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. 2025 Sep 8;20(9):e0330345. doi: 10.1371/journal.pone.0330345

Refining the diagnostic approach to latent tuberculosis Infection with Quantiferon gold plus: A retrospective analysis of borderline results

Alba Ruedas-López 1,2,#, Juan María Herrero-Martínez 3,#, Alhena Reyes 1, Beatriz González-Blanco 1, Paula López-Roa 1,*
Editor: Frederick Quinn4
PMCID: PMC12416684  PMID: 40920768

Abstract

The Quantiferon Gold Plus (QFT) test, a widely used interferon-γ release assay (IGRA), diagnoses latent tuberculosis infection (LTBI) with a positivity threshold of ≥0.35 IU/mL. Results near this cut-off can be challenging to interpret due to variability from immunological, pre-analytical, and technical factors, prompting recommendations for a borderline range to refine diagnosis and reduce overtreatment. This retrospective study analyzed QFT results from 9,944 patients (2019–2023), establishing ranges: < 0.2 IU/mL as negative, 0.2–0.35 IU/mL as borderline negative, 0.35–0.7 IU/mL as borderline positive, and >0.7 IU/mL as positive. Borderline results occurred in 7.6% of patients, particularly in those born in Africa or South America, and in older individuals. Of 64 patients retested, 60.9% reverted to negative, while 17.1% of borderline negatives later converted to positive or borderline positive. Notably, no active TB cases emerged among those who reverted to negative on repeat testing. These findings emphasize the need for cautious interpretation of borderline QFT results, as their link to active TB progression differs from clear results. The study supports repeat testing of borderline cases to enhance LTBI diagnostic accuracy and inform treatment decisions.

Introduction

The Quantiferon Gold Plus (QFT) test is one of the two commercially available interferon-γ release assays (IGRAs) used for diagnosing latent tuberculosis infection (LTBI). Compared to its predecessor, the QuantiFERON-TB Gold In-Tube (QFT-GIT), the QFT-Plus has demonstrated improved sensitivity [1], while maintaining comparable or, in some studies, improved specificity [2,3]. Additionally, QFT-Plus may offer greater sensitivity for predicting active TB [4]. However, IGRA tests are more expensive than the tuberculin skin test (TST), which may remain a cost-effective option in some high TB burden countries. Factors such as cost-effectiveness and local epidemiological conditions, including TB prevalence and resource availability, are critical when choosing between these tests [3]. In our clinical practice, we have adopted the QFT-Plus as the primary diagnostic tool for LTBI. As per the manufacturer’s guidelines [5], a QFT result is classified as positive if it is ≥ 0.35 IU/mL [6].However, interpreting IGRA results can be challenging due to issues such as indeterminate and equivocal results, including conversions (from negative to positive) or reversions (from positive to negative) during repeat testing [7].

Longitudinal studies have observed variability in IGRA results over time, particularly when the outcomes are near the threshold. These studies report a significant occurrence of conversions and reversions in borderline results [8,9]. This fluctuation is believed to arise from pre-analytical, technical, or patient-related factors [7].

Given the high frequency of conversions and reversions around the 0.35 IU/mL threshold, some researchers have recommended establishing a borderline or equivocal range [10]. This approach aims to reduce unnecessary treatment for individuals with unstable positive results while ensuring appropriate treatment for those close to the threshold who may convert to positive on retesting [11]. Suggested borderline ranges vary across studies, with some recommending 0.20–0.70 IU/mL and others extending up to 1.0 IU/mL [8,12–14].

In addition to borderline results, QFT outcomes may be uninterpretable if the positive (mitogen) or negative control tubes fail to provide valid responses. Such results are labeled as indeterminate, often necessitating a repeat test. Under routine conditions, the rate of indeterminate outcomes has been reported to range between 3.4% and 10.2% [15,16].

In our setting, limited data exist on the frequency of borderline IGRA results and associated risk factors under routine clinical conditions. The objective of this study was to determine the proportion of QFT tests that yield neither clear positive nor negative outcomes in clinical practice and to evaluate the results of repeating equivocal tests.

Methods

A retrospective review of patients with QFT results between 2019 and 2023 was conducted, including demographic data such as age, gender, and country of birth. These variables have been associated, along with borderline test results, with both the likelihood of conversions and reversions of Interferon γ Release Assays [7]. The study was conducted in a routine clinical setting at a tertiary hospital in Madrid, focusing on patients of all age groups who underwent QFT testing. Children were not excluded, as previous research suggests that the QFT assay is accurate in pediatric practice, with good sensitivity [17]. The hospital is a 1,300-bed acute teaching hospital that serves as a referral centre for a population of 462,000 [18], of whom 33.6% are foreign-born [19]. The population is comprised of 69% individuals between the ages of 14 and 64 years, 11% between 65 and 79 years, and 6% over 80 years. The incidence of tuberculosis in the population of the three health districts attached to the hospital is among the highest in the Community of Madrid, ranging from almost 19 cases per 100,000 inhabitants in the districts of Usera or Villaverde, to 14.8 per 100,000 in Carabanchel, compared to the Community average of 8.59 per 100,000 [20].

Data for research purposes were accessed from January 2024 to May 2024. Patients were tested based on clinical indications, such as suspected latent or active tuberculosis, or for screening purposes. Repeat testing was carried out when deemed necessary by the attending physicians, in consultation with microbiologists. This occurred in cases where there were inconsistencies with expected results, a high clinical suspicion of TB, or indeterminate results.

The assay was performed using chemiluminescence on the Liaison XL platform (DiaSorin S.p.A., Saluggia, Italy), with results interpreted according to the recommended cut-off point (0.35 IU/mL).

We aimed to assess the proportion of QFT tests yielding equivocal results and to evaluate the outcomes of retesting these cases by establishing a defined borderline range. The results were categorized as follows: negative result: < 0.2 IU/mL; borderline negative: ≥ 0.2 and <0.35 IU/mL; borderline positive: 0.35–0.7 IU/mL; positive: > 0.7 IU/mL.

We employed a logistic regression model to assess the association between demographic variables and QFT results.

Ethical statement

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Hospital 12 de Octubre (reference number: CEIm 23/569), ensuring compliance with international ethical standards. All procedures adhered to the hospital’s biosafety regulations and were approved by the Local Institutional Review Board. Given the retrospective nature of the study, all data were fully anonymized prior to access. Additionally, due to the retrospective design, the Ethics Committee waived the requirement for written informed consent, as no identifiable patient information was included in the analysis.

Results

We identified a total of 9944 individuals who underwent at least one QFT test, with 5020 (50.5%) being male. The study population comprised individuals aged from 0.05 to 98.52 years, with a mean age of 51.64 years (SD = 19.24). Focusing on younger subgroups, individuals under 18 years of age accounted for 4.7% (466 patients), while those under 5 years represented only 1.2% (122 patients) of the total population. The average age was 51.7 years (SD = 19.4) for men and 51.6 years (SD = 19.0) for women. Information about the country of birth was available for 9040 participants (90.9%), among whom 2684 (29.7%) were born outside the country.

Using the standard cut-off of 0.35 IU/mL, 1805 (18.1%) tests were classified as positive, 7872 (79.2%) as negative, and 267 (2.7%) as indeterminate. When introducing a borderline range, 7506 (75.5%) results fell into the negative category, 1413 (14.2%) were positive, 392 (3.9%) were classified as borderline positive, and 366 (3.7%) as borderline negative. In total, 7.6% (758/9944) of those tested had values within the borderline range (Fig 1).

Fig 1. Distribution of test results by cut-off ranges.

Fig 1

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Compared to individuals born in Europe, those born in Africa and South America were significantly more likely to have borderline results. A slight increase in age was also associated with a higher probability of borderline outcomes, while no notable differences were found between males and females. Details are provided in Table 1.

Table 1. Analysis of variables associated with a borderline result*.

Variable Results** N (%) Adjusted OR (all borderline vs. all definitive positive or negative results)
Negative BL-neg. BL-pos. Positive ORa (95% CI) p
Place of Birth (OR, relative to Europe) Europe 5163 (79.6) 224 (3.5) 258 (4.0) 844 (13.0) – –
Africa 311 (57.5) 28 (5.2) 33 (6.1) 169 (31.2) 2.05 (1.54-2.74) <0.001
Asia 99 (71.7) 5 (3.6) 8 (5.8) 26 (18.8) 1.62 (0.90-2.91) 0.1
North America 312 (82.5) 10 (2.7) 13 (3.4) 43 (11.4) 1.12 (0.72-1.73) 0.6
South America 924 (73.2) 69 (5.5) 44 (3.5) 226 (17.9) 1.59 (1.27-1.98) <0.001
Unknown 697 (80.3) 30 (3.5) 36 (4.2) 105 (12.1) 1.26 (0.96-1.66) 0.1
Female 3899 (80.9) 177 (3.7) 183 (3.8) 560 (11.6) – –
Male 3607 (74.3) 189 (3.9) 209 (4.3) 853 (17.6) 1.11 (0.95-1.28) 0.2
Age (years, median (IQR)) 50.6
(36.8-62.7)		 56.6
(44.0-71.8)		 59.4
(47.4-73.6)		 58.5
(46.5-71.1)		 1.02(1.02-1.03) <0.001
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Note. “BL-pos.”: borderline positive results; “BL-neg.”: borderline negative results.

* Patients with indeterminate results were excluded from the analysis. N = 9677.

** Negative: < 0.2 IU/mL; borderline negative: ≥ 0.2 and <0.35 IU/mL; borderline positive: 0.35–0.7 IU/mL; positive: > 0.7 IU/mL.

Among 64 patients with an initial borderline result who underwent repeat testing, the outcomes were as follows: 3 (4.7%) were indeterminate, 39 (60.9%) negative, 9 (14.1%) borderline negative, 6 (9.4%) borderline positive, and 7 (10.9%) positive. For patients initially classified as borderline negative, 17.1% converted to positive (9.8%) or borderline positive (7.3%) on retesting. In contrast, among those initially categorized as borderline positive, 73.9% reverted to negative (52.2%) or borderline negative (21.7%) upon retesting. Categorical outcomes of repeat testing are detailed in Table 2 and both categorical and demographic data, including continent and mean age, are presented in S1 Table.

Table 2. Categorical distribution of follow-up QFT results when retesting those with initial results in the borderline range (0.20–0.70 IU/ml).

Initial result Total Retested
N (%)		 Results of follow-up QFT test* N (%) Months until retesting
Ind. Negative BL negative BL positive Positive Mean (SD) Range
BL negative 366 41 (11.2) 3 (7.3) 27 (65.9) 4 (9.8) 3 (7.3) 4 (9.8) 15.1 (10.7) 0.1 - 49.2
BL positive 392 23 (5.9) 0 (0) 12 (52.2) 5 (21.7) 3 (13.0) 3 (13.0) 10.94 (9.4) 0.03 - 29.9
All borderline 758 64 (8.4) 3 (4.7) 39 (60.9) 9 (14.1) 6 (9.4) 7 (10.9) 13.6 (10.4) 0.03 - 49.2
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Note. BL: borderline, Ind.: Indeterminate. * Negative: < 0.2 IU/mL; borderline negative: ≥ 0.2 and <0.35 IU/mL; borderline positive: 0.35–0.7 IU/mL; positive: > 0.7 IU/mL.

During the study period, 90 individuals were diagnosed with active tuberculosis (TB) among those included in our study. QFT and culture results for these cases are presented in Table 3.

Table 3. Mycobacterial culture according to Quantiferon test result.

QFT results Positive culture results
Co-prevalent (<3 months) Incident (>3 months) Total
Indeterminate 4 (5.1%) 0 (0.0%) 4 (4.4%)
Negative 9 (11.4%) 1 (9.1%) 10 (11.1%)
BL-Neg 3 (3.8%) 2 (18.2%) 5 (5.6%)
BL-Pos 2 (2.5%) 1 (9.1%) 3 (3.3%)
Positive 61 (77.2%) 7 (63.6%) 68 (75.6%)
Total 79 (87.8%) 11 (12.2%) 90 (100%)
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Note. QFT, Quantiferon; BL-Neg, borderline negative; BL-Pos, borderline positive.

Excluding those with indeterminate or negative results, there was a significantly higher proportion of patients developing active TB in those with positive results >0.7 IU/mL (4.8%; 68/1413) compared to individuals with values within the borderline range of 0.20–0.7 IU/mL (1.1%; 8/757, p < 0.001).

Among individuals with initial QFT results in the 0.20–0.7 IU/mL range who underwent retesting, 3.1% (2/64) progressed to active TB. One patient initially classified as borderline negative and another as borderline positive later tested positive during follow-up. For those with borderline positive results (0.35–0.7 IU/mL), reversions to negative were observed in 52.2% (12/23), and no cases of active TB were detected among individuals who reverted.

Discussion

In this evaluation of borderline QFT results obtained under routine clinical conditions in a low-endemicity setting involving 9,944 individuals, nearly 8% of tests yielded results within the borderline range. Interestingly, among those retested after an initial borderline-range result, 60.9% showed convincingly negative results (<0.20 IU/ml). Additionally, 73.9% of individuals initially classified as borderline positive reverted to either negative or borderline negative, with no new TB cases identified in this group. However, 17.1% of those initially categorized as borderline negative progressed to borderline positive or positive results, suggesting a potential underestimation of latent TB risk. These findings highlight the challenge of interpreting single borderline results, which can result in both unnecessary treatments and missed LTBI cases.

Variability in QFT results may stem from immunological, pre-analytical, and technical factors [21]. This variability has been extensively studied in the context of serial testing among healthcare workers (HCWs). For example, studies in large HCW populations in the USA showed that 65–75% of individuals who converted from negative to positive reverted to negative upon repeat testing [22,23]. Such fluctuations have prompted recommendations to introduce a borderline range to aid interpretation. A German study, applying a borderline zone of 0.2–0.7 IU/mL, demonstrated a reduction in reversion rates from 37% to 19% [8]. Similarly, 43% of HCWs in Sweden with borderline QFT results (0.30–0.99 IU/mL) showed reversion upon retesting [14]. However, beyond HCW-focused studies, limited data exist on the prevalence of borderline results and their associated risk factors in routine settings. Our analysis revealed that approximately 8% of QFT tests resulted in borderline findings, with the progression risk to active TB likely differing from clearly positive or negative results.

Consistent with previous studies [24–26], our results suggest that borderline QFT values, particularly those that revert to negative, may reflect false-positive elevations rather than genuine immune responses to TB antigens. This hypothesis is supported by the absence of active TB cases in individuals whose borderline results reverted to negative.

Our multivariate analysis identified that individuals born in Africa or South America were significantly more likely to obtain borderline results. Age was also found to slightly increase the likelihood of borderline results, while no significant sex-based differences were observed. The higher percentages of borderline QFT results observed in Africa and South America may be linked to the higher prevalence of TB in these regions. While this is a plausible explanation, it remains speculative, as TB prevalence was not included in our regression model. It is also important to note that certain species of non-tuberculous mycobacteria (NTM), such as M. kansasii, M. marinum, and M. szulgai, can cause positive QFT results [5]. However, due to the limited availability of comprehensive data on the prevalence of these NTMs across different continents, it is not possible to determine whether this factor influences the observed differences in borderline percentages. Nonetheless, regardless of the underlying cause, there appears to be added value or greater indication for retesting borderline results in these populations.

Interpreting diagnostic test results requires careful consideration of the clinical and epidemiological context. The risk of developing active TB varies among individuals with positive IGRA reactions. Studies have demonstrated a correlation between IGRA result magnitude and the incidence of active TB, including cases within the borderline range, suggesting a dose-response relationship [27]. However, in regions with low TB prevalence, individuals presenting with borderline QFT results often experience reversion to negative upon retesting. A study involving 58,539 subjects in a low-endemicity country found that among those with initial borderline results who were retested, 38% reverted to negative, with no cases of incident active TB reported within two years. This finding suggests that a significant proportion of initial borderline results may represent false positives [26]. Consequently, the risk of missing a true LTBI in such cases is relatively low, and avoiding unnecessary treatment minimizes the potential for adverse effects and disruptions to daily life.

Conversely, in populations at higher risk for TB progression, such as immunosuppressed individuals, the emphasis shifts toward maximizing test sensitivity, even at the expense of specificity. Failure to diagnose and treat LTBI in these patients can lead to severe outcomes, including the development of active TB disease. Therefore, integrating other risk factors—such as recent exposure to active TB cases, underlying comorbidities, and the degree of immunosuppression—along with underlying determinants such as access to healthcare, socioeconomic status, and level of vulnerability is essential in guiding clinical decisions [28].

Contrasting evidence suggests that most borderline QFT results reflect true antigen-specific responses rather than random variability, and that the presence of various non-IGRA risk factors and parameters of TB infection showed a gradient along increasing quantitative QFT results [29,30]. This study provided interesting data on QFT results, but it is important to note that the study was conducted within the context of a contact study, and not in other settings, where the interpretation of the results and the risk of progression to active TB may vary significantly. We agree with Wikell et al. [26] that the inherent variability of the technique should be given due consideration, and that further study is required to ascertain the clinical significance of borderline results in terms of the development of active TB in a range of clinical settings.

Furthermore, given the proportion of borderline QFT results observed in our study and the low incidence of TB progression within this subgroup, one could argue that clinical follow-up alone may suffice, with treatment initiated as necessary. However, refining diagnostic classification strategies would enhance the efficient allocation of healthcare resources and could also reduce the stigma associated with a positive diagnosis. Targeting preventive treatment to individuals most likely to benefit not only optimizes cost-effectiveness but also reduces unnecessary interventions in those at low risk for disease progression.

From a public health perspective, any modifications to TB screening protocols must consider the availability of resources—both material and personnel—to ensure appropriate patient follow-up. Implementation strategies should be tailored to local epidemiological conditions and healthcare infrastructure to maximize feasibility and impact.

Given the significant morbidity and mortality associated with TB, further research is essential to determine which individuals with IGRA conversion or reversion are most likely to benefit from TB preventive treatment, and more studies are warranted across diverse geographical regions and ethnic populations. A deeper understanding of the clinical significance of borderline results for specific patient populations will be crucial in refining LTBI management strategies and improving patient outcomes.

This study has several limitations. First, it relied on retrospectively collected clinical data, and not all equivocal or indeterminate results were retested. Second, data on the reasons for testing and whether LTBI treatment was initiated were unavailable. Third, the observed relationship between QFT results and active TB development should be interpreted cautiously due to the retrospective design and the small number of borderline cases progressing to active TB. Additionally, some patients may have been diagnosed with TB at other institutions, potentially affecting the results.

In conclusion, approximately 8% of patients had borderline QFT results, with these findings being more prevalent among individuals born in South America or Africa, as well as older individuals. Patients with borderline positive results showed a high reversion rate (about 50%), and no cases of TB were identified among those who reverted. These findings emphasize the importance of retesting and cautious interpretation of borderline QFT results in clinical practice. While repeat testing offers valuable insights, it may not always be feasible, and further studies are needed to assess the long-term clinical outcomes of borderline QFT results.

Supporting information

S1 Table. Mean age and categorical distribution of follow-up QFT results when retesting those with initial results in the borderline range (0.20–0.70 IU/ml), by continent.

(DOCX)

pone.0330345.s001.docx (23.6KB, docx)

Acknowledgments

This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project “PI21/01738” and co-funded by the European Union.

Data Availability

The data that support the findings of this study are openly available in Figshare. The data can be accessed at Figshare using the following DOI link: https://doi.org/10.6084/m9.figshare.28659293.v1.

Funding Statement

This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project ‘PI21/01738’ and co-funded by the European Union.

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  • 29.Uzorka JW, Kroft LJM, Bakker JA, van Zwet EW, Huisman E, Knetsch-Prins C, et al. Proof of concept that most borderline Quantiferon results are true antigen-specific responses. Eur Respir J. 2017;50(5):1701630. doi: 10.1183/13993003.01630-2017 [DOI] [PubMed] [Google Scholar]
  • 30.Uzorka JW, Bossink AWJ, Franken WPJ, Thijsen SFT, Leyten EMS, van Haeften AC, et al. Borderline QuantiFERON results and the distinction between specific responses and test variability. Tuberculosis (Edinb). 2018;111:102–8. doi: 10.1016/j.tube.2018.06.002 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Xiangwei Li

12 Feb 2025

PONE-D-24-52042Refining the Diagnostic Approach to Latent Tuberculosis Infection with Quantiferon Gold Plus: A Retrospective Analysis of Borderline ResultsPLOS ONE

Dear Dr. Lopez Roa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

Reviewer #6: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Congratulations for submitting this manuscript and for completing this study as part of the body of knowledge in the field of TB diagnosis.

Generally, the paper is clear and straightforward in addressing the research objective. In the methods section, you may specify the general objective you mentioned in the introduction. For instance, you could have explained more why did you choose the demographic variables and further describe what could be its implications when you include it in your study as far as QFT is concerned. In the resuls section, please be consistent with the use of proper formatting of tables based on the guidelines of the PLOS. Another suggestion would be to include contrasting studies (aside from consistent ones), whenever available. Additional references in the discussion section further improves the credibility of the findings. Lastly, you have a good conclusion.

May you keep on working on this kind of studies that would possibly inform future policy. God bless.

Reviewer #2: This retrospective review study aims to assess the (1) clinical characteristics of those with borderline tests and (2) the clinical applicability of borderline QFT-gold test results by analyzing the results of repeat testing (ie conversions/reversions) for the 7-8% of patients with borderline results on the initial test. Given the number of patients in the borderline category to reverted or converted, this study highlights the challenge of interpreting single borderline results and argues for repeat testing over time. This study did demonstrate that the risk of progressing to active disease in the borderline group (~5%) was different from that of both the negative (~0.1%) and positive group (~5%), though it should be noted that this study is retrospective in nature and sample numbers are small in the borderline group.

1. Did this study include children? It was not clear from the Methods section and given the emphasis on age as a correlate of borderline QFT results, I would recommend being explicit about the age range included in the study, especially if those <5yo are included.

2. Only 64/758 borderline patients were retested, but the clinical characteristics of this subset are not included in the study nor is the re-testing interval. While not all data may be available, it would be helpful to know if these patients had risk factors or other clinical characteristics different from the overall cohort that prompted the closer follow-up (for example, heavier exposure to TB, country of origin, symptoms of active TB, etc) compared to those that were not re-tested? Additionally, is it confirmed whether these 64 patients are treatment-naive given treatment data was not included as part of the study?

3. While re-testing of borderline QFT results may provide additional diagnostic clarity, retesting ~8% of patients is an additional health care cost and risks losing patients to follow-up, some of whom would go on to develop active TB, but has the advantage of potentially avoiding unnecessary LTBI treatment in a subset of individuals. It may be helpful to include in the discussion potential positive and negative consequences of initiating this intervention.

4. It would also be helpful to include in the discussion the effects of serial testing on public health in both low endemicity and high endemicity settings.

Reviewer #3: The manuscript entitled "Refining the Diagnostic Approach to Latent Tuberculosis Infection with Quantiferon

Gold Plus: A Retrospective Analysis of Borderline Results" needs these revisions

-Please check keywords according to MESH browser; delete QuantiFERON-TB Gold Plus and borderline results from keywords

-Please write about the age range of patients

-What are the rules for repeat testing, what is the minimum time interval for repeat testing

Reviewer #4: The manuscript is well written and highlighted the important subject of diagnosing LTBI, and more importantly to the borderline outcomes interrupting the results which alters in the retesting. Applause to the authors for extracting and analysing such data to develop this manuscript.

There are a couple of minor comments/suggestions to this.

Methods:

Line 75 - 86: this section could explore further the setup of health facility which the study was conducted, such as TB facility or any other, how patients were selected to test, QTF testing criteria, criteria for retesting of borderline results, and treatment and follow up or further care after LTBI diagnosis.

The demographic variables are only age, gender and country of birth. Are there any other variables included in the regression model such as previous TB history, HIV coinfection, or other comorbidities? It will be interesting to see the relation with such variables. Or are they not available as a limitation of the retrospective study.

Reslts:

Line 188 - 124: this is an interesting outcome after repeated testing. What is the time interval between initial testing vs repeated testing?

Reviewer #5: There is a very small percentage of patients who fall within the borderline range . Of these your classification improves the predictability of the classification. However only 1.1% of the cases develop TB during followup. It may not be too difficult to just follow-up those who fall in the border line range. So what is the added value of your improved Classification

You have said that the Percentages are more in the countries like Africa? Why is this so? Would the prevalence of NTM have something to do with it? This point could be addressed more in the discussion

Reviewer #6: In the introduction, it would be beneficial for the authors to highlight any existing studies that have evaluated and compared the results between QFT-Plus and QFT-GIT. Discussing any observed differences, such as costs and immune responses, would provide valuable insights, and including relevant references would enhance the credibility of the review.

In the materials and methods section, it is important to clearly indicate whether the patients are immunocompromised, part of a frail population, or have comorbidities. This information is essential, as these factors can significantly influence the interpretation of QFT results.

Furthermore, it is critical to emphasize that clinicians are not just interested in laboratory evaluation ranges, but in how these ranges are defined by population stratification, particularly by age. For instance, in Europe, the age demographic of the Caucasian population may differ. More importantly, the patient's overall health status plays a pivotal role in interpreting laboratory data. A holistic approach to clinical management should involve evaluating the population based on race and understanding the type of immune response, factoring in essential epidemiological and clinical variables such as cancer, autoimmune disorders, protein intake, BMI, coinfections, and access to healthcare, which can differ across countries.

**********

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Reviewer #1: Yes:  Marlon L. Bayot

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Reviewer #6: Yes:  none

**********

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PLoS One. 2025 Sep 8;20(9):e0330345. doi: 10.1371/journal.pone.0330345.r002

Author response to Decision Letter 1

25 Mar 2025

Reviewer #1:

Congratulations for submitting this manuscript and for completing this study as part of the body of knowledge in the field of TB diagnosis. Generally, the paper is clear and straightforward in addressing the research objective.

Reviewer comment 1: In the methods section, you may specify the general objective you mentioned in the introduction.

Response: Thank you for your suggestion. We have addressed this by adding the following sentence to the Methods section “We aimed to assess the proportion of QFT tests yielding equivocal results and to evaluate the outcomes of retesting these cases by establishing a defined borderline range”

Reviewer comment 2: For instance, you could have explained more why did you choose the demographic variables and further describe what could be its implications when you include it in your study as far as QFT is concerned.

Response: Thank you for your comment. We agree that clarifying the importance of the selected demographic variables is relevant. To address this, we have added the following sentence to the Methods section: “These variables have been associated, along with borderline test results, with both the likelihood of conversions and reversions of Interferon γ Release Assays [1].”

Reviewer comment 3: In the resuls section, please be consistent with the use of proper formatting of tables based on the guidelines of the PLOS.

Response: Thank you for your feedback. We have reviewed and updated the formatting of the tables in the Results section to ensure they are consistent with the PLOS guidelines.

Reviewer comment 4: Another suggestion would be to include contrasting studies (aside from consistent ones), whenever available. Additional references in the discussion section further improves the credibility of the findings. Lastly, you have a good conclusion. May you keep on working on this kind of studies that would possibly inform future policy. God bless.

Response: Thank you for your valuable suggestion. We have include contrasting studies and expanded the discussion section with additional references. This has improved the contextualisation and overall understanding of the article.

“Interpreting diagnostic test results requires careful consideration of the clinical and epidemiological context. The risk of developing active TB varies among individuals with positive IGRA reactions. Studies have demonstrated a correlation between IGRA result magnitude and the incidence of active TB, including cases within the borderline range, suggesting a dose-response relationship [2]. However, in regions with low TB prevalence, individuals presenting with borderline QFT results often experience reversion to negative upon retesting. A study involving 58,539 subjects in a low-endemicity country found that among those with initial borderline results who were retested, 38% reverted to negative, with no cases of incident active TB reported within two years. This finding suggests that a significant proportion of initial borderline results may represent false positives [3]. Consequently, the risk of missing a true LTBI in such cases is relatively low, and avoiding unnecessary treatment minimizes the potential for adverse effects and disruptions to daily life.

Conversely, in populations at higher risk for TB progression, such as immunosuppressed individuals, the emphasis shifts toward maximizing test sensitivity, even at the expense of specificity. Failure to diagnose and treat LTBI in these patients can lead to severe outcomes, including the development of active TB disease. Therefore, integrating other risk factors—such as recent exposure to active TB cases, underlying comorbidities, and the degree of immunosuppression—along with underlying determinants such as access to healthcare, socioeconomic status, and level of vulnerability is essential in guiding clinical decisions [4].

Contrasting evidence suggests that most borderline QFT results reflect true antigen-specific responses rather than random variability, and that the presence of various non-IGRA risk factors and parameters of TB infection showed a gradient along increasing quantitative QFT results [5, 6]. This study provided interesting data on QFT results, but it is important to note that the study was conducted within the context of a contact study, and not in other settings, where the interpretation of the results and the risk of progression to active TB may vary significantly. We agree with Wikell et al. [3] that the inherent variability of the technique should be given due consideration, and that further study is required to ascertain the clinical significance of borderline results in terms of the development of active TB in a range of clinical settings.”

Reviewer #2

This retrospective review study aims to assess the (1) clinical characteristics of those with borderline tests and (2) the clinical applicability of borderline QFT-gold test results by analyzing the results of repeat testing (ie conversions/reversions) for the 7-8% of patients with borderline results on the initial test. Given the number of patients in the borderline category to reverted or converted, this study highlights the challenge of interpreting single borderline results and argues for repeat testing over time. This study did demonstrate that the risk of progressing to active disease in the borderline group (~5%) was different from that of both the negative (~0.1%) and positive group (~5%), though it should be noted that this study is retrospective in nature and sample numbers are small in the borderline group.

Reviewer comment 1. Did this study include children? It was not clear from the Methods section and given the emphasis on age as a correlate of borderline QFT results, I would recommend being explicit about the age range included in the study, especially if those <5yo are included.

Response: We agree that clarifying the age range is important. We have included this information in both the Methods and Results sections:

“The study was conducted in a routine clinical setting at a tertiary hospital in Madrid, focusing on patients of all age groups who underwent QFT testing. Children were not excluded, as previous research suggests that the QFT assay is accurate in pediatric practice, with good sensitivity [7]”

“The study population comprised individuals aged from 0.05 to 98.52 years, with a mean age of 51.64 years (SD = 19.24). Focusing on younger subgroups, individuals under 18 years of age accounted for 4.7% (466 patients), while those under 5 years represented only 1.2% (122 patients) of the total population.”

Reviewer comment 2. Only 64/758 borderline patients were retested, but the clinical characteristics of this subset are not included in the study nor is the re-testing interval. While not all data may be available, it would be helpful to know if these patients had risk factors or other clinical characteristics different from the overall cohort that prompted the closer follow-up (for example, heavier exposure to TB, country of origin, symptoms of active TB, etc) compared to those that were not re-tested? Additionally, is it confirmed whether these 64 patients are treatment-naive given treatment data was not included as part of the study?

Response: Thank you for your valuable comment. Unfortunately, due to the retrospective nature of the study, we were unable to obtain data on the patients' risk factors, symptoms, or comorbidities. To address the time interval between the initial testing and the repeated testing, we have added the "Months until retesting Mean(SD)" and "Range" columns to Table 2. Additionally, we have included Supplementary Table 1, where the data for continent and mean age can be found in detail. We hope these additions provide greater clarity on the patients' demographics. Moreover, all individual-level data for each patient and result will be made publicly available in a repository.

Reviewer comment 3. While re-testing of borderline QFT results may provide additional diagnostic clarity, retesting ~8% of patients is an additional health care cost and risks losing patients to follow-up, some of whom would go on to develop active TB, but has the advantage of potentially avoiding unnecessary LTBI treatment in a subset of individuals. It may be helpful to include in the discussion potential positive and negative consequences of initiating this intervention.

Reviewer comment 4. It would also be helpful to include in the discussion the effects of serial testing on public health in both low endemicity and high endemicity settings.

Response to comments 3 and 4: Thank you for your valuable contributions. We have added some paragraphs addressing these topics in the discussion section:

“Interpreting diagnostic test results requires careful consideration of the clinical and epidemiological context. The risk of developing active TB varies among individuals with positive IGRA reactions. Studies have demonstrated a correlation between IGRA result magnitude and the incidence of active TB, including cases within the borderline range, suggesting a dose-response relationship [2]. However, in regions with low TB prevalence, individuals presenting with borderline QFT results often experience reversion to negative upon retesting. A study involving 58,539 subjects in a low-endemicity country found that among those with initial borderline results who were retested, 38% reverted to negative, with no cases of incident active TB reported within two years. This finding suggests that a significant proportion of initial borderline results may represent false positives [3]. Consequently, the risk of missing a true LTBI in such cases is relatively low, and avoiding unnecessary treatment minimizes the potential for adverse effects and disruptions to daily life.

Conversely, in populations at higher risk for TB progression, such as immunosuppressed individuals, the emphasis shifts toward maximizing test sensitivity, even at the expense of specificity. Failure to diagnose and treat LTBI in these patients can lead to severe outcomes, including the development of active TB disease. Therefore, integrating other risk factors—such as recent exposure to active TB cases, underlying comorbidities, and the degree of immunosuppression—along with underlying determinants such as access to healthcare, socioeconomic status, and level of vulnerability is essential in guiding clinical decisions [4].

Contrasting evidence suggests that most borderline QFT results reflect true antigen-specific responses rather than random variability, and that the presence of various non-IGRA risk factors and parameters of TB infection showed a gradient along increasing quantitative QFT results [5, 6]. This study provided interesting data on QFT results, but it is important to note that the study was conducted within the context of a contact study, and not in other settings, where the interpretation of the results and the risk of progression to active TB may vary significantly. We agree with Wikell et al. [3] that the inherent variability of the technique should be given due consideration, and that further study is required to ascertain the clinical significance of borderline results in terms of the development of active TB in a range of clinical settings.

Furthermore, given the proportion of borderline QFT results observed in our study and the low incidence of TB progression within this subgroup, one could argue that clinical follow-up alone may suffice, with treatment initiated as necessary. However, refining diagnostic classification strategies would enhance the efficient allocation of healthcare resources and could also reduce the stigma associated with a positive diagnosis. Targeting preventive treatment to individuals most likely to benefit not only optimizes cost-effectiveness but also reduces unnecessary interventions in those at low risk for disease progression.

From a public health perspective, any modifications to TB screening protocols must consider the availability of resources—both material and personnel—to ensure appropriate patient follow-up. Implementation strategies should be tailored to local epidemiological conditions and healthcare infrastructure to maximize feasibility and impact.

Given the significant morbidity and mortality associated with TB, further research is essential to determine which individuals with IGRA conversion or reversion are most likely to benefit from TB preventive treatment, and more studies are warranted across diverse geographical regions and ethnic populations. A deeper understanding of the clinical significance of borderline results for specific patient populations will be crucial in refining LTBI management strategies and improving patient outcomes.”

And we also have added a brief statement in the conclusions of the study:

“While repeat testing offers valuable insights, it may not always be feasible, and further studies are needed to assess the long-term clinical outcomes of borderline QFT results.”

Reviewer #3:

The manuscript entitled "Refining the Diagnostic Approach to Latent Tuberculosis Infection with Quantiferon Gold Plus: A Retrospective Analysis of Borderline Results" needs these revisions

Reviewer comment 1: Please check keywords according to MESH browser; delete QuantiFERON-TB Gold Plus and borderline results from keywords.

Response: Thank you for your valuable comment. We have revised the keywords and have replaced "QuantiFERON-TB Gold Plus" and "borderline results" with more appropriate terms according to the MESH browser.

Reviewer comment 2: Please write about the age range of patients

Response: We agree that clarifying the age range is important. We have added this information in results section:

“The study population comprised individuals aged from 0.05 to 98.52 years, with a mean age of 51.64 years (SD = 19.24). Focusing on younger subgroups, individuals under 18 years of age accounted for 4.7% (466 patients), while those under 5 years represented only 1.2% (122 patients) of the total population.”

Reviewer comment 3: What are the rules for repeat testing, what is the minimum time interval for repeat testing.

Response: Thank you, we agree with your suggestion and have expanded on this topic in the Methods section:

“Patients were tested based on clinical indications, such as suspected latent or active tuberculosis, or for screening purposes. Repeat testing was carried out when deemed necessary by the attending physicians, in consultation with microbiologists. This occurred in cases where there were inconsistencies with expected results, a high clinical suspicion of TB, or indeterminate results.”

To clarify the time intervals for repeat testing, we have included two new columns in Table 2: 'Months until retesting (Mean ± SD)' and 'Range'. This addition provides a clear overview of the minimum time intervals and variability between initial and repeated testing.

Reviewer #4:

The manuscript is well written and highlighted the important subject of diagnosing LTBI, and more importantly to the borderline outcomes interrupting the results which alters in the retesting. Applause to the authors for extracting and analysing such data to develop this manuscript. There are a couple of minor comments/suggestions to this.

Reviewer comment 1: Line 75 - 86: this section could explore further the setup of health facility which the study was conducted, such as TB facility or any other, how patients were selected to test, QTF testing criteria, criteria for retesting of borderline results, and treatment and follow up or further care after LTBI diagnosis.

Response: Thank you for your valuable comment. We agree with your suggestion and have expanded on this topic in the Methods section. The following additional information has been included:

“A retrospective review of patients with QFT results between 2019 and 2023 was conducted, including demographic data such as age, gender, and country of birth. These variables have been associated, along with borderline test results, with both the likelihood of conversions and reversions of Interferon γ Release Assays [1]. The study was conducted in a routine clinical setting at a tertiary hospital in Madrid, focusing on patients of all age groups who underwent QFT testing. Children were not

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Decision Letter 1

Frederick Quinn

31 Jul 2025

Refining the Diagnostic Approach to Latent Tuberculosis Infection with Quantiferon Gold Plus: A Retrospective Analysis of Borderline Results

PONE-D-24-52042R1

Dear Dr. Roa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Frederick Quinn

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

Reviewer #6: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: (No Response)

Reviewer #5: Congrajulations to the authors on having successfyllly and adequately addressed all the coments given by the reviewesr. Though uch more could be done with the data set, the present manuscript is well written and complete in itself.. It is an important topic and the study needs to be published as soon as possible

I wonder if you can do a followup paper focussing on only the children below 15 or 18 and on those to 10 years of age and see if there is a difference in the rates of conversion in those two age group. This information will be very helpful in deciding policy regarding preventive therapy.

Reviewer #6: The authors have revised the manuscript based on the reviewers' suggestions, and it is now ready. for publication

**********

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Reviewer #4: No

Reviewer #5: Yes:  Manjula Datta

Reviewer #6: Yes:  Paola Di Carlo

**********

Acceptance letter

Frederick Quinn

PONE-D-24-52042R1

PLOS ONE

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Mean age and categorical distribution of follow-up QFT results when retesting those with initial results in the borderline range (0.20–0.70 IU/ml), by continent.

    (DOCX)

    pone.0330345.s001.docx (23.6KB, docx)
    Attachment

    Submitted filename: F_Response to reviewers and editor.docx

    pone.0330345.s002.docx (41.9KB, docx)

    Data Availability Statement

    The data that support the findings of this study are openly available in Figshare. The data can be accessed at Figshare using the following DOI link: https://doi.org/10.6084/m9.figshare.28659293.v1.

    Articles from PLOS One are provided here courtesy of PLOS

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