The ANRS-12140 Pediacam Study
ANRS-12140 Pediacam is a prospective cohort study of HIV-infected children included between November 2007 and October 2011 from three referral hospitals in Cameroon: the “Centre Mère et Enfant de la Fondation Chantal Biya” (CME-FCB) and “Centre Hospitalier d’Essos” (CHE), both in Yaounde, and the “Hôpital Laquintinie de Douala” (HLD) in Douala. Pediacam comprised two phases and has been described previously (37,38). Briefly, during the first phase, infants born to HIV-infected mothers and those born to HIV-uninfected mothers were matched according to gender and site of recruitment during the first week of life and followed until the fourteenth week. All infants received routine vaccines according to the Expanded Program of Immunization at birth, and 6, 10, and 14 weeks. HIV-exposed infants were tested for HIV using molecular tools at six weeks of age. HIV test results were available at 10 weeks with retesting for HIV-positive diagnoses. HIV-negative breastfed infants were retested six weeks after weaning. All HIV-infected infants and subsamples of uninfected HIV-exposed and HIV-unexposed infants were eligible for the second phase of follow-up, planned to continue until five years of age. During the above inclusion period, direct inclusion into the phase 2 follow-up was proposed to the mothers of HIV-infected infants identified and diagnosed after the first week but before seven months of life. Overall, 210 HIV-infected infants were included in this second phase. ART was systematically proposed as soon as the HIV status was confirmed, according to the Cameroonian guidelines at the time of the study: zidovudine (or abacavir or stavudine) + lamivudine, for all infants, combined with ritonavir-boosted lopinavir or nevirapine, depending on the previous history of nevirapine used for prevention of mother-to-child transmission (PMTCT). HIV viral load was measured by RT PCR (Biocentric, Bandol, France) using plasma specimen, with 300 copies/mL as lower limit of quantification from 2007 to 2011. Thereafter, new devices for measuring viral load were acquired with the threshold of 60 copies/mL as lower limit of quantification. Data concerning biological and clinical parameters collected at inclusion and every three months after ART initiation until the age of two years and every six months thereafter. At the same time-points, a standardized questionnaire was administered to caregivers to collect data on family living conditions and adherence to ART. Children whose HIV viral load remained high (≥ 1000 copies/mL) for at least three consecutive medical visits were selected for HIV genotypic resistance test to ARVs performed with 1 mL of plasma using the French ANRS (French National Agency for Research on AIDS and Viral Hepatitis) protocol (39). The targeted regions were Protease and Reverse transcriptase of the HIV polymerase gene. The exams and transport fees were paid by the Pediacam project.
Study population
Among the 210 HIV-infected children included in phase 2 of the Pediacam study, 149 who initiated ART no later than the age of one year were alive at two years and considered for this analysis (Fig. 1). Data were analyzed from the second to fifth year of ART.
Endpoints
The main outcome variable for this study was virologic success (VS), defined on the one hand as the achievement of viral load < 400 copies/mL at least once during the period from two to five years after ART initiation, and one the other as the achievement of viral load < 400 copies/mL after five years of ART initiation. This threshold had also been reported in previous studies (9,24,40–42). We also studied mortality and the immune status, defined by the percentage of CD4 lymphocytes, at five years after ART initiation.
Virologic, immunological, and clinical data were analyzed during the visits scheduled every six months from 24 months after ART initiation: month 24 (M24), month 30 (M30), month 36 (M36), month 42 (M42), month 48 (M48), month 54 (M54), and month 60 (M60). A window of three months before and after these time points was accepted for data collection. The monitoring values used were the nearest measurements within these ± 3-month intervals. The children still alive who did not attend the scheduled visit were classified as virologic failure with respect to the corresponding visit.
Exposure variables
We mainly considered characteristics at M24 to identify factors associated with VS at M60: calendar period, ART regimen, site of ART delivery, age and clinical presentation of the child, attainment of confirmed VS (viral load (VL) < 1000 copies/mL) within the first two years after ART initiation (43), virological status at M24 (viral load < or ≥ 1000 copies/mL), and immunological status (percentage of CD4 lymphocytes). We also considered variables related to living conditions at M24: people living in the household, access to running water, access to electricity, and availability of a functional refrigerator at home. Finally, we considered the reporting of caregiver adherence (recall of missed doses in the past three days) collected at M60. Children were classified as non-adherent if at least one dose was missed during the past three days (18,36).
Statistical analysis
The study population was initially divided into two groups by distinguishing their virological status at two years of ART: 1) children who had a viral load < 400 copies/mL at M24 (Group 1) and 2) children who had a viral load ≥ 400 copies/mL or whose viral load had not been measured at M24 (15 children of whom the last viral load measured was ≥ 400 copies/mL at M12) (Group 2).
The probability of maintaining VS from M24 to M60 for group 1 was estimated using the Kaplan-Meier model and the probability of achieving VS for group 2 was estimated using a competing risk regression curve (with death as the competing event).
The association of exposure variables with viral load < 400 copies/mL (versus a viral load ≥ 400 copies/mL) at M60 for children who were still alive was assessed in univariate and multivariate analysis using logistic regression. The site of ART delivery, virological status at M24, ART regimen at M24, and reporting of caregiver adherence (recall of missed doses in the past three days) at M60 were included in all multivariate models (a priori risk factors). Other exposure variables, with p ≤ 0.20 in univariate analysis, were also included in the initial multivariate model. The final model was built using backwards elimination, comprising a priori risk factors and other exposure variables with p ≤ 0.20. All statistical analyses were performed using STATA © 13.0 (Stata Corp, College Station, TX).