To assess the possibility of differential subtype sensitivity by the Amplicor Monitor, we compared the ratios of subtype-independent virion-associated RT activity as measured by the Amp-RT assay with the Amplicor Monitor viral load at the first determination following EDS. Similar ratios of Amp-RT to Amplicor Monitor viral load [medians of 0.10 (Q1–Q3 range, 0.028–0.31) for 21 subtype B specimens and 0.095 (Q1–Q3 range, 0.024–0.38) for 85 subtype E specimens tested (Wilcoxon rank sum test, P = 0.99)] suggested no significant assay bias by subtype.
Mean and median CD4 counts were similar for infected persons regardless of subtype during the entire 24 month period following EDS (Table 3). The CD8 counts were slightly higher for persons infected with subtype E, especially during the first 3 months after EDS, but the differences were not statistically different (Table 3). The NK cell percentages were significantly lower for persons infected with subtype E than B during the first 12 months but later became less significant (Table 3).
From one of the largest and best characterized cohorts of HIV-1 seroconverters in Asia and one of the few cohorts worldwide with substantial proportions of more than one infecting HIV-1 subtype, the most notable finding was the significantly higher viral load observed during the early period following seroconversion among persons infected with subtype E compared with those infected with subtype B. Although viral RNA levels remained higher in persons infected with subtype E, the magnitude of the intersubtype difference was not statistically significant during the period from 12 to 24 months.
Observed differences in inter-subtype viral loads can be the result of a number of factors. Therefore, it is important to evaluate the likelihood of any systematic biases prior to considering causative factors. One potential bias is the possibility of variable viral quantitation assay sensitivity for different HIV-1 subtypes. However, we believe that differential assay sensitivity is unlikely to account for the inter-subtype difference for several reasons. Although earlier versions of viral load assays were primarily based on subtype B sequences from North America and Europe and were less sensitive in quantifying viral load for certain non-B subtypes such as subtype E, any differential assay sensitivity would more likely result in underestimation rather than overestimation of viral load levels in subtype E-infected persons [30,31]. Furthermore, newer versions of viral load assays such as the Amplicor Monitor version 1.5 have improved sensitivities for non-B subtypes, such that sensitivities now seem comparable for the major HIV-1 subtypes [30–32]. In addition, as the magnitude of inter-subtype viral load differences decreased over time, a time-dependent bias in assay sensitivity seems unlikely. Finally, similar ratios and distributions of Amp-RT (which measures reverse transcriptase activity independent of which subtype is present) to Amplicor Monitor for both subtypes B and E also support the absence of any significant assay bias by subtype and thus support the validity of higher early viral loads in subtype E infections.
Another potential bias that may artefactually influence measured intersubtype viral load differences would be the systematic sampling of persons infected with one subtype closer to their actual time of seroconversion than those with the other subtype. The present results show that the times between the last seronegative and first seropositive test results were very similar. Although the proportion of subtype B specimens that were RNA-positive at their last seronegative visit was slightly higher, this difference was not significant, and when those seronegative polymerase chain reaction-positive specimens were excluded, the early viral load difference between subtypes B and E persisted. In the absence of any strong systematic biases, our observed findings more likely reflect the true higher early viral load levels for persons recently infected with subtype E than with B.
In general, our viral load measurements over time were similar in magnitude or slightly higher (0.5 to 1.0 log10) than those reported from other well-controlled prospective cohort studies of seroconverters from North America or Europe [6,33–36]. However, comparisons of early viral load between studies are difficult due to differences in the seroconversion intervals. On the other hand, CD4 lymphocyte counts among seroconverters in the present study were similar or lower than those reported from earlier cohort studies [33,37]. However, evidence of lower baseline CD4 counts and higher NK cell numbers in Asian populations compared with non-Asians has been reported [38,39].
The observation that intersubtype viral load differences decrease over time in the context of similar CD4 counts in persons infected with either subtype suggest that the host immune system can respond in a similar general manner to infections with distinct HIV-1 subtypes. Nevertheless, a trend in immunologic parameters, such as the higher early CD8 counts, although not statistically significant, and the significantly lower NK cell percentages in persons infected with subtype E as compared with B, may be consistent with slight differences in the early host immune response to higher viral loads associated with subtype E [40,41].
Very few prospective studies have been conducted to specifically address the issue of differences in clinical and immunologic progression by HIV-1 subtype. Although significant differences in viral load and disease progression between HIV-1 and HIV-2 have been well documented, analogous evidence of significant inter-subtype differences is far from clear [42,43]. Not only is there relatively little information on the clinical progression of HIV-1 infection in most developing countries, where the predominant subtypes are not subtype B [42,44,45], studies of otherwise similar persons infected with different subtypes living in similar settings are limited. Although one study from West Africa reported that women infected with subtype A appeared less likely to develop AIDS at 5 years post-seroconversion than women infected with subtypes C, D, and G, this study did not report viral load comparisons . A prospective study of female sex workers in northern Thailand infected primarily with HIV-1 subtype E showed similar or slightly faster progression compared with populations infected with subtype B in Western countries before highly active antiretroviral therapy was available . Continued follow-up of our cohort will be useful to compare our data with other studies in Thailand [11,45] to assess potential clinical progression differences by subtype or transmission mode as well as to confirm whether higher initial viral loads in persons infected with subtype E are correlated with subsequent intersubtype differences in disease progression.
It is unclear what factors contribute to the higher observed viral loads associated with subtype E infection. Although research has shown that viral genetic variation can influence phenotypic properties such as cell-tropism, co-receptor usage, and the ability to form syncytia [46–50], these properties have generally not been associated with genetic subtype [4,49]. For example, although initial reports had suggested that isolates of HIV-1 env subtypes E infected Langerhans cells (found in the genital tract) more readily than isolates of subtype B , two subsequent studies failed to show any subtype-specific differences [51,52]. On the other hand, recent characterization of subtype B and E viruses from our cohort of seroconverters showed major inter-subtype differences in the proportions of different envelope V3 motifs as well as predicted co-receptor usage and phenotype from genetic sequence data . In addition, reported inter-subtype differences in the function of long terminal repeat (LTR) transcriptional promoters suggest that subtype E may replicate more efficiently than subtype B in certain circumstances .
Although subtype-specific biologic characteristics may account for our observed viral load differences, it is possible that higher viral loads associated with subtype E may also be influenced by the dynamics of the HIV-1 epidemic among IDU in Bangkok. As subtype E was introduced more recently with a higher incidence in this population of IDU, one might predict a higher proportion of persons with recent infection and hence higher mean viremia among the reservoir of source partners infected with subtype E than with subtype B [54,55]. Indeed, our own cohort data indicate that the proportion with higher viral loads among persons infected with subtype E is greater than those with subtype B. Thus, if these recently infected persons in our study are similar to those serving as source partners, then regardless of the reasons for it, the subtype E reservoir of infection sources is richer in higher viral loads than for the subtype B reservoir. As higher viral load has been associated with not only higher rates of transmission through a variety of modes [15,56] but higher subsequent viral loads in the recipient in animal models [57,58], we hypothesize that a person infected by someone with subtype E would be more likely to have encountered someone with a higher viral load or inoculum, which in turn would result in higher initial viral loads in the seroconverter, which is consistent with our findings. Further research will be helpful in understanding the dynamics of transmission and the public health significance of inter-subtype viral load differences. In addition to providing very important information on the clinical progression among persons infected with two different HIV-1 subtypes, this study will provide useful background data within the context of an ongoing phase III HIV vaccine efficacy trial in Thailand .
The authors gratefully thank the participants of the study and all of the staff affiliated with this study from the Bangkok Metropolitan Administration, the HIV/AIDS Collaboration (HAC), Mahidol University, the World Health Organization, the Joint United Nations Programme on HIV/AIDS, and the Centers for Disease Control and Prevention for administrative, clinical, laboratory, and data management support. Specifically, we would like to acknowledge the study coordinators from HAC: Pongsri Virapat, Wanitchaya Kittikraisak, and Natapakwa Skunodom, Nartlada Chantharojwong (HAC) and Robert Nelson (CDC) for data management, Vedapuri Shanmugam for performing the Amp-RT assays, Marie Morgan (CDC) for editorial assistance and Timothy Dondero (CDC) for critical review of the manuscript and overall support.
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