Impact of blood volume, tube shaking, and incubation time on the reproducibility

JCM Accepts, published online ahead of print on 21 August 2013 J. Clin. Microbiol. doi:10.1128/jcm.01627-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Impact of blood volume, tube shaking, and incubation time on the reproducibility of QuantiFERON-TB Gold In-Tube assay 3 Rajiv L. Gaur a, Madhukar Pai b, and Niaz Banaei a,c,d# 4 5 6 7 8 9 10 11 12 13 14 a Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. b Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada. c Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, USA. d Clinical Microbiology Laboratory, Stanford University School of Medicine, Stanford, CA, USA. Running Title: Sources of QuantiFERON-TB Gold In-Tube variability # To whom correspondence should be addressed. 3375 Hillview Ave, Room 1106 15 Palo Alto, CA 94304 16 Phone: 650-736-8052 17 Fax: 650-725-5671

18 E-mail: niazbanaei@stanford.edu 19

20 Abstract 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Interferon gamma release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). Yet, studies are showing non-reproducible IGRA results. In this study we investigated the effect of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle vs. vigorous), and incubation duration (16, 20, and 24 hour) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results in 50 (33 uninfected and 17 infected) subjects. The median IFN-γ TB response (TB Antigen (Ag) minus Nil) was significantly higher with 0.8 ml blood (1.04 IU/ml) compared to 1.0 ml (0.85 IU/ml; P=0.002) and 1.2 ml (0.49 IU/ml; P<0.001) in subjects with LTBI. Compared to 0.8 ml (11.8%), there were higher proportions of false negative results with 1.0 ml (29.4%; P=0.2) and 1.2 ml (41.2%; P=0.05) of blood in infected subjects. Blood volume did not significantly change the proportion of positive results in uninfected controls. Compared to gentle shaking, vigorous shaking increased the median IFN-γ response in Nil (0.04 vs. 0.06 IU/ml; P<0.001) and TB Ag (0.12 vs. 0.24 IU/ml; P=0.004) tubes and increased TB response (TB Ag Vigorous minus Nil Gentle ) (0.02 vs. 0.08, P=0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.

42 43 Introduction 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 There are 2 billion individuals worldwide with latent tuberculosis infection (LTBI) (1). Treatment of LTBI is a proven strategy for preventing progression of LTBI to active disease (2, 3). Interferon gamma (IFN-γ) release assays (IGRAs) are relatively new assays developed as an alternative to tuberculin skin test (TST) for diagnosis of LTBI. IGRAs are functional assays that measure T cell response to Mycobacterium tuberculosis-specific antigens, such as ESAT-6, CFP-10 and TB7.7, in whole blood or blood-derived mononuclear cells (4). Two Food and Drug Administration-approved commercial IGRAs are currently available: the QuantiFERON-TB Gold In-Tube assay (QFT-GIT; Qiagen, Carnegie, Australia) and the T-SPOT.TB assay (Oxford Immunotec, Abingdon, United Kingdom). IGRAs have improved specificity and offer logistical advantages as compared to TST (4). In recent years, IGRAs have replaced TST for annual screening of health care workers (HCWs) in many occupational health programs in North America (5, 6). In addition, IGRA conversion rate is now being used as a secondary measure of vaccine efficacy in tuberculosis (TB) vaccine trials (7). Despite much optimism for commercial IGRAs, studies in the past decade have revealed significant variability in IGRA sensitivity and reproducibility (4-6, 8-10). The sensitivity of IGRAs in culture-positive active TB cases has ranged from 65% to 100% (4, 8, 9). In contact investigation studies, the sensitivity of IGRAs for LTBI in patients that progressed to active TB ranged from 40% to 100% (10).

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 Furthermore, studies conducted in HCWs in low incidence settings have shown highly variable IGRA results with serial testing. The rate of conversions (negative to positive result) using the manufacturer recommended cut-offs ranged from 2% to 15% and the rate of reversions (positive to negative result) ranged from 20% to 40% (6, 11). The conversion rates in these studies were higher than expected based on the risk of TB infection in low incidence settings and TST conversion rates. Recent studies have identified several sources of variability related to assay manufacturing, preanalytical processing, analytical testing, and immunological boosting and modulation (12-16). However, causes of the broad IGRA variability in sensitivity and reproducibility are not fully understood. The suboptimal reproducibility and accuracy of IGRAs have important implications for occupational health and vaccine trials performing serial testing. In HCWs, false-negative results can lead to nosocomial outbreaks and false positives can result in excessive treatment and additional testing (17). In vaccine trials, false results can exaggerate or underestimate the protective efficacy of a vaccine candidate. Therefore, it is critical to identify and eliminate or standardize the predictable sources of variability. In this study we investigated the effect of blood volume, the extent of tube shaking and the duration of incubation of tubes on TB response with QFT-GIT assay in a cohort of healthy HCWs both with and without LTBI. 84

85 Methods 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 Study Design. In a prospective study recruiting HCWs, we compared quantitative and qualitative QFT-GIT results after varying (i) blood volume, (ii) tube shaking, and (iii) incubation duration. A schematic overview of the study design is shown in Figure 1. This study was approved by the Stanford University IRB. Study Subjects. Healthy HCWs were recruited from the Stanford University Medical Center Clinical Laboratories. After obtaining consent, subjects were asked to complete a standardized self-questionnaire including information on age, gender, ethnicity, country of birth, countries visited for at least 6 months, BCG vaccination history, prior TST and QFT-GIT results, known exposure to M. tuberculosis, chest X-ray results, and immune status. Prior QFT-GIT results were confirmed in the laboratory information system. Volunteers were invited to enroll if they met criteria for (i) having LTBI (prior history of positive TST ( 10 mm) or QFT-GIT and 1 TB risk factor) or (ii) being an uninfected control (US born with prior negative TST or QFT-GIT and no TB risk factors). Volunteers were excluded if they reported signs or symptoms of active TB. The demographic data and prior results are presented in Table 1. 103 QuantiFERON-TB Gold In-Tube testing. Except for the experimental 104 105 106 modifications described below, all tests were performed according to the QFT- GIT package insert (18). Blood was collected by a trained phlebotomist in a 10 ml Kendall Monoject Green Stopper tube and 1 ml was immediately transferred

107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 to each QFT-GIT tube. Tubes were shaken gently by inverting the tubes 10 times according to package insert and immediately incubated for 24 hours. Plasma was separated and stored at room temperature up to 8 hours before a quantitative enzyme-linked immunosorbent assay (ELISA) for IFN-γ, measured in IU/ml, was performed on a DSX automated system (Dynex Technologies, Chantilly, VA). One IFN-γ ELISA plate was designated for all testing for each subject. Interpretation of results was done by the software provided by the QFT- GIT manufacturer. A positive result was defined per manufacturer as TB response (TB Ag minus Nil) value 0.35 IU/ml and 25% of Nil value. Blood volume. One milliliter of blood was transferred into each of the QFT-GIT blood collection tubes. In addition, 0.8 and 1.2 ml of blood was transferred into two additional TB Ag tubes. IFN-γ TB response was determined for each blood volume using the same Nil result. The volume of blood drawn directly into QFT- GIT tubes was also determined. In randomly selected group of volunteers (n=30), blood was drawn directly into a Nil and TB Ag vacutainer tubes of the same lot number at sea level until the tube appeared filled close to the indicator line, according to the package insert. Immediately after, the blood volume in each tube was measured using a calibrated pipette. 125 126 127 128 Tube Shaking. One milliliter of blood was transferred into each of the two sets of Nil and TB Ag tubes. One set was mixed gently by inverting the tubes 10 times according to package insert, ensuring that the entire inner surface of the tube was coated with the blood. The second set was held in one hand and mixed by

129 130 131 132 vigorously shaking the tubes in an up-and-down motion for 10 seconds. The blood transfer and tube shaking were performed by the same technologist for all study subjects. Immediate incubation of tubes and ELISA were performed as described above. 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 Incubation period. One milliliter of blood was transferred into three sets of QFT- GIT tubes. After mixing the content according to package insert, the sets were incubated at 37 C either for 16, 20, or 24 hours, respectively. ELISA was performed as described above. Statistical analysis. A non-parametric test, the Wilcoxon signed-rank test of medians, was used to compare differences between paired results. The chisquared test was used to determine the statistical significance of the difference between proportions. All statistical tests were computed for a two-sided type I error rate of 5%. Statistical analyses were performed using the Prism software (GraphPad, San Diego, CA). Results Effect of blood volume on TB response. According to the package insert, the standard QFT-GIT vacutainer tubes are calibrated to draw between 0.8 ml and 1.2 ml blood at altitudes from sea-level to 2,650 feet. To determine the distribution of blood volume drawn into QFT-GIT tubes, blood volume drawn into

149 150 151 152 the Nil tube and TB Ag tube from 30 subjects was measured. As shown in Figure 2A, the blood volume in the Nil and TB Ag tubes ranged from 0.785 ml to 1.0 ml (median, 0.923 ml; CV, 5.8%) and 0.810 ml to 1.010 ml (median, 0.930 ml; CV, 4.7%), respectively. 153 154 155 156 157 158 159 160 161 162 163 164 165 166 To investigate the effect of blood volume variability on QFT-GIT results, IFN-γ TB response (TB Ag minus Nil) with 0.8, 1.0, and 1.2 ml of blood per TB Ag tube and 1.0 ml per Nil tube was measured. Results were available for the three blood volumes in 50 (33 uninfected and 17 infected) subjects (Table 2). In uninfected volunteers, the median TB response was significantly higher with 0.8 ml blood (0.01 IU/ml) compared to 1.0 ml (0 IU/ml; P=0.03) and 1.2 ml (0 IU/ml; P=0.04) (Figure 2B). One subject turned positive with 1.2 ml blood, but there was no significant difference in the proportion of positive results between the three blood volumes. In infected subjects, the median TB response was significantly higher with 0.8 ml blood (1.04 IU/ml) compared to 1.0 ml (0.85 IU/ml; P=0.002) and 1.2 ml (0.49 IU/ml; P<0.001) (Figure 2C). 88.2% (15/17) of infected subjects were positive with 0.8 ml blood, 70.6% (12/17) with 1.0 ml blood, and 58.8% (10/17) with 1.2 ml blood (Table 2). The proportion of subjects with positive results was significantly lower with 1.2 ml compared to 0.8 ml (P = 0.05). 167 168 169 170 Effect of tube shaking on TB response. To investigate the effect of blood mixing variability on QFT-GIT results, tubes inoculated with equal volume of blood were mixed either gently or vigorously. Results were available for 40 (23 uninfected and 17 infected) subjects. The median IFN-γ concentration with

171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 gentle and vigorous shaking was 0.04 and 0.06 IU/ml in the Nil tube and 0.12 and 0.24 IU/ml in the TB Ag tube, respectively (Table 3). Compared to gentle shaking, there was a significant increase in median IFN-γ level in the Nil and TB Ag tube with vigorous shaking (P<0.001 and P=0.004, respectively; Figure 3). Although the median TB response did not change significantly between gentle (0.02 IU/ml) and vigorous (0.03 IU/ml) shaking, the median TB response increased significantly when vigorously-shaken TB Ag response was paired with gently-shaken Nil response (Tb Ag Vigorous minus Nil Gentle ; P<0.001) and decreased significantly when gently-shaken TB Ag response was paired with vigorously-shaken Nil response (Tb Ag Gentle minus Nil Vigorous ; P=0.004) (Table 3 and Figure 3). There was no significant difference between the proportion of positive results with gentle (32.5%) and vigorous shaking (35.0%) but the proportion of positive results increased to 42.5% for Tb Ag Vigorous minus Nil Gentle and decreased to 27.5% for Tb Ag Gentle minus Nil Vigorous (Table 3). Effect of incubation duration on TB response. According to the package insert, the QFT-GIT tubes should be incubated between 16 and 24 hours. To determine the effect of incubation duration on TB response, QFT-GIT tubes from 50 (33 uninfected and 17 infected) subjects were incubated at 37 C for 16, 20, and 24 hours. The median TB response in uninfected subjects was 0, 0, and 0 IU/ml and in infected subjects was 0.66, 0.96, and 0.85 IU/ml for 16, 20, and 24- hour incubations, respectively (Table 4 and Figure 4). Except for 16 compared to 24 hours (P=0.04) in the uninfected group, the median TB response was not significantly different between the three incubation times in the uninfected or

194 195 infected subjects (Table 4 and Figure 4). No reversions or conversions were observed in the infected and uninfected subjects (Figure 4). 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 Discussion In this study we investigated the role of blood volume, tube shaking, and incubation duration in reproducibility of the QFT-GIT assay. We showed that blood volume and tube shaking represent novel preanalytical sources of variability that likely contribute to discordant IGRA results in individuals undergoing serial testing. The impact of blood volume variability on TB response is most concerning for false-negative results in patients with TB infection. We showed that compared to 0.8 ml, inoculation of TB Ag tube with 1.0 and 1.2 ml of blood, which are within the range reported by the manufacturer, resulted in 17.6% and 29.4% reduction, respectively, in positive results in infected subjects. Although blood volume correlated with TB response in uninfected subjects, there were no false positive results with 0.8 ml blood in uninfected subjects. Thus, in addition to improving QFT-GIT reproducibility, our findings suggest standardizing blood volume to 0.8 ml may also increase assay sensitivity. In a recent study that included culture-confirmed adult patients, it was shown that 0.3 ml blood per QFT microtube, a modified version of QFT-GIT that requires 0.3 ml blood per tube, resulted in a TB response similar to that obtained with 1.0 ml blood added to standard QFT-GIT tubes (19). Interestingly, TB response with 0.3 ml blood was significantly higher than with 1.0 ml when higher antigen concentration (3

216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 μg/ml) was used in the microtube. Altogether, these findings suggest a smaller blood volume (or increased antigen concentration) may improve detection of TB response in infected individuals. Further studies are needed to determine the optimum blood volume for maximizing QFT-GIT sensitivity without compromising specificity. A limitation of this study includes not including an assessment of the impact of blood volume on the IFN-γ concentration in the Nil tube. We also confirmed heterogeneity in the volume of blood drawn into TB Ag tubes (range, 0.810 ml to 1.010 ml; CV, 4.7%). Factors that contribute to blood volume variability include blood pressure of the individual at the time of blood draw, the position of vacutainer tube relative to venipuncture site, and altitude where blood is being drawn. Blood pressure has not been correlated with TB response in previous reproducibility studies but it may be an important factor that accounts for some of the within-subject variability observed in serial testing studies. The manufacturer of QFT-GIT produces two types of blood collection tubes; one tube type is calibrated for low altitude (sea-level to 2,650 feet) blood collection while the second type is produced for high (3,350 and 6,150 feet) altitude use. Users outside these altitude ranges (between 2,650 and 3,350 feet or above 6,150 feet) are instructed to collect blood using a syringe and transfer 1 ml to each of the three tubes. Future studies are needed to investigate the effect of altitude on blood volume drawn into standard and high altitude QFT-GIT tubes. 236 237 The impact of rigorous tube shaking on QFT-GIT results would depend on whether the Nil and TB Ag tubes are shaken identically or not. We showed that

238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 vigorous shaking caused a significant increase in TB response when vigorouslyshaken TB Ag response was paired with gently-shaken Nil response and a significant decrease in TB response when gently-shaken TB Ag response was paired with vigorously-shaken Nil response. These results imply that if the Nil and TB antigen tubes are shaken separately as blood is sequentially drawn into the Nil and TB Ag tube, respectively, there is a possibility that differential shaking will result in an either false positive or false negative QFT-GIT result, especially when TB response is bordering the assay cut-off of 0.35 IU/ml. In the absence of a preventative TB vaccine (7), LTBI treatment remains the only proven strategy for preventing progression of latent infection to active disease (2). Epidemic modeling studies suggest that eradication of LTBI in high burden countries is necessary for successful elimination of TB (20, 21). Given the critical role IGRAs serve in identifying those individuals that need prophylactic treatment, it is imperative that we apply the knowledge gained from reproducibility studies to improve the sensitivity and reproducibility of IGRAs. Emphasis on the rigorous standardization of all controllable variables, two of which are highlighted in this study, is necessary to optimize the QFT-GIT assay. For example, blood volume variability can be avoided by collecting blood using a syringe and transferring an equal volume of blood into each QFT-GIT tube. A less standardized, but more practical, approach may include direct blood draws in the QFT-GIT tubes with close monitoring of the blood level relative to the 1.0 ml indicator line. Variable shaking can be minimized if all three tubes are simultaneously mixed gently in one hand. Alternatively, a roller mixer instrument may prove more effective for

261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 standardization of mixing. Incubation delay is another preanalytical source of variability that has been shown to negatively impact TB response in IGRAs (12, 22-25). Immediate incubation of QFT-GIT can be achieved with the use of a portable incubator in the field or placement of an incubator in the phlebotomy station as reported previously (12, 26, 27). However, not all sources of variability can be eliminated through assay standardization. There is a need for a borderline zone to account for unpredictable variation that occurs due to random sources of error such as analytical (13). The contribution of each of the three preanalytical variables to IGRA variability in this study proved to be mechanistically counter-intuitive. For example, one might hypothesize that higher blood volume would yield a greater TB response due to higher number of antigen presenting cells (APCs) and T cells. To the contrary, we found that 0.8 ml of blood produces a significantly higher TB response compared to 1.0 and 1.2 ml of blood. This phenomenon is likely related to higher ratio of TB antigen to APCs and or T cells. The importance of antigen availability for T cell activation has been previously described for initiation of adaptive immunity but its effect on stimulation of effector T cells in IGRAs is not documented (28). Similarly, one might also hypothesize that the length of time T cells are stimulated with TB antigens correlates with IFN-γ release; therefore longer incubation of QFT-GIT tubes would lead to greater TB response. To the contrary, we did not find a significant change in TB response in infected subjects when QFT-GIT tubes were incubated for 16, 20, or 24 hours. Our finding, however, is inconsistent with an unpublished study showing a significantly lower

284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 TB response with shorter incubation times (16-17 hours vs. 23-24 hours) (29). The enhanced IFN-γ release with vigorous shaking of the Nil and TB Ag tubes was also unexpected given that, until recently, the QFT-GIT package insert instructed users to mix the tubes by SHAKING VIGOROUSLY for 5 seconds to ensure that the entire inner surface of the tube has been coated with the blood. The mechanism through which vigorous shaking stimulates T cells to release IFN-γ is not known, but it may occur through direct and or indirect activation of blood cells. Shear forces are well known stimuli for dose and time dependent activation of platelets (30). In summary, we identified blood volume and tube shaking as previously unrecognized preanalytical sources of IGRA variability. With many sources of variability becoming known, studies are now needed to quantify the improvements in IGRA reproducibility and accuracy after elimination or standardization of systematic sources of variability. Acknowledgements We thank Mady Slater for reviewing the manuscript and the study subjects for participating in this study. 302 303 References

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413 414 415 416 417 418 419 420 Figure 1. Schematic overview of the study design. Subjects underwent QuantiFERON-TB Gold In-Tube (QFT-GIT) testing to investigate the effects of blood volume variability, shaking variability, and incubation duration variability on results. TB Ag, TB Antigen. 421

422

423 424 425 426 427 428 429 430 431 432 Figure 2. The effect of blood volume variability on QuantiFERON-TB Gold In- Tube (QFT-GIT) results. (A) Distribution of blood volume drawn into Nil and TB Ag tubes in 30 subjects. TB Ag, TB Antigen; CV%, coefficient of variance. (B and C) IFN-γ TB Ag-Nil values for 33 uninfected (B) and 17 infected (C) subjects tested with the indicated blood volumes in TB Ag tube. The assay cut-off for positive results (TB Ag-Nil value 0.35 IU/ml) is marked with a dashed line. Values <0 IU/ml are shown as 0 IU/ml. Wilcoxon signed-rank test was used to compare differences in medians. Downloaded from http://jcm.asm.org/ on April 10, 2018 by guest

433 434 435 436 437 438 439 440 Figure 3. The effect of tube shaking on QuantiFERON-TB Gold In-Tube results. (A and B) IFN-γ values in Nil (A) and TB Ag (B) tube with gentle and vigorous shaking. (C) IFN-γ TB Ag-Nil values with gentle and vigorous shaking. Data is representative of 40 (23 uninfected and 17 infected) subjects. The assay cut-off for positive results (TB Ag-Nil value 0.35 IU/ml) is marked with a dashed line. Values <0 IU/ml are shown as 0 IU/ml. Wilcoxon signed-rank test was used to compare differences in medians. TB Ag, TB Antigen; Gen, gentle; Vig, vigorous. 441

Downloaded from http://jcm.asm.org/ 442 443 444 445 446 447 448 Figure 4. The effect of incubation duration variability on QFT-GIT results. (A and B) IFN-γ TB Ag-Nil values for 33 uninfected (A) and 17 infected (B) subjects tested with indicated duration of incubation. The cut-off for positive results (TB Ag-Nil value 0.35 IU/ml) is marked with a dashed line. Values <0 IU/ml are shown as 0 IU/ml. Wilcoxon signed-rank test was used to compare differences in medians. on April 10, 2018 by guest

Table 1. Demographic data and LTBI risk factors for the study subjects. Parameters a Number (%) of subjects Sex Male 19 (38) Female 31 (62) Age 44 ± 13.7 b Ethnicity White 22 (44) Asian 18 (36) African/African American 1 (2) Hawaiian/Pacific Islander 3 (6) Indian Continent 5 (10) Native American 1 (2) Place of Birth USA 19 (38) Foreign Country 31 (62) Previous TST result Negative 29 (58) Positive 17 (34) NA 4 (8) QFT-GIT result Negative 29 (58) Positive 17 (34) NA 4 (8) a NA, not available. b Mean age (in years) ± standard deviation

Table 2. QuantiFERON-TB Gold In-Tube results with variable blood volume. Subjects TB Ag Nil Median (Range) 0.8 ml 1.0 ml 1.2 ml P value % Positive TB Ag Nil Median (Range) % Positive TB Ag Nil Median (Range) % Positive 0.8 vs. 1.0 ml 0.8 vs. 1.2 ml Uninfected (n=33) 0.01 (-0.03-0.13) 0 0.00 (-0.10-0.08) 0 0.00 (-0.15-0.59) 3 0.03 0.04 0.06 1.0 vs. 1.2 ml Infected (n=17) 1.04 (0.26-13.59) 88.2 0.85 (0.06-8.58) 70.6 0.49 (0.02-8.67) 58.8 0.002 <0.001 <0.001 TB Ag, TB Antigen; P value is for TB Ag-Nil Downloaded from http://jcm.asm.org/ on April 10, 2018 by guest

Table 3. QuantiFERON-TB Gold In-Tube results with variable tube shaking. TB Response Median (range) P value* % Positive (n=40) P value* Nil Gen 0.04 (0.02-0.29) - - - Nil Vig 0.06 (0.02-9.16) <0.001 - - TB Ag Gen 0.12 (0.02-18.28) - - - TB Ag Vig 0.24 (0.02-31.81) 0.004 - - Tb Ag Gen - Nil Gen 0.02 (-0.098-18.22) - 32.5 - Tb Ag Vig - Nil Vig 0.03 (-0.99-22.65) 0.35 35.0 0.81 Tb Ag Gen - Nil Vig 0.00 (-7.08-17.48) <0.001 27.5 0.63 Tb Ag Vig - Nil Gen 0.08 (-0.04-31.60) 0.004 42.5 0.36 Vig, vigorous; Gen, gentle *Compared to gentle shaking: Nil Gen, Tb Ag Gen and Tb Ag Gen - Nil Gen

Table 4. QuantiFERON-TB Gold In-Tube results with variable incubation duration. Subjects TB Ag Nil Median (Range) 16 hr 20 hr 24 hr P value % Positive TB Ag Nil Median (Range) % Positive TB Ag Nil Median (Range) % Positive Uninfected (n=33) 0.00 (-0.15-0.08) 0 0.00 (-0.11-0.07) 0 0.00 (-0.10-0.08) 0 0.53 0.04 0.28 Infected (n=17) 0.66 (0.04-8.97) 70.6 0.96 (0.05-8.58) 70.6 0.85 (0.06-8.58) 70.6 0.25 0.98 0.17 TB Ag, TB Antigen; hr, hours; P value is for TB Ag-Nil 16 vs. 20 hr 16 vs. 24 hr 20 vs. 24 hr Downloaded from http://jcm.asm.org/ on April 10, 2018 by guest