Tuberculin and Anergy Testing in HIV-Seropositive and HIV-Seronegative Persons

Human immunodeficiency virus (HIV) is playing a substantial role in the resurgence of tuberculosis in the United States. Particularly affected are people in urban areas, where there are large populations of HIV-infected persons [1-8]. Urban subpopulations with a high prevalence of HIV infection, such as intravenous drug users (a group already at increased risk for tuberculosis before the appearance of the acquired immunodeficiency syndrome [AIDS]), have the highest tuberculosis attack rates [3]. Unlike other AIDS-associated opportunistic pathogens, Mycobacterium tuberculosis is readily communicable among persons with all levels of immunity. Recently, tuberculosis outbreaks, some with multidrug-resistant strains, have occurred among HIV-positive patients with transmission to HIV-negative patients and health care workers [9-13]. Prevention strategies rely heavily on the use of tuberculin purified protein derivative (PPD) to identify persons harboring M. tuberculosis [14]. Anergy, a consequence of HIV infection, undermines these strategies in persons at the highest risk for tuberculosis infection and subsequent active disease [15-18]. A negative PPD test result in this setting could be attributable to a true lack of exposure to tuberculosis or simply to the incapacity of the patient to manifest an appropriate cell-mediated immune response. To reduce the measured prevalence of anergy and thereby increase the proportion of tuberculin nonreactors who can be considered truly PPD negative, the Centers for Disease Control and Prevention (CDC) has recommended the additional use of at least two delayed-type hypersensitivity control antigens (mumps antigen plus Candida antigen or tetanus toxoid) when screening HIV-infected patients. Thus, persons from populations with a prevalence of tuberculous infection of 10% or more and who are tuberculin negative but not anergic may be spared preventive therapy with isoniazid [19]. However, the ability of control antigens to predict the likelihood that a negative PPD test result is truly negative in this highly anergic population is unknown. To improve approaches to tuberculosis prophylaxis, more data are needed about the relations among delayed-type hypersensitivity responsiveness, the prevalence of tuberculosis, and the waning immunity associated with progressive HIV infection. In an ongoing multicenter study of the natural history of the pulmonary complications associated with HIV infection, we have been examining these factors prospectively in a cohort of 1353 persons in 6 U.S. geographic areas. Recently, we evaluated baseline delayed-type hypersensitivity responses in this cohort of HIV-seropositive and HIV-seronegative persons and identified variables associated with tuberculin reactivity and anergy. Methods Patients and Study Design The Pulmonary Complications of HIV Infection Study is a multicenter study designed to prospectively describe the frequency, types, and effect of pulmonary complications in HIV-infected persons, both before and after the development of AIDS. All diagnoses, treatments, and outcomes are recorded and monitored in a common database. Because our purpose was to evaluate longitudinally both the early and late pulmonary manifestations of HIV infection, each center attempted to recruit about 170 HIV-seropositive participants, half with CD4 lymphocyte counts of 400 cells/mm3 or more and no HIV-related symptoms and half with fewer than 400 CD4 cells/mm3 or symptomatic HIV-infection (defined by a temperature of 38 C or more for at least 2 weeks, involuntary weight loss of 10% or more from baseline, diarrhea of at least a 1-month duration, oral candidiasis, or oral hairy leukoplakia). Within each group, participants were drawn from one of three HIV transmission categories (homosexual men, male and female intravenous drug users, and women with heterosexually acquired HIV infection) to reflect their approximate distribution at each clinical site. About 30 HIV-seronegative homosexual men and intravenous drug users were also recruited at each site to serve as controls. Participants had to be willing and able to comply with the protocol and were required to give informed consent. The study was reviewed and approved by the institutional review board at each site. Exclusion criteria included Centers for Disease Control and Prevention (CDC)-defined AIDS [20], severe non-HIV-related disease likely to affect survival, lung disorders likely to interfere with the required evaluations, acute pulmonary processes, immunosuppressive therapies within the previous 6 months, and treatment for active tuberculosis within the past 12 months. From November 1988 through February 1990, we enrolled 1353 persons in the study, of whom 1171 were HIV seropositive and 182 were HIV seronegative. Human immunodeficiency virus serologic status was confirmed at study entry using a licensed enzyme-linked immunosorbent assay and a Western blot assay. Further baseline evaluation included a complete medical history, a physical examination, hematologic and biochemical studies, T-lymphocyte subset analysis, delayed-type hypersensitivity testing, a chest roentgenogram, and pulmonary function measurements. Measurement of Delayed-Type Hypersensitivity Response We tested delayed-type hypersensitivity with the following antigens: mumps antigen (Connaught Laboratories, Inc., Swiftwater, Pennsylvania); Dermatophytin 0 (Candida) at 1:100 dilution (Hollister-Stier, Spokane, Washington); Dermatophytin (trichophytin) at 1:100 dilution (Hollister-Stier); and tuberculin PPD at a strength of 5 tuberculin units per 0.1-mL dose (Connaught Laboratories, Ltd., Willowdale, Ontario, Canada). Tests were administered by intradermal injection of 0.1 mL of antigen (Mantoux method) and read by a trained observer 48 to 72 hours after application in most participants (the interval exceeded 4 days in 18 persons). Response was recorded as the greatest diameter of induration. We used the current standard operational criteria for a positive response: induration of at least 5 mm for all antigens except PPD, for which an induration of 10 mm was required among HIV-uninfected persons [19]. Anergy was defined as 0 mm of induration for all delayed-type hypersensitivity antigens administered in a given panel. Unless otherwise specified, a test battery of tuberculin PPD, mumps antigen, and Candida antigen was used to define anergy. At one site, however, the investigators did not distinguish between induration and erythema for the mumps, Candida, and trichophytin tests, documenting reactions to these antigens in terms of millimeters of erythema. When examined by zone diameter, their measurements were generally consistent with those from the other centers. Furthermore, the results of multivariate analyses with and without the data from this site were similar. Other investigators have shown a high degree of correlation between induration and erythema with these antigens [21]. Thus, for the purposes of our analysis, responses were recorded in millimeters of induration. Because trichophytin elicited a positive reaction in only 14.0% of those tested, it was dropped from the delayed-type hypersensitivity battery midway through the enrollment period. Although lot numbers varied, the skin tests used at the centers were supplied by the same manufacturers, with a single exception: At one site, investigators used a different Candida preparation. The Candida test results for this center (247 participants) were excluded from all analyses involving this antigen. Determination of Lymphocyte Subsets Lymphocyte subsets were determined for CD3, CD4, and CD8 receptor-bearing cells by the same laboratory at each site. All laboratories participated in the flow cytometry quality control program sponsored by the National Institute of Allergy and Infectious Diseases [22]. Statistical Analysis All analyses are based on data collected during the baseline evaluation. Statistical significance for comparisons of proportions was determined by chi-square or Fisher exact test [23]. For comparisons among nonindependent groups, repeated-measures analysis for categorical outcomes was used to determine statistical significance [24, 25]. Logistic regression models were used to study the relation between PPD positivity or anergy and potential risk factors [26]. Risk factors considered were HIV status; CD4 count among HIV-seropositive persons; intravenous drug use; race or ethnicity; a history of a positive PPD test result, tuberculosis, or BCG vaccination; age; gender; and socioeconomic status. Seventy-seven participants, including women with heterosexually acquired infection and persons who were not white, black, or Hispanic, were excluded from all multivariate analyses because of small sample sizes. Initial models included HIV status (positive or negative), intravenous drug use (presence or absence), race or ethnicity (white, black, or Hispanic), a history of a positive PPD test result, and age, as well as interaction terms, to determine whether the effect of HIV positivity varied among these groups or whether the effect of drug use varied by race. No statistically significant interactions were observed. All odds ratios presented were derived from subsequent models containing main effects only. A dichotomous variable indicating the 12% of the cohort who did not have a high school diploma was used as an index of socioeconomic status and was included in all final models. All tests were two sided. A P value of 0.05 was considered statistically significant. Ninety-five percent CIs are given when appropriate. Results Patient Characteristics During the 16-month enrollment period, 1171 HIV-seropositive and 182 HIV-seronegative persons entered the study. The two groups were similar with regard to age, sex, race, transmission category, and tuberculosis-associated history (Table 1). Of the 1165 men, 966 (82.9%) were homosexual; of the 188 women, 132 (70.2%) were intravenous drug users.