IGRA tests perform similarly to TST but cause no adverse reactions: pediatric experience in Finland
© Tavast et al; licensee BioMed Central Ltd. 2008
Received: 25 November 2008
Accepted: 15 January 2009
Published: 15 January 2009
Two commercial interferon gamma release assays (IGRAs) (QuantiFERON®-TB Gold in Tube and T SPOT®-TB) to detect a contact with M. tuberculosis have recently become available. The majority of studies agree that the sensitivity and specificity of these methods are superior to the Tuberculin Skin Tests (TSTs) in detecting an exposure to bacteria in latently infected individuals and in clinical tuberculosis. However, the data in children remains limited.
Consecutively collected samples from children (n = 99) representing age range from zero to 18 years were analyzed in a retrospective non-blinded study. The two IGRAs were modified and adapted to the needs of Finland, a country of a low tuberculosis incidence. For 27 children, both tests were performed simultaneously and compared with the TST and clinician's diagnosis. The sensitivity, specificity, and accuracy of both IGRAs was determined. QuantiFERON TB Gold and T SPOT-TB performed (respectively) as follows: sensitivities 0.92 (95% confidence interval, CI, 0.67–0.99) and 0.85 (0.64–0.95); specificities 0.91 (0.77–0.97) and 1.00 (0.93–1.00); accuracies 0.91 (0.80–0.97) and 0.96 (0.88–0.99). This compares favorably to the TST whose known figures are 0.90, 0.95, and 0.95, respectively. The agreement between the IGRAs was high, k = 0.89. Finally, both methods agreed well with the TST, k = 0.86 for TST/QuantiFERON-TB Gold and k = 0.76 for TST/T SPOT-TB.
The sensitivity and specificity of IGRA methods compares well with the TST without the inconveniences and complications associated with TST, including exaggerated delayed type hypersensitivity reactions. These properties place them as acceptable substitutes for TST.
Diagnosis of childhood tuberculosis (TB) remains challenging . Two commercial IFN-γ release assays (IGRAs), namely T-SPOT.TB and QuantiFERON®-TB Gold In-Tube (QFGT), have been recently developed. These methods show acceptable diagnostic accuracy for active TB in adults  and have better correlation with tuberculosis exposure than the TSTs in contact tracing for revealing latent infections (LTBI) [3, 4]. However, pediatric data are limited, and studies evaluating the performance of IGRAs in children have been called for especially in low-incidence countries [2, 5, 6].
In Finland the incidence of TB is low, 5.6/100 000 inhabitants in 2006 . Only zero to six child TB diagnoses per annum has been registered during the last ten years . Universal BCG vaccination of newborns was practiced until 2006 
Between September 2004 and January 2007 our laboratory adapted the tests to the general needs in Finland. After completion of the field evaluation  the methods became available for routine diagnostics. We then evaluated the performance of IGRAs in children and compared them to the TST and clinical diagnoses.
Characteristics of children enrolled in the study (n = 99)
Age (yrs), median (min-max)
BCG vaccination status unknown
Born outside Finland or at least one of the parents is from a country with endemic TB:
➢ Somalia (several families)
➢ Peru (1 family)
➢ Afghanistan, Angola, Azerbadzan, Burma Burundi, Estonia, India, Congo, Sudan, Thailand, Turkey, Uganda
12 (one each)
➢ Siblings who live in Russia
TST test performed
TST not performed
Culture or/and Nucleic acid amplification (NAA) method performed
➢ acid fast staining positive
2/6 (also culture pos)
➢ culture positive (M. tuberculosis)
➢ NAA positive
2/6 (also culture pos)
Radiological examination performed
➢ pulmonary TB
➢ enlargement of the hilus zone (Hodgkin lymphoma, TB lymphadenitis, one mild in LTBI, one mild in salmonelosis)
4 (one each)
➢ interstitial changes (vasculitis)
➢ pleural effusion (pleuritis and chylothorax)
Indications for IGRA-tests
➢ exposure to TB
➢ clinical signs compatible with TB
Ly-TbSpot procedure and quality control
The Ly-TbSpot, a modified version of T-SPOT.TB (Oxford Immunotec, Oxford, UK) , was performed using standard operation procedures (SOP). Firstly, the results were expressed as a number of reactive spots/million lymphocytes. The lymphocyte count from isolated peripheral blood mononuclear cell (PBMC) preparation was calculated with an automated hematologic analyzer (Advia® 60, Bayer, Germany). Secondly, duplicate ELISPOT wells were used for each antigen and media. Thirdly, Purified Protein Derivative (PPD) (Statens Serum Institut, Copenhagen, Denmark) was used as an additional positive control. Fourthly, we adopted a double cut-off policy, i.e. the responses below 25 spot/million lymphocytes were considered as non-reactive according to the guidelines by the manufacturer; the responses between 25 and 55 spots were considered borderline and over 55 spots were interpreted as reactive. An internal control of cryopreserved cells was run with each new batch of reagents.
B-TbIFNγ procedure and quality control
Method of TST
TST was performed with two TU, purified protein derivative (PPD, RT23, Statens Serum Institut, Copenhagen, Denmark) according to the Mantoux technique. The result was recorded after 48–72 h. A cut-off for positivity was defined as 10 mm or more in BCG-vaccinated and 5 mm or more in non-vaccinated children. If a child had a negative TST result, but was examined because of contact with an infectious TB case, the test was repeated .
For methods evaluation we used conventional interpretations of definitive and probable TB and of LTBI.
The results of the tests were considered eligible if the reactivities to the mitogen were as suggested by the manufactures. Seven samples were excluded from the ELISPOT analysis because of high non-specific background.
The concordances between the IGRAs, and of each test with the TST, were assessed in three categories by describing proportions of agreement (PA) and with the Cohen's kappa statistic with linear weighting. 95% confidence intervals (CI) were calculated with the Wilson efficient-score method which was corrected for continuity. Using a conservative approach, the cases interpreted in the grey zone were placed into categories of false positives or false negatives for the calculations of specificity, sensitivity and accuracy.
Final diagnosis of the participants
Final diagnoses of the patients
TST performed, number
TST, mm median (range)
TB detected, n = 23
➢ 1 acid fast staining and culture pos
➢ 4 with radiologic findings, history of contact and positive TST
➢ 4 lymphadenitis (one HIV+), 3/4 culture positive
➢ 1 with clinical presentation and positive TST
No TB detected, n = 76
Healthy contacts of TB cases
Abscesses of diverse localizations
Pneumonia or acute respiratory infection
Hypersedimentation due to obesity
Various infectious or inflammatory conditions, one each
The major indication for performing IGRA was recent contact with an infectious case of TB (62 of 99 patients). Thirty-seven children were examined because of symptomatic illness, with TB considered a diagnostic modality. Ten children were diagnosed with TB; five had pulmonary TB, only one of them culture positive; five were diagnosed with extrapulmonary TB. One patient also had an HIV infection, and one patient was HIV infected but had no TB.
Anamnestic data and the observed kinetics of immunological conversion in a 13-year-old boy.
A visitor in the family with pulmonary TB(AFB+)
The grandparent diagnosed with culture-positive TB
Ly-TbSpot (reactive cells/106 lymphocytes.
The max response is shown
Performance characteristics of the IGRAs
Performance characteristics of the IGRA-methods.
Sensitivity (95% CI§)
Specificity 95% CI)
PPV† (95% CI)
NPV‡ (95% CI)
Accuracy (95% CI)
n = 72
(0.64 – 0.95)
(0.93 – 1.00)
(0.82 – 1.00)
(0.87 – 0.99)
(0.88 – 0.99)
n = 41
(0.67 – 0.99)
(0.77 – 0.97)
(0.76 – 1.00)
(0.84 – 0.99)
(0.80 – 0.97)
Agreement between Ly-TbSpot, B-TbIFNγ and the TST
Agreement between Ly-TbSpot, B-TbIFNγ and the TST
Proportions of agreement (95% CI)
κ (95% CI)
0.89 (0.70 – 0.97)
0.89 (0.77 – 1.00)
0.89 (0.77 – 0.95)
0.76 (0.59 – 0.92)
0.90 (0.77 – 0.97)
0.86 (0.72 – 1.00)
Analysis of non-interpretable results
Non-specific background activation of cells for IFNγ production was observed in seven samples analyzed with the Ly-TbSpot. Of those, six samples were from members of a family newly immigrated from Ethiopia. These patients showed reactivity without stimulation with an added antigen, the size of the spots being very small. This non-specific reactivity was interpreted as the reactivity of other than effector T-cell populations, most probably of NK cells. All results obtained with the B-TbIFNγ-method were acceptable.
The influence of the cut-off levels on performance characteristics of the Ly-TbSpot
We present results of a retrospective, non-blinded study of two modified IGRAs for the diagnosis of childhood TB. The modifications were aimed to avoid false positive interpretations. Because no method can guarantee 100% sensitivity and specificity, we made a pragmatic decision to offer the best possible specificity even at the cost of sensitivity. Changing of the cut-off levels of the commercial IGRA methods has been suggested by other investigators. Lee at al  suggested lowering the cut-off in the QFGT method. This change would have resulted in a drop of specificity from 91.6% to 87.0% which we, contrary to Lee, do not regard as a minimal loss. Arend et al  also recommended dropping the cut-off of the QFGT to achieve a detection rate of potentially infected persons similar to that of TST. The recommendation is not acceptable for the reason that TST is by no means a gold standard. The sensitivity of QFGT could instead be improved e.g. by using more sensitive EIA techniques. In fact, although there was high agreement between IGRAs and TST in our studies, three earlier studies found a lower agreement: two studies [16, 17] performed on pediatric samples in low risk countries observed concordant results between the IGRAs but inferior specificity of the TST, and one large cohort study of TB disease in African children  found lower agreement with the TST. In another cohort study of contact tracing, performed on Gambian children, the clinical sensitivity of the TST was found superior to that of the ELISPOT in diagnosing LTBI . In that study, however, the discordance between the tests was not significant . In a recent study from Gambia  comparing the new IGRAs to TST in contact tracing, ELISPOT was found more sensitive than the QFGT in the diagnosis of TB disease while equally sensitive in the diagnosis of LTBI. That study showed no significant discordance between IGRAs but the results of the TST were influenced by the BCG vaccination status. The observed discrepancies in the estimation of the three methods are most probably attributed to the differences in the research population, their vaccination status and exposure to other mycobacteria, and most importantly, in the variability of the methods' performance using variable threshold levels.
Our study raises several important issues. When performing TST, intradermal inoculation of PPD in children exposed to M. tuberculosis may produce an excessive delayed type hypersensitivity reaction. Secondly, demonstration of the test variability around the cut-off zone in real laboratory settings convinces that interpretation of immunodiagnostic methods should take into account the method imprecision and utilize a grey zone.
The sensitivity and specificity of IGRA methods compares well with TST but do not cause exaggerated delayed type hypersensitivity reactions.
Tuberculin Skin Tests
Interferon Gamma Release Assays
Latent Tuberculosis Infection
Standard Operation procedure
- Positive (PPV) and (NPV):
Negative Predictive Values
Proportions of Agreement
Enzyme-linked Immunosorbent Spot Assay
Enzyme-linked Immunosorbent Assay
QuantiFeronGold in Tube
Natural Killer cells
Purified Protein Derivative
Human Immunodeficiency Virus.
We wish to thank for financial support in the evaluation of new IGRA-methods the following private Finnish foundations: Finnish Lung Health Association (Filha ry), Pulmonary Association Heli, and the Tuberculosis Association of the University of Tampere.
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