Participants

All patients were seen routinely for their infections at one of four academic centers in Thailand or Taiwan and provided written informed consent according to the protocol, which was approved by the National Institute of Allergy and Infectious Diseases and all local sites. The first author vouches for the completeness and accuracy of the data and for the fidelity of the study to the protocol.

At baseline, complete histories were obtained and physical examinations with routine clinical laboratory tests were performed in all patients. Data were recorded on standardized case-report forms. Patients had no history of cancer, immunodeficiency, or immune suppression within 4 weeks before enrollment or diagnosis of their infections.

We enrolled the participants in five groups: patients with disseminated, rapidly or slowly growing, nontuberculous mycobacterial infection (group 1); patients with another opportunistic infection (e.g., infection with Cryptococcus neoformans, Histoplasma capsulatum, or Penicillium marneffei; disseminated salmonellosis; or severe varicella–zoster virus infection), with or without nontuberculous mycobacterial infection (group 2); patients with disseminated tuberculosis (group 3); patients with pulmonary tuberculosis (group 4); and healthy controls (group 5).

In groups 1, 2, and 3, disseminated disease was defined as infection in two noncontiguous, sterile sites, at least one of which was extrapulmonary. Patients in groups 1 and 2 were eligible for enrollment if they had an active disseminated opportunistic infection or a history of culture-proven disseminated opportunistic infection. In groups 1 and 2, HIV testing was performed with the use of up to three different rapid enzyme immunoassays, as specified by World Health Organization guidelines.19 Group 3, which was composed of patients with disseminated tuberculosis, was an exploratory group that was not included in the predefined statistical analysis. HIV testing was performed in group 3, and all patients were HIV-negative.

Patients with pulmonary tuberculosis, who were recruited as controls with mycobacterial disease (group 4), had culture-proven tuberculosis or smear-positive results for acid-fast bacilli and an appropriate response to directed antituberculous therapy. They were not routinely screened for HIV in the absence of an overt clinical suspicion of HIV infection, since Thailand and Taiwan are regions with a high burden of tuberculosis.20 Infections were categorized as active or inactive at enrollment on the basis of clinical evidence, including the ongoing need for antimicrobial agents. Healthy controls (group 5) were anonymized blood donors who were enrolled from one site each in Thailand and Taiwan. They provided written informed consent separately and were compensated for their participation. Only age, sex, and race or ethnic group were recorded for participants in group 5; HIV testing was not performed.

Clinical Laboratory Tests and Immunophenotyping

Blood specimens were separated at each local site into plasma and peripheral-blood mononuclear cells (PBMCs) by means of density-gradient centrifugation. PBMCs were stimulated as described previously for assessment of cell-intrinsic interferon-γ synthesis and response.10 Immunophenotyping by means of flow cytometry was performed at the local site, but raw data were analyzed centrally with the use of FSC Express, clinical version 3 (De Novo Software), and FlowJo, version 9.1 (Treestar) (for details, see the Supplementary Appendix, available with the full text of this article at NEJM.org). Clinical laboratory tests included a complete blood count with a differential count, assessment of liver and kidney function, antinuclear antibody testing, and quantitative measurement of serum immunoglobulin levels.

Anticytokine Autoantibodies

The detection of autoantibodies against cytokines with the use of Luciferase Immunoprecipitation Systems has been reported previously.4 Autoantibodies were evaluated against 41 targets: interferons γ, α1, β1, ε, λ1, λ3, and ω; interleukins 1α and 1β; the interleukin-1 receptor antagonist; interleukins 2, 3, 4, 6, 7, 8, 10, 12p35, 12p40, 15, 17A, 17F, 18, 21, 22, 23p19, 27p28, 32, and 33; Epstein–Barr virus–induced gene 3 protein (interleukin-27b); granulocyte colony-stimulating factor (G-CSF); granulocyte–macrophage colony-stimulating factor (GM-CSF); TNF-α; tumor necrosis factor β; B-cell–activating factor; a proliferation-inducing ligand; the Fas ligand (FasL); the CD40 ligand; erythropoietin; transforming growth factor β; and the extracellular domain of the CD4 receptor. Additional methodologic details are described in the Supplementary Appendix; a detailed protocol and video describing the Luciferase Immunoprecipitation Systems technique are also included in an article by Burbelo et al.21

Anti–interferon-γ–specific autoantibody isotype and IgG subclasses were determined with the use of a particle-based assay, as described previously22; total IgG subclasses were determined with the use of the Bio-Plex isotype kit (Bio-Rad Laboratories) according to the manufacturer's instructions. Interferon-γ–specific IgG was purified by fractionating total IgG on protein G columns (Ab SpinTrap, GE Healthcare) and applying the total IgG fraction to an interferon-γ column.

Interferon-γ–Induced Signaling and Cytokine Production

PBMCs (at a concentration of 1×106 cells per milliliter) were cultured in complete RPMI 1640 medium consisting of 2 mM glutamine, 20 mM HEPES buffer, 100 U of penicillin per milliliter, 100 μg of streptomycin per milliliter, and 10% patient or control plasma. Cultures were left unstimulated or were stimulated with interferon-γ (1000 U per milliliter, InterMune) or interferon-α2b (1000 U per milliliter, Schering) for 15 minutes at 37°C. Monocytes were identified with CD14+ surface staining. Intranuclear staining was performed as described previously11 with the use of anti–phospho–signal transducer and activator of transcription 1 (STAT1) (tyrosine 701) antibody (BD Pharmingen).

Data were collected with the use of FACSCanto flow cytometry (BD Biosciences) and analyzed with the use of FlowJo (Treestar). The methods for the detection of interferon-γ–induced TNF-α are described in the Supplementary Appendix.

Statistical Analysis

Group differences were examined with the use of Fisher's exact test for categorical variables and with the use of analysis of variance for continuous variables. Mean differences between each group of patients and the group of healthy controls were examined by means of Wald tests, with Holm's procedure used to correct for multiple comparisons. Tests for between-group differences in the 41 anticytokine autoantibodies used a Bonferroni-adjusted P value (P=0.0012). Skewed laboratory data were log-transformed, and counts were offset by one half to avoid logarithms of zero.

The normal range for the anti–interferon-γ–autoantibody concentration was defined by the 99th percentile for the patients with pulmonary tuberculosis (group 4) and the healthy controls (group 5) combined and was estimated with the use of the log-normal distribution. Outlying concentrations were classified as positive for anti–interferon-γ autoantibodies. Differences in biologic function of antibodies and in interferon-γ–induced phospho-STAT1 production according to interferon-γ–autoantibody status were examined with the use of the Wilcoxon rank-sum test.

Statistical tests were two-sided and, unless otherwise noted, performed at the 0.05 level. Statistical analysis was performed with the use of R software (www.r-project.org).