title: Comparative Analysis of Cell-Associated HIV DNA Levels in Cerebrospinal Fluid and Peripheral Blood by Droplet Digital PCR.
abstract: BACKGROUND
  Measurement of HIV DNA-bearing cells in cerebrospinal fluid (CSF) is challenging because few cells are present. We present a novel application of the sensitive droplet digital (dd)PCR in this context.
  
  
  METHODS
  We analyzed CSF cell pellets and paired peripheral blood mononuclear cells (PBMC) from 28 subjects, 19 of whom had undetectable HIV RNA (<48 copies/mL) in both compartments. We extracted DNA from PBMC using silica-based columns and used direct lysis on CSF cells. HIV DNA and the host housekeeping gene (RPP30) were measured in CSF and PBMC by (dd)PCR. We compared HIV DNA levels in virally-suppressed and-unsuppressed subgroups and calculated correlations between HIV DNA and RNA levels in both compartments using non-parametric tests.
  
  
  RESULTS
  HIV DNA was detected in 18/28 (64%) CSF cell pellets, including 10/19 (53%) samples with undetectable HIV RNA. HIV DNA levels in CSF cell pellets were not correlated with RPP30 (p = 0.3), but correlated positively with HIV RNA in CSF (p = 0.04) and HIV DNA in PBMC (p = 0.03). Cellular HIV DNA in CSF was detected in comparable levels in HIV RNA-suppressed and unsuppressed subjects (p = 0.14). In contrast, HIV DNA levels in PBMC were significantly lower in HIV RNA-suppressed than in unsuppressed subjects (p = 0.014). Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001).
  
  
  CONCLUSIONS
  Despite low mononuclear cell numbers in CSF, HIV DNA was detected in most virally suppressed individuals. In contrast to PBMC, suppressive ART was not associated with lower HIV DNA levels in CSF cells, compared to no ART, perhaps due to poorer ART penetration, slower decay of HIV DNA, or enrichment of HIV DNA-bearing mononuclear cells into the CSF, compared to blood. Future studies should determine what fraction of HIV DNA is replication-competent in CSF leukocytes, compared to PBMC.
authors.affiliation: University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Michelli Faria
authors.initials: MF
authors.lastname: de Oliveira
authors.affiliation: University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Sara
authors.initials: S
authors.lastname: Gianella
authors.affiliation: HIV Neurobehavioral Research Center, University of California, San Diego, San Diego, California, United States of America.
authors.forename: Scott
authors.initials: S
authors.lastname: Letendre
authors.affiliation: University of California, San Diego, La Jolla, California, United States of America; Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Konrad
authors.initials: K
authors.lastname: Scheffler
authors.affiliation: University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Sergei L
authors.initials: SL
authors.lastname: Kosakovsky Pond
authors.affiliation: University of California, San Diego, La Jolla, California, United States of America; Veterans Affairs San Diego Healthcare System, San Diego, United States of America.
authors.forename: Davey M
authors.initials: DM
authors.lastname: Smith
authors.affiliation: University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Matt
authors.initials: M
authors.lastname: Strain
authors.affiliation: HIV Neurobehavioral Research Center, University of California, San Diego, San Diego, California, United States of America.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Ellis
mesh_terms: D019380:Anti-HIV Agents / Q000627:therapeutic use; D023241:Antiretroviral Therapy, Highly Active; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002555:Cerebrospinal Fluid / Q000166:cytology; D004279:DNA, Viral / Q000097:blood* / Q000134:cerebrospinal fluid*; D015658:HIV Infections / Q000097:blood / Q000134:cerebrospinal fluid / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000134:cerebrospinal fluid; D012189:Retrospective Studies; D019562:Viral Load; D014766:Viremia / Q000188:drug therapy / Q000821:virology*; D014779:Virus Replication
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral; D012367:RNA, Viral