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Low levels of HIV-1 RNA detected in the cerebrospinal fluid after up to 10 years of suppressive therapy are associated with local immune activation.
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AIDS advance pub July 2014
Dahl, Viktor; Peterson, Julia; Fuchs, Dietmar; Gisslen, Magnus; Palmer, Sarah; Price, Richard W.
"In conclusion, in this study, we show that HIV-1 RNA can be detected in 17% CSF samples during suppressive therapy, which is significantly less than the 57% plasma samples that were positive. The concentration of HIV-1 RNA in CSF was also lower than in plasma. We found that HIV-1RNA could be detected in the CSF up to after 10 years of suppressive therapy and is associated with elevated neopterin levels. This suggests that HIV-1 virions continue to drive immune activation and possibly neurological injury during suppressive therapy. The findings of HIV-1 RNA in the CSF during suppressive therapy also indicate that the CNS could be a possible reservoir for HIV-1 during suppressive therapy."
Abstract
Objective and design: Though combination antiretroviral therapy reduces the concentration of HIV-1 RNA in both plasma and cerebrospinal fluid (CSF) below the detection limit of clinical assays, low levels of HIV-1 RNA are frequently detectable in plasma using more sensitive assays. We examined the frequency and magnitude of persistent low-level HIV-1 RNA in CSF and its relation to the central nervous system (CNS) immune activation.
Methods: CSF and plasma HIV-1 RNA were measured using the single-copy assay with a detection limit of 0.3 copies/ml in 70 CSF and 68 plasma samples from 45 treated HIV-1-infected patients with less than 40 copies/ml of HIV-1 RNA in both fluids by standard clinical assays. We also measured CSF neopterin to assess intrathecal immune activation. Theoretical drug exposure was estimated using the CNS penetration-efficacy score of treatment regimens.
Results: CSF HIV-1 RNA was detected in 12 of the 70 CSF samples (17%) taken after up to 10 years of suppressive therapy, compared to 39 of the 68 plasma samples (57%) with a median concentration of less than 0.3 copies/ml in CSF compared to 0.3 copies/ml in plasma (P < 0.0001). CSF samples with detectable HIV-1 RNA had higher CSF neopterin levels (mean 8.2 compared to 5.7 nmol/l; P = 0.0085). Patients with detectable HIV-1 RNA in CSF did not differ in pretreatment plasma HIV-1 RNA levels, nadir CD4+ cell count or CNS penetration-efficacy score.
Conclusion: Low-level CSF HIV-1 RNA and its association with elevated CSF neopterin highlight the potential for the CNS to serve as a viral reservoir and for persistent infection to cause subclinical CNS injury.
HIV-1 enters the central nervous system (CNS) early during systemic infection and can then be detected in the cerebrospinal fluid (CSF) throughout the subsequent course of untreated infection in most individuals [1-3]. The concentration of HIV-1 RNA in the CSF averages about one-tenth of that in plasma during chronic infection, though the CSF : plasma virus ratio varies among individuals and also may change over time within individual patients [1,2,4,5] related to both the stage of systemic infection and the origins of the CSF virus [2,6].
Through the course of infection, HIV-1 is accompanied by local inflammation and immune activation that is reflected in CSF lymphocytic pleocytosis and elevated concentrations of a number of CSF inflammatory biomarkers, including neopterin (a marker for macrophage activation), and chemokines CCL2 and CXCL10 [5,7].
Later in its course, infection may also lead to brain injury reflected in changes in CSF biomarkers of neuronal injury including neurofilament light (NFL) chain, tau and amyloid proteins [8-10]. In the era before effective treatment, 20-30% of untreated patients with advanced immunosuppression developed overt HIV-associated dementia (HAD) [11,12].
Combination antiretroviral therapy (cART) effectively reduces HIV-1 RNA concentrations in both CSF and plasma to levels below the lower level of detection of assays used in clinical settings (<20-50 copies/ml) [2,4,13,14]. However, using very sensitive assays such as single-copy assay (SCA) that allows HIV-1 RNA quantification down to less than one copy of viral RNA/ml, it is possible to detect HIV-1 RNA regularly in plasma and less commonly in CSF during suppressive therapy [15,16].
In addition to reducing the HIV-1 RNA concentrations, cART also reduces intrathecal inflammation and ongoing neural injury [14,17-19]. When it is widely available, cART has greatly reduced the incidence of HAD and opportunistic CNS infections [20-23]. However, recent studies indicate that less severe cognitive impairment is common in patients on cART despite plasma virus suppression, related either to earlier injury before treatment was initiated or to persistent low-level CNS infection accompanied by local immune activation [22,24-26].
In this study, we examined paired CSF and plasma samples from 45 patients on long-term suppressive therapy who volunteered for lumbar puncture. We used SCA to determine the frequency and magnitude of HIV-1 RNA detection in the CSF compared to plasma during suppressive cART, and also measured CSF neopterin to assess whether low concentrations of HIV-1 in CSF was associated with CNS immune activation.
Discussion
These results show that low-level HIV-1 RNA could be measured by SCA in CSF in a substantial proportion of treated individuals with 'undetectable' viral loads in both CSF and plasma by conventional clinical assays. Additionally, detection of CSF HIV-1 RNA was associated with higher CSF neopterin concentrations, suggesting that the presence of CSF virus may either be a cause or a response to local immune activation.
This is the first study using SCA to measure the low HIV-1 RNA concentrations in CSF in a substantial number of patients and samples. We found that HIV-1 RNA could be detected in 17% (12 of 70 CSF samples) during suppressive therapy. This was significantly less than the 57% (39/68) plasma samples that were positive. Both the proportion of positive samples and the measured concentrations were consistent with our previous study using the same method for a smaller group of participants enrolled in a treatment intensification study [15]. The lower concentration of HIV-1 RNA in CSF than in plasma is consistent with the relationship observed in untreated HIV-1 infection where the levels of HIV-1 RNA in CSF are usually 10-fold lower than in plasma albeit with considerable variation [31]. We found CSF HIV-1 RNA after up to 10 years of suppressive therapy. This is in agreement with smaller previous intensification studies wherewe have found CSF HIV-1RNAafter years of therapy using SCA and other methods [15,32]. By comparison, 57% of the plasma samples were positive with a median of 0.3 copies of HIV-1 RNA copies/ml and plasma HIV-1 RNA could be detected after up to
11 years of therapy which is comparable to what has previously been described [16].
We examined several factors that might have influenced CNS infection, but none appeared to favor the capacity for detecting CSF HIV-1 RNA in this group. CSF HIV-1 RNA did not correlate with that of plasma. This could suggest two separate reservoirs, one in the CNS and one in the periphery, which release HIV-1 virus particles independently. In favor of this explanation is that HIV-1 variants found in the CSF and the plasma during suppressive therapy can be genetically different variants [33]. Less likely, HIV-1 RNA found in the CSF is actually 'spill over' from the periphery, but because of temporal dispersion of viral peaks and very low levels in CSF, the associations were not seen and it would require analysis of more samples from more patients to discern this association. Whereas concentrations of plasma HIV-1 RNA found during suppressive therapy have previously been linked to pretherapy plasma HIV-1 concentrations [34], we did not find any association between the occurrence of on-therapy CSF HIV-1 RNA and pretherapy plasma HIV-1 concentrations. Likewise, because nadir CD4 T-cell counts have been linked to neurocognitive impairment in patients on cART [35], we examined the relationship of cell count nadir to detection of CSF HIV-1 RNA, but found none. Finally, since reduced drug exposure might also predispose a person to CNS infection, we examined whether there was an association with aggregate drug penetration estimates using the CPE score, but again we found no association. Hence, we were unable to identify patient background features that predisposed to appearance of detectable CSF HIV-1 RNA in these patients with plasma and CSF HIV-1 concentrations below the conventional laboratory limits.
In the light of the lack of these associations, it is of interest that detection of CSF HIV-1 RNA was associated with CSF neopterin levels. CSF neopterin has proved to be a useful biomarker of local, intrathecal immune activation [28]. It is produced predominantly by cells of the monocyte-macrophage lineage and likely also by astrocytes in response to interferon gamma and other signals [36]. CSF neopterin is elevated in untreated viremic patients with particularly high levels in HAD patients. Likewise, suppressive cART reduces these levels rapidly, though not fully to normal, and a previous study showed that immune activation in the CNS as measured by CSF neopterin continued even after years of suppressive therapy [37]. It has also been demonstrated that asymptomatic CSF 'virological escape' (measurable HIV-1RNA in CSF by standard assays during cART) was associated with elevated levels of CSF neopterin above that of patients without escape [38]. Additionally, another study using a different assay for HIV-1 RNA quantification showed that patients with CSF HIV-1 RNA concentrations above 2 copies/ml had higher levels of CSF neopterin than those with undetectable CSF HIV-1 RNA by this assay [39]. We also used CSF neopterin as our outcome biomarker for CSF immune activation. As in the previously mentioned studies, we found that the presence of HIV-1 RNA at very low levels was linked to higher levels of CSF neopterin, indicating that measuring these low levels of CSF virus is associated with an independent biological measurement. We also found that the patients who had detectable CSF HIV-1 RNA at one time point did not have elevated levels of CSF neopterin at other time points. This suggests that these patients may not have had 'chronic' immune activation in the CSF, but rather that release of these very low levels of HIV-1 in the CNS may lead to macrophage activation and neopterin production even during suppressive therapy. Alternatively, the sequence of events may be reversed macrophage activation in the CNS, provoked by some other factor, which could lead to the
production of virions. This additionally raises the question of whether these 'blips' of detectable CSF HIV-1 RNA, either by standard assays or by SCA, might also have neuropathological consequences through this increase in the local immune activation. The association between elevated levels of CSF neopterin and detectable CSF HIV-1 RNA also suggests that CSF neopterin might be used as a surrogate marker for low CNS-grade infection in evaluating treatment regimens and various strategies for CNS viral eradication. Whether the low, but clearly abnormal, levels of neopterin detected in these patients indeed reflect a level of local immune activation sufficient to chronically impair neurological function and damage CNS integrity is uncertain. Addressing this issue will require longitudinal study of the predictive value of low levels of CSF neopterin on neurological function. The study highlights the CNS as a potential reservoir for HIV-1 during suppressive therapy. It has been suggested that the CNS is a 'sanctuary site' where HIV-1 replication can persist during suppressive therapy by replicating at a low level since some drugs penetrate less well across the blood-brain barrier. In this study, we found that there was no association between the estimated CPE and the occurrence of CSF HIV-1 RNA. We have previously examined this question and found that treatment intensification did not affect CSF HIV-1 RNA or CSF immune activation, suggesting that there is no, or very little, ongoing replication in the CNS during suppressive therapy with undetectable HIV-1 RNA concentrations in both CSF and plasma by standard assays [15,32]. In addition, when we sequenced CSF HIV-1 RNA, we found little evidence of viral evolution in the CSF during therapy [33]. These findings taken together do not entirely rule out ongoing replication in the CSF during suppressive therapy. Perhaps, additional drugs are not enough to inhibit the ongoing replication in the CNS or the methods used were not sensitive or precise enough to measure these small changes. The range of CPE values in our study was narrow, and the patients were selected on the basis of both plasma and CSF viral suppression below 40 copies/ml. In this setting, the CPE score may not provide a sufficiently precise measure of CNS drug effectiveness. Nevertheless, there is no evidence in support of ongoing replication in the CNS during suppressive therapy. Hence, whether the presence of small amounts of HIV-1 RNA in the CSF during suppressive therapy is the result of production of HIV-1 visions within the CNS by cells that were infected before the initiation of therapy or relates to transport of visions across the blood-brain barrier or migration of infected cells into the CNS at the time of sampling needs to be further studied.
One limitation of our study was that the SCA only amplifies HIV-1 RNA from approximately 80% of HIV-1 B strains due to primer and probe mismatch [27].We did not have access to pretherapy samples to test for primer and probe match. Our HIV-1 RNA concentration determinations are therefore likely to be an underestimate, though with a similar effect on CSF and plasma samples.
In conclusion, in this study, we show that HIV-1 RNA can be detected in 17% CSF samples during suppressive therapy, which is significantly less than the 57% plasma samples that were positive. The concentration of HIV-1 RNA in CSF was also lower than in plasma. We found that HIV-1RNA could be detected in the CSF up to after 10 years of suppressive therapy and is associated with elevated neopterin levels. This suggests that HIV-1 virions continue to drive immune activation and possibly neurological injury during suppressive therapy. The findings of HIV-1 RNA in the CSF during suppressive therapy also indicate that the CNS could be a possible reservoir for HIV-1 during suppressive therapy.
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