Investigation Of ELISPOT And IP-10Detection In The Diagnosis Of Tuberculosis

Mycobacterium tuberculosis (Mtb) infection causes tuberculosis (TB) in lungs and many other organs. The incident of TB increases due to human immunodeficiency virus (HIV) infection, movement of population, drug-resistant bacteria, and use of immunosuppressive agent. There are an estimated8million new cases of TB globally, and3million people died of TB each year. It is the second leading cause of death from an infectious disease worldwide. There are an estimated5million TB patients in China, and0.13million deaths every year. The golden criterion of TB diagnosis is detection the AFB in the sputum and/or pathological samples. Sputum-positive tuberculosis patients are the main source of infection of tuberculosis, however, about74%of active TB patients are sputum-negative. TB is often confused with lung common diseases (such as bacterial pneumonia and lung cancer) in case that AFB was not found in the samples. Tuberculous meningitis is a severe form of tuberculosis, and its prognosis is significantly different depending upon the time of diagnosis and treatment. Traditional techniques such as bacteriological or pathological examination has low diagnostic value for tuberculous meningitis. Misdiagnosis or delayed diagnosis not only caused poor prognosis, also increasing group pressure on tuberculosis patients.Memory T cells (Th1) are generated in vivo after infected With Mycobacterium tuberculosis (Mtb). IFN-y is produced when stimulated with Mtb-specific antigens. ELISPOT is developed for diagnosing both latent TB infection and TB disease in people by detecting IFN-y producing cells. ESAT-6and CFP-10are Mtb complex-specific antigens, which avoid interference of BCG or NTM. Many studies demonstrate high sensitivity and specificity of the ELISPOT in the diagnosis of pulmonary tuberculosis. Previous studies show that tuberculosis antigen-specific T cells were gathered in lesions of infection, and there were more ESAT-6and CFP-10-specific cells in the infected body fluids. We speculated that detection of IFN-y secreting cells in cerebrospinal fluid by ELISPOT might have a higher sensitivity and specificity in diagnosis of tuberculous meningitis. In this study, IFN-y secreting cells in cerebrospinal fluid and blood were detected by ELISPOT. The relationship between the ELISPOT results and the disease progress were analyzed. The sensitivity and specificity were compared among ELISPOT, acid-fast staining, mycobacterial culture, quantitative PCR, tuberculosis antibody detection. The results showed that the CSF-ELISPOT had higher sensitivity and specificity, and it was a useful assay for tuberculous meningitis diagnosis. The study provides the basis for early diagnosis and treatment of tuberculous meningitis.IP-10is a chemokine belonging to the CXC subfamily, and it is induced by IFN-y or lipopolysaccharide. IP-10plays an important role in the activation of lymphocytes at sites of inflammation. The expression of IP-10is closely related with the production of IFN-y. Since IP-10is the chemokine induced by IFN-y, it can expand the signal of IFN-y. Studies had found that high concentration of IP-10was induced by PBMCs from patients with TB when stimulated by Mtb-specific peptides. Ruhwald et al reported that combined detection of IP-10and IFN-y could improve the sensitivity of Mtb infection. In this study, PBMCs were purified from tuberculsosis patients, healthy controls and patients suffering common lung diseases, and were stimulated with Mtb-specific antigens ESAT-6and CFP-10. The culture supernatant was collected after20hours, and the concentration of IP-10and IFN-γ were dectected by FlowCytomix. The results suggested the sensitivity and specificity for the diagnosis of tuberculosis can be significantly improved by combined detection of Mtb-specific IP-10and IFN-y, which could be used as an auxiliary examination for diagnosis of tuberculosis.ObjectiveTo investigate the value of cerebrospinal fluid ELISPOT in the diagnosis of tuberculous meningitis; To investigate the value of combined detection IP-10and IFN-y in the diagnosis of tuberculosis.Methods1. IFN-y secreted cells in CSF and blood were detected by ELISPOT.2. The concentration of Mtb-specific IP-10and IFN-y secreted by PBMCs from tuberculosis patients and patients suffering other common lung diseases was detected by FlowCytomix.3. The diagnostic value of cytokine release assay, AFB staining, mycobacterial culture, quantitative PCR and tuberculosis antibody test results were compared.Results1. The sensitivity and specificity of CSF-ELISPOT were67.3%and89.2%respectively, fit degree was73.3%, positive predictive value was94.3%, and had a higher diagnostic value.2. The sensitivity and specificity of blood-ELISPOT test were85.9%and41.9%respectively, fit degree was74.1%, positive predictive value was80.2%. The specificity of the blood-ELISPOT was lower than CSF-ELISPOT, which was associated with extra-brain tuberculosis.3. The sensitivity and specificity of combined detection of CSF and blood-ELISPOT were91.1%and96.4%respectively.4. Comparison of CSF-ELISPOT with tuberculosis antibody detection, quantitative PCR, AFB staining and mycobacterial culture indicated that CSF-ELISPOT was significantly more sensitive in the diagnosis of tuberculous meningitis.5. The concentration of IP-10and IFN-y in PBMCs culture supernatant was significantly higher in tuberculosis patients than in other lung diseases patients and healthy controls.6. The concentration of IP-10in PBMCs culture supernatant was positive associated with IFN-y in tuberculosis patients. The sensitivity and specificity of the diagnosis of tuberculosis could significantly improve by combined detection of Mtb-specific IP-10and IFN-γ.Conclusion1. CSF-ELISPOT had higher sensitivity and specificity and could be used as an auxiliary test for early diagnosis of tuberculous meningitis. Combined CSF-and blood-ELISPOT examinations could significantly improve the sensitivity for the diagnosis of tuberculous meningitis.2. Combined detection of Mtb-specific IP-10and IFN-y could significantly improve the diagnositic sensitivity and specificity for tuberculosis.