Abstract:

Objective To evaluate the application of recombinant Mycobacterium tuberculosis 11 kDa protein (relative molecular mass 11000) in screening for latent tuberculosis infection and identification of BCG vaccination. Methods Recruited volunteers in Changping District, Huairou District, and Daxing District of Beijing from July 2014 to March 2016, signed informed consent, and recruited 3001 healthy volunteers after qualified medical examination, using the Mantoux method to carry out the two-arm skin test of recombinant 11 kDa protein and TB-PPD, and intradermal injection of different forearms 0.1ml of recombinant 11 kDa protein (10μg/ml) and 0.1ml of tuberculin pure protein derivative (TB-PPD) (50 U/ml), and observed the skin reaction 72 hours after injection. Before the skin test, venous blood of volunteers was collected for in vitro interferon gamma release assay (IGRA) detection. The “consistency rate (%)” and “the first-order agreement coefficient (AC₁)” were used to compare the results of the recombinant 11 kDa protein skin test, TB-PPD test and IGRA results. Three thousand and one volunteers were tested by 3 methods, choosing 475 of subjects with negative results of all the three test, to sign an informed consent. According to the random number table double-blind principle (in the ratio of 2∶1), they were vaccinated with BCG (318 subjects) and placebo (147 subjects). After 3 months of inoculation, they were again subjected to parallel recombinant 11 kDa protein skin test, TB-PPD skin test and IGRA. Results In the screening of latent tuberculosis infection in healthy volunteers, the difference between 11 kDa (32.2%, 966/3001) and IGRA (34.4%, 1033/3001) results was statistically significant (χ²=22.787, P<0.001); the consistency rate of IGRA and 11 kDa was 93.4% (2804/3001), and the Gwet’s AC₁ value was 0.882 (95%CI=0.866-0.898), the two had good consistency. Compared with the result of 11 kDa (32.2%, 966/3001) and TB-PPD (47.7%, 1430/3001), the positive rate of TB-PPD was significantly higher than that of 11 kDa. The difference between the result of TB-PPD and 11 kDa was very significant (χ²=182.146, P<0.001). The consistency rate of TB-PPD and 11 kDa was 60.6% (1819/3001), and its Gwet’s AC₁ value was 0.243 (95%CI=0.207-0.279). The consistency between the two was poor. In the BCG vaccination identification study, there was no statistically significant difference between the 11 kDa (1.3%, 4/318) and IGRA (3.1%, 10/318) results in the vaccination group (χ²=2.571, P=0.109). The agreement rate was 95.6% (304/318), and the Gwet’s AC₁ value was 0.954 (95%CI=0.929-0.979), the two had good consistency. The difference between 11 kDa (1.3%, 4/318) and TB-PPD (56.3%, 179/318) results was very significant (χ²=167.350, P<0.001). The consistency rate of TB-PPD and 11 kDa was 42.5% (135/318), and its Gwet’s AC₁ value was 0.025 (95%CI=-0.106-0.155). The consistency between the two was poor. In the placebo group, there was no significant difference in the positive rate of 11 kDa (4.1%, 6/147) and IGRA (1.4%, 2/147) (χ²=2.667, P=0.109); the consistency rate was 95.9% (141/147), and the AC₁ value was 0.957 (95%CI=0.921-0.993), which had good consistency. The positive rate of TB-PPD (17.7%, 26/147) was higher than that of 11 kDa (4.1%, 6/147), the difference was statistically significant (χ²=15.385, P<0.001), the consistency rate was 82.3% (121/147), and the AC₁ value was 0.781 (95%CI=0.690-0.871). Conclusion Recombinant Mycobacterium tuberculosis 11 kDa protein has a good consistency with IGRA in screening for latent tuberculosis infection and BCG vaccination, suitable for large-scale screening, and can be used as one of the candidate diagnostic reagents for the screening of latent infected persons and the differentiation of BCG vaccination.

Key words: Recombinant proteins, Latent tuberculosis, Reagent kits, diagnostic, Skin tests, BCG vaccine, Diagnosis, differential