Limits...
Comparative Analysis of Cell-Associated HIV DNA Levels in Cerebrospinal Fluid and Peripheral Blood by Droplet Digital PCR.

de Oliveira MF, Gianella S, Letendre S, Scheffler K, Kosakovsky Pond SL, Smith DM, Strain M, Ellis RJ - PLoS ONE (2015)

Bottom Line: Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001).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.

View Article: PubMed Central - PubMed

Affiliation: University of California, San Diego, La Jolla, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Levels of HIV DNA and HIV RNA within and between blood and CSF.(A) Association of HIV DNA levels between PBMC and CSF. (B) Association between HIV RNA levels between blood plasma and CSF. (C) Association of between HIV RNA in blood plasma and HIV DNA levels in PBMC. (D) Association of between HIV RNA and HIV DNA levels in CSF. Kendall’s rank correlation is indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4592012&req=5

pone.0139510.g002: Levels of HIV DNA and HIV RNA within and between blood and CSF.(A) Association of HIV DNA levels between PBMC and CSF. (B) Association between HIV RNA levels between blood plasma and CSF. (C) Association of between HIV RNA in blood plasma and HIV DNA levels in PBMC. (D) Association of between HIV RNA and HIV DNA levels in CSF. Kendall’s rank correlation is indicated.

Mentions: Next, we investigated if levels of HIV DNA in PBMC were associated with levels of HIV DNA in CSF cell pellets. Overall, there was a significant positive correlation between levels of HIV DNA measured in PBMC and CSF cell pellets (τ = 0.3; p = 0.033, Fig 2A), but 10 subjects (35.7%) had undetectable HIV DNA in their CSF cell pellets despite having high levels of HIV DNA in their PBMC. When repeating this analysis including only the 19 subjects with undetectable HIV RNA in blood plasma, this association was no more significant (τ = 0.3; p = 0.2).


Comparative Analysis of Cell-Associated HIV DNA Levels in Cerebrospinal Fluid and Peripheral Blood by Droplet Digital PCR.

de Oliveira MF, Gianella S, Letendre S, Scheffler K, Kosakovsky Pond SL, Smith DM, Strain M, Ellis RJ - PLoS ONE (2015)

Levels of HIV DNA and HIV RNA within and between blood and CSF.(A) Association of HIV DNA levels between PBMC and CSF. (B) Association between HIV RNA levels between blood plasma and CSF. (C) Association of between HIV RNA in blood plasma and HIV DNA levels in PBMC. (D) Association of between HIV RNA and HIV DNA levels in CSF. Kendall’s rank correlation is indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592012&req=5

pone.0139510.g002: Levels of HIV DNA and HIV RNA within and between blood and CSF.(A) Association of HIV DNA levels between PBMC and CSF. (B) Association between HIV RNA levels between blood plasma and CSF. (C) Association of between HIV RNA in blood plasma and HIV DNA levels in PBMC. (D) Association of between HIV RNA and HIV DNA levels in CSF. Kendall’s rank correlation is indicated.
Mentions: Next, we investigated if levels of HIV DNA in PBMC were associated with levels of HIV DNA in CSF cell pellets. Overall, there was a significant positive correlation between levels of HIV DNA measured in PBMC and CSF cell pellets (τ = 0.3; p = 0.033, Fig 2A), but 10 subjects (35.7%) had undetectable HIV DNA in their CSF cell pellets despite having high levels of HIV DNA in their PBMC. When repeating this analysis including only the 19 subjects with undetectable HIV RNA in blood plasma, this association was no more significant (τ = 0.3; p = 0.2).

Bottom Line: Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001).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.

View Article: PubMed Central - PubMed

Affiliation: University of California, San Diego, La Jolla, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus