Hepatitis C Virus Infection among Pregnant Women in Ibadan, Nigeria: Prevalence, Correlates and Co-infection with HIV

Aim: Vertical transmission of Hepatitis C virus, often enhanced in the presence of HIV co-infection, results in unidentified perinatally infected children who present in adulthood with long-term complications of chronic liver disease. This study was set out to determine the prevalence of chronic HCV infection, co-infection with HIV and associated risk factors among pregnant women in Ibadan, Nigeria. Study Design: A cross sectional study. Method: A total of 180 pregnant women attending the ante-natal clinic of the University College Hospital Ibadan, from March to August 2013, were screened for HCV using third generation Original Research Article Anaedobe et al.; IJTDH, 14(1): 1-11, 2016; Article no.IJTDH.23584 2 Enzyme Linked Immuno-absorbent Assay (ELISA) and confirmatory assay using nucleic acid tests were done on positive samples. Anti-HIV-1 antibodies were identified using qualitative immunoassay determine test strips. Pretested validated questionnaire were used to obtain bio-data on sociodemographic characteristics and presence of possible risk factors for HCV infection. Data analyses was done using SPSS version 20. Results: Overall seroprevalence of anti-HCV antibody was 1.7% (3/180) and anti-HIV-1 antibody was 20.6% (37/180). All HCV positive samples had HCV RNA identified in them but no detectable viraemia. No co-infection between HCV/HIV was observed. Low level of education, marital status, and positive history of surgical procedures, blood transfusion and jaundice was significantly associated HCV infection. Conclusion: Hepatitis C virus infection is less prevalent than HIV infection among pregnant women in Ibadan and its co-infection with HIV is uncommon. The sexual behavioural risk factors identified in this study were not predictors of HCV infection; however, these factors may predispose these pregnant women to other sexually transmitted infections (STIs).


INTRODUCTION
Hepatitis C virus, a blood borne pathogen found worldwide, is the leading cause of chronic liver disease and the commonest indication for liver transplant [1,2]. It was first characterized in 1989 as an enveloped single-stranded RNA virus of the Flaviviridae family and genus Hepacivirus, with about 6 major genotypes and over 50 subtypes identified [3,4]. HCV is five times as widespread and ten times as infectious as HIV [5] and recent estimates of HCV disease burden show that about 185 million people worldwide are chronically infected [6], over 80% of whom are asymptomatic and 55-85% progress to chronic liver disease in a slow and insidious manner [4][5][6][7]. This silent epidemic has become a major public health problem globally and deaths from liver disease secondary to HCV infection will continue to increase over the next 20 years [8]. In resource-poor countries particularly in Sub-Saharan African region with a high HCV prevalence rate, HIV pandemic, and very limited access to accurate diagnosis and treatment; the morbidity and mortality will continue to be on the rise despite treatment outcomes that are comparable to those in well-resourced setting [4,9,10].
Pregnant women and their infants fall among the groups less often discussed when considering the burden of HCV infection. The worldwide prevalence of HCV in the pregnant population ranges from 0.15% to 2.4% in the developed countries and estimated to be as high as 8.6% in the black populations [9][10][11][12][13][14]. Factors known to increase the risk of perinatal transmission include high maternal HCV viral loads, HIV co infection [4,14], transfusion of unscreened blood products, intravenous drug use [4,15], concurrent alcoholism and unsafe surgical procedures [4,16]. Sexual transmission is infrequent but can be facilitated by concurrent sexually transmitted diseases, and traumatic sexual practices [4,14]. Universal HCV screening among pregnant women is not in practice and considered unjustified given the absence of intervention to prevent vertical transmission of HCV, the absence of HCV vaccines or approved therapy to prevent HCV infection during pregnancy [1,14]. Nevertheless, antenatal HCV testing will help identify asymptomatic women with chronic hepatitis at risk of transmitting the virus to their child and household, those who may benefit from antiviral therapy, monitoring of infected mother and child. There are no licensed treatments or guidelines for the treatment of infants or children infected with HCV infection [10,17].
In view of the HIV pandemic which has its peak in sub Saharan Africa, HCV co-infection with HIV is very likely as they share similar route of transmission, leading to increased risk of transmission of these viruses and hence, a vicious cycle. In addition, maternal HCV and HIV co-infection has shown that vertical transmission is 3-to 4-fold higher compared to maternal HCV alone and transmission during breastfeeding occurs more often in patients co-infected with HIV [14,18].
Vertical transmission in HCV RNA-negative pregnant women is about 0% to 3% [11], about 4% to 15% in HCV RNA-positive women [1,2], but can be as high as 19% in HIV-coinfected mothers [11,18]. The role of confirmatory diagnosis of chronic HCV infection by HCV RNA detection is pivotal in predicting risk of transmission and progression of disease. While some patients positive for HCV antibodies, who also have persistently normal liver enzymes have spontaneously cleared their acute HCV infection, they continue to have detectable HCV antibodies in their blood stream [19], giving a false positive result.
Pregnant women at high risk for HCV infection ought to be screened for anti-HCV antibodies and if positive, must have HCV RNA testing done, however these are not practiced in Nigeria. There is a dearth of published data on confirmed chronic HCV infection among pregnant women in Nigeria and limited knowledge on actual risk of transmission from mother to child. Given that most perinatal HCV infections are silent with long-term complications presenting later in adulthood, with increased morbidity, mortality, and significant financial burden on the society, there is need for accurate and early detection, prevention and control practices to reduce the risk of vertical transmission from infected mothers.
This study was set out to determine prevalence of Hepatitis C virus infection among pregnant women, its co-infection with HIV infection as well as risk factors which serves as identifiers of women at high risk of HCV infection. The findings are expected to preempt the review of obstetrics policies in Nigeria with respect to testing, counseling and follow-up of pregnant women positive for HCV infection as well as their infants.

Study Design
This was a cross-sectional study in which consenting pregnant women, attending Ante natal clinic at the University College Hospital in southwestern Nigeria were recruited between March 2013 and August 2013 and their blood samples taken for analysis. Semi structured questionnaires which had been pretested and validated, was used to obtain socio-demographic characteristics and risk factors for HCV infections.

Study Area and Population
The University College Hospital, the pioneer tertiary health institution in Nigeria was established in 1957 with the aim of research, training and provision of health services. The hospital is located in Ibadan, south-western Nigeria, about 437 km from Abuja the Federal Capital Territory. The participants were pregnant women attending antenatal clinic in the University College Hospital and hailed from different parts of the country but largely comprised of the indigenes of Southern Nigeria. The practices of the people such as polygamy, early age at first sexual intercourse, female circumcision, are seen among these women who also visit local beauty parlor where tattooing, manicure, pedicure and fixing of artificial hair are done using shared needles and possibly contaminated sharp instruments. These women are also possible recipients of unscreened blood and blood products.
Sample size was calculated to give a 95% confidence level, a margin of error of +/-5%, using a previous survey of HCV seroprevalence found among pregnant women attending antenatal clinic in a Medical Centre in Benin City, in southern Nigeria which was taken as 5% [20]. A total of a hundred and eighty consenting pregnant women with ages ranging between 22 and 44 years were recruited. A written informed consent was obtained after careful explanation, in a clear language, of the concept of the study to each pregnant woman before their inclusion in the study. Ethical clearance was sought and obtained from the Joint Ethical committee of the University of Ibadan and University College Hospital Ibadan before the commencement of the study.

Specimen Collection and Handling
About 5mls of venous blood was collected aseptically by venipuncture into plain bottles and allowed to clot. The sera was separated by centrifugation at room temperature at 3000 rpm and stored in aliquots in the freezer at -20°C. This was done on every visit to the Ante natal clinic.

Laboratory Investigations
All samples were screened, using a sandwich third generation enzyme linked immunosorbent assay ELISA for anti HCV antibody (DIAPRO Diagnostic Bioprobes Milano Italy) and anti-HIV antibody using Uni-Gold Recombigen and ALERE determine. All samples found to be positive for anti HCV antibody were re-tested with a second third generation ELISA and further analyzed for HCV nucleic acid identification and quantification using a real time quantitative polymerase chain reaction (Light cycler 2000 by Roche). All tests were carried out according to the manufacturer's instructions as outlined in the package inserts.

HCV and HIV-1 detection by enzyme linked immunoassay
A third generation enzyme linked immunoassay was used to identify presence of anti-HCV antibody (DIAPRO ITALY) as well as anti-HIV-1 antibodies (Uni-Gold Recombigen and ALERE determine). It is a solid-phase simultaneous sandwich immunoassay, which employs specific monoclonal antibodies and polyclonal antibodies. Protocol for the measurement was done according to the manufacturer's instruction and reading was done at O.D. of 450 nm with an EIA plate reader. The tests ran were validated and results were interpreted according to the manufacturer's instruction.

Protocol for HCV RNA extraction, detection and quantification
RNA extraction from serum was done using the miTotal RNA extraction miniprep system (Green bioresearch) in accordance with manufacturer's instructions. The Bioneer's AccuPower® HCV quantitative RT-PCR Kit was used, which was composed of target specific primers, fluorogenic probe, DNA polymerase, dNTPs, reaction buffers and stabilizers. The reaction mixture for RT-PCR was prepared in a single tube and contained: HCV pre mix (50 mM KCl, 10 mM Tris-HCl, 0.01 mM EDTA, 60 nM Passive Reference [pH 8.3]), 5 mM MgCl 2 , 20 pmoles/ μl of primer, each deoxynucleoside triphosphate at a concentration of 0.3 mmol, 0.4 U of RNase inhibitor/ μl, 0.025 U of Taq Gold Polymerase/ μl) was added in 45 μl of Dep C water and vortexed, thereafter 2.5 μl of extracted RNA was added to 22.5 μl of premix to make 25 μl. About 2.5 μl each of the 5 standards (positive) controls were loaded and the mixture was incubated at 45°C for 15 minutes to convert the RNA into cDNA. About 20 μl of the cDNA was added to the RT-PCR capillary tubes, labeled and loaded into the machine. Amplification was done with the following protocol; pre denaturation at 95 °C for 5minutes, then 60 cycles of denaturation at 95 °C for 7secs, annealing at 5 2°C for 7 secs, extension at 72 °C for 7secs and cooling at 42 °C for 2 secs. 5 standards were used to calculate the viral load from 200 IU/ml to 2×10 6 IU /ml of sample.

Data Analysis
Data collected were subjected to descriptive and inferential statistical analysis using SPSS version 20. (SPSS Inc. Illinios, USA). The Mean, standard deviation and test of comparison using student's t-test was derived for continuous variables, while categorical variables were summarized as proportions, and further analyzed using Chi square and Fisher's exact test to assess association between the variables. Test of association using logistic regression was done to describe the relationship between the predictor variables (risk factors for maternal infection found to be statistically significant) and the outcome variable (anti-HCV antibody). P values ≤ 0.05 was considered significant.

Sociodemographic Characteristics of the Participants
The mean age of the one hundred and eighty participants finally enrolled was 32 (SD 4.

Prevalence of HCV and Co-infection with HIV
Anti-HCV antibody was detected in three (1.7%) of the pregnant women, all of whom had HCV RNA identified in their serum but had undetectable viral load levels (LLD of 200 IU/ml). The overall HIV seroprevalence was 20.6% (37/180). No co-infection between HCV/HIV was observed among the participants. The OD450nm values of the HCV positive samples are shown in Table 2, as well as the cut off values for the controls.

Risk Factors for HCV Infection among the Participants
The mean age of anti HCV sero-positive pregnant women was 29. 3 =10.733, P-value=0.005) respectively. However, no significant positive association was found between factors such as religion, gestational age and parity and the likelihood of having Hepatitis C virus infection. This is further illustrated in Table 3.  Table 4.

DISCUSSION
A hospital-based cross sectional study was carried out at the ante-natal clinic of the University College Hospital Ibadan in southwestern Nigeria. One hundred and eighty pregnant women were recruited, their serum analyzed for anti-HCV antibody, HCV RNA, and anti-HIV-1 antibody, while socio demographic characteristics and risk factors for HCV infection was assessed with pretested questionnaires.
The prevalence of HCV infection was found to be 1.7% which is consistent with the 2.1% quoted for Nigeria in the Hepatitis C global prevalence data, published in 2010 [2] and similar to the 1.86% found among pregnant women in University of Benin Teaching Hospital by Onakewhor et al. [12]. It is however lower than the 5% reported in Benin City by Duru et al. [20], while lower rates of 0.5% reported in Gwagwalada by Agarry et al. [21] and 0.4% in Calabar by Mboto et al. [22] were observed in Nigeria. When compared with findings from other African countries, it is similar to the prevalence rate of 1.8% found among pregnant women in Cameroon [5], the 2%, 2.1% and 2.6% reported in Burkina Faso, Gabon and Côte d'Ivoire respectively [23][24][25]. However it is lower than 17-26% in Egypt and 5% in Pakistan [9,21]. In other parts of the world, it is higher than the 0.3% reported by Todd et al. [26] in Afghanistan.
Hepatitis C virus is transmitted vertically in 5-15% of all pregnancies in HCV seropositive women [1,2]. In this study, all participants positive for anti-HCV antibody had HCV-RNA identified in their serum suggesting high rate of active infection and increased risk of vertical transmission among these women. All anti-HCV positive participants in this study had undetectable viral load, a finding not uncommon in sub-Saharan Africa [18]. Fluctuation in HCV RNA levels, low sensitivity of the PCR machine, technicalities in sample processing/ storage, and the heterogeneity and complex nature of the virus may account for the low rate of detectable viraemia; these are few of the challenges encountered in sub-Saharan Africa. Determination of HCV viraemia in pregnancy permit more accurate prediction of infant outcome as it is likely that women found consistently PCR negative during pregnancy have a near zero risk of vertical transmission [14].    This brings to mind the need for high quality diagnostic methods such as nucleic acid testing for accurate identification of women with asymptomatic viral infections who are likely to transmit the virus to their newborn and household. It is also vital in the management and prognostication of infected individuals and will help reduce incidence of false positive results, thereby giving a more robust prevalence rate reports. It should be established in every tertiary institution across the country, however very few health care institutions have facilities/equipment for molecular diagnosis of viral infections in Nigeria, this may be due to limited resources and lack of trained personnel.
The mean age of 29.3(±5.51) was found among the anti-HCV antibody sero-positive women, all of whom were below 35 years of age. Age is a known risk factor for hepatitis C infection; seropositivity has been reported to increase until the age 40 and then declines over time [24]. The finding in the study is in keeping with similar studies observed in Gabon, Burkina Faso [24], as well as Japan [1] and can be explained by the greater probability of exposure of these women to risk factors such as multiple sexual partners, multiparty, surgical procedure and blood transfusion.
Majority (66.1%) of the pregnant women tested had tertiary education. This may be because this study was tertiary hospital-based and in an urban center. Increasing level of education was noted to be inversely related with HCV infection, this may be explained by the fact that a higher educational status is associated with greater awareness of the infection, its route of transmission and prevention, indicating the positive influence of education and public enlightment/ awareness on the carrier rate of HCV infection.
The presence of cosmetic alterations in the form of body piercing or tattooing, cultural practices that allow female circumcision and scarification marks as well as unsafe injection practices should be taken into consideration whenever assessing the risk of an individual having HCV infection. The presence of these risk factors were increased among the sero-positive women in the study, however they were not statistically significant. This might be due to a decline in these practices as a result of the nationwide public enlightenment and higher educational level. Nevertheless, with the strength of the sample size, and the urban setting of the study, caution must be applied, as the findings might not be transferable to larger studies done in rural communities where these cultural practices persists and patronage of chemist manned by unqualified medical personnel is common.
Blood transfusion was a significant risk factor for the acquisition of Hepatitis C infection in this study. This was also observed in Congo as  [1,2,11,21], and some experts are of the opinion that HCV and HIV are not transmitted via the same routes in sub-Saharan Africa [18]. Some studies have reported very low intra-spousal transmission of HCV among monogamous couples and the CDC does not recommend the use of barrier precautions among heterosexual monogamous couples to prevent HCV transmission [1,2,11]. Nevertheless the role of sexual transmission in the spread of HCV should no longer be overlooked and there is increasing need for education of the general population on the evils of risky sexual behaviour.
A previous history of jaundice has been identified as independent risk factor for HCV infection in this study. This is in agreement with studies that have identified contact with persons with Jaundice as independent risk factor to HCV infection [11,15,21]. It is therefore important to adequately screen all pregnant women with history of jaundice. Past surgical history was found significant on univariate analysis and this is in order in view of the various route of transmission viz a viz blood transfusion, use of poorly sterilized instruments, that comes with surgical procedures. antibodies against other pathogens such as HCV, thus HCV RNA or viral load test will be required to identify those with HCV infection. This is important because once the use of antiretroviral drugs is commenced, the phase of immune-competence is initiated, and with time, the course of Hepatitis C is aggravated and more often than not will go undetected leading to significant morbidity and mortality. Hence, HCV-HIV co-infection is of grave concern in sub-Saharan Africa where these viral infections are endemic. If unchecked, the increased vertical transmission, accelerated liver disease progression and increased rate of chronicity and hepatocellular cancer would enforce the HCV pandemic with devastating consequences.
While some countries practice selective screening of "high risk" women in pregnancy, experts are advocating universal HCV screening in pregnancy as some studies have demonstrated the failure of selective antenatal screening policy to identify some previously undetected HCV infections [23]. In developing countries with limited resources, nonidentification of asymptomatic HCV infection may be due to failure to identify and screen all women at high risk of HCV infection, and refusal to HCV test. In Nigeria, there is need for a nationwide survey, with high quality diagnostic method, to accurately determine the prevalence and risk factors for HCV and HIV infections among pregnant women, as well as a review of obstetrics policies as regards antenatal screening.
There are a few limitations in our study. The study was not a community based study, but rather a hospital based study carried out among pregnant women attending the antenatal clinic (ANC) of the hospital, and thus may not be used as a true reflection of Ibadan as a whole. However, University hospital provides ANC services to majority of Ibadan pregnant women. The selection of participants for the study was limited to the first 180 consenting pregnant women who met the eligibility criteria and this may have introduced a bias. It is unlikely that bias was introduced, however, owing the use of a randomized sampling technique in selection of participants.

CONCLUSION
There is a low prevalence of chronic HCV infection among these women and HCV-HIV coinfection is uncommon despite a high prevalence of HIV infection. While parenteral transmission including blood transfusion and unsafe surgical procedures, possibly plays a major role in HCV acquisition, sexual transmission of HCV among pregnant women seems to be limited. It is important to keep educating the masses on mode of spread of these viruses, implementation of universal precautions and avoidance of highrisk sexual behavior. Preventing exposure to infected blood or blood products, safe sexual practices, the use of condom during sexual contact, adequate sterilization of hospital equipment and materials, needle-exchange programs for injecting drug users may help to limit the spread of HCV infection. There is need for larger surveys with nucleic acid testing for more robust prevalence estimates to inform development of health care policies on prevention, control and treatment programmes. Pregnant women at high risk for HCV infection should be screened for anti-HCV, and if positive, HCV RNA, and HIV testing should be performed. Infants of women with hepatitis C infection should be screened and followed up [11].

CONSENT
Consent was obtained from all the study participants.

ETHICAL APPROVAL
The study protocol was approved by the Institutional Review Board of the University of Ibadan, and the University College Hospital both in Oyo State, Nigeria.