Journal of Immunological Methods 370 (2011) 55–64 Contents lists available at ScienceDirect Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m Research paper Detection of proliferative responses to ESAT-6 and CFP-10 by FASCIA assay for diagnosis of Mycobacterium tuberculosis infection Emilie Borgström a,⁎, Peter Andersen b, Lena Andersson c, Inger Julander a, Gunilla Källenius d, Markus Maeurer e, Maria Norrby f, Ida Rosenkrands b, Teghesti Tecleab c, Judith Bruchfeld a, 1, Hans Gaines c, 1 a Unit of Infectious Diseases, Institution of Medicine, Karolinska Institutet, Karolinska University Hospital, Solna, 171 77 Stockholm, Sweden Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark c Swedish Institute for Infectious Disease Control, Nobels väg 18, 171 82 Solna, Stockholm, Sweden d Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, 118 83 Stockholm, Sweden e Microbiology and Tumor Biology Center, Karolinska Institute, 171 77 Stockholm, Sweden f Clinic of Infectious Diseases, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden b a r t i c l e i n f o Article history: Received 12 November 2010 Received in revised form 23 February 2011 Accepted 23 May 2011 Available online 30 May 2011 Keywords: CFP-10 Diagnostic methods ESAT-6 FASCIA Tuberculosis a b s t r a c t There is a large and growing worldwide need for reliable tests to diagnose active and latent tuberculosis (TB). Improved methodology for identifying individuals with true latent TB (LTBI), particularly those with a recent infection, would pave the way for targeted prophylactic treatment. The traditionally used tuberculin skin test (TST) is unspecific and impractical. Interferon gamma release assays (IGRA) are more specific than the TST but, like that test, cannot discriminate either between recent and remote TB infection, or between these and a mere immunological memory of previous TB infection. The Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) combines long-term antigen stimulation of whole blood and flow-cytometric analysis with quantification of the expanded T-lymphoblasts and can also be employed for measurement of cytokine responses. © 2011 Elsevier B.V. All rights reserved. Abbreviations: Ag, antigen; BCG, bacille Calmette–Guerin vaccine; CFP10, culture filtrate protein 10; Elispot, enzyme-linked immunosorbent spot; EPTB, extra pulmonary TB; ESAT-6, early secretory antigen 6; FASCIA, Flowcytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood; HIV, human immunodeficiency virus; IGRA, interferon gamma release assay; IQR, inter-quartile range; LTBI, latent tuberculosis infection; M tb, Mycobacterium tuberculosis; PTB, pulmonary tuberculosis; Spearman's rho, Spearman's rank correlation coefficient; TB, tuberculosis; TST, tuberculin skin test; QFT, QuantiFERON TB Gold In-Tube. ⁎ Corresponding author at: Clinic of Infectious Diseases, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. Tel.: + 46 8305615. E-mail addresses: emilie.wahren-borgstrom@karolinska.se (E. Borgström), PA@ssi.dk (P. Andersen), lena.andersson@smi.se (L. Andersson), inger.julander@bredband.net (I. Julander), gunilla.kallenius@ki.se (G. Källenius), markus.maeurer@ki.se (M. Maeurer), maria.norrby@karolinska.se (M. Norrby), IDR@ssi.dk (I. Rosenkrands), teghesti.tecleab@smi.se (T. Tecleab), judith.bruchfeld@ki.se (J. Bruchfeld), hans.gaines@smi.se (H. Gaines). 1 Shared last authorship. 0022-1759/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2011.05.008 We established a FASCIA test for the detection of cellmediated immune responses to TB antigens ESAT-6 and CFP-10 and evaluated this assay in 21 TB non-exposed controls as well as in 161 patients referred to the Karolinska University Hospital for suspected active TB. This resulted in the diagnosis of 33 pulmonary TB (PTB) cases and 21 extra pulmonary TB (EPTB) cases verified by microbiological methods, histopathology or a clinical suspicion of disease. For all the patients with active TB, the TB FASCIA achieved an overall sensitivity of 86% and a specificity of 91%. In patients with PTB, FASCIA was positive significantly more often in patients with Mycobacterium tuberculosis (M tb) culture-verified disease than in M tb culture negative patients (20/25 vs. 2/8; p b 0.008 by Fisher's exact test). Patients with EPTB displayed significantly higher responses in FASCIA than patients with PTB to CFP-10 (p= 0.037) and close to significance to ESAT-6 (p= 0.076) (Wilcoxon's signed rank test). The sensitivity and specificity of FASCIA were 56 E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 similar to that of IGRAs, performed in a subset of individuals, with mainly concordant results. The development of a test for immune response to TB is, however, intended not so much for active TB as for the diagnosis and management of true latent TB. In keeping with what was done initially for TST and IGRAs, the performance of the TB FASCIA has now been assessed in active TB and TB nonexposed controls; the assay can be used for the study of LTBI, including the exploration of other TB antigens and immune markers such as cytokines and chemokines. 1. Background In connection with the human immunodeficiency virus (HIV) epidemic and socio-economic factors, tuberculosis (TB) has re-emerged as a major global health threat. It is possible that an estimated one-third of the world's population is infected with Mycobacterium tuberculosis (M tb) (Corbett et al., 2003), which constitutes a source of latent TB infection (LTBI) that can lead to symptomatic, often contagious TB disease. Several methods are available for diagnosing active TB disease on the basis of identifying tubercular bacilli. In LTBI, however, the bacterial burden is too low for it to be possible to culture the bacilli and to date there is no truly golden standard for diagnosing this condition (Pai et al., 2010). The existing indirect diagnostic tests for LTBI are the traditional tuberculin skin test (TST) (Huebner et al., 1993) and the more recently developed interferon gamma release assays (IGRAs) (Lalvani et al., 2001a, 2001b; Mazurek et al., 2005). The latter are blood tests based on overnight incubation of patient samples with specific M tb antigens, whereby an immunological response with production of gamma-interferon is detected. The TST is unspecific because of cross-reaction to antigens present also in bacille Calmette– Guerin (BCG) vaccine strains (Ravn et al., 1999; Brock et al., 2001) (Lalvani et al., 2001a, 2001b) and in environmental mycobacteria (Arend et al., 2005). The IGRAs are more specific (Richeldi, 2006) but, like the TST, cannot discriminate between different clinical entities, such as active TB, recent or remote LTBI or a mere immunological memory of previous TB disease (Mack et al., 2009; Kabeer et al., 2010). An important component in TB control in low TB endemic areas is the detection of recently TB-infected individuals who run an increased risk of progressing to active disease (Pai, 2010); the need to find new antigens and immunological markers for this patient category is urgent. With adequate prophylactic treatment, these patients will not develop symptomatic disease and the chain of transmission can be broken (Rieder et al., 1996). Our aim with the present study was to establish the already accredited method, Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA), with TB antigens for future research in the area of latent TB immunology and diagnostics. The assay is based on a seven-day incubation, enabling proliferative responses to the specific M tb antigens ESAT-6 and CFP-10, and can be employed for the detection of proliferative cells as well as cytokine responses. Another advantage of long-term cultures is that weak responses, which may not reach the level of detection for methods such as the IGRAs that use only over-night culture, can be amplified by cell proliferation and thus become detectable (Gaines et al., 1996; Gaines and Biberfeld, 2000; Svahn et al., 2003; Lagrelius et al., 2006). The FASCIA technology is based on an ordinary flow cytometric (FACS) method, with staining for CD4+ cells, but it is also possible to use monoclonal antibodies for the detection of other surface antigens. In this study, in the absence of a golden standard for LTBI, FASCIA was assessed for the diagnosis of TB in patients with suspected active TB disease. Patients with microbiologically verified disease were used as positive controls, and healthy controls with no exposure to or risk factor for TB were used as negative controls to determine a cut-off level for positive reactions and calculate the new assay's sensitivity and specificity for TB diagnosis. 2. Methods 2.1. Patients, controls and samples Consecutive patients referred to the TB center at the Clinic of Infectious Diseases at Karolinska University Hospital, Stockholm, Sweden, were recruited to the study between 2006-11-07 and 2008-03-12 after verbal and written informed consent. The inclusion criterion was the attending physician's sufficient suspicion of active TB to warrant taking a specimen for mycobacterial analysis, including microscopy for acid-fast bacilli, M tb complex polymerase chain reaction (PCR) and mycobacterial culture. An exclusion criterion was ongoing TB treatment for more than one week. Patients were examined with standardized questionnaires regarding their clinical status with symptom screening, including cough, fever, haemoptysis and weight loss, BCG status, age, gender, origin and immunosuppression. In addition, TB investigation was performed according to clinical routines, with radiology and 3 sputum samples for smear microscopy, polymerase chain reaction for the M tb complex and mycobacterial culture with BACTEC. Bronchoscopy and histopathological examination of tissue biopsies were performed when appropriate. In most cases the TST was performed and read before FASCIA and IGRA testing. Venous blood samples were drawn and then coded and analyzed in a blinded fashion. For FASCIA, 5 ml blood was collected in a sodium heparin tube; for T-SPOT.TB and QuantiFERON-TB Gold In-Tube (QFT), blood was collected in prefabricated tubes according to the manufacturer's instructions. The patients were categorized into subgroups on the basis of clinical data. Case definitions: a) and b) Verified active pulmonary TB (PTB) and extra pulmonary TB (EPTB), respectively: patients with suggestive clinical symptoms and microbiological verification by either smear microscopy, M tb PCR, mycobacterial culture or histopathological diagnosis of biopsy material from lymph nodes with granulomatous lymphadenitis. c) Clinical TB: patients with suggestive clinical symptoms, suspicious radiology and recovery after TB treatment, but without microbiological verification of the disease. d) Previous TB: patients with a history of TB disease or suggestive radiology for remote TB disease but without E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 any signs of TB activation by mycobacterial culture and radiology. No distinction was made between previously treated and untreated patients. e) LTBI: patients with TST ≥10 mm and with exposure to TB disease or originating from a TB high endemic country (TB incidence N100/100,000) (http://www.who.int/en/) but with no radiological evidence of ongoing or previous active TB in the clinical work-up. f) TB negative: patients with TST 6 mm or less, no exposure to TB, no history of earlier active TB disease and negative TB investigation. g) “Other diagnoses”: patients who did not fit into any of the above groups. They could not be classified as TB negative because they were either exposed to TB or not tested with TST. Controls: Students (n = 17) and laboratory personnel (n = 4) were consecutively recruited to the study as healthy controls after verbal and written informed consent. Inclusion criteria were no previous TB history, no contact with a TB patient, no stay in a TB endemic country for more than 3 months (outside western Europe, north America or Australia), no environmental mycobacterium infection and not worked for more than one year in a hospital, a prison, with asylum-seekers or in similar institutions. Blood samples from these individuals were used to establish cut-off levels for positive reactions by FASCIA. 2.2. Laboratory procedures The FASCIA was employed as earlier described (Gaines et al., 1996; Gaines and Biberfeld, 2000; Svahn et al., 2003; Lagrelius et al., 2006) with the modification that proliferative results were measured as number of CD3 + CD4+ lymphoblasts generated per μl peripheral blood. The blood was diluted 1:8 in RPMI 1640 (Gibco/BRL, UK) supplemented with 10,000 IU/ml penicillin (Gibco/BRL), 10,000 μg/ml streptomycin (Invitrogen, Stockholm, Sweden), and glutamax [RPMI medium]. 400 μl of the diluted blood and 100 μl of antigen or medium only were added to 12 × 75 mm polystyrene round-bottom tubes with caps (Falcon 2058, Becton Dickinson Labware, NJ) and incubated for 7 days in a humidified atmosphere at + 37 °C with 5% CO2 in air. Negative (medium only), positive (phytohemagglutinin, PHA) and tuberculin-control cultures were run in parallel with specific stimulations. After incubation, the tubes were centrifuged at 300 x g and the supernatants were removed and kept at −80 °C until required for analysis of cytokine concentrations. The pellets were stained with anti-CD3-Fitc and anti-CD4 PerCP [Becton Dickinson Immunocytometry Systems (BDIS), Stockholm, Sweden] for 10–15 min in room temperature. A lysing solution (1.0 ml Pharmlyse, BDIS) was added for 5–10 min at room temperature, followed by centrifugation, removal of the supernatants, additional lysing if needed, washing with phosphate buffered saline (PBS), and resuspension in 450 μl PBS with 5% paraformaldehyde. The samples were stored in the dark at +4 °C for no more than 4 hours and then analyzed on a FACScalibur (BDIS) using CellQuest software (BDIS). The instrument was calibrated to acquire 60 ± 6 μl per minute and set for four-color analysis using FACSComp software (BDIS) in conjunction with 57 CaliBRITE (BDIS). Ten per cent of the sample was acquired and saved as list-mode data for analysis, using a previously described method (Gaines et al., 1996; Gaines and Biberfeld, 2000) with modifications as indicated, as shown in Fig. 1. From SSI (Statens Serum Institut) we received a peptide pool of ESAT-6 defined by the following overlapping ESAT-6 peptides: ESAT-6 1-20, ESAT-6 10-25, ESAT-6 16-40, ESAT-6 31-55, ESAT-6 46-70, ESAT-6 61-85 and ESAT-6 71-95, and a CFP-10 peptide pool defined by the overlapping CFP10 peptides CFP10 1-25, CFP10 16-40, CFP10 30-55, CFP10 46-70, CFP10 61-85 and CFP10 76-100 (SSI). Optimal concentrations of M tb antigens were determined before the study by testing with samples from TB positive and TB negative controls. Concentrations between 1 and 5 μg/ml of ESAT-6 and CFP10 per peptide were studied and an optimal concentration of 2 μg/ml was chosen for both antigens. The commercially available QuantiFERON TB Gold In-Tube (QFT) from Cellestis and the T-SPOT.TB from Oxford Immunotec were performed according to the manufacturer's instructions and evaluated together with FASCIA in our study. These methods employ whole blood (QFT) or PBMC (T-SPOT-TB) incubated for 16–24 hours with the specific M tb antigens ESAT-6, and CFP-10. The QFT-TB Gold IT test also includes TB7.7. After incubation, interferon-γ (IFN-γ) responses are measured by ELISA in QFT and by ELISPOT in T-SPOT.TB. The TST was performed and read with the Mantoux method by nurses specifically trained for the technique. Two units of purified protein derivative (Tuberculin PPD RT23, Statens Serum Institute, Copenhagen, Denmark) were injected intradermally and cutaneous induration was measured with a ruler after 72 hours. A positive TST was defined as an induration of 10 mm or more and TST conversion as a test converting from negative to positive with an increase in size of at least 10 mm. Smear microscopy for acid-fast bacilli by auramine-fluorescence staining was done after concentration, polymerase chain reaction for the M tb complex using the COBAS AMPLICOR MTB test (Roche, Branchburg, NJ, USA) and mycobacterial culture with the BACTEC 960 MGIT system (BD, Sparks, MD, USA) on conventional Löwenstein-Jensen media. HIV testing was done in verified TB cases at the Microbiology Department, Karolinska Hospital, with chemiluminescence detection of both antibodies and viral antigens using Architect HIV Combo (Abbott Scandinavia AB, Stockholm, Sweden). 2.3. Radiology The radiographs were read independently by two radiologists, not blinded to clinical information, since this was a study in a clinical setting and the definition of active TB was based on microbiological rather than radiographic criteria. Apical involvement, cavities with acinonodular foci, miliary pattern, localized small fibronodular foci, unilateral hilar and/or mediastinal adenopathy with or without localized alveolar opacities and pleural effusion were interpreted as radiographic changes suggestive of active TB. Solitary calcified nodules were interpreted as radiographic changes suggestive of previous primary TB infection. Previously active but healed TB was suspected when there was fibrotic scarring of the upper lung lobes and loss of lung volume as well as apical pleural scarring. 58 E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 A B C D Fig. 1. FACS cell proliferation with the FASCIA method with positive and negative controls in one patient with and one patient without active TB, after stimulation with ESAT-6 and CFP-10. Assessment of TB-specific CMI using FASCIA for the detection of CD3+, CD4+ lymphoblasts. Diluted whole blood from a TB negative subject (columns A + B) and a subject with active TB (columns C + D) was cultured in the presence of medium only, PHA, CFP-10 or ESAT-6. Dot plots in the lefthand column for each patient display cell size (x-axis) vs. cell granularity (y-axis) [forward scatter, FSC vs. side scatter, SSC] and large granular lymphoblasts (LGLs) are recovered by region R1. Cells from R1 are examined using dotplots in the right hand column displaying CD3+, CD4+ lymphoblasts [FL-1 vs. FL-3] which are recovered by region R2. The events (cells) displayed on dotplots and logically gated by both R1 and R2 are derived from acquisition of exactly 1/10 of a culture including 50 μl blood and thus, the counted number of cells is divided by five to provide the FASCIA unit of measurement of CD3+, CD4+ lymphoblasts generated per μl blood. Finally, net results are calculated by subtracting the background results for cultures with medium only from the results for cultures with mitogen/ antigen. 59 E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 2.4. Statistical methods Comparisons of the proliferative responses for ESAT-6 and CFP-10 antigens in FASCIA were performed within and between all groups and subgroups of patients in the study with Wilcoxon's signed rank test (two-tailed). Comparisons of test results from TST, QFT, T-SPOT.TB and FASCIA in terms of positive/negative results were made by head-to-head comparisons with McNemar's test. P values b0.05 were considered significant. The correlation between the numerical values for the TST in millimeters and the FASCIA proliferative response after tuberculin stimulation was investigated using Spearman's rho (Spearman's rank correlation coefficient) since simple histogram plots indicated that data may not be normally distributed. A correlation coefficient of p b 0.05 was considered significant. Analyses were conducted using SAS v.9.1. 2.5. Ethical considerations The Regional Ethics Committee in Stockholm approved the study. Patients were included after giving their verbal and written informed consent when the nature and possible consequences of the study had been fully explained. Laboratory samples were coded and analyzed in a blinded fashion. If IGRAs were needed in the diagnostic work-up, additional blood tests for that purpose were taken and handled outside the protocol of the study. 3. Results 3.1. FASCIA method set-up FASCIA results were measured as net number of CD3 + CD4 + lymphoblasts generated per μl blood, as demonstrated in Fig. 1. The results from parallel negative (in the presence of medium only) and positive (with the mitogen PHA) control cultures were employed to determine whether a subject's FASCIA results should be accepted or interpreted as indeterminate. The percentile 0.05-0.95 (5–95%) for medium control, with samples from all patients was used to set the highest acceptable background, which gave results from 0.64-12.3 cells/μl. If the background was above 12 cells/μl, the test was considered indeterminate and these results were excluded if their PHA tube result divided by the medium tube result was not N10 (stimulation index N 10). In FASCIA, the median value for background controls with medium was 3 (IQR (inter-quartile range) 4) cells/μl for all the 161 patients tested. The median value for positive controls (PHA) was 1282 (IQR 1546) cells/μl and for tuberculin 2158 (IQR 3505) cells/μl. When results from the positive control sample, either PHA or tuberculin, were divided by results from the negative control sample, the outcome was deemed positive (stimulation index N20) if it was 20 or more; results below 20 were considered indeterminate. The cut-off for FASCIA was set by analysis of blood samples from 21 healthy TB disease-negative controls. Median results for the specific M tb antigen stimulation plus 3 standard deviations gave 4.72 for CFP-10 and 5.10 for ESAT-6. The cut-off for a positive test was thus set at 6 cells/μl in either CFP-10 or ESAT-6; this gave 100% specificity among the controls. IGRAs were not performed in these patients. The median values for CFP-10 and ESAT-6 for clinically negative students/laboratory personnel were 1 cell/μl (IQR 2 and 1 cell/μl, respectively). 3.2. Patients 179 patients were recruited to the study but 18 of them were excluded because of missing or contaminated samples, missing medical records or, for two patients, duplicate inclusion by mistake. Demographic and clinical data for the remaining 161 study subjects are given in Table 1. Ninety-one (57%) were women and 70 (43%) were men. Their nationalities represented all continents except Australia. Eighty-four (52%) came from TB high-endemic areas and the single largest group, 39 patients (24%), came from Somalia. The 161 patients studied were categorized according to the study definitions as 54 cases of active TB (34%), 22 with previous TB (14%) (previously treated for TB, n = 16, and untreated, n = 6), 62 with LTBI (39%), 11 TB negative (7%) and the other 12 as other diseases (7%). The 54 patients with active TB consisted of 33 (61%) patients with PTB (2 of them with miliary TB and dissemination) and 21 (39%) patients with EPTB (13 with lymph node TB, 1 with gastrointestinal TB, 2 with urogenital TB, 4 with skeletal TB and 1 with ocular TB). Active TB was verified microbiologically by mycobacterial culture in 24/33 PTB and 14/21 EPTB, or by PCR only in 1/33 PTB and 1/21 EPTB, while the remaining 8 PTB cases were diagnosed by suggestive radiology, clinical symptoms and response to TB treatment and the remaining 6 EPTB cases were diagnosed by suggestive histopathology (n = 3) or clinical suspicion (n = 3). Table 1 Demographic and clinical background data. Values given are numbers of patients in each category (percentage in parentheses). Average age in years as x (range). Low endemic origin ≤1/100,000, high endemic origin N100/100,000 TB incidence. Immunosuppression includes potentially immunosuppressed patients (diabetes mellitus, treatment with immunosuppressant drugs, transplanted patients, renal insufficiency, rheumatic diseases, alcoholism and pregnancy). Patient category Pulm TB n = 25 Explum TB n = 18 Clinical TB n = 11 TB neg n = 11 Latent TB n = 62 Previous TB n = 22 Other n = 12 Female gender n (%) Age x (range) BCG n (%) Origin n(%) Low-endemic High-endemic 12 (48) 13 (72) 8 (73) 6 (55) 31 (50) 11 (50) 10 (83) 34 (20–66) 39 (19–76) 43 (20–79) 41 (27–26) 42 (18–67) 49 (25–84) 49 (24–69) 17 8 5 8 48 12 7 9 (36) 4 (22) 8 (73) 6 (55) 19 (31) 11 (50) 8 (67) 16 (64) 14 (78) 3 (27) 5 (45) 43 (69) 11 (50) 4 (33) (68) (44) (45) (73) (77) (55) (58) Immunosuppression n(%) HIV+/HIV tested (%) Culture + or PCT + n(%) 5 (20) 3 (17) 5 (45) 2 (18) 11 (11) 4 (18) 6 (50) 2/19 0/14 0/6 0/6 0/15 1/8 0/7 25 (100) 15 (83) 0 (0) – – – – (11) (0) (0) (0) (0) (13) (0) 60 E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 Fig. 2. FASCIA proliferative results in cells/μl for ESAT-6 and CFP-10 in 43 patients with pulmonary or extra pulmonary TB verified by microbiological methods or histopathology. The 11 TB negative cases were diagnosed with psoas abscess (n = 1), rheumatic disease (n = 1), sarcoidosis (n = 2), pneumonia (n = 3), erythema nodosum (n = 1), fibroma molle (n = 1), reactive lymphadenitis (n = 1) and bronchiectasias (n = 1), while the 12 patients with other diseases were diagnosed as ordinary pneumonia (n = 3), lung tumor (n = 1), chronic obstructive lung disease (n = 1), bronchitis (n = 1), rheumatic diseases (n = 3) and unspecified cough (n = 3). significant differences between the antigens. Neither was there any no significant difference between ESAT-6 and CFP10 when all groups were analyzed together (p = 0.11). FASCIA was positive in 38/62 (61%) patients with LTBI (including 2 of 4 subjects with evidence of recent TST conversion). FASCIA was positive, displaying very low reactivity (7 cells/μl), in only one of 11 patients classified as TB negative; thus, 10 of 11 were negative in FASCIA, which gave a specificity of 91%. The outcome of FASCIA in the group of “other diagnoses” reflects the large number of patients in our clinic who originated in TB high-endemic areas. 3.3. FASCIA results Eight of 161 samples (5%) were indeterminate by FASCIA; 5 for too high reactivity in the negative control and 3 for too low reactivity in the positive control. Altogether, 36 patients with potentially immunosuppressive disorders or treatment (diabetes mellitus, pregnancy, corticosteroid treatment, kidney failure, lymphoma, leukemia, recent varicellae and rheumatoid disease) were tested. Another 3 patients were HIV infected. Three patients (two with HIV, one with acute leukemia) had indeterminate FASCIA results due to positive control failure. Responses to CFP in FASCIA were significantly higher in patients with extra pulmonary TB than in those with pulmonary TB (p = 0.037) and the difference in responses to ESAT-6 was close to significance (p = 0.076) (Wilcoxon's signed rank test), as shown in Fig. 2. The responses to ESAT-6 and CFP-10 did not differ significantly between any of the other groups of patients studied. The FASCIA outcomes for the patient groups are shown in Table 2. FASCIA was positive significantly more often in patients with microbiologically verified PTB than in those with clinical PTB (20/25 vs. 2/8; p b 0.008 by Fisher's exact test), suggesting that some of the latter may have been misdiagnosed. A comparison of ESAT-6 and CFP-10 responses in the subgroups of patients (PTB; p = 0.48, PTB; p = 0.17, previous TB; p = 0.28 and LTBI; p = 0.35) showed no 3.4. FASCIA versus IGRA FASCIA was performed head-to-head with IGRAs in a limited number of patients. Both IGRAs had a specificity of 100%, but this was tested in the very small group of TB negative patients; QFT (n = 6) and T-spot (n = 3). The outcomes of IGRAs in the patient groups are shown in Table 2. Three of 48 (6%) tested samples were indeterminate in T-SPOT.TB and 5 of 87 (6%) were indeterminate in QFT according to the manufacturer's instructions. High concordance was found between proliferative responses measured in long-term culture (7 days) by FASCIA and those measured in short-term culture (overnight) for active and latent TB by T-SPOT.TB and QFT; concordance was even higher when indeterminate results for all tests were excluded from the analysis (Fig. 3). None of the tests was significantly better than the others in diagnosing verified active TB when assessed by head-tohead comparisons using McNemar's test: FASCIA versus QFT (p = 1.0), FASCIA versus T-SPOT TB (p = 0.32) and FASCIA versus TST (p = 0.18). The tests’ sensitivities were similar but a combination of the results for QFT and FASCIA gave detection of 96% (27/28) of all the patients with active TB who were tested with both tests. Like FASCIA, QFT tended to be positive more often in patients with microbiologically verified pulmonary TB than in 61 E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 Table 2 Frequency of positive TST, QFT, T-SPOT.TB and FASCIA in different patient groups. Test (number of patients tested) TST (n = 127) QFT (n = 85) T-SPOT (n = 49) FASCIA (n = 182) All verified TB n = 43 Pulm TB n = 25 EPTB n = 18 Clinical TB n = 11 Previous TB n = 22 Latent TB n = 62 TB negative n = 11 Other diagnoses n = 12 Healthy controls n = 21 26/28 14/16 12/12 7/8 8/11 62/62 0/11 0/7 – 18/20 10/12 8/8 2/5 9/12 23/37 0/6 2/5 – 11/13 (85%) 8/8 (100%) 3/5 (60%) 2/4 (50%) 4/7 (57%) 5/17 (29%) 0/3 (0%) 1/5 (20%) – 37/43 20/25 17/18 5/11 16/22 38/62 1/11 4/12 0/21 (93%) (88%) (100%) (88%) (73%) (100%) (0%) (0%) (90%) (83%) (100%) (40%) (75%) (62%) (0%) (40%) (86%) (80%) (94%) (45%) (73%) (61%) (9%) (33%) (0%) For the definitions of groups, see Methods section. those with clinical TB (Fisher, p = 0.06) but the numbers were too small to confirm this statistically. The T-SPOT.TB results from this group were so few that no conclusions could be drawn. In EPTB the test results did not differ between the groups with or without microbiological verification. As T-SPOT.TB gave two indeterminate results in this small group, the sensitivity was only 60%. 3.5. TST results Of the 161 patients who were tested with TST, 127 (79%) had the expected results for the respective group (Table 2). The mean value was 14 mm (range 0–38 mm). Using a webbased algorithm, we estimated on the basis of origin that 81% (50/62) in the LTBI group had been BCG vaccinated only once in infancy, thus increasing the positive predictive value of the TST (Menzies et al., 2008). The concordance of positive and negative results was 46/64 (72%) between TST and QFT-TB Gold IT, 25/38 (66%) between TST and T-SPOT.TB and 83/105 (79%) between TST and FASCIA. The correlation between the TST in mm and FASCIA tuberculin results for all patients (n = 127) tested with both tests was 0.67 (Spearman's rho) (p b 0.0001); for patients with verified active TB (n = 31) the correlation was 0.77 (Spearman's rho) (p b 0.0001). Fig. 3. Concordance between FASCIA and T-SPOT.TB or QFT in active and latent TB (concordance calculated excluding indeterminate results). 62 E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 4. Discussion In this study we have established the already accredited FASCIA method with the specific TB antigens ESAT-6 and CFP-10 in a group of patients with active TB, and have compared the results with non-exposed controls. Our aim in establishing the method for TB antigens is to proceed with research in the area of latent TB immunology and diagnostics by examining new cytokine markers for different entities of LTBI and studying suggested new “TB latency antigens”. The TB FASCIA is employed to measure long-term proliferative lymphoblast responses with specific TB antigens; it has the additional advantage of allowing production and measurement of extracellular cytokines at a higher level, compared to overnight incubation tests (Butera et al., 2009). Clonally expanded T cells are crucial in immune surveillance against M tb (Tully et al., 2005) and have consequently been studied with flow cytometry with or without TB antigen short-term stimulation in human whole blood (Tena-Coki et al., 2010; Sester et al., 2004, 2006; Dinser et al., 2008), in separated lymphocytes of mice (Billeskov et al., 2007) and in animal models with separated lymphocytes for TB vaccine research (Aagaard et al., 2009). Long-term stimulation in whole blood (between 6 and 9 days) has been performed with M tb RD1 and atypical mycobacterial antigens with IFN-γ detection by ELISA (Butera et al., 2009; Black et al., 2001; Leyten et al., 2007) and in separated PBMCs after M tb antigen stimulation with detection of IFN-γ producing cells by ELISPOT and FACS (Goletti et al., 2006). The commercially available IGRAs are based on overnight incubation of patient samples with the specific TB antigens ESAT-6 and CFP-10. 4.1. Correlation between FASCIA results and disease The difference in proliferative responses between TB negative and verified TB patients using FASCIA represented a specificity of 91% and a sensitivity of 86%. The FASCIA proliferative responses in the EPTB group were significantly higher than in the PTB group for CFP-10 and close to significant for ESAT-6. The difference in cell response could be due to the patients with PTB being more severely ill and thereby relatively more immunocompromised, or having a redistribution of specific CD4+ lymphoblasts from blood/lymphatic organs to the lungs, with lower levels of cells in peripheral blood. We also found significant differences within the PTB group, where patients with microbiologically verified PTB were positive significantly more often in FASCIA compared to the small group of patients with clinical PTB. Such differences were not observed between the patients with microbiologically verified EPTB and those with EPTB indicated by histopathology. The majority of patients with EPTB had lymph node manifestations; the histopathology of lymph node TB with granuloma formation and giant cells is rather specific for TB. It is commonly accepted that a certain percentage of both PTB and EPTB patients are culture negative and diagnosis is based on other methods, such as histopathology, radiographic abnormalities and response to treatment (Lange et al., 2009). The evidence for EPTB is often stronger, for instance histopathological diagnosis, while the evidence for culturenegative PTB is less solid. Thus, a hypothesis for the differences found in our study is that in some cases the diagnosis of culture-negative PTB may have been incorrect. These diverse results indicate the need for a reliable golden standard when evaluating new diagnostic tests. There was a trend for the group with verified active TB (EPTB and PTB) to have higher FASCIA cell proliferation results than the group with LTBI, but the numbers were too small to confirm this statistically. This will be an area for further studies with FASCIA cytokine analyses to investigate differences in immunological response between active disease and LTBI, as well as between recent and remote TB infection. 4.2. FASCIA is as specific and sensitive as QFT and T-SPOT.TB In this study we found that the concordance between FASCIA and IGRAs is high in both active TB and LTBI. Using a web-based algorithm (Menzies et al., 2008), we estimated on the basis of origin that 81% (50/62) in the LTBI group had been BCG vaccinated only once in infancy, thus increasing the positive predictive value of the TST (Farhat et al., 2006). Using the cut-offs set for the control groups in this study, the 91% specificity for the TB negative group using FASCIA is comparable to the results from a meta-analysis of ELISPOT (92%), QFT (97%) and TST (98%) in non-BCG vaccinated subjects (Menzies et al., 2007). The sensitivity for verified active TB was fairly high for all the investigated tests in our study but still too low for separate use as a diagnostic tool. Although the combination of FASCIA and QFT gave a sensitivity of 96% in patients with verified active TB, the results of the two tests need to be compared in a larger cohort to see if they really can function complementarily. However, the inability of various tests to differentiate between active TB disease and LTBI has prompted the suggestion that currently available immunological methods should be used primarily to rule out suspected active TB (Lange et al., 2009; Lange and Mori, 2010). 4.3. IGRA test results are comparable to results from previous studies The IGRA results in this study showed 90% sensitivity for QFT and 85% for T-SPOT.TB in total for verified active TB (PTB and EPTB); this is comparable with previous studies of IGRAs in active TB disease. The sensitivity of ELISPOT in groups of HIV negative and HIV positive individuals with culture-verified TB was 100% and 90%, respectively (Chapman et al., 2002). In a large meta-analysis of commercial IGRAs (Diel et al., 2010), the pooled sensitivity for QFT was 81%; in a large review (Pai et al., 2008) it was somewhat lower, 78%. The latter results may have to do with the diagnostic procedures for categorizing patients as TB versus non-TB (culture/PCR or clinical diagnosis). The results for T-SPOT TB differed, with 85% sensitivity in our study compared to 88% in the meta-analysis and 90% in the review; however, the number of T-SPOT TB tested patients in the present study was limited (n= 13) in the verified TB group. The rate of indeterminate test results for IGRAs in this study was higher than in the meta-analysis by Diehl et al. (Diel et al., 2010), with 6% for both QFT and T-SPOT.TB compared to 2.13.8% in the meta-analysis. However, the indeterminate test results were higher in the meta-analysis in the subgroup of immunosuppressed hosts, 4.4-6.1%, which may be explained by the fact that many of the patients in our cohort were severely ill E. Borgström et al. / Journal of Immunological Methods 370 (2011) 55–64 with underlying immunosuppressive disease, even though the number of patients with concomitant HIV was low. The FASCIA showed good performance in the group of moderately immunosuppressed patients, such as those with pregnancy, lymphoma, diabetes mellitus, rheumatic disease and kidney failure. However, the results were indeterminate in two patients with severe immunosuppression (leukemia and AIDS). 4.4. FASCIA and TST The sensitivity of TST in culture-verified active TB was high, 93%, compared to 70% in the above-mentioned meta-analysis. This difference can partly be explained by a bias in the study, in that patients with suspicion of active TB were often included because of a positive TST combined with suggestive TB symptoms. TST and FASCIA with positive/negative results were well correlated in both PTB and EPTB cases, immunosuppressed as well as immunocompetent, which suggests that in future testing, FASCIA analysis alone could suffice for comparing different mixtures of TB antigens to obtain the most accurate results, as compared to studies with TB antigen mixtures in animals and humans with TST or IGRA (Andersen et al., 2000; Bergstedt et al., 2010). In all patients tested with both methods, the numerical results of TST in millimeters also correlated very well with the tuberculin FASCIA cell proliferation number, which makes the FASCIA test trustworthy and is helpful when setting a cut-off for FASCIA. 5. Conclusion In summary, the results confirm that the new TB FASCIA test is reliable and therefore useful in further studies regarding TB immunity. The next step will be to analyze cytokine results from different categories of TB patient, with more TB antigens, for example latency antigens, to see if these results can lead to more accurate diagnostic markers for differentiating the various clinical entities of TB disease as well as recent and remote latent TB infection, as has been suggested by other authors (Goletti et al., 2010). Competing interests statement The authors declare no competing financial interests; P.A. is co-inventor of a patent related to ESAT-6, but all rights belong to Statens Serum Institut. 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