| Background and objectiveRD2region (Region of Deletion, RD) of M.tb genome encodes both CFP21andMPT64antigens, which are only present in the genome of M.tb and several substrainsof BCG distributted worldwide. RD1region of M.tb genome encodes both CFP10andESAT-6antigens, which express in the early culture filtrate of M.tb but not express inBCG and most of NTM. HspX is a protein over-expressed during the phase ofdormancy or latent M.tb infection (LTBI). Previously, we constructed and expressedrHspX, rCM (the fusion protein of CFP21and MPT64antigens) and rEC (the fuionprotein of CFP10and ESAT-6antigens) proteins using E.coli expression system,respectively. In this study, whole-blood IFN-γ releasing assay methods based on thesethree proteins were established. Their diagnostic value for M.tb infection wereevaluated.Methods1. Purifition，quantification and identification of rCM, rEC and rHspX proteins;2. Screening and identifying three kinds of population including health, LTBI andSPTB, based on questionare, physical examination, chest X-ray, TST, sputumsmear and acid-fast staining, and clinical dignosis; 3. Each sample was detected by rCM-WBIA, rEC-WBIA and rHspX-WBIA,respectively. The diagnosis value for M.tb based on these methods were analyzedwith spss17.0software.Results1We successfully expressd and purified rCM, rEC and rHspX proteins. Afterfreeze-dried, proteins were dissolved in1mL1×PBS. The proteins were filtered forsterilization and kept at-20oC until futher use. The size and purification of proteinswere identified by SDS-PAGE and the results were confirmed as expected.2All participants were recruited from Tongji Medical College, Xinxiang and JiningInstitution of Prevention and Control of TB.120healthy individuals were classifiedaccording to the healthy standards such as normal physical examination, normal chestX-ray and TST negative.92LTBI individuals were defined according to the healthystandards such as normal physical examination, normal chest X-ray, TST positive andsputum smear negative.43individuals were identified as SPTB with obvious clinicalsigns, abnormal chest X-ray, TST positive and sputum smear positive.3IFN-γ levels (Mean±sd) in the whole blood of Health, LTBI and SPTB afterstimulated by rCM were117.3±116.8pg/mL，790.7±876.5pg/mL and728.5±672.0pg/mL, respectively; there are statistically significant between SPTB andHealth (p﹤0.001) as well as between LTBI and Health (p＜0.001). IFN-γ levels(Mean±sd) in the whole blood of Health, LTBI and SPTB after stimulated by rECwere119.3±105.0pg/mL，961.3±1112.8pg/mL and1050.9±917.6pg/mL, respectively;there are statistically significant between SPTB and Health (p＜0.001) as well asbetween LTBI and Health (p＜0.001). IFN-γ levels (Mean±sd) in the whole blood ofHealth, LTBI and SPTB after stimulated by rHspX were25.8±76.7pg/mL，86.5±251.7pg/mL and130.2±208.5pg/mL, respectively; there are statisticallysignificant between SPTB and Health(p＜0.001) as well as between LTBI and Health(p＜0.001). IFN-γ levels (Mean±sd) in the whole blood of Health, LTBI and SPTB after stimulated by PHA were1274.1±1665.0pg/mL，2604.4±1805.7pg/mL and2456.2±2985.8pg/mL, respectively; there is statistically significant only betweenSPTB and Health (p＜0.05).4The sensitivity and specificity of rCM-WBIA and rEC-WBIA to diagnose M.tbinfections in total group were64.4%and100%,74.8%and98.3%, respectively. Theirdiagnosis standards were based on AUCs0.855and0.877with cut-off values393.4pg/ml and352.2pg/ml, respectively. In order to raise the sensitiveity ofdiagnosis, rCM-WBIA and rEC-WBIA in series and in parallel to diagnose M.tbinfections in total group were used with sensitivity51.1%and87.4%, specificity100%and98.3%, respectively.rCM-WBIA and rEC-WBIA were used to diagnose SPTB from M.tb infections,with sensitivity62.8%'86.0%, and specificity35.9%and30.4%, respectively. Inorder to raise the sensitivity and specificity, rCM-WBIA and rEC-WBIA in series andin parallel were used to diagnose SPTB from M.tb infections with sensitivity60.5%and88.4%, and specificity53.3%and13.0%, respectively.5The consistency between rCM-WBIA and rEC-WBIA to diagnose M.tb infectionsfrom total population is general with coincidence rate63.7%and κ=0.16. Theconsistency between rCM-WBIA and rEC-WBIA to diagnose SPTB from M.tbinfections is extremely low with coincidence rate72.1%and κ=0.316.6Cut-off value of rHspX-WBIA to diagnose LTBI from M.tb infections was54.2pg/ml with a sensitivity of23.8%and a specificity of52.6%.Conclusions1In order to obtain the best sentivity and specificity of diagnosing M.tb infectionsfrom total population, rCM-WBIA and rEC-WBIA may be used in parallel;2In order to obtain the best sentivity and specificity of diagnosing SPTB from M.tbinfections, rCM-WBIA and rEC-WBIA may be used in series;3rHspX-WBIA has no diagnostic value in diagnosing LTBI from M.tb infections. |
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