Diagnostic Value of ELISPOT Technique for Osteoarticular Tuberculosis

Clin. Lab. 2014;60:1865-1870 Copyright ORIGINAL ARTICLE Diagnostic Value of ELISPOT Technique for Osteoarticular Tuberculosis XUEQIONG WU 1, *, YUANZHENG MA 2, *, LAN WANG 1, DAWEI LI 2, YOURONG YANG 1, MING HU 2, YAN LIANG 1, HAIBIN XUE 2, JUNXIAN ZHANG 1 * These authors made equal contributions to this study 1 Army Tuberculosis Prevention and Control Key Laboratory, Institute of Tuberculosis Research, Beijing 100091, China 2 Department of Orthopaedics, the 309th Hospital of Chinese PLA, Beijing 100091, China SUMMARY Background: Osteoarticular tuberculosis (TB) is a common severe form of extrapulmonary tuberculosis. Early diagnosis and treatment can decrease the deformity of spine and limbs and joint dysfunction. Methods: We compared and evaluated two commercially available rapid test kits based on the ELISPOT assay for the diagnosis of osteoarticular disease. Results: The diagnostic sensitivity and specificity of the FS-SPOT assay (50.0% and 85.7%) were similar to those of the T-SPOT-TB assay (45.5% and 81.0%). When the two test wells in the T-SPOT-TB assay were both positive, the test wells in FS-SPOT assay were usually positive with the number of SFCs exceeding those in the negative control wells by more than 30. The sensitivities, specificities, PPV, NPV, and agreement of FS-SPOT assay results in 99 TB cases and 54 non-tb disease cases were 55.6%, 83.3%, 84.7%, 52.9%, and 66.0%, respectively. SFC counts from test wells in the TB group were significantly higher than those from the non-tb group (p < 0.001). Conclusions: Higher numbers of SFCs in the ELISPOT assay suggest higher risk of active TB. ELISPOT may be a diagnostic aide for active osteoarticular TB. (Clin. Lab. 2014;60:1865-1870. DOI: 10.7754/Clin.Lab.2014.140110) Correspondence: Xueqiong Wu Army Tuberculosis Prevention and Control Key Laboratory Institute of Tuberculosis Research the 309th Hospital of Chinese PLA Beijing 100091, China Fax: +86 10 80115555 ext. 768415 Email: wu-xueqiong@263.net Manuscript accepted May 5, 2014 KEY WORDS ELISPOT assay, T-SPOT-TB assay, osteoarticular tuberculosis, diagnosis INTRODUCTION Osteoarticular tuberculosis (TB) is typically caused by dissemination of M. tuberculosis through the blood and common in secondary extrapulmonary tuberculosis, accounting for 5% to 10% of all TB patients in China [1, 2]. 95% of osteoarticular TB arises from pulmonary tuberculosis and about 48-87% occurs in the spine, mainly in the lumbar and thoracic vertebrae [3]. Early symptoms are usually mild. The disease is slowly progressive, with systemic symptoms gradually increasing in severity, eventually leading to deformity of spine and limb and joint dysfunction. Therefore, the early diagnosis and early treatment are very important for osteoartic- Clin. Lab. 11/2014 1865

XUEQIONG WU et al. ular TB. Currently, early diagnosis of osteoarticular TB is usually based on imaging examination, for example with X-ray, CT scan, magnetic resonance imaging, etc.; however, it is difficult to differentiate TB from other bacterial, fungal, and cancer diseases of bones and joints. Clinical samples are often difficult to obtain for pathological examination and many cases yield paucibacillary specimens. Immunological diagnosis using blood samples has some promise to contribute to early differential diagnosis in these cases. Although purified protein derivative (PPD) skin test is widely used to detect TB infection and aid in the diagnosis of TB, it has broad cross-reactivity with non-tb mycobacteria and the vaccine strains of M. bovis BCG. Further, the PPD skin test can produce a false-negative result in patients with advanced TB or who have decreased immune function [4]. The enzyme-linked immunospot (ELISPOT) technique is a new T-cell-based gamma interferon (IFN-γ) release assay. The TB ELISPOT assay uses M. tuberculosis early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) peptides or proteins as a stimulus, since these are absent from all strains of M. bovis BCG and most non-tuberculosis mycobacteria [5-7]. It is becoming widely used to aid clinical diagnoses of TB (pulmonary TB and extrapulmonary TB) [8-12]. Two kits have been manufactured and commercialized: T SPOT-TB kit uses pools of ESAT-6 and CFP-10 peptides as a stimulus (Oxford Immunotec, Oxford, United Kingdom) [13]; the FS-SPOT kit uses a fusion protein of CFP-10 and ESAT-6 as a stimulus (ShangHai Mingyuan Health-digit Biochips Co., Ltd Shanghai, China). In this study, we compared and evaluated these two rapid test kits based on the ELISPOT assay for their diagnostic value in Chinese cases of osteoarticular disease. MATERIALS AND METHODS Participants 144 participants were recruited at the 309th Hospital of the Chinese PLA, Beijing 100091, China from December 2009 through December 2011. Ethical approval for the study was granted by the 309th Hospital of the Chinese PLA Research Ethics Committees. Informed consent was obtained from 144 participants. The Ziehl- Neelsen staining smear or BACTEC 960 mycobacterial culture of pathology sample tissues or aspiration pus from the patients were examined in our laboratory according to the protocols in the Chinese Laboratory Science Procedure of Diagnostic Bacteriology in Tuberculosis [14]. Patients were untreated or had received less than 2 weeks of therapy at the time of venipuncture for the ELISPOT assay. The clinical data were surveyed retrospectively and the selected patients were divided into two groups: TB and non-tb. The final diagnoses of 90 TB cases had been determined according to the following criteria: (1) tuberculosis symptoms and signs: for example, fever, night sweats, fatigue, loss of appetite, weight loss, pain, dysfunction, swelling, deformity, etc.; (2) TB lesion on X-ray or CT; (3) positive smear or positive culture; (4) strongly positive PPD skin test; (5) biopsy of the affected tissue and pathological examination; (6) anti-tb chemotherapy and/or operation treatment was effective [15]. The TB group contained 90 TB cases, mean age 43.5 ± 16.5, 46 males and 44 females, in which 29 patients were retrospectively confirmed smear-positive and/or culture-positive. The non-tb disease group contained 54 subjects, mean age 43.5 ± 17.6, 34 male and 20 female. Table 1 shows the detailed information of the selected participants in our study. T-SPOT-TB Assay The T-SPOT-TB assay (Oxford Immunotec, Oxford, United Kingdom) for human IFN-γ was performed according to the manufacturer s recommendations [13]. Briefly, 250,000 peripheral blood mononuclear cells (PBMCs) were plated for 18-20 hours on 96-well plates, which had been pre-coated with a mouse anti-human IFN-γ antibody per well. The cells were left unstimulated (negative control) or were stimulated with 50 μl PHA (positive control) or with 50 μl of ESAT-6 peptides and CFP-10 peptides in separate wells. The number of spot-forming cells (SFCs) in each well was automatically counted with a CTL-ImmunoSpot S5 Versa Analyzer (Cellular Technology Ltd USA). The response of stimulated cultures was considered positive when one or both test wells contained at least six more spots than the negative control wells or had at least twice as many as SFCs as the negative control wells. FS-SPOT Assay The FS-SPOT assay (ShangHai Mingyuan Health-digit Biochips Co., Ltd Shanghai, China) for human IFN-γ was performed according to the manufacturer s recommendations. For each person, 4-5 ml of venous blood sample was collected in a heparinized glass tube, and then PBMCs were isolated and quantified. The cells were aliquoted to 250,000 cells per well, and the cells were incubated with 50 μl of recombinant CFP-10/ ESAT-6 fusion protein antigen at 37 C with 5% CO 2 for 18-20 hours. PBMCs in medium alone were used as the negative control, and 50 μl phytohemagglutinin (PHA) was used as the positive control. The number of SFCs in each well was automatically counted with a CTL-ImmunoSpot S5 Versa Analyzer (Cellular Technology Ltd USA). The response of the stimulated culture was considered positive when the test well contained at least 20 more spots than the negative control wells or had at least twice as many SFC as the negative control wells. Data Management and Statistical Analysis All data were recorded in a Microsoft Office Excel file and the means and standard deviations were calculated. The difference between groups was analyzed by Student's T tests. All of the significance tests were twosided, and a p value of < 0.05 was considered to be statistically significant. The sensitivities, specificities, pos- 1866 Clin. Lab. 11/2014

ELISPOT FOR OSTEOARTICULAR TUBERCULOSIS Table 1. Study participants. Participants Number 1. TB patients 90 Spine TB 5 Cervical vertebra TB 2 Thoracic vertebra TB with or without paravertebral abscess 32 Thoracolumbar vertebra TB 3 Lumbar vertebra TB 41 Lumbosacral vertebrae TB 3 Sacral vertebra TB 2 Femoral trochanteric TB 2 2. Non-TB disease 54 Lumbar intervertebral disc herniation 19 Cervical vertebra disease 7 Intraspinal tumor 6 Spinal metastatic cancer 6 Suppurative spondylitis 7 Fracture of lower limb 5 Fracture of lumbar vertebra 1 Spinal lesions 1 Multiple myeloma 1 Vertebral hemangioma 1 Total 144 Table 2. Comparison of T-SPOT-TB and FS-SPOT assays used for patients with osteoarticular diseases. Groups No. of cases T SPOT - TB No. of positive (positive rate %) ESAT-6 and/or CFP-10 peptides ESAT-6 and CFP-10 peptides FS-SPOT No. of positive (positive rate %) SFC 20 SFC 30 TB 22 10 (45.5) 5 (22.7) 11 (50.0) 8 (36.4) Non-TB 21 4 (19.0) 2 (9.5) 3 (14.3) 2 (9.5) Total 43 Table 3. Detection of cases with osteoarticular diseases by FS-SPOT assay. Groups No. of cases No. of positive (positive rate %) mean number of SFC standard deviation SFC 20 SFC 30 PHA CFP10/ESAT6 protein TB 90 50 (55.6) 35 (38.9) 289.0 231.9 & 77.9 130.6 # Non-TB 54 9 (16.7) 5 (9.3) 267.9 217.1 10.7 14.3 Total 144 & - p > 0.05 vs. the non-tb disease subjects, # - p < 0.001 vs. the non-tb disease subjects. Clin. Lab. 11/2014 1867

XUEQIONG WU et al. Figure 1. Representative results of T-SPOT-TB assay (lane 1 to 4) and FS-SPOT assay (lane 5 to 7) from 4 patients. Figure 1A shows a negative result from a non-tb disease patient with fracture of a lumbar vertebra. Figure 1B, 1C, and 1D show positive results from 3 patients with lumbar vertebra TB. In the T-SPOT-TB assay, Figure 1B shows a positive result in an ESAT-6 peptide well, Figure 1C shows positive results in ESAT-6 peptide well and CFP-10 peptide well, but SFC count of the CFP-10 peptide well was much more than the ESAT-6 peptide well; Figure 1D shows positive results in ESAT-6 peptide and CFP-10 peptide wells, in which there are similar SFC counts. Lane 1 - negative control, in which 2 x 10 5 PBMCs were unstimulated, Lane 2 - positive control, in which 2 x 10 5 PBMCs were stimulated by PHA, Lane 3 - test well A, in which PBMCs were stimulated by ESAT6 peptides, Lane 4 - test well B, in which PBMCs were stimulated by CFP10 peptides, Lane 5 - negative control, in which 2 x 10 5 PBMCs were unstimulated, Lane 6 - positive control, in which 2 x 10 5 PBMCs were stimulated by PHA, Lane 7 - test well, in which PBMCs were stimulated by recombinant CFP10/ESAT6 protein antigen. itive predictive values (PPV), negative predictive values (NPV) and agreement between the two assay methods were calculated with reference to the clinical final diagnosis. RESULTS Comparison of T-SPOT-TB Assay and FS-SPOT Assay Using clinical data from our retrospective survey, we selected 22 confirmed cases with osteoarticular TB and 21 cases with non-tb osteoarticular disease and compared available data from T-SPOT-TB and FS-SPOT assays that were done at the same time on the same samples. The results are shown in Figure 1 and Table 2. The diagnostic sensitivity and specificity of the FS- SPOT assay (50.0% and 85.7%) was similar to the T- SPOT-TB assay (45.5% and 81.0%). When the two test wells in the T-SPOT-TB assay were both positive, the SFCs of test wells in the FS-SPOT assay were usually 30 greater than those in negative control wells. TB cases gave significantly higher SFCs than non-tb disease cases (p < 0.001). Clinical Diagnostic Value of the FS-SPOT Assay for Osteoarticular TB Using the clinical data from the retrospective survey, 99 TB cases and 54 non-tb cases with available data from the FS-SPOT assay were evaluated. The results are shown in Table 3. The sensitivity, specificity, PPV, NPV, and agreement of FS-SPOT assay were 55.6%, 83.3%, 84.7%, 52.9%, and 66.0%, respectively. SFC counts of PHA wells in the TB group were similar to the non-tb group (p > 0.05). SFC counts of test wells in the TB group were significantly higher than in the non-tb group (p < 0.001). DISCUSSION Early diagnosis and early treatment are effective measures to reduce the disability induced by the osteoarticular TB. In the early stage, its clinical manifestations are not obvious, and its imaging features are not always typical. While the pathology examination requires a preoperative or intraoperative biopsy to obtain pathological tissue, there are frequently only low amounts of bacteria present, there may be nonspecific inflammatory reaction, and there may be low sensitivities of acid-fast 1868 Clin. Lab. 11/2014

ELISPOT FOR OSTEOARTICULAR TUBERCULOSIS staining smears and mycobacterial cultures. Bacteriological confirmation of osteoarticular TB is therefore difficult. In this study, there only 30.2% (29/90) patients were smear-positive and/or culture-positive. The early, rapid diagnosis of the osteoarticular TB is a significant problem in clinical practice. Cell-mediated immune responses play a key role in host defense against M. tuberculosis infection [16]; hence, diagnostic methods based on cellular immunology can have utility in the diagnosis of extrapulmonary TB [10-12]. At present, the commercial T-SPOT-TB and FS- SPOT kits aim to provide auxiliary diagnosis of TB because the M. tuberculosis-specific IFN-γ release is not influenced by prior BCG vaccination, and there is low cross-reactivity to non-tb mycobacteria [5-7]. There have been many reports about the value of ELISPOT techniques in the diagnosis of latent TB infection and pulmonary TB [8,17], but few studies focused on extrapulmonary TB [10-12] and only two papers concerned osteoarticular TB [12,18]. Therefore, the actual clinical performance of the ELISPOT technique for the diagnosis of osteoarticular TB needed further evaluation. In this study, we compared the results of two kits in detecting active osteoarticular TB. Two of 43 subjects had results that differed between the T-SPOT TB and FS- SPOT assays (Table 2). The implied differences in immune responses to peptide antigens and the fusion protein might result from differences in structure between peptides and the fusion protein, leading to the presentation of different epitopes. Nevertheless, the kit sensitivity and specificity did not differ significantly. The FS- SPOT assay is much cheaper than the T-SPOT TB assay. Therefore, we evaluated the sensitivity and specificity of the FS-SPOT assay using confirmed cases of TB and controls. PHA is a nonspecific stimulus to activate T cells to secrete cytokines. The SFC counts in PHA wells did not significantly differ between the TB group and the control group, which suggests that the patients with osteoarticular TB had no significant changes in general cellular immunity. The SFCs (77.9 ± 130.6) and positivity rate (55.6%) of the FS-SPOT assay in the TB group were significantly higher than in the control group (10.7 ± 14.3, 16.7%), consistent with specific IFN-γ release from effector T-cells facilitating detection of current or more active TB infection [19-21]. Jafari C et al. [8] found that although there were no significant differences in lymphocyte subpopulations (total CD4+ or CD8+ T-cells) between patients with pulmonary TB and control patients with alternative pulmonary pathologies, the patients with pulmonary TB had more ESAT6- and CFP10-specific lymphocytes. Among the 54 control non-tb patients, 9 had positive ELISPOT results (range 21-57 SFC). We hypothesize that these subjects may have actually had TB infection. The specificity (83.3%) in the 54 controls was similar to pulmonary TB (generally 80-90%); however, the sensitivity in 90 subjects with osteoarticular TB was lower than the sensitivity in those with pulmonary TB (generally 70-90%) [9]. This may be because osteoarticular TB is a localized extrapulmonary disease, where there are fewer M. tuberculosis-specific lymphocytes in lesion sites, and this may elicit a lower immune response than observed in patients with pulmonary TB. Although the ELISPOT assay cannot distinguish active TB from latent TB infection, the high prevalence of latent TB infection in China may result in false positive ELISPOT results in the diagnosis of active TB. The higher numbers of SFCs produced by the more TB-specific effector T-cells suggest a higher risk of active TB or recent or active TB infection [19-21]. Our results imply that a positive ELI- SPOT result may help to diagnose active osteoarticular TB, but a negative ELISPOT result cannot exclude the diagnosis of active osteoarticular TB. Hence, the ELI- SPOT assay has potential utility in the diagnosis of osteoarticular TB. But confirmative diagnosis is still needed to link with the clinical manifestation. Compared with the results of Cho et al. [12] and Lai CC et al. [18] who used the T-SPOT-TB to diagnose active osteoarticular TB, the sensitivity of our FS-SPOT assay was lower (55.6% vs. 100% and 86.7%), but its specificity was higher (83.3% vs. 58% and 61.9%). 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