We enrolled 389 consenting adult patients, whose demographic characteristics are shown in . Blood was unavailable from 3 patients, tuberculin skin test results were unavailable in 67 patients, and 15 ELISpot assays (4%) were indeterminate (). The 67 patients without tuberculin skin test results were similar to those with skin test results in terms of age, sex, ethnicity, presence of tuberculosis, site of disease, and presence of comorbid conditions (data provided on request). We classified 154 patients (40%) as having culture-confirmed tuberculosis, 40 (10%) as having highly probable tuberculosis, and 39 (10%) as clinically indeterminate; we excluded active tuberculosis in 154 patients (40%) ( and , available at www.annals.org
). Twenty-eight percent of tuberculosis cases were extrapulmonary; shows the clinical subtypes of tuberculosis and alternative diagnoses for patients without tuberculosis.
Demographic and clinical characteristics of the study population n=389
Figure 1 Study flow diagram. BCG = bacille Calmette–Guérin; ELISpot = enzyme-linked immunospot assay incorporating early secretory antigenic target-6 and culture filtrate protein-10; ELISpotPLUS = enzyme-linked immunospot assay incorporating early (more ...)
Final diagnoses of study participants
For culture-confirmed and highly probable cases, diagnostic sensitivities were 89% (95% CI, 84% to 93%) with ELISpotPLUS, 85% (CI, 79% to 90%) with standard ELISpot, 79% (CI, 72% to 85%) with 15-mm threshold tuberculin skin testing, and 83% (CI, 77% to 89%) with stratified 10-mm threshold tuberculin skin testing (). The ELISpotPLUS assay was more sensitive than tuberculin skin testing with the 15-mm threshold (P = 0.01) but not with the stratified 10-mm threshold (P = 0.10). The ELISpotPLUS assay was more sensitive than tuberculin skin testing with a 15-mm threshold in the 297 patients with results from both tests (89% [CI, 83% to 94%] versus 81% [CI, 73% to 87%]; P = 0.04), but not with stratified-10 mm threshold (85% (CI, 78% to 90%, P = 0.2). The sensitivity of ELISpotPLUS was 4.2% higher than that of standard ELISpot (P = 0.02). Seven patients with active tuberculosis had ELISpot responses to novel peptides but not to early secretory antigenic target-6 or culture filtrate protein-10, of whom 4 were positive to Rv3873, 3 to Rv3878, and all 7 to Rv3879c.
Diagnostic accuracy for the diagnosis of active TB disease
In a sensitivity analysis to investigate whether inclusion of highly probable cases (category 2, which was biased in favor of the tuberculin skin test because a diagnosis of highly probable tuberculosis was based in part on skin test results) affected performance estimates, we re-estimated sensitivity by using only culture-confirmed cases (category 1). Sensitivities were 91% (CI, 85% to 95%) with ELISpotPLUS, 87% (CI, 80% to 92%) with standard ELISpot, 79% (CI, 71% to 86%) with tuberculin skin testing using the 15-mm threshold, and 82 % (CI, 74% to 89%) with the stratified 10-mm threshold. The ELISpotPLUS assay was more sensitive than tuberculin skin testing using either the 15-mm (P = 0.005) or the stratified 10-mm threshold (P = 0.03) ().
Tuberculin skin testing and ELISpot had higher sensitivity when used in combination. For culture-confirmed or highly probable diagnoses of tuberculosis, sensitivity of ELISpotPLUS
with tuberculin skin testing was 99% (CI, 95% to 100%) and standard ELISpot with tuberculin skin testing was 97% (CI, 93% to 99%) (). A negative result on both tests was associated with likelihood ratios of 0.02 (CI, 0 to 0.06) for ELISpotPLUS
with tuberculin skin testing and 0.04 (CI, 0.01 to 0.10) for standard ELISpot with tuberculin skin testing. Positive results from both tests provided likelihood ratios of 5.5 (CI, 3.2 to 9.5) for ELISpotPLUS
with tuberculin skin testing and 5.7 (CI, 3.3 to 9.9) for standard ELISpot with tuberculin skin testing, whereas discordant results (likelihood ratios, 0.93 to 1.56) had no diagnostic value. shows sequential likelihood ratios when ELISpot assays are followed by tuberculin skin testing, and (available at www.annals.org
) shows corresponding results when tuberculin skin testing is used first.
Figure 2 Likelihood ratios, sensitivities, and specificities of tests used in combination, using ELISpot or ELISpotPLUS first. Data are for patients in whom results on both tests were available (n = 265). Except where stated, values are likelihood ratios with (more ...)
The high sensitivity of the combined tests reflects the fact that false-negative tuberculin skin test results and false-negative ELISpot results occurred in different patient populations. In univariate analysis among culture-confirmed and highly probable cases, the presence of factors associated with cutaneous anergy (5
) or risk of progression from latent infection to active tuberculosis (35
) was associated with false-negative tuberculin skin test results (P
= 0.001) but not with false-negative ELISpot results (P
= 0.55). Specifically, 28% (CI, 11% to 44%) of patients with false-negative tuberculin skin test results had factors associated with cutaneous anergy (3 had diabetes and 3 were HIV-positive), compared with 5.0% (CI, 1.1% to 8.8%) of those with true-positive results. Eleven patients with tuberculosis were deemed immunocompromised on the basis of being HIV-positive (8 patients) or receiving long-term corticosteroid treatment (3 patients); 10 had positive ELISpot results and all 11 had positive ELISpotPLUS
results. Neither age nor presence of any comorbid condition was associated with false-negative tuberculin skin test or ELISpot results (P
> 0.11 in all cases).
The sensitivity of culture was 79% (CI, 73% to 85%) for confirmed and highly probable tuberculosis, which was lower than that of both ELISpot assays; this difference was significant for ELISpotPLUS (P = 0.005). Microscopy had a sensitivity of 39% (CI, 32% to 46%). Microbiological techniques had lower sensitivity in extrapulmonary disease than in pulmonary disease (culture, 53% [CI, 39% to 67%] vs. 89% [CI, 83% to 94%], respectively [P < 0.001]; microscopy, 14% (CI, 5.5% to 26%) vs. 49% (CI, 40% to 58%), respectively [P < 0.001]), whereas ELISpotPLUS showed a nonsignificantly higher sensitivity in extrapulmonary disease (94% [CI, 84% to 99%]) than in pulmonary disease (88% [CI, 81% to 93%]) (P = 0.19). Among patients with suspected pulmonary tuberculosis, the sensitivity of sputum microscopy with ELISpotPLUS was 94% (CI, 89% to 98%) for culture-confirmed and highly probable cases, generating a negative likelihood ratio of 0.10 and a negative predictive value of 90%, compared with a sensitivity of 78% (CI, 69% to 85%) for microscopy combined with tuberculin skin testing (P < 0.001).
Specificity of ELISpotPLUS was 69% (CI, 61% to 77%) for active disease in all category 4 patients, which was lower than the specificity of tuberculin skin testing (81% [CI, 73% to 87%]; P = 0.03). Although ELISpotPLUS gave more diagnostic negative likelihood ratios and negative predictive values than tuberculin skin testing, positive likelihood ratios and positive predictive values were somewhat lower, most likely because of detection of latent infection in category 4 patients; this finding is consistent with the 84% (CI, 60% to 97%) specificity of ELISpotPLUS calculated by using category 4D patients (who were considered least likely to have latent infection). Specificity of standard ELISpot was similar to ELISpotPLUS (). Three of 21 category 4D patients had false-positive results by standard ELISpot, and 3 of 19 had false-postive results by ELISpotPLUS. All 3 individuals falsely-positive by ELISpotPLUS were United Kingdom-born white persons with 0 mm of induration on the Mantoux test, and 2 were 75 years of age or older. Only 2 category 4D patients who were negative by standard ELISpot were positive to any of the new antigens; both responded to Rv3873 alone. Thus, inclusion of Rv3879c alone in ELISpotPLUS enhanced diagnostic sensitivity without compromising specificity.
Our comparison of the Heaf test and the Mantoux test with a 15-mm induration threshold for patients with culture-confirmed and highly probable cases of tuberculosis revealed a nonsignificant increase in sensitivity for the Heaf test (86% [CI, 74% to 82%] for the Heaf test vs. 76% [CI, 66% to 84%] for the Mantoux test; P = 0.14) and a higher specificity for the Mantoux test (66% [CI, 46% to 82%]) for the Heaf test vs. 85% [CI, 77% to 92%] for the Mantoux test; P = 0.02) (subgroup data provided on request). Receiver-operator characteristic analysis indicated that the area under the curve for both skin test formats was identical at 0.84. Subgroup analysis of combined use of standard ELISpot or ELISpotPLUS followed by Mantoux testing (for the 184 patients who received Mantoux testing) gave similar final likelihood ratios to those observed in the whole study population tested with Mantoux or Heaf methods (265 patients; subgroup data provided on request).
Overall, 4.7% of positive results on standard ELISpot or ELISpotPLUS were dependent on a single peptide pool response of 5 to 10 spot-forming cells per well. These results might therefore be considered borderline positive.