This study is based on data generated from the follow-up of the clinical HIV-1peri-exposure prophylaxis with lopinavir-ritonavir ANRS12174 PROMISE- PEP trial (ClinicalTrials.gov, number NCT00640263) [26]. The ANRS12174 PROMISE- PEP trial is described in detail in a previous paper [26]. The trial conducted between 2009 and 2013, was a multi-center randomized trial including pregnant women with HIV-1 , recruited at gestational age of 28-40 weeks at antenatal clinics in four African sites; Ouagadougou, Burkina Faso; East London, South Africa; Mbale, Eastern Uganda; and Lusaka, Zambia. HIV-1 infected pregnant women intending to breastfeed were referred for further assessment of inclusion criteria and again with their HIV uninfected children for enrolment and randomization at day 7 post-partum. Infants were eligible for inclusion if they were: a singleton; breastfed at day seven by their mothers; had a negative HIV-1 DNA PCR and had received any PMTCT. Further inclusion criteria was mother aged 18 years or older, intending to continue breastfeeding, HIV-1 infected, and not eligible for ART (either clinically or because CD4 count >350 cells/μL at that time). All eligible mothers and infants followed the routine national mother-to-child transmission prophylaxis (PMTCT) with antepartum zidovudine, intrapartum nevirapine, zidovudine-lamivudine for mothers and nevirapine for infants for 7 days postpartum.
In Uganda, 278, seven-day old uninfected children born to HIV-1 infected women, were randomized to receive infant prophylaxis (either lamivudine, 3TC or boosted lopinavir-ritonavir, LPV/r daily) throughout the breastfeeding period from day 7 to 50 weeks. The primary outcome was mother to child HIV-1 transmission, diagnosed every 3rd months with HIV-1 DNA PCR between 7 days and 50 weeks post-delivery. Infection rates, and clinical and biological severe adverse events did not differ between the two drug regimens suggesting that infant HIV-1 prophylaxis with either drug was not superior as both led to very low rates of HIV-1 postnatal transmission during 50 weeks of breastfeeding [26].
In 2017, 244 out of 278 mothers with HIV-1 infected and their uninfected children were eligible for re-enrollment in the follow-up study; the PROMISE-PEP Mechanism Safety study (PROMISE-PEP M&S Ritonavir ANRS12341). Of the 166 HEU children re-enrolled, 2 were excluded due to HIV-1conversion. Thus, 68% of the eligible cohort of HEU children (164/244) was followed up with 32% (n= 112) missed due to attrition. A comparison group of 199 HUU children matched on age and sex as well as their HIV uninfected mothers were recruited of which 19 were excluded due to a positive HIV-1test result leaving 181 HUU children and their uninfected mothers enrolled. This study uses information from interviews and clinical oral examinations of 164 HIV-1 infected caregiver-HEU child pairs and 181 age-and sex- matched HUU children and their caretakers at the follow-up in 2017.
Interviews with mothers of the HEU and HUU children at follow-up
Trained interviewers performed face-to-face interviews with caregivers before the children underwent oral clinical examination using semi-structured interviews in the local language Lumasaba. The interview was constructed in English and translated into Lumasaba for use in the field. The schedule had been reviewed previously by project staff for semantic, experiental and conceptual equivalence of the source version. Sensisitivity to culture and selection of appropriate words were considered [27]. Caretakers responded to questions about themselves and their children. Information was documented on case record forms (CRFs) and electronically with Capture software System.(Clinsight) and Epidata progaram www.epidata.dk for the clinical oral examinations.
Socio-demographic characteristics of caretakers were assessed in terms of level of education, type of income and marital status. Level of education was categorized into: primary school (1), end of primary (2) higher education (3). Marital status was categorized: divorced (1), cohabiting/married (2), single (3), widowed (4) and recoded into single/divorced/widow (0) and married/cohabiting (1). Type of income was categorized into (1) no regular income (2) regular income. Child’s characteristics were assessed in terms of dental care utilization, tooth brushing, ever toothache, and breastfeeding duration. Mother’s behavioral characteristics were assessed in terms of tooth brushing frequency (Categories of the socio-demographic and behavioral covariates are shown in Table 1).
Child impact section, (CIS) of the early childhood oral health impact scale (ECOHIS) scale for child’s oral health related quality of life was assessed using six of its original nine questions [28]. The ECOHIS scale has previously been tested for its psychometric properties in the context of Ugandan pre-school children [29]. Care givers were asked “has your child: ever had tooth ache, ever had swollen/bleeding gums, ever cried because of pain in mouth, ever failed to sleep because of pain in mouth, ever refused to eat because of pain in mouth, and ever refused to play because of pain in mouth. Dummy variables (0= no, 1= yes) were summed into a count variable (range 0-6) and dichotomized into 0= no child impacts, 1= at least one child impact. Family impact section, FIS, was assessed using the four original questions. “Because of child’s dental problems, how often have you or the other parent: taken time off from work, been upset, felt guilty and had financial problems? Response categories were rated on a 5 point scale from 0= never to 4= almost daily. Each item was dichotomized and the dummy variables were summarized into a count variable (range 0-4). The sum score was dichotomized into 0= no family impacts and 1= at least one family impact. A total ECOHIS score was constructed by adding the Child impact- and Family impact scores.
Clinical oral examination of children and mothers at follow up
Two experienced and calibrated dental surgeons (NB and MM) performed a full-mouth oral clinical examination among children and their caregivers. Duplicate examinations, for inter-rater and intra-rater reliability were performed with 26 children not part of the ANRS12341-PROMISE- PEP trial cohort.
Dental caries was assessed on fully erupted primary/permanent teeth in children and caregivers using the decayed, missing, and filled teeth indexes (dmft/DMFT) in accordance with the World Health Organization (WHO) guidelines for field conditions [30]. A tooth was documented as decayed if it was visually cavitated with the aid of a dental mirror and periodontal probe (Michigan O probe), and recorded as missing when extracted due to caries, as confirmed by the caregiver. For analyses, dental caries was denoted in two ways: presence or absence of dmft/DMFT and the total number of decayed, missed and filled teeth in caregivers and their children with dmft/DMFT scored 0 and 1 for presence /absence of caries experience in children and caregivers. Number of erupted (fully emerged recorded as free occlusal /incisal surface) permanent teeth and number of primary teeth maintained were counted for children with mixed dentition. Since dental caries in the permanent dentition of children was very limited, DMFT was not calculated for children’s permanent teeth.
Maternal and child HIV-1status at baseline and follow-up
At follow-up, HIV-1status of the HEU children was assessed using the HIV-1 DNA polymerase chain reaction from dried bloodspots. Mothers and children in the comparison group were tested for their HIV-1 status using serial and parallel HIV rapid testing with Determine, Stat-Pak and Uni-Gold, three test algorithm as recommended by the Ugandan Ministry of Health [31].
Anthropometric status for caregiver-child pairs at follow -up
At the five year follow-up, anthropometric measurements for all the participants (caregivers - child pairs) in terms of weight and height were collected twice according to WHO guidelines (http: //www. who. int/ childgrowth/ training/ en/) using Seca- brand scales and Stadiometers to the nearest decimal place.
Statistical analysis
STATA 15 (College Station, Texas 77845 USA) was used for data analysis. Chi-square tests for (categorical) variables and independent sample T-tests (continuous variables) were used to assess differences in baseline characteristics between HEU children lost to follow-up and those who retained in the cohort, and for the crude associations between HIV-1exposure status, covariates and the outcome variables. The data presented had a clustered two-level hierarchical structure with individuals clustered as matched HEU/HUU pairs. Ignoring that observations in a cluster are correlated (that is intra cluster dependency) usually leads to an underestimation of the standard errors, too narrow confidence intervals and higher Type 1 error rates. As clustering in these data were considered quite limited, multiple variable analyses were conducted with ordinary logistic regression, OLR, providing population averaged estimates. Secondly, multilevel random intercept logistic models (RIM) were fitted using mixed effects logistic regression. The RIM model explicitly allows for clustering by including both intra- and inter cluster variation in the model. Behavioral and clinical covariates statistically significantly associated both with HIV-1exposure status and the outcomes of dental caries and oral quality of life were included in the multilevel logistic regression models as potential confounding variables. The effect of clustering (HEU/HUU pairs) was assessed by calculating intra class correlation coefficients, ICC, expressing variations between clusters as a proportion of the total variance (within plus between cluster variance). ICC varies from 0, which implies independent observations within cluster to 1 indicating no within cluster variation. Hence, high values of ICC implies dependency between observations within cluster. A likelihood –ratio test was calculated to test the null hypothesis that ICC equals 0. Rejection of the null hypothesis implies that the multilevel model is preferable.