The significance of different mediators of HIV-associated NCI may differ across the age spectrum and, to date, studies have not described a consensus marker focused on older patients. Determining mediators and biomarkers involved in the development of HIV-associated NCI, particularly among older adults taking suppressive ART, is essential for the design of future treatment and prevention strategies. To address this gap, our study was designed to investigate biomarkers specifically associated with NCI among older and younger HIV-infected adults.

In a first step, we compared neurocognitive functioning in older and younger HIV-infected and matched HIV-uninfected adults. We did observe a significant effect of HIV infection on NCI in both younger and older HIV infected groups compared to HIV negative age-matched controls, demonstrating that HIV infection can affect NC functioning independently of age.

Second, we observed that older HIV-infected individuals present higher levels of inflammatory markers in blood and CSF (in particular, sCD163, which is a marker of monocyte activation) compared to the younger individuals, but have similar albumin CSF/serum ratios. This suggests that the higher levels of monocyte activation observed in older subjects might be a consequence of a local inflammatory process in both compartments rather than a disruption of the brain blood barrier. Interestingly, none of these inflammatory markers were associated with worse NCI in the older population, which is in line with previous reports suggesting that inflammation might play a less central role in the development of NCI during suppressive ART26.

On the other hand, we found that older adults with higher levels of HIV DNA presented more severe NCI, particularly in the domain of executive functions (e.g., cognitive flexibility, planning). This effect was not evident in the younger HIV-infected population, despite similar levels of HIV DNA, neurocognitive functioning and cognitive reserve (i.e. years of education and verbal IQ) between age groups. This difference could be a consequence of enhanced transmigration of activated HIV-infected cells through the blood brain barrier, resulting in CNS inflammation, oxidative stress and neuronal injury27,28 and deserves further investigation.

Levels of HIV DNA in peripheral blood have previously been associated with NCI10,21,22. In these studies, which included subjects from an Aging cohort in Hawaii, individuals with HIV–associated dementia presented higher levels of HIV DNA in PBMC than individuals with normal cognition, including a subset of 13 individuals with undetectable viral load11. Interestingly, no difference was noted between mildly impaired individuals and individuals with normal cognition21. Also, in the Hawaiian studies, levels of HIV DNA were significantly associated with cognitive impairment in all domains, except visuospatial ability21,22. Our data demonstrated a specific association between HIV DNA levels and the domain of executive functions, which is potentially important because executive dysfunction is increasingly prevalent during the ART era, among HIV+ individuals with well-managed disease (e.g., Heaton et al., 2011). Executive dysfunction is also a robust predictor of poor everyday functioning outcomes (see Blackstone et al., in press). In contrast with the previously published studies, our cohort includes only cases of non-severe NCI and subjects with normal cognition on suppressive ART, which is more relevant to the current HIV epidemics in the US; moreover, the main strength of our analyses is the design based on age categories, which is unique and relevant considering the ongoing changes in the HIV epidemiology characteristics.

Furthermore, in our study, we used the highly sensitive and precise digital droplet PCR (ddPCR) technology, which is particularly suitable for detection of low levels of HIV DNA, as expected to be the case in virally suppressed subjects.

Our study has several limitations. First, the small sample size and the cross-sectional design limited our ability to perform more detailed analyses on subgroups and longitudinally. For example, the lack of association between nadir CD4 count and NCI (previously reported by our group) could be a consequence of the limited sample size. Additionally, the observational study design did not allow inferring causality from the observed associations. Second, we normalized the HIV DNA levels based on the percentage of CD4 positive cells, despite the fact that some HIV DNA could have originated from non-CD4 expressing cells (particularly macrophages). Unfortunately, as a consequence of our retrospective study design, we were not able to measure HIV DNA levels in the monocyte population. Similarly, we were not able to measure HIV DNA levels using stored CSF cellular pellets, due the low number of cells in the majority of samples. Future studies should obtain freshly collected samples and possibly larger volumes of CSF in order to investigate the role of HIV-infected monocytes in the CNS and, ideally, in brain tissue from autopsy.

In summary, our study found that higher levels of peripheral CD4-associated HIV DNA was the main predictor of NCI among older HIV-infected adults on suppressive ART and that this association was driven by a deficit in the Executive Functions domain. These findings add to emerging evidence that the correlates of NCI differ in older and younger HIV infected adults and suggest that future treatment and prevention strategies for HIV-associated neurocognitive disorders may need to be tailored on the basis of age. Strategies to reduce the HIV DNA reservoir will likely positively affect neurocognitive functioning in the HIV-infected population. For example, early initiation of ART might be particularly important for older individuals to limit CNS damage and prevent development of NCI. Future studies are needed to confirm our findings in a larger cohort and to investigate possible mechanisms underlying this association.