C-Tb skin test to diagnose Mycobacterium tuberculosis infection in children and HIV-infected adults: A phase 3 trial.

BACKGROUND: C-Tb, an ESAT-6/CFP-10-based skin test, has similar sensitivity for active TB compared to tuberculin skin test (TST) and QuantiFERON-TB-Gold-In-Tube (QFT). However, data are limited in children and HIV-infected persons. METHODS: Asymptomatic South African contacts <5 years (n = 87; HIV-uninfected), or symptomatic individuals of all ages presenting to clinics with suspected TB (n = 1003; 30% HIV-infected) were recruited from eight South African centres. C-Tb and TST were allocated to either forearm double blinded. Samples for QFT were collected in parallel, and test-positivity rates were compared. RESULTS: In participants with microbiologically confirmed TB (n = 75; 45% HIV-infected) sensitivity of C-Tb, TST and QFT were similar (72% versus 75% versus 73%; p>0.5). All 3 tests had similar positivity rates in HIV-infected participants with active TB, however, positivity rates were reduced when CD4 counts were <100 cells/µL. In participants where active TB was excluded (n = 920), C-Tb (41%), TST (43%), and QFT (44%) also had similar test-positivity rates. Among asymptomatic contacts aged below five, 32% (28/87) tested positive with C-Tb and 32% (28/87) with TST (concordance 89%). Overall, C-Tb and TST showed a similar safety profile. CONCLUSION: C-Tb was safe and showed similar test-positivity rates, compared to TST and QFT, in children and HIV-infected persons with active or latent M. tuberculosis infection. These data inform the utility of C-Tb in clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov NCT01642888. EudraCT 2011-005078-40.

Safety and efficacy of the C-Tb skin test to diagnose Mycobacterium tuberculosis infection, compared with an interferon γ release assay and the tuberculin skin test: a phase 3, double-blind, randomised, controlled trial.

BACKGROUND: Targeted screening and treatment of Mycobacterium tuberculosis infection substantially reduces the risk of developing active tuberculosis. C-Tb (Statens Serum Institute, Copenhagen, Denmark) is a novel specific skin test based on ESAT-6 and CFP10 antigens. We investigated the safety and diagnostic potential of C-Tb compared with established tests in the contact-tracing setting. METHODS: Negative controls, close contacts, occasional contacts, and patients with active pulmonary tuberculosis were enrolled at 13 centres in Spain. We compared C-Tb with the QuantiFERON-TB Gold In-Tube ([QFT] Qiagen, Hilden, Germany) interferon γ release assay (IGRA) and the purified protein derivative (PPD) RT 23 tuberculin skin test ([TST] Statens Serum Institute). All participants older than 5 years were tested with QFT. Some participants in the negative control group received C-Tb without the TST to test for potential interactions between C-Tb and PPD RT 23. The rest were randomly assigned in blocks of ten and tested with both C-Tb and TST, with five in each block receiving injection of C-Tb in the right arm and the TST in the left arm and five vice versa. The primary and safety analyses were done in all participants randomly assigned to a group who received any test. This trial is registered with ClinicalTrials.gov, number NCT01631266, and with EudraCT, number 2011-005617-36. FINDINGS: From July 24, 2012, to Oct 2, 2014, 979 participants were enrolled, of whom 263 were negative controls, 299 were occasional contacts, 316 were close contacts, and 101 were patients with tuberculosis. 970 (99%) participants completed the trial. Induration sizes were similar for C-Tb and TST, but TST positivity was affected by BCG vaccination status. We found a strong positive trend towards C-Tb test positivity with increasing risk of infection, from 3% in negative controls to 16% in occasional contacts, to 43% in close contacts. C-Tb and QFT results were concordant in 785 (94%) of 834 participants aged 5 years and older, and results did not differ significantly between exposure groups. The safety profile of C-Tb was similar to that for the TST. INTERPRETATION: C-Tb delivered IGRA-like results in a field-friendly format. Being unaffected by BCG vaccination status, the C-Tb skin test might provide more accurate treatment guidance in settings where the TST is commonly used. FUNDING: Statens Serum Institut.

Sensitivity of C-Tb: a novel RD-1-specific skin test for the diagnosis of tuberculosis infection.

C-Tb, a novel Mycobacterium tuberculosis and 6-kDa early secretory antigenic target/10-kDa culture filtrate protein (ESAT-6/CFP-10)-specific skin test, has high specificity in bacille Calmette-Guerin-vaccinated healthy controls. However, the sensitivity of C-Tb has hitherto not been determined. The objective was to determine the sensitivity of C-Tb in patients with active tuberculosis (TB) in comparison with the tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT).C-Tb and TST were randomly administered in a double-blinded fashion to one or the other forearm in 253 patients with active TB with or without HIV co-infection. QFT-GIT testing was performed prior to skin testing.Using a receiver operating characteristic curve-derived cut-point of 5 mm, C-Tb sensitivity was similar to QFT-GIT (73.9 (95% CI 67.8-79.3) versus 75.1 (95% CI 69.3-80.2)), and similar in HIV-infected and HIV-uninfected patients (76.7 (95% CI 69.0-83.3) versus 69.5 (95% CI 59.2-78.5)). However, sensitivity was significantly diminished in HIV-infected patients with CD4 counts <100 cells·mm(-3). C-Tb and QFT-GIT combined had significantly higher sensitivity than C-Tb alone (p<0.0001). C-Tb was safe with no significant adverse events. The 5 mm cut-point corresponded to that found in the previously published specificity study (TESEC-04).C-Tb has similar sensitivity compared with QFT-GIT for the diagnosis of M. tuberculosis infection. Sensitivity was reduced only in HIV-infected patients with severe immunosuppression. Further studies in different settings are required to validate the proposed 5 mm cut-point.

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

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.

First-in-man open clinical trial of a combined rdESAT-6 and rCFP-10 tuberculosis specific skin test reagent.

BACKGROUND: Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity. METHODOLOGY: In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1ratio1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 microg or 0.1 microg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN gamma response just below the Quantiferon(R) cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded. CONCLUSION: The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT00793702.

Risk of sensitization in healthy adults following repeated administration of rdESAT-6 skin test reagent by the Mantoux injection technique.

Limited specificity of the tuberculin skin test incited the development of the intradermal Mycobacterium tuberculosis-specific rdESAT-6 skin test. Animal studies have shown, however, that there is a possible risk of sensitization when repeated injections of rdESAT-6 are given. The aim of this phase 1 open clinical trial was to assess the sensitization risk and safety of repeated administration of rdESAT-6 reagent in 31 healthy adult volunteers. Three groups of volunteers received two fixed doses of 0.1 microg rdESAT-6 28, 56 or 112 days apart, respectively. After the second injection, the diameter of induration and/or redness at the injection site was measured and taken as a possible sensitization reaction if >5mm. In vitro interferon gamma (IFN-gamma) responses were measured as supportive evidence. Local adverse reactions at the injection site and adverse events were recorded. One out of 31 (3%) volunteers showed a positive skin reaction (sensitization) upon a second injection of rdESAT-6 after 28days and an increased IFN-gamma response to ESAT-6. For 7 (23%) of the volunteers, local adverse reactions related to the product were registered, but all reactions were mild and predictable. In conclusion, repeated injections of the rdESAT-6 skin test reagent are safe, and sensitization occurs at a low rate, especially if the time span between succeeding doses is wide.

Double-blind randomized Phase I study comparing rdESAT-6 to tuberculin as skin test reagent in the diagnosis of tuberculosis infection.

Limited specificity of the tuberculin skin test incited the development of in vitro assays based on Mycobacterium tuberculosis-specific antigens such as ESAT-6 that are lacking in Bacillus Calmette Guérin (BCG). In animal studies, intradermal ESAT-6 was safe and induced specific skin test responses. The aim of the study was to assess the safety of intradermal recombinant dimer ESAT-6 (rdESAT-6) compared with tuberculin and to determine the human dose. The study design was a double-blind Phase I study with intra-subject randomization to the left and right forearm, comparing 2 Tuberculin Units (TU) intradermal tuberculin (RT23) with 0.01, 0.1, 1 or 10 microg rdESAT-6 in groups of five healthy controls or treated tuberculosis (TB) patients. The risk of sensitization after skin testing was assessed in healthy volunteers. All doses were tolerated well by healthy volunteers and responses to rdESAT-6 were limited to transient redness after 24 h only at the highest dose. No sensitization was observed. Because 1 microg rdESAT-6 induced large responses with local side effects in some TB patients, the 10 microg dose of rdESAT-6 was not tested. Mean responses to 0.01, 0.1 and 1 microg rdESAT-6 measured 14.0, 19.8 and 38.8 mm of redness, respectively, and 7.0, 13.4 and 14.6 mm of induration. The response to tuberculin was similar to the responses to 0.1 microg rdESAT-6. Mild local side effects due to tuberculin and rdESAT-6 were observed in 8/15, respectively, 6/15 patients, more pronounced at the highest rdESAT-6 dose. In conclusion, this pilot Phase I study of safety, feasibility and dose finding of intradermal rdESAT-6 provides proof of principle of a specific skin test for human use. No serious adverse events were observed but the study was not sufficiently powered to demonstrate complete safety. Intradermal rdESAT-6 did not seem to sensitize healthy volunteers. In treated TB patients, responses to rdESAT-6 were optimal at 0.1 microg. Further studies are needed to evaluate sensitization after repeated doses and to study the effect of additional CFP-10 on the sensitivity of a TB-specific skin test.

Safety of ESAT-6.

A recombinant dimer of the Mycobacterium tuberculosis (MTb) 6 kDa early secreted antigenic target (ESAT-6) was produced in Lactococcus lactis. Pharmacodynamic and safety studies were carried out in guinea pigs, rats, mice and dogs with intradermal (id), subcutaneous (sc) and intravenous (iv) administration of the antigen. In contrast to tuberculin purified protein derivative (PPD) the recombinant dimer (rdESAT-6) was able to discriminate MTb infection from BCG vaccination in vivo. In guinea pigs sensitized by infection with MTb, 1 microg rdESAT-6 gave a mean delayed-type hypersensitivity (DTH) response of 22 mm, a significantly stronger reaction than in animals sensitised by the environmental mycobacteria M. kansasii, M. szulgai and M. marinum. rdESAT-6 proved to be a safe tuberculin reagent in a dose range of 1-1000 microg with no or only minor local reactions.