RDT BASED DETECTION OF HEPATITIS B SURFACE ANTIGEN (HBsAg) AMONG BLOOD DONORS AT SPECIALIST

Hepatitis B infection is a major global health problem and a potentially life-threatening infection that attacks liver cells caused by Hepatitis B virus and it is characterised by liver cirrhosis, liver scarring, liver failure or cancer of the liver as the disease progresses. This study was carried out to determine theRDT based detection of hepatitis B surface antigen (HbsAg) among blood donors at specialist hospital, Sokoto, Sokoto State Nigeria. Two hundred (200) centrifuged sera from blood donors who have given prior verbal consent to participate in the study were screened for HBsAg using rapid diagnostic test (RDT). The result obtained shows that, 51 out of the 200 blood donors which represented (25.5%) were reactive while 149 were non-reactive. With Chi-square analysis only blood donors that are none previous blood donors showed a significant association with HBV

Hepatitis B infection is a major global health problem and a potentially life-threatening infection that attacks liver cells caused by Hepatitis B virus and it is characterised by liver cirrhosis, liver scarring, liver failure or cancer of the liver as the disease progresses. This study was carried out to determine theRDT based detection of hepatitis B surface antigen (HbsAg) among blood donors at specialist hospital, Sokoto, Sokoto State Nigeria. Two hundred (200) centrifuged sera from blood donors who have given prior verbal consent to participate in the study were screened for HBsAg using rapid diagnostic test (RDT). The result obtained shows that, 51 out of the 200 blood donors which represented (25.5%) were reactive while 149 were non-reactive. With Chi-square analysis only blood donors that are none previous blood donors showed a significant association with HBV infection, (p-value 0.05). This study however, indicates that, hepatitis B infection occurs in one out of every four blood donors. It also observed that the virus antigen is highly endemic among blood donor and these blood donors can serve as potential reservoir for the transmission of the virus to susceptible host individuals in the study area.

…………………………………………………………………………………………………….... Introduction:-
Hepatitis B infection is a major global health problem and a potentially life-threatening infection that attacks liver cells caused by Hepatitis B virus and it is characterised by liver cirrhosis, liver scarring, liver failure or cancer of the liver as the disease progresses (Isahet al., 2015). The disease is highly contagious and has been found to be fifty to one hundred (50-100) times more contagious and infectious than humans immunodeficiency virus and it is ten (10) times more infectious than hepatitis C virus, because it replicates profusely and produces high titter in the blood (10 8 -10 10 virions/ml). The virus can resist antibody product by the defence system, only people who have been vaccinated successfully or those who have developed HBs antibodies after hepatitis B infection are immune to hepatitis infection (Kaojeet al., 2018). Hepatitis B virus enters the liver through the bloodstream and its replication occurs only in the liver tissues (cytoplasm of the hepatocytes) thereby interfering with the liver function (Samuel, 2010). Hepatitis B virus has been detected in peripheral mononuclear cells, tissues such as pancreas, spleen, kidney and body fluids like blood, saliva, semen, sweat, breast milk, faeces, urine and vaginal secretion (Isahet al., 2015).

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There are high risk groups for hepatitis B virus infection; these include parenteral drug users, institutionalized parsons, health care personnel, organ transplant patients, blood transfusion and haemodialysis patients and staff that are highly promiscuous and infants born to infected mothers (Isahet al., 2015). Hepatitis B virus infection is mostly asymptomatic but in few cases the infection is symptomatic often with high morbidity and mortality (WHO, 2012). The virus (HBV) may establish a chronic infection especially in those infected as infants. Signs and Symptoms of an infection include: itchy skin, body aches, joint pains, fatigue, loss of appetite, nausea, vomiting, dark urine and jaundice (yellow colouration of skin and eyes). The highest concentrations of infectious Hepatitis B virus are found in blood and serum. However, other body fluids, such as semen and saliva have also been reported to be infectious (Colins et al., 2016).
There is a high level of occurrence of blood demanding health conditions in many parts of sub-Saharan Africa. In Nigeria, the increase in road accidents, pregnancy-related haemorrhages, armed robbery attacks, and violent events, increase the possibility of the transmission of HBV (and other blood-borne pathogens) through contaminated blood as reported by United Nations System in Nigeria (UNSN 2001) (Unekeet al., 2005). Blood transfusion service (BTS) is an integral and indispensable part of the healthcare system. The priority objective of blood transfusion service (BTS) is to ensure safety, adequacy, accessibility, and efficiency of blood supply at all levels (Islam, 2009). Transfusion of blood and blood components, as a specialized modality of patient management saves millions of lives worldwide each year and reduces morbidity. It is well known that blood transfusion is associated with a large number of complications, some are only trivial while others are potentially life threatening, demanding for meticulous pre-transfusion testing and screening. The use of unscreened blood and ineffective blood screening transfusion keeps the patient at risk of acquiring many transfusion transmitted infections (TTI) like hepatitis viruses (HBV, HCV), human immune-deficiency viruses (HIV), syphilis, malaria (Khan et al., 2007). Hepatitis B virus infection is a potentially life threatening liver infection caused by the hepatitis B virus, in some people, the hepatitis B virus can cause chronic liver infection and puts people at high risk of death from cirrhosis and liver cancer. However, some people have acute illness with symptoms that last several weeks, including yellowing of the skin and eyes (jaundice), dark urine, extreme fatigue, nausea, vomiting and abdominal pain (Idioha et al., 2010).

Materials and Methods:-
The study was carried out for a period of four month (February to April) in 2018. The study was conducted in Specialist Hospital Sokoto. The hospital is located in Sokoto South Local Government, Sokoto state. According to Sokoto State Diary SSGOD(2015) the state is located between latitudes 11 0 30`and 13 0 50`N and longitudes 4 0 and 6 0 E. Sokoto state shares border with Niger Republic to the north eastern part of the state. It shares common boundaries with Kebbi state to the west and Zamfara state to the south. The total land area is about 32,000 square kilometers, with total population, according to provisional figures of 2006 census, of 3,696,999 people (SSGOD, 2015). The state is characterized by alternating wet and dry seasons with a short spell of cold period (Harmattan) which usually lasts from December to February, with 3 to 4 months rainfall (from June to September or October). The duration and intensity of rainfall increase from north to south and environmental temperature ranges from 21 to 42 0 C and highest of (47 O C) recorded in April and the lowest 13 0 C recorded in January (Newtrack india.com, 2018). Culturally, the state is homogenous. The people of the state are Muslims and Islamic religion provides them with code of conduct and behaviours, their mode of dressing is also in line with Islamic tenets (Garba, 2015). The state has an estimated population of more than 4.5 million as according to census, (NPC, 2006)

Study population
The study included male and female blood donors which comprises two hundred (200) participants during the study time. Individuals with no history of hepatitis B infection or prior treatment against hepatitis B virus infection attending Sokoto State Specialist Hospital for blood donation were recruited and screened for the study.

Ethical consideration
Ethical approval was obtained from Institutional Ethical Review Committee of the Specialist Hospital and the Ministry of Health, Sokoto State.

Inclusion criteria and exclusion criteria
The blood donors of both sexes aged (18 and above years) who have given prior verbal consent to participate in the study and attendees off specialist hospital sokoto were included.

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Theexclusion criteria were the individuals who are less than 18 and are non-attenders of specialist hospital, sokoto were excluded.

Sample collection
Blood samples were collected aseptically using venepuncture method, a tourniquet were applied about two inches above the elbow or above the forearm and the site of puncture was disinfected using 70 % ethyl alcohol vigorously and allowed to dry (Jeon et al., 2011).Five millilitre of blood was collected using a 5 ml syringe into a properly labelled vacutainer plane bottles and transported to the central research laboratory, Usmanu Danfodiyo University for analysis.

Sample processing
Blood specimens were allowed on the bench for 30 minutes to allow coagulation after which the serum was separated by centrifuged at 1000 rpm for 5 minutes. Pasteur pipettes were used to carefully transfer the supernatant (sera) to plane cryovials. All serum samples were analysed for HBsAg using rapid strip test based on the principle of one-step test (Aspen, one strip Hepatitis B-surface antigen Test strip, USA),

Hepatitis B virus rapid strip test
Hepatitis B virus (HBsAg) strips test were used to detect the reactive and non-reactive hepatitis B virus surface antigen (HBsAg) among blood donors, the samples were analysed following standard procedures as described by the manufacturer. Each serum was screened for the presence of hepatitis B virus antigen (HBsAg) using one step test strip, which is a rapid chromatographic immunoassay for qualitative determination of HBV antigen in the serum

Principles of the test
This test device is a chromatographic lateral-flow immunoassay, including membrane pre -coated with anti-HBsAg antibodies and a pink coloured pad with colloidal gold reagents labeled with anti-HBsAg antibodies. The membrane has two coated lines: a test line (T line) and a control line (C line). At the T line, a pink coloured band develops if HBsAg is present in the specimen tested. If antigen is not present, no T line will develop. At the C line, a pink colored band should appear regardless of the presence of HBsAg. The C line serves as an internal qualitative control indicating that an adequate volume of solution added to the test system (according to manufacturer specification Instant-View HBsAg One-step serum test. ALFA Scientific Design USA).

Assay Procedure
The test strips were removed from the sealed pouch it was vertically immersed in the serum for 10 seconds. Then, removed and placed on a clean, flat, non-absorbent surface were allowed to stand for 15 minutes. The result was read between control and patient line (according to manufacturer specification).

Statistical analysis
Data obtained were recorded on excelspread sheet which was subsequently analysed using statistical Graph Pad software. Descriptive statistics of socio-demographic variable were computed. The prevalence was determined at 95% confidence interval (CI). A p value less than 0.05 was calculated to be statistically significant. The statistical difference was also evaluated by applying the Chi-square test. All statistical analysis was done using the Statistical Package for Social Sciences (SPSS) software package version 10 (SPSS Inc. Chicago, USA).

Interpretation of result
Observation for band information as done and results recorded. At the T line, a pink coloured band developed in the present of HBsAg in the specimen tested. But in the absence of antigen, no T line was developed. At the C line, a pink coloured band should appear regardless of the presence of HBsAg.
A total of two hundred (200) of milliter blood sample of blood donors were analysed for seroprevalence of hepatitis b surface antigen which indicate the presence of HBV in blood. The result of this study revealed the overall prevalence of hepatitis B Sero-antigen among the blood donors to be 25.5%.were 149 tested negative for hepatitis B surface antigen.
Result obtained in relation to gender shows that, 51 were positive cases, 48 among them were male while 3 were female with 27.9% and 10.7 respectively (Table 4.1).

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Distribution of hepatitis b sero-antigen among blood donors in relation to marital status shows that, out of two hundred (200) samples subjected to general screening using rapid strips test, one hundred and forty-one 141 were married while 59 were unmarried. Out of the 141 married study subjects, 35 were positive for HBV (24.8%) of the 59 unmarried donors 16 were positive for HBV (27.1%), (Table 4.2).
Hepatitis B sero-antigen detected in serum sample of intravenous drugs users, were 6/22 (27.27%) were intravenous drug user blood donors while one hundred and seventy-eight 178 blood donors were non intravenous drugs user. Out of which 45 were positive cases of hepatitis B sero antigen with 25.2% prevalence (Table 4.3).
Out of the two hundred study subject, 123 had no history of previous blood donation, and 31 were positive for 27.2% prevalence while 77 had history of blood donation, out of which 20 were positive cases with an incidence of 25.9% respectively ( Table 4.4).
The age distribution of Hepatitis B virus sero-antigen among blood, among (200) subjects, subjected to general screening using rapid strips test base on the principle. It was observed that, majority belong to the age group of 15-30 years with proportion percentage of 32.0%, flowed by age group 31-45 years and 45 above with percentage of 21.5% and 21.6% respectively (   Statistically using chi-square shows that x 2 = 0.115, DF = 1, p-value 0.734, old ration 01.127 and at 95% C I = 0.5 -2.246 not significance The result shows that previous blood donation were 20/77 (25.97%) positive and Never blood donation were 31/123 (27.20%). Chi-square shows that x 2 = 0.903, DF = 1 and p-value 0.015, old ration 1.041and C I 0.543-1.999 is significance.

Discussion:-
Hepatitis B virus is considered one of the most common viral infection spread through blood transfusion; therefore, Safety of blood products is a critical factor for successful blood transfusion as it decreases the incidence of post transfusion hepatitis B infection. Out of two hundred (n-200) samples screened using HBsAg rapid strip test, 51 study subjects tested reactive, giving an overall prevalence of 25.5%. This implies that hepatitis B is positive in one out of every four blood donors in the study site. The value obtained in the present study is regarded as high as described by World Health Organisation classification of assessment of the severity sof hepatitis B virus infection in hepatitis B virus endemic countries. WHO defines low prevalence to be less than 2%, moderate 2-8% and high when values are greater than 8% (WHO, 2012). The result obtained in this study is similar to 25.7% obtained by Bada (1996) Musa et al.,(2013) in an prevalence of hepatitis B in Nigeria. These differences might be by using difference methods, it might also be related to the fact that, the infection tend to vary from one locality to another and from one country to another and also differ in associated risk factors. The value obtained in this study is far less than 42.1% reported by Kristen et al.
(1999) and 38.1% reported by Alkali et al. (2017), in Sokoto, these could be attributed to the differences in the sampling population and associated risk factors. For instance, Alkali et al. (2017) studied in inmates in correctional facilities who were at high risk of harbouring infectious diseases, unlike the study subject individuals sampled in this study who are more likely blood donors. Thus, it is not unexpected if the incidence rate is lower in this population. Gender specific prevalence of hepatitis B virus infection among the study subject blood donors, revealed out the prevalence of 12.0% (03/28) for females and 38.7% (48/172) for male. Therefore, there was tune out of male individuals with high frequency in blood donation than female during the period of this study. The Male might be a carrier of this infection which might be easily transferred during blood donation, in considering they were in high frequency in blood donation than females in the study area. This finding agrees with that of Kouassiet al. (2011) and Pennap(2011) in a tertiary institution in Nigeria where results obtained showed a higher prevalence of HBsAg 193 among male than female. This result is higher than an incidence of 24.7% male and 28.2% incidence obtained by Mustapha and Jibrin (2004) in Gombe, it also deferred from the prevalence of 93.6% male and 6.4% in females reported by Wasfi and Sadeket al. (2011) in Alexandria Egypt. This could be attributed to the higher percentage of male to females study subjects in the study. The result obtained in this study is higher observation made by Lavanyaet al. (2012) WHO reported an incidence of 3.5% among all the study subject male individuals, but no female donors were positive for the virus. In a similar study by Uneka (2005) in Jos Nigeria, 12.9% for female and 14.3% for male were observed to be positive. The differences might be due to differences in the inclusion criteria. Therefore, the finding in this study may be explained by the fact that male preponderance common and more obvious in blood donation than female.
In relation to Marital status, highest prevalence was recorded among Single 27.11% (16/59)  . This may be explained by the fact that promiscuity and unprotected sexual behaviours among single unmarried individuals might be higher than among the married in the study area. Therefore, increasing the risk of acquiring the viral infection is due to their inability to stick to only one sexual partner. The implication is that, it will be easier for them to transmit the hepatitis B sero antigen during blood donation.  27.2%. Therefore, using ELISA here may serve as confirmatory test, it will serve as a factor of transferring the infection from blood donors during blood donation.
Three (3) out of the eleven Elisa samples showed HBsAg profile. Two of the samples showed;HBeAb, HBcAb and HBsAg positive, which indicates that the presence of the virus may be as a result of vaccination or the patient is in the recovery state from a recent infection while among all subjected samples viral envelop antigen HBeAg not presence, it shows that no viral in blood circulation while the remaining seven sample were negatives for HBeAg.

Conclusion:-
It is clear from this study we found that sero-proportion percentage of Hepatitis B viral antigen among blood donors to be 25.5% using RDT, it indicate that hepatitis B infection occurs in one out of every four blood donors, and one out of every eight none reactive donors from the RDT screening. The study has also observed that the virus antigen is highly endemic there are many unvaccinated blood donor, based on this, these blood donors can serve as potential reservoir for the transmission of the virus to susceptible host individuals in the study area.

Recommendations:-
From this study, following recommendations are suggested; General vaccination, mass immunization public health education should be carried out and compulsory blood screening of Hepatitis B virus of blood donors before blood transfusion is very necessary and should be maintained as routine.

Conflict of interest;
The authors declare no conflicts of interest in this work.