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Urgent improvements in the diagnosis and management of Mycobacterium tuberculosis infection are required to reach End TB goals. Conventional interferon-gamma release assays (IGRAs), such as QuantiFERON-TB Gold Plus (QFT-Plus), require substantial laboratory infrastructure and large blood volumes, limiting use in high-burden settings. The QIAreach QuantiFERON-TB (QIAreach QFT) was developed to overcome these challenges but has not previously been evaluated in field conditions in a low-income, high-burden country, or at scale in children. We performed a diagnostic evaluation of QIAreach QFT against QFT-Plus, in a cross-sectional IGRA survey in Blantyre, Malawi. We recruited a population-representative sample of children aged 1-4 years and adolescents and adults aged 10-40 years, from households and primary care. We calculated sensitivity, specificity, and Cohen's kappa for QIAreach QFT against QFT-Plus, and constructed Bayesian hurdle-categorical models to compare quantitative test results. A total of 1,049 participants were recruited (64%: 1-4 years; 13%: 10-19 years; and 23%: 20-40 years). More participants had a positive QIAreach QFT result (32%) compared to QFT-Plus (15%). Over half of positive QIAreach QFT results had time-to-positivity of exactly 20 min, the assay cutoff. There was minimal agreement between QFT-Plus and QIAreach QFT results ( κ = 0.26), which was lowest in children aged 1-4 years ( κ = 0.13). Sensitivity and specificity of QIAreach QFT relative to QFT-Plus were 62% and 74%, respectively, with poor correlation between quantitative results. The suboptimal performance of QIAreach QFT, particularly in young children, suggests that it cannot currently be recommended for wider use and that the urgent need for an accessible test of Mtb infection remains unmet., Importance: Almost a quarter of the world's population has evidence of Mycobacterium tuberculosis (Mtb) infection. Monitoring and addressing this substantial burden of so-called "latent" tuberculosis (TB) infection will be critical to reach End TB targets. However, current interferon-gamma release assays (IGRAs) for Mtb infection are costly, and require a large volume of venous blood and significant laboratory processing, which are major barriers to their wider use in low-income countries. The novel QIAreach QuantiFERON-TB (QIAreach) assay has been designed as a more accessible alternative. We sought to evaluate it against a reference standard of QuantiFERON-TB Gold Plus, in a large cross-sectional survey in Blantyre, Malawi. To our knowledge, this is the first diagnostic evaluation of QIAreach QFT to be performed in a population-based survey in a low-income high-incidence setting, and to specifically focus on young children (a priority group for interventions targeting Mtb infection). In contrast to previous studies in other settings, we observed poor performance of QIAreach QFT, particularly in young children where there was little correlation between the novel test and the reference standard. This leads us to conclude that this test cannot be widely recommended for use in its current form; indeed manufacture is currently suspended. We believe our findings are of urgent importance to policymakers, clinicians, and researchers and underscore the importance of careful evaluation of new diagnostics in the contexts where they are intended to be used., Competing Interests: The manufacturers of QFT-Plus and QIAreach QFT (QIAGEN) provided tests at reduced or no cost for the purposes of research and technical support for quality control but played no role in study design, collection, analysis, or dissemination of findings.

Immunological tests, including the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, represent an important aid for diagnosing active tuberculosis (TB) and latent TB infections in children, but concerns about their use in children <5 years of age persist. This is a multicenter retrospective study comparing a population of 226 children to 521 adults with pulmonary or extrapulmonary TB. The aim was to evaluate the QFT-IT performance, analyzing both qualitative and quantitative results, according to age, birthplace, and disease localization. Compared to culture, QFT-IT sensitivity was 93.9%, 100%, and 94.4% in children ≤2, 2 to 5, and 5 to 16 years of age, respectively, and was significantly higher than that in adults (81.0%) ( P  < 0.0001). The rate of indeterminate test results for children (2.2%) was significantly lower than that for adults (5.2%) ( P  < 0.0001). In children, QFT-IT sensitivity was not affected by disease localization or birthplace (Italy born versus foreign born). Interferon gamma (IFN-γ) values in response to TB antigen and mitogen were significantly higher in children than in adults (TB antigen, median of 10 versus 1.66 IU IFN-γ/ml; mitogen, median of 10 versus 6.70 IU IFN-γ/ml; P  < 0.0001). In summary, this study supports the use of QFT-IT as a complementary test for the diagnosis of pediatric TB even under 2 years of age. Our observations could be applicable to the new version of the test, QuantiFERON-TB Gold Plus, which has recently been shown to have similar sensitivity in active TB, although data in children are still lacking., (Copyright © 2019 American Society for Microbiology.)

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 abscessu s. 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., (Copyright © 2018 American Society for Microbiology.)

QuantiFERON-TB Gold Plus (QFT-Plus) is a new-generation QuantiFERON-TB Gold In-Tube (QFT-GIT) assay which has two antigen-coated tubes called TB1, which contains long peptides derived from ESAT-6 and CFP-10, and TB2, which contains the same components as TB1 and additional short peptides which potentially stimulate CD8 + T cells through the presentation of major histocompatibility complex class I. This is the first study to compare QFT-Plus and QFT-GIT for use in the diagnosis of latent tuberculosis infection (LTBI) among immunocompromised patients in the Republic of Korea. Among 317 consecutive patients who underwent screening for LTBI before solid organ or hematopoietic stem cell transplantation and tumor necrosis factor alpha inhibitor treatment, LTBI was identified in 92 (29.0%) and 88 (27.8%) patients by QFT-GIT and QFT-Plus, respectively. The rate of concordance between QFT-GIT and QFT-Plus was 93.7% (κ value, 0.860), and the indeterminate rate (3.2%) was similar between QFT-GIT and QFT-Plus. Of 20 (6.3%) samples with discordant results, 11 (55.0%) and 7 (35.0%) were positive by QFT-GIT alone and QFT-Plus alone, respectively, and 2 (15.0%) were indeterminate by each assay. The interferon gamma level in samples with discordant results ranged from 0.39 to 1.10 IU/ml, except for one sample, in which the gamma interferon level was 2.97 IU/ml only in TB2. Conclusively, there was a high degree of agreement between the results of QFT-GIT and QFT-Plus for the screening of immunocompromised patients for LTBI. The reactivity in TB2 contributed substantially to the difference between QFT-GIT and QFT-Plus, particularly in solid organ transplant candidates. The significance of the discrete responses in TB1 and TB2 of QFT-Plus needs to be explored further by means of an immunological and clinical approach in different patient groups and clinical settings., (Copyright © 2018 American Society for Microbiology.)

We investigated the prevalence of latent tuberculosis infection (LTBI) among the residents in seven long-term care facilities (LTCFs) located in different regions of Taiwan and compared the performance of two interferon gamma release assays, i.e., QuantiFERON-TB Gold In-Tube (QFT-GIT) and QuantiFERON-TB Gold Plus (QFT-Plus) for screening LTBI. We also assessed the diagnostic performance against a composite reference standard (subjects with persistent-positive, transient-positive, and negative results from QFTs during reproducibility analysis were classified as definite, possible, and not LTBI, respectively). Two hundred forty-four residents were enrolled, and 229 subjects were included in the analysis. The median age was 80 years (range, 60 to 102 years old), and 117 (51.1%) were male. Among them, 66 (28.8%) and 74 (32.3%) subjects had positive results from QFT-GIT and QFT-Plus, respectively, and the results for 215 (93.9%) subjects showed agreement. Using the composite reference standard, 66 (28.8%), 11 (4.8%), and 152 (66.4%) were classified as definite, possible, and not LTBI, respectively. For definite LTBI, the sensitivity, specificity, positive predictive value, and negative predictive value of QFT-GIT were 89.4%, 95.7%, 89.4%, and 95.7%, respectively, and those for QFT-Plus were 100.0%, 95.1%, 89.2%, and 100.0%, respectively. The sensitivity of QFT-GIT decreased gradually with patient age. Compared to QFT-GIT, QFT-Plus displayed significantly higher sensitivity (100.0% versus 89.4%, P = 0.013) and similar specificity (95.1% versus 95.7%). In conclusion, a high prevalence of LTBI was found among elders in LTCFs in Taiwan. The new QFT-Plus test demonstrated a higher sensitivity than QFT-GIT in the older adults in LTCFs., (Copyright © 2018 American Society for Microbiology.)

The QuantiFERON-TB Gold Plus (QFT-Plus; Qiagen, Germantown, MD) interferon gamma release assay (IGRA) received FDA clearance in 2017 and will replace the prior version of the assay, the QFT-Gold In-Tube (QFT-GIT). Here, we compared performances of the QFT-Plus assay and the QFT-GIT version in a diverse patient population, including patients undergoing evaluation for or follow-up of latent tuberculosis infection (LTBI; n = 39) or active TB infection ( n = 3), and in health care workers (HCWs; n = 119) at Mayo Clinic (Rochester, MN). Compared to the QFT-GIT, the QFT-Plus assay showed 91.2% (31/34) positive, 98.4% (124/126) negative, and 96.6% (156/161) overall qualitative agreement among the 161 enrolled subjects, with a Cohen's kappa value of 0.91 (excellent interrater agreement). Among the 28 patients diagnosed with LTBI at the time of enrollment, the QFT-GIT and QFT-Plus assays agreed in 24 (85.7%) patients; in all four discordant patients, the positivity of the QFT-GIT or QFT-Plus IGRA was associated with low-level interferon gamma (IFN-γ) reactivity, ranging from 0.36 IU/ml to 0.66 IU/ml. Additionally, we document a high degree of correlation between IFN-γ levels in the QFT-GIT TB antigen tube and each of the two QFT-Plus TB antigen tubes, as well as between the QFT-Plus TB1 and TB2 tubes (Pearson's correlation coefficients [ R ] > 0.95). Overall, we show comparable results between the QFT-GIT and QFT-Plus assays in our study population composed of subjects presenting with a diverse spectrum of TB infections. Our findings suggest that the necessary transition to the QFT-Plus assay will be associated with a minimal difference in assay performance characteristics., (Copyright © 2018 American Society for Microbiology.)

Gamma interferon (IFN-γ) 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). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-γ TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml; P = 0.002) or 1.2 ml (0.49 IU/ml; P < 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger 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 for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-γ response in nil (0.04 versus 0.06 IU/ml; P < 0.001) and TB Ag (0.12 versus 0.24 IU/ml; P = 0.004) tubes and increased TB responses (TB Agvigorous minus nilgentle) (0.02 versus 0.08 IU/ml; 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.