br Methods br Results Plasma NFL concentrations were

Methods
Results Plasma NFL concentrations were within the linear range of the assay in all samples, Fig. 1a. It was thus possible to quantify plasma NFL in all subjects analyzed, despite more than 50-fold lower concentrations than in CSF. This included the HIV-negative controls and other subjects with low normal CSF NFL levels, Table 2. CSF concentrations were likewise within the quantitative range of the standard assay, Fig. 1b. Notably, plasma NFL concentrations were highly correlated with those of CSF (r=0.89, p<0.0001), Fig. 1c. Fig. 1 provides general context for the NFL results, showing plasma and CSF HIV RNA (d and e) and CSF neopterin (f) concentrations are also shown in Fig. 1. The patterns of plasma and CSF NFL concentration changes across the HIV-infected groups were very similar. In agreement with previous studies, the CSF NFL elevations were greatest in the HAD groups but there was also a high prevalence of abnormal levels in untreated neuroasymptomatic subjects with low CD4+ T-cell counts (Jessen Krut et al., 2014; Peterson et al., 2014). Using the laboratory age-related cut-offs for CSF NFL, all of the HAD patients had elevated CSF NFL concentrations, and 75% of untreated neuroasymptomatic subjects with CD4+ T-cell count below 50cells/μL and 58% with 50–199 CD4+cells/μL had abnormal CSF NFL concentrations; additionally 24% of subjects with 200–349 CD4 hiv fusion inhibitors and 16% of those with CD4 cell counts above 350cells/μL had increased levels—indicating a high level of clinically unappreciated ongoing CNS injury that varied with degree of systemic disease progression. By comparison, in the treated group, and in HIV-negative subjects, the frequency was much lower, 4 and 5%, respectively. There also were moderate elevated CSF NFL levels in a large proportion of subjects with PHI, 54%, in accordance with previous reports (Spudich et al., 2011). Unlike CSF, age-related reference values have not yet been established for plasma NFL, but the pattern of changes in the subject groups was very similar to that of CSF NFL. Again the HAD had highest plasma NFL concentrations, with all values exceeding the 90th percentile of the HIV negative control levels. Like CSF, there were also elevations of plasma NFL in a substantial portion of the neuroasymptomatic subjects with low CD4+ T cell counts. The reason for the similarity in these patterns was the strong correlation between plasma and CSF NFL (Fig. 1c) and thus the plasma levels appear to reflect concentrations in CSF and, hence, the severity of CNS injury. Statistical comparisons between groups using One-way ANOVA with Tukey\'s multiple comparison tests are listed in Table 3. The plasma and CSF NFL concentrations in the HAD group were significantly elevated compared to all other subgroups. The neuroasymptomatic CD4<50 group were also elevated above the other groups, except compared to neuroasymptomatic CD4 50–199, and, for CSF only, also to the PHI group. CSF NFL increases with age in the absence of HIV infection (Jessen Krut et al., 2014) and, as anticipated, there was a significant correlation between age and CSF NFL in the group of 19 HIV-negative controls (r=0.67, p=0.002). Similarly, plasma NFL was strongly associated with age in HIV-negative controls (r=0.79, p<0.0001). In Fig. 2, plasma (b) and CSF (c) NFL are plotted against age in the different groups studied. The slopes of the plasma and CSF NFL values against age in HIV-negative controls were similar to what has been reported previously in CSF (Jessen Krut et al., 2014), Fig. 2a. Plasma and CSF NFL were also significantly correlated with CD4+ T cell count, plasma and CSF HIV RNA levels, blood and CSF neopterin, and the albumin ratio. Correlation coefficients were similar for CSF and plasma NFL (Table 4).
Discussion We found elevated levels of NFL, both in CSF and plasma, in patients with HAD and in some neuroasymptomatic HIV-infected patients, mainly in subjects with low CD4+ cell count. This is in agreement with previous studies on CSF NFL, reflecting an ongoing subclinical axonal injury in these subjects (Jessen Krut et al., 2014). CSF NFL can predict subsequent development of HAD and we have previously shown that CSF NFL was increased 1–2years before overt symptoms of dementia in patients developing HAD in the pre-ART era (Gisslen et al., 2007). The incidence of HAD has decreased substantially during recent years in the developed world when the majority of HIV-infected patients are on effective ART (Lescure et al., 2011). Nowadays, HAD develops almost exclusively in so called late presenters with low CD4+ T cell counts at time of diagnosis. ART effectively prevents HAD, and severe neurocognitive disease rarely develops in patients on effective ART. Exceptions are rare cases with ‘neurosymptomatic CSF escape’ characterized by new progressive CNS disease in patients on ART with undetectable or low HIV RNA in plasma but disproportionately increased viral loads in CSF (Canestri et al., 2010; Peluso et al., 2012). Although CSF NFL can be used to detect active neuronal injury in these settings, it is limited by the need for CSF sampling that sometimes could be an obstacle. While more severe CNS disease is uncommon in treated HIV, mild neurocognitive impairment is frequently found also in patients on ART (Antinori et al., 2007; Heaton et al., 2010). In these treatment settings, plasma NFL may be a useful marker to help discriminate active ongoing neuronal injury from neurological or cognitive symptoms related to sequelae of prior CNS damage that occurred before treatment initiation (Munoz-Moreno et al., 2008), so called inactive disease, though this important issue needs further direct study. This distinction between active and static injury may have important implications for treatment, the first more amenable to intervention either with additional antiviral or adjuvant, anti-inflammatory, interventions. Previous efforts at mitigating neurocognitive impairment of treated-suppressed individuals may have been underpowered because of failure to distinguish between active and static neural injury. Importantly, a plasma assay of injury might prove valuable to assess neurological impact of treatment trials that have not included neurological assessments or lumbar puncture, opening a large experience with different treatment strategies and drug regimens to neurological outcome analysis.