IFN-γ Immunospot Assay Of Bronchoalveolar Lavage Fluid For Rapid Diagnosis Of Active Pulmonary Tuberculosis

Objective: In order to improve the clinical diagnostic ability of active pulmonary tuberculosis patients using Mycobacterium tuberculosis antigen specific gamma interferon enzyme-linked immunospot assay of bronchoalveolar lavage fluid.Methods: 414 adults with suspected TB were enrolled in Shenzhen third people’s hospital from August 2017 to August 2018.The basic clinical information of the subjects was collected,including name,gender,age,contact information and medical history,orbidity,comorbidities,imaging diagnosis,bacteriological results,pathological test,samples with5 ml peripheral blood and 30 ml alveolar lavage fluid from all patients were collected at the same time,then bronchoalveolar lavage fluid mononuclear cell enzyme-linked immunospot assay（BALMC-Elispot）was test to evaluate diagnostic ability in patients of active pulmonary tuberculosis,meanwhile,peripheral blood mononuclear cell enzyme-linked immunospot assay（PBMC-Elispot）,BALF-acid fast bacilli（BALF-AFB）,BALF-M.tuberculosis culture（BALF-M.tb culture）and BALF-nucleic acid amplification（BALF-Gene Xpert assay）were used as controls;In order to further confirm that Mycobacterium tuberculosis antigen-specific T cells are more abundant in alveolar lavage fluid,29 peripheral blood specimens and 11 lavage fluid samples were randomly selected from all specimens,then mononuclear cells（PBMC/BALMC）isolated from which were surface stained with anti-CD4-PE-cy7 and anti-CD3-percp antibodies at 4℃for 30 minutes.After incubation at 4 C for 30 minutes,4%paraformaldehyde was added to fix for 30 minutes,then membrane disruptor was added to break the membrane.After centrifugation at10 000 rpm for 30 seconds,anti-IFN-γ APC-cy7 antibody was added to stain the IFN-γ,then the sample was read on a BDcanto II instrument and analyzed using Flow Jo software.Results: A new method for the enzyme-linked immunospot detection of bronchoalveolar lavage fluid was successfully constructed;Compared with the non-pulmonary tuberculosis patients group,the production of antigen-specific interferon gamma（IFN-γ）in the lavage fluid of the tuberculosis patients group was significantly increased,and the difference was statistically significant.The two specific antigens of M.tuberculosis-Early Secretory Antigen Target 6000 Protein（ESAT6）、Peptides of Early Secretory Antigen Target 6000 Protein and culture filtrate protein 10（ESAT6/CFP10 peptide）can stimulate T cells to produce an immune response,and the number of effector T cell is related to the type of specimen;The number of T lymphocytes from alveolar lavage fluid is much more than that of the paired peripheral blood in sputum culture negative or positive tuberculosis,which is consistent with the results of flow-staining.The number of antigen-specific T cells in the lavage fluid is related to bacterial amount,and the difference is statistically significant;Compared with the other routine four tests（including BALF-AFB,BALF-M.tb culture,BALF-Gene Xpert,PBMC-Elispot）,the sensitivity,specificity,positive predictive value,negative predictive value,positive likelihood ratio,negative likelihood ratio of BALMC-Elispot were 91.60%,81.50%,95.30%,70.20%,4.95,0.1,respectively,the sensitivity of which was higher than other tests and negative likelihood ratio was lower than that of other tests.The result of BALMC-Elispot is not affected by bronchial tuberculosis or diabetes,and can improve diagnostic ability of tuberculosis patients.Conclusion: Alveolar lavage fluid enzyme-linked immunospot assay can be used to diagnose active pulmonary tuberculosis and has good clinical diagnostic value due to it did not be affected by the diabetes or EBTB.