Comparative performance evaluation of QIAreach QuantiFERON-TB and tuberculin skin test for diagnosis of tuberculosis infection in Viet Nam

Current WHO-recommended diagnostic tools for tuberculosis infection (TBI) have well-known limitations and viable alternatives are urgently needed. We compared the diagnostic performance and accuracy of the novel QIAreach QuantiFERON-TB assay (QIAreach; index) to the QuantiFERON-TB Gold Plus assay (QFT-Plus; reference). The sample included 261 adults (≥ 18 years) recruited at community-based TB case finding events. Of these, 226 underwent Tuberculin Skin Tests and 200 returned for interpretation (TST; comparator). QIAreach processing and TST reading were completed at lower-level healthcare facilities. We conducted matched-pair comparisons for QIAreach and TST with QFT-Plus, calculated sensitivity, specificity and area under a receiver-operating characteristic curve (AUC), and analyzed concordant-/discordant-pair interferon-gamma (IFN-γ) levels. QIAreach sensitivity and specificity were 98.5% and 72.3%, respectively, for an AUC of 0.85. TST sensitivity (53.2%) at a 5 mm induration threshold was significantly below QIAreach, while specificity (82.4%) was statistically equivalent. The corrected mean IFN-γ level of 0.08 IU/ml and corresponding empirical threshold (0.05) of false-positive QIAreach results were significantly lower than the manufacturer-recommended QFT-Plus threshold (≥ 0.35 IU/ml). Despite QIAreach’s higher sensitivity at equivalent specificity to TST, the high number of false positive results and low specificity limit its utility and highlight the continued need to expand the diagnostic toolkit for TBI.


Discussion
Our study found that QIAreach had a high sensitivity when compared to QFT-Plus (reference standard).In direct comparison with TST using a 5 mm induration threshold, the assay also performed well, achieving a significantly higher sensitivity with a statistically equivalent specificity.However, the assay produced a high number of false positive results, which resulted in a significantly lower specificity compared to TST using a 10 mm induration threshold, which was the standard of care for the majority of persons with TBI according to national guidelines in Viet Nam at the time of the study 22 .The assay's high sensitivity was concordant with recent studies that reported similar high sensitivity versus QFT-plus ranging from 99.1 to 100% [23][24][25] .Moreover, QIAreach displayed a high diagnostic performance in our study, which may qualify the assay as sufficiently accurate for a diagnostic test 26 .As studies have shown a better predictive ability of IGRAs than TSTs, the level of concordance with QFT-Plus and diagnostic accuracy suggests Table 3. Sensitivity, specificity and ROC AUC of QIAreach.Sensitivity, specificity, positive predictive value, negative predictive value, Area under the Receiver Operating Charactistics curve (ROC AUC) of QIAreach were calculated using manufacturer instructions with QFT-Plus as the reference standard (n = 261).To our knowledge, this was the first study to include a matched comparison between QIAreach and TST.The latter currently represents the programmatic standard for TBI diagnosis in Viet Nam as well as many other HBCs 28 .However, there is urgent need to validate new TBI diagnostic tools following the detection of substantial variability in TST positivity despite sourcing the tuberculin from the same manufacturer (PPD-Bulbio) 10 .This variability has been reflected in a decline in positivity and subsequent increase in resource requirements for meeting national TPT targets 29 .In response, the Viet Nam NTP issued new rapid guidance in September 2022 to lower the positivity threshold to 5 mm, and improve positivity and programmatic scale-up of TPT 30 .Thus, our analysis plan included both 10 mm and 5 mm induration sizes to accommodate the latest developments with www.nature.com/scientificreports/ the former being the standard of care at the time of the study.As the NTP has shifted its focus towards the more aggressive strategy of higher sensitivity in exchange for lower specificity, in the scenario of a 5 mm threshold our results show that QIAreach may serve as a viable clinical alternative to TST.This is also one of the first published studies to validate QIAreach outside of the health facility setting as per its intended purpose of expanding access to IGRA closer to the point of care.Operationally, QIAreach offers many of the similar key advantages of QFT-Plus compared to TST.Only one visit is required to complete the test, while TST still requires one visit for testing and a second for interpretation 31,32 .In our research, almost a quarter of recruited participants did not agree to conduct the test or did not present for results interpretation.These data exemplify the hindrance created by the inconvenience of the TST.Clinically, the T-Cell-based QIAreach offers protection from the array of confounders commonly affecting TST results such as age, nutrition, immunology, genetics, BCG vaccination, and cross-reactivity with non-tuberculous mycobacteria 33 .
Simultaneously, QIAreach also aims to resolve key challenges of the QFT-Plus assay by offering simplified operating procedures, faster turnaround time, and greater flexibility of deployment.QFT-Plus requires a laboratory infrastructure, technical expertise, and expensive equipment, while the QIAreach eStick-eHub architecture aims to emulate the success of other cartridge-based diagnostic tools for TB in low-resource settings that have little to no access to sophisticated laboratory capacity.The rapid turnaround time that characterizes the eStick-eHub design also enables higher throughput of up to 24 samples per eHub per hour 34 .
Nevertheless, our study also exposed key performance issues in our setting that will need to be evaluated further to facilitate uptake of this new tool.QFT-Plus needs four BCTs (TB1, TB2, Nil and mitogen) to optimize results interpretation.Meanwhile, QIAreach relies only on a single BCT, which is designed to maximize sensitivity with antigens optimized to stimulate CD4 and CD8 T-cells.Past studies have found this method to possibly increase IFN-γ levels which would inflate the number of results considered positive 17,35 .These factors may have contributed to the low specificity of QIAreach observed on this study as evinced in the analysis of uncorrected (TB2) and corrected (TB2-Nil) mean IFN-γ levels.Specifically, the uncorrected and corrected IFN-γ levels were 1.6-1.7 times higher in participants testing negative on QFT-Plus than on QIAreach.Thus, the absence of the negative (Nil) control likely contributed to the impaired specificity, especially if QIAreach-positivity was calibrated using QFT-Plus thresholds.
The lower specificity on our study was discordant with the limited available evidence base.Two hospitalbased studies in Italy and Japan detected high concordance between QIAreach and QFT-Plus performance and specificity in particular.The Italian study observed a specificity versus QFT-Plus of 93.4% and an overall concordance with QFT-Plus of 95.7% (κ = 0.96) among 130 persons with confirmed TB and 174 healthy controls 23 .Similarly, the Japanese study conducted in 41 persons with active TB and 42 healthy individuals recorded a specificity of 97.6% among the TB patient cohort with an overall concordance of 98.8% versus QFT-Plus in the sample (κ = 0.98) 24 .Moreover, the study highlighted that the IFN-γ concentration cutoff point for QIAreach was similar to that of the QFT-Plus assay (0.35 IU/mL) for the active TB population 24 , which our study did not corroborate.Based on these data, a hypothesis to explain the low specificity of QIAreach compared QFT-Plus may be the study setting.Contrary to these two examples, our study recruited participants in the community with a comparatively lower rate of TB infection and disease.This setting may also be exposed to confounding and bias reflected in the greater variance in the QFT-Plus results as seen in the high indeterminate rate (17/278 = 6.1%).
It is evident that more work is needed to specify the utility and role that QIAreach can play in the global scale-up of TPT.Studies should also incorporate economic and market analyses once the product moves towards commercialization to address the most common criticism of IGRAs-their costs 36 -as health economic analyses have estimated IGRAs to be more cost-effective than TST 37 .For now, the test is not recommended in national guidelines.At the current diagnostic accuracy, it may only find limited application in priority groups with elevated risk of progression and settings where the benefits of aggressive intervention outweigh the health system and patient costs of unnecessary treatment.An example of such a priority group may consist of household and close contacts of MDR-TB patients as a recent study reported a strong correlation between results from TST and QFT-Plus when detecting TBI in MDR-TB contacts, concluding TST could be used in place of QFT-Plus 38 .
Our study was limited in a number of ways that may affect its generalizability.The aforementioned lack of a formal health economic analysis prevents the ability to build an investment case for policymakers and multilateral funding agencies.Another key limitation of the study was a lack of comparison between QIAreach and bacteriologically-confirmed individuals with TB, including children, which precluded the determination of "true" sensitivity and specificity of the assay in Viet Nam.In addition, large gaps in the TBI cascade for TST resulted in a smaller sample size, which may have deleteriously affected the statistical comparison between QIAreach and TST resulting in their respective specificities showing no significant difference at a 5 mm threshold.These drops in the cascade may have also introduced bias into the results.

Conclusions
Currently, TST and IGRA are the only recommended diagnostics for TBI.However, the limitations of these methods still impair the accuracy, effectiveness and uptake of these tools and scale-up of TPT overall.This study provided evidence on the performance and accuracy of QIAreach assay compared to TST and QFT-Plus.Our results showed high sensitivity and AUC classification, but also exposed a suboptimal specificity, thereby potentially affecting its value and utility, particularly in low-resource, high-burden settings.Thus, more evidence for this new IGRA assay and other new diagnostic tools for TBI remain urgently needed.Nevertheless, this study was among the first to evaluate QIAreach along several dimensions and contributes to the available evidence base to inform future research, programmatic implementation and policy development towards reducing the global seedbed of TB.

Table 2 .
QIAreach, QFT-Plus and TST results.Contingency tables of QIAreach and TST results compared to the reference standard.TST results were presented at 5 mm and 10 mm induration thresholds.Chi-squared tests were used to calculate p-values; p < 0.05 was considered statistically significant.¥ McNemar's test used to compare QIAreach and QFT-Plus with TST at a threshold of 5 mm and 10 mm.

Table 4 .
Sensitivity, specificity and concordance of QIAreach compared to TST in individuals with a valid TST result (n = 200).Sensitivity, specificity, positive predictive value, negative predictive value, AUC of Tuberculin skin test (TST) with 5 mm and 10 mm induration threshold values for the subset of individuals with a TST result with QFT-Plus as reference standard (n = 200).Chi-squared tests were used to calculate p-values; p < 0.05 was considered statistically significant.