| [Objective]To assess the clinical diagnostic validity, reliability, and diagnostic value of enzyme-linked immunospot assay (ELISPOT) based on ESAT-6 and CFP-10 peptide and tuberculin skin test (TST) in the rapid diagnosis of active tuberculosis(TB) in a developing country with high prevalence. To approcach feasibility of ELISPOT in rapid diagnosis of active TB patients with negative sputumn culture of MTB and negative acid fast bacilli(AFB) smear results, children TB patients and extra-pulmonary patients.[Methods]A prospective study was conducted from March to August 2011 at the Nanning Fourth Hospital(NFH), Guangxi province, an area with high prevalence of TB.140 patients with bacteriologically or biopsy specimen confirmed TB(90 cases were culture positive and 36 were culture negative), and 98 patients with non-TB illness attending the NFH during the study period were tested by ELISPOT on peripheral blood mononuclear cells(PBMCs) and TST (Tuberculin skin test) simultaneously. Sensitivity, specificity, positive predictive value(PPV), negative predictive value(NPV), Positive Likelihood Ratio, and Negative Likelihood Ratio were used to assess the diagnostic validity and diagnostic value of ELISPOT and TST based on final clinical and micrological diagnosis. Concordance between test results were used to assess the reliability of ELISPOT and TST. Non conditional univariate and multivariate logistic regression was used to analysis effective factors for sensitivity and specificity of ELISPOT and TST.[Results](1)SFCs distribution of ELISPOT:①The numbers of ESAT-6 SFCs in PBMC were significantly different between patients with active TB and controls (median number of SFCs was 74 and 11 out of 250,000 PBMCs, respectively, P＜0.001). The numbers of CFP-10 SFCs in PBMC were significantly different between patients with active TB and controls (median number of SFCs was 100 and 9 out of 250,000 PBMCs, respectively, P＜0.001).②The numbers of ESAT-6 SFCs in PBMC were significantly different between patients with positive and negative AFB active TB and controls (median number of SFCs was 92,74 and 11 out of 250,000 PBMCs, respectively, P<0.001). The numbers of CFP-10 SFCs in PBMC were significantly different between patients with positive and negative AFB active TB and controls (median number of SFCs was 150,100 and 9 out of 250,000 PBMCs, respectively, P＜0.001).③FCs produced by ESAT-6 and CFP-10 were associated with age, Body mass Index and anti-TB treatment significantly(P＜0.05), but not with sex, BCG vaccination, previous history of TB, household contact with TB patients, and immunosuppress(P＞0.05).(2)Distribution of TST induration:①Size of TST induration were significantly different between patients with active TB and controls (median number of induration was 7.00mm and 4.50mm respectively, P=0.007). The size of TST induration for active TB patients with AFB positive, active TB patients with AFB negative and control was 8.0mm(0～14.0mm),7.0mm(0～27.0mm) and 4.5mm(000～15.0mm), respectively. Distribution of TST induration between active TB patients with AFB smear positive, active TB patients with AFB smear negative and control were significantly different(x2=7.912, P=0.023). However, distribution of TST induration between active TB patients with AFB positive and active TB patients with AFB smear negative were not significantly different(P>0.05). Distribution of TST induration between active TB patients with AFB smear positive and control, and between active TB patients with AFB smear negative and control were were significantly different(P＜0.05).②In 14 TB patients and 16 controls who were children, size of TST induration were significantly different between patients with active TB and controls (median number of induration was 10.8mm and 3.5mm, respectively, P=0.008).(3)Dignostic validity and value of ELISPOT and TST:①The sensitivites, specificity, Youden's index, PPV and NPV of ELISPOT were 89.3%(95%CI: 83.1-93.4),82.7%(95%CI:74.0-88.9),0.72,88.0%(95%CI:81.7-92.4) and 84.4%(95%CI:75.8-90.3), respectively.②The sensitivites, specificity, Youden's index, PPV and NPV of TST(≥5mm) were 68.3%(95%CI:59.6-75.9), 51.0%(95%CI:41.3-60.7),0.193,63.6%(95%CI:55.2-71.4), and 56.2%(95%CI: 45.8-66.0, respectively; The sensitivites, specificity, Youden's index, PPV and NPV of TST(≥10mm) were 35.8%(95%CI:27.9-44.6),91.8%(95%CI: 84.7-95.8),0.276,84.6%(95%CI:72.5-91.2) and 53.3%(95%CI:45.7-60.6), respectively.③The sensitivites, specificity, PPV and NPV of ELISPOT were higher than that of TST(≥5mm) with significant differences(P＜0.001). Sensitivity and NPV of ELISPOT were higher than that of TST(≥10mm) (P＜0.001), and no significant difference were observed in specificity and PPV between ELISPOT and TST(≥10mm) (P＞0.05).③In patients with a positive ELISPOT, TST(≥5mm) and TST (≥10mm) results, the diagnostic OR for active TB were 39.71(95%CI:18.78-83.93),2.24(95%CI:1.30-3.88) and 6.27(95%CI:2.78-14.11), repectively。 (4)Sensitivity of ELISPOT and TST in TB patients with positive and negative culture results:①The sensitivity in TB patients with positive results of acid fast stain or sputum bacteriological culture for ELISPOT and TST were 97.8%(95%CI,88.7-99.6) and 90.0%(95% CI,74.4-96.5), respectively. Sensitivity for ELISPOT was higher than that of TST with significant differences(P=0.33).②ensitivity for ELISPOT and TST in TB patients with negative results of acid fast stain or sputum bacteriological culture were 86.7% (95% CI,78.1 to 92.2) and 59.6%(95% CI,49.2-69.2), respectively. The sensitivity of ELISPOT was higher than that of TST with significant difference (P＜0.001)in cases who were negative in results of acid fast stain or sputum bacteriological culture.(5)Sensitivity of ELISPOT and TST in extra-pulmonary TB patients: 24/28(85.7%) and 17/24(70.8%) cases of extra-pulmonary TB were positive in ELISPOT and TST respectively. Similarly,4/4 (100%) and 2/4 (50.0%) cases of cerbral tuberculosis were positive in ELISPOT and TST respectively.(6)Sensitivity of ELISPOT and TST in children TB patients:①Among 14 children cases and 16 children controls, the sensitivity for ELISPOT and TST was 85.7%(95%CI:60.1-95.0) and 71.4%(95%C7:45.4-88.3), respectively.②Specificity for ELISPOT and TST was 93.8%(95%CI:71.7-98.9)and 56.3%(95%CI:33.2-76.9), respectively.③Sensitivity of ELISPOT was higher than that of TST, although no significant difference was observed(P＞0.05). Specificity of ELISPOT was higher than that of TST with with significantly different(P＜0.05).(7)Concordance between ELISPOT and TST:①Very poor agreement was observed between ELISPOT and TST in all participants(K=0.24). In patients with active TB, agreement between ELISPOT and TST was poor (κ=0.071), similarly, agreement between ELISPOT and TST was poor (κ=0.15). Disconcordance test results were most often ELISPOT positive yet TST negative in patients with active TB (33 cases), and ELISPOT negative yet TST positive in patients with non TB illness (36 controls).②or agreement was observed between ELISPOT and TST in children participants(K=0.35). In children patients with active TB, agreement between ELISPOT and TST was poor (κ=0.18), similarly, agreement between ELISPOT and TST was poor (κ=0.16) in control children. Disconcordance test results were most often ELISPOT positive yet TST negative in patients with active TB (3 cases), and ELISPOT negative yet TST positive in patients with non TB illness (7 controls).(8)Influence factors of sensitivity of ELISPOT and TST:①Non conditional univariate and multivariate logistic regression analysis showed that sensitivity of ELISPOT was not affected by sex, age, Body Mass Index, BCG vaccination, previous TB history, household contact with patients known to have TB, and immunosupress(P＞0.05).②Multivariate logistic regression analysis showed that Body Mass Index less than 18.5(OR=0.22,95% CI:0.08～0.59), BCG vaccination(OR=4.36,95% CI:1.25～15.26), household contact with patients known to have TB(OR=6.61,95% CI:1.69～25.93), and immunosupress(OR=0.076,95% CI:0.016～0.37) were independent affected factors for sensitivity of TST(P＜0.05). It was suggested that malnutrition and immunosupress tend to decrease sensitivity of TST, however, BCG vaccination and household contact with patients known to have TB tend to increase sensitivity of TST.(9)Influence factors of specificity of ELISPOT and TST:①evious TB history(OR=7.89,95% CI:1.15～54.02) and household contact with patients known to have TB(OR=12.71,95% CI:2.56～63.22) were independent affected factors for false positive rate of ELISPOT according to multivariate logistic regression analysis(P＜0.05). It was suggested that previous TB history and household contact with patients known to have TB tend to decrease specificity of ELISPOT.②Body Mass Index less than 18.5(OR).18,95% CI:0.04～0.82), BCG vaccination(OR=12.73,95% C7:3.49～46.50), previous histroy of TB (OR=83.85,95% CI:6.77～1038.38)and household contact with patients known to have TB(OR=21.69,95%CI:2.24～210.29) were independent affected factors for false positive rate of ELISPOT according to multivariate logistic regression analysis(P＜0.05). It was suggested that malnutrition tend to increase specificity of TST, however, BCG vaccination, previous histroy of TB and household contact with patients known to have TB tend to decrease specificity of TST.[Conclusion](1)High sensitivity, specificity, PPV, NPV and diagnostic OR of ELISPOT based on ESAT-6 and CFP-10 peptide are observed in high prevalance background. Diagnositic validity and value of ELISPOT is higher than that of tuberculin skin test. The diagnositic value of TST is limitted.(2)The sensitivity of ELISPOT is less affected by those factors which included age, malnutrition, history of BCG vaccination, previous history of TB, household contact of TB, or immunolsuppress status, and the specificity is not decreased by BCG vaccination.The detection results of ELISPOT appears to be more reliable than TST.(3)ELISPOT have obviously drawback:it can't distinguish active TB patients and patients with LTBI obviously, its'specificity is decline by the household contact of TB and previous history of TB, false negative results can not be excluded due to limitation of human leucocyte antigen, and T-SPOT-TB kit is too costly to diagnosis active TB and screen TB in a large scale in area with low income. It is not neccessarly to perform ELISPOT detection for TB patients with TST induration larger than 10 mm and positive results of AFB smear. (4)ELISPOT is a reliable supplementary means for rapid diagnosis of active TB with negative AFB smear results, extra-pulmonary and children cases in country with high prevalence of TB, which would lead to rapid diagnosis and treatment and reduction of medical cost. |
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