METHODS
macrocytosis
REGRESSION, SECONDARY, ARRESTED, MACROCYTOSIS
ODYSSEY (NCT02259127) is an open‐label, 96‐week randomized, multicentre, non‐inferiority trial comparing dolutegravir‐based ART (DTG arm) with standard‐of‐care (SOC arm) in children starting first‐line ART (ODYSSEY A) or second‐line ART (ODYSSEY B) in Africa, Europe and Southeast Asia [Children in three Ugandan sites (Joint Clinical Research Center, Baylor Uganda and Makerere University‐Johns Hopkins University Research Collaboration), aged ≥6 years, were enrolled in folate and B12 sub‐study and had folate and vitamin B12 levels measured and analysed by the randomized arm. MCV comparison was a secondary analysis for the sub‐study which allowed us to explore the differences between the randomized arms in the whole trial.We limited the sub‐study to children ≥6 years, as the majority of children in the main ODYSSEY trial were 6 years and older (679/707 [96%]), and folate and B12 normal levels tend to be higher and have wider normal ranges in younger children [For the sub‐study sample size calculation, we considered a difference of half a standard deviation or more between randomized groups in RBC folate at week ≥96 and/or plasma folate at week 4 (adjusting for baseline) as clinically relevant. We estimated we would require 140 children to show these differences with 80% power, at a 5% level of significance, assuming a correlation between pre‐ and post‐treatment levels of plasma folate of 0.3, and allowing for 10% unanalysable samples. The sample size calculation is described in the ODYSSEY protocol [In statistical analysis, laboratory measurements below the 1st percentile were recoded as equal to the 1st percentile, and above the 99th percentile as equal to the 99th percentile. Linear regression was used to compare cross‐sectional RBC folate and vitamin B12 between arms at week ≥96, and change from enrolment (adjusted for baseline) in plasma folate to weeks 4 and ≥96 and MCV to weeks 4, 24, 48, 72 and 96. Estimates were adjusted for site, sample date (using restricted cubic splines with knots at the 10th, 50th and 90th percentiles, included because of an observed non‐linear trend over time by sample date), time between sample taken and sample analysis, and randomization stratification factors (first‐/second‐line ART, site availability of routine resistance testing and NRTI backbone chosen pre‐randomization). Heterogeneity of treatment effects by first‐ and second‐line and by site were assessed using interaction terms.Zidovudine competes with natural deoxynucleoside triphosphates causing impairment of mitochondrial DNA biosynthesis and mitochondrial dysfunction, which leads to arrested cell growth of erythrocyte precursors and macrocytosis [In sensitivity analyses of plasma and RBC folate and of serum vitamin B12, data in children with a treatment change prior to outcome measure were excluded, with treatment change defined as: (i) any time off third agent in the 3 months prior to the sample collection (where a third agent is a third antiretroviral drug co‐administered with two NRTIs used in the ART regimen), or (ii) time on non‐dolutegravir regimen for those in the DTG arm or on dolutegravir for those in the SOC arm prior to the sample collection. An interaction term between body weight at week ≥96 as a continuous covariate and treatment effect was considered as an indicator of the difference by dolutegravir dose per kilogram (99% of children were receiving the adult 50 mg dose).The proportions of children with levels of plasma folate and RBC folate below and above the expected ranges were compared between trial arms, based on the lower and upper end of the reference ranges (normal ranges 3.89 to 26.8 ng/ml for plasma folate and 523 to 1257 ng/ml for RBC folate) [Caretakers and children gave written informed consents and assents as appropriate for participation in the ODYSSEY trial and the sub‐study. The folate and B12 sub‐study was included in the ODYSSEY main protocol v5.0, which was approved by ethics committees for all ODYSSEY trial sites (Germany, Thailand, Portugal, Spain, South Africa, Uganda, UK and Zimbabwe).