The demographic constitution of the cohort used in this study is summarized in . The quality and quantity of DNA were adequate from all of the specimens. The distributions of HIV DNA were skewed, as reflected in the standard deviations and differences between mean and median HIV DNA values (, ).
Demographic data of the study population (n = 49).
Parameters by diagnostic category.
Individuals with HAD had a median [interquartile range (IQR)] of 9.11 (37.20) HIV DNA per 1 × 106 PBMC compared to 0.49 (0.89) HIV DNA per 1 × 106 PBMC in individuals with NC (). In an unadjusted logistic regression model, the association of HIV DNA with HAD resulted in an odds ratio (OR) of 2.83 (IQR, 1.57–5.08; P < 0.001). The analyses were repeated after controlling for VL, CD4 cell count, HAART (71% of the participants were on HAART), duration of infection, education, age, and ethnicity which continued to show significance (P < 0.01). To evaluate the possibility that these findings were driven primarily by a few participants with very high HIV DNA values, we excluded the participants with the five highest values. This did not substantially change our results. We repeated the univariate analysis in the subset of individuals who had undetectable VL (HAD, 11; NC, 13) to determine whether the effect we observed was attributable to plasma VL. This also did not substantially change the results (OR, 2.76; IQR, 1.28–5.94; P < 0.01) ( and ).
Of particular interest were two individuals with HAD who had relatively high HIV DNA and undetectable VL: 114 and 618 HIV DNA per 1 × 106
PBMC. In an attempt to understand potential differences in these two individuals, analyses were performed on these two, and three other specimens, to ascertain HIV DNA in subsets of PBMC. PBMC from the five individuals (three with HAD, including the two with highest HIV DNA per 1 × 106
PBMC, and two individuals with NC) were obtained and separated into CD14/CD16 and CD14-negative subsets. A representative flowcytometer plot is shown in where 11–15% of the monocytes demonstrate CD14/CD16 phenotype. The CD14+/ CD16− subset was not recovered. HIV DNA was measured from each of the retained subsets (). From the two individuals with relatively high HIV DNA in PBMC, the HIV DNA per 1 × 106
PBMC was the highest in the CD14/CD16 subset (activated M/MΦ) compared to the CD14-negative subset. By flow cytometry, the CD14-negative subset included CD4 T cells. The number of cells lysed from the isolated subsets used to obtain DNA varied depending on the total number of cells originally obtained, ranging from 30 000 to 500 000. Therefore, the HIV DNA per 1 × 106
PBMC was extrapolated from the number of cells used to isolate DNA for each of the specimens. For low levels of HIV DNA (below 1 copy number per million cells), such as the CD14-negative subsets in , the values were extrapolated based on the standard curves generated by the iCycler IQ Software Program, version 3.0 (BioRad). Representative graphs of the β-globin and HIV-1 PCR standard curves and unknown samples are shown in , as described previously [32
]. From the β-globin and HIV-1 PCR standard curves, the equivalent cell number and HIV-1 copy number, respectively, are calculated for each unknown specimen. The ratio of HIV DNA copy number per 1 × 106
cells is then acquired. At the lower limits of the standard curves, if limited amount of a specimen is available, the IQ Software Program extrapolated the HIV DNA copy number per 1 × 106
cells, which assumes a linear pattern.
Representative immunofluorescence analysis of CD14/CD16 subset
HIV DNA in peripheral blood mononuclear cell (PBMC) subsets.
Representative real-time PCR standard curves with unknown examples