HIV infection has transitioned from a fatal to a chronic disease. Sub-Saharan Africa has 10% of the world's population, but 68% of adults and 90% of children with HIV infection . Combination antiretroviral therapy (ART) guidelines have evolved over time toward earlier initiation at higher CD4+ cell counts in adults and for treatment of all children regardless of age, disease stage or CD4+ cell count. The early data of the children with HIV early antiretroviral (CHER) trial, released in 2007, was pivotal in changing pediatric ART guidelines. Infants at a median age of 7.4 weeks (interquartile range, 6.6–8.9) and a CD4+ percentage of 35.2% (interquartile range, 29.1–41.2) were randomized to immediate or deferred ART. Early ART was associated with reduction in mortality by 76% and HIV progression by 75% compared with deferred ART [2,3]. A neurodevelopmental substudy  found that infants started on early ART had significantly higher locomotor and general scores on the Griffiths Mental Development Scales at a median age of 11 months compared with infants on deferred ART. However, long-term effects of timing of ART initiation relative to acute or chronic HIV infection and long-term effects of ART on neurodevelopment and neurocognition remain unclear [4–6].
The developing brain is especially vulnerable to HIV infection because of rapid and critical brain development in the first 2 years of life . Pediatric HIV and ART-associated damage has been primarily investigated from neuropsychological [4,5] and clinical perspectives . Neuroimaging can potentially describe the mechanisms underpinning neurobehavioral outcomes by quantifying the structural, functional, and chemical condition of the brain [9–11]. Few neuroimaging studies have assessed HIV-infected study participants stable on ART, particularly among young children .
Proton magnetic resonance spectroscopy (1H-MRS) noninvasively evaluates localized brain metabolism, providing insights into brain health, development, and the consequences of both long-term HIV infection [9,12] and ART. The archetypal metabolites measured include: N-acetyl-aspartate (NAA), a marker of neuronal integrity and viability; creatine-phosphocreatine (CrPCr), involved in the Krebs cycle; choline (GPCPCh), which comprises glycerophosphocholine (GPC) and phosphocholine (PCh), reflecting cell membrane integrity and turnover primarily in macrophages and microglia; glutamate (Glu), a primary neurotransmitter and myo-inositol (Ins) involved in cell transport and hormone-sensitive neuroreception.
The basal ganglia play important roles in cerebral development, controlled movement, hand–eye coordination, and motivation  but remain among the most HIV-affected regions [14,15]. 1H-MRS studies of HIV-infected adults [16,17] and children [10,15,18,19] report altered basal ganglia neurometabolism, irrespective of symptoms. Adult studies documented reduced basal ganglia NAA levels or NAA/creatine ratios with HIV-dementia progression, as well as increased GPCPCh levels or GPCPCh/creatine ratios and Ins/creatine ratios as markers of increased neural inflammation and gliosis. However, findings in children are conflicting, possibly because of small sample sizes, wide age ranges over several developmental phases, different ethnicities, different treatment regimens and late therapy initiation, as well as study-based focus on HIV-encephalopathies and other malignancies [18,19].
We aimed to investigate the effects of different ages of ART initiation and disease severity in infancy on metabolite levels in the basal ganglia at age 5 years. We hypothesized that children who initiated ART early would show neurometabolic advantages as a proxy for brain health at preschool age compared with delayed treatment, and that poorer immune health in infancy would be associated with poorer neurometabolic outcomes at age 5 years, indicating developmental delay/damage. Strengths of this study include prospective follow-up, early access to standardized ART, uniform sociodemographics, and all children assessed within a narrow age range (5–6.4 years).
Participants were 62 HIV-infected Xhosa children from the randomized CHER trial in follow-up at the Children's Infectious Diseases Clinical Research Unit, Tygerberg Children's Hospital, Cape Town [2,3].
Zidovudine (ZDV, AZT, Retrovir) was administered from 34 weeks gestation and to neonates for 7 days. Additionally, a single dose nevirapine (NVP, Viramune) was given to mothers in labor and to infants shortly after birth [2,3]. HIV infection was confirmed by a positive polymerase chain reaction (PCR; Roche Amplicor Version 1.5 RNA) test for HIV-1 DNA and plasma viral load (PVL) more than 1000 HIV-1 RNA copies/ml. All children with CD4+ percentage (CD4%) at least 25% were randomized to one of the following three treatment arms: ART-Def (ART Deferred until CD4% < 25% in first year or CD4% < 20% thereafter, or if clinical disease progression criteria presented); ART-40W (ART initiated ‘early–i.e., before 12 weeks of age’ – and interrupted after 40 weeks); and ART-96W (ART initiated early and interrupted after 96 weeks) [2,3].
As our aim was to investigate potential benefits of early ART on neurometabolism at age 5 years, and some children in the ART-Def arm met criteria for almost immediate initiation of ART, the CHER children were grouped here into those who received ART at or before 12 weeks of age (early ART) and those who received ART after 12 weeks (late ART), irrespective of treatment arm.
All children underwent comprehensive clinical and immunological follow-up until 5 years of age. Additionally, a neurological examination was conducted prior to MRI scanning at ages 4.8–5.9 years. The ART regimens comprised ZDV + lamivudine (3TC, Epivir) + lopinavir-ritonavir (LPV/r, Kaletra). Clinical follow-up occurred every 3 months. HIV-1 RNA was monitored using the Amplicor HIV-1 Monitor PCR test (Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) over the range of 400–750 000 copies/ml of plasma.
Seventeen HIV-uninfected Xhosa children were recruited from an interlinked vaccine trial  as controls. They comprised HIV-exposed but uninfected (HEU) children born to HIV-infected mothers but who tested HIV-negative (PCR) at baseline and 30 days after the third dose of vaccine, and HIV unexposed and uninfected children born to HIV-seronegative mothers (tested after 24 weeks gestation) who remained seronegative at enrollment .
Children received neuroimaging on the 3T Allegra MRI Scanner (Siemens, Erlangen, Germany) at the Cape Universities Brain Imaging Centre (Tygerberg Children's Hospital) between ages 5–6.4 years, without sedation, according to protocols approved by the Human Research Ethics Committees of the Universities of Cape Town and Stellenbosch. Parents/guardians provided written informed consent; children provided oral assent.
The protocol included a high-resolution T1-weighted acquisition for voxel placement and single voxel spectroscopy in the right basal ganglia (Fig. 1a) using a real-time motion and B0 corrected point resolved spectroscopy (PRESS) sequence (1.5 × 1.5 × 1.5 cm3 voxel; TR 2000 ms, TE 30 ms, 64 averages) . The voxel comprised the frontal limb of the internal capsule and part of the caudate nucleus, putamen, and globus pallidus. Water unsuppressed 1H-MRS measurements were obtained at different TE's (TE = 30/50/75/100/144/500/1000 ms, TR = 4000 ms, 2 averages) to estimate tissue fraction composition .
Absolute metabolite levels of the corrected water-suppressed spectra were quantified using Linear Combination Modelling software (LCModel) . The water signals of the unsuppressed spectra were modeled (Sigma Plot, San Jose, California, USA) to estimate the tissue fractions in the voxel [11,23]. Tissue content values and relaxation correction factors were utilized to obtain absolute quantification values [11,22] from offline frequency and phase-corrected data.
Data were excluded if the full-width at half maximum line widths of NAA exceeded 0.075 ppm and if the SNR was below 7 [11,24]. Study participants were excluded if the mean of the SNR-dependent Cramér–Rao minimum variance bounds (CRMVB) for the chosen metabolites – NAA, CrPCr, GPCPCh, Glu, and Ins – was above 20%. A senior neuroradiologist reviewed all structural scans.
Statistical analyses were performed in SPSS 22 (IBM, Armonk, New York, USA). Demographic variables and clinical measures were compared between groups using analysis of variance (ANOVA). Among infected children, differences in metabolite levels between children receiving early and late ART were examined using a student's t-test. We also investigated possible group differences in metabolite levels between children who had normal vs. abnormal neurological examinations, and among the children receiving early ART between those with treatment interruption vs. continuous treatment. As a proxy for inflammation, we compared metabolite levels and clinical variables between infected children who had any instances of virological breakthrough following ART initiation and those in whom PVL remained suppressed.
Any confounders related to a given outcome at P less than 0.10 were entered into an analysis of covariance (ANCOVA) to determine whether group differences remained significant after controlling for these measures. Further, since ART interruption is characterized by a subsequent drop in CD4+ cell count and increased PVL that may negate the potential benefits of early ART, we also controlled for duration of ART interruption.
Pearson's and Spearman's rank (ρ) correlations were used to assess metabolites with clinical measures recorded at enrollment and at time of scanning – CD4+ cell count, CD4%, CD8+ cell count, CD8%, ratio of CD4+/CD8+ cell counts – as well as cumulative period on ART, age at ART initiation, and the age at which PVL first dropped below 400 copies/ml (first PVL suppression). We did not examine associations with PVL as the range was concatenated. Multiple regression analyses were used to examine the effect of confounding variables related to the outcome at P < 0.10 on the associations between metabolite levels and clinical measures.
We performed correction for multiple comparisons using the Benjamini–Hochberg false discovery rate method .
Of 79 children recruited, 26 were excluded for the following reasons: 15 for scanner claustrophobia, one because of poor data quality because of excessive head movement and 10 because of incomplete water reference spectra (necessary for tissue fraction estimation for quantification of absolute metabolite levels). No study participants were excluded because of NAA line widths exceeding 0.075 ppm, SNR below 7 or mean CRMVBs above 20%. We thus present results for 38 HIV-infected children (age 5.0–6.3 years; 18 male) comprising 12 who received late ART and 26 who received early ART, as well as 15 uninfected controls (age 5.1–6.4 years; seven male). Of the 26 who received early ART, 10 remained on continuous ART in line with clinical criteria governing interruption. Treatment was interrupted in 16, two of whom had not met ART restart criteria at the time of scan as they remained clinically well with CD4% of at least 20%. Twelve of 15 control children (80%) were HEU.
Radiological reports were normal in all except seven children who were referred for follow-up. Only in one child who manifested right basal ganglia malformation, (ventriculomegaly and colpocephaly) was left basal ganglia spectra obtained. Comparative analyses, including and excluding this study participant were performed. For the neurological examinations; 38 children were normal, 11 children had brisk limb reflexes (two late ART, eight early ART, one control), three children had lower limb spasticity (two late ART, one early ART), and one control child did not undergo examination.
There were no significant differences in demographics between controls and infected children in the early or late treatment groups, and no differences in clinical measures at enrollment or at scan between children receiving early and late ART (Table 1). Owing to the design of the CHER study, treatment-related measures differed between the groups. Children who initiated ART early were younger when viral load was first suppressed, but tended to be older at their nadir CD4% and peak CD8+. Notably, nadir CD4+ and peak CD8+ measures typically occurred after interruption in the early treatment children, whereas nadir CD4% and peak CD8+ cell count occurred around time of ART initiation in the late ART group.
There were no significant differences in demographic or clinical variables among the early ART children in those with and without interruption, except that cumulative period on ART was longer in children on continuous treatment (mean ± standard error = 283 ± 6 weeks compared with 219 ± 14 weeks, P = 0.001) and CD8% at enrollment tended to be higher in those receiving continuous treatment compared with those in whom treatment was interrupted (36.2 ± 4.5 vs. 27.1 ± 2.3, P = 0.07). Interestingly, children with abnormal neurological signs had higher CD8+ cell count at scan compared with neurologically normal children (1291 ± 153 vs. 856 ± 82, P = 0.01). Instances of virological breakthrough occurred in 19 children after ART initiation. Total period on ART was shorter in these children (235 ± 10 weeks compared with 263 ± 8 weeks in children without virological breakthrough, P = 0.03), and they tended to be younger at ART initiation (13.5 ± 2.6 weeks, compared with 23.0 ± 5.0 weeks in children without virological breakthrough, P = 0.09).
Metabolite level group comparisons
The children who initiated ART after 12 weeks of age had lower levels of CrPCr, GPCPCh, and Glu than those initiating treatment at or before 12 weeks (Table 2). Of the potential confounders considered, only birth weight had a significant association with GPCPCh (r = 0.26, P = 0.05). Differences between early and late ART groups remained significant after controlling for ART interruption and the potential confounding effects of birth weight on GPCPCh. In Figure 1b, we show mean and 95% confidence intervals of basal ganglia metabolite levels for early and late ART groups together with values for uninfected controls. Unexpectedly, uninfected children had lower levels of NAA (Student's t-test P = 0.006) and GPCPCh (P = 0.03) than children in the early ART group. The GPCPCh difference remained significant after controlling for birth weight. There were no significant differences in metabolite levels between uninfected children and children in the late ART group.
Notably, GPCPCh was higher in children with abnormal neurological examinations (1.12 ± 0.02 vs. 1.04 ± 0.02, P = 0.02), whereas none of the other metabolites differed significantly (all P > 0.2). Controlling for abnormal neurological signs did not alter the group difference in GPCPCh between children who initiated ART early and late (P = 0.05).
No metabolite levels differed between children on continuous early ART vs. interrupted early ART (all P > 0.2), nor between children with or without virological breakthrough (all P > 0.13).
Associations between metabolite levels and clinical measures in HIV-infected children
Higher CD8%, as well as lower CD4+ cell count and lower CD4+/CD8+ ratio, all at enrollment, were associated with reduced NAA and GPCPCh. Higher baseline CD8+ cell count was also associated with lower NAA (Table 3). The aforementioned associations with GPCPCh remained significant after controlling for birth weight. Figure 1c shows the relations of NAA and GPCPCh with CD4+/CD8+. In contrast, metabolite levels were ‘not’ associated with any of the clinical measures at time of scan or cumulative time on ART. Similar to the higher CrPCr levels observed in children initiating ART before 12 weeks, younger age at ART initiation and at first PVL suppression were both associated with increasing CrPCr levels at age 5 years. Controlling for presence of abnormal neurological signs did not alter the associations between clinical measures and GPCPCh (partial r = 0.35, P = 0.04).
Partial correlation analysis was used to determine the degree to which the effects of immunocompromise during infancy on GPCPCh might be attributable to poorer neuronal integrity, as measured by NAA. After controlling for NAA, the relation of CD4+/CD8+ at enrollment to GPCPCh was no longer significant (partial r = 0.12, P = 0.48), indicating that the effects of poor immune health in infancy on GPCPCh may be attributable to its effect on NAA.
After Benjamini–Hochberg false discovery rate correction for multiple testing, only the relationships between NAA and CD8% at enrollment as well as NAA and CD4+/CD8+ at enrollment survived correction with P 0.003 or less at α = 0.05 .
This is the first study to use metabolite levels as markers of neurodevelopment and brain health [5,9,11,15] in young HIV-infected children in a narrow age range who initiated ART at different stages in infancy, of whom 95% have suppressed viral loads and 66% are neurologically normal. Notably, children who received ART before 12 weeks of age had significantly higher mean creatine, GPCPCh, and Glu levels at age 5 years, compared with those in whom ART was initiated after 12 weeks, irrespective of ART interruption, which may have negated some of the benefits of early ART. The higher metabolite levels imply that early ART initiation affords newborns neurometabolic advantages, and by extension, neurodevelopmental benefits [4–6,11,15,17,26], which are detectable even at later ages.
In contrast, independent of when treatment was initiated, poorer immune health in infancy (baseline CD4+/CD8+) was associated with reduced neuronal integrity at age 5 years. This result suggests that early effects of HIV exposure and immunocompromise in utero or during the first 6–8 weeks of life may lead to either delayed neurodevelopment or brain damage that persists into early childhood [27,28]. The CD4+/CD8+ ratio may be a more sensitive marker than CD4+ depletion from 8 weeks  as the ratio reflects both CD4+ depletion and CD8+ expansion characterized by acute HIV infection [30,31]. These results are consistent with previous studies reporting that neurological damage sustained in utero and/or during the first 6–8 weeks of life persists into early childhood, irrespective of ART, likely through toxicity, apoptosis, synaptic injury, and neuronal pruning [9,14]. One study  postulated that HIV-associated damage is reversible through ART mitigation via observed NAA recovery. Longitudinal studies are needed to establish whether recovery occurs.
The association between GPCPCh and baseline CD4+/CD8+ was lost after controlling for NAA, suggesting that the effect of poor immune health in infancy on GPCPCh levels is attributable to its effect on neuronal cell density, and that HIV-1 may target neurons rather than neuronal support cells.
Although creatine levels are widely regarded as stable, there is evidence of HIV-associated decreases in the basal ganglia [15,32]. Consistent with Keller et al., we found that the younger the children at time of first viral load suppression (<400 copies/ml plasma), the higher their CrPCr levels at age 5 years. This finding was supported by higher CrPCr levels in children in the early ART group in whom PVL would have been suppressed earlier in life. CrPCr is essential for the Krebs cycle, involving cellular transport of high-energy phosphate from mitochondria . Decreased CrPCr levels may reflect decreased total neurometabolism suggesting neural cell loss [15,17] and is associated with advanced neurological disease  or late stage dementia  in HIV-infected adults.
GPCPCh is a precursor to the neurotransmitter acetylcholine, which influences neurogenesis, stem cell differentiation, myelin formation, and synaptogenesis during early neurodevelopment . Reduced levels of GPCPCh-containing compounds in children indicate lower neural cell density . Elevated GPCPCh levels together with higher CD8+ cell count at scan observed in children with abnormal neurological signs, however, provides evidence of acute inflammation. The fact that reduced GPCPCh levels in children initiating ART after 12 weeks remain significant after controlling for abnormal neurological signs, suggests that this reduction is attributable to lower neural cell density rather than acute inflammation in the early ART children. It is striking that reduced GPCPCh presumably sustained from delayed ART initiation persists into early childhood, despite children being stable on ART at the time of scanning with high CD4+ cell counts and viral load suppression.
Lower Glu levels suggest decreased neurotransmitter levels, found in excitatory glutamatergic neuronal mitochondria. Reasons for Glu alterations are complex, but HIV-driven neuronal mitochondrial excitotoxic dysfunction is a plausible explanation for the reduced Glu levels in ART-delayed children .
Using instances of virological breakthrough as a proxy for inflammatory activity after initial ART, the similarity in metabolites between children with virological breakthrough vs. without virological breakthrough, provides evidence that the differences seen here between children initiating ART early and late are not attributable to inflammatory activity after ART initiation.
Although metabolite levels of uninfected controls were mostly similar to children receiving early ART, NAA, and GPCPCh levels were lower in controls than children in the early ART group, similar to findings by Keller at al. . This unexpected result may be attributed to 80% of the uninfected children being HEU, thus exposed to both HIV and ART, similar to observations of Cortey et al.. A limitation of this study was the low number of HIV-unexposed and uninfected children recruited.
Contrary to our expectations, only baseline CD8% tended to be higher in children on early continuous ART vs. early interrupted ART, likely reflecting more severe disease at baseline and already reaching end point criteria prior to planned treatment interruption. Furthermore, interruption did not affect the similarity in metabolites between early ART and late ART, but the relation between CD4+/CD8+ and GPCPCh strengthened (r = 0.56, P = 0.005), affirming our observation that the effects of immunocompromise (decreased CD4+/CD8+ ratio) before 6–8 weeks persist into early childhood, irrespective of treatment strategy .
There were limitations in our study, such as the selectivity of MRS in measuring certain metabolites (unlike other compounds like glucose via other modalities) as well as clinical limitations such as measurement of additional lymphocyte inflammatory markers, such as CD38 and HLA-DR expression, all which would also have been beneficial. Some of these limitations can be explained by ethical and clinical considerations since the study was in children stable on standardized ART, 73% neurologically normal, and over a narrow age range.
Our findings support an emerging philosophy of administering ART at birth to HIV-exposed infants, as seen in the ‘Mississippi Baby’  and ‘Canadian Newborns’ case series , as well as the CHER and neurodevelopmental substudy findings [2–4,6].
Although various studies have shown that HIV infection causes neurometabolic alterations, these changes and their effects on different aspects of neurodevelopment remain unclear. MRS offers a sensitive and powerful tool for measuring specific metabolite levels known to relate to neuronal and cellular integrity. Our findings support the benefits of early ART initiation into early childhood. In addition, the association of poor baseline immune health in infancy with compromised neuronal integrity at age 5 years suggests that early brain damage or developmental delay may persist into childhood, despite early ART and viral load suppression.
Author contributions: M.F.C. was part of the team that designed the CHER trial. E.D. and B.L. managed the patients in the Children's Infectious Diseases Clinical Research Unit and contributed to protocol development. B.L. was principal investigator of the neurodevelopmental substudy to the CHER trial. A.J.W.K., B.L., and E.M. were principal investigators, designed the imaging study, oversaw protocol development, data collection, data analyses, and interpretation of the findings, as well as sourced funding from the US National Institute of Child Health and Human Development, the US National Institute of Mental Health, and the RSA National Research Foundation. A.T.H. developed the scanning sequences and contributed to the data management and processing operations under supervision from A.J.W.K. and E.M. K.K.M. performed data analyses and wrote the primary report under supervision of M.J.H. and E.M. F.L. provided statistical expertise and E-M.R. contributed to the reviewership of the report.
Participating members: We extend our gratitude to Kennedy Otwombe from the Perinatal HIV Research Unit and CIPRA-SA and Christie Heiberg formerly of the Desmond Tutu HIV Foundation for assisting with clinical data requests; Lindie du Plessis (MIRU, UCT) for assistance with processing spectral data; the CUBIC radiographers Marie-Louise de Villiers and Nailah Maroof; research staff Thandiwe Hamana, Rosy Khethelo, and Anita Janse van Rensburg of Stellenbosch University for assisting with data collection. We would also like to thank Dr Christelle Ackerman, Stellenbosch University, for the neuroradiological reporting.
Support and funding: Support was provided by the NRF/DST South African Research Chairs Initiative; US National Institute of Allergy and Infectious Diseases (NIAID) through the CIPRA network, Grant U19 AI53217; NIH grants R01HD071664 and R21MH096559; NRF grant CPR20110614000019421, and the South African Medical Research Council (SAMRC). The Departments of Health of the Western Cape and Gauteng, South Africa and ViiV Healthcare/GlaxoSmithKline plc provided additional support for the CHER study.
The content of this paper does not reflect the views or policies of NIH, NRF, or SAMRC, nor does mention of trade names, commercial projects, or organizations imply endorsement by any organization or government.
None of the funding sources had any role in the study design; the collection, analysis, and interpretation of data; the writing of the report; and in the decision to submit the paper for publication.
Conflicts of interest
There are no conflicts of interest.
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