Interferon gamma release assays (IGRAs) are important tools in identifying prior tuberculosis exposure. The new-generation QuantiFERON-TB Gold Plus (QFT-Plus) assay, recently approved for use in the United States, differs from the current-generation QFT Gold-In-Tube (QFT-GIT) assay with the addition of a second antigen tube that also contains novel CD8+ T-cell-stimulating peptides.
KEYWORDS: diagnostic, nontuberculous mycobacterial infection, latent tuberculosis infection
ABSTRACT
Interferon gamma release assays (IGRAs) are important tools in identifying prior tuberculosis exposure. The new-generation QuantiFERON-TB Gold Plus (QFT-Plus) assay, recently approved for use in the United States, differs from the current-generation QFT Gold-In-Tube (QFT-GIT) assay with the addition of a second antigen tube that also contains novel CD8+ T-cell-stimulating peptides. The QFT-Plus assay has increased sensitivity in immunocompromised populations, and we sought to assess the specificity of QFT-Plus compared to that of QFT-GIT in low-risk individuals. We enrolled adults without tuberculosis risk factors, including a subgroup with pulmonary nontuberculous mycobacterial (NTM) disease due to Mycobacterium avium complex (MAC) or Mycobacterium abscessus. The primary outcome measures included specificity, interassay concordance, and agreement between the QFT-Plus and QFT-GIT assays. Of 262 participants enrolled, 51 had pulmonary NTM. The median age was 39 years (age range, 18 to 78 years); 73% were female. Among the 262 individuals who were enrolled, 5 (1.9%) individuals had positive QFT-Plus results, and 3 of these individuals also had positive QFT-GIT results. The two individuals with discordant results (QFT-Plus positive/QFT-GIT negative) had only one tube positive in the QFT-Plus assay. The overall specificity of QFT-Plus and QFT-GIT was 98.1% (95% confidence interval [CI], 95.6, 99.4%) and 98.9% (95% CI, 96.7, 99.8%), respectively. The QFT-Plus specificity was similar in both the NTM (98.0% [95% CI, 89.4, 99.9%]) and non-NTM (98.1% [95% CI, 95.2, 99.5%]) groups. QFT-Plus has a high specificity, similar to that of the QFT-GIT assay, including in patients with pulmonary MAC or M. abscessus disease.
INTRODUCTION
Despite its diminishing incidence in the United States, tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. In the United States, recent efforts have focused on eliminating the reservoir of those with latent TB infections (LTBI) who might later progress to active TB (1). Recent guidelines advocate using interferon gamma (IFN-γ) release assays (IGRAs) and/or the tuberculin skin test (TST) to identify those with latent infection. Two IGRAs are available for screening, including the 3rd-generation QuantiFERON-TB Gold (QFT-GIT; Qiagen, Hilden, Germany) test and the T-SPOT TB test (Oxford Immunotec, UK).
The 3rd-generation QFT-GIT test measures responses to the Mycobacterium tuberculosis antigens ESAT-6, CFP-10, and 7.7 (2). Recently, a 4th-generation assay, the QuantiFERON-TB Plus (QFT-Plus) assay, which utilizes two antigen tubes, has been developed and approved. The first tube contains only ESAT-6 and CFP-10 (7.7 has been removed from the assay), meant to elicit a CD4+ response, and the second tube includes these same antigens plus additional proprietary peptides designed to stimulate TB-specific CD8+ T lymphocytes (3). Recent studies have found that both CD4+ and CD8+ T lymphocytes produce IFN-γ in the presence of M. tuberculosis antigens. M. tuberculosis-specific CD8+ responses have been detected in patients with LTBI as well as those with active TB (4–7). Moreover, ESAT-6- and CFP-10-specific CD8+ T lymphocytes are described as being detected more often in patients with active TB disease than in those with LTBI, which may be related to a more recent exposure to M. tuberculosis (8–10). M. tuberculosis-specific CD8+ T cells producing IFN-γ have also been detected in active TB patients with HIV coinfection (11, 12) as well as young children with TB disease (13).
Few studies have evaluated active nontuberculous mycobacterial (NTM) disease causing false-positive IGRA results. Evidence indicates that IGRAs are not affected by most clinically relevant NTM, as there is little overlap between ESAT-6 or CFP-10 and most NTM species (14). A recent study found that children in Denmark with at least one positive NTM culture (positive for either M. avium complex [MAC], M. abscessus-M. chelonae, M. celatum, M. fortuitum-M. peregrinum, M. gordonae, M. malmoense, or M. xenopi) had no positive or indeterminate IGRA results, indicating a specificity of 100% (15). However, infection with either M. marinum or M. kansasii, both of which express ESAT-6 and CFP-10, has been shown to cause positive IGRA results (16, 17). The cross-reactivity of the proprietary antigens within the secondary tube of the QFT-Plus assay with these or other clinically relevant NTM is unknown. How the removal of 7.7 or the addition of CD8-stimulating peptides affects the sensitivity or specificity in diagnosing LTBI is unclear. In the present study, we sought to compare the specificity of the new QFT-Plus assay with that of the QFT-GIT assay in a population of patients lacking TB risk factors, including a subset of individuals with pulmonary NTM disease.
MATERIALS AND METHODS
Participants.
We prospectively enrolled individuals aged ≥18 and ≤70 years at the Oregon Health & Science University (OHSU) in Portland, OR, USA. Participants were enrolled into one of two groups; group 1 consisted of individuals with no recognized TB risk factors, and group 2 consisted of patients also lacking TB risk factors but who met American Thoracic Society (ATS)/Infectious Diseases Society of America (IDSA) case criteria for nontuberculous mycobacterial (NTM) disease (18). The TB risk factors considered included a history of bacillus Calmette-Guérin vaccination; a documented TB diagnosis or completion of empirical TB therapy; prior contact with a TB case; a history of living or traveling in India, China, sub-Saharan Africa, or Southeast Asia or in an area with a current active TB prevalence of >50 per 100,000 (defined as an area where TB is endemic) for more than 1 month; birth in a country where TB is endemic; and exposure to high-risk environments for TB exposure for more than 1 month (e.g., a nursing home, hospital, or homeless shelter) (19, 20). For group 1 recruitment, we advertised via OHSU's intrainstitutional website for potential study participants. Individuals in group 1 were local to Portland, OR, and were associated with OHSU (nonclinical staff and students). Study patients for group 2 were identified from a chronic chest infection clinic at OHSU where NTM patients were screened for participation. All participants were enrolled on a first come-first served basis until the target enrollment numbers were met. Inclusion criteria for both groups 1 and 2 necessitated an absence of TB risk factors. Individuals were excluded if they had resided or traveled within areas where TB is endemic (defined as residence or travel within a region having a TB incidence of >50/100,000 for >30 days), a prior diagnosis of TB, prior exposure to someone with active TB, a history of living or working for more than 1 month in a jail or a homeless shelter, a history of health care work interacting directly with patients at risk for TB, or a history of an immunosuppressive condition, such as human immunodeficiency virus infection, cancer, or systemic immunosuppressant usage. In addition to the blood sample, we collected demographic, epidemiological, and clinical information pertaining to past TB exposure and previous skin test or QFT-TB test status.
Previous data suggested that the specificity of QFT-GIT is 99.2% (95% confidence interval [CI], 97.7, 99.8%) (QuantiFERON-TB Gold In-Tube product insert [document no. US05990301L], Cellestis Inc., March 2013). We hypothesized that the specificity of QFT-Plus would be similar. Our prespecified noninferiority margin was 3%, providing a lower bound of 96.2% specificity to be necessary to conclude that QFT-Plus was noninferior. Two-hundred three study subjects were needed to ensure a power of 80%, assuming a specificity of 99.0% for the current-generation QFT-GIT assay. Informed consent was obtained prior to enrollment. The Oregon Health & Science University's Institutional Review Board approved the study protocol.
QFT-GIT and QFT-Plus assays.
The assays were performed according to the instructions in the manufacturer's (Qiagen) QuantiFERON-TB Gold In-Tube and QuantiFERON-TB Plus package inserts (2, 3). Whole-blood samples were collected by normal venipuncture and placed into a 9-ml sterile sodium heparin tube for both the QFT-GIT and QFT-Plus assays. One milliliter of blood was transferred to antigen-nil, mitogen, and QFT-GIT antigen tubes and QFT-Plus tube 1 and tube 2 and was incubated within 16 h of the blood draw. There was one set of antigen-nil and mitogen tubes used for the two assays (QFT-GIT and QFT-Plus) in this study. Following a 16- to 24-h incubation period at 37°C, the blood tubes were centrifuged at 2,500 relative centrifugal force (2,500 × g) for 15 min, and the plasma was harvested. Samples were immediately frozen at −70°C and later batch tested.
The QuantiFERON-TB (QFT) enzyme-linked immunosorbent assay procedure was performed by the manual method to detect and measure the amount of IFN-γ produced in each QFT blood collection tube. The results were compared to those obtained by both a 4-point and an 8-point standard curve analysis using Qiagen software and are reported as the number of international units (IU) per milliliter. The 8-point standard curve was used for data analysis for this study. A cutoff value (the value for the IFN-γ antigen tube minus the value for the antigen-nil tube control) of 0.35 IU/ml was used. The QFT-GIT assay was considered positive if the measured antigen response minus the background (antigen-nil) response was >0.35 IU/ml. For the QFT-Plus assay, a positive result was defined as a single antigen tube (either tube 1 or tube 2) with an antigen response of >0.35 IU/ml. A mitogen tube-minus-antigen-nil tube control value of <0.5 IU/ml or an antigen-nil tube value of >8.0 was classified as indeterminate.
Statistical analyses.
We calculated the median, mean, and interquartile range (IQR) for all antigen and negative-control response tubes. We calculated specificity as the proportion of negative cases out of the total patient cohort with the 95% confidence interval (CI). We calculated P values between specificity values for each assay overall and per population, using McNemar's test for 2-by-2 tables. P values for mean IFN-γ concentrations were calculated by the t test. We assessed the agreement between QFT-GIT and QFT-Plus using the overall percentage of concordant results with 95% confidence intervals and calculated Cohen's kappa values using the categorical variables (positive/negative) based on the cutoff values for IFN-γ (21). We performed the analyses using Stata (version 13) software (StataCorp LP, College Station, TX).
RESULTS
Study populations.
The demographic and clinical characteristics of the two study populations are shown in Table 1. There were similar comorbidities between the NTM and non-NTM populations, with the exception of bronchiectasis, which was more common among NTM patients. Patients with NTM disease had predominately M. avium-M. intracellulare (90.2%), with M. abscessus (9.8%) being the other species.
TABLE 1.
Demographic and clinical characteristics
| Characteristic | Values for: |
|
|---|---|---|
| NTM group (n = 51) | Non-NTM group (n = 211) | |
| No. (%) of females | 37 (72.5) | 157 (73.7) |
| Median (range) age (yr) | 65 (18–78) | 34 (18–75) |
| No. (%) of subjects by race | ||
| American Indian or Alaska Native | 1 (1.9) | 1 (0.5) |
| Asian | 13 (6.1) | |
| Black or African American | 2 (0.9) | |
| Multiracial | 1 (1.9) | 13 (6.1) |
| White | 49 (96.1) | 178 (83.6) |
| Unknown or not reported | 6 (2.8) | |
| No. (%) of subjects by Hispanic or Latino ethnicity | ||
| Hispanic or Latino | 1 (1.9) | 10 (4.7) |
| Not Hispanic or Latino | 50 (98.0) | 194 (91.9) |
| Unknown or not reported | 9 (4.3) | |
| No. (%) of subjects infected with the following NTM species: | ||
| M. avium-M. intracellulare | 46 (90.2) | |
| M. abscessus | 5 (9.8) | |
| No. (%) of subjects with the following comorbidities: | ||
| Lung cancer | 1 (2.0) | 1 (0.5) |
| Sjogren's syndrome | 1 (2.0) | 1 (0.5) |
| Type 2 diabetes | 1 (2.0) | 2 (0.9) |
| Gastrointestinal reflux disease | 8 (15.7) | 9 (4.3) |
| Inflammatory bowel disease | 1 (2.0) | 3 (1.4) |
| COPDa | 8 (15.7) | 2 (0.9) |
| Cystic fibrosis | 3 (5.9) | |
| Bronchiectasis | 35 (68.6) | |
| Rheumatoid arthritis | 3 (5.9) | |
COPD, chronic obstructive pulmonary disease.
Test results and assay specificity.
Of 263 enrolled participants, 1 (0.4%; an NTM patient) had an indeterminate result with mitogen values of <0.2 IU/ml for both the QFT-GIT and QFT-Plus assays and was excluded from further analysis. Of the remaining 262 individuals, 5 (1.9%) had positive QFT-Plus results, and 3 of these 5 also had positive QFT-GIT results; no other subjects tested positive by QFT-GIT. Of the three individuals with concordant QFT-Plus-positive/QFT-GIT-positive results, both tube 1 and tube 2 within the QFT-Plus assay were positive. Conversely, the two individuals with discordant QFT-Plus-positive/QFT-GIT-negative results had only one tube positive in the QFT-Plus assay. One of these individuals had only tube 1 positive, while the second individual had only tube 2 positive.
Overall, the specificity of QFT-Plus was 98.1% (95% CI, 95.6, 99.4%). This was similar to that of QFT-GIT (specificity = 98.9% [95% CI, 96.7, 99.8%]; P = 0.45). The kappa value between the two assays was 0.75 (95% CI, 0.41, 1.00), which is in line with previously calculated kappa values (22). The QFT-Plus specificity was similar in both the NTM and non-NTM groups (Table 2).
TABLE 2.
Assay specificity
| Population | % specificity (95% CI) by test |
P value | |
|---|---|---|---|
| QFT-GIT | QFT-Plus | ||
| Total study population (n = 262) | 98.9 (96.7, 99.8) | 98.1 (95.6, 99.4) | 0.45 |
| Non-NTM subgroup (n = 211) | 99.1 (96.6, 99.9) | 98.1 (95.2, 99.5) | 0.67 |
| NTM subgroup (n = 51) | 99.5 (97.4, 100) | 98.0 (89.6, 100) | 0.17 |
Out of the 211 non-NTM subjects enrolled, 4 (1.9%) subjects had positive QFT-Plus results, and 2 of these 4 subjects also had positive QFT-GIT results. No other subjects tested positive by QFT-GIT. The kappa value was 0.66 (95% CI, 0.22, 1.00), indicating a 99.06% agreement between the two tests.
For the 51 NTM patients enrolled, there was 1 patient positive by both assays; in addition, both antigen tubes of QFT-Plus were concordantly positive for this patient (Table 3). No other patients had positive test results with either assay. The kappa value between the two assays was 1.00, indicating a 100.0% agreement between the two tests.
TABLE 3.
Stimulated TB antigen-nil IFN-γ concentration by population
| Population | Mean (IQR) IFN-γ concn (IU/ml) |
P valuea | ||
|---|---|---|---|---|
| QFT-GIT | QFT-Plus |
|||
| Tube 1 | Tube 2 | |||
| Total study population (n = 262) | 0.016 (−0.08, 0.29) | 0.018 (−0.08, 0.37) | 0.043 (−0.15, 0.46) | 0.27 |
| Non-NTM subgroup (n = 211) | 0.046 (−0.05, 0.22) | 0.021 (−0.08, 0.32) | 0.051 (−0.14, 0.34) | 0.28 |
| NTM subgroup (n = 51) | 0.015 (−0.05, 0.02) | 0.005 (−0.04, 0.03) | 0.011 (−0.03, 0.03) | 0.77 |
P value for the interassay comparison between tube 1 and tube 2.
Quantitative IFN-γ values.
The mean (IQR) amounts of stimulated antigen-nil IFN-γ (TB nil) for each tube (tube 1, tube 2, and QFT-GIT) is shown in Table 3. For both the NTM and non-NTM groups, there were no significant differences between tube 1, tube 2, or QFT-GIT antigen-nil tube IFN-γ concentrations. The means for the QFT-GIT antigen-stimulated IFN-γ response (TB antigen nil) are reported in Table 3 (overall and by NTM diagnosis). The results for the individuals with positive assay results are reported in Table 4.
TABLE 4.
Stimulated TB antigen-nil IFN-γ concentration for participants with at least one positive test result
| Participant | NTM infection status | Mean (IQR) IFN-γ concn (IU/ml)a |
||
|---|---|---|---|---|
| QFT-GIT | QFT-Plus |
|||
| Tube 1 | Tube 2 | |||
| 1 | No | 0.09 | 0.4a | −0.24 |
| 2 | No | 0.01 | 0.01 | 5.44a |
| 3 | No | 0.56a | 0.66a | 0.73a |
| 4 | No | 0.43a | 0.37a | 0.46a |
| 5 | Yes (MAC) | 1.25a | 0.54a | 0.77a |
The value is above the 0.35-IU/ml cutoff point considered positive.
DISCUSSION
We evaluated the QFT-Plus IGRA among a cohort of individuals lacking TB risk factors, including patients with pulmonary NTM disease. Our findings suggest that QFT-Plus has a high specificity similar to that of the current-generation QFT-GIT assay. Further, we found a high specificity and strong agreement between the two tests among both NTM and non-NTM individuals in our cohort, suggesting that the new QFT-Plus assay is unlikely to be false positive in most cases of pulmonary NTM.
To date, the specificity of the new QFT-plus assay has received little evaluation. Barcellini et al. recently evaluated QFT-Plus (7) and found a specificity of 97.17% among 106 low-risk persons and zero indeterminate results. The calculated specificity was similar to that found in our study, and interestingly, among the three positive patients in that study, only one had concordant positive results between tube 1 and tube 2 QFT-Plus antigen tubes. The remaining two study subjects had discordant tube 1/tube 2 results, similar to our study, in which two of the five subjects with positive results lacked concordant tube 1/tube 2 results, and both of these cases were negative by the QFT-GIT assay. This discordance suggests the possibility that such results were false positive. While the lack of a gold standard makes such a conclusion speculative, we believe that enrollees with concordant tube 1/tube 2 results, in addition to a positive QFT-GIT result, are more likely to have LTBI. Consistent with this thinking, a recent study from Stanford University (22) among low-risk health care workers examined the use of a conservative definition of a positive QFT-Plus conversion by requiring positive results in both the tube 1 and tube 2 antigen tubes (rather than the manufacturer's use of either tube being positive). Similar to our study, the Stanford University study found among 626 health care workers with no LTBI risks that the QFT-GIT and QFT-Plus specificity was 97.9% and 97%, respectively. Using the conservative definition necessitating concordant positive tube 1 and tube 2 results, the QFT-Plus specificity increased to 99% (P = 0.07). In our study, if we used this approach and classified the two individuals with discordant positive tube 1/tube 2 results as being overall test negative, then the specificity would be similar at 98.9%.
There is a lack of data assessing IGRA specificity in the population of patients with NTM (15, 23), and to our knowledge, no prior study has evaluated IGRAs within a cohort of patients with pulmonary M. avium or M. abscessus infection. Together, these two species account for more than 90% of cases of pulmonary NTM disease in the United States (24, 25). Among our NTM cohort, only 1 of 51 patients was positive by either the QFT-GIT or QFT-Plus assay, and they were concordantly positive by both assays, with, in addition, both tube 1/tube 2 being positive as well. This individual was infected with MAC and had concurrent cystic fibrosis, and after repeated interviews, absolutely no TB risk factors could be elicited. The subject was retested outside this protocol with an additional QFT-GIT test and remained positive. Lastly, there was one patient with indeterminate results, which were concordant in both assays due to a low mitogen control result. This individual was receiving hydroxychloroquine (200 mg daily) for lupus that was not initially reported.
ATS, IDSA, and the U.S. Centers for Disease Control and Prevention have jointly published new recommendations/guidelines for the diagnosis of M. tuberculosis infection, including LTBI (26). Their new recommendations for LTBI diagnostic testing are based on the likelihood of infection with M. tuberculosis and, if the individual is infected, the likelihood of progression to TB disease. The new testing strategy recommends dual testing in some situations, and with dependence on the probability of prior exposure and the risk of progression if the individual is infected, utilization of the results from two tests (IGRA or TST) is recommended. This strategy is in line with prior recommendations (27, 28). The results from our study might suggest the possibility that the new QFT-Plus assay could assist in such a two-test strategy. If two antigen tubes of QFT-Plus are considered dual IGRA testing within a single assay, it would follow that result interpretation could take the same risk-stratified approach suggested by current LTBI testing guidelines. Results from both the study of Barcellini et al. (7) and the Stanford University study (22) support this risk-stratified approach, whereby requiring positivity in both tubes could maximize specificity and potentially minimize false-positive results. However, if the patient had a risk of exposure and a high risk of progression, then accepting either one as positive in defining a positive assay result would provide maximal sensitivity.
Our study was limited by its relatively small size and the lack of a gold standard assay for LTBI with which to compare assay results. Our study provides reassurance that the new QFT-Plus assay will not cross-react with most clinically important NTM, such as MAC and M. abscessus. We were not able to assess this question in regard to other NTM species, like M. marinum or M. kansasii, which contain ESAT-6 and CFP-10 and are known to cause positive IGRA results (29–31).
In summary, our study found the performance of the QFT-Plus test to have a similar and acceptable specificity in a low-risk population in comparison to the specificity of the QFT-GIT assay. Our study adds to the small body of literature on IGRA specificity in the NTM population. Future studies should evaluate the outcomes for individuals with discordant tube 1/tube 2 results among both high-risk and low-risk populations. It is possible that a definition of overall test positivity that necessitates interassay concordance between tube 1 and tube 2 could have higher utility in screening low-risk populations, whereby specificity is preferred.
ACKNOWLEDGMENTS
We thank the participants who graciously consented to participate in this study. We acknowledge Megan Wardrop for her help.
We all contributed to the study design, interpretation of the analyses, and critical revision of the manuscript.
This work was supported by Qiagen, Inc.
K.L.W. has received an investigator-initiated grant from Qiagen, Inc.
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