Sero prevalence of Hepatitis B Virus (HBV) in general population of Lakki Marwat, Khyber Pakhtunkhwa- Pakistan

A research study was conducted with the aim to investigate the frequency of Hepatitis B Virus positivity in the general population of district Lakki Marwat. Blood samples were collected from 2000 individuals in various hospitals and diagnostic/ medical laboratories of the study area. All the blood samples were evaluated for HBV by Liver function tests (LFTs), Immunochromatographic tests (ICT) and PCR. Among the total, 108 blood samples were found positive for the virus with a prevalence rate of 5.4%. A higher prevalence rate (4.15%) was found in males as compare to 1.25% in females. Among the total HBV positive samples 83 (76.85%) were male (from 4 years to 75 years) and 25 (23.19%) were female (from 5 years to 60 years). The HBV positive data was also analyzed for various age groups i.e. 1-15 years, 16-30 years, 31-45 years, 46-60 years and above 60 years. It was found that disease prevails more in males than in females and is predominant in the age group 16-30 (57%), followed by age groups 3145 (23%), 1-15 (10.18%), 46-60 (7%) and >60 (3%) respectively. Various risk factors were found involved in the spread of HBV in the study area like medical injections (59.26%), dental procedures by medical quacks (12.04%), hair dressing and shaving salons/ barber shops (11.11%), transfusion of blood and blood based products (5.56%), mother to children transmission (3.70%), occupational risk groups (2.78%), sexual transmission (2.78%), tattooing (0.92%), circumcision (0.92%) and shared injection by drug abuser (0.92%).


Introduction
Hepatitis which literally means inflammation of the liver; is caused by six different types of viruses, which are hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV). These viruses are very distinct from each other and the similarities exist only in their organ specificity and epidemiology. Hepatitis A and hepatitis E viruses cause water and food borne diseases while hepatitis B, hepatitis C, hepatitis D and hepatitis G viruses cause blood-borne diseases [1,2]. Various dreadful diseases like chronic hepatitis, hepatic liver cirrhosis (HLC) and hepatocellular carcinoma (HCC) are caused by Hepatitis B Virus (HBV) of the family hepadnaviridae. The virus has a circular partially double stranded DNA genome [3]. The virus infects about 400 million people from all around the world with majority (75%) patients from Southeast Asia and Fareast Asia, where it causes one million per annum deaths [4]. There are reservations and uncertainties about the HBV origin and its association with humans. Relationship of HBV with humans started some 33600 years ago [5]. In another work, the researchers sequenced the avian hepadna virus in Zebra finches and claimed that viruses of the Hepadnaviridae evolved some 40-80 million years ago [6]. On May 2018, Barbara Mühlemann and others reported that they have discovered ancient hepatitis B virus in 4,500 years old human remains. They analyzed 12 ancient HBV genomes that were obtained from 800 to 4,500 years old human remains from Europe and Asia. According to them the HBV association with humans started some 8.6 to 20.9 thousand years ago [7]. In another study the complete genome sequence of HBV from an Italian child mummy of mid 16 th century was recovered [8].

Data collection
A 3ml venous blood sample was collected from each of 2000 individuals who came to visit the hospitals and diagnostic/ medical laboratories of the district Lakki Marwat for various purposes. Blood sample was collected after taking their consensuses. The blood samples were accumulated in small veils having anticoagulant EDTA and were labeled with natural numbers. Basic information like name, father's name/ husband name, sex, age, contact number and locality against each number was recorded on separate sheet. For sera separation, the samples were centrifuged at 5000 rpm for 15 minutes.

Screening of samples
Liver Function Tests were performed on all blood samples and HBV positive blood samples were tested by Immunochromatography tests and then by PCR for confirmation of hepatitis B infection.

Bio-chemical diagnosis
The Liver Function Tests (LFTs) especially ALT (Alanine Aminotransferase Test) and AST (Aspartate Aminotranferase Test) were performed by using Microlab 300 (Merck, USA), ALT and AST kit (Diasys Diagnostic System Germany).

Immuno-chromatography tests (ICT)
Immuno-Chromatography Tests were performed for confirmation of HBsAg in the HBV patients. InTec (China) and Determine (Abbot) ICT kits were used. The reason for using these kits was their coast effectiveness and 98% sensitivity [24]. HBV-DNA detection by PCR The ICT positive blood samples were confirmed by PCR. . All blood samples were tested by Liver Function Tests (LFTs), and then by Immuno-chromatography tests (ICTs). For further conformation the positive blood samples were evaluated by PCR and 108 blood samples were found HBV positive ultimately with a prevalence rate of 5.4% in the general population of the study area (Table 1). Among the total HBV positive samples 83 individuals (76.85%) were male (4 years to 75 years) and 25 individuals (23.19%) were female (5 years to 60 years). The occurrence of disease in males was found 4.15% and in females it was quite low i.e. 1.25%. The HBV positive data was also analyzed for various age groups i.e. 1-15 years, 16-30 years, 31-45 years, 46-60 years and more than 60 years. It was found that disease is more prevalent in males than in females and predominated in age group 16-30 (57.41%), followed by age groups 31-45 (23.15%), 1-15 (10.18%), 46-60 (6.5%), and >60 (2.8%) respectively (Table  2). To know about the risk factors the patients above age 16 years were investigated that "what would be the possible reason to get this disease in their view?" The above said question was asked from the parents of kids (≤ 15 years of age). Their responses were analyzed and it was found that medical injections (59.26%), dental procedures (12.04%), hair dressing & shaving in barber shops (11.11%) and transfusion of blood & blood based products (5.56%) are the major risk factors of HBV spread in the study area while mother to children transmission (3.07%), occupational risk groups (2.78%), sexual transmission (2.78%), Circumcision (0.92%), Tattooing (0.92%) and injecting abused drugs (0.92%) are least significant risk factors (Table 3).  The reason of relatively high prevalence of disease in the general population of district Lakki Marwat than Dir, Charsadda and Swat districts may be due to the fact that the study area is a suburb of war stricken areas which show a higher prevalence. Hepatitis B victimizes the individuals of rural area 3.8 times more than urban areas and similarly the disease is 2.2 times more prevalent in the individuals with poor economic conditions. Since the study area fulfills both the conditions, therefore it might be the reason of relatively higher HBV prevalence in the area [25].

Hepatitis B Virus spreads by vertical and horizontal
transmissions. Vertical transmission means transfer of HBV from mother to her children while horizontal transmission means proliferation of disease by body fluids (blood and lymph etc). Various risk factors prevailed in the study areas are following.

Medical injection
It is the one of the major risk factor of HBV spread in the area. Patients usually prefer injectable over oral medicines because they consider them quick relief provider. The use of medical injections is very common practice in the district, especially in the rural area, where people usually consult medical quacksalvers. These quacksalvers use contaminated needles and they use the same syringe to give many injections to different patients. Due to low literacy rate the patients are unaware of the fact that these contaminated injections are the major source of HBV transmission. Some authors consider the use of medical injections as a potential threat of hepatitis B in Pakistan [42, 43].

Dental surgery
Dental treatment demands a great care and hygiene. Since qualified dentists and oral surgeons claim high charges, so people prefer to take services from dental quacks with unsterilized equipment which may cause dreadful diseases like hepatitis B [44, 45].

Barber shops
At the barber shops, the procedures of work are not safe. They do not use disposable razors/ blades rather use the same razor/ blade to shave many customers. HBV infected razor may transmit this virus to others [46].

Transfusion of blood and blood based products
Blood banks usually acquire blood from paid blood donors and amongst them many are drug abusers. Drug abusers are mostly hepatitis patients and hence may be a potential source of the disease [47, 48].

Mother to child and intra-familial transmission
Vertical transmission of HBV from infected mother to her children is a wellknown route of hepatitis B transmission [49]. Transmission of the virus among family members is also a potential source of the disease [50, 51]. Due to low literacy rate and prevailing poverty in the area, people do not take necessary steps to avoid this disease.

Sexual transmission
As HBV is found in body fluids (blood and semen etc) so it transmits from one partner to the others during sexual process [52][53][54][55].
Inhabitants of the area usually do not follow the precautionary measurement even when they come to know that the other partner is HBV positive and hence this risk factor prevails significantly in the district. 7. Occupational risks Professionals of health services, prostitution, and haircutting and shaving are at greater risk of catching HBV than general population [54, [56][57][58][59][60][61]. A study conducted on 75 health care workers of three major hospitals of the KP province and reported a 2.18% hepatitis B prevalence rate [62]. In another report [63] 11.65% occurrence rate of HBV in the female sex workers of Lahore was observed.

Tattooing
To express love and affection with someone many youngster of the district make tattoos. They imprint the names of their special one, shapes of heart or alphabet letters of their beloved on their hands and arms. Many females of the district tattoo a traditional green, blue or black spots (called Khaal) on chins, foreheads and between the eye brows. Tattoo making is also a potential source of HBV [64][65][66]. So precautions and safety measurements are vital to avoid the cruel clutches of hepatitis B.

Circumcision
In the rural areas of the district circumcision is mostly done by local barbers and medical quacks. People prefer them due to easy access and low fee. This is also a contributor of HBV spread in children. Different studies also reconcile the HBV transmission by this procedure [