Tuberculosis (TB), the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, is one of the top-ten causes of preventable death globally.1 The World Health Organization (WHO) estimates that in 2019 alone, around 10 million people suffered from TB and 1.4 million people died from the disease,1 with one-third of humans having M. tuberculosis infection.2

Diagnosis and treatment of TB infection are core conceptual elements of the TB elimination strategy,3-6 as reflected in WHO's emphasis on TB prevention in its End TB Strategy.1

Few tests are available to detect TB infection. The century-old tuberculin skin test (TST) is based on delayed type hypersensitivity reaction in the skin upon intradermal injection of purified protein derivative (PPD) from mycobacterial culture. Although TST is still widely utilized, it has several limitations7-12 that interferon-γ (IFN-γ) release assays (IGRAs) have been developed to overcome.13 IGRAs are in vitro blood assays that measure the levels of IFN-γ released by T lymphocytes stimulated with antigenic peptides of M. tuberculosis.3,4,12-17 Two WHO-endorsed IGRAs are commonly used to detect TB infection: T-SPOT®.TB (Oxford Immunotec, Abingdon, UK) and QuantiFERON®-TB Gold Plus (QFT-Plus, QIAGEN, Hilden, Germany).18,19 The QFT-Plus assay, the fourth generation of QuantiFERON®-TB, is designed to measure IFN-γ released by both CD4 and CD8 T cells.20,21 To date, both WHO-endorsed IGRA tests need quality laboratory support, potentially limiting their use in peripheral and/or limited resource settings.

Lateral flow immunoassays (LFAs) are portable, easy to use outside specialized laboratory environments, and provide a quick readout, making them ideal point-of-care (POC) tests.22 QIAGEN has recently developed a new diagnostic test for TB infection, the QIAreachTM QuantiFERON-TB (QIAreach QFT) assay. This novel digital fluorescence LFA uses nanoparticle technology to measure the levels of IFN-γ in plasma released from both CD4 and CD8 T cells, thus eliminating the need for enzyme-linked immunosorbent assay (ELISA).23 QIAreach QFT, which uses the same test tube as the TB2 tube of QFT-Plus, is an easy-to-use rapid test requiring less instrumentation and blood volume than QFT-Plus. Key characteristics of the QIAreach QFT assay compared to QFT Plus are presented in Table 1.

No previous study has compared the new QIAreach QFT test against the established (FDA-approved and CE-marked) QFT-Plus test in detecting TB infection. Furthermore, the potential role of QIAreach QFT as a diagnostic test for TB infection has never before been evaluated.

The aims of this study were to 1) compare the QIAreach QFT and QFT-Plus tests to detect TB infection; 2) evaluate the clinical performance of QIAreach QFT for detection of TB infection by analyzing plasma samples from patients with active TB disease and healthy or low-TB-risk individuals in a clinical setting; and 3) conduct a preliminary evaluation of the QIAreach QFT test in immunocompromised individuals.

This is the first evaluation of a new diagnostic test, QIAreach QFT, in detecting TB infection compared with the QFT-Plus assay and as a screening tool for TB infection.

This new IGRA test is based on digital fluorescence LFA with nanoparticle technology. Similar to the QFT-Plus assay, it measures the levels of IFN-γ secreted from both CD4 and CD8 T lymphocytes in response to stimulation with M. tuberculosis-specific antigens, with advantages in use as point-of-care-test.24

The QIAreach QFT test exhibited high clinical performance: 100% sensitivity, 97.6% specificity, and 98.8% overall concordance using QFT-Plus as the reference standard. Sensitivity for detection of active TB was also 100%. The specificity and sensitivity of the QIAreach QFT assay reported here are comparable to those previously reported for the QFT-Plus assay. An assessment of the performance of the QFT-Plus assay among active TB patients and healthy individuals in Japan reported 96.2% sensitivity and 96.7% specificity.25 According to a meta-analysis of 15 published reports, the QFT-Plus assay had a pooled sensitivity of 94% for active TB patients and a pooled specificity of 96% for healthy individuals.26 A multicenter investigation into the performance of QFT-Plus test at three U.S. sites and two Japanese sites found the test to have 93.0% sensitivity in adult TB patients.27

Notably, the values of the IFN-γ levels shown in Fig. 1.1 and 1.3 are the uncorrected and corrected values of TB2 tubes of the QFT-Plus assay for all the samples tested—i.e., the level of IFN-γ in each TB2 tube without and with subtracting the level of IFN-γ of the Nil tube (background tube). Our results demonstrated that the cut-off point of IFN-γ concentration for QIAreach QFT assay might be similar to that of the QFT-Plus assay (0.35 IU/mL). We did find a statistically significant relationship between levels of uncorrected and corrected IFN-γ in plasma of active TB patients and TTR (natural logarithms conversion of each) with a linear regression analysis (r = -0.913, p < 0.001 and r = -0.918, p < 0.001, respectively) (Fig. 1.3 and 1.4). This study's results suggest that the higher the IFN-γ level of the sample, the shorter the TTR, which could be used as a surrogate marker of IFN-γ concentration in plasma when using QIAreach QFT assay. Like QFT-Plus, however, QIAreach QFT may have variations in measured IFN-γ values near the cutoff (i.e., 0.2 - 0.7 IU/mL), so there is a relatively high possibility of false negatives and/or false positives for the QIAreach QFT results with a TTR close to 1200 seconds.

Seven cases in the active TB group who were immunocompromised (CD4 <200/μL) returned positive results on QIAreach QFT, suggesting this assay could be considered a promising digital fluorescence LFA for detecting TB infection among immunocompromised patients. Larger studies on representative cohorts are needed to confirm its performance in those immunocompromised.

Our assessment of this cohort of plasma samples revealed a single false positive from a healthy individual. This was a sole sample with a high triglyceride level (1588 mg/dL) and was turbid following the freeze and thaw processes. Testing on this individual was repeated by obtaining a new sample exhibiting a high triglyceride level (1288 mg/dL) that was not subjected to freeze and thaw processes. A negative result was obtained. Various factors, such as sample viscosity, may have affected the development speed of the sample solution on the nitrocellulose membrane of the LFA system. Of note, the false-positive results of QIAreach QFT assay could be caused by milky plasma as well as autoimmune disease.28

The QIAreach QFT assay offers a number of workflow advantages over more complex laboratory-based assays, such as QFT-Plus (Table 1). The QIAreach QFT assay is objective—reporting test results as either positive or negative—and it requires only 1 mL of blood from each patient, compared with 4 mL of blood required for the QFT-Plus assay. In addition, the QIAreach QFT test results can be obtained within a relatively short time of up to approximately 20 min for each specimen analyzed. In contrast, the QFT-Plus test based on ELISA requires at least 150 min to obtain a readout. Moreover, the QIAreach system can be used for a single test or up to eight tests at a time for each eHub being used. The QIAreach QFT testing system and hardware can be used anywhere, like the QIAreach anti-SARS-CoV-2 total test.23 Implementing the QIAreach QFT testing system does not require any specialized instruments (e.g., an automated ELISA workstation), trained laboratory officers to perform ELISA or a dedicated laboratory space; importantly eHub is battery operated allowing its use in remote areas with limited electricity supply. These features make the QIAreach QFT a suitable and highly attractive test to detect TB infection in decentralized settings.

A TB diagnostic test with these characteristics is of particular value for screening efforts in countries with high TB prevalence.24 In 2019, TB cases in countries in Southeast Asia and Africa accounted for 69% of the total TB cases worldwide.1 However, these settings often lack the resources for maintenance and calibration, the infrastructure for complex instrumentation, and the specialized laboratory staff needed for older laboratory-based assays. Many of these low-resource settings that need TB infection screening are outside of major urban centers. The QIAreach QFT test will also be useful for TB infection screening among special groups, including—among others—immigrants,19 inmates,29 and children.

The study has clear limitations, including the fact it was conducted in a single center and on a convenience sample (i.e., being a preliminary study, no sample size calculation was performed), thus requiring larger studies to confirm the findings.

In terms of clinical performance, the QIAreach QFT assay displayed 100% sensitivity, 97.6% specificity, and 98.8% overall concordance compared with the QFT-Plus test. This assay is objective, quicker to perform than QFT-Plus, and requires only a small amount of blood (1 mL) per test. The test offers flexibility in that it can be easily performed anywhere and is not restricted to a laboratory environment. This novel assay can be useful in screening for TB infection in high-TB-burden, low-resource countries and, also, for screening of immunocompromised patients. Larger studies are necessary to confirm these preliminary findings.

Authors’ contributions were as follows: study conception and design (FK, KT), acquisition of data (FK, AK, KA, KT), and analysis and interpretation of data (FK, KT, SN, MH). All authors have contributed substantially to drafting and revising the article critically for important intellectual content. All authors approved the submitted version of the article. K. Fukushima: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing. K. Akagi: Data curation, Writing - original draft, Writing - review & editing. A. Kondo: Data curation, Writing - original draft, Writing - review & editing. T. Kubo: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing. N. Sakamoto: Formal analysis, Investigation, Writing - original draft, Writing - review & editing. H. Mukae: Formal analysis, Investigation, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. All authors approved the submitted version of the article.