We have demonstrated that genetically compartmentalized R5 T cell-tropic and macrophage-tropic HIV-1 populations are independently found in the CSF of subjects diagnosed with HIV-1-associated dementia. Our study was limited to a cohort of eight subjects with neurological disease who were receiving lumbar punctures at the time of HAD diagnosis and while initiating therapy, with samples available prior to therapy in two subjects followed longitudinally. In spite of a relatively small sample size we were able to link several features that distinguish two different virological states associated with severe neurological dysfunction. Compartmentalized HIV-1 populations in the CSF with R5 T cell-tropic entry phenotypes were separated phylogenetically from plasma virus populations, and were associated with clonal amplification of the CSF viral population. Replication in CD4+ T cells is consistent with both the rapid decay of compartmentalized virus in the CSF after the initiation of therapy in these subjects, and the migration of immune cells into the CNS/CSF as indicated by the presence of elevated CSF pleocytosis. Compartmentalized macrophage-tropic HIV-1 populations were associated with more genetically diverse viral populations in the CSF, and the slow decay of virus in the CSF after the initiation of therapy, indicative of viral replication in a long-lived cell. However, given the small sample size we cannot provide an accurate estimate of the relative frequency of each type of virologic state other than to note that they appeared with similar frequency in this cohort of eight subjects (three with compartmentalized R5 T cell-tropic virus, five with compartmentalized macrophage-tropic virus). Overall we detected a significantly compartmentalized CSF population in seven of these eight subjects suggesting that virus outgrowth in the CNS, whether macrophage-tropic or R5 T cell-tropic, will be a feature in a majority of HAD cases.
HIV-1 replication in the CNS is thought to occur in perivascular macrophages and/or microglia within the brain parenchyma 
. We found that both R5 T cell-tropic and macrophage-tropic HIV-1 populations are independently associated with clinical dementia. This indicates a more complex interaction between HIV-1 and the CNS since the genetically compartmentalized R5 T-cell tropic viruses are unlikely to be replicating in macrophages or microglia given their requirement for high levels of CD4 to enter target cells. During simian immunodeficiency virus (SIV) infection of macaques, CNS infection is associated with the presence of infiltrating SIV-specific CD8+
T cells in the brain, but infiltrating CD4+
T cells have not been detected 
. Trafficking of CD4+
T cells has been reported in the CNS during infection of other neurotropic viruses 
. We propose that the presence of viral antigen, especially during periods of increased HIV-1 replication in the CNS/CSF compartment, could drive the migration of both CD8+
T cells into the CNS/CSF (resulting in elevated CSF pleocytosis) and lead to persistence of compartmentalized virus through replication in the CD4+
T cells, and thus the apparent loss of this cell type. Consistent with the loss of these cells is the rapid decay of virus in the CSF during the initiation of therapy, which is considered a marker of viral replication in activated T cells 
The pathological determinants of HAD are poorly understood. Some subjects with dementia exhibit HIV-1 encephalitis (HIVE) characterized by the presence of multinucleated giant cells of the macrophage/microglia origin and immunohistochemical evidence of viral replication 
. Although the incidence of HIVE has decreased during the HAART era, neuropathological changes in brain tissue, including glial activation and monocyte/macrophage infiltration 
, are still common. Future studies examining HIV-1 populations in paired blood, CSF, and brain tissue from HAD subjects with and without neuropathological findings will help determine whether there are physiological differences in brain pathology between subjects with R5 T cell-tropic versus macrophage-tropic HIV-1 variants as the predominant CSF population. Also, the appearance of macrophage-tropic viruses largely restricted to the CNS/CSF compartment is most consistent with the appearance of these viruses late in the infection time course of HIV-1, representing an expanded host range of the virus that is initially replicating in activated T cells. Although severe neurological disease associated with HIV-1 infection has declined in the HAART era, milder forms of neurological disease are increasing. In this study we detected a significantly compartmentalized macrophage-tropic HIV-1 population in the CSF of one subject with more mild neurological dysfunction (subject 4013; ), illustrating the potential importance of understanding the correlates of HIV-1-associated neurological dysfunction with CNS/CSF viral population phenotypes.
HIV replication in the CNS can contribute to neurocognitive decline, so the ability to detect features of the CSF viral population associated with viral replication in the CNS may provide new opportunities to guide interventions prior to the development of overt neurological disease. In our study, one subject with longitudinal sampling (subject 7115) had macrophage-tropic variants present as a minor CSF population prior to the diagnosis of severe dementia (). The application of sequencing technologies with greater capacity to sample a large number of viral genomes would allow the identification of minor CSF population variants, but this approach would rely on genotypic determinants of macrophage tropism rather than the phenotypic determinants used in our study. Several sequence determinants in env
have been reported to be associated with macrophage tropism 
; however, none of these determinants distinguishes the CSF-derived macrophage-tropic viruses from the paired blood-derived T cell-tropic viruses for the subjects in our study. Thus, the evolution of macrophage-tropic virus likely occurs through multiple pathways that will require a larger catalog of env
sequences to allow reliable genotypic identification. It remains a possibility that the clonal amplification of R5 T cell-tropic viruses we detected in three HAD subjects is obscuring a smaller population of macrophage-tropic CNS virus, a question that could be addressed using more sensitive sampling methods. Developing a more complete understanding of the virological markers of CNS replication, and utilizing deep sequencing technologies to find minor populations, will provide opportunities to examine the use of CSF for information about viral replication in the CNS as a potential predictor of neurological involvement in the pathogenic process.