Validation of ante mortem TB tests in Camelids Shelley Rhodes & Martin Vordermeier TB Research Group AHVLA - Weybridge funded by and performed on behalf of the British Alpaca Society, the British Llama Society & British Camelids Ltd (Sept 2010 Sept 2011)
TB Research Group AHVLA-Weybridge Shelley Rhodes, Tom Holder & Martin Vordermeier CERA AHVLA-Weybridge Robin Sayers TB Policy AHVLA Ricardo de la Rua-Domenech AHVLA-Langford Lucy Waring Animal Services Unit AHVLA-Weybridge Derek Clifford & Ian Dexter TB Diagnostics LSD AHVLA-Weybridge Jacky Brewer AHVLA-Starcross Tim Crawshaw AHVLA-Worcester Steve Gillgan Chembio Diagnostic Systems, USA Konstantin Lyashchenko IDEXX Reference Laboratories, Canada John Lawrence Laboratory testing, data analysis, reporting Statistical advice and analysis Advice, policy support, coordination Laboratory testing Blood sampling from volunteered animals Bacteriology Post-mortem co-ordination Volunteer herd PM co-ordination Supply of diagnostic kits under development Supply of diagnostic kit under development
Definitions: Sensitivity and Specificity Sensitivity The sensitivity of a test is the proportion of truly infected animals that are detected with a test. For example, 90 % sensitivity means that out of 100 infected animals, 90 will be correctly identified (true-positives), and 10 will be missed (false-negatives)
Definitions: Sensitivity and Specificity Specificity The specificity of a test is the proportion of truly uninfected animals are correctly classified as test-negative. For example, 90 % specificity means that out of 100 truly negative animals, 90 will be test-negative (true-negatives) whilst 10 will test positive (false-positives)
What can ante-mortem diagnostic tests be used for: Herd-level test: Test is designed to detect herds with infection, or to assign disease-free-status to a herd (for example OTF). Examples: Surveillance operations, herd certifications Signature test: Tuberculin skin tests for cattle Individual animal test: Test is designed to detect the maximal number of infected individuals (maximise sensitivity as defined by the OIE and EU directives for cattle). Examples: Clear-up of infected herds to remove sources of infection, premovement trade test, post-movement trade test, tracing test Signature test: Interferon-gamma test used in cattle
Summary of TB Immunology: Why different tests can behave differently Tuberculin Skin Test Immune Response Gamma Interferon Test (IFN-γ) Antibody Response Increasing bacterial load Increasing pathology Infectivity
1. Validate IFNγ assay AIMS of the study: 2. Compare IFNγ with Serological Tests; STAT-PAK and DPP rapid tests (Chembio, USA) IDEXX ELISA Enferplex ELISA (no data, optimization issues in ROI) Apply tests to; Problem herds (relative Sensitivity) how many truly infected animals identified? (target 50 VL alpacas) TB-free herds (Specificity) how many false-positives do the tests identify (target 300 TB-free alpacas)
Tuberculin PPD and defined antigens: Tuberculin is the name given to extracts of Mycobacterium tuberculosis, M. bovis, or M. avium that is used in skin testing in animals and humans to identify a tuberculosis infection. Several types of tuberculin have been used for this, of which purified protein derivative (PPD) is the most important. PPD is a poorly-defined, complex mixture of antigens. An antigen is any substance that causes the immune system to produce antibodies (serology) or T cell responses (IFN-γ, skin test) against it. For bovine tuberculosis diagnosis, bovine and avian PPD is used Bovine PPD is made from M. bovis Avian PPD is made from M. avium We and others have defined components within bovine PPD that are the active components (such as ESAT-6 and CFP-10) that can be synthesised chemically and are therefore defined
Test Principle; Interferon-gamma release assay 1. Bleed animals and supply blood to test lab within 24 hours. 2. Culture separated blood lymphocytes in the presence of antigen (PPD-A and PPD-B, ESAT6/CFP10) for 3 days at 37 o C. Antigen (e.g. PPD) IFN-γ T cell Macrophage 3. Harvest cell-free supernatants and measure IFN-γ using ELISA.
Sensitivity and Specificity of IFN-γ in cattle (GB data) Diseased animals Cut-off % Sensitivity % Specificity SICCT +ve 0.1 89.7 96.5 SICCT -ve 0.1 91.4 96.5 + and - combined 0.1 90.9 96.5 874 TB-free cattle from the GB Specificity Trial 431 Diseased cattle: 107 SICCT+ : 324 SICCT- *Schiller et al., Clin. Vaccine Immunol., 2009, vol. 16(8): 1196-1202
STAT-PAK rapid antibody test (Chembio USA) Dean et al (2009) Veterinary Record Llamas with confirmed Mycobacterium bovis infection Sensitivity of 63.4%, rising to 100% if llamas skin-tested within previous 30 days. Recent study: llamas and alpacas: Sensitivity 71, Specificity 94% (Lyashchenko et al., 2011, CVI 18(12): 2143-2147)
DPP (dual path platform, Chembio USA) Martin Vordermeier (TB Research) and Konstantin Lyashchenko (Chembio) Recent published data study: llamas and alpacas: Sensitivity 74%, Specificity 98% (Lyashchenko et al., 2011, CVI 18(12): 2143-2147)
IDEXX M. bovis Ab Test (IDEXX) Shelley Rhodes (TB Research), John Lawrence (Idexx) Test developed for cattle, but we have together with IDEXX modified it so that it also works in camelids
Animals Diseased/infected: 59 VL alpacas from 10 separate culture-confirmed herds, of which; 55VL were tested with IFNγ 52 were tested with antibody tests 48 were tested with both IFNγ and antibody tests TB-free: 257 alpacas from 17 distinct premises across 9 counties 49 serum samples from USA tested with antibody tests Total of 306 TB-free tested with antibody tests 257 tested with both IFNγ and antibody tests
IFNγ Results (55VL & 257 TB-free) 1. Observed results % Sensitivity [95%CI] % Specificity [95%CI] PPD+ 63.6 [50.9-76.3] 89.1 [85.3-92.9] PPD+ and EC+ 34.6 [22-47.2] 98.1 [96.4-99.8] PPD+ or EC+ 80 [69.4-90.6] 80.9 [76.1-85.7] (Cut-offs: PPDB-PPDA>0.1; EC-nil>0.1) 2. Results adjusted for High Specificity % Sensitivity [95%CI] % Specificity [95%CI] PPD+ 29.1 [17.1-41.1] 96.9 [94.8-99] PPD+ and EC+ 16.4 [6.6-26.1] 100 [N/A] PPD+ or EC+ 41.8 [28.8-54.8] 93.8 [90.9-96.7] (Cut-offs: PPDB-PPDA>0.7272; EC-nil>0.3797)
Antibody Test Results (52VL & 306 TB-free) % Sensitivity [95%CI] % Specificity [95%CI] STAT-PAK 67.3 [54.5-80.8] 97.4 [95.6-99.2] DPP 57.7 [44.3-71.1] 96.7 [94.1-98.4] IDEXX 69.2 [56.7-81.7] 97.4 [95.6-99.2] (ROC Cut-offs: DPP>9.05; IDEXX>0.34)
IFNγ and Antibody Test Combinations for maximum sensitivity in TB-breakdown herds (48VL & 257 TB-free) 1. Antibody Test Combinations % Sensitivity [95%CI] % Specificity [95%CI] STAT-PAK/IDEXX 81.3 [71-91.6] 95.8 [93.3-98.2] IDEXX/DPP 72.9 [61.1-84.6] 94.9 [92.2-97.6] STAT-PAK/DPP 75 [63.6-86.4] 94.6 [91.8-97.4] STAT-PAK/IDEXX/DPP 81.3 [71-91.6] 93 [89.9-96.1] (ROC Cut-offs: DPP>9.05; IDEXX>0.34)
IFNγ and Antibody Test Combinations for maximum sensitivity in TB-breakdown herds (48VL & 257 TB-free) 2. IFNγ and Antibody Test Combinations % Sensitivity [95%CI] % Specificity [95%CI] IFNγ/STAT-PAK 93.8 [87-100] 79.2 [74.2-84.2] IFNγ/IDEXX 91.7 [83.8-99.5] 78.6 [73.6-83.6] IFNγ/DPP 91.7 [83.9-99.5] 77.4 [72.3-82.5] IFNγ/STAT-PAK/IDEXX 100 [N/A] 77.8 [72.7-82.9] IFNγ/STAT-PAK/DPP 100 [N/A] 76.7 [71.5-81.9] IFNγ/IDEXX/DPP 93.8 [87-100] 75.9 [70.7-81.2] ROC Cut-offs: IFNγ: PPD+ or EC+ ; PPDB-PPDA>0.1008, EC-nil>0.1003 DPP>9.05 IDEXX>0.34
Summary 1. IFNγ test can be applied with high sensitivity, but one would need to accept lower specificity 2. Application of IFNγ test should depend upon the outcome required e.g. High Sensitivity using PPD or EC option High Specificity using PPD and EC option 3. Animals with M. microti infection will give a positive IFNγ result to PPD 4. Antibody tests can provide high specificity and good sensitivity, but; - they are dependent upon skin test or on animals having severe/advanced pathology - they will not (even in combinations) remove all infected animals 5. Combination of IFNγ and antibody tests maximises detection of infected animals
High level summary: 1. More tools in the tool box (= tests of defined performance) 2. Informed/evidence-based policy discussions now possible 3. Evidence that your industry takes btb seriously 4. Application of the blood tests as individual animal tests plays to their strengths: clean-up of infected herds, tracing, trade/movement test Options of possible application in this role (subject to a full appraisal of costs and benefits and discussions between stakeholders and government): Anamnestic StatPak (and/or IDEXX), as a follow-up to the skin test, offers the best combination of Se and Sp for pre-/post-movement TB testing, pre-export testing, and also for tracings from infected herds. To resolve TB outbreaks in known infected herds one could use skin and anamnestic StatPak (or IDEXX) serology as a minimum to lift restrictions, with additional voluntary IFN-γ testing to boost Se, if the herd owner so wishes. 5. Surveillance use: Blood tests are probably not applicable
Future work? 1. Define skin tests performances: Single intradermal comparative test, single intradermal test (as potential surveillence/herd test) 2. Add more defined antigens such as ESAT-6/CFP-10 to IFN-γ test to increase the sensitivity of this test in the serial/combination application that has high specificity but low sensitivity: SE SPE PPD+ and EC+ 16.4 100 3. Define M. microti -specific antigens to allow better differential diagnosis (theoretically possible)