Multiple Cytokines Are Released When Blood from Patients with Tuberculosis Is Stimulated with Mycobacterium tuberculosis Antigens

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone

Assessment of the QuantiFERON-TB Gold In-Tube test for the detection of <i>Mycobacterium tuberculosis</i> infection in United States Navy recruits

<div><p>Background</p><p>Immunologic tests such as the tuberculin skin test (TST) and QuantiFERON^®-TB Gold In-Tube test (QFT-GIT) are designed to detect M<i>ycobacterium tuberculosis</i> infection, both latent <i>M</i>. <i>tuberculosis</i> infection (LTBI) and infection manifesting as active tuberculosis disease (TB). These tests need high specificity to minimize unnecessary treatment and high sensitivity to allow maximum detection and prevention of TB.</p><p>Methods</p><p>Estimate QFT-GIT specificity, compare QFT-GIT and TST results, and assess factor associations with test discordance among U.S. Navy recruits.</p><p>Results</p><p>Among 792 subjects with completed TST and QFT-GIT, 42(5.3%) had TST indurations ≥10mm, 23(2.9%) had indurations ≥15mm, 14(1.8%) had positive QFT-GIT results, and 5(0.6%) had indeterminate QFT-GITs. Of 787 subjects with completed TST and determinate QFT-GIT, 510(64.8%) were at low-risk for infection, 277(35.2%) were at increased risk, and none had TB. Among 510 subjects at low-risk (presumed not infected), estimated TST specificity using a 15mm cutoff, 99.0% (95%CI: 98.2–99.9%), and QFT-GIT specificity, 98.8% (95%CI: 97.9–99.8%), were not significantly different (p>0.99). Most discordance was among recruits at increased risk of infection, and most was TST-positive but QFT-GIT-negative discordance. Of 18 recruits with TST ≥15mm but QFT-GIT negative discordance, 14(78%) were at increased risk. TB prevalence in country of birth was the strongest predictor of positive TST results, positive QFT-GIT results, and TST-positive but QFT-GIT-negative discordance. Reactivity to <i>M</i>. <i>avium</i> purified protein derivative (PPD) was associated with positive TST results and with TST-positive but QFT-GIT-negative discordance using a 10 mm cutoff, but not using a 15 mm cutoff or with QFT-GIT results.</p><p>Conclusions</p><p><i>M</i>. <i>tuberculosis</i> infection prevalence was low, with the vast majority of infection occurring in recruits with recognizable risks. QFT-GIT and TST specificities were high and not significantly different. Negative QFT-GIT results among subjects with TST induration ≥15 mm who were born in countries with high TB prevalence, raise concerns.</p></div

Associations between selected subject characteristics and tuberculin skin test or QuantiFERON^®-TB Gold In-Tube test results.

<p>Associations between selected subject characteristics and tuberculin skin test or QuantiFERON^®-TB Gold In-Tube test results.</p

Outcomes of the QuantiFERON^®-TB Gold In-Tube test versus the tuberculin skin test among 856 U.S. Navy recruits who had blood collected and skin test placed.

<p>Outcomes of the QuantiFERON^®-TB Gold In-Tube test versus the tuberculin skin test among 856 U.S. Navy recruits who had blood collected and skin test placed.</p

Diagram of study participants and testing.

<p>QFT = QuantiFERON^®-TB test; QFT-G = QuantiFERON^®-TB Gold test; QFT-GIT = QuantiFERON^®-TB Gold In-Tube test; TST = tuberculin skin test.</p

Test agreement.

<p>Test agreement.</p

Results for various cohort subsets.

<p>Results for various cohort subsets.</p

Once Weekly Azithromycin Therapy for Prevention of Mycobacterium avium Complex Infection in Patients with AIDS: A Randomized, Double-Blind, Placebo-Controlled Multicenter Trial

We conducted a randomized, double-blind, placebo-controlled multicenter trial of azithromycin (1,200 mg once weekly) for the prevention of Mycobacterium avium complex (MAC) infection in patients with AIDS and a CD4 cell count of <100/mm3. In an intent-to-treat analysis through the end of therapy plus 30 days, nine (10.6%) of 85 azithromycin recipients and 22 (24.7%) of 89 placebo recipients developed MAC infection (hazard ratio, 0.34; P = .004). There was no difference in the ranges of minimal inhibitory concentrations of either clarithromycin or azithromycin for the five breakthrough (first) MAC isolates from the azithromycin group and the 18 breakthrough MAC isolates from the placebo group. Of the 76 patients who died during the study, four (10.5%) of 38 azithromycin recipients and 12 (31.6%) of 38 placebo recipients had a MAC infection followed by death (P = .025). For deaths due to all causes, there was no difference in time to death or number of deaths between the two groups. Episodes of non-MAC bacterial infection per 100 patient years occurred in 43 azithromycin recipients and 88 placebo recipients (relative risk, 0.49; 95% confidence interval, 0.33–0.73). The most common toxic effect noted during the study was gastrointestinal, reported by 78.9% of azithromycin recipients and 27.5% of placebo recipients. Azithromycin given once weekly is safe and effective in preventing disseminated MAC infection, death due to MAC infection, and respiratory tract infections in patients with AIDS and CD4 cell counts of <100/mm3

Comparisons of IFN-γ responses to different antigens measured by commercial ELISA among patients with culture-confirmed tuberculosis.

<p>Median IFN-γ responses (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026545#pone-0026545-t002" target="_blank">Table 2</a>) to early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), TB7.7, and combinations of these antigens for patients with culture-confirmed tuberculosis (patients) are listed with p values in parenthesis calculated by Mann-Whitney U Rank Sum test. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

Cytokine concentrations and responses measured with a microarray.

<p>Median background cytokine concentrations (Nil, pg/mL) and cytokine responses (pg/mL) to phytohemagglutinin (Mitogen Response), early secreted antigenic target-6 (ESAT-6 Response), culture filtrate protein 10 (CFP-10 Response), TB7.7 (TB7.7 Response), and combinations of these <i>M. tuberculosis</i> (<i>Mtb</i>) antigens (ESAT-6+CFP-10 Response and ESAT-6+CFP-10+TB7.7 Response) are listed with ranges in parentheses. Cytokine concentrations were measured by a commercial quantitative immuno-microaray (microarray) for patients with culture-confirmed tuberculosis (patients) and subjects at low risk for <i>Mtb</i> exposure (controls). Responses were calculated by subtracting the background cytokine concentration in plasma from blood incubated with saline (Nil) from the cytokine concentration in plasma from blood incubated with mitogen or <i>Mtb</i> antigens. Cytokine responses for some patients and controls were not determined due to poor performance of the respective standard curves. N = 5 or 6 for patients subjects and N = 6 or 7 for controls unless indicated (*n = 4, **n = 5). <i>P</i> values were calculated by Mann-Whitney U Rank Sum test comparing cytokine concentrations or responses for patients with controls. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p