Clinical Evaluation Of An Enzyme-linked Immunospot Assay For The Diagnosis Of Spinal Tuberculosis

BackgroudTuberculosis (TB) is one of the most important infectious disease in the world. TB is a disease that can result in very high mortality, especially in developing countries. The incidence of TB has become increasingly serious because of several reasons: the biological characteristics of tuberculosis, the floating of the population and an epidemic of Multi-drug resistant and the AIDS. What’s more, delayed diagnosis and treatment can increase the risk for dissemination of M. tuberculosis and decrease survival for some subgroups of TB patients. TB has been an important public health problem, which will become a priority of disease control. According to the World Health Organization (WHO) surveillance study, there were about8.8million new TB cases around the world in2010. According to recent statistics, China is one of the leading countries with a high incidence of TB. Due to of people’s living conditions, social and economic development, the differences of the health habits, the morbidity of tuberculosis is high. TB prevention and control work in Guangdong province has made considerable progress and achievements, but also very difficult task. The number of the TB patients always stands in the top list of A or B class infectious diseases in the whole province. According to recent statistics,46thousand people were infected by TB in Guangdong province2010, with about2000reported multidrug resistant. The first group is farmer, occupied70%of TB patients.According to traditional chinese medicine, osteoarticular TB was called "Gulao".Osteoarticular TB usually presents in a slowly indolent manner with nonspecific clinical presentations, so the diagnosis of tuberculous arthritis is often difficult and delayed, and remains a great challenge for clinicians. Osteoarticular TB accounts for3-5%of all forms of TB and approximately35-50%of cases with extrapulmonary TB. Spinal TB is found in1%to3%of all TB cases and in50%to60%cases of osteoarticular TB. The typical clinical presentations of spinal TB includes:lower back stiffness, night sweats, anorexia, hot flushes and weakness. At physical examination of the patients, kyphosis and neurological deficit was also seen in the late change of the spinal TB. Actually, spinal TB are a great challenge to physicians because of the nonspecific and wide spectrum of clinical presentations that result in delay of diagnosis and the risk of significant potential morbidity and mortality due to several complications. Early diagnosis and treatment is the key to avoiding this long-term disability.As we know, if the doctor can make an early diagnosis and treatment for spinal TB, they could cure the TB patients without surgery and help the country save a lot of medical costs. According to recent statistics, the morbidity of atypical spinal TB was approximately2.1-12%of cases with spinal TB. The misdiagnosis rate of this disease was32.2%. Definite diagnosis is generally considered to require microbiological confirmation; however, the bacteriological gold standard result, including solid media-based techniques using Lowenstein-Jensen slants and Middlebrook7H10/7H11agar and liquid media-based methods, such as with the BACTEC960MGIT system, usually takes several weeks to be available. These may lead to delay in diagnosis and initiation of treatment. Therefore, we urgently need a faster, more sensitive, and specific test for the diagnosis of spinal TB in clinical practice.Recently, an enzyme-linked immunospot (ELISPOT) assay using pools of early secretory antigenic target6(ESAT-6) and culture filtrate protein10(CFP-10) peptides was manufactured and commercialized. It is based on the detection of interferon-gamma (IFN-y) released by activated T lymphocytes. The ELISPOT assay has been demonstrated to be useful for the diagnosis of pulmonary TB. However, few studies have investigated the sensitivity and specificity of this assay for diagnosing atypical spinal TB.Objectives1.To assess performance of a laboratory-developed recombinant CFP-10/ESAT-6fusion protein (rCFP-10/ESAT-6)-based ELISPOT assay in diagnosis of spinal tuberculosis.2.To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the diagnosis of spinal tuberculosis.3.To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.Methods1.To assess performance of a laboratory-developed recombinant CFP-10/ESAT-6fusion protein (rCFP-10/ESAT-6)-based ELISPOT assay in diagnosis of spinal tuberculosis.Suspected spinal TB patients were prospectively recruited recruited in two hospitals (Nanfang Hospital, Southern Medical University; Guangzhou thoracic hospital) from May2011to July2012. Data on clinical characteristics of the patients and conventional laboratory results were collected. The specific data were collected on age, sex, underlying diseases, radiographic image examination, lymphocyte count, pathology, microbiological results, and follow-up observations.4-5ml of venous blood samples from the volunteers were taken in heparinized glass tubes, and peripheral blood mononuclear cells (PBMC) were isolated and quantified the second day after the patients went into hospital. This study aimed to assess performance of a laboratory-developed rCFP-10/ESAT-6-ELISPOT assay in diagnosis of spinal TB.2. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT.TB) for the diagnosis of spinal TB.Suspected spinal TB patients were prospectively recruited recruited in two hospitals (Nanfang Hospital, Southern Medical University; Guangzhou thoracic hospital) from May2011to August2012. Data on clinical characteristics of the patients and conventional laboratory results were collected. The specific data were collected on age, sex, underlying diseases, radiographic image examination, lymphocyte count, pathology, microbiological results, and follow-up observations. In addition to conventional tests for TB, we used ELISPOT assays to measure the IFN-y response to ESAT-6, CFP-10, rCFP-10/ESAT-6, Rv2041c, Rv1419and Rv0057-1352in T-cells in samples of peripheral blood mononuclear cells (PBMC).3. To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.From March2011to September2012, a total of29patients with atypical spinal TB were enrolled. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for T-SPOT.TB assay. The aim of this study was to assess the diagnostic value of T-SPOT.TB assay in clinically atypical spinal TB.4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.A sequential subgroup of spinal TB patients was enrolled for evaluation of responses of T-SPOT.TB assay on admission, two days before and after surgery. All these spinal TB patients underwent focal cleaning or one-stage anterior debridement, bone graft plus anterior or posterior instrumentation.Results1. The performance of a laboratory-developed rCFP-10/ESAT-6-based ELISPOT assay in diagnosis of spinal TB.①Patient characteristicsA total of102patients with suspected spinal TB were prospectively recruited during the study period.11patients were excluded from the study, among which5did not complete the rCFP-10/ESAT-6-ELISPOT assay and6had no final diagnosis. The remaining91patients were ultimately included for ELISPOT analyses. The spinal TB group contained52cases. The non-TB disease group contained39subjects.47patients with spinal TB had available biopsy or surgical specimens for histopathological examination.42patients with spinal TB had AFB staining and40patients had cultures for M. tuberculosis, respectively.②Spread of SFCs above the negative control/2.5×105peripheral blood mononuclear cells (PBMCs) in response to recombinant CFP-10/ESAT-6fusion protein in spinal TB patients and non-spinal TB patients. The mean number of SFC in spinal TB patients was significantly higher than that of non-spinal TB patients.③Clinical diagnostic value of the ELISPOT assay for spinal TBAmong the spinal TB patients and non-TB disease patients, the overall sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the PPD skin test were61.5%,46.2%,60.4%and47.4%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the Antibody detection were55.8%,61.5%,65.9%and51.1%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the ELISPOT assay were82.7%,87.2%,89.6%and79.1%, respectively. The overall sensitivity of ELISPOT is higher than that of PPD skin test and antibody detection test (P<0.05).④Comparsion of spinal tuberculosis patients detected by PPD skin test, TB antiboby test, Histopathology diagnosis and ELISPOTWhen we assessed the concordance between the histopathology detection and the ELISPOT assay using the κ coefficient, we found moderate agreement between the two tests.⑤Clinical characteristics associated with the ELISPOT results in spinal TB patientsIn this study, we found that age and emaciation were associated with the number of SFC or positive rate of ELISPOT in spinal TB patients.2. Clinical value of an enzyme-linked immunospot assay (T-SPOT.TB) for the diagnosis of spinal tuberculosis.①Patient characteristicsA total of70patients with suspected spinal TB were prospectively recruited during the study period.5patients were excluded from the study.The remaining65patients were ultimately included for T-SPOT.TB analyses. The spinal TB group contained34cases. The non-TB disease group contained31subjects.②Clinical diagnostic value of the T-SPOT.TB assay for spinal TBAmong the spinal TB patients and non-TB disease patients, the overall sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the PPD skin test were61.8%,58.1%,61.8%and58.1%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the Antibody detection were58.8%,70.9%,68.9%,61.1%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the T-SPOT.TB assay were85.3%,77.4%,80.6%and82.8%, respectively. The overall sensitivity of T-SPOT.TB is higher than that of PPD skin test and antibody detection test (P<0.05).③Comparsion of spinal tuberculosis patients detected by PPD skin test, TB antiboby test, Histopathology diagnosis and T-SPOT.TBWhen we assessed the concordance between the histopathology detection and the T-SPOT.TB assay using the κ coefficient, we found moderate agreement between the two tests.④The determination of test condition about Rv2041c-ELISPOT, Rv1419-ELI SPOT and Rv0057-1352-ELISPOTTo change the condition of experiment, and find that the best numbers of cell is2×105, the best incubated time is20hour and the best density of proteinum is20μg/ml.④Clinical diagnostic value of different antige response to the ELISPOT assay for spinal TB The sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the CFP-10/ESAT-6-ELISPOT assay were82.4%,83.9%,84.8%and71.9%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the T-SPOT.TB were85.3%,77.4%,74.4%and80.8%.When we assessed the concordance between these two tests using the κ coefficient, we found moderate agreement between the two tests.3. To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.①Clinical presentationBack pain was the most common clinical complaint (24patients,82.8%).②Imaging study findingsThrough the imaging study, worm-eaten destruction of vertebral endplate was seen in5patients. Destruction of centricity of the single vertebral body or concentric collapse of single vertebral body were seen in5patients.15patients presented contiguous or skipped vertebral body destruction. Tuberculous abscess with no identifiable osseous lesion were visible by CT and MRI in4patients③Clinical diagnostic value of the T-SPOT.TB assay for atypical spinal TBWe found that37.5%,78.3%of patients were positive for AFB smear and cultures for M. tuberculosis, respectively. The overall sensitivity, specificity, positive predictive value, and negative predictive value for atypical spinal TB diagnosis by the PPD skin test were58.6%,59.4%,56.7%and61.3%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for atypical spinal TB diagnosis by the T-SPOT.TB assay were82.8%,81.3%,80.0%and83.9%, respectively. The overall sensitivity of the T-SPOT.TB assay was higher than that of PPD skin test (P<0.05). 4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.①Patient characteristicsAdditional responses were evaluated in a consecutive subset of12spinal TB patients on two days before and after surgery.3spinal TB patients only underwent focal cleaning. The remaining9spinal TB patients underwent one-stage anterior debridement, bone graft plus anterior or posterior instrumentation.②Responses to ESAT-6, CFP-10before and after SurgeryThe median ESAT-6(30spots per250,000PBMCs) and CFP-10(44spots per250,000PBMCs) response were slightly lower after surgery than those before surgery (ESAT-6:36spots per250,000PBMCs; CFP-10:57spots per250,000PBMCs), but those failed to reach statistical significance (P>0.05).Conclusions1. A laboratory-developed rCFP-10/ESAT-6-based ELISPOT assay is a useful adjunct to current tests for diagnosis of spinal TB.2. The T-SPOT.TB assay is a promising tool for the clinical evaluation of spinal TB and merits additional assessment in a diagnostic trial.3. The recombinant Rv1419protein of mycobacterium tuberculosis has relatively high value in the immunological diagnosis of tuberculosis.