Randomised clinical trial investigating the specificity of a novel skin test (C-Tb) for diagnosis of M. tuberculosis infection

Abstract

Background: Tuberculin skin testing is simple and relatively inexpensive, but the specificity of PPD is affected by BCG vaccination.

Objective: Determine optimal dose and specificity of recombinant ESAT-6 and CFP-10 (C-Tb) produced in Lactococcus lactis for diagnosis of M. tuberculosis infection.

Methods: In a dose finding phase I trial 0.01 or 0.1 µg preserved and unpreserved C-Tb was injected by Mantoux technique in 38 patients with active tuberculosis and induration responses measured. In a phase II specificity trial in 151 uninfected, BCG vaccinated participants 0.1 µg C-Tb was compared to 2 TU PPD.

Results: 0.1 µg C-Tb gave a median induration of 15 mm after 2 days. Phenol preservation did not affect the response. The specificity of C-Tb was 99.3% (95% CI 96-100%) regarding indurations ≥5 mm as a positive outcome. This was higher than the specificity of PPD (63% using a cut-off of 5 mm or 92% using a cut-off of 15 mm to adjust for non-specific BCG responses). Local adverse reactions following C-Tb injection included transient itching and discomfort as expected components of the immune response.

Conclusion: C-Tb offers a simple and convenient skin test to diagnose M. tuberculosis infection using a single, universal cut-off unaffected by BCG vaccination.

Trial registration: ClinicalTrials.gov NCT01033929 and NCT01241188.

Conflict of interest statement

Competing Interests: PA is co-inventor of a patent related to ESAT-6 but all rights belong to Statens Serum Institut. Patent name: Tuberculosis vaccine. No: WO9501441. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Flow diagram of dose-finding trial.

TB patients received C-Tb (unpreserved and phenol preserved) in either arm. 3/11 (0.01 µg C-Tb) and 13/24 (0.1 µg C-Tb) had a culture or PCR confirmed TB at inclusion, the rest were smear (2/11 and 2/24) or IGRA (6/11 and 9/24) positive.

Figure 2. Flow diagram of specificity trial.

BCG vaccinated, healthy, QFT-IT negative participants received 0.1 µg C-Tb and 2 TU PPD RT 23 SSI in either arm.

Figure 3. Time course of the induration response to preserved C-Tb in patients with active TB.

The median induration response (line within boxes) is shown for the first 4 days after injection of TB patients with 0.01 µg (white; N = 11) or 0.1 µg C-Tb (grey; N = 24) in dose-finding trial. The boundaries of boxes are 25th and 75th percentiles. Error bar indicate 90th percentile.

Figure 4. Induration 2 days after injection of unpreserved and phenol preserved C-Tb (0.1 µg) to TB patients.

Line of identity is shown. Area of dot indicates number of patients with dot at (0.0) representing 7 participants. N = 24.

Figure 5. QFT-IT and C-Tb responses in patients with active TB.

QFT-IT positive-negative result (panel A) and baseline concentration of IFNγ (panel B) at screening versus induration diameter 3 days after injection of 0.1 µg preserved C-Tb to TB patients (N = 24). Indeterminate (Ind) results are omitted in panel B. Dotted lines represents cut-off values. Regression line is shown in panel B.

Figure 6. Distribution of induration responses and specificity of C-Tb and PPD in healthy BCG-vaccinated adults.

The induration responses 2–3 days after testing with 0.1 µg C-Tb (black bars) and 2 TU PPD RT 23 SSI (grey bars) are shown in panel A. The resulting specificity of C-Tb (solid line) and PPD RT 23 SSI (broken line) as a function of cut-off is shown in panel B. N: 147.