Study design, setting and population
The study was a hospital based cross-sectional descriptive study, covering the period from October 2014 to March 2015.
In all, three hundred and eighty (380) women attending routine Cervicare Clinics at the Kumasi South Regional Hospital, Kumasi, Ashanti Region, Ghana and Ridge Regional Hospital, Accra, Greater Accra Region in Ghana were enrolled in the study.
The participants were women who had come to Cervicare centers for visual inspection with acetic acid or to perform Papanicolaou (Pap) smear test. The Cervicare centers were established by Ghana Health Service in selected regional hospitals and health facilities where regular public announcements are carried out to invite the women to participate in screening programs.
Sampling and data collection
Convenient sampling protocols were followed to recruit the required sample size. The sample size was calculated by StatCalc application of EpiInfo 3.5.3. The approach used here to calculate sample size emphasized adequate precision of reported sample statistics: that is the ability to estimate sample statistics that do not differ from the true population parameter by more than a preset limit of confidence. Therefore, assuming a prevalence of HSV of 67% in the general population of women, and a sufficiently large population of women attending cervical screening clinics, a maximum sample size of 340 women ensured that the study had adequate precision (here we have set the desired level of precision at ±5%) to provide statistics close enough to the true population parameters. The required sample size was pegged at 380 to cater for missing and incomplete data entries and other unforeseen circumstances.
To mitigate bias in the sample, researchers conducted public health awareness campaigns within the catchment of the hospitals: on radio and at market centers and encouraged women to present themselves for screening at no cost. Additionally, a separate day was set aside the regular clinic days to enroll study participants. At recruitment, all volunteers gave informed consent by signature or thumbprint. A questionnaire was administered through one-on-one interview for data collection on socio-demographic and gynecological characteristics, sexual exposure, medical history and knowledge of HSV infection. No participants had symptoms of cervical ulcer from gynecological examination or orofacial ulcer at the time of recruitment.
Inclusion and exclusion criteria
Participants who were more than 20 years old, non-pregnant and who had written informed consent and had gone through a pre-consented interview were included in the study. Participants, who were less than 20 years old, had previously undergone a cervical examination, were pregnant, had refused to sign an informed consent and were unable to undergo a pre-consented interview were not included in the study.
Sample collection
Five milliliters (5 ml) of venous blood was drawn from all subjects to determine the presence of HSV-1 IgG and HSV-2 IgG. The samples were allowed to clot before centrifugation. Serum obtained by centrifugation was aliquoted into eppendorf tubes for storage at − 20° C till analyzed.
Laboratory analysis
The serum HSV-1 IgG and HSV-2 Ig G were determined by ELISA method using commercial test kits from Calbiotech Inc., CA, USA. The manufacturer’s instructions were followed for the analyses. Briefly, 10 μl of serum was diluted with 200 μl of diluent and incubated at room temperature for 5 min. 100 μl of the sample diluent (as a reagent blank), calibrator, negative and positive controls, as well as patients’ serum were then aliquoted into microplate wells in duplicate, and incubated at room temperature for 20 min. Three cycles of washing were performed using 1X washing buffer and 100 μl of anti-IgG conjugate was added and incubated for 20 min. The washing procedure was repeated for another three cycles and 100 μl of substrate solution was added and incubated in the dark at room temperature for 10 min after which the reaction was stopped with 100 μl of stopping solution. The absorbance was measured at 450 nm within 15 min using a reference wavelength of 600 nm – 650 nm. The Antibody (Ab) Index of each determination was calculated by dividing the mean OD value of each sample as well as negative and positive controls by the cut-off value. The cut-off value was calculated as Calibrator OD × Calibrator Factor. Calibrator factor value was indicated on the calibrator bottle.
Wells with patient antibody index greater than 1.1 were conventionally considered positive for the various antibodies tested and those between 0.9 and 1.1 were considered equivocal. While those wells with antibody index less than 0.9 were considered negative for the different antibodies tested. All equivocal samples were retested with reagents of the same kit lot number.
Statistical analysis
The data collected from the questionnaire responses was stored using Microsoft Excel 2007 software (Microsoft Corporation, Redmond Campus, Washington DC, USA).
Quantitative variables were tested for normal distribution and reported as means ± standard deviation. Qualitative variables were presented as count (percentages). The Chi-square test was used to investigate the association between sero -prevalence of HSV type 1 and type 2, and socio-demographical and behavioral factors using the Statistical Package for the Social Scientists (SPSS) version 22. Statistical significance was conventionally set at p < 0.05.
