Influence Factors of T-SPOT.TB assay in TB Patients

Analysis of Influence Factors of T-SPOT.TB assay in Active Tuberculosis Patients

Keywords interferon-gamma release assays, T-SPOT.TB, tuberculin skin test, pulmonary TB, extrapulmonary TB


Objective: To evaluate the influence factors of interferon-gamma release assays (IGRAs) for active tuberculosis (TB) in previously Bacillus Calmette-Guerin (BCG)-vaccinated patients.

Methods: 456 patients underwent the T-SPOT.TB test including 402 active pulmonary TB (PTB) and 52 active extrapulmonary TB (EPTB). Correlations between the positive rate of T-SPOT.TB assays and lung lesion severity, positive smear grade, disease site and the durations of anti-TB treatments were evaluated.

Results: The positive rate of the T-SPOT.TB assay for active TB was 76.66%. The positive rate was higher in PTB cases than that in EPTB cases (P = 0.01). The positive rate in active TB cases during anti-TB treatment was significantly lower than without treatment (P = 0.002) and the positive rate gradually decreased with the treatment durations (P = 0.01).

Conclusion: Age > median age of 50-years and anti-TB treatment as well as male gender turned out to be three independent factors related to negative test results, whereas PTB was related to positive test results.


Tuberculosis (TB) remains unique among the major infectious diseases in lacking accurate and rapid tests. In 2012, approximately one third of people with TB are not diagnosed according to a report from the World Health Organization. The gold standard for diagnosis of pulmonary tuberculosis (PTB) is the identification of Mycobacterium Tuberculosis (MTB), but cultures are time consuming and lack sensitivity in addition to difficulties for obtaining clinical isolates especially in case of extrapulmonary TB (EPTB). Development of molecular technologies is most advanced, but their sensitivity can only reach the level of sputum culture at most and facing the same problem of obtaining clinical isolates. Clinicians often have to rely on immunoassays. However, the diagnostic value of tuberculin skin tests (TST) is limited due to the cross-reactivity of purified protein derivate from the Bacillus Calmette-Guerin (BCG) strain and Nontuberculous Mycobacteria (NTM) (3). The introduction of T-SPOT.TB assay (Oxford Immunotec, Abingdon, UK) based on the specific antigens early secreted antigenic target (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) absent in BCG or most of the NTM strains is a promising approach for the diagnosis of active TB (ATB) and latent TB infection (LTBI). Since the gold standard for the diagnosis of LTBI is absent, those healthy individuals with positive T-SPOT.TB assay could be defined as having potential LTBI. But what is the interpretation of ATB with negative T-SPOT.TB assay? In order to apply T-SPOT.TB assay to better service for clinicians, it is significant to elucidate the impact factor of T-SPOT.TB assay. Although there have been a number of studies evaluating the performance of T-SPOT.TB assay (11-13), studies about the influence factors of T-SPOT.TB assay in ATB patients are limited. In this present study, we compared 102 ATB patients with negative T-SPOT.TB assay with 354 ATB patients with positive T-SPOT.TB assay in peripheral CD4+ T-cell counts, lung lesion severity, positive smear grade and the durations of anti-TB treatments, concomitant diseases, the duration of symptoms and some laboratory indexes to analysis the influence factor of T-SPOT.TB assay.


Study population

In our prospective study, 456 active TB were enrolled from December 2011 to September 2012 at Shanghai Pulmonary Hospital in Shanghai, China. All subjects were HIV-negative and have been BCG vaccinated in early childhood or during adolescence. The 456 patients were T-SPOT.TB tested at enrollment. All patients had a symptom assessment, a full clinical assessment, and sputum was sent for smear microscopy and mycobacterial culturing. Chest radiographs and/or CT scans were performed. Venous blood samples were used to perform the T-SPOT.TB assay and CD4+ T-cell counts. A standard questionnaire has been completed for each patient, including basic demographic data, previous TB, during anti-TB treatment or not as well as underlying diseases and concurrent immunosuppressive therapies. Written informed consent was obtained from each of subjects and the study was approved by the Ethics Committee of Shanghai Pulmonary Hospital.

Definitions and Diagnosis

Final diagnosis was obtained through analysis of patients’ medical records and laboratory results. A diagnosis of “confirmed TB” was made on the positive culture of M. TB from sputum or the presence of caseating granuloma in biopsy specimen. A diagnosis of “clinical TB” was made if there were radiographic appearances in accordance with TB and appropriate response to anti-TB treatment. Patients classified as “active TB” had either “confirmed TB” or “clinical TB”. Patients with EPTB in combination with PTB were included into the PTB group.

X-ray criteria for lesion severity scores

The chest X-rays of the tuberculosis patients were divided into six lung fields (Figure 1) and the severity of the lung lesions was scored based on (a) the range of lung field foci and (b) the number/size of cavities (Table 1). The final lesion severity score was the sum of six lung scores fields (every lung field = a+b of Table 1) and ranked as ≤ 2.5 mild, 2.5-6 as moderate and ≥ 6 points as severe.

The white arrows indicate the lesions and cavities. A: Field 1, ≥50% of area affected = score 2; Field 2, <50% of area affected = score 1. B: Field 1, single cavity, <2cm diameter = score 0.25. C: Field 1, single cavity, 2-4cm diameter = score 0.5; Field 3, single cavity, >4cm diameter = score 1. D: Field 1, multiple cavities, the largest <2cm diameter = score 0.5; Field 2, multiple cavities, the largest 2-4cm diameter = score 1. E: Field 3, multiple cavities, the largest >4cm diameter = score 2.

Sputum smear grade

The sputum smear was graded as smear 3+, which was ≥ 10 acid-fast bacilli (AFB) per 1 high-power field (HPF), 1–9 AFB per 1 HPF was smear 2+ and 10–99 AFB per 100 HPF was smear 1+.

T-SPOT.TB assay

The T-SPOT.TB test was performed following the manual of the assay kit (Oxford Immunotec Ltd., Oxford, United Kingdom). Blood was collected just prior to the tests in order to avoid potential interferences and we recommend the patients who received blood transfusions or did PET-CTs within 1 week to retest two weeks later. Briefly, peripheral blood mononuclear cells (PBMCs) were seeded on pre-coated IFN-γ ELISPOT plates and incubated with media without antigen (as a negative control), media containing peptide antigens derived from ESAT-6 (labeled panel A) or peptide antigens derived from CFP-10 (labeled panel B) and media containing phytohemagglutinin (as a positive control) in a 5% CO2 atmosphere at 37°C for 20 h. After counting spot-forming cells (SFCs), the results of the T-SPOT.TB assay were either considered positive if panel A or panel B or both had six or more spots than the negative control or if this number was at least two times greater than that of the negative control.

Statistical analyses

Statistical analyses were conducted using SPSS statistical software (SPSS Statistics 17.0; SPSS Inc., Chicago, IL). Assumed a positive rate of T – SPOT tests in 75% of patients with active tuberculosis, we needed 133 cases of patients with tuberculosis as sample size for a power test. Pearson’s chi-square test were used for proportional comparisons among different subgroups. Wilcoxon rank test was used for comparison of continuous variables. All p-values were two-sided with α = 0.05. Risk factors were evaluated using odds ratios (ORs) in a univariate analysis. In a multiple regression analysis model, the TSPOT.TB test was used as a dependent variable while over median age (>50-year), sex, clinical symptoms, anti-TB treatment, disease site (PTB or EPTB) and diabetes were selected as independent variables.


Demographic profiles of the patients

A total of 456 consecutive patients, who required a TB differential diagnosis were initially enrolled in this study and included 404 active PTBs (including 89 patients undergoing anti-TB treatment) and 52 active EPTBs (including 6 cases of osteoarticular tuberculosis, 13 cases of tuberculous lymphadenitis and 33 cases of tuberculous pleurisy) (Figure 2). 354 ATB showed positive T-SPOT.TB assay and102 ATB showed negative T-SPOT.TB assay. The average age of the patients was 41±16 years. Sixty-eight percent of the cohort was male. Almost 10% PTB patients presented with diabetes mellitus and other complications were few. For other characteristics see Figure 2.

Factors associated with positive and negative test results

we found that 76.66% ATB patients were T-SPOT.TB test positive, which included 77.97% PTB and 69.23% EPTB patients. This implied that most ATB patients had positive T-SPOT.TB test results. In addition, we found the positive T-SPOT.TB test results in the ATB subgroup were related with positive results of bacterial cultures and smear or biopsy-confirmed diagnoses (p=0.023), but the test sensitivity was higher in the ‘Smear+/Culture+/clinical test’ cases than in the ‘biopsy-confirmed’ cases. Next, we investigated the performance of T-SPOT.TB stratified in disease site and the sensitivity was lower in EPTB compared to PTB cases. Notably, in contrast to osteoarticular tuberculosis, a higher sensitivity was observed in tuberculous pleurisy and tuberculous lymphadenitis. The difference between the three groups was significant (X2=9.1974, P = 0.01) (Table 2). Moreover, the performance of T-SPOT.TB stratified with duration of anti-TB treatment showed, that the test sensitivity in ATB cases during treatment (65.17%, 58/89) was significantly lower than without treatment (80.62%, 208/258) (X2=8.8292, P = 0.003) and gradually decreased with the treatment duration. The test differences among the treatment time less than 1 month, less than 1 year and treatment time more than 1 year was significant (X2=8.5883, P = 0.01) (Table 4). Factors associated with positive and negative test results in the ATB group were evaluated by a multivariate analysis. Age > median age of 50-years and anti-TB treatment as well as male gender turned out to be three independent factors related to negative test results, whereas PTB was related to positive test results (Table 3).



The diagnosis of tuberculosis in patients with negative bacteriological results is still a problem to be resolved in clinical settings. Nonetheless, because the cross-reaction of the TST with BCG vaccination attenuates the specificity, alternative fast and specific diagnostic tools are desirable to replace the TST. The aim of this study performed in a country with a high prevalence of TB diseases and BCG vaccination rates was to determine the influence factors of the T-SPOT.TB test in routine clinical practice. We observed that the positive rate of the T-SPOT.TB test in active PTB and EPTB were 77.97% and 69.23% and much lower than previously reported (14-16). The lower sensitivity obtained in our analysis may be due to the included patients, who were undergoing anti-TB treatments. The positive rate of the T-SPOT.TB assay in ATB patients with anti-TB treatment was significantly less compared with patients without treatment (P=0.002). The longer the treatment time was, the lower was the positive rate, a phenomenon that might be related to decreased antigen burden, which is in agreement with several prior studies (17-20). In order to confirm the assumption, that the T-SPOT.TB assay results might be directly associated with the actual bacterial load, we further investigated the correlation between sensitivity and disease severity plus smear grade. The positive rate gradually decreased with the reduction of the disease severity and increase of the smear grade, which might be due to the small sample size of our study. However, some studies have demonstrated persistently high of positive T-SPOT.TB test results even after treatments. Ribeiro et al. showed, that only 10% of individuals with a baseline reactive test reverted to negative at completion of 24 weeks treatment (19). Thus, the hypothesis that the T-SPOT.TB assay in ATB patients during anti-TB treatment could serve as an effective predictor of therapeutic efficacy requires further investigation. There has been considerable debate about the diagnostic value of IGRAs for EPTB. Fan et al. noted a pooled sensitivity of 90% (95% CI, 86-93%) (15). However, Cho et al. reported that the T-SPOT.TB was more sensitive in patients with chronic forms of EPTB such as lymph node or osteoarticular TB (93%) than in patients with acute EPTB forms such as TB meningitis or miliary TB (79%) (21). Liao et al. stated an overall sensitivity of 79.8%, and the sensitivity ranged from 100% for tuberculous meningitis, tuberculous pericarditis and intestinal TB to 95% for lymphadenitis and 42.9% for tuberculous peritonitis (22). Our study revealed a 69.23% overall positive rate, much lower than for PTB, and the positive rate ranged from 78.79% for tuberculous pleurisy to 69.23 % for tuberculous lymphadenitis and 16.67% for osteoarticular tuberculosis. The positive rate differences between affected sites was significant (P = 0.01). Our findings add strength to the hypothesis that IFN-γ producing T-cell responses vary according to the location of the disease, but further investigation is necessary to elucidate this. As the positive rate of T-SPOT.TB test was associated with anti-TB treatments in our results, for patients during anti-TB treatment the T-SPOT.TB assays might be of diagnostic value for treatment outcome assessments.