1. Assay Parameters Affecting Variability Of QuantiFERON®-TB Gold In-Tube Assay Results Whitworth WC1, Mazurek GH1, Goodwin DJ2 1Centers for Disease Control and Prevention (CDC), Division of TB Elimination, Atlanta, GA, USA; 2United States Air Force School of Aerospace Medicine, Wright-Patterson Air Force Base, OH, USA
Introduction
Results
The QuantiFERON®-TB Gold In-Tube test (QFT-GIT), designed to detect Mycobacterium tuberculosis (Mtb) infection, measures the interferon gamma (IFN-γ) response to specific Mtb peptides using an enzyme-linked immunosorbent assay (ELISA). Variability due to differing test parameters may limit test utility. We hypothesized that 1) a lower incubation temperature, 2) a longer delay from blood collection to incubation, and 3) a shorter incubation duration would result in lower IFN-γ levels.
158 subjects were enrolled (Figure 1)
155, 102, 152, and 149 had paired ELISA results available for the control, incubation temperature, incubation duration, and delay to incubation comparisons, respectively.
A lower number of subjects were available for the temperature assessment because this objective was added after 50 subjects had been enrolled into the study.
Figure 1. Subject enrollment and study design. Experimental parameters are shown in blue.
QUALITATIVE RESULTS
Results from qualitative comparisons of test interpretations are shown in Tables 2-4 A (crosstabs of interpretations) and Tables 2-4 B (line lists of subjects with discordant results). There was one indeterminate result for the delay (11-12 hr test), duration (16-17 hr test), and control (Test 2) comparisons due to low mitogen responses (same subject for the duration and control comparisons).
Tables 5A & 5B: Control.
5A. Crosstab B. Discordants.
Test 2
Test 1
Test 2 ID TB
Nil
TB Resp.
Interp. 70
0.775
0.184
0.591
0.496
0.171
0.325
pos/neg
109
0.589
0.186
0.403
0.388
0.138
0.250
146
1.934
0.107
1.827
0.068
0.075
-0.007 37
0.363
0.166
0.197
0.559
neg/pos
103
0.366
0.035
0.331
0.612
0.073
0.539
140
0.338
0.046
0.292
0.675
0.044
0.631
147
0.440
0.116
0.324
0.633
0.558
pos
neg 27 3 30 4
120
124 31
123
154
Methods
Test 1
Tables 2A & 2B: Incubation temperature.
2A. Crosstab B. Discordants.
Subject Selection
Subjects recruited at two sites as part of a larger study investigating QFT-GIT reproducibility
Inclusion: self-reported prior-positive TST to enrich the number of positives (prior unpublished assessments in similar cohorts have found that 40% to 50% of persons with self-reported prior-positive TST results were positive by QFT-GIT vs. <3% for the general U.S. population)
Exclusion: TST in the past 3 years or < 18 years old
QFT-GIT Testing and Experimental Design
Subjects were seen at 2 visits: 1) Visit 1, 6 to 9 am on Day 1 and 2) Visit 2, 6 to 9 pm on Day 7
At each visit, blood was collected into three sets of QFT-GIT tubes (three tests), each set consisting of one Nil (Nil), one Mitogen (phytohemagglutinin A), and one TB antigen (TB) tube for each test
Tubes mixed with Stuart rock and roll mixer (Scitech Instruments, Franklin, NJ) for 3 minutes at 33 RPM
ELISAs performed on a Triturus automated ELISA workstation (Grifols USA, Miami, FL), using eight
IFN-γ calibrators (8, 4, 2, 1, 0.5, 0.25, 0.125, and 0 IU/mL) in duplicate to create standard curves
[IFN-γ] (IU/mL) calculated from standard curve using software developed at the CDC, and test results interpreted as indicated in the Cellestis package insert and CDC guidelines
“TB Response” calculated by subtracting [IFN-γ] from unstimulated blood (Nil) from [IFN-γ] from Mtb antigen-stimulated blood (TB)
“Mitogen Response” (positive control) was calculated by subtracting [IFN-γ] from unstimulated blood (Nil) from [IFN-γ] from mitogen-stimulated blood
Three assay parameters were intentionally varied and included a control. Paired comparisons of test results using an experimental parameter vs. those using a reference parameter were performed. For each comparison, all test parameters were identical except for the experimental parameter being assessed. Reference parameters were a) 37.0 (± 1.0) oC incubation temperature, b) ≤ 1 hour delay to incubation (at ambient temperature, range 18 oC to 24 oC), and c) 23 to 24 hour incubation duration.
1) Incubation temperature assessment
Reference parameter: 37.0 (± 1.0) oC
Experimental parameter: 35.0 (± 1.0) oC
Both tests using reference delay to incubation of ≤ 1 hour and 23 to 24 hour incubation duration
2) Delay from blood collection to incubation assessment
Reference parameter: ≤ 1 hour at ambient temperature
Experimental parameter: 11 to 12 hours at ambient temperature
Both tests using reference incubation temperature of 37 oC and 23 to 24 hour incubation duration
3) Incubation duration assessment
Reference parameter: 23 to 24 hours
Experimental parameter: 16 to 17 hours
Both tests using reference incubation temperature of 37 oC and delay to incubation of ≤ 1 hour
4) Control (reproducibility, 2 tests from 2 samples collected at the same visit using same parameters)
All 3 reference parameters; 1st test also used as reference comparator in the temperature assessment
Statistical Methods
Subjects with incomplete tests were excluded from each comparison, and subjects with indeterminate
results were excluded from qualitative analyses
Qualitative (comparison of +/- test interpretations between reference and experimental parameter groups)
Comparison of percent positive of all subjects by each test using McNemar’s test
Percent discordant and percent positive among discordants
Percent of discordant subjects with results the near the QFT-GIT cutoff (±0.25 IU/mL)
Quantitative (comparison of IFN-γ concentrations between reference and experimental parameter groups)
Distributional differences in Nil, TB, and TB Response using Wilcoxon signed-rank test
268 persons encountered
158 subjects consented
55 persons declined
55 persons not eligible
37 oC
35 oC ID TB
Nil
TB Resp.
Interp. 70
0.775
0.184
0.591
0.527
0.262
0.265
pos/neg 85
0.651
0.080
0.571
0.392
0.061
0.331
145
1.996
0.099
1.897
0.068
0.094
-0.026
146
1.934
0.107
1.827
0.060
0.047 83
2.136
1.990
0.146
3.394
1.602
1.792
neg/pos
138
0.519
0.234
0.285
0.823
0.373
0.450
140
0.338
0.046
0.292
0.524
0.052
0.472
155
0.296
0.040
0.256
0.560
0.073
0.487
35 oC
19.5% (30/154) positive with Test 1 vs % (31/154) positive with Test 2, p = 0.36
Discordance = 4.5% (7/154)
Among discordants:
42.9% (3/7) positive with Test 1 vs. 57.1% (4/7) positive with Test 2
71.4% (5/7) with both TB Responses within IU/mL of cutoff (red)
pos
neg 17 4 21 77 81
102
37 oC
Significantly more subjects were positive with a delay to incubation of ≤ 1 hour than with a delay of 11 to 12 hours (25% vs. 21%, p = 0.03).
The widest disparities between the two discordant categories, showing more positives for the reference parameters than the experimental parameters, were seen for the delay (8 vs. 2) and duration (6 vs. 2) comparisons.
Among discordants, the delay and duration comparisons had fewer subjects with both TB Response values near the QFT-GIT cutoff than the temperature and control comparisons, suggesting a lower likelihood of these effects being attributable to test variation alone.
Parameter
Visit 1 (Day 1, am collection)
Visit 2 (Day 7, pm collection)
Temperature
37 oC
35 oC
Delay
≤ 1 hr
≤ 1hr
11-12 hrs
Duration
23-24 hrs
16-17 hrs
20.6 % (21/102) positive with 37 oC incubation vs % (21/102) positive with 35 oC incubation, p = 1.00
Discordance = 7.8% (8/102)
Among discordants:
50.0% (4/8) positive with 37 oC incubation vs % (4/8) positive with 35 oC incubation
62.5% (5/8) with both TB Responses within IU/mL of cutoff (red)
Control
(Reproducibility)
n = 155
Incubation
Temperature
n = 102
Duration
n = 152
Delay to
n = 149
Tables 3A & 3B: Delay to incubation.
3A. Crosstab B. Discordants.
QUANTITATIVE RESULTS
Quantitative results are shown in Table 6.
Table 6. Medians and means of test results, IU/mL IFN-γ.
Comparisons:
≤ 1 hr
11-12 hr ID TB
Nil
TB Resp.
Interp. 5
0.405
0.040
0.364
0.132
0.037
0.095
pos/neg 66
0.493
0.069
0.424
0.299
0.053
0.246 81
0.869
0.071
0.798
0.360
0.291 85
0.691
0.114
0.577
0.086
0.274
123
0.475
0.100
0.375
0.217
0.055
0.162
138
0.957
0.289
0.668
0.521
0.232
140
0.989
0.079
0.910
0.285
0.087
0.198
147
0.845
0.161
0.684
0.241
0.105
0.136 37
0.312
0.236
0.076
0.655
0.154
0.501
neg/pos 83
2.154
3.900
-1.746
1.319
0.074
1.245
11-12 hr
pos
neg 29 8 37 2
109
111 31
117
148
≤ 1 hr
Nil TB
TB Response
p-value
Parameter
Median
Mean
TB Resp.
Temperature
37 oC
0.08
0.11
0.12
1.09
0.05
0.98
0.23
0.57
35 oC
1.12
0.02
1.00
Delay
≤ 1 hr
0.15
1.38
0.04
1.23
0.0009
11-12 hr
0.07
0.10
1.13
0.03
1.03
Duration
23-24 hr
1.45
1.30
0.25
0.002
16-17 hr
1.21
1.08
Control
Test 1
0.13
0.93
0.82
0.77
0.69
Test 2
0.96
0.84
Subject characteristics of the 158 subjects are shown in Table 1.
Mean/median age of 45/46 years (range 24 to 74)
Most born in the United States or Canada (72%) and had lived abroad for ≥ 1 year (58%)
Most received therapy for LTBI (72%)
Table 1. Subject characteristics, N = 158.
25.0 % (37/148) positive with ≤ 1 hr delay vs. 20.9% (31/148) positive with hr delay, p = 0.03
Discordance = 6.8% (10/148)
Among discordants:
80.0% (8/10) positive with ≤ 1 hr delay vs % (2/10) positive with hr delay
30.0% (3/10) with both TB Responses within IU/mL of cutoff (red)
Category
n (%)
Age, years
16 (10.1%)
Therapy for LTBI
Yes
114 (72.2%)
35 (22.2%) No
43 (27.2%)
45 (28.5%)
Unknown
1 (0.6%)
42 (26.6%)
≥ 60
20 (12.7%)
Exposure to Active TB
60 (38.0%)
74 (47.1%)
Gender M
70 (44.3%)
24 (15.2%) F
88 (55.7%)
BCG
32 (20.3%)
Race/Ethnicity
White, non-Hispanic
79 (50.0%)
112 (70.9%)
Black, non-Hispanic
37 (23.4%)
14 (8.9%)
Asian/Pacific
18 (11.4%)
Hispanic
15 (9.5%)
Native American
Region of Birth
United States/Canada
113 (71.5%)
Other
8 (5.1%)
Asia
Central America/Caribbean
12 (7.6%)
Africa
7 (4.4%)
Last Positive TST
Europe/Russia
4 (2.5%)
Pacific
3 (1.9%)
Southeast Asia
2 (1.3%)
Middle East
65 (41.1%)
South America
54 (34.2%)
Years Lived Outside USA
None
66 (41.8%)
1 - 10
63 (39.9%)
Therapy for TB
154 (97.5%)
Tables 4A & 4B: Incubation duration.
4A. Crosstab B. Discordants.
IFN-γ levels for TB and TB Response were significantly lower with longer delay and shorter incubation.
23-24 hr
16-17 hr ID TB
Nil
TB Resp.
Interp. 5
0.405
0.040
0.364
0.110
0.034
0.076
pos/neg 81
0.869
0.071
0.798
0.309
0.060
0.249 85
0.691
0.114
0.577
0.336
0.098
0.238
119
2.221
2.145
0.387
0.059
0.328
123
0.475
0.100
0.375
0.134
0.039
0.095
151
0.622
0.099
0.523
0.468
0.217
0.251 83
2.154
3.900
-1.746
4.822
1.402
3.420
neg/pos
109
0.411
0.277
0.670
0.158
0.512
16-17 hr
Conclusions
pos
neg 33 6 39 2
110
112 35
116
151
23-24 hr
Lower QFT-GIT IFN-γ TB Response levels are observed with longer delays from blood collection to incubation (11 to 12 hrs vs. ≤ 1 hr) despite being within the limits recommended by the manufacturer of within 16 hours,
p =
These differences translate to a greater number of positive results with a shorter delay of ≤ 1 hr (25%) than with a longer delay of 11 to 12 hrs (21%), p = 0.03.
Lower QFT-GIT IFN-γ TB Response levels are observed with shorter incubation duration (16 to 17 hrs vs. 23 to 24 hrs) despite being within the limits recommended by the manufacturer of 16 to 24 hours, p =
These differences translate to a greater number of positive results with a longer duration of 23 to 24 hrs (26%) than with a shorter duration of 16 to 17 hrs (23%), but this difference was not significant, p = 0.08.
25.8 % (39/151) positive with hr incubation vs. 23.2% (35/151) positive with hr incubation, p = 0.08
Discordance = 5.3% (8/151)
Among discordants:
75.0% (6/8) positive with hr duration vs % (2/8) positive with hr duration
37.5% (3/8) with both TB Responses within IU/mL of cutoff (red)
Contact
William C. Whitworth, MPH
(404)
Approved for public release; distribution is unlimited. Case Number: 88ABW , 27 Apr 2012, USAF