Article Text
PDF
Abstract
Aim To identify clinical risk factors for obtaining an ‘equivocal’ T-SPOT.TB result in patients consistent with tuberculosis (TB)-associated uveitis (TAU).
Methods Prospective cohort study of consecutive new patients, with ocular signs consistent with TAU at a single tertiary centre enrolled over 15 months. All subjects underwent ocular and systemic evaluation, and investigations to rule out underlying disease. Subjects with underlying disease and interdeterminate T-SPOT.TB results were excluded. Patients were followed up for 1 year from enrolment.
Results 15 of 155 subjects (9.67%) obtained ‘equivocal’ T-SPOT.TB results. Mean age was 52.2 (range 12–77) years. Most of the subjects were Chinese (n=8, 53.3%) with no sex dominance (seven male, eight female). None were immunocompromised. Patients aged <13 or >55 years old were found to be more likely to have an ‘equivocal’ T-SPOT.TB result (OR 21.2; 95% CI 3.7 to 121.6; p=0.001), while adjusting for possible confounders including sex, race, history of diabetes mellitus, disease duration, type of uveitis and tuberculin skin test positivity. These patients are more likely to be QuantiFERON-TB Gold In-tube negative (OR 14.7; 95% CI 1.2 to 179.9; p=0.035).
Conclusion An ‘equivocal’ T-SPOT.TB result is associated with patients aged >55 years. Such patients are likely to have a negative QuantiFERON-TB Gold In-tube result.
- Tuberculosis
- interferon γ release assay
- uveitis
- infection
Statistics from Altmetric.com
Introduction
Tuberculosis (TB)-associated uveitis (TAU) is difficult to diagnose as it may present with a wide spectrum of ocular manifestations.1 These may represent a true ocular infection or an immune-mediated reaction to an unseen or latent TB infection (LTBI).1–3 Interferon γ (IFN-γ)-release assays (IGRAs) are new tests used to diagnose TB. These tests are based on in vitro detection of IFN-γ released by T cells in response to antigens specific to Mycobacterium tuberculosis (MTB).4 As these antigens are absent from Bacille Calmette–Guérin (BCG) vaccinations and most strains of non-tuberculous mycobacteria, IGRAs offer improved specificity over the time-honoured tuberculin skin test (TST), which uses a crude extract of proteins from MTB (purified protein derivative (PPD)).5 6 Commercially available IGRAs include the T-SPOT.TB (Oxford Immunotec, Oxford, UK) and QuantiFERON-TB Gold In-tube (QFT) (Cellestis Incorporated, Carnegie, Victoria, Australia).7 In studies comparing both IGRAs, the T-SPOT.TB was found to be more specific and sensitive than the QFT.5–7
In populations where TB is not endemic, the sensitivity of IGRAs has been reported to be lower than that of TST in diagnosing LTBI8–10 and TAU.11 12 Many studies have explored the use of IGRAs in TAU.11–19 However its role is still uncertain.19 In our centre we use both the T-SPOT.TB and QFT to diagnose TAU. An ‘equivocal’ test result is particular to the T-SPOT.TB because of a range of cut-off values.8 This occurs when the numbers of ‘spots’ produced are more than the negative cut-off value but less than the positive cut-off value.6 As the ‘equivocal’ T-SPOT.TB is inconclusive, there are currently no clear guidelines on how to interpret the result or what clinicians should do next. Most ophthalmologists either disregard the result, repeat the T-SPOT.TB test or perform QFT.
In this study we aim to determine risk factors for obtaining an ‘equivocal’ T-SPOT.TB result in patients suspected of a tubercular aetiology and to study the clinical implications of such a test result in Singapore, where TB infection is moderately endemic.20
Materials and methods
We prospectively reviewed all consecutive new patients with uveitis who presented to Singapore National Eye Center Ocular Inflammation and Immunology Service (from 1 September 2008 to 31 December 2009) with clinical ocular signs consistent with TAU as described by Gupta et al. and Tabarra.21–23 Briefly, all study subjects in our cohort study underwent a full systemic review, ocular examination and standard baseline investigations. All patients also had blood taken for T-SPOT.TB before a TST was performed. Patients were excluded if they had any other possible underlying aetiology or a T-SPOT.TB result that was ‘indeterminate’. Patients who had ‘equivocal’ T-SPOT.TB results had a QFT performed. All patients who had a positive T-SPOT.TB or TST result were referred to the infectious disease physicians at Singapore General Hospital for review and prescribed anti-TB therapy (ATT) as indicated. Patients were followed up every 2 weeks for the first 8 weeks, then every 2 to 3 months (or more frequently as required) to monitor response to therapy. All patients were followed-up for 1 year from enrolment.
Each subject had a heparinised blood sample collected by venous puncture for whole blood IGRA, before administration of TST. All patients had standard baseline investigations: complete blood count, erythrocyte sedimentation rate, liver enzyme panel, infectious disease screen (which included the Venereal Disease Research Laboratory (VDRL) test for syphilis, TST and urine microscopy) and a chest x-ray. Other tests such as QFT, acid-fast bacillus (AFB) smears from throat swabs or PCR assays for TB DNA or viral DNA of ocular samples were performed where indicated. The PCR assays performed for TB were as previously described, using primers corresponding to IS6110 region and using known positive and negative controls.24 Tetraplex PCR analysis of all aqueous samples were tested for the presence of cytomegalovirus, herpes simplex virus, varicella-zoster virus, rubella virus and toxoplasmosis genomic DNA.25
TST was performed by using the standard Mantoux method via intradermal injection of 2 tuberculin units (TU) of PPD (RT23 SSI, 2 TU/0.1 ml; Statens Serum Institut, Copenhagen, Denmark). The induration was measured at 72 h with a ruler by an independent observer and considered positive if it measured more than or equal to 15 mm. The cut-off value of 15 mm was used as recommended by WHO guidelines and validated in our moderately endemic Singapore population.26 27
T-SPOT.TB was performed according to the manufacturer's instructions.28 For each patient, 8 ml of blood was collected in lithium heparin tubes and was processed within 8 h from sampling. Peripheral blood mononuclear cells were prepared by density gradient centrifugation over Ficoll-Paque Plus (GE Healthcare, Singapore, Republic of Singapore). Two-hundred and fifty thousand cells were seeded in each of four wells of the assay plate. The cells were stimulated for 16–20 h (under 5% carbon dioxide at 37°C) with GIBCO (Carlsbad, CA, USA) AIM-V medium (nil control), phytohaemagglutinin (mitogen-positive control) or the TB-specific peptide antigens (peptide pools for early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) in separate wells) in a total volume of 150 μl per well. Two readers quantified the number of IFN-γ spot-forming T cells visually, and a third reader was consulted if the results were disparate. The T-SPOT.TB test was considered positive if there were >8 spots compared with the negative control well; negative if there were <4 spots compared with the control well; or equivocal if the test wells had 5–7 spots more than the control. If the negative control well had >10 spots and/or <20 spots in the mitogen-positive control wells, the result was considered to be indeterminate. In patients with an ‘equivocal’ T-SPOT.TB, a QFT test was performed.
QFT was performed according to the recommended guidelines.29 Whole blood from each patient was divided into three tubes of 1 ml each (nil control, positive control and TB-specific antigens (ESAT-6, CFP-10 and TB7.7)). Samples were incubated with the stimulating antigens for 16–24 h at 37°C. Plasma samples were then harvested and the amount of IFN-γ released was measured via ELISA. The result obtained in the nil control was subtracted from the mitogen control and the antigen-stimulated samples. The result was considered positive if the response to the specific antigens was ≧0.35 IU/ml, regardless of the level of the positive control; negative if the response to the specific antigens was <0.35 IU/ml and the IFN-γ level of the positive control was ≧0.5 IU/ml; and indeterminate if both antigen-stimulated samples were <0.35 IU/ml and the level of the positive control was <0.5 IU/ml.
Statistical analysis included descriptive statistics, where the mean and SD were calculated for the continuous variables, while frequency distribution and percentages were used for categorical variables. We compared features of patients with ‘equivocal’ T-SPOT.TB results with those with positive or negative T-SPOT.TB results as controls. Comparisons between categorical variables were conducted by Fisher's exact tests, whereas the one-way ANOVA test was used for means. We analysed risk factors associated with ‘equivocal’ T-SPOT.TB results while controlling for possible confounders using backward stepwise logistic regression. We included factors in our model that were clinically important although not statistically significant on univariate analysis, such as demographic variables and clinical features including type, chronicity and disease duration, systemic diseases including diabetes and previous TB infection (which have been shown to affect T-SPOT.TB), and test results of TST and QFT which we used to diagnose latent TB. The upper age limit of 55 years was chosen as that is considered to be the age of onset of immunosenescence,30 while age <13 years defines the paediatric age group studied in our population for TST.27 The estimate of OR and its associated 95% CI were calculated. A p value <0.05 was considered statistically significant. All analyses were performed using STATA version 11 (StataCorp LP, Texas, USA).
Results
A total of 179 patients met our inclusion criteria and were enrolled in our study. Twenty-one patients (13.0%) were subsequently found to have underlying non-tuberculous aetiology, while three patients (1.9%) had ‘indeterminate’ T-SPOT.TB results and all were excluded from our study analysis. Patients excluded were HLA-B27-positive (n=10), VDRL-positive (n=5), or diagnosed with herpetic anterior uveitis (n=4) and sarcoidosis (n=2). All three patients with ‘indeterminate’ T-SPOT.TB results had unilateral, acute anterior uveitis that was successfully treated with topical steroids with no recurrence of inflammation at the end of our study period.
Of the remaining 155 patients, 15 (9.7%) had ‘equivocal’ T-SPOT.TB results. Demographics, clinical characteristics, investigation results and final diagnoses for these 15 patients are described in table 1. The mean age of these patients was 52.2 (range 12–77) years. Most were Chinese (n=8, 53.3%) with no sex dominance (seven male, eight female). One patient had diabetes mellitus and one patient had a history of past pulmonary TB infection. None of the patients had a history of malignancy or any immunosuppressive disease. None of the patients were on systemic steroid or immunosuppressive therapy at the time of presentation and testing.
Clinical features and investigation results of patients with equivocal T-SPOT.TB results
Results of the comparison of demographics, clinical profiles and investigation results between patients with ‘equivocal’ T-SPOT.TB results and controls are described in table 2. In most of these patients, the final aetiology of the disease was unknown (idiopathic) (n=11, 73.3%). Among the patients with ‘equivocal’ T-SPOT.TB, only one patient was presumed to have TAU as defined by previous studies.22 23 He had a positive TST and a positive QFT, with chest x-ray findings supportive of TB, and also responded positively to ATT without relapse. All other patients (14/15, 93.3%) had negative QFT (p<0.001) and 13 (86.7%) had negative TST (p=0.002). Four patients had ocular fluid samples tested negative for viral DNA via PCR. None of the patients had neutropenia. We also compared the demographics and frequency of clinical signs associated with TAU21 between these patients and the controls but did not find any significant differences. However, we found that patients being <13 or >55 years old was an independent risk factor for an ‘equivocal’ T-SPOT.TB result in our study cohort (OR 21.2; 95% CI 3.7 to 121.6; p=0.001) while adjusting for potential confounders including sex, race, past medical history of diabetes mellitus, immunosuppression, disease duration, type of uveitis and TST (table 3). These patients were also more likely to have a negative QFT result (OR 14.8; 95% CI 1.2 to 179.9; p=0.035).
Comparison of characteristics between patients with ‘equivocal’ T-SPOT.TB results (n=15) and remaining patients in study cohort as controls (n=140)
Factors for obtaining an ‘equivocal’ T-SPOT.TB result
Discussion
In our study, patients aged <13 years and >55 years were 21 times more likely to have an ‘equivocal’ T-SPOT.TB result independent of sex, race, medical history or clinical features of uveitis. These patients were also approximately 15 times more likely to have a negative QFT. The CIs associated with the ORs are expectedly wide due to the small number of patients with ‘equivocal’ results. As the incidence of ‘equivocal’ T-SPOT.TB results is low (9.7% in our study and 5.0% in the study of Bienek and Chang31), recruiting the required number of subjects in our prospective study would be a challenge. Nevertheless, our results suggest that QFT is a more useful test than T-SPOT.TB in the older age group and that patients with ‘equivocal’ T-SPOT.TB results are unlikely to have LTBI.
Our findings are consistent with reports that IGRAs may not be useful in patients 2–9 years old.32–34 Although the exact mechanism is uncertain, it has been hypothesised that this is due to an immature immune system.33 The variable T cell IFN-γ response in children has lead to reports that IGRAs may be inferior in accuracy to the TST in certain paediatric age groups.35 The utility of IGRAs is also limited by increasing age due to the waning of T cell function.36 Ageing is thought to affect the immune system by causing a decline in the functional activity of the peripheral blood mononuclear cells and a reduced diversity of T cells.30 As immunosenescence commences at the age of 50–55 years,30 cytokine-based tests such as IGRAs may be less useful in older patients. In addition, a number of studies have described an age-related decreasing trend in IFN-γ production from T cells in response to mitogen and MTB antigens in whole blood.6 31 36 We observed that the QFT was not affected by age in our study. This may be because the T-SPOT.TB test is a more sensitive test, producing an intermediate result that does not reach the positive threshold.5 7
Although largely similar, there are some fundamental differences between QFT and T-SPOT.TB. T-SPOT.TB counts the number of IFN-γ-producing T cells after stimulating isolated peripheral blood mononuclear cells with ESAT-6 and CFP-10, using an enzyme-linked immunospot assay (ELISPOT).31 On the other hand, QFT is a whole blood assay that quantifies IFN-γ produced by T cells in response to ESAT-6, CFP-10 and TB7.7 using an ELISA.29 Thus, T-SPOT.TB has better resolution of samples that would usually give ‘indeterminate’ QFT results due to inadequate T cell numbers (eg, blood samples from immunocompromised individuals).37 T-SPOT.TB therefore produces fewer ‘indeterminate’ results than QFT in these patients.38 The rate of ‘indeterminate’ results range from 5.0% to 40.0% for QFT and 0% to 5.4% for T-SPOT.TB.6 Factors that have been reported to contribute to this result include age, immunosuppression and the use of steroids.7 37 38 In our study, we found that the incidence of ‘indeterminate’ T-SPOT.TB results was 1.89% (3/158).
Yet another significant difference between QFT and T-SPOT.TB is the distinct cut-off value in QFT versus the range of cut-off values in T-SPOT.TB (negative <4 spots and positive >8 spots). An ‘equivocal’ T-SPOT.TB result is reported if the number of spots falls in the range of 5–7. We hypothesise that this result could represent a trend towards a false positive, which is corroborated by our study findings. Most of these patients (13/15, 86.7%) had a negative TST (<15 mm). Using a cut-off value of 10 mm, our results remained unchanged. This is because all 13 patients (83.7%) with equivocal T-SPOT.TB results had TST of <10 mm compared with 52 patients (37.4%) with valid results (p<0.001), which suggests that using this cut-off would further strengthen our hypothesis and outcome. Even though patients with ‘equivocal’ T-SPOT.TB had their blood sampled for QFT after the TST (which potentially could lead to a ‘boosting’ effect), QFT was still negative in 14/15 patients (93.3%). These results taken collectively enabled us to comfortably rule out LTBI in these 14 patients and prescribe appropriate immunosuppressive treatment without the need for ATT. All these patients had resolution of uveitis after discontinuation of immunosuppression, without recurrence during the 1-year follow-up.
The diagnosis of extra-pulmonary TB, including TAU, is fraught with difficulty because of the lack of confirmatory tests such as AFB smears or MTB cultures. Our findings are useful in helping ophthalmologists decide on the course of action for patients with ‘equivocal’ T-SPOT.TB results. Although repeating T-SPOT.TB in such patients is recommended, the clinician may order QFT instead. Our study showed that there is a high likelihood that QFT will be negative. In addition, QFT should be preferred to the T-SPOT.TB in adults >55 years old. The main limitation of our study is its small sample size, which resulted in the enrolment of only one patient aged <13 years with an equivocal T-SPOT.TB result. We were thus unable to conclude with certainty that T-SPOT.TB may be less useful in younger patients. We were also unable to study the effects of immunosuppression on T-SPOT.TB, as we lacked such patients. QFT should ideally be performed before the TST to prevent a potential boosting effect. However, the findings of our study and those of others have demonstrated that the TST is unlikely to affect the QFT result.29
In conclusion, we found that an ‘equivocal’ result, which is particular to the T-SPOT.TB, is commonly encountered in patients aged >55 years old. However, these patients are highly likely to have a negative QFT test result in a population in which TB is moderately endemic. Nonetheless, larger clinical studies are needed to confirm our findings.
References
Footnotes
Funding Singapore National Eye Centre Health and Research Endowment Fund.
Competing interests None declared.
Patient consent Obtained.
Ethics approval This study was approved by the Singapore Health Services Institutional Review Board in accordance with their guidelines.
Provenance and peer review Not commissioned; externally peer reviewed.
Linked Articles
- At a glance