To the Editor:
Delayed hypersensitivity skin testing (DTH) is a widely available, cost-effective, and relatively easy-to-use tool for detection of cell-mediated immunity (CMI). A response to DTH signifies intact CMI, whereas a negative response may represent a possible defect in CMI or a lack of previous exposure. In HIV infection, DTH predicts clinical progression (1,2) and has been shown to correlate with CD4 cell count (1,3). Highly active antiretroviral therapy (HAART) has significantly improved clinical progression of HIV infection (4,5). HAART with protease inhibitors (PIs) has also rapidly restored immunologic abnormalities in HIV infection (6,7). Although studies have shown the virologic efficacy of non-PI HAART (8–10), data on the recovery of immunologic functions with these regimens is scarce. Indeed, it has been postulated by some that PI-based regimens may result in better immune reconstitution compared with regimens without PIs.
In this prospective, nonrandomized, cross-sectional study, HIV-infected subjects with and without HAART were recruited between August 1 and September 30, 2001. Recruitment was done at the Thai Red Cross AIDS Research Center and the Immune Clinic at Chulalongkorn Hospital in Bangkok, Thailand. Inclusion criteria for subjects on HAART were viral load (VL) <50 copies/mL for at least 6 months and maintenance on one of these regimens: triple nucleoside reverse transcriptase inhibitor (NRTI), dual NRTI plus nonnucleoside reverse transcriptase inhibitor (NNRTI), or dual NRTI plus PIs during the period of viral suppression. HIV-infected untreated subjects served as controls. The most recent CD4 cell count within 6 months of the recruitment date was used. The multitest CMI (11) used in this study includes the following antigens:Candida, Trichophyton, Proteus mirabilis, old tuberculin, Streptococcus group C, diphtheria, and tetanus (MULTITEST CMI; Aventis Pasteur Thailand, Ltd, Thailand). The DTH was placed by trained personnel and read at 48 to 72 hours after placement by 2 immunologists. The average of combined horizontal and vertical diameters in millimeters represented the DTH response to each antigen. Subjects with response of ≥5 mm to tuberculin were advised to obtain chest radiography (CXR). Subjects with normal CXR were advised to take isoniazid for 9 months, whereas subjects with abnormal CXR were advised to visit their physician for further investigation for tuberculosis. The primary outcome was the summed DTH response, defined as the summation of induration in millimeters to each antigen divided by the number of antigens with induration. Furthermore, the percentage of subjects with intact CMI, defined as a positive DTH response (≥2 mm) to at least two antigens, and the ability of individual antigens to elicit a DTH response were investigated. For the groups of subjects compared (PI-HAART, non-PI–HAART, HAART-treated, and HAART-untreated), stratified analysis was performed based on a CD4 count of <350 or ≥350 cells/mm3, according to the current recommendation for the initiation of HAART (12). Differences between groups were calculated using the Mann-Whitney U test at an α value of 0.05. For three-group comparison, the Kruskal-Wallis H test was performed. Multiple linear regression was used to determine factors related to DTH response.
A total of 142 subjects underwent DTH. Ninety-three subjects were treated with HAART, and 49 were untreated. Subjects on HAART were divided into a group of PI-HAART (n = 22) and a group of non-PI–HAART (n = 71; 10 triple NRTI and 61 dual NRTI plus NNRTI). There were significant differences between HAART-untreated and HAART-treated groups in age (younger in untreated group, 33.2 ± 7.9 years versus 38.1 ± 5.4 years in treated group;p < .01) and median CD4 cell count (lower in the <350 cells/mm3 strata for untreated subjects, 149 (53–225) versus 242 (176–291) for treated subjects;p < .002). Between the HAART-treated groups, baseline differences were Centers for Disease Control and Prevention (CDC) clinical class (more subjects in CDC class A for non-PI–HAART, 55.1% versus 22.7% for PI-HAART;p < .001) and time on HAART (longer for patients with PIs, 2.41 ± 0.7 years versus 1.67 ± 0.92 years without PIs;p < .01). Otherwise, the groups were well balanced. Table 1 demonstrates the summed DTH response and the percentages of subjects with intact CMI and with a positive old tuberculin test according to HAART status, CD4 categories, and treatment regimens. Overall, there was no statistically significant difference between the summed DTH response in subjects on HAART with and without PIs. This also applied when subjects were stratified within the <350 cells/mm3 and ≥350 cells/mm3 groups. The percentage of subjects with intact CMI was higher in the non-PI group, however. When comparing subjects on HAART with subjects without HAART, summed DTH response was significantly better in the CD4 <350 cells/mm3 category for subjects with HAART. Overall, DTH response and chance for intact CMI were significantly better in subjects who had CD4 ≥350 cells/mm3 compared with subjects with CD4 <350 cells/mm3. In the univariate linear regression analysis, summed DTH response was significantly and positively associated with a viral load <50 copies/mL, the absolute CD4 cell count, and HAART. In the multivariable analysis, only the absolute CD4 cell count remained significantly and positively associated with the summed DTH response, although there was a trend for the association between summed DTH response and viral load <50 copies/mL (p = .149). The antigens that elicited DTH response from largest to smallest were as follows: diphtheria, tetanus, old tuberculin, Candida, Proteus mirabilis, Streptococcus group C, and trichophyton.
Our results show that the immune recovery measured by summed DTH response is independent of the HAART regimen. These findings are in accordance with recent studies showing that immune reconstitution achieved after therapy with a PI-sparing or PI-containing regimen was similar (13) and independent of virologic efficacy (13,14). Summed DTH response in our study was positively associated with increasing CD4 cell count independent of the HAART regimen or any antiretroviral treatment. A positive correlation between CD4 cell count and DTH response is well known; however, the CD4 cutoff differs between studies (1,3,15,16). Brown et al. (3) reported complete anergy in 38% of 73 subjects with CD4 counts of 0 to 200 cells/mm3 and in 6% of 78 subjects with 201 to 400 cells/mm3. Suwanagool et al. (16) showed that tuberculin reactions of HIV-seropositive patients were 6.4 mm versus 11.0 mm among those with CD4 counts of 200 to 299 cells/mm3 and ≥300 cells/mm3, respectively. Intact CMI was found more frequently in subjects with PI-sparing HAART. There were higher percentages of subjects in the non-PI–HAART group with less advanced clinical status and with positive tuberculin reactions compared with the PI-HAART group, however. Interestingly, in the subgroup of subjects with a CD4 cell count <350 cells/mm3, the DTH response was significantly better in subjects treated with HAART compared with untreated subjects. This was probably due to the higher median CD4 count in the treated subjects in this subgroup. Indeed, French et al. (17) showed that even a small increase in CD4 cell count achieved by azidothymidine monotherapy had a positive effect on DTH response. It has been shown that, at least in advanced HIV infection, viral replication impairs immune functions (18). Our data show a positive trend between summed DTH response and VL <50 copies/mL. Diphtheria, tetanus, and tuberculin were the most likely antigens to elicit differences in DTH response between the two CD4 groups, suggesting that in a resource-constrained setting, the choice of antigens can be limited to these antigens.
In summary, no differences between PI-based and non-PI–based HAART in in vivo immune reconstitution can be found in subjects who have achieved virologic undetectability and an identical CD4 cell response.
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