Testing for TB. Bart Van Berckelaer Territory Manager Benelux. Subtitle
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1 Testing for TB Bart Van Berckelaer Territory Manager Benelux Subtitle
2 Agenda TB infection pathway TB immunisation Testing options Pre lab considerations of the whole blood ELISA test The T-SPOT.TB test reliable by design Conclusion
3 TB infection pathway
4 Immunisation for TB Immunisation programmes offer some protection against TB. However, the BCG vaccination is being used in fewer countries as its long-term efficacy has been questioned Protection has been shown to last for 10 to 15 years World Health Organisation (1999) cited by The Green Book (2011) After this time protection against TB may diminish
5 Test options Overall, without treating latent TB, about 5 to 10% of infected persons will develop TB disease at some time in their lives Centers for Disease Control and Prevention Available at (accessed on 24 September 2014) Therefore, it is important that we test for latent TB infection and treat it Test Sputum smear Culture X-ray Tuberculin Skin Test (TST) Interferon Gamma Release Assay s (IGRA s) Suitable in LTBI?
6 Test choice considerations False positive results can occur because of the sensitising effect on the immune system of either prior BCG vaccination or opportunist environmental mycobacteria. False negative results can occur due to anything reducing immunity, particularly co-infection with HIV, but also treatments such as cytotoxics, or immunosuppression. Extensive tuberculosis (pulmonary or miliary) can itself also temporarily depress the immunity, and can lead to a paradoxically negative Mantoux test. NICE (2011) The tuberculin skin test (TST) has several limitations TST is known to have less than optimal sensitivity and specificity False-negatives are common amongst those with culture-confirmed TB and in immunocompromised patients False-positives are common in those with prior BCG vaccination and/or non-tuberculous mycobacterial infection Results are open to interpretation Up to a third of patients fail to return to have their test read Huebner et al., Clinical Infectious Diseases (1993)
7 Test choice considerations IGRA s offer a 21 st century alternative to the TST. They are generally accepted to be more specific than the TST as, unlike the TST, they both use antigens that are TB-specific (i.e. not present in BCG vaccination or most non-tuberculous mycobacteria) Two tests are commercially available, both measuring interferon gamma. However, they have different characteristics, particularly in test reliability 1. The T-SPOT.TB test Simplified ELISPOT test using purified cells 2. ELISA test using whole blood
8 Test choice considerations Sample collection (phlebotomy) Phlebotomy steps The T-SPOT.TB test Whole blood ELISA test Collection tubes Standard heparin tube No training required Uses specialised blood collection tubes 1 Three tubes for each individual test 1 Requirement to draw specific blood volume Precise volumes of blood are not required >2mL (children up to 2 years old) >4mL (children 2-9 years old) >6mL (adults and children 10 years old and over) Each tube is manufactured to draw 1 ml of blood and performs optimally within the range of 0.8mL and 1.2mL 1 Under or over filling may lead to erroneous results 1 Shake collection tube Not required As the tubes only collect 1 ml of blood, thorough mixing is essential to solubilize the tubes contents, which are coated on the inner wall. This is best achieved by shaking the tubes ten (10) times, just firmly enough to ensure the entire inner surface of the tube is coated with blood, immediately after filling tubes. 2 Over-energetic shaking can result in erroneous results and must be avoided! 1 Logistics Ship overnight Test validated for 32 hours following blood draw Un-incubated samples can be stored for up to 16 hours before incubation 2 although incubation as soon as possible after blood collection is preferable 2 1 Cellestis sponsored by Qiagen, QFT Instructional Videos : 2 Qiagen & QuantiFERON, Frequently Asked Questions, QuantiFERON -TB Gold :
9 Pre lab considerations of the whole blood ELISA test Shaking the collection tube can affect the interferon gamma levels of the whole blood ELISA test The study suggests that interferon gamma levels are affected by mild or vigorous tube shaking Table 2: Effects of shaking methods of QFT-3G tubes on test results (Verification in Nil tubes) Healthy volunteers No. Mild shaking How to shake tubes IFN- y production (IU/ml) Harada et al., Kekkaku (2011) Vigorous shaking
10 Pre lab considerations of the whole blood ELISA test Inconsistent shaking of the collection tubes can affect the results of the whole blood ELISA test Key findings IFN-y levels are affected by gentle or vigorous tube shaking Results can vary if the antigen and nil tubes are shaken inconsistently Amount of shaking % Positive (n=40) Tb Ag Gen - Nil Gen 32.5% Tb Ag Vig - Nil Vig 35.0% Tb Ag Gen - Nil Vig 27.5% Tb Ag Vig - Nil Gen 42.5% Gaur et al., Journal of Clinical Microbiology (2013)
11 Pre lab considerations of the whole blood ELISA test Different volume draws can affect the result of the whole blood ELISA test Key findings TB Antigen minus Nil response was affected by blood draw volume Increasing blood volume decreased test positivity in infected subjects Blood volume (ml) Response IU/mL +ve tests in subjects with LTBI % (15/17) % (12/17) % (10/17) We showed that the blood volume and tube shaking represent novel preanalytical sources of variability that likely contribute to discordant IGRA results for individuals undergoing serial testing. Gaur et al., Journal of Clinical Microbiology (2013)
12 Pre lab considerations of the whole blood ELISA test Incubation delay can cause false negatives in the whole blood ELISA test Incubation delay Whole blood ELISA test # of indeterminate % Discordant 0 hours 0/41 N/A 6 hours 10% (2/20) 22.2% (4/18) 12 hours 17.1% (7/41) 20.6% (7/34) We found that incubation delay significantly increased the frequency of indeterminate results. Other causes for indeterminate QFT-IT results include preanalytical errors such as storage of tubes outside the recommended temperature, overor underfilling of tubes with blood, and insufficient mixing. Incubation delay Whole blood ELISA test Mean IFN produced % Reversions 0 hours 0.77 IU/mL N/A 6 hours 0.35 IU/mL 19% 12 hours 0.19 IU/mL 22% delays in incubation of QFT-GIT blood samples cause false negative results in a significant fraction of subjects with risk factors for LTBI Herrera et al., Journal of Clinical Microbiology (2010) Doberne et al., J of Clinical Microbiology (2011)
13 Pre lab considerations of the whole blood ELISA test Incubation delay cont d granulocytes inhibit T cell function in aged blood. Therefore, preventing granulocyte activation in blood specimens is critical to maintain optimal T cell function McKenna et al., Journal of Immunological Methods (2009) The T-SPOT.TB test performed with the T-Cell Xtend reagent removes granulocytes and prevents their activated state affecting the result of the assay.
14 The T-SPOT.TB test Processing a sample is simple with the T-SPOT.TB test 1. Fill one standard lithium heparin blood collection tube 2. Send to a) Oxford Diagnostic Laboratories (ODL ), the only expert national laboratory dedicated to running the T-SPOT.TB test b) Your chosen laboratory for processing
15 Assay methodology PURIFY: White blood cells are extracted and washed to remove contaminating IFN-y from whole blood, resulting in lower background noise STANDARDISE: The T-SPOT methodology corrects low cell counts prior to processing so that each test uses approximately 250,000 white blood cells. This normalises patients with different levels of immunosuppression COUNT: The output of the T-SPOT.TB assay is the numbers of individual T cells secreting IFN-γ. This single-cell resolution allows weak signals to be detected, maximising the chance of obtaining a result in patients with immune deficiencies These advanced steps are critical to improved IGRA performance T-Cell Xtend
16 Sensitivity - having a test that minimises false-negative results Sensitivity in head to head studies in subjects with culture confirmed TB Publications The T-SPOT.TB test Whole blood ELISA test Detjen et al., Clinical Infectious Diseases (2007) 92.9% (26/28) 92.9% (26/28) Dominguez et al., Clinical and vaccine immunology (2008) 92.3% (36/39) 78.6% (33/42) Goletti et al., PLoS ONE (2008) 84.1% (58/69) 75.4% (52/69) Chee et al., Journal of Clinical Microbiology (2008) 94.1% (254/270) 83.0% (224/270) Lai et al., Diagnostic Microbiology and Infectious Diseases (2011) 87.8% (86/98) 65.3% (64/98) Kobashi et al., Internal Medicine (2012) 95.5% (21/22) 90.5% (19/21) The T-SPOT.TB s consistently superior sensitivity is likely explained by the required 250,000 PBMC in each of its test wells, whereas the measurement of IFN-γ in the supernatant of whole blood in the QFT-IT test would adversely affect this assay s performance in immunosuppressed persons with low T-cell counts. Chee et al., Journal of Clinical Microbiology 2008 Quantitative summary of all head-to-head studies between the T-SPOT.TB test and In-Tube in culture confirmed active TB who received less than 1 month of TB therapy, excluding indeterminate results. Data compiled December Cut-off date for publication 31 st October 2012.
17 Comparison of positivity rates among diagnostic tests for LTBI 166 military recruits from a TB endemic region (Nepal) were tested with TST, T-SPOT.TB and the whole blood ELISA test on day 0, day 7 and day 200 Positivity of tests Day 0 Day 7 TST 21/166 (12.9%) Not done T-SPOT.TB 21/163 (12.9%) 23/163(14.1%) Whole blood ELISA test 8/166 (4.8%) 23/166 (13.8%) Note: n=163 due to the exclusion of three indeterminate results In the light of the findings presented in this study the UK Ministry of Defence has altered its policy for screening in Gurkha recruits to the use of a single T-SPOT.TB IGRA O Shea et al., PLOS ONE (2014)
18 Specificity - having a test that minimises false-positive results IGRA specificity in head-to-head studies with low risk controls The T-SPOT.TB test ELISA blood test Higuchi et al. IJTLD (2012) 99.1% (110/111) 99.0% (96/97) Mancuso et al. AJRCCM (2012) 98.7% (1336/1354) 98.8% (1338/1354) Quantitative summary of all head-to-head studies between the T-SPOT.TB test and In-Tube in truly low risk control subjects. Data compiled November 2012, Cut-off date for publication 31 st October Other papers of interest: Lee et al., ERJ (2006)
19 T-SPOT.TB test specificity of 99.1% Both IGRAs have excellent specificity and are unaffected by prior BCG vaccination Study population: 108 subjects from Ohio State University and the Nationwide Children s Hospital in the USA The specificity of the T-SPOT.TB study results for the control arm was 99.1% (107/108) Wang et al., Scandinavian Journal of Infectious Diseases (2010)
20 T-SPOT.TB test specificity of 98.9% Study population: 326 Naval recruits in the USA Detailed questionnaire used to exclude subjects with risk factors for TB infection It appears that the estimated specificity of both IGRAs is excellent for the diagnosis of TB in a population at low risk of M. tuberculosis exposure Bienek et al., International Journal of Tuberculosis Lung Disease (2009)
21 Conclusion There are important differences in the design and methodology of the two IGRAs which have significant clinical and logistical consequences The T-SPOT.TB test reliable by design Easier blood collection enables easier adoption of testing Control over assay performance is wholly within lab Excellent sensitivity & specificity Immunosuppression has little effect on performance Thank you
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