(A and B) Longitudinal analysis of cell-associated HIV-1 DNA (A) (determined by ddPCR) and genome-intact or defective proviral sequences (B) (determined by single-genome, near–full-length next-generation sequencing) in neonates with antepartum (AP; n = 9) and peripartum (PP; n = 1) HIV-1 infection at indicated time points after initiation of ART. Data from children with delayed treatment initiation (Controls; median of 93 weeks after treatment initiation, n = 10) and from long-term ART–treated adults (median of 16 years after treatment initiation, n = 31) are shown for comparison in (A). (C) Cross-sectional comparison of the relative frequency of genome-intact and defective proviral sequences in children with early treatment initiation (EIT; week 84/96 after beginning of ART, n = 9), delayed treatment initiation (Controls; median of week 93 after beginning of ART, n = 10), and ART-treated adults with chronic HIV-1 infection (median of 16 years after beginning of ART, n = 31). Box-and-whisker plots in (A) reflect median, minimum, maximum, and interquartile ranges. Dot plots with mean and SEM are shown in (B) and (C). Significance was tested using a two-sided Kruskal-Wallis with post hoc Dunn’s multiple comparison test between longitudinal time points or groups and a Wilcoxon test between pairs. LOD, limit of detection, calculated as 0.2 (ddPCR) or 0.5 (single-genome near–full-length PCR) copies per maximum number of cells tested without target identification (see Materials and Methods for details). (D) Decay kinetics of intact and defective proviral HIV-1 sequences during the first 24 weeks after treatment initiation. Significance was calculated using a Wilcoxon signed-rank test. (E) Longitudinal analysis of viral reservoir composition in EIT neonates, as determined by single-genome near–full-length, next-generation sequencing in samples from indicated time points after treatment initiation. Data from control children (Controls) and ART-treated adults with chronic HIV-1 infection are shown for comparison. Each pie chart reflects relative contribution of HIV-1 amplification products with defined defects or genome-intact sequences. Total number of individual sequences included in each diagram is listed in the middle of each pie chart. (F and G) Bar diagrams reflecting proportions of individual proviral sequences detected once (nonclonal sequences) or detected more than once (clonal sequences) at a given time point. (F) Data from all (intact and defective) proviral sequences. (G) Data from genome-intact proviral sequences only. The total number of individual proviral sequences analyzed at each time point is listed above each bar.