of HRP-conjugate was added to each well except the blank and were mixed by tapping the plate gently.Covered the plate with glue slip and was incubated at 37°C for one hour.After incubation, the glue slip was removed from each well and washed five times with diluted buffer.50 μL of chromogen solution A and 50 μL of chromogen solution B were dispensed into each well including the blank and were mixed by tapping the plate gently for 15 seconds.The plate was then incubated at 37°C in the dark for 15 minutes without shaking.50 μL of Stop solution was added to stop the reaction.The absorbance of controls and specimens were determined within 15 minutes by spectrophotometer.The enzymatic reaction between the chromogen solutions and HRP-conjugate produced a blue color in positive control well and HBsAg positive sample wells before the addition of stop solution.After addition of stop solution, the blue color in positive control well and HBsAg positive wells changed to yellow color; Negative samples have clear water like appearance before and after addition of stop solution.Absorbance values of Specimen equal to or greater than the cut-off value i.e. (2.00) were considered HBsAg positive and Specimen with absorbance values less than the cut-off value were considered HBsAg negative [20].Results A total of 460 male blood donors with age range 15-55 years were tested for HBsAg by Immunochromatographic technique (ICT) and 3 rd generation enzyme-linked immunosorbent assay (ELISA).Out of 460 blood samples, 11(2.39%) were found positive for HBsAg by ICT, and 13(2.82%) by ELISA.The mean seroprevalence of HBV from both ICT and ELISA was (2.60%), (Table 1).The highest seroprevalence (46.15%) was found among participants within the age range of 21-30 years followed by (30.76%) among 15-20 years, (15.38%) among 31-40 years and lowest seroprevalence (7.69%) among 41-55 years (Figure 1).Blood transfusion (30.76%) was the most apparent influencing risk factor for HBV followed by dental treatment (23.07%), Sexual partner positive for HBV infection (15.38%), surgery and shave by the barbers (7.69%) for each and unknown reason (15.38%) (Figure 2).

Discussion
The purpose of the current study was to investigate the seroprevalence of HBV infection and its possible risk factors associated with HBsAg positivity through ICT and 3 rd generation ELISA among healthy blood donors of district Charsadda, Khyber Pakhtunkhwa Pakistan.All the blood donors included in this study were males because the majority of females in Pakistan do not donate blood due to traditions, especially in Khyber Pakhtunkhwa.There is also an urgent need to increase relevant guidelines for counseling and management of HBs Ag-positive blood donors.It is necessary to implement the strict standard selection criteria to defer and discourage the blood donors with certain high-risk factors.Vaccination for HBV should be adopted at national level.ELISA assay should be adopted in routine blood screening to prevent the transmission of HBV infection and to ensure the safe blood transfusion

Figure 1 .Figure 2 .
Figure 1.Percentages of the different age groups of blood donors from district Charsadda, KP Pakistan.The blue color in the figure showed positive samples and red color showed the positive percentage of different age groups