HIV-exposed uninfected (HEU) infants have poorer growth than HIV-unexposed infants [1–3]. Impaired linear growth is associated with increased mortality, reduced neurodevelopment and lower human capital. Although the pathogenesis of growth failure remains unclear, systemic inflammation and coinfections such as cytomegalovirus (CMV) may contribute [4,5]. The growth hormone (GH) axis regulates infant growth through hepatic synthesis of insulin-like growth-factor-1 (IGF-1) , which is disrupted by chronic inflammation  and acute infections , including CMV . We recently showed that Zimbabwean HEU infants have elevated inflammatory markers, a high prevalence of CMV infection in early life, and higher CMV viral loads compared with HIV-unexposed infants . Here, we tested the hypothesis that these factors suppress the GH axis and contribute to impaired growth in HEU infants.
The ZVITAMBO trial was undertaken prior to maternal or infant antiretroviral therapy (ART) availability (1997–2001) . Briefly, 14 110 mother–infant pairs were recruited at birth and followed for 1–2 years. Exclusion criteria included birthweight less than 1500 g, plans to leave Harare, multiple pregnancy, and life-threatening medical conditions. Mothers underwent longitudinal HIV testing . Longitudinal blood samples from children were cryopreserved; the last available sample was HIV-tested by DNA PCR (Roche Amplicor, v1.5; Roche Diagnostics, Alameda, California, USA) or ELISA (GeneScreen; Sanofi Diagnostics Pasteur, Lyon, France) depending on age. HEU infants in this study were defined as infants born to HIV-positive mothers who remained HIV-negative through 6 months of age.
We selected all HEU infants in whom mother and infant survived through 6 months, with available infant anthropometry and IGF-1 measurement from 6 weeks of age. A random selection of 100 HIV-unexposed infants meeting these criteria provided comparative data.
Weight, length and head circumference were measured at birth, 6 weeks, 3 months and 6 months using previously described methods , and converted to Z-scores using WHO standards .
Plasma IGF-1 and C-reactive protein (CRP) were measured by ELISA (R&D Systems, Minneapolis, Minnesota, USA). Viral nucleic acid was extracted from plasma using the QIAamp DSP Virus Spin Kit (Qiagen, Hilden, Germany), and CMV was detected by quantitative PCR using the Abbott RealTime CMV Amplification Reagent Kit (Abbott Laboratories, Lake Bluff, Illinois, USA) on the Abbott m2000rt platform .
We used unpaired t tests and Mann–Whitney tests to compare continuous variables, and Fisher's exact tests for categorical variables. Univariable linear and logistic regression was used to determine associations between IGF-1 and growth. Where there was a possible interaction with HIV exposure (P < 0.2), results were stratified by maternal HIV status. We used univariable linear and logistic regression to determine associations between IGF-1 and CRP and CMV, and to determine the modifying effect of HIV exposure. Where there was a possible interaction with HIV exposure (P < 0.2), analyses were stratified, and multivariable linear regression was used to adjust for maternal HIV disease severity. Analyses were undertaken using Prism v6.0 (Graphpad, La Jolla, California, USA) and STATA v15.1 (StataCorp, College Station, Texas, USA).
Mothers provided written informed consent. The trial and this substudy were approved by the Medical Research Council of Zimbabwe, Johns Hopkins Bloomberg School of Public Health Committee on Human Research, and Montreal General Hospital Ethics Committee.
A total of 243 HEU and 100 HIV-unexposed infants were included. Baseline characteristics are shown in Supplementary Table 1, https://links.lww.com/QAD/B799. HEU and HIV-unexposed infants were similar at baseline, but HEU infants were born to mothers with lower mid-upper arm circumference (25.6 vs. 26.2 cm; P = 0.04) and haemoglobin (113 vs. 121 g/l; P = 0.005), and there were differences in breastfeeding . Although there were no statistically significant differences in growth between HEU and HIV-unexposed infants in this substudy (Supplementary Table 1, https://links.lww.com/QAD/B799), analysis of the whole ZVITAMBO cohort (N = 14 110) demonstrated significantly poorer growth amongst HEU compared with HIV-unexposed infants .
Insulin-like growth-factor-1 and growth
Mean IGF-1 at 6 weeks was lower in HEU infants [29.5 ng/ml (SD 10.1) than HIV-unexposed infants] [32.6 ng/ml (10.4); P = 0.014; Fig. 1a]. Table 1 shows associations between IGF-1 and growth from univariable linear regression analyses. Six-week IGF-1 was associated with length-for-age Z-score (LAZ) between 6 weeks and 6 months (Fig. 1b). The effect of IGF-1 on LAZ was not modified by infant HIV exposure status (all time-points P > 0.2). IGF-1 was positively associated with weight-for-age Z-score at all time-points, and not modified by HIV exposure (P > 0.2). IGF-1 was positively associated with weight-for-length Z-score (WLZ) at 6 weeks; there was some evidence that this association was modified by HIV exposure at 3 and 6 months of age. We therefore stratified results by HIV exposure status, and found associations between IGF-1 and WLZ in HIV-unexposed but not HEU infants at 3 and 6 months. IGF-1 was not significantly associated with head circumference-for-age Z-scores (HCZ) between birth and 3 months. However, there was some evidence that the association between IGF-1 and HCZ was modified by HIV exposure at 6 months; when results were stratified, there was an association in HIV-unexposed infants (β = 2.39 × 10–2; P = 0.04) but not HEU infants (β = 0.28 × 10–2; P = 0.72).
Taken together, IGF-1 at 6 weeks was lower in HEU compared with HIV-unexposed infants. IGF-1 was associated with future linear growth in HEU infants and with both linear and ponderal growth in HIV-unexposed infants.
Inflammation and insulin-like growth-factor-1
We hypothesized that a proinflammatory state is associated with lower circulating IGF-1. CRP was measured in 324/343 (94%) infants. On univariable linear regression, there was an inverse relationship between log CRP and IGF-1 at 6 weeks of age (β = −0.84; P = 0.03; Fig. 1c), which was not modified by HIV exposure status (P = 0.97). CRP was not directly associated with anthropometric measures (data not shown).
Cytomegalovirus DNAemia and insulin-like growth-factor-1
We hypothesized that CMV affects growth by disrupting the GH axis. Plasma CMV was measured in 331/343 (97%) infants at 6 weeks of age. 81.4 and 74.0% of HEU and HIV-unexposed infants had CMV DNAemia. We found no difference in mean (SD) IGF-1 between infants with [30.1 (10.1) ng/ml) or without (31.5 (11.4) ng/ml] CMV DNAemia (P = 0.30), regardless of HIV exposure (interaction P = 0.25). However, among infants with CMV DNAemia, there was an inverse relationship between log CMV viral load and IGF-1 (β = −1.04; P = 0.01), which was modified by HIV exposure (P = 0.16): when disaggregated by HIV status, the relationship between log CMV viral load and IGF-1 was significant in HEU (β = −1.16; P = 0.008; Fig. 1d) but not HIV-unexposed (β = 0.21; P = 0.83) infants. The relationship in HEU infants was not confounded by maternal HIV disease severity: the association between log CMV viral load and IGF-1 in HEU infants persisted after adjusting for maternal CD4+ cell count and HIV viral load (β = −1.25; P = 0.02). The relationship was also not mediated through CRP (after adjusting for CRP, β = −1.00; P = 0.016).
Taken together, detection of CMV was not associated with IGF-1 levels, but among those with CMV DNAemia, CMV viral load was inversely associated with IGF-1 in HEU, but not HIV-unexposed infants; this finding was not explained by maternal HIV disease severity or the inflammatory milieu in HEU infants.
Effect of trial arm
Because this substudy was nested in a placebo-controlled trial of maternal and/or infant vitamin A, data were also analysed with adjustment for trial arm, with no meaningful effects on findings (Supplementary Table 2, https://links.lww.com/QAD/B800).
Delineating the mechanisms underlying growth failure in HEU infants is necessary to design interventions aiming to promote healthy growth and development in this expanding population . We have previously hypothesized that drivers of growth impairment include inflammation, viral coinfections and maternal HIV disease . The current study has four key findings. First, HEU infants had modest reductions in IGF-1 compared with HIV-unexposed infants at 6 weeks of age; second, IGF-1 concentrations at 6 weeks were associated with subsequent linear growth through 6 months amongst HEU children; third, IGF-1 was inversely related to CRP, suggesting that systemic inflammation may reduce IGF-1 levels ; and, fourth, CMV viral load was inversely related to IGF-1 in HEU infants but not in HIV-unexposed infants. Collectively, these findings suggest independent effects of early-life CMV acquisition and systemic inflammation in disruption of the GH axis in HEU infants.
GH is an important regulator of infant growth through hepatic synthesis of IGF-1 . Here, we show that IGF-1 is approximately 10% lower in HEU compared with HIV-unexposed infants as early as 6 weeks. Six-week IGF-1 was associated with length and weight at most subsequent time points, suggesting that lower concentrations of IGF-1 could plausibly contribute to growth impairment in HEU infants. There was evidence that HIV exposure modified the association between IGF-1 and WLZ and HCZ, such that the effect was more pronounced in HIV-unexposed infants. This may indicate a greater, or different, range of drivers of growth failure in HEU compared with HIV-unexposed infants , and should be investigated further.
In a previous study among HIV-unexposed infants from this cohort, we reported that stunting is characterized by chronic inflammation, which appears to disrupt the GH axis , as in other inflammatory diseases . We also reported raised inflammatory markers among HEU infants from as early as 6 weeks of age . Here, we extend these findings by showing that the inverse relationship between IGF-1 and CRP is not modified by HIV-exposure status, meaning inflammation in HEU infants likely contributes to growth impairment through reduced IGF-1 as in HIV-unexposed infants. Whether raised inflammatory markers persist in the modern era of prevention of mother-to-child transmission (PMTCT) remains unknown and warrants investigation.
The ZVITAMBO trial reported increased frequency and severity of common childhood infections in HEU infants  and a failure to effectively control CMV infection . We have also shown in the same cohort that acute clinical infections disrupt the GH axis . CMV has previously been associated with impaired growth among HIV-exposed and HIV-unexposed children in Zambia . Here we hypothesized that CMV viraemia contributes to disruption of the GH axis. The presence of CMV DNAemia at 6 weeks had no association with IGF-1; however, among HEU (but not HIV-unexposed) infants with CMV viraemia, there was an inverse relationship between CMV viral load and IGF-1 concentrations. This is consistent with our previous finding that HEU infants have impaired handling of infections [10,15], and suggests this may have consequences for growth. Importantly, the relationship between CMV viral load and IGF-1 was not explained by severity of maternal HIV, or by elevated inflammatory markers in those infants with higher CMV viral loads. Collectively, these findings indicate two distinct potential mechanisms underlying growth failure in HEU infants: chronic inflammation and impaired control of viral infections.
The current study has strengths and limitations. HIV-exposed infants were known to be HIV-uninfected to at least 6 months of age, meaning the group was not contaminated by infants becoming postnatally HIV-infected through breastfeeding. The trial took place before ART availability, meaning positive or negative effects of ART on growth were eliminated. We included all available HEU infants in this analysis; however, it is possible that the study was underpowered to identify true modifying effects of HIV exposure. Measuring CRP only may not have fully captured the inflammatory milieu, including CMV-related inflammation. We were unable to delineate causal relationships, but our findings warrant further mechanistic exploration.
In summary, we find evidence for greater disruption of the GH axis in HEU compared with HIV-unexposed infants by 6 weeks of age, which suggests a potential role for reduced IGF-1 in mediating growth impairment in HEU infants. The previously reported proinflammatory state of HEU infants likely represents one pathway underlying growth impairment, but we also found an independent association between greater CMV viraemia and reduced IGF-1, suggesting that both chronic inflammation and poor handling of infections might contribute to stunting in HEU infants. These pathways provide an opportunity to evaluate targeted interventions for HEU infants, to promote healthy growth and development.
C.E. and A.J.P. designed the analysis. C.E., S.R., M.G. and K.M. carried out the laboratory assays. C.E., B.C. and R.N. carried out the statistical analyses. C.E. wrote the first draft of the article. All authors critically reviewed and revised the article. J.H.H. designed and recruited to the original trial and R.N. was a coinvestigator on the original trial.
The current work was funded by a grant from Barts Charity, awarded to C.E. (MGU0300), and by the Wellcome Trust awarded to A.J.P. (093768/Z/10/Z and 108065/Z/15/Z). C.E. was funded by the Wellcome Trust (210807/Z/18/Z) and the National Institute for Health Research. A.J.P. was funded by the Wellcome Trust (093768/Z/10/Z and 108065/Z/15/Z). The ZVITAMBO trial was supported by the Canadian International Development Agency (CIDA) (R/C Project 690/M3688), United States Agency for International Development (USAID) (cooperative agreement number HRN-A-00-97-00015-00 between Johns Hopkins University and the Office of Health and Nutrition – USAID) and a grant from the Bill and Melinda Gates Foundation, Seattle WA. Additional funding was received from the SARA Project, which is operated by the Academy for Educational Development, Washington DC and is funded by USAID's Bureau for Africa, Office of Sustainable Development under the terms of Contract AOT-C-00-99-00237-00, the Rockefeller Foundation (NY, NY) and BASF (Ludwigshafen, Germany).
Previous presentations: the current work was presented at the Conference on Retroviruses and Opportunistic Infections (CROI), Boston, MA, 2018.
Conflicts of interest
There are no conflicts of interest.
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