Delayed-Type Hypersensitivity Skin Testing Predicts Progression to AIDS in HIV-infected Patients

Methods

Patients

All HIV-infected USAF personnel undergo an extensive staging evaluation, at least annually, including DTH skin testing to a purified protein derivative of tuberculin and four control antigens as part of the USAF HIV Natural History Study. In addition, each patient undergoes T-cell surface marker determinations. The data for this study were derived from 889 patients undergoing their first staging evaluation from 1985 through August 1990.

Delayed-Type Hypersensitivity Skin Testing

Each patient received the standard Mantoux type of intradermal skin test. The antigens and concentrations used were as follows: purified protein derivative of tuberculin (Connaught), 5TU; mumps (Connaught), 40 colony-forming units per milliliter full strength; trichophyton (Holister-Stier), 1:500 dilution; candida (Holister-Stier), 1:500 dilution; and tetanus toxoid (Lederle, 10 Lf/mL), 1:5 dilution.

Test results were assessed at 48 hours. Tuberculin skin test results were considered positive when 10 10 mm or greater induration was presentthe standard recommendation at the time of this study. Smaller areas of induration were not recorded. Control skin test results were considered positive when 5 5 mm or greater induration was present as defined by the Walter Reed staging system [14].

T-Cell Phenotypes

The CD4 T-cell subset was identified by laser-based flow cytometric analysis using single-color direct labeling techniques on ethylene diamine tetra-acetic acid-anticoagulated whole blood. Specimens were drawn consistently between 7:00 and 9:00 a.m. to minimize effects of diurnal variation on absolute CD4+T-cell counts [15]. Labeling of samples for CD4 (OKT4A-FITC, Ortho Diagnostic Systems, Inc., Raritan, New Jersey) in parallel with manufacturer's matched MslgG2a isotypic control was done using an NH_4Cl whole blood lysis technique (Ortho Diagnostic Systems, Inc.) with paraformaldehyde fixation [16]. Flow cytometric analysis was done on an EPICS Profile (Coulter Electronics, Hialeah, Florida); quality control and data analysis were done as described previously [16]. Absolute CD4+T-cell counts were calculated by multiplying the flow cytometry-determined percentage of CD4 cells by the total lymphocyte count obtained, using either a Coulter S-PLUS II or STKR hematology analyzer (Coulter Electronics).

Follow-up

Persons enrolled in the USAF HIV Natural History Study were clinically evaluated and staged by the Walter Reed staging system at 6-to 12-month intervals while on active duty and at 12- to 18-month intervals when medically retired.

Statistical Analysis

Chi-square analysis for trend was used to compare the proportion of persons responding to DTH skin tests by sequential CD4+T-cell groups. Analysis of variance (ANOVA) was used to evaluate the mean CD4+T-cell count as a function of the number of skin test responses. Tukey paired comparisons were used to evaluate differences in the mean CD4+T-cell count between paired DTH skin test response groups.

Progression to Walter Reed stage 6 (presence of an AIDS-defining opportunistic infection) was examined as a function of DTH response at first evaluation. The DTH responses were coded as categoric variables based on the number of positive skin tests (zero, one, two or more positive results). Kaplan-Meier survival curves were constructed to graphically illustrate progression to Walter Reed stage 6 for these three groups. A Cox proportional-hazards regression model was used to determine whether DTH response is an independent predictor of progression when controlling for initial Walter Reed stage and absolute CD4+T-cell count.

Results

The median age of patients in this study was 27.8 years (range, 15 to 74 years). Fifty-four percent of patients were white, 39% were black, 5% were Hispanic, and 2% were from other ethnic groups. Ninety-four percent of patients were men. Twenty-eight percent of this cohort received zidovudine at some time during the follow-up period of the study. Seven hundred ten patients with at least two evaluations over time were available for longitudinal analysis. Twenty patients had missing data points and were eliminated from the final analysis, resulting in a cohort of 690 patients. The mean follow-up time for this group was 28.5 months (SD, 12.3 months).

Table 1 shows the percentage of HIV-infected patients exhibiting anergy, a positive response to individual skin tests, response to any single skin test, and positive DTH responses to two or more skin tests when patients were stratified by absolute CD4 count in increments of 200 cells/mm³. Nearly 60% of patients with fewer than 200 CD4+T cells/mm³ were completely anergic. In contrast, 94% of the patients with more than 400 CD4+T cells/mm³ were able to mount a cutaneous DTH response to at least one skin test (P < 0.001, comparing all patients with CD4+T-cell counts less than 400/mm³ [113 of 168 patients] with those with CD4+T-cell counts greater than 400/mm³ [680 of 721 patients]). In addition, 86% (618 of 721) persons with greater than 400 CD4+T cells/mm³ were able to respond to at least two DTH skin tests, compared with only 45% (76 of 168) of those with less than 400 CD4+T cells/mm³ (P < 0.001).

The single best control skin test for distinguishing patients among these CD4 groups in our previously vaccinated population appeared to be tetanus toxoid, with statistical differences in the percentage of responders between each CD4 group up to 800 CD4+T cells (Table 1). The least discriminating skin test appeared to be trichophyton, with no statistical differences seen in the percentage of responders between CD4 groups. Mumps and candida skin testing appeared to be of intermediate utility in separating persons in these CD4+T-cell groups.

Tuberculin skin test reactivity (defined as 10 mm induration) in our population ranged between 1.1% and 2.9% among the various CD4 groups. No patient with fewer than 200 CD4 cells was found to have a positive tuberculin test result. None of these differences in tuberculin skin test reactivity was statistically significant, however, including a comparison of patients having CD4 counts less than 400/mm³ (2 of 175) with those having CD4 counts greater than 400/mm³ (14 of 729) (P > 0.2).

Figure 1 compares the mean CD4+T-cell count to the ability to mount a DTH skin test response to none, one, two, three, or all of the four control skin tests. This analysis showed that the number of positive skin test responses increased as the CD4+T-cell count increased (P < 0.001, ANOVA). The mean CD4+T-cell count for anergic patients was lower than that of patients with one or more positive skin test results (P < 0.05). In addition, patients who were able to mount only a single positive skin test response had a lower mean CD 4+ T-lymphocyte count than did those who were able to mount two or more DTH responses (P < 0.05). No statistical differences were noted between the mean CD4+T-cell counts for patients showing two, three, or four positive skin test responses on this battery of four skin test antigens.

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Figure 1. Mean CD4+T-cell counts (CD4+T cells per mm ) for patients responding to zero, one, two, three, or four of the four control skin tests ( < 0.0001 by ANOVA; see text for specific comparisons). CD4+T-cell count as a function of number of skin test responses.3P

Figure 2 shows Kaplan-Meier survival curves for progression to Walter Reed stage 6 for patients exhibiting complete anergy (n = 47), a single positive skin test response (n = 74), or two or more positive skin test responses (n = 589) at first evaluation. These curves graphically show that DTH skin test response is a predictor of progression in this cohort of patients (P < 0.001, Mantel-Cox). The median time to progression for each skin test group was 43.4 months for anergic patients, 50.6 months for patients with a single positive skin test response, and 54.9 months for patients with two or more skin test responses.

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Figure 2. Kaplan-Meier survival curves for progression to Walter Reed stage 6 based on delayed-type hypersensitivity skin test result at first evaluation for the entire cohort ( < 0.0001, Mantel-Cox). Progression to Walter Reed stage 6 for the entire cohort.P

The relative risk for progression to Walter Reed stage 6, at 5 years of follow-up, was 2.5 (Breeland-Robins 95% CI, 1.2 to 5.2) for anergic patients compared with those responding to a single DTH skin test. Similarly, patients responding to a single DTH skin test had a relative risk for progression of 3.0 (CI, 1.4 to 6.2) compared with persons responding to two or more skin tests. When anergic patients were compared with those responding to two or more skin tests, the relative risk for progression was 7.3 (CI, 4.1 to 13.2).

To determine whether the prognostic utility of DTH skin testing was independent of CD4+T-cell count, separate Kaplan-Meier survival curves were constructed for patients with more than 400 CD4+T cells (n = 613) (Figure 3) or fewer than 400 CD4+T cells (n = 97) (Figure 4) at initial evaluation. The DTH response remained a useful predictor of progression for both early- (CD4 count, >400/mm³; P < 0.001) and late- (CD4 count, <400/mm³; P < 0.001) stage HIV-infected persons.

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Figure 3. Kaplan-Meier survival curves for progression to Walter Reed stage 6 based on delayed-type hypersensitivity skin test result at first evaluation for patients with more than 400 CD4+T cells/mm ( < 0.0001, Mantel-Cox). Progression to Walter Reed stage 6 among patients with a baseline CD4 count greater than 400/mm³.3P

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Figure 4. Kaplan-Meier survival curves for progression to Walter Reed stage 6 based on delayed-type hypersensitivity skin test result at first evaluation for patients with fewer than 400 CD4+T cells/mm ( < 0.001, Mantel-Cox). Progression to Walter Reed stage 6 among patients with a baseline CD4 count less than 400/mm³.3P

To determine whether anergy status is an independent predictor of progression to AIDS when controlling for both initial CD4+T-cell count and Walter Reed stage, we did a stepwise Cox model proportional hazards regression analysis using these variables and their interactions. This analysis (Table 2) showed that each of these variables provided independent predictive value for progression of disease in this cohort. The interactive terms for these variables did not add significant prognostic information. The adjusted relative risks shown in (Table 2) provide an estimation of the relative contribution of each of these variables in predicting progression, per unit of change for each variable, in this multivariable model. For example, the adjusted relative risk for progression for DTH skin test response is 1.85 (CI, 1.39 to 2.30) for each loss of one skin test response from two or more to zero skin tests.

Discussion

We have shown that the ability to respond to DTH skin test antigens correlates with the degree of immune dysregulation in HIV-infected persons, as measured by CD4+T-cell counts. Previous studies have shown that most patients with AIDS are completely anergic [17-21] and that HIV-infected persons without AIDS have decreased responsiveness to purified protein derivative skin testing compared with non-HIV-infected persons of a similar epidemiologic background [22-24]. These studies, however, have not stratified patients by stage of disease, nor have they reported concomitant DTH controls. A recent study by Graham and colleagues [25], however, examined a group of intravenous drug users and showed that DTH skin test responses depend significantly on disease stage. Our cross-sectional analysis of DTH, as measured by the response to four control skin tests, also showed a significant increase in the percentage of patients exhibiting complete anergy as CD4+T-cell counts decrease. This increase was particularly dramatic in patients with fewer than 200 CD4+T cells/mm³, of whom 60% were anergic. In the group with 201 to 400 CD4+T cells/mm³, however, DTH responses were also abnormal (16% anergic) compared with historical series in which anergy rates of 5% to 10% have been reported in non-HIV-infected controls using various skin test antigens and concentrations [26-29]. In contrast, HIV-infected persons with more than 400 CD4+T cells/mm³ appear to have nearly normal DTH responses, with only 5.4% showing complete anergy and only 14% unable to respond to at least two DTH antigens.

In evaluating individual control skin tests by CD4 count, tetanus toxoid and mumps (at the dilutions used in this study) gave the best discriminating power, with significant differences in the percentage of responders among each CD4+T-cell group up to 800 CD4+T cells/mm³ and 600 CD4+T cells/mm³, respectively. Historical series reporting responses to tetanus toxoid among normal controls have shown response rates of 50% to 90%, depending on age, antigen dose, and time since last tetanus immunization [30-32]. Patients in our population were universally vaccinated against tetanus during induction to the military, and those with CD4 counts greater than 400/mm³ appeared to have response rates similar to those of healthy controls (75% to 88%) [32]. Similarly, normal control responses to mumps antigen have been reported in the range of 52% to 75% [26-28], similar to the range seen in our patients with more than 400 CD4+T cells/mm³ (57% to 75%). With respect to candida antigen, normal response rates in historical controls have been in the range of 52% to 92% [26, 28, 30, 31], depending on the dilution of antigen used. In this study, we used a relatively low concentration of candida antigen (1:500 dilution) compared with that used in these historical series, and this fact is reflected in the response rates (55% to 63%) for our patients with more than 400 CD4+T cells/mm³. Historical control responses to trichophyton skin testing have ranged from 28% to 53% in healthy persons [27, 28, 30], a figure that is similar to the 33% to 37% response seen in our patients with more than 400 CD4+T cells/mm³. Unlike tetanus and mumps skin testing, however, we did not find significant stepwise decreases in the percentage of patients responding to the trichophyton antigen in the CD4 groups with fewer than 400 CD4+T cells/mm³. Overall, based on this comparison with historical norms, our patients with more than 400 CD4+T cells/mm³ appeared to have a nearly normal ability to respond to DTH skin test antigens at the concentrations used in this study. At levels less than 400 CD4+T cells, however, the ability to mount DTH skin test responses was significantly impaired. Our inability to detect a difference in tuberculin skin test reactivity based on CD4 count probably represents a type II error due to the low prevalence of tuberculous infection in our sample. In addition, our use of a 10-mm cut point to define a positive tuberculin skin test result may have obscured differences in rates of reactivity among CD4+T-cell groups.

In addition to evaluating the percentage of patients responding to DTH antigens by CD4+T-cell count, we determined mean CD4+T-cell counts based on the number of skin tests to which each patient was able to respond. From this analysis, it appears that patients can be stratified into three groups: 1) anergic patients, with the lowest mean CD4+T-cell counts; 2) patients responding to a single DTH skin test, with intermediate mean CD4+T-cell counts; and 3) patients responding to two or more DTH skin tests, with the highest mean CD4+T-cell counts. The concept that a single positive DTH skin test result may indicate an incomplete DTH response has been incorporated into the Walter Reed staging classification for HIV infection [14] and has been called partial anergy. This differential point is used to separate patients with fewer than 400 CD4+T cells/mm³ into Walter Reed stages 3 through 5. Specifically, Walter Reed stage 3 patients are defined as having intact DTH responses (two or more positive skin test results), Walter Reed stage 4 patients as having partial anergy (a single positive skin test result), and Walter Reed stage 5 patients as having complete anergy. Our ability to stratify patients into three groups by DTH response, each showing distinct mean CD4+T-cell counts, lends support to the concept of partial anergy having a valid immunologic basis.

In a longitudinal analysis of our cohort, completely anergic patients progressed most rapidly to AIDS, whereas those exhibiting responses to a single DTH skin test had an intermediate prognosis compared with anergic patients and those responding to two or more skin tests. This result supports our cross-sectional analysis that DTH is not an all or nothing phenomenon. Patients responding to a single DTH skin test had intermediate CD4+T-cell counts as well as an intermediate prognosis for progression to AIDS. In addition, the utility of DTH skin testing as a marker of disease progression does not appear to be limited to either early- or late-stage disease. Our analysis of DTH response in persons with more than or fewer than 400 CD4+T cells/mm³ shows that DTH is a significant predictor of progression for both of these patient groups.

Previous studies have identified several cellular and serologic markers that predict progression of disease in HIV-infected persons [6-12]. This is the first study, however, in which DTH status has been shown to predict progression to AIDS, even when controlling for CD4 count and Walter Reed stage in a multivariate analysis. Because DTH is thought to represent an integrated measure of the cellular and cytokine function of the cell-mediated immune response, determination of DTH status in HIV-infected patients should provide critical information regarding the risk for progression to AIDS as well as the risk for false-negative tuberculin skin test results. In addition, as a functional measure of cellular immunity, DTH status may also be a useful surrogate marker of disease progression for clinical trials evaluating new therapeutic modalities for the treatment of HIV infection. Studies evaluating the effect of antiretroviral therapy on DTH response have shown that DTH function may be restored in patients treated with antiretroviral agents [33, 34]. Further work is needed in this area to better define the characteristics of serial DTH skin testing in patients treated with antiretroviral therapy.

Because DTH is clearly impaired by HIV infection, several recommendations have been made for the management of HIV-infected persons at risk for tuberculosis. The Centers for Disease Control (CDC) and the American Thoracic Society currently recommend that tuberculin reactions greater than 5 mm be considered positive in HIV-infected patients and that these persons be considered for isoniazid chemoprophylaxis [13]. The optimal management of HIV-infected persons with the potential for false-negative tuberculin skin test results, however, remains unclear. Guidelines from the CDC and from a recent clinical study recommend DTH skin testing with controls at the time of tuberculin skin testing and consideration of preventive therapy for those persons who are completely anergic and who have a high risk for tuberculous infection (> 10% prevalence) [13, 35]. This approach makes the assumption that a single positive control skin test response (defined in the CDC guidelines as any degree of induration) indicates a normal DTH response, with a presumed low risk for false-negative tuberculin skin test results. The data presented in this study, however, show that response to one skin test only (using a more rigorous definition of a positive result requiring 5 mm of induration) correlates with a significantly more advanced stage of immunosuppression as measured by lower CD4+T-cell counts, and a more rapid progression to AIDS, compared with persons responding to two or more DTH skin tests. Based on these findings, we suggest the possibility that HIV-infected persons manifesting a single positive control skin test response (partial anergy) may still be at risk for false-negative tuberculin skin test results despite tuberculous infection. Unfortunately, the potential magnitude of this problem is difficult to predict because latent tuberculous infection can be shown only by tuberculin skin testing.

Another approach to the problem of false-negative tuberculin skin test results in HIV-infected persons, as suggested by Graham and colleagues [25], is to decrease the cut point for a positive tuberculin skin test from 5 mm to 2 mm of induration. This suggestion was based on the tuberculin positivity rate in HIV-negative injecting drug users as the criterion for a true infection rate. This approach, however, necessarily decreases the specificity of tuberculin skin testing and may lead to the treatment of many persons with nonspecific cross-reactions due to nontuberculous mycobacteria. In addition, significant technical problems may occur in reliably measuring 2 mm of induration [36].

Application of our results to a population of intravenous drug users must be made with caution, however, because we do not have reliable risk data from our cohort of patients, due to military mandates against homosexual behavior and intravenous drug use. In an anonymous survey conducted by the Army on a population similar to ours, fewer than 12% of the cohort reported intravenous drug use as a risk behavior for HIV infection [37]. Evidence suggests that non-HIV-infected intravenous drug users have decreased DTH responsiveness compared with non-HIV-infected homosexual or bisexual men [38]. Further studies on the prognostic utility of DTH skin testing in intravenous drug users are necessary to validate DTH status as an independent surrogate marker for disease progression in that population.

Based on our results, we concur with the CDC recommendations that all HIV-infected persons be evaluated with DTH skin testing, not only to assess tuberculin status but also to determine the risk for progression to AIDS. Future studies should assess the risk for development of active tuberculosis among tuberculin-negative HIV-infected persons who exhibit partial anergy. In addition, future multivariate models for predicting the risk for progression to AIDS should evaluate the contribution of DTH status in determining the prognosis for HIV-infected patients.

The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Defense or other departments of the U.S. Government.