Abstract:

Objective: To evaluate the rationality of different result reading time points and positive criteria for the recombinant Mycobacterium tuberculosis fusion protein (ESAT6/CFP10, EC) skin test based on data from a phase Ⅳ clinical trial among community population, and analyze its diagnostic agreement with the interferon-gamma release assay T-SPOT.TB, to provide evidence for standardized reading and interpretation of the EC skin test. Methods: From April 17, 2023 to May 13, 2024, a prospective, multi-cohort phase Ⅳ clinical trial of the EC skin test targeting community residents aged ≥6 months was carried out by Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention at two study sites: Liuzhou Center for Disease Control and Prevention and Liuzhou Liujiang District Center for Disease Control and Prevention. According to study setting, participants were divided into two cohorts. Cohort 1 received T-SPOT.TB before skin testing, followed by intradermal injection of EC and tuberculin purified protein derivative (TB-PPD) into separate forearms of the same person. This cohort was used to evaluate the reading time and positive criteria of EC, as well as its diagnostic agreement with T-SPOT.TB. Cohort 2 received one dose of EC from the same batch on the same day, and was used to observe the 48-72 h reading results and safety of EC skin test in large-scale community population. Results: A total of 7498 subjects were enrolled according to inclusion and exclusion criteria, including 500 subjects in Cohort 1 and 6998 subjects in Cohort 2. Among the 498 evaluable participants in Cohort 1, the diagnostic agreement rates between EC skin test results and T-SPOT.TB results at 24 h, 48 h and 72 h (91.06% (Kappa=0.62), 94.39% (Kappa=0.80), and 96.05% (Kappa=0.86), respectively) showed a gradual upward trend as the reading time was prolonged. However, the diagnostic agreement rate within the 48-72 h reading window (95.84%, Kappa=0.86) was basically consistent with that obtained by single reading at 72 h. When the large of the mean diameter of erythema and induration was ≥5 mm (as the positive criterion), the diagnostic agreement rates between EC skin test and T-SPOT.TB (95.84% (Kappa=0.86) and 95.63% (Kappa=0.84), respectively) were superior to those of TB-PPD (54.26% (Kappa=0.20) and 65.70% (Kappa=0.29), respectively). In addition, the diagnostic agreement rates between EC skin test read at 48-72 h and T-SPOT.TB under skin reaction thresholds of 3-6 mm and ≥7 mm were both roughly consistent (95.84% (Kappa=0.86) and 95.63% (Kappa=0.85), respectively). Subgroup analysis demonstrated that among key populations including elderly adults aged ≥65 years, close contacts of bacteriologically confirmed pulmonary tuberculosis patients and patients with diabetes mellitus, the EC skin test exhibited high diagnostic agreement with T-SPOT.TB results in the 498 evaluable participants of Cohort 1, with diagnostic agreement rates ranging from 86.67% (Kappa=0.72) to 100.00% (Kappa=1.00). The lower and upper bounds of this range was higher than those of the TB-PPD skin test, which varied from 60.00% (Kappa=0.18) to 76.67% (Kappa=0.52). In Cohort 2, the positive rate of EC skin test read at 48-72 h was 20.04% (1398/6975). The corresponding positive rates of EC skin test in people aged ≥65 years, individuals aged under 18 years, close contacts of bacteriologically confirmed pulmonary tuberculosis patients, patients with diabetes mellitus, and people living with HIV were 29.94% (595/1987), 2.16% (23/1066), 27.43% (65/237), 28.98% (91/314) and 2/6, respectively. The incidence of adverse reactions to EC skin test was 4.13% (289/6998), predominantly mild-to-moderate local reactions. The incidence of Grade ≥3 adverse reactions was 0.04% (3/6998). No serious adverse events related to the EC skin test were observed. Conclusion: Local erythema and induration after EC skin test administration reflect specific immune responses, and both demonstrate higher diagnostic agreement with T-SPOT.TB results compared with TB-PPD skin testing. Using the large of the mean diameter of erythema and induration ≥5 mm as positive criterion and reading results within 48-72 h achieves favorable diagnostic consistency and clinical operability, the EC skin test shows good tolerability and safety across the general population, elderly individuals, and special populations, making it suitable for use in primary medical institutions and for large-scale population screening.

Key words: Mycobacterium tuberculosis, Recombinant fusion proteins, Skin tests, Clinical trials, phase Ⅳ as topic