Age at antiretroviral therapy initiation and cell-associated HIV-1 DNA levels in HIV-1-infected children

Abstract

Background: The latent viral reservoir is the major obstacle to achieving HIV remission and necessitates life-long antiretroviral therapy (ART) for HIV-infected individuals. Studies in adults and children have found that initiating ART soon after infection is associated with a reduction in the size of the HIV-1 reservoir. Here we quantified cell-associated HIV-1 DNA in early-treated but currently older HIV-infected children suppressed on ART.

Methods: The study participants comprised of a cohort of 146 early-treated children with HIV-1 RNA <50 copies/ml enrolled as part of a clinical trial in Johannesburg, South Africa. A stored buffy coat sample collected after a median 4.3 years on ART and where HIV-1 RNA was <50 copies/ml was tested for cell-associated HIV-1 DNA levels. An in-house, semi-nested real-time quantitative hydrolysis probe PCR assay to detect total HIV-1 subtype C proviral DNA was used. Children were followed prospectively for up to 3 years after this measurement to investigate subsequent HIV-1 RNA rebound/failure while remaining on ART. Age at ART initiation, HIV-1 RNA decline prior to HIV-1 DNA measurement and other factors were investigated.

Results: A gradient between age at ART initiation and later HIV-1 DNA levels was observed. When ART was started <2 months of age, the lowest levels of cell-associated HIV-1 DNA (median 1.4 log10copies/106 cells, interquartile range [IQR] 0.95-1.55) were observed compared to ART started at 2-4 months (median 1.68, IQR 1.26-1.97) or 5-14 months of age (median1.98, IQR 1.69-2.25). A low CD4 T-cell count pre-treatment predicted higher levels of HIV-1 DNA on later testing. The probability of HIV-1 RNA rebound >50 copies/ml whilst on ART within 3 years after the DNA measurement was 2.07 (95% CI: 1.352-3.167) times greater if the HIV-1 DNA level was above the median of 55 copies/106 cells.

Conclusions: Cell-associated HIV-1 DNA levels measured after more than 4 years on ART were lower the younger the age of the child when ART was initiated. This marker of the size of the viral reservoir also predicted subsequent viral rebound/treatment failure while ART was sustained. The results provide additional evidence of the benefits of prompt diagnosis and early ART initiation in newborns and infants.

Fig 1. Comparison of reverse transcriptase-based single round qPCR (a and b) and sn-qPCR (c and d) assays designed to quantify subtype C HIV-1 proviral DNA.

Amplification curves (a and c) of ten-fold diluted plasmid (p8MJ4) DNA harboring subtype C HIV-1 gag-pol gene and standard curves generated (b and d) from respective curves. Grey zone shows that the cycle threshold (C_T) region where 10⁵ plasmid DNA copies are amplified in the single round qPCR (a) is equivalent to the C_T region where a single copy (10°) is amplified using the sn-qPCR assay. Eff = PCR efficiency.

Fig 2. Associations between age at starting ART and cell-associated HIV-1 DNA levels after a median of 4.3 years of treatment among 146 HIV-infected children.

a: Scatterplot of HIV-1 DNA log₁₀ copies/10⁶ cells (Y-axis) by age at ART start in months (x-axis). LOcally WEighted Scatter-plot Smoother (LOWESS) and linear regression prediction lines are shown in blue and red, respectively; and the grey area shows the 95% prediction elipse. b: Box and whisker plot of HIV-1 DNA log₁₀ copies/10⁶ cells by age at ART start groups; c: Histograms and estimated normal and kernel distributions of HIV-1 DNA log₁₀ copies/10⁶ cells among those who started ART <4.5 and ≥4.5 months of age.

Fig 3. Scatterplots showing associations between HIV-1 DNA log₁₀ copies/10⁶ cells and a: Time on ART in years; b: Pre-ART HIV-1 RNA copies/ml and c: Pre-ART CD4 count in cells/mm3.

LOcally WEighted Scatter-plot Smoother (LOWESS) and linear regression prediction lines shown in blue and red, respectively.; grey area shows the 95% prediction elipse.

Fig 4. Histograms and estimated normal and kernel distributions of HIV-1 DNA log₁₀ copies/10⁶ cells by reactive or non-reactive/low HIV-1 antibody results among 74 HIV-infected children who initiated ART < 4.5 months of age.