Chronic HIV-1 infection of the central nervous system (CNS) begins with primary infection.1 The unique CNS environment, which differs from the periphery by reduced immune surveillance and specific target cell characteristics, may result in compartmentalization of HIV-1 replication in the CNS-producing genotypes and phenotypes associated with neurological impairment.2–4 Furthermore, reduced drug penetration may induce distinct drug resistance mutations in cerebrospinal fluid (CSF).5,6
In general, HIV infection of the CNS responds well to combined antiretroviral therapy (cART).7–9 However, various case reports, and retrospective and cross-sectional studies reported detectable HIV-1 RNA in CSF in individuals with undetectable viremia.10–16 Because several antiretroviral drugs do not efficiently cross the blood–brain barrier, some experts suggest that CSF penetration of antiretrovirals needs to be considered when a treatment is started.17,18
Our aims were to determine HIV-1 RNA in CSF of patients on successful cART in a longitudinal study and to evaluate if treatment combinations with a higher CNS penetration-effectiveness (CPE) achieve better viral suppression in CSF. Furthermore, we explored whether CSF concentrations of atazanavir, lopinavir, and efavirenz have a direct impact on CSF viral suppression.
Study Design and Patients
This study was embedded in a randomized controlled study evaluating the efficacy and safety of monotherapy with lopinavir/ritonavir (MOST-study; Clinical trial: NCT00531986).19 All patients were recruited from the Swiss HIV Cohort study, a prospective multicenter cohort study.20 The study was performed in 5 HIV clinics in Switzerland (Zurich, St Gallen, Bern, Geneva, and Lausanne) and was approved by local ethics committees. Written informed consent from all participants was obtained.
Study participants had to be on cART [defined as 2 nucleoside/tide reverse transcriptase inhibitors (NRTI) plus either a nonnucleoside reverse transcriptase inhibitor (NNRTI) or a boosted or unboosted protease inhibitor (PI), or 3 NRTI alone] for more than 6 months with plasma HIV-1 RNA <50 copies per milliliter for at least 3 months. No history of treatment failure or any PI resistance was allowed. The study included clinical examinations, neuropsychological assessments with a test battery (Colour Trial 1 and 2, EWIA DIGIT Symbol test, and Grooved pegboard test with dominant and nondominant hand), phlebotomies, and lumbar punctures (LPs) for all patients at baseline, at week 48, and at study termination. In the MOST trial, the participants were randomised either to monotherapy with lopinavir/ritonavir or to continuation of triple therapy. In the present study, we analyzed all data of the MOST participants on continued triple cART (all data at baseline, plus week 48 for the patients on continued triple therapy). LPs were performed between May 2007 and November 2008.
CSF and Blood Measurement
Plasma HIV viral load (VL) was quantified in local laboratories by automated reverse transcriptase–polymerase chain reaction (COBAS AmpliPrep/COBAS TaqMan HIV-1, version 1; Roche Diagnostic Systems, Basel, Switzerland), with a detection limit of 40 copies per milliliter. Similarly, the CSF HIV-1 RNA was measured in the laboratory of the University Hospital of Geneva in samples frozen at −80°C after collection. CSF cell count, protein and glucose measurements, and peripheral CD4 cell counts were performed locally using routine methodology. We determined the plasma and CSF drug concentrations of the most frequently prescribed PIs lopinavir and atazanavir and the most frequently used NNRTI efavirenz. The measurements were performed in the Laboratory of Clinical Pharmacology in Lausanne, by reversed-phase liquid chromatography coupled to tandem triple-stage quadripole mass spectrometry (LC-MS/MS) on a TSQ Quantum (Thermo Co, San Jose, CA), using an adaptation of the previously described method.21 The LC-MS/MS method for CSF measurements was calibrated using matrix-matched samples prepared in artificial CSF (glucose 0.8 g/L, albumin 0.2 g/L, NaCl 7.3 g/L, KCl 0.3 g/L, NaHCO3 1.9 g/L, pH adjusted to 7.5 with NaHPO4 buffer), as previously reported.22 The lower limit of detection for lopinavir, atazanavir, and efavirenz in CSF is 1, 1, and 2.5 ng/mL, respectively. For pharmacokinetic analyses, the times of last drug intake, phlebotomy, and LP were recorded.
Patient-specific lopinavir, atazanavir, and efavirenz trough levels (Cmin) were extrapolated from the concentrations in CSF measured at their clinical visits. These extrapolations were based on mean terminal t1/2 established in population pharmacokinetic models developed on the basis of a systematic review of published literature.23 As one-compartment models, they assume that equilibrium between plasma and all-body area, including CSF, is essentially instantaneous and that the drugs are eliminated by a first-order process.
CPE Scores for Antiretroviral Medications
The CPE was calculated by adding up predefined scoring points for each component of the antiretroviral regimen; a higher CPE indicates better effectiveness. We assessed the CPE using 2 different methods proposed by Letendre et al,18,24 which are summarized in Tables 1 and 2. The score depends on physicochemical, pharmacokinetic, and pharmacodynamic properties, and on drug effectiveness in the CNS found in clinical studies. The revised version of the CPE score incorporated data from recent pharmacokinetic and pharmacodynamic analyses and was reported to correlate more strongly with CSF VLs than the initial CPE score.
Differences of CPE and other covariables for patients with detectable versus undetectable CSF VL were evaluated using marginal logistic regression models, with exchangeable correlation structure and robust SEs to account for repeated measures per patient. Statistical analyses were performed using STATA version 12.1 (StataCorp LP, Lakeway Drive, TX) and graphs were drawn using Graph Pad Prism version 5.
At baseline, all patients included in the MOST study underwent LP. The LPs were repeated in all patients at week 48 and at study termination. For the current study, we included 60 baseline LPs and 28 LPs at week 48 of the patients randomized to continued triple therapy. Because in 1 CSF sample HIV-1-RNA measurement was missing, 87 CSF samples from 60 patients on cART were analyzed.
The participants' demographic and clinical characteristics at baseline are summarized in Table 3. At time of LP, plasma HIV RNA was suppressed for a median of 41 [interquartile ratio (IQR) 21–75] months. Sixty-four samples were obtained from patients treated with PI (all but 1 boosted with ritonavir), 20 with NNRTI, and 3 with NRTI only. The most commonly used regimen (n = 24) was 3TC, zidovudine, and lopinavir/ritonavir.
Characteristics of Participants With Detectable HIV-1-RNA in CSF
In 56 (93.3%) subjects, the CSF VL was always undetectable (<40 copies/mL). In 4 subjects, VL in CSF was detectable in 1 measurement, 3 of which had detectable VL at baseline and 1 at week 48. Only in 2 of the 3 patients with detectable CSF VL at baseline, the LPs were repeated on cART after 48 weeks; both CSF showed undetectable VLs. The other patient with detectable VL at baseline was randomised to monotherapy with lopinavir/ritonavir. His CSF VL increased from 43 to 250 copies per milliliter after 37 weeks when the study was prematurely terminated, although full HIV suppression in plasma was maintained in his case.19 However, the values of the LP on monotherapy were excluded from further analysis in our study. Characteristics of the patients with detectable VL in CSF are summarized in Table 4. Two patients had a pretreatment plasma HIV-1 RNA >100,000 copies per milliliter, and 2 had >300,000 copies per milliliter. Three patients had a CD4-nadir <200 cells per microliter. Two patients were in Centers for Disease Control and Prevention stage C3, with a history of tuberculosis and Kaposi sarcoma, respectively. Current CD4 counts were >300 cells per microliter in all patients. In 1 participant, plasma VL had been suppressed for 3 months, and in 3, for more than 2 years. Neither CD4-nadir, current CD4 count, maximal VL, nor time since suppression differed in participants with detectable VL and patients with undetectable VL in CSF.
cART and CPE of Patients With Detectable HIV-1 RNA in CSF
All 4 patients with detectable CSF VL were on emtricitabine plus tenofovir combined with efavirenz (n = 1), atazanavir/ritonavir (n = 2), or lopinavir/ritonavir (n = 1) (Table 4). No patient on an NRTI-backbone with zidovudine (n = 39) or abacavir (n = 18) had detectable VL in CSF at any time. The 2008 and 2010 CPE scores of the patients with detectable VL in CSF, respectively, were 1 and 6 in 3 patients and 1.5 and 7 in 1 patient (Table 4). The median CPE of patients with suppressed CSF HIV-1 RNA was 2.3 (range, 1.0–3.5) and 8 (range, 5–12) (Table 5). Hence, the CPE for the 4 regimens with detectable HIV-1-RNA in CSF was significantly lower for both 2008 and 2010 scores (P = 0.011 and 0.022, respectively) (Fig. 1) compared with the 83 regimens with undetectable VL. Our data suggest a threshold of 2 for the 2008 CPE resulting in a sensitivity of 100% and a specificity of 63%, and a threshold of 7 for the 2010 CPE resulting in a sensitivity of 75% and a specificity of 82% for VL suppression in CSF.
Drug Concentrations of Lopinavir, Atazanavir, and Efavirenz in Plasma and CSF
Drug concentration was measured in 71 plasma (45 lopinavir, 10 atazanavir, 16 efavirenz) and 72 CSF samples (42 lopinavir, 12 atazanavir, 18 efavirenz). Individual values are depicted in Figure 2.
Median plasma concentration of lopinavir was 5435 ng/mL (IQR 4049–7816) at a median of 6 hours (IQR 5–9) postdose, corresponding to 3566 ng/mL (IQR 2579–5388) extrapolated at trough (Cmin,12h). Median concentration in CSF was 23.0 ng/mL (IQR 17.5–40.0) at 6.8 hours (IQR 4.8–8.9) postdose, corresponding to 18.4 ng/mL (IQR 10.5–29.3) at Cmin,12h. In 41 of 42 (98%) CSF samples, the extrapolated Cmin,12h of lopinavir were above the 50% inhibitory concentration (IC50) for wild-type HIV-1 of 1.9 ng/mL.26,28 The patient with detectable VL in CSF taking lopinavir/ritonavir had a plasma concentration of 2970 ng/mL at 6 hours postdose, which lies below the 10th percentile (Fig. 2, stars); however, the concentration in CSF was 14 ng/mL at 5.4 hours postdose, corresponding to 8.3 ng/mL at Cmin,12h, which is above the IC50.
Median atazanavir concentration was 700 ng/mL (IQR 470–964) in plasma at 13.8 hours (IQR 11.4–16.0) postdose, corresponding to 265 ng/mL (IQR 177–447) at Cmin,24h. The median concentration in CSF was 14.5 ng/mL (IQR 1.9–17.5) at 15.5 hours (IQR 11.5–22.9) postdose, corresponding to 7.3 ng/mL (IQR 1.9–10.4) at Cmin,24h. Only in 3 of 12 (25%) CSF samples, the extrapolated Cmin,24h for atazanavir was clearly above the presumed IC50 for wild-type HIV-1 of approximately 11 ng/mL in plasma. However, the presumed IC50 in a protein-free medium is probably 10-fold less, meaning 1 ng/mL. As CSF contains some protein, the IC50 is estimated to lie somewhere between 1 and 11 ng/mL.27 In 2 of 12 (17%) CSF samples, the atazanavir drug level was even below the lower limit of detection of 1 ng/mL. Both patients with detectable HIV-1 RNA in CSF on atazanavir/ritonavir had low CSF concentrations: 36 ng/mL at 5.3 hours postdose and 1.6 ng/mL at 22.5 hours postdose; corresponding to 8 and 1.4 ng/mL at Cmin,24h, respectively, therefore in the range of the presumed IC50 in CSF.
The median concentration of efavirenz in plasma was 936 ng/mL (IQR 382–1116) at 14.4 hours (IQR 13.2–15.7) postdose, corresponding to 752 ng/mL (IQR 379–901) at Cmin,24h. The median concentration in CSF was 10 ng/mL (IQR 7.0–14.0) at 15.4 hours (IQR 13.2–16.3) postdose, corresponding to 8.3 ng/mL (IQR 6.0–12.0) at Cmin,24h. In 14 of 18 (78%) CSF samples, Cmin,24h were above the published IC50 of 0.51 ng/mL.25 The patient with detectable CSF VL had an efavirenz concentration in CSF of 10 ng/mL at 16.2 hours postdose, corresponding to 8.7 ng/mL at Cmin,24h and therefore well above the published IC50.
In summary, the extrapolated trough levels in CSF for lopinavir and efavirenz were above the IC50 in the majority of samples. In contrast, the trough levels in CSF for atazanavir were above the presumed IC50 in only one quarter of the analyzed samples. Accordingly, the 2 patients with detectable CSF HIV-1 RNA on atazanavir/ritonavir had extrapolated trough concentrations in CSF just in the range of the reported IC50, whereas the 2 patients on lopinavir/ritonavir and efavirenz had extrapolated trough levels above their respective IC50.
Other CSF Markers
Protein, glucose, and cell count were normal in 86 of 87 CSF samples. One subject with suppressed CSF VL had elevated white blood cells (9 cells/µL). White cell count and protein in CSF of the patients with detectable CSF VL were slightly but not significantly higher (Table 5).
None of the patients with detectable CSF VL complained about neuropsychological disturbances. There was no difference in tests assessing the speed of processing information between patients with undetectable and detectable VL in CSF. Two patients with detectable CSF HIV-1 RNA showed slower motor speed than the average; however, because of the small sample size and the older age of one of these individuals, no statistical association between test results and detectable CSF VL can be established (data not shown).
Successful cART correlated with full suppression of HIV-1 RNA in a vast majority of CSF samples. The 4 patients with detectable CSF HIV-1 RNA were on treatments with significantly lower CPE scores. The majority of patients on lopinavir/ritonavir and efavirenz, including the 2 patients with detectable CSF HIV-1 RNA, had extrapolated CSF trough levels above the respective IC50. It must be cautioned, however, that the exact IC50 in CSF for efavirenz and lopinavir is not well established.25,26 Further pharmacodynamic studies assessing this question are definitively warranted. In general, the IC50 of wild-type HIV viruses are known to be relatively variable,29 and finding information on the exact medium used in the published phenotypic assays (regarding protein concentration) is far from easy. The IC50 levels chosen as target in our study have to be considered as an order of magnitude, rather than as a precise value.
The extrapolated CSF trough levels for atazanavir were below the presumed IC50 for plasma in 75% of the samples. The 2 patients on atazanavir/ritonavir with detectable HIV-1 RNA in the CSF had extrapolated trough levels just in the range of the IC50 in CSF. This finding confirms the observation from the CHARTER group that atazanavir in CSF does not consistently exceed the wild-type IC50.27 Accordingly, relatively low rankings in the CPE score are attributed to atazanavir.18,24
Viral suppression in CSF may also significantly depend on the backbone drugs whose activity depends on intracellular phosphorylation rather than measured extracellular drug level. Hence, there are no established target concentrations for these drugs. The fact that all 4 patients with detectable VL in CSF were on treatment with tenofovir provides further evidence for the low score of 0 and 1 attributed to tenofovir. In contrast, all patients on zidovudine or abacavir, with scores of 1 and 4 or 1 and 3, respectively, did not have detectable VL in CSF at any time point. The benefit of zidovudine on the CNS was described already in the beginning of the antiretroviral therapy era,30,31 and its use is still recommended in the current EACS guidelines for treatment of patients suffering from HIV-associated dementia or HIV-associated neurocognitive disorder.32
In summary, our data support the hypothesis that the composition of an antiretroviral regimen and the corresponding CPE, integrating all drug components including NRTIs, is a better predictor for treatment potency and viral suppression in CSF than the sole concentration of a single PI or NNRTI in plasma or CSF.
Besides low CSF drug concentration, weak host immunity may account for a detectable CSF VL. Several studies have described an association between low CD4 counts and neurocognitive impairment.33,34 The 4 patients of our study with detectable HIV-1 RNA in CSF had high pretreatment HIV-1 RNA in plasma and 3 of them had a CD4 nadir <200 cells per microliter. However, in our study, these values did not significantly differ from the patients with undetectable CSF VL.
The emergence of discordant HIV suppression in plasma and CSF is only beginning to be recognized. Edén et al12 described viral escape in CSF in 7 (10%) of the 69 successfully treated subjects. In that study, the subjects with detectable VL had longer treatment durations, a higher number of viral blips, and more treatment interruptions. The CPE did not correlate with detectability of HIV-1 RNA in CSF. Compared with our study, the median CPE was lower (1.7 versus 2.3), the rate of detectable CSF HIV-1 RNA was higher (10% versus 4.6%), and the measured VLs levels were higher (121 versus 51 copies/mL). The lower number of patients with detectable CSF HIV-1-RNA in our study may be because of superior adherence; none of our patients had treatment failure.
In our study, no individual on triple therapy had a detectable CSF VL on 2 occasions. CSF cell counts and protein levels were not elevated. We speculate that slightly diminished treatment efficacy—not measurable in terms of elevated plasma VLs—might lead to residual intrathecal low-level viral replication without detectable inflammation. We could not demonstrate a clinical impact of detectable CSF VLs because the study participants neither reported any neurocognitive impairments nor could a clear impairment in neuropsychological tests be documented. In contrast to this finding, Canestri et al13 described 11 patients who developed neurological symptoms with detectable HIV-1-RNA in CSF despite undetectable viremia. Their median CPE was 2 (range, 1–3) and thus comparable to our study. However, the median CSF HIV RNA level was approximately one magnitude higher than in our patients (880 versus 51 copies/mL). This difference may well explain the unequal occurrence of neurological symptoms.13 A recently published analysis of the ALLRT cohort with 2636 participants suggests that optimized treatment regimens with higher CPE scores could improve neurocognitive function.35 It is conceivable that—although our patients did not complain about neurological symptoms—low-level HIV-1-RNA in CSF may have a clinical impact over time.
Currently, in the Swiss HIV Cohort Study, 19% of the patients are on treatment with tenofovir, emtricitabine, and efavirenz and 10% on tenofovir, emtricitabine, and atazanavir (data on file). These regimens are frequently used because of their high potency, favorable adverse drug reaction profiles, and once-daily administration. However, both combinations have low CPE of 1 and 6.
Our study has strengths and limitations. Strengths include the inclusion of patients within the well-defined study setting of the Swiss HIV cohort study with repeated LP after 48 weeks in a random 50% sample of patients. This allowed overcoming the inherent shortcomings of cross-sectional studies that are mostly conducted if CSF needs to be investigated. Because of the excellent adherence of our study population with 93% of all patients reporting a 100% drug intake that was assessed by validated self-reported questionnaires,36 we could demonstrate a suppression of HIV-1 RNA in CSF of a vast majority of patients. We repeatedly performed standardized neuropsychological assessments and measured drug levels in blood and CSF in the majority of samples. Another strength is that we used the CPE score, as proposed by Letendre et al, that has been successfully validated in various analyses: higher CPE score was reported to be more effective in controlling CSF viral replication,37 to be associated with improved neuropsychological performance,35,38,39 and longer survival after diagnosis of HIV encephalopathy40 or opportunistic infection of the CNS.41
One important limitation is the relatively low number of 60 analyzed patients in consequence of the highly demanding study protocol. In these highly motivated individuals who agreed to undergo sequential LPs and who had never failed a previous treatment regimen, the fraction of patients with detectable CSF VL and the magnitude of the detected VLs in CSF most likely are lower than in the general HIV-1–infected population under treatment.
In summary, our study provides further evidence that triple antiretroviral treatment taken with good adherence suppresses HIV-1 RNA in a vast majority of CSF samples. However, treatment regimens with low intracerebral efficacy may lead to detectable HIV-1-RNA in the CSF. In our study, the estimation of the intracerebral efficacy by the CPE score as described by Letendre et al integrating all drug components including NRTIs was a better predictor for treatment potency and viral suppression in CSF than the sole concentration of a single PI or NNRTI in plasma or CSF. The long-term impact of an intermittent presence of low-level HIV-1 RNA in CSF associated with some of the most frequently used cART regimens with low CPE scores are currently unclear and should be further evaluated in longitudinal studies.
The authors are grateful to the patients participating in this very demanding study and thank the physicians and study nurses for excellent patient care, the laboratories for high-quality data, the data center in Lausanne for excellent data management, and Marie-Christine Francioli for administrative assistance. The members of the Swiss HIV Cohort Study are Barth J, Battegay M, Bernasconi E, Böni J, Bucher HC, Burton-Jeangros C, Calmy A, Cavassini M, Cellerai C, Egger M, Elzi L, Fehr J, Fellay J, Flepp M, Francioli P (President of the SHCS), Furrer H (Chairman of the Clinical and Laboratory Committee), Fux CA, Gorgievski M, Günthard H (Chairman of the Scientific Board), Haerry D (deputy of “Positive Council”), Hasse B, Hirsch HH, Hirschel B, Hösli I, Kahlert C, Kaiser L, Keiser O, Kind C, Klimkait T, Kovari H, Ledergerber B, Martinetti G, Martinez de Tejada B, Metzner K, Müller N, Nadal D, Pantaleo G, Rauch A, Regenass S, Rickenbach M (Head of Data Center), Rudin C (Chairman of the Mother and Child Substudy), Schmid P, Schultze D, Schöni-Affolter F, Schüpbach J, Speck R, TafféP, Tarr P, Telenti A, Trkola A, Vernazza P, Weber R, Yerly S.
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