PREVALENCE OF HEP ATITIS B AND C VIRUS ANTIBODIES AMONG S TUDENTS INVOLVED IN RIS KY S EXUAL B EHAVIOR IN A NIGERIAN TERTIARY INS TITUTION .

Oluboyo Bernard Oluwapelumi, Sunday Samuel Ik o-Ojo, Akinseye Janet Funmilayo, Akele Richard Yomi. Oluboyo Adeola Olanrewaju 1 , Adewumi Funmilayo. 1. Department of Medical Laboratory Science, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti. 2. Department of Microbiology and Parasitology, College of Medicine, Ekit i State University, Ado -Ekiti. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 09 August 2018 Final Accepted: 11 September 2018 Published: October 2018

Sexual contact is one of the ways hepatitis B and C can be transmitted. Limited investigations have been recorded on the prevalence of HBV and HCV antibodies among students involved in risky sexual behavior in tertiary institutions in Nigeria. This study was conducted to determine the prevalence of Hepatitis B and C antibodies in such group of people. Structured questionnaires were administered to obtain data on sexual behavior of the participants. A total of 91 students consisting of 46 males and 45 females between ages 16 and 25 years were examined using enzyme lin ked immunosorbent assay (ELISA) technique. Liver en zy mes of ELISA positive samples were assayed. Prevalence of HBV and HCV antibodies among students involved in risky sexual behavior were 23.1% and 0% respectively. There was no significant difference between the prevalence of HBV antibodies among gender. Although higher prevalence was recorded among students between ages 16-20, in relation to students in ages 21-25, the difference is not statistically significant (>0.05). The prevalence of HBV antibodies among students involved in mult iple sex partnership was significantly h igher (p <0.05) than those involved in single sex partnership. The prevalence was significantly higher (p<0.05) in noncondom users than in condom users. Six (6.0%) of the students recorded raised liver en zy mes. Students involved in risky sexual behavior are at high risk of HBV infection especially in cases of mu ltip le sex partnership. Condom usage may not provide risk-free sex for users. Enlightenment campaign against risky sexual behaviors in higher institutions is advocated.

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Hepatitis B is frequently acquired when blood, semen, or other body fluids fro m an infected person gain entrance into the body of an uninfected person (WHO, 2017). Workers in healthcare centers, blood transfusions centers, dialysis rooms, people living with an infected person and travelers to countries where the infection rate is high are all classified as risk group to the viral infection (CDC, 2011). Hepatitis B virus is said to survive outside the body for at least 7 days and remains highly infective if it enters the body of a person who is not protected by the vaccine (WHO, 2017). Hepatitis B virus (HBV) and hepatitis C virus (HCV) are spread mainly through contaminated blood and blood products, sexual contact and contaminated needles (Rikab i et al., 2009). The virus replicates profusely and produces high titre in the blood such that any parenteral or mucosal exposure to infected blood poses a high risk of infection (Pennap et al., 2011). HBV and HCV are both associated with jaundice, liver cancer, and liver damage (Ayolabi et al., 2006).
Although much is known about the epidemiology of HBV in Nigeria, limited investigations have been published on HBV among students in tertiary institutions in Nigeria involved in risky sexual activ ities. Studies carried out by various authors have shown that HBV infections are highly prevalent among Nigerians (Imarenezor et al., 2016). Researchers focused on infections in various groups such health care workers such as blood donors and medical waste handlers; there is little informat ion on the prevalence of the virus among the youths at risk of infection because of their sexually act ive stage. This work is therefore aimed at determining the seroprevalence of Hepatit is B and C viral in fection among students involved in risky sexual act ivities in a Nigerian tertiary institution of learning.
Sexual t ransmission of hepatitis C has been a subject of debate, however, some researchers opined that sexual practices that involve higher levels of trauma to the anogenital mucosa, such as anal penetrative sex, or that occur when there is a concurrent sexually transmitted infection, includ ing HIV or genital ulceration, do present a risk (Tohme and Ho lmberg, 2010). There is scarce information on the epidemio logy of HCV in Nigeria; however, it has long been suspected that it may be endemic. So me studies in Nigeria reported prevalence of 12.3% among adult blood donors (Ejiofo r et al., 2010), 11.0% among doctors and dentists (Olubuyide et al., 1997).
Youth sexual behavior is highly significant to different public health problems. Unprotected vaginal sexual intercourse among youth has contributed to unwanted pregnancies, abortions, pregnancy related complicat ions, and sexually transmitted infections (Tang et al., 2011). Substantial increase in the proportion of undergraduate students who report sexual activity wh ile at school in Nigeria has been observed. Studies have reported this among the adolescents (Owuamanam 1995;Ugoji 2008). Sexual behaviors particularly among undergraduate students has become alarming and worrisome (Ugoji 2014) in view of the associated short and long term effects. Risky sexual behaviors among undergraduates involve the practice of mult iple sex partnership, group sex, sex withou t condom, anal and oral sex.These actions expose students to sexually transmitted infections.
The prevalence of chronic HBV ranges from less than 2 percent in low-prevalence areas (e.g. United States, Canada, Western Europe) to 2-7 percent in intermediate-p revalence areas (e.g. Mediterranean countries, Japan, Central Asia, Middle East, and parts of South America) to ≥8 percent in high -prevalence areas (e.g. Western Africa, South Sudan) (Ott et al., 2012;Zhang et al., 2016). Nigeria is classified among the high-prevalence areas.
In Nigeria, the risk of acquiring HBV in fection is considerable; as many as 75% of the population may be exposed (Ola et al., 2002). So me reports have shown varying risk group-specific estimates. A prevalence of 10-15% in the average risk Nigerian population has been reported (Emechebe et al., 2009), researchers have reported high HBV prevalence among surgeons (25.7%), voluntary blood donors (23.4%), and infants (16.3%) (Musa et al., 2015). Information on the prevalence of HBV and HCV among students in tertiary institution involved in risky sexual behavior will be an added contribution to reports on risk specific groups.

Study area
This study was carried out among students a tertiary institution in Ado -Ekit i, Nigeria. Ado-Ekit i is a city in southwest Nigeria that lies between latitude 7 o 35` and 7 o 38 North of the equator and Longitude 5 o 10 and 5 o 15` East of the Greenwich Meridian (Adebayo and Jegede, 2010),

Eligibility criteria
659 Students between ages 15 and 35 years involved in sexual acts who consented to the study were elig ible for the study. Students who have been transfused with blood, intravenous drug users and those with tattoo marks on their body were excluded. Students without sexual experience were also excluded fro m the study. Ethical considerati on Ethical clearance was obtained from the research and ethics committee of the institution under study. The study participants were informed about the purpose of the study and consented to the study.

Sample size
The sample size was determine using the following formu la (Smith, 2013) using an estimated prevalence of HBV of 6.0% in Nigeria tertiary institution (Imarenezor et al., 2016) at a confidence level of 95 and desired precision of 0.05. Ninety one (91) students were enrolled for the study. These comprised of 45 females and 46 males.

Sample collection
A semi structured questionnaire was used to obtain informat ion on student's sexual behaviors. Blood was collected aseptically fro m consenting individuals by venipuncture, allowed to clot and the serum was separated by centrifugation at room temperature for 3000 revolution per minute (rp m) fo r 10 minutes into screw-capped plain bottles, stored at -20 o C, until ready for analysis ( Tammen et al., 2005).

Sample anal ysis
The serum samp les were analyzed using ELISA technique for identificat ion of antibodies to Hepatitis B and C.

Hepatitis B surface anti body (HBsAb) ELIS A test
AccuDiag TM HBsAb ELISA (DIA GNOSTIC AUTOMATION INC., Woodland Hills, CA) is a solid phase enzyme lin ked immunosorbent assay based on the principle of double antigen sandwich technique for the detection of antibodies to HBsAg in human serum or plas ma (Hoofnagle, and Di-Bisceglie, 1991). During the assay, the test specimen and horseradish peroxidase-ABsAg (HRP-HBsAg) conjugates are incubated simu ltaneously with the coated micro wells. HBsAb, if present in the specimen, reacts to the HBsAg coated on the micro well surface as well as the HRP -HBsAg conjugate, forming sandwich complex conju gates. Unbounded conjugates are then removed by washing. The presence of the complexed conjugates is shown by a blue color upon additional incubation with 3, 3', 5', 5'-tetramethylebenzid ine (TM B). The manufacturer's procedure was strictly followed. The optical density was measured at 450n m wavelength using a plate reader (MARVOTECH PLATE READER, China).

Hepatitis C virus (HCV) anti body ELIS A test
The test principle is based on indirect enzyme immunoassay. Micro titer wells are coated with certain amount o f HCV reco mb inant antigens. Then serum samples are allo wed to react with solid phase antigens. If HCV-specific antibodies (IgG and IgM) are presented in the serum they will b ind to HCV antigens through their indiv idual Fab section. After incubation, the wells are washed to remove unbound antibodies and anti-human antibodies (IgG/IgM) conjugated with HRP is added into the wells following another incubation and wash step. A solution of chromogen 3,3',5',5'-tetramethylebenzidine (TM B) is added and incubated for 15 minutes, resulting in the development of a blue color. The color develop ment is stopped with the addition of stop solution, and the color is changed to yellow and measured spectrophotometrically at 450 n m in a MARVOTECH PLATE READER (China).

Alani ne ami notransferase (ALT) estimation
Alanine aminotransferase (ALT) is incubated at 37 °C for exactly 30 minutes in a pH 7.4 buffered substrate containing alanine and alpha-ketoglutarate. ALT catalyzes the transfer of the amino group from alanine to ketoglutarate, forming pyruvate and glutamate. The pyruvate reacts with 2, 4 -dinit rophenylhydrazine (DNPH) to form 2, 4-din itrophenylhydrazone which in an alkaline mediu m gives a red -brown colour. The absorbance of the colour produced is measured in a spectrophotometer (PIOWA Y, Ch ina) at 546 n m wavelength (Young, 1997). The corresponding concentration of the optical density was recorded from the plotted graph of ALT activity chart. Reference range of A LT is 7-56 international units/Litre (IU/ L) (Shivaraj et al., 2009).

Aspartate ami notransferase (AS T) esti mation
The enzy me aspartate aminotranceferace (AST/ GOT) cataly zes the transfer of an amino group fro m L-aspartate to 2-o xoglutarate to give L-glutamate and o xalacetate. The o xalacetate reacts with reduced Nicotinamide adenine dinucleotide (NADH) in the presence of Malate dehydrogenase (MDH) to produce Malate and NAD. The decrease 660 in NADH concentration, measured at 340 n m, is proportional to the AST/GOT activity. Lactate dehydrogenase (LDH) is present in the substrate to convert endogenous pyruvate to lactate prior to measuring the AST/GOT level (Young, 1997). The concentration of AST in the serum was obtained from the plotted graph of AST activity chart. Seru m AST reference range is 0-35 IU/ L (Shivaraj et al., 2009).

Result:-
Students having multip le sex partners numbered 30 (33%) as against 61(67%) recorded by students having a single sex partner. Condom users were 59 (64.8%) while none condom users were 32 (35.2%). Of the 91 part icipants, 21 (23.1%) were positive and 70 (76.9%) were negative for Hepatit is B virus (HBV) antibody. None (0.0%) of the participants was positive for HCV antibody. Prevalence of HBV antibody was significantly higher (p < 0.05) among mult iple sex partners in relation to single sex partners (Table 1). Non-condom users recorded a significantly (p < 0.05) higher rate of infect ion than condom users ( Table 2). There was no significant difference (p > 0.05) between HBV infect ivity among the gender (Table 3). There was also no significant difference (p > 0.05) between the age ranges of the student in relation to HBV infectivity ( Table 4). Six of the ELISA positive samples recorded A LT and AST above the upper limits of the reference ranges giving a total prevalence of suspected active infection as 6.0%.

Discussion:-
An overall prevalence rate of 23.1% was recorded for HBV antibody and 0% was recorded against HCV antibody in this research. This result for HBV may be regarded as high according to WHO classification for assessing severity of HBV infections in HBV endemic countries. WHO defines low prevalence to be < 2%, moderate to be 2-8% and high prevalence to be > 8% HBV positivity (WHO, 2010). Records are scanty on the prevalence of HBV antibody among tertiary students involved in sexual act ivities. Ho wever, seroprevalence rate for HBV antibodies recorded in random samp ling of students irrespective of their sexual status are more available. Such records include prevalence of 6.0% recorded among students of the Federal university of wukari, Taraba state, Nigeria (Imaren ezor et al., 2016), 4.5% recorded among students of university of Maiduguri in Nigeria (Dawurang et al., 2012) and 12.5% reported among asymptomat ic students in Ahmadu Bello Un iversity, Zaria (A minu et al., 2013). Also, the prevalence rate of 9.5% reported in a population of students at Ladoke Akintola Un iversity of Technology in south west Nigeria (Mabayoje et al., 2010), 9.2% recorded in Ahmadu Bello university, Zaria, Kaduna State, Nigeria (Isa et al., 2015); and 13.2% recorded in Nassarawa State Univers ity in Keffi, Nigeria (Pennap et al., 2010) for HBsAg are all lower than the prevalence rate recorded in this study for HBV antibodies. The reason is that while these researchers investigated randomly selected subjects irrespective of risk exposure, this s tudy focused only on a group of subjects with sexual risk behavior only . Thus, risky sexual behavior is an important risk factor in the transmission of HBV virus in tertiary institution of learn ing in Nigeria.
No case of anti-HCV antibody was recorded in this study. This is consistent with WHO report that transmission of HCV through sex is uncommon (WHO, 2017). As this study excluded other risk factors for HCV infection such as intravenous drug use, blood transfusion, sharing of blades and contaminated sh arp objects, reuse of contaminated needles were excluded fro m the study. These factors may be responsible for the prevalence of HCV recorded previously by other researchers.
Risky sexual behavior among the students (and perhaps, youth generally) is an important factor in the transmission of HBV and other sexually transmitted diseases. A study found out that 80% of the males and 73% of the females had experienced heterosexual intercourse in Un ited States (Reinisch et al., 1995). Another study showed that 74% of university students reported ever having had sexual intercourse in Turkey (Gokengin et al., 2003). The situation is the same among youth irrespective of their race. Young people are at the age of greatest sexual activities thus enhancing sexual transmission of microorganisms (Dawaki and Kawo 2006). The focus on sexual transmission as the sole risk factor in the prevalence of HBV and HCV among students revealed risky sexual behavior among students as a major risk factor in the transmission of HBV.
Of the 30 students who admitted having multip le sex partners, 21 (70 %) tested positive to HBV antibodies. This supports the reports published in other literature that involvement in mult iple sex partnership increases the risk of HBV infect ion (Adekanle et al., 2010). Having unprotected vagina sex may also be a reason for the relat ively h igher prevalence recorded in this study. Condom usage was the preferred means of prevention of pregnancy and infection by some students investigated in this. Kabir et al (2004) reported in a study on sexual behavior among students in tertiary institutions in Kano, northern Nigeria that 71.7% of students preferred the use of condoms as a means of conferring protection against unwanted pregnancy and the risk of sexually transmit ted infections. However, 32 of the investigated participants in this study were non-condom users and of these, 30 (94%) tested positive to HBV 662 antibodies. Among the condom users only 4.8% tested positive to HBV antibodies. The prevalence of HBV infection among non-condom users is significantly higher (p < 0.05) than condom users. This means that the risk of acquiring HBV infection is higher among non-condom users as compared to condom users. This is consistent with the findings fro m other studies that unprotected sexual intercourse is a major route of transmitting the infection (Okonkwo et al., 2010;Dawaki and Kawo, 2006). In an attempt to determine the level of protection conferred on an individual against HBV through the use of condoms in this study, the risk percentage was calculated among condom users (1 in 59). The risk percentage was 1.7%. Th is means that out of every 100 students that make use of condoms in an attempt to protect against acquiring HBV infection, about 2 of them will contact the infection. Though significant numbers of students were protected from HBV through condom usage, it does not confer absolute protection against HBV infection.
Age or gender has no direct relationship with HBV infect ivity. Both sexes are infected at the same rate. No particular age appeared to be more susceptible to HBV infection than the other.
A raised liver en zy me (ALT and AST) is suggestive of active infection in some of the students. The percentage of students having raised liver enzy mes (6.0%) is of serious concern as they may develop into liver diseases. More studies focusing on risk group of students such as those involved in risky sexual behavior in many more tertiary institutions of learn ing will further expose the danger confronting this group of students and help in campus campaign against such behavior.

Conclusion:-
This study found the prevalence of HBV and HCV antibodies among students involved in sexual risk behavior to be 23.1% and 0% respectively. Seventy percent (70%, 21 out of 30) of students involved in multip le sex partnership recorded positive result to HBV ant ibodies while none of the students involved in single sex partnership tested positive to HBV antibodies. Only 1.7% of condom users tested positive to HBV antibodies as against 62.5% of noncondom users that recorded positive result to HBV antibodies. 28.9% of females tested positive to HBV antibodies while 17.4% of the males tested positive to the virus. This difference is however not statistically significant. 27.1% of students in ages between 16 and 20 tested positive to HBV infection as against 18.6% in ages between 21 and 25. This difference is equally not statistically significant. 6.6 % o f the students recorded increased liver enzy mes suggestive of active infection.
This study has provided informat ion on the burden of HBV among students involved in risky sexual behavior in a Nigerian tertiary institution of learning. Campaign against risky sexual behavior in Nigerian tertiary institutions of learning will help in reducing the rate of spread of HBV.