Delayed diagnosis and treatment can increase the risk for dissemination of M
and decrease survival for some subgroups of TB patients (13
). Thus, new technologic developments, which facilitate rapid diagnosis, are needed for successful control of this disease. Besides the development of nucleic acid amplification assays for rapid detection of M
complex, attempts have been made to exploit the T-cell response for rapid diagnosis of M
). The major problem with tuberculin skin testing (TST) is cross-reactivity with antigens in other mycobacteria, such as the M
BCG vaccine strain and environmental mycobacterial species. This cross-reactivity leads to false-positive results and decreased PPV, especially in BCG-vaccinated persons and in areas of high incidence of NTM disease, such as Taiwan. In Taiwan in 2001, 2.74% of preschool children were TST positive, whereas active TB developed in only 2.29/100,000 children 5–9 years of age (1
). Use of ESAT-6 and CFP-10, two antigens encoded in the region of difference 1, which distinguishes M
from other mycobacteria, has increased the specificity and PPV of IFN-γ ELISPOT assays (10
). Our study showed that the sensitivity, specificity, PPV, and negative predictive values (NPV) of the ELISPOT assay were >80% in the diagnosis of active TB in clinically suspected patients. Results were also available ≈45 days earlier than those obtained with mycobacterial culture.
The genes coding for ESAT-6 and CFP-10 are absent from most environmental mycobacteria, except for M
, and M
). Whether ESAT-6 or CFP-10 is present in M
and the M
complex has not yet been determined. Although PPV is associated with pretest probability of active TB in a cohort, our results showed that the ELISPOT can accurately discriminate TB from NTM disease and other respiratory diseases. All 3 patients with false-positive ELISPOT results had NTM disease. The 3 AFS-positive TB patients with false-negative ELISPOT results had other diseases (2 had diabetes mellitus and 1 had AIDS), which could weaken the T-cell response (26,27
). However, neither of the 2 AFS-negative ELISPOT false-negative TB patients had another disease. Because HIV status was not routinely tested, the possibility of asymptomatic HIV infection that potentially influenced the ELISPOT results cannot be excluded.
Consistent with previous reports (sensitivity 80.7%–94.4%) (20
), assays detecting secretion of IFN-γ caused by stimulation with ESAT-6 or CFP-10 for diagnosis of TB have a sensitivity >80%. However, specificity (45.5%–69.2%), PPV (65.4%–85.4%), and NPV (53.6%– 90.0%) were highly variable, which was probably due to different criteria for patient selection and diagnosis of active TB. In a study conducted in Japan (20
), only patients with culture-confirmed infection were considered to have active TB. Thus, culture-negative TB patients would be classified into a non-TB group, but some showed positive test results, which resulted in decreased specificity and PPV. In the study conducted in Denmark (28
), several risk factors predisposing persons to recent M
infection were observed in the 10 patients with false-positive results, including a history of recent exposure, immigration from a highly disease-endemic area, intravenous drug use, and HIV positivity. In the study conducted in Brazil (30
), controls were medical students, who were at high risk for nosocomial exposure; 50% of them were ELISPOT positive, which resulted in low specificity and PPV.
Many of our patients without active TB were ELISPOT negative. In a study in Taiwan in 2001, 2.74% of preschool children were TST positive, and the annual estimated infection rate was 0.43% (1
). Therefore, it is unlikely that all of our ELISPOT-negative patients had never been infected with M
. Furthermore, the results with samples from HCWs decrease the possibility that acute illness caused a false-negative result. Previous studies with sequential testing showed that responses of ESAT-6– or CFP-10–specific T cells decay progressively with treatment for TB (9
). Our ELISPOT-negative patients may not have been recently infected with M
; thus, levels of their circulating ESAT-6– or CFP-10–specific effector T cells, rather than memory T cells, decreased and failed to yield a positive ELISPOT result (35,36
). Further long-term follow-up study of ELISPOT-positive TB patients is needed to better understand the dynamic changes in ESAT-6– or CFP-10–specific effector T cells.
Patients with AFS-negative TB should be further investigated because this type of TB is usually diagnosed late and has been reported to be responsible for ≈17% of TB transmission (37,38
). Our study showed that all AFS-negative ELISPOT-positive patients had true cases of TB, i.e., PPV = 100%. Only 2 patients with TB pleurisy and negative sputum culture for M
showed false-negative ELISPOT results. The cause of this finding is not known because the current hypothesis for the pathogenesis of TB pleurisy is that the caseous material from a subpleural focus ruptures into the pleural space 6–12 weeks after a primary infection. This material then interacts with previously sensitized T cells, which results in a delayed hypersensitivity reaction and accumulation of fluid (39-40
). The 2 patients with false-negative ELISPOT results might have been at a early stage of primary TB, and their sensitized T cells had not yet returned to the systemic circulation before sampling was conducted. Further investigation is needed to assess the performance of the ELISPOT assay in patients suspected of having TB with negative AFS results.
The resurgence of TB has prompted the need for sensitive, accurate, and fast methods for laboratory detection of M. tuberculosis infection. Although previous studies demonstrated that the ELISPOT assay for INF-γ is a powerful tool for detecting latent M. tuberculosis infection, our results showed that in patients who were previously vaccinated with BCG, the diagnostic value of this test in detecting active TB approached 90% in sensitivity, specificity, PPV, and NPV, even in an area with a high incidence of NTM disease.