The development of highly active antiretroviral therapy (HAART) for the treatment of HIV-1 has been associated with improvements in prognosis for patients living with HIV infection. Despite effective therapies, challenges remain in the management of chronic HIV-1 infection. Infection of the central nervous system (CNS) with HIV-1 can lead to the development of HIV-1-associated dementia or HIV-1 encephalitis, and in the HAART era, the neurological disorders in HIV patients remain prevalent.
The mechanisms leading to cognitive impairment and dementia are complex and require further investigations. The issue of whether HIV-1 in cerebrospinal fluid (CSF) originates from viral replication sources in CNS or from peripheral blood is still a subject of debate. The CNS may serve as an HIV-1 reservoir owing to the long-lived nature of CNS-infected cells and poor penetration of certain antiretroviral (ARV) therapies [1–4]. Indeed, the ARVs are different in regards to their capacity to efficiently penetrate the blood–brain barrier and attain optimal concentrations in the CNS to prevent the viral replication . The viral populations that are found in the CNS can differ from plasma quantitatively and also qualitatively.
The goal of this work was to characterize HIV-1 compartmentalization between CSF and plasma for patients with HIV-1 encephalitis and to determine the relationship between genetic distance and other virological factors.
At the time of neurological disorders, blood and CSF samples were collected in order to explore the cause of the suspected neurological disease. The HIV-1-associated encephalitis was considered as a diagnosis of exclusion based on clinical symptoms, CSF analysis, computed tomography and/or MRI. The HIV-1 viral load, bulk genotypic resistance tests and predicted tropism were determined on paired CSF and plasma samples, as described earlier . For DNA amplification, extraction was previously realised on 200 μl of whole blood. The reverse transcriptase and protease genes were analysed by population sequencing according to the Agence Nationale de Recherche sur le SIDA (ANRS) consensus protocol (www.hivfrenchresistance.org). The genotypic susceptibility score (GSS) was calculated according to the last ANRS AC-11 genotype interpretation algorithm (www.hivfrenchresistance.org): 0 or 1 if resistant or susceptible to the drugs of the current therapy, respectively. The protease reverse transcriptase viral diversity was evaluated by calculating the genetic distance among the representative sequences using Kimura's two-parameter model taking into account transitional and transversional substitution rates (MEGA 4.0 software) [7,8]. The ARV penetration was estimated by revised CNS penetration effectiveness (CPE) score . The CPE score was also balanced as follows: when the molecule was resistant on CSF genotype, the score of penetration was not assigned. Statview software, version 5.0 (SAS Institute Inc., Cary, North Carolina) was used for statistical analyses.
The samples of 39 patients diagnosed with HIV encephalitis were studied. The median age was 44 years and men were predominant (54%). The median CD4 T-cell count was 293 cells/μl (range 38–2369) and the median CD4 nadir was 77 cells/μl (range 4–656). The median HIV RNA was 3.3 log10 copies/ml in CSF (range 2–5.7 log10 copies/ml) and 3.2 log10 copies/ml in plasma (range 1.6–6 log10 copies/ml). Fourteen patients had a viral load less than 40 copies/ml in plasma. The median of CPE score was 8 (3–14). The majority of viruses were R5-tropic in CSF (29 of 30 with a successful tropism determination).
The viral diversity was evaluated by calculating the genetic distance between plasma RNA or cellular DNA and CSF RNA viruses. The genetic distance between HIV in plasma and CSF was statistically lower in patients with plasma viral load higher than 40 copies/ml than in patients with plasma viral load lower than 40 copies/ml (median 0.006 versus 0.011 for the viral load >40 and <40 copies/ml, respectively, P = 0.038). The CSF GSS of the current treatment and the CPE score were not associated with the genetic distance (P = 0.156 and P = 0.441, respectively). However, the CPE, taking into account the GSS of the current treatment (also named balanced CPE score), was associated with the genetic distance, showing a higher score when the genetic distance was low (P = 0.007).
In the present study, the low diversity between plasma and CSF viruses was linked to plasma viral loads higher than 40 copies/ml, suggesting that plasma and CSF viruses were similar, the CSF viruses were probably deriving from plasma by diffusion across the blood–brain barrier. Conversely, a high diversity was linked to plasma viral loads lower than 40 copies/ml. Thus, the viruses were different between both compartments, suggesting a self-viral replication in the CNS. Studies have revealed that HIV-1 genetic populations in CSF and peripheral blood plasma are often compartmentalized, implying that at least a portion of the CSF viral population can be produced locally within the CNS [10–13]. Functional compartmentalization has also been shown with respect to drug resistance  and cell tropism . Another group speculated that the level of blood and CSF compartmentalization is a reflection of the amount of local CNS viral replication studied by heteroduplex assay, which displays coexisting genetic variants . The HIV-1 population diversity could also be determined by PCR/cloning technique or single genome sequencing .
Interestingly, the genetic distance was also related to the CPE taking into account the GSS of the current treatment and not to the CPE score nor the GSS alone, arguing to an improvement of the CPE score by considering the ARV resistance. Indeed, when the ARVs penetrated into the CNS (high score), the genetic distances tend to be lower, suggesting that viruses would not replicate in the CNS and would derive from the plasma. The low-balanced score were in favour of poorer penetration of ARV drugs or penetration in CNS with low possibility of action owing to the resistance allowing a replication in the CNS. It is known that HIV-1 replication may persist in the CNS during ARV therapy because of the insufficient CNS penetration by several drugs .
In conclusion, a low HIV genetic distance between blood and CSF viruses in patients with HIV encephalitis suggested that viruses of both compartments were similar and that CSF viruses were probably derived from plasma. The detectable viral loads in plasma might favour virus diffusion across the blood–brain barrier and high-balanced CPE score could prevent the viral replication in the CNS compartment. A high genetic distance associated with undetectable plasma viral load and low-balanced CPE score implicated a compartmentalization of HIV-1 replication. Thus, the ARV treatment may impact the diversity of HIV.
This study was supported by the ANRS (National French Agency for AIDS Research). The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013), under the project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’ – grant agreement number 223131 and ARVD (Association de recherche en Virologie et en Dermatologie).
C.S. was in charge of virological analysis, data management and writing of the manuscript; S. F., S. L.-N. and L. M.-J. were in charge of virological analysis; R.T., A.C., P.-M. G. and C.K. were responsible for patient management; and V.C. and A.-G.M. were responsible for study coordination.
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