Study design, setting, and population
We undertook a cross-sectional study in the Southwest region of Cameroon, where we recruited participants over a period of four months (January to April 2019). Our target population was infants attending Infant Welfare Clinics (IWC) in the Buea Regional Hospital (BRH) and Limbe Regional Hospital (LRH). These hospitals are the major hospitals in the Southwest region of Cameroon, and they each receive averagely 80–100 infants per month for routine vaccination visits. These facilities have well equipped laboratories with the materials needed for sample collection, adequate storage, and analysis. Even though the LRH has multiple IWC visit days per week and a larger general population compared to BRH, we did not intend to compare results between the two hospitals.
Sampling method
All infants aged 6–9 months with documented evidence of receiving all three doses of the HBV vaccine and last dose received at least a month prior to the study were sampled for this study. Infants were met at the various IWC Centres and the study explained to the parents or guardians. Those who consented to participation orally then signed a written consent and were enrolled. We excluded all infants with documented evidence of HBV infection and further excluded infants with insufficient blood samples.
Sample size estimation
Our minimum sample size of 114 participants was calculated using the Cochran’s formula [18]:
\({\varvec{n}}=\frac{{{\varvec{Z}}}^{2}{\varvec{p}}(1-{\varvec{p}})}{{{\varvec{d}}}^{2}}\) where n = minimum sample size, Z = Standard normal Variation which was set at 1.96 (p value: 0.05 and 95% confidence interval), p = anti-HBs seroprevalence in immunized infants. A study carried out in Dakar (Senegal) and Yaoundé (Cameroon) revealed a prevalence of 92% [19]. Thus, we assumed a p = 92% = 0.92, and d = precision at 0.05 at 95% confidence interval.
Study procedure
We approached parents at the IWC, gave talks on general infant welfare and about the HBV. Participants with infants between 6 and 9 months were approached, study explained to them, and consent obtained.
Laboratory analysis
Specimen collection, transportation, and storage
Following participants enrolment, venous blood samples were collected using a 5-ml syringe into a 4-ml serum separating tube. These samples were then transported within 30 min following collection in a blood transport box to the laboratory where samples were centrifuged, serum extracted and stored at −20 °C. ELISA kits were stored at 4 °C.
Assessment of Hepatitis B surface antibody (anti-HBs) titres
Commercial quantitative ELISA Foresight® (ACON Laboratories, Inc., USA) was used to determine the presence of antibodies to HBsAg. Tests were performed according to the kit manufacturer’s instructions using sandwich ELISA method Foresight® (ACON Laboratories, Inc., USA).
Reagents and samples were removed and allowed to thaw prior to testing. We then prepared the working wash buffer (Tris–HCl). For each micro titre plate used, well A1 was left as blank. Calibrators (Standards obtained from the manufacturer containing varying known concentrations of anti-HBs and negative for HBsAg, HCV, and HIV) preserved in 0.1% ProClin™300 were added into wells B1 to C2. The colour of wells containing calibrator 1–5 gradually changed from yellow to Blue. Starting from D2 we added our samples. We added 50 µL of conjugate containing purified HBsAg bound to peroxidase to each well. We then mixed gently the plate and incubated at 37 °C for 30 min. Each well was then washed 5 times with 350 µL of working wash buffer and dried. To each well, 50 µL of substrate A (citrate phosphate buffer containing hydrogen peroxide) and B (buffer containing tetramethylbenzidine [TMB]) were added and incubated at 37 °C for 15 min. A blue coloration was noticed in positive wells. The reaction was then stopped by adding 50 µl of the stop solution (0.5 M H2SO4) to each well. The positive wells that initially developed blue colour changed to yellow while the negative wells (that were initially colourless) remained colourless. All calibrators, buffer, conjugate, substrates and stop solution were from ACON Laboratories, Inc., USA.
The optical densities of the wells were determined immediately using the spectrophotometer at 450 nm–700 nm. The concentration of the standards and their optical densities were used to plot a standard curve from which corresponding concentration of the optical densities of the samples were extrapolated.
In order to identify those with HBsAg carriage, all children who had not developed anti-HBs were screened for HBsAg using Wondfo rapid test (Guangzhou Wonfo Biotech Inc., China).
Additional data
Data collection
A semi-structured interview form was used to obtained sociodemographic data and medical information to assess variables such as vaccination status of infants [number of doses, interval between doses, route of administration, use of birth dose of vaccine and use of hepatitis B immunoglobulin (HBIG)], vaccination timeliness, mother’s hepatitis B status prior to parturition and immune status. Infants’ medical histories (gestational age, birthweight, and past illnesses) were obtained from past records and current anthropometric parameters were measured and recorded to complete the questionnaire by the investigator.
Data analysis
Participants were identified using unique codes and data were inputted into a secured computer using participants codes only. These data will be deleted at the end of statistical analysis. Data obtained were entered into EpiData version 3.1 and the database exported to IBM SPSS version 25 software (IBM Corporation, Somers, New York) and analysed. The primary study outcome was infants with anti HBs titres > 10.0 IU/µL (seroprotection). Continues variables such as age, gestational age and birthweight were discretized. Participant characteristics were summarised using proportions, frequencies and percentages as well as mean and standard deviation (SD) or median and 25th–75th percentiles as appropriate. Spearman correlation test was performed on all the variables to find out if there was any significant relationship between any two variables. Differences between means of quantitative data were tested using Students’ t-test while association between the categorical variables was determined using chi squared (χ2) or Cramer’s V test. A p value < 0.05 was considered statistically significant. We used a Firth logistic regression model with backward deletion for our multivariate analysis since events in our outcome variable were rare.