Laboratory Updates on IGRA Testing

Transcription

1 Laboratory Updates on IGRA Testing Edward A. Graviss, PhD, MPH, FIDSA August 18, 2017 EXCELLENCE EXPERTISE INNOVATION Edward A. Graviss, PhD, MPH, FIDSA, has the following disclosures to make: No conflict of interests No relevant financial relationships with any commercial companies pertaining to this educational activity 1

2 IGRAs in Clinical Practice - Update (Half Full and Half Empty) Aker/Zvonkovic Photography Edward A. Graviss, PhD, MPH, FIDSA eagraviss@houstonmethodist.org Conflict of Interest Statement Qiagen: PI initiated research; QFT-Plus assay tubes and kit donation Director of the Houston Methodist Hospital Molecular TB Laboratory: Perform both the QFT-GIT and T-SPOT.TB assays 2

3 Objectives TB infection natural history and diagnosis Issues with IGRA Interpretation With-in subject variability High INF-γ responses High conversion rates Low mitogen values Overview of QFT-Plus: the future of QFT QFT-Plus: recent studies (published papers) Natural History of TB Infection Canadian Tuberculosis Standard, 7 th edition, Chapter 2,

4 TBI Diagnosis Two currently accepted methods for screening Mtb-exposed individuals for TB infection. TST Tuberculin Skin Test 100+ years of experience, cheaper, more widely used worldwide IGRA - Interferon Gamma Release Assay 10+ years of experience, moderately expensive, used extensively in the developed world QuantiFERON T-SPOT.TB In-vitro and In-vivo Diagnostic Tests Injection of PPD antigens below epidermal layer in vivo causes infiltration of antigen-specific lymphocytes and inflammatory cytokines PBMCs from the peripheral blood are stimulated in vitro and production of INF-gamma from sensitized T cells is measured by ELISA or ELISPOT Andersen P et al., Lancet 2000;356:

5 TST versus IGRA comparison Test Invasiveness Antigen Used *Specificity TST in vivo (skin test) PPD (a polyvalent antigen mixture 200+ antigens) 97% in non-bcg vaccinated 60% in BCG vaccinated QuantiFERON-TB Gold InTube in vitro (2.4 ml blood draw total) ESAT, CFP-10, TB7.7 (Mtb specific antigens) TSPOT.TB in vitro ( 4ml blood draw) (possibly less for children, 1 compromised based on CBC) ESAT, CFP-10 (Mtb specific antigens) >95% in non-bcg vaccinated >95% in non-bcg vaccinated (not affected by BCG vaccination) (not affected by BCG vaccination) *Sensitivity 80% (diminished in HIV+ and children) 80% 90% (diminished in HIV+ and children) (diminished in HIV+ and children) Clinic Visits Timing hours hours hours Error Possibility High (manipulation and reading errors) Low Mod : ELISA test (Pre-analytical sources) Low : ELISPOT assay BCG Boosting Effect Yes No (yes in TB infected) No History 100+ years * Pai M et al. Clin Microbiol Rev 2014; 27: years (FDA approved May 2005) 9 years (FDA approved July 2008) 1) Cruz AT et al., Pediatrics Jan;127(1):e31-8. Issues with IGRA Result Interpretation Significant within-subject variability at least 6 retrospective and prospective studies. High INFγ responses in the control unstimulated assay tube, high nils High IGRA conversion rates (4-7%) in serial tested health care workers when compared to historical or concurrent TST conversion rates ( %) Other Issues Low mitogen values 5

6 Within-Subject Variability Metcalf JZ et al Am J Respir Crit Care Med. 2013; 187: consecutive patients with QFT ordered from 8/1/2010 7/31/2011, that had repeat testing per clinical lab s SOP (>0.25 IU/mL) Metcalf JZ et al., AJRCCM 2013;187: Within-Subject Variability (Conversions and Reversions) 543 eligible patients 177 indeterminate = 366 positive and negative replicates Metcalf JZ et al., AJRCCM 2013;187:

7 Within-Subject Variability In this dataset a borderline TBI response was defined as a IFNgamma concentration of IU/mL For test results close to the 0.35 IU/ml cut point, the normal withinsubject variability = ±0.24 IU/mL Metcalf JZ, et al., AJRCCM 2013;187: Palo Alto Study 1 : IU/mL 80% risk of reversion IU /ml 75% risk of reversion 1. Thanasssi W, et al., Pulm Med 2012; Sources of IGRA Variability Pai, M., et al., Clin Microbiol Rev 2014; 27:3 20 7

8 IGRA Sources of Variability Effect on Assays Pai, M., et al., Clin Microbiol Rev 2014; 27:3 20 Levels of agreement differed between individual technicians and automated reader from moderate to very good Commercial ELISpot reader and manual counting have good agreement Agarwal S et al., Tuberculosis (Edinb) 2016; S92-S98. 8

9 IGRA Sources of Variability Effect on Assays Pai, M., et al., Clin Microbiol Rev 2014; 27:3 20 Serial Testing HCW Seroconversions? Conversion n/n (%) Stable conversion n (%) Transient conversion n (%) TST 21/2293 (0.9) 1/12 (8) 11/12 (92) QFT 138/2263 (6.1) 25/106 (24) 81/106 (76) TSPOT 177/2137 (8.3) 27/118 (23) 91/118 (77) Stable conversion: Change from Neg to Pos, followed by Pos test at next 6-month f/u Transient conversion: Change from Neg to Pos, followed by Neg test at next 6-month f/u Dorman SE, et al., AJRCCM 2014; 189:

10 Summary Points: Conversions (1) Proportion of participants who underwent a conversion from negative to positive was higher when assessed by either IGRA than by the TST (7 9X) Approximately 75% of IGRA conversions were transient (after 6 months and no treatment) Compared with transient T-SPOT.TB converters, stable T- SPOT.TB converters had higher median (Ag-nil) values at conversion (10 vs 13) but there were other quantitative test results that differentiated stable vs transient converters Conversions (by any test method) were not associated with TB exposure variables assessed Dorman SE, et al., AJRCCM 2014; 189: Summary Points: Conversions (2) The likelihood of IGRA conversion increased as the baseline quantitative result increased toward the positivity cut-point, but proportionately few conversions were accounted for by individuals with baseline quantitative results just below the cut point. Test variability explained only a small proportion of apparent IGRA conversion (QFT: 5.2% 7.5%; T-SPOT: 6.0% - 8.1%) In a sub-study in which QFT plasma were rerun immediately, half of the apparent conversions were not conversions on the re-runs Similar proportions of participants had IGRA conversions over intervals of 0 days, 2 weeks, and 6 months Dorman SE, et al., AJRCCM 2014; 189:

11 Conclusions For BCG-vaccinated individuals, use of an IGRA at the time of initial TBI testing will reduce the number of TBI diagnoses compared with using TST alone (the assumption is that IGRAS will correctly label as TBI-negative those people with a TST that is positive due to BCG sensitization, although there is no data is available support or refute this assumption) In this group of HCWs undergoing period TBI screening, a large % of apparent IGRA conversions did not represent a true change in biological status with respect to TB infection Strategies for discerning true vs false IGRA conversions are required; until then caution should be used in interpreting a single newly positive test in an individual with one or more prior negative tests For QFT, doing repeat ELISA on the existing stimulated plasmas may be helpful Repeating the IGRAs should be considered, although we did not formally assess this as a strategy Dorman SE, et al., AJRCCM 2014; 189: Graviss Lab Statistics (7/31/2017) QFT Overall (+) 13.26% (5149/38830) QFT Overall (-) 81.70% (31726/38830) QFT Indeterminate 5.03% (1955/38830) All HMH Indeterminate 25.27% (996/3942) HMH TMC Only Indeterminate 25.46% (712/2797) T-Spot Overall (+) 8.30% (1327/15997) T-Spot Overall (-) 83.78% (13402/15997) T-Spot Borderline 2.67% (427/15997) T-Spot Fail 5.23% (836/15997) T-Spot No Test Performed.03% (5/15997) T-spot Fail excluding No Test Performed (n = 5) and Low Cell Count 2.72% (423/15579) 11

12 Overview of 4 th generation IGRA QuantiFERON-TB Gold Plus QFT-Plus is FDA approved July 8, Package insert not available at current time. Evolution of QFT Technology QuantiFERON-TB to QuantiFERON-TB Gold 1 st Generation 2 nd Generation 3 rd Generation QuantiFERON-TB 2001 U.S. FDA approval Measured cell-mediated immunity to the same tuberculin purified protein derivative (PPD) used for the tuberculin skin test (TST; M. avium) QuantiFERON-TB Gold 2004 U.S. FDA approval Liquid antigen version Antigens specific for M. tuberculosis complex organisms to measure cellmediated immunity 99% specificity No cross reactivity with BCG QuantiFERON-TB Gold In tube 2007 U.S. FDA approval Scalable and easily automated Logistical advantage remote incubation RD1 antigens and TB7.7 coated inside tubes >1300 peer reviewed publicatoins 12

13 QuantiFERON-TB Gold Plus (QFT-Plus) Desired characteristics Enhanced performance Increased sensitivity Sustained high specificity Improved performance in high-risk groups People who are immunocompromised People living with HIV/AIDS Potential to provide additional clinical information Risk-based algorithms Better to assist patient assessment and management Improve risk prediction? Harmonization of workflow options globally 1-tube blood collection (optional) 4-point standard curve QFT-Plus EU Package Insert QFT In Tube QFT-Plus Intended Use No change QFT is an in vitro diagnostic test using a peptide cocktail stimulating ESAT-6, CFP-10 proteins to stimulate cells in heparinized whole blood. Detection of interferon-γ (IFN-γ) by Enzyme- Linked Immunosorbent Assay (ELISA) is used to identify in vitro responses to these peptide antigens that are associated with Mycobacterium tuberculosis infection. QFT is an indirect test for M. tuberculosis infection (including disease) and is intended for use in conjunction with risk assessment, radiography and other Medical and diagnostic evaluations. Blood Collection Tubes 3 Tube test 4 Tube test LITHIUM HEPARIN SINGLE TUBE OPTION Blood collected in single tube (5 ml) Transported at (22 C ± 5 C) 16 hrs to incubation time Transferred to tube at lab Control of pre-analytical error Interpretation Of Results Test value IU/mL QFT result TB-Nil (IU/ml) 0.35 Positive Test value IU/mL QFT-Plus result TB1-Nil (IU/ml) 0.35 Positive TB2-Nil (IU/ml) 0.35 Positive Both TB1 - TB2-Nil (IU/ml) 0.35 Positive 13

14 Differences between (QFT-GIT and QFT-Plus) Number of tubes used and volume (ideal volume) QFT-GIT = 3 tubes (CD4 + ) = 2.4 μl QFT-Plus = 4 tubes (CD4 + / CD8 + ) = 3.2 μl Polypeptides used in assays: QFT-GIT (ESAT-6 Rv3874, CFP-10 Rv3875, TB 7.7 Rv3875c ) QFT-Plus (ESAT-6 Rv3874, CFP-10 Rv3875 ) Size of polypeptides QFT-GIT: 20-mer (18pp with 10aa overlap) QFT-Plus: 20-mer in TB1; 20-mer and smaller in TB2 ELISA Standards: Used for standard curves QFT-GIT: 26 pts with 2 sets of 8-standards QFT-Plus: 22 pts with 2 sets of 4-standards CD8+ T cells and role in TB immunity New CD8 + antigens: Why? Mtb-specific CD8 + T cells secrete IFN and other soluble factors to (1 3): Suppress Mtb growth Kill infected cells Directly lyse intracellular Mtb 1. Immunology 1996; 87, Eur. J. Immunol. 2003; 33, Science 1998; 282,

15 CD8+ T cells and role in TB immunity Biomarker for intracellular TB burden TB-specific CD8 + T cells that produce IFN have been: More frequently detected in those with active TB disease vs. latent infection (4, 5) Associated with recent exposure to TB (6) Detectable in active TB subjects with HIV coinfection and young children (7, 8) Observed to decline when patients are exposed to anti-tuberculosis treatment (9) 4. J. Immunol. 2011; 187, Eur. J. Immunol. 2013; 43, Diagn. Microbiol. Infect. Dis. 2013; 75, J. Infect. 2014; 69, Am. J. Respir. Crit. Care Med. 2012; 185, PLoS ONE 2014; 8, e Epub. QFT-Plus Clinical Performance Published Data 15

16 First Independent Evaluation QFT-PLUS Letter to the Editor Study design: Prospective multi-center study, 6 sites Italy and UK, enroll 11/ /2015: 119 active TB cases; 106 low-risk controls, Non-BCG vaccinated pool; evaluated with QFT-PLUS Sensitivity: 88% (102/116), 100% in subjects co-infected with HIV/TB (n=4) Specificity: 97% (103/106) Indeterminate: 1.3% (3/225) overall Three indeterminates were from the active TB cohort (2.5% - 3/119) Correlation with QFT-GIT (in a sub-cohort of 73 where QFT-GIT results were available): 94% Barcellini L., et al. Eur Respir J. 2016;47: Barcellini et al., 2016 ERJ: Authors conclusions Sensitivity Advantage Sensitivity (88%) which was higher than culture-confirmed active TB patients in a 2011 meta-analysis for QFT-GIT (1) CD8 Potential Difference between TB2 and TB1 was higher in smear-positive compared to smear-negative patients (significant difference). Magnitude of difference in agreement with flow studies (2) Advantage in Immunosuppression The increased IFNγ release by combined stimulation of CD4+ and CD8+ T-cells observed in the TB2 antigen tube might be advantageous for improving the assay s accuracy in patients with low CD4+ T-cell counts 1. Sester M, et al., Eur Respir J. 2011;37: D Ambrosio L et al., Eur Respir J. 2014; 43:

17 First evaluation of QuantiFERON-TB Gold Plus performance in contact screening 1 Study design: Prospective recruitment of TST-positive adult contacts (TST 5 mm) at a single hospital in Milan Mean age = 39 (30-79). 51.3% (n=61) non-european born, 78.8% (n=82) BCG vaccinated 9.24% (n=11) were immunocompromised (HIV and other) Contact screening based on NICE TB guidelines 2011 and Italian guidelines 1. Barcellini L et al., Eur Respir J. 2016; 48; Barcellini et al, 2016: Results continued Overall agreement: ĸ = 0.8 (QFT-GIT vs QFT-Plus) Discordant results n=12 All 12 results were negative QFT-GIT and positive QFT-Plus All but one discordant result had positive TST result >10 mm Two conversions occurred with QFT-GIT upon retesting at weeks, both initially positive by QFT-Plus QFT-Plus had a stronger risk association (univariate analysis) QFT-Plus showed a stronger risk association to aggregate exposure time than QFT-GIT (>12 hours/day): Odds ratio 14.8 QFT-Plus vs. 6.8 QFT-GIT. QFT-Plus showed a stronger risk association to index case proximity than QFT-GIT (sleeping in the same room): Odds ratio vs. 5.6 QFT- Plus vs 4.0 QFT-GIT Barcellini L et al., Eur Respir J. 2016; 48;

18 Barcellini et al, 2016: Results continued TB2-TB1 differential as a surrogate measure for CD8 + stimulation 15% of 119 contacts had TB2-TB1 values >0.6 IU/mL Significantly associated with proximity to the index case (p = ) Significantly associated with European origin (p = ) [QFT-Plus performance] suggests a role for the differential value between the TB2 and TB1 tubes as a proxy for recent infection. Barcellini et al, Eur Respir J Jul 7. pii: ERJ doi: / Epub ahead of print] Barcelleni L et al., Eur Respir J. 2016; 48; First characterization of the CD4 + and CD8 + T- cell responses to QuantiFERON-TB Plus Study design: Prospectively enrolled 57 non-hiv pulmonary TB and TBI individuals within 7 days of treatment, at 1 site in Rome, Italy. 27 active TB = 23 culture positive and 4 clinically confirmed. 30 TBI = 18 remote contact (>3 years), 12 recent contact (<3m) QFT-GIT, QFT-Plus and flow specimen drawn together Goal: Evaluate by flow cytometry CD4 + and CD8 + responses to the Mtb antigens contained in the QFT-Plus. Petruccioli, E, et al. J Infect 2016; 73:

19 Characterizing CD4/CD8 Response with Flow Data Petruccioli, E, et al. J Infect 2016; 73: CD4/CD8 Response with Disease Severity Petruccioli, E, et al. J Infect 2016; 73:

20 Petruccioli E. et al take home TB1 peptides were designed to stimulate CD4+ cells and TB2 designed to elicit CD4+ and CD8+ responses. CD8+ responses were seen following TB1 stimulation in a few folks (not all) TB2 CD8+ T-cell responses were associated with disease severity (radiologically) Across all evaluated groups TB2 CD8+ T-cell responses were associated with CD4 specific responses No association in the difference in INFγ production between TB2 and TB1, thus no differential CD8+ response. CD8+ T-cell responses are preferentially induced by TB2 which are mainly associated with active TB, thus potentially useful for CD4 impaired individuals Sensitivity of QFT-GIT and QFT-Plus are similar Petruccioli, E, et al. J Infect 2016; 73: Evaluation of QuantiFERON-TB Gold Plus for Detection of Mycobacterium tuberculosis Infection in Japan Lina T et al. Scientific Reports; 2016; 6:30617 Inferiority Study: QFT Plus vs QFT-GIT; multi-site (6 hospitals in Japan); 88% BCG+ Lina T. et al., Sci Reports; 2016; 6:

21 Evaluation of QFT-Plus in Japan Concentration of INFγ (IU/mL) measured using the different antigen-containing tubes in patients and in low-risk subjects Different parameters calculated using the different antigen-containing tubes. Lina T. et al., Sci Reports; 2016; 6:30617 Evaluation of QFT-Plus in Japan Results: IFNγ concentration of QFT-Plus was lower than that of QFT-GIT in TB patients (p < 0.001), but no difference in performance ROC curves showed both assays had AUC values over 0.99 without significant difference. QFT-Plus: sensitivity of 91.1% and specificity 97.6% QFT-Plus was as accurate as QFT-GIT despite a lack of TB7.7 IFNγ values associated with age in QFT-GIT (p = 0.035) but not in QFT-Plus TB2 had significantly higher IFNγ concentrations compared TB1 (p < 0.001) Lina T. et al., Sci Reports; 2016; 6:

22 Study design: Direct comparison study: QFT-GIT vs. QFT-Plus; single hospital in Munich, Germany. Consented HCWs thru annual screening (n = 77) and TB suspects (n = 86) from 7/2015 1/2016. Suspects = 24/86 (27.9%) culture confirmed; 33/86 (38.4%) clinical TB but 10/86 post-specific CXR anomalies. Hoffman H et al., Clin Microbiol Infect; 2016; 22: Hoffmann H et al, 2016: Results continued 9/163 = 5.5% Pooled positive rates TB cases 89% compared to ~ 80% [1] Sensitivity rates similar between QFT-GIT and QFT-Plus Authors comment INFγ concentrations in QFT-GIT significantly exceeded those measured in the TB1 tube though identical antigens used. 1. Sester M et al Eur Respir J 2011; 37:

23 QFT-Plus: a plus in variability? Evaluation of New Generation IGRA in Serial Testing of Students with a Migration Background in Germany Study design: Small cohort of migrant students, 11 QFT-GIT or QFT-Plus positive, 30 QFT-GIT and QFT-Plus negative serial tested weekly over a month (4x); Lubeck, Germany between 2/2016 3/2016. Exposed students offered prophylaxis after r/o TB disease and 2 serial positive results. Knierer et al., J Occ Med Toxicol 2017;12:1 QFT-Plus: a plus in variability? Evaluation of New Generation IGRA in Serial Testing of Students with a Migration Background in Germany A total of 163 serial measurements over a 4 week period - 41 students with 1 mis-draw. QFT-Plus: 4 conversions and 2 reversions Conversion rate of 4.3% (4/93 possible conversions, 95% CI %) Reversion rate of 6.9% (2/29 possible reversions, 95% CI %) QFT-G: 2 conversions and 1 reversion Conversion rate: 2.2% (2 of 91, 95% CI %) Reversion rate: 3.2% (1 of 31, 95% CI %) Agreement between the two IGRAs was 95.1% (κ = 0.89) Variance attributed to the individuals was low (QFT-Plus: ICC = 0.88) Knierer et al., J Occ Med Toxicol 2017;12:1 23

24 Evaluation of QuantiFERON-TB Gold-Plus in Health Care Workers in a Low-Incidence Setting Study design: Cross-sectional single-center study, Stanford Health Care Occ Health, enrollment :7/8/ /15/2015; 989 random HCWs during annual and new employee TBI screening; Risk factor assessed via questionnaire; comparison of QFT-GIT and QFT-Plus Positivity Rate QFT-GIT = 4.3% QFT-Plus = 6.4% TB1 = 4.2% TB2 = 5.2% Moon H-W et al., J Clin Microbiol 2017;55: Evaluation of QuantiFERON-TB Gold-Plus in Health Care Workers in a Low-Incidence Setting Moon H-W et al., J Clin Microbiol 2017;55:

25 The Sensitivity of the QuantiFERON TB Gold Plus in Zambian Adults with Active Tuberculosis Study design: Consecutive AFB smear or Xpert positive pulmonary suspected TB patients, Lusaka, Zambia, enrollment :6/2015 3/2016; 108 adults. Blood drawn (2 days) prior to treatment in 1 LiHep tube transferred to lab within 8 hours of draw, risk factor assessed via methodology unknown (med rec, questionnaire?) Telisinghe L. et al., Int J Tuberc Lung Dis 2017;21(6): The Sensitivity of the QuantiFERON TB Gold Plus in Zambian Adults with Active Tuberculosis PLOS ONE :e2489 Telisinghe L. et al., Int J Tuberc Lung Dis 2017;21(6):

26 Study design: Cross-sectional (prevalent) study of 134 FB students and young professionals from high TB incidence countries 125:100,000; enrolled from 2 3/2016 in Lϋbeck, Germany. Goal: Estimate prevalence and risk factors of FB students/young professionals and compare results of QFT-GIT and QFT-Plus 5.2:100,000 (2012) 7.3:100,000 (2015); 1648 TB cases No surveillance of universities - 236K FB students between 2014 and 2015 Self-admin risk questionnaire; QFT-GIT, QFT-Plus (via Liheparin); IGRA positive = retest after 1 week Gallegos-Morales et al., J Occ Med Toxicol 2017;12:12 Gallegos-Morales: Results QFT-Plus positive = 11/134 (8.2%); QFT-Plus + QFT-GIT positive = 13/134 (9.7%) ; ĸ = students positive by both assays; 2 QFT-GIT+/QFT-Plus -; 2 QFT-GIT - /GFT-Plus No TB cases identified No data on repeat draw (n = 11) other than: positivity confirmed in at lest 1 of 2 IGRA test methods in all cases Gallegos-Morales et al., J Occ Med Toxicol 2017;12:12 26

27 Gallegos-Morales: Discussion Students in high burden countries have a high TBI prevalence (6.9% Rio 45.1% in Uganda vet student; students in low burden countries have a low TBI prevalence (0.1% [med students Italy], 2.1% [RN Germany] and now students from High burden country in a low burden country setting (8.2% - Germany) High burden country = major risk factor for TB and higher risk of progression for TBI FB individuals Currently no regulations regarding TBI screening in immigrants nor students in Germany (ACHA association requires TBI screening for incoming students [2016]). Authors recommend risk questionnaire first then potential IGRA screening based on risks (e.g. targeted testing). Agreement good between QFT-GIT and QFT-Plus Gallegos-Morales et al., J Occ Med Toxicol 2017;12:12 Summary: QuantiFERON-TB Gold Plus (QFT-Plus) QFT-Plus: the latest evolution of QFT technology same test principle-procedure as the QFT-GIT QFT-Plus is an improved version of QFT-GIT : Sensitivity of >95% in registration trials, lower in published trials Higher specificity of any test for TB infection, but comparison to T-SPOT.TB unknown Innovative CD8 + T-cell technology Optimized for CD4 + and CD8 + response CD8 correlation to active TB, new infection and burden of TB One Li-Heparin tube draw for transfer into QFT-Plus tubes 27

28 Questions? HMH IGRA Lab Justin Lew, BS Ngan Ha, BS Sophie Im, BS Katie Ta, BS Effect of Tuberculosis Treatment on Newly Developed QuantiFERON-TB Gold Plus Kamada, Arisu et al, National Hospital Organization Hokkaido Medical Center, Sapporo, Japan P1174, 2016 ATS N = 38 non-mdr TB patients (successfully treated) Significant decreases of interferon gamma responses were observed during treatment of active TB in both TB1 and TB2 tubes in the first 3 months. The delta (TB2-TB1) between tubes was statistically significant in the latter half and throughout treatment (P<0.05). The finding suggests CD8+ responses (represented by the difference between the 2 tubes) declined with TB treatment 28

29 Preliminary Data on the Precision of QFT-TB Plus Performance (In-house validation) Compared coefficient of variation (CV) between the QFT-Plus and QFT 2 donors, one with TB infection and the other, without. 10 blood samples were drawn into the standard tubes from each subject (20 samples in total) Statistical analysis: The coefficient of variation (CV) of each batch of subjects on each analyzer was calculated based on the mean value for TB antigen/tb1/tb2 minus the nil Qualitative Results: Concordance - 100% and no indeterminate results Quantitative Results: QFT-Plus ELISA vs. QFT mean CV: 9.60% vs.18.25% Discussion: There was no variation in qualitative results between the two assays; however, when looking at the in-batch quantitative data, the QFT- Plus CV was ~50% lower than QFT. Potential Impact Reduction of reversions or conversions near the 0.35 IU/mL cut point Gallagher D. et al., Eur Respir J 2016; 48: