Perinatal vertical HIV transmission is the common mode of HIV transmission in children. Intervention programs aimed at reducing the mother-to-child transmission of HIV have successfully led to a decreasing number of new perinatal HIV infections, resulting in a growing population of children who are HIV-exposed uninfected (CHEU) with 15.4 million CHEU reported globally in 2020 (Slogrove et al., 2020). CHEU are often exposed to HIV and/or antiretroviral therapy (ART) during perinatal brain development; therefore, it is important to understand the impact of perinatal HIV and/or ART exposure on early neurodevelopment in CHEU.

During early perinatal development, important cellular neurodevelopmental processes such as neurogenesis, neuronal migration, and synaptogenesis occur (Silbereis et al., 2016; Dehorter and Del Pino, 2020), which form the basis upon which optimal brain health and function are built (Hertzman and Wiens, 1996). Given that perinatal exposure to viral and bacterial agents can affect fetal brain development (al-Haddad et al., 2019), exposure to maternal HIV and/or ART during early neurodevelopment may adversely affect functional brain development and subsequent long-term neurological functioning. Brain developmental outcomes have previously been investigated in CHEU, with studies reporting significantly worse functional neurodevelopmental outcomes in areas of language and motor function in CHEU when compared to children who are HIV-unexposed (CHU) during the first 2 years of life (Wedderburn et al., 2022). In a South African birth cohort of 2-year-old CHEU, receptive and expressive language function was found to be delayed (Wedderburn et al., 2019). In addition, significantly delayed gross motor development in CHEU at the age of 6 months has been reported (le Roux et al., 2018). Significantly worse neurofunctional outcomes have also been reported in older CHEU. For example, a study in Zambia found 8-year-old CHEU to have poorer math scores when compared to CHU (Nicholson et al., 2015). Another study conducted in Thailand reported CHEU to have significantly lower IQ and memory scores at an average age of 7 years (Kerr et al., 2014). Although studies found poorer cognitive outcomes in school-aged CHEU, little is known about motor development at this age. Using magnetic resonance imaging (MRI) techniques as quantitative measures of brain development in CHEU, studies have reported significant neurostructural alterations, including reduced gray matter volumes, in infants who are HIV-exposed uninfected compared to CHU (Wedderburn, 2022) and compromised white matter integrity in infants (Tran et al., 2016) and older children (Jankiewicz et al., 2017).

Magnetic resonance spectroscopy (MRS) is a technique used to quantify specific metabolites in the brain by detecting the proton signals from metabolites within a given volume of interest (VOI) (Hajek and Dezortova, 2008). Using MRS, a study reported significant neurometabolite alterations in the basal ganglia in CHEU at the age of 9 years compared to matched CHU (Graham et al., 2020). In the same cohort of CHEU, similar changes in metabolite concentrations were found in the mid-frontal gray matter and peritrigonal white matter at the age of 11 years (Robertson et al., 2018). At both age points, reduced concentrations of N-acetyl aspartate (NAA) and Glutamate (Glu) were observed, suggesting that perinatal exposure to HIV and/or ART may affect regional neuronal integrity and glutamatergic pathways in the developing brain. Furthermore, a neurometabolic pattern identified through factor analysis was found to be a predictor of HIV exposure status in young 2-year-old (Bertran-Cobo et al., 2022) CHEU enrolled in the Drakenstein Child Health Study (DCHS). The neurometabolic pattern identified was dominated by myo-inositol (Ins) concentrations, a marker of inflammatory processes across parietal gray and white matter. Overall, regional neurometabolite patterns support findings from individual neurometabolite concentrations, providing a better understanding of how individual neurometabolites interact with each other within the context of perinatal HIV exposure.

Although regional neurometabolite concentrations and their association with functional neurodevelopmental outcomes have been investigated in the context of pediatric HIV infection (Keller et al., 2004), the link between regional neurometabolite profiles and functional neurodevelopmental outcomes in CHEU has not yet been explored. Metabolites in the brain are tightly regulated as metabolites mediate numerous metabolic and signaling pathways underlying various brain functions, including perception (Kandel et al., 2000), motor control (Kandel et al., 2000), homeostasis (Boison et al., 2017), and cognition (Nichols and Newsome, 1999). Therefore, alterations in neurometabolite concentrations in the context of perinatal HIV exposure could be involved in the pathophysiological mechanism underlying impaired functional neurodevelopmental symptomology in CHEU. To this end, we compared MRS-quantified neurometabolite concentrations between school-aged CHEU and CHU enrolled in the DCHS, building on prior findings at 2 years of age, and explored the association between regional neurometabolite profiles and functional neurodevelopmental outcomes.

Our findings implicate alterations to regional neurometabolic profiles in the context of perinatal HIV and/or ART exposure in young school-aged children. In this study, CHEU had reductions in absolute Glu and relative tCho concentrations in the parietal gray matter and parietal white matter, respectively. Furthermore, we found an underlying parietal gray matter neurometabolic pattern in CHEU to be associated with HIV exposure status and found this neurometabolic pattern to be correlated with ELOM scores of gross motor development in CHEU, with significant correlations between parietal white matter Ins concentrations and ELOM scores of fine motor development also observed. This suggests a potential neurometabolic mechanism that may underlie impaired functional neurodevelopmental outcomes in CHEU.

Our findings of regional alterations in Glu concentrations in young school-aged CHEU are consistent with results from previous MRS studies reporting reduced Glu concentrations in the basal ganglia and frontal gray matter of 9-year-old (Robertson et al., 2018) and 11-year-old (Graham et al., 2020) CHEU, respectively. In contrast, in the same cohort, no Glu neurometabolic differences were reported at 7 years of age (Robertson et al., 2018); however, this may be due to larger variability in the data at this age. Furthermore, in our cohort of CHEU at the age of 2 years, elevated Glu/Cr concentrations were reported in the parietal white matter (Bertran-Cobo et al., 2022). One of the key aspects of MRS studies that requires consideration is that of voxel tissue composition. Differences in neurometabolic trends between the gray and white matter have been observed under both normal and pathophysiological conditions (Krukowski et al., 2010) and are expected, especially since these tissues differ in terms of their chemical composition. Contradictory outcomes may therefore also be attributed to age differences with Glu elevations reported in toddlers who are HIV exposed, while reductions are found in school-aged CHEU.

Glu serves as both an excitatory neurotransmitter and an amino acid, playing a crucial role in normal cellular physiology and neurotransmission (Ramadan, 2013). Neuronal Glu is released during synaptic transmission into the extracellular space where it gets taken up by astrocytes and converted into Gln (Meldrum, 2000). Since the concentration of extracellular Glu is much lower than intracellular Glu, the Glu signal detected through MRS is primarily intracellular Glu (Ernst et al., 2010). The dysregulation of the Glu-Gln cycle could result in incomplete Glu recycling contributing to excessive extracellular Glu concentrations that may lead to neurotoxic Glu-mediated excitotoxicity (Vázquez-Santiago et al., 2014). Reduced intracellular Glu, as reported in our study, reflecting excessive extracellular Glu has commonly been implicated in studies on HIV-associated brain injury, and there are several hypotheses for the biological basis and interpretation of this finding: (i) the attenuated ability for HIV-infected astrocytes to take up Glu due to excessive tumor necrosis factor-alpha (TNF-α) release from HIV-infected microglial (Tilleux, 2008), (ii) elevated protein synthesis to repair damage in the brain due to potential exposure to HIV may be contributing to the reduced pool of intracellular Glu (Ernst et al., 2010), and (iii) mitochondrial dysregulation due to certain PMTCT ARV medications (Aldrovandi et al., 2009).

MRS-detected tCho signals consist of a combination of choline-containing compounds predominately found in myelin and neuronal cell membranes and serve as a marker of membrane density; thus, reduced tCho ratios to creatine concentrations in parietal white matter as reported in our study potentially reflect delayed myelination due to in utero and early postnatal exposure to HIV (Riley, 2018). An interpretation of reduced tCho concentrations in CHEU as reflecting regional myelin loss in the parietal white matter is broadly supported by findings from previous neuroimaging studies. For example, in 7-year-old CHEU increased fractional anisotropy on DTI was found (Jankiewicz et al., 2017), which is consistent with axonal matrix complexity reductions (Ackermann et al., 2016).

Neurometabolite concentrations of Glu and Ins have both previously been implicated individually in the context of perinatal exposure to HIV (Robertson et al., 2018; Bertran-Cobo et al., 2022). For the first time, we found a neurometabolic pattern implicating both Glu and Ins concentrations. Based on existing scientific literature, neurometabolite patterns dominated either by Glu or Ins have previously been implicated in factor analysis studies on neurochemical signatures in the context of HIV (Lentz et al., 2008). Based on MRS findings from our cohort of CHEU at 2 years of age, a cross-regional parietal matter pattern dominated by Ins across the parietal gray and white matter was found to be associated with HIV exposure status, suggesting a longitudinal signal for neuroinflammation spanning from 2 to 6 years of age (Bertran-Cobo et al., 2022). Given this finding, we hypothesized that at 2 years of age, there is a parietal neurometabolic Ins dominated factor associated with HIV exposure and that as the CHEU gets older, i.e., at the age of 6 years, there is a Glu emergence, resulting in the parietal gray matter Glu and Ins dominated factor as identified in this study.

Given that the parietal lobe is somewhat involved in motor function, including the integration of incoming motor information (Fogassi, 2005), this may explain the associations between neurometabolic profiles and motor development in CHEU. Ins is a marker of neuroglia, primarily those serving as neuroimmune cells. Correlations between parietal white matter Ins ratios to tCr+PCr in CHEU and ELOM scores in fine motor are consistent with a study by Chang et al., where lower regional Ins concentrations in the thalamus were found to be significantly associated with poorer performance in visual motor integration tasks, in children who were exposed to methamphetamine during in utero development (Chang, 1995). Furthermore, the association between the HIV-associated parietal gray matter Glu and Ins dominated neurometabolic patterns and gross motor developmental scores suggests that an increase in parietal gray matter Glu and Ins dominated factor scores is associated with lower gross motor developmental scores in CHEU, which have been previously reported. In a recent meta-analysis of neurodevelopmental outcomes (Wedderburn et al., 2022), CHEU had poorer gross motor function when compared to CHU. In addition, this association is further supported by a prior study from the DCHS cohort showing that in CHEU, infant serum inflammatory markers were associated with poorer motor development at the age of 2 years (Sevenoaks et al., 2021).

Findings from our study contribute to a growing body of literature on the regional neurometabolic signatures in children exposed to HIV and/or ART perinatally to the best of our knowledge and are the first to provide some insight into the potential role of parietal neurometabolic profiles in pathophysiology governing impaired functional neurodevelopmental symptomology observed in CHEU. The inclusion of well-characterized participants from a sub-Saharan African LMIC setting with a rich body of clinical, socioeconomic, and demographic information available and the fact that covariates were considered in our analyses are some strengths of this study. We recognize that our study had some limitations which could have affected our findings and thus our interpretation thereof. First, we had a small sample size and thus reduced statistical power, especially in the correlation analyses. Second, neurometabolite concentrations were only quantified from voxels located in particular regions in the parietal matter, thus limiting the brain metabolite findings to this region of interest. Third, despite taking covariates into account, we recognize that there may be underlying covariates not corrected in our analysis that could have influenced our findings.

Future research should be undertaken to investigate these neurometabolic profiles in other brain regions, such as frontal and limbic regions. Furthermore, further research focusing on investigating neurochemical outcomes in CHEU as adolescents or young adults is warranted, where neurometabolic findings could perhaps be investigated in relation to neurological outcomes relevant to individuals emerging into adulthood. Given that gross motor development in CHEU was associated with an neurometabolic pattern in this study and inflammatory serum markers in another study (Sevenoaks et al. 2021), a future study could perhaps focus on exploring the link between regional neurometabolites and peripheral or central inflammatory markers in CHEU. In addition, future research could also be geared toward pre-clinical animal studies exploring the exact mechanism by which tCho and Glu neurometabolite alterations may be occurring in CHEU to gain a better understanding of the pathophysiology governing neurochemical outcomes in CHEU. This could also be useful in the development of therapeutic interventions aimed at improving functional neurodevelopmental outcomes in children who are exposed to HIV and/or ART during in utero and/or early development.

In conclusion, by assessing individual relative and absolute neurometabolite concentrations in CHEU and CHEU, significant brain neurometabolite alterations were observed in both parietal gray matter and parietal white matter in this cohort study of CHEU. Using an exploratory factor analysis approach, underlying regional brain metabolite patterns were identified, with an intra-regional neurometabolic pattern found to be significantly associated with HIV exposure status. Furthermore, a parietal gray matter Glu and Ins dominated factor as well as parietal white matter Ins/tCr+PCr ratios were significantly correlated with motor development in CHEU. Findings from this study support the notion that neurometabolic alterations are prevalent in the context of exposure to maternal HIV/ART perinatally and highlight a potential pathophysiological brain neurometabolic mechanism that may be contributing to motor developmental impairment described in CHEU.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Western Cape Provincial Health Research Committee and Faculty of Health Science, Human Research Ethics Committee at the University of Cape Town. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

SW: data collection, methodology, gathering literature for the manuscript, formal analysis and interpretation, visualization, drafting the original manuscript, and review and editing. FR: methodology, supervision, and writing—review and editing. CW contributions to analysis, interpretation, and writing—review and editing. JR, LB, and CN: data collection, methodology, and writing—review and editing. NH and SJ: methodology and writing—review and editing. HZ: conceptualization, methodology, resources, and writing—review and editing. DS: supervision, conceptualization, methodology, resources, and writing—review and editing. KD: conceptualization, methodology, investigation, resources, supervision, and writing—review and editing. All authors contributed to the article and approved the submitted version.