We studied the frequency of positive PBMC cultures in a subset of subjects enrolled in a phase III clinical trial which compared triple therapy with indinavir and two nucleosides to dual-nucleoside therapy alone. In this study, we limited our analyses to subjects who had positive PBMC cultures at baseline. Cultures were performed in real time with freshly isolated patient PBMCs according to a standard procedure in which cultures were defined as positive by using uniform criteria (10
). Of note is that the culture method used did not include CD8 cell depletion. Cultures were held for a minimum of 18 days before they were determined to be negative, although there was some variability in the length of time beyond 18 days that negative cultures were incubated. This study was prompted in part by reports of negative PBMC culture results, determined by using standard PBMC culture methods, for subjects with HIV-1 RNA suppression. Those studies demonstrated that depletion of CD8 cells from patient and donor PBMCs dramatically increased the positive culture rate (13
Surprisingly, we found that approximately 40% of samples with a plasma HIV-1 RNA concentration of <50 copies/ml yielded a positive PBMC culture. Since cultures with negative results were not incubated for 28 days at all sites, we believe that our isolation rate of 40% in these samples is an underestimate. We suspect that the higher rate of HIV isolation in our study than in previously published work is related both to differences in the patient populations studied and to technical differences in the standard culture method used. Samples from patients in our study were obtained for HIV-1 culture between 8 and 40 wks after initiation of treatment, whereas patients who were previously studied usually had achieved virologic suppression for longer periods of time. Technical differences in the culture methods that could have led to higher isolation rates in our study included the number of patient PBMCs used, the duration of culture incubation, and our performance of cultures in real time with freshly isolated patient PBMCs. Previously published studies have demonstrated that freshly isolated PBMCs give higher culture yields than methods using cryopreserved PBMCs (1
). We do not have data on whether adding a CD8 cell depletion step would further increase the sensitivity of the ACTG culture assay used in our study. Since few cultures were incubated for more than 28 days, we also do not know whether the rate of HIV isolation would be significantly increased by a further increase in the duration of culture incubation.
We studied in detail the association of treatment and viral load with culture yield. We found that there were significantly lower rates of positive cultures in the triple-therapy arm than in the dual-nucleoside arm. Because the two treatment arms differed in the frequency of suppression of plasma HIV-1 RNA levels, we reasoned that samples with lower plasma HIV-1 RNA levels would have a lower rate of positive cultures. We found that, when our analysis was limited to subjects in the indinavir triple-therapy arm, the frequency of positive PBMC cultures was strongly correlated with plasma HIV-1 RNA concentration but that a substantial proportion of subjects with suppressed HIV-1 RNA in plasma still had positive PBMC cultures.
The finding that PBMC cultures are positive in approximately 40% of samples that have plasma HIV-1 RNA levels below the current limits of detection further supports growing evidence that reservoirs of replication-competent virus continue to exist despite the apparent suppression of HIV-1 replication (2
). In addition, at a more pragmatic level, our findings suggest that obtaining an HIV isolate may be possible in a substantial proportion of patients with viral load suppression, without resorting to CD8 cell depletion. We have demonstrated that positive PBMC cultures from samples with lower plasma HIV-1 RNA concentrations turn positive at a later time than those from samples with higher plasma HIV-1 RNA concentrations. Based on the results from this study, the ACTG consensus protocol for HIV PBMC culture now recommends a minimum incubation time of 28 days, rather than the originally recommended 21 days, before a culture is declared negative (6
Because not all patients in the indinavir arm of the study developed negative PBMC cultures or achieved virologic suppression, we postulated that we might be able to detect an association between the PBMC culture result and the subsequent virologic outcome. We did find that a negative PBMC culture result at week 8 was associated with an increased likelihood of virologic suppression at week 24. However, the PBMC culture result did not provide additional predictive value regarding those subjects with a week 8 HIV-1 RNA concentration of <50 copies/ml. This finding suggests that PBMC culture does not provide predictive information in addition to that provided by the viral load, although the small sample sizes limited our power to evaluate this question.
In summary, we have demonstrated that HIV-1 can be isolated from a substantial proportion of patients with viral loads below the limits of detection of currently available assays, even when a standard PBMC culture technique is used. Lower viral loads result in delays in a detectable rise of p24 antigen, and it is likely that prolonging the incubation time for negative cultures to 28 days will improve the sensitivity of PBMC cultures to some extent. A negative PBMC culture at 8 weeks of therapy appears to provide some predictive power for subsequent virologic suppression, although this finding did not persist when controlling for the week 8 plasma HIV-1 RNA concentration.