Long-term virological outcome in children receiving first-line antiretroviral therapy

During every review, the child’s well-being, adherence to ART and anthropometry were recorded. Adherence to ART was assessed by self-reporting by the caregiver, pill count and on-time hospital attendance by the child or his/her caregiver. Blood was drawn for complete blood count, CD4 percentage, absolute CD4⁺ T cell counts, and HIV-1 viral load, once every 6 months. CD4⁺ T cell percentage and CD4⁺ T cell counts were measured using the FACSCount flow cytometer (Beckton Dickinson Biosciences, San Jose, CA) and plasma viral load was estimated using the Roche COBAS AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0. Stored plasma samples from children with at least 12 months of follow-up and proven virologic failure were analyzed for HIV drug resistance mutations (DRM) by HIV-1 RNA using the QIA amp viral RNA extraction kit (Qiagen, Valencia, CA) and sequencing the relevant portion of the HIV pol gene by adopting previously published HIV drug resistance testing protocol [14]. The sequences were assembled and analyzed using Seqscape software V2.6 (Applied Biosystems). HIV DRMs were defined using the Stanford University HIV drug resistance database.

Patient retention-rate was defined by the proportion of children who were still receiving care at the study sites. Attrition consisted mainly of mortality and loss to follow-up. If a child missed less than 4 doses of ART in a month, he/she was considered as > 95% adherent to ART. Similarly, if the child missed 5–12 doses of ART in a month they were considered only 80–95% adherent to ART, while missing more than 12 doses of ART in the last 1 month, put their adherence to < 80%. Loss to follow-up was defined as children not attending the scheduled follow-up visits for three consecutive visits, and all attempts to retrieve the patient for follow-up failed. The child also had not made any unscheduled visits during this period. The WHO Global Database on Child Growth and Malnutrition recommends a cut-off z-score of < − 2 standard deviation (SD) to classify low weight-for-age (WAZ) (underweight), low height-for-age (HAZ) (stunting), and low weight-for-height (WHZ) (wasting) as moderate, and a Z-score of < − 3 SD to define severe undernutrition [15]. Immunologic Failure (IF) was defined as either fall of CD4 count to pre-therapy baseline or below (2) > 30% fall of absolute CD4 count from the on-treatment peak value or (3) persistent CD4 levels below 100 cells/mm³; while immunologic recovery (IR) was defined as an increase in CD4⁺ T cell count of ≥ 25% from pre-therapy baseline levels [16, 17]. Virologic suppression was defined as plasma HIV-1 RNA < 400 copies/ml after 12 months of ART while virologic failure (VF) was defined as HIV-RNA over 1000 copies/ml after 12 months of ART in patients who had achieved initial virologic suppression [10, 18].

The primary endpoints were virologic and immunologic failure at 12 months after initiation of first-line ART. The first analysis was done for all children who were on regular follow-up and completed 12 months of ART. Subsequently, a second analysis was done for those who went on to complete 24-months of ART. Data analysis was performed using SPSS software, version 19.0. We also calculated the changes in weight, height, and few laboratory parameters from baseline to weeks 48 and 96. We compared the continuous normally distributed data using Student t-test and proportions by Chi square test or McNemear test, as appropriate, to determine the change in anthropometry, immunology and virological parameters between pre-therapy baseline versus 12 months and pre-therapy baseline versus 24 months. We considered p < 0.0025 for statistical significance for comparing variables like stunting and underweight, moving from worst to better or normal category. This was done to avoid multiple comparisons for different time points. We performed a multivariate logistic regression analysis for all variables using enter method (all variables entered at the same time regardless of their significance level in univariate analysis), to identify independent predictors of virological failure at year 1. We considered age, gender, height and weight-for-age z scores, TB status, viral load, and CD4⁺ T cell count at the time of ART initiation, type of ART (nevirapine vs efavirenz), poor adherence (< 80% drug intake) to treatment, not having a parent as the primary caregiver, living in residential institutions as potential predictors of virological failure by entering all variable.

The study was approved by the Institutional Ethics Committee at both the institutions and informed written consent was obtained from the parent/guardian and assent from the child, wherever applicable.

Of the 393 children initiated on ART, 74% of children were started on nevirapine-based first-line ART while 24% on efavirenz-based first-line ART along with lamivudine and stavudine or zidovudine, (based on their hemoglobin levels > or < 6 gm/dl). The remaining 2% were on abacavir with lamivudine and stavudine or zidovudine. 378 children came for regular study-related follow-ups and further investigations. At the end of 12 months of ART, 328 of 393 children (83%) were still on follow-up with an overall retention rate of 87%. Self-reported adherence to ART at 12 months was > 90% drug intake in 83% (276/331) of children, 80–90% in 7% and < 80% in 10% of children on ART. There were—lost to follow-ups and missed visits during the follow-up period. By the end of 18 and 24 months of ART, there were 268 and 234 children on follow-up respectively. Figure 1 shows the study population along with reasons for the decrease in numbers during follow-up (care cascade).

After 12 months of ART, significant improvement was noticed in the weight-for-age and height-for-age z-scores from baseline (all p < 0.001). Though a significant number of children had moved from the worse stunting category to better and normal category, 21% of children remained stunted (HAZ score < − 2) and 22% undernourished for their age (WAZ score < − 2) (Table 2). The immunologic response was good; among children > 5 years of age, 90% [245/273] at 6 months, 91% [238/261] at 12 months, and 91% [176/193] at 24 months had an increase in their absolute CD4⁺ T cell count to more than 350 cells/mm³ when their baseline CD4 count was below 350 cells/mm³. A similar trend of increasing proportion of children showing a favorable response in CD4% with duration was seen as shown in Fig. 2. Immunologic failure was seen in 11% [28/261] and 9% [17/193] of children at 12 and 24 months respectively.

Viral load data were available for 348 children at the end of 6 months, for 328 children at the end of 12 months and 218 children at the end of 24 months of ART. All the children (except 28) had a high viral load in the pre-ART period and showed a good clinical response after initiation of ART. 78% of children achieved undetectable viral load (< 400 copies/l as detected by our viral load machine) by 6 months of ART. This proportion decreased with increasing duration of ART—67% at 12 months and 69% at 24 months had undetectable plasma viral loads. (Table 2).

After 12 months of ART, a quarter of the children in this cohort (94/328) showed virological failure. Majority of failure (62/94) were seen in the age group of 7–12 years and in children receiving nevirapine-based ART (74/94, 79%) as compared to efavirenz-based ART (17/94, 18%) and other groups (3/94, 3%) (p = 0.192). The proportion of virologic failures remained similar at 24 months of ART too. 62% of children with VF reported > 90% adherence to ART.

Of the 94 children with virological failure after 12 months of ART, drug resistance mutation (DRM) results were available for 65 children. At the time of virologic failure, 20% (13/65) of children had no DRM while 80% (52/65) of the children had multiple NNRTI-associated mutations.

Among the children with DRMs, 85% (44/52) had both NRTI and NNRTI DRMs while 15% (8/52) had only major NNRTI DRMs. M184V/I was present in 79% (41/52) of the children, and 27% (14/52) had Thymidine analog mutations (TAMs). K103N (48%), Y181C (37%), G190A/S (25%), Y188C/L (10%), V106M/A (8%), K65R (8%) and L100I (4%) were the major NNRTI DRMs observed in these 52 children (Fig. 3). A retrospective DRM testing showed that only 6.4% (5/78) of the children had K103N as the major DRM at baseline (pre-ART). DRM results were available for 3 of these children at 12th month and they had either M184V or M184I mutation in addition to K103N at this time point.

Of the 94 children with virologic failure at 12 months, only 36 children showed immunologic failure. Correlation between virologic and immunologic failure was absent even at 24 months of ART (VF in 63 children versus IF in 39 children). Sensitivity (95% CI) of immunologic failure to detect virologic failure was only 7% (2.0–12.0), specificity 97% (92.4–98.9), PPV 44% (13.7–78.8) and NPV was 72% (65.0–77.9), highlighting the fact that immunologic failure cannot detect virologic failure accurately and hence cannot be a good tool to assess the treatment response in children on ART.

At the time of virologic failure, none of the children were switched to second-line ART but continued on first line regimen as their CD4⁺ T cell counts were high and they did not fit into the criteria for switching to second-line ART as per the national guidelines [19].

In univariate analysis, the type of ART regimen showed a trend towards positive correlation with virologic failure. But in multivariate analysis, there were no identifiable predictors to detect children who will develop virologic failure while on treatment (Table 3).