Gates Open Res Gates Open Research 2572-4754 F1000 Research Limited London, UK 10.12688/gatesopenres.13191.3 Research Article Articles Comparing QuantiFERON-TB Gold Plus with QuantiFERON-TB Gold in-tube for diagnosis of latent tuberculosis infection among highly TB exposed gold miners in South Africa

[version 3; peer review: 2 approved, 1 approved with reservations]

 Ntshiqa Thobani Conceptualization Formal Analysis Methodology Project Administration Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-0752-3192 a 1 Chihota Violet Conceptualization Funding Acquisition Investigation Methodology Project Administration Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-1444-9266 1 2 Mansukhani Raoul Data Curation Formal Analysis Software Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-9456-5859 3 Nhlangulela Lindiwe Methodology Project Administration Writing – Original Draft Preparation Writing – Review & Editing 1 Velen Kavindhran Conceptualization Formal Analysis Methodology Project Administration Writing – Original Draft Preparation Writing – Review & Editing 1 Charalambous Salome Conceptualization Investigation Methodology Project Administration Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 2 Maenetje Pholo Investigation Methodology Project Administration Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 Hawn Thomas R. Formal Analysis Funding Acquisition Investigation Methodology Supervision Writing – Original Draft Preparation Writing – Review & Editing 4 Wallis Robert Conceptualization Funding Acquisition Investigation Methodology Writing – Original Draft Preparation Writing – Review & Editing 1 Grant Alison D. Conceptualization Formal Analysis Funding Acquisition Investigation Methodology Project Administration Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-2437-5195 2 3 5 Fielding Katherine Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Software Supervision Validation Writing – Original Draft Preparation Writing – Review & Editing 2 3 Churchyard Gavin Conceptualization Funding Acquisition Investigation Methodology Project Administration Resources Software Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 2 Implementation Research Division, The Aurum Institute, Johannesburg, Gauteng, 2193, South Africa School of Public Health, University of the Witwatersrand, Johannesburg, Gauteng, 2193, South Africa Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, United Kingdom, WC1E 7HT, UK Department of Medicine, University of Washington, Seattle, Seattle, New York, 98195, USA Africa Health Research Institute, Laboratory Medicine & Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban, KwaZulu-Natal, 4041, South Africa thobanintshiqa@yahoo.com

No competing interests were disclosed.

 18 8 2022 2021 5 66 4 8 2022 Copyright: © 2022 Ntshiqa T et al. 2022 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: QuantiFERON-TB-Gold-in-tube (QFT-GIT) is an interferon-gamma release assay (IGRA) used to diagnose latent tuberculosis infection. Limited data exists on performance of QuantiFERON-TB Gold-Plus (QFT-Plus), a next generation of IGRA that includes an additional antigen tube 2 (TB2) while excluding TB7.7 from antigen tube 1 (TB1), to measure TB specific CD4+ and CD8+ T lymphocytes responses. We compared the agreement between QFT-Plus and QFT-GIT among highly TB exposed goldminers in South Africa.

Methods: We enrolled HIV-negative goldminers in South Africa, aged ≥33 years with no prior history of TB disease or evidence of silicosis. Blood samples were collected for QFT-GIT and QFT-Plus. QFT-GIT was considered positive if TB1 tested positive; while QFT-Plus was positive if both or either TB1 or TB2 tested positive, as per manufacturer's recommendations. We compared the agreement between QFT-Plus and QFT-GIT using Cohen’s Kappa. To assess the specific contribution of CD8+ T-cells, we used TB2−TB1 differential values as an indirect estimate. A cut-off value was set at 0.6. Logistic regression was used to identify factors associated with having TB2-TB1>0.6 difference on QFT-Plus.

Results: Of 349 enrolled participants, 304 had QFT-Plus and QFT-GIT results: 205 (68%) were positive on both assays; 83 (27%) were negative on both assays while 16 (5%) had discordant results. Overall, there was 94.7% (288/304) agreement between QFT-Plus and QFT-GIT (Kappa = 0.87). 214 had positive QFT-Plus result, of whom 202 [94.4%, median interquartile range (IQR): 3.06 (1.31, 7.00)] were positive on TB1 and 205 [95.8%, median (IQR): 3.25 (1.53, 8.02)] were positive on TB2. A TB2-TB1>0.6 difference was observed in 16.4% (35/214), with some evidence of a difference by BMI; 14.9% (7/47), 9.8% (9/92) and 25.3% (19/75) for BMI of 18.5-24.9, 18.5-25 and >30 kg/m 2, respectively (P=0.03).

Conclusion: In a population of HIV-negative goldminers, QFT-Plus showed high agreement with QFT-GIT, suggesting similar performance.

 Latent Tuberculosis Infection QFT-GIT QFT-Plus TST Performance Goldmines South Africa Gates Foundation OPP1116635 This study was funded by the Gates Foundation [OPP1116635]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 2

The difference between the current and previously published versions is the removal of the word “performance” in the study title. The study title now reads as follows: Comparing QuantiFERON-TB Gold Plus with QuantiFERON-TB Gold in-tube for diagnosis of latent tuberculosis infection among highly TB exposed gold miners in South Africa. The word performance was also removed from the introduction, objectives, and methods sections both in the abstract and main manuscript to reflect that agreement between QFT-Plus and QFT-GIT was measured rather than performance.

 Introduction

Latent tuberculosis infection (LTBI) is the seedbed from which tuberculosis (TB) cases arise. LTBI is defined as an asymptomatic state characterized with a persistent immune response to stimulation by Mycobacterium tuberculosis (Mtb) antigens with no evidence of active TB 1– 3 . LTBI is typically characterized by a positive tuberculin skin test (TST) in vivo, involving intradermal injection of purified protein derivative from Mtb strain and/or a positive interferon-gamma release assay (IGRA) 4, 5 . Better tests are needed to identify persons at increased risk of developing TB disease.

IGRAs measure released interferon-gamma from cluster of differentiation (CD) T-lymphocytes specific to Mtb complex antigens but not produced by Mycobacterium bovis BCG vaccine strains 6 . QuantiFERON-TB Gold In-Tube assay (QFT-GIT) is designed to elicit interferon-gamma response from CD4+ helper T lymphocytes in a single TB antigen tube containing long peptides from ESAT-6, CPF-10 and TB7.7 antigens (Qiagen, Germantown, MD) 5– 7 . QuantiFERON-TB Gold Plus assay (QFT-Plus) is a next generation IGRA that contains peptides from only the ESAT-6 and CFP-10 antigens comprising a TB1 tube, identical to the QFT-GIT, with the exception of TB7.7, and stimulates CD4+ T cells, and an additional antigen tube, TB2, which has a cocktail of both long and short ESAT-6 and CFP-10 peptides to elicit interferon-gamma release from both CD4+ helper T lymphocytes and CD8+ cytotoxic T lymphocytes 5– 8 .

CD8+ cytotoxic T lymphocytes stimulating peptide was included in the QFT-Plus assay to improve on the sensitivity of QFT-GIT. Currently, limited data exist comparing the performance of these assays in high TB burdened settings. We compared the agreement between QFT-Plus and QFT-GIT among highly Mtb exposed goldminers in South Africa.

 Methods Study setting and population

The study was conducted in the South African goldmines at the Occupational Health Centre (OHC), in Orkney, North West Province among goldminers, attending for their annual medical examination between July 2015 and December 2016.

 Study design and procedures – parent study

In a cohort study, herein described as the parent study, we enrolled miners to identify those who were uninfected with Mtb despite being highly exposed to Mtb, to compare epidemiological factors between Mtb uninfected and infected miners and to collect specimens from Mtb uninfected and infected miners to determine gene expression and immunological profiles associated with being Mtb uninfected in future analysis.

Goldminers, attending OHC for their annual medical examination were pre-screened for the study to identify those aged 33–60 years who had worked in the mining industry for at least 15 years. Following informed consent, a full screen was conducted. Miners were included if they did not have symptoms suggestive of TB, no prior or current history of treatment for active TB disease, no history of or not currently taking isoniazid preventive therapy, no silicosis, had body mass index (BMI) >18.5, no serious medical conditions, HIV negative and no current treatment for cancer, no treatment with steroid tablets, inhalers or injections.

Blood samples were collected intravenously by trained professional phlebotomy nurses amongst those who met the inclusion criteria for QFT (QFT-Plus and QFT-GIT; Qiagen, Hilden Germany), peripheral blood mononuclear cell (PBMC), and transcriptomic (PAXgene) testing. Under the first version of the protocol (enrolments from 10 July 2015 to 29 October 2015), participants gave blood samples for QFT at enrolment.

A sputum sample was also collected for mycobacterial culture testing (BACTEC MGIT 960 system, BD Diagnostic Systems, Sparks, MD, USA) to exclude subclinical TB. A questionnaire was administered in a private room by a trained research assistant to collect demographic characteristics and information on factors associated with being TB uninfected. Blood samples collected were tested at the Aurum Clinical Research laboratory for LTBI using QFT-GIT and QFT-Plus. A 6 ml of whole blood sample was collected intravenously from each participant into a single lithium heparin tube. Samples were then transported to Aurum Clinical Research laboratory where they were aliquoted into 1 ml tubes: three tubes for QFT-GIT test kit (QFT-GIT nil, QFT-GIT TB, QFT-GIT mitogen with catalogue numbers 0594-0201 and 0594-0501) and four for QFT-Plus (QFT-Plus nil, QFT-Plus TB1, QFT-Plus TB2, QFT-Plus mitogen with catalogue number 622120). Tubes were then placed in a pre-heated 37°C portable incubator for 16–24 hours, within eight hours of collection. Subsequently, samples were centrifuged to separate plasma for same day testing. Alternatively, samples were stored at -80°C for up to 48–72 hours prior testing. Plasma was tested by interferon-gamma enzyme-linked immunosorbent assay (ELISA), performed using Biotek microplate reader model EL x 800 using Gen 5 software. Biotek microplate washer model EL x 508 and Thermostar shaker were also used.

 Study design – sub-study

In this sub-study, using cross-sectional data from the parent study, we compared the agreement between QFT-Plus and QFT-GIT using QFT measurements which were all done at baseline.

 Study definitions

QFT-GIT was considered positive if TB1 tested positive; while QFT-Plus was positive if both TB1 and TB2 tested positive or if either TB1 or TB2 tested positive, as per manufacturer's recommendations. To assess the specific contribution of CD8+ T-cells, we used TB2−TB1 differential values as an indirect estimate. A cut-off value was set at 0.6 in order to reduce the bias of the intrinsic variability of the test 9 .

 Statistical methods

All eligible participants from the parent study were included in the analysis. Binary outcomes (positive/negative) from QFT-Plus and QFT-GIT were compared using the percentage agreement and Kappa statistic. TB1 and TB2 responses for those QFT-Plus positive were summarized by QFT-GIT status using median and interquartile range (IQR) and percentage positive (measurement minus nil response>0.35). Logistic regression was used to identify factors associated with having TB2-TB1 >0.6. Results were summarized using odds ratios (OR) with their corresponding 95% confidence intervals (CI) and p-values. Due to a small number of outcomes a multivariable analysis was not conducted. Data were analyzed using Stata version 15 (StataCorp. 2017. Stata Statistical Software: Release 15. College Station, TX: StataCorp LP.

 Ethical statement

The study received ethical clearance from the University of Witwatersrand Human Research Ethics Committee (WHREC Ref: 150217), London School of Hygiene & Tropical Medicine, UK (LSHTM Ethics Ref: 9279), University of Washington, USA (IRB number 33335) and North West Health Research and Ethics Committee (DOH-27-0515-4991). We sought informed consent from all study participants using written informed consent and information sheets available in the most commonly used local languages. Participants who were unable to read or write were asked to make a mark or thumbprint in the presence of a witness. This study was conducted according to Good Clinical Practice guidelines, in accordance with the requirements of the funders and respective ethics committees.

 Results Process flow

We approached 25,627 miners, 17,030 (66.5%) agreed to be pre-screened, of whom 3,534 (20.8%) satisfied the pre-screen criteria and were eligible for full screening, following informed consent ( Figure 1). Overall, 2,980 (84.3%) were offered consent, of whom 1,749 (58.7%) consented and 1,231 (41.3%) declined to take part in the study; 554 (15.7%) were not offered consent as they were lost in the OHC queue.

Participant flow chart at enrolment.

*Data from the daily attendance register. Includes repeat attendances during this time period. §Screen out questions were asked in stages; if screened out at a stage no screening was conducted for subsequent stages. Within a stage more than one screen-out reason may apply. Yrs, years; IPT, isoniazid preventive therapy; BMI, body mass index; QFT-Plus, QuantiFERON-TB Gold-Plus; QFT-GIT, QuantiFERON-TB Gold In-Tube assay,.

Following the full screen, 349/1,749 (20.0%) met the inclusion criteria and were enrolled into the study. Of the 349, 304 had a baseline QFT-Plus and QFT-GIT.

 Demographic characteristics

Of the 349 participants enrolled into the study, the median age was 48 years (IQR 45, 53 years), median years in the workface was 24 (IQR 18, 28 years) and 98.6% (344) were male ( Table 1). Overall, 92.0% (321) were of Black/African ethnicity, 72.5% (253) had a BCG scar present, 66.8% (233) were born in South Africa and a minority lived in a mine hostel (28.7%; 100). Subsample of the 349 participants who had a baseline QFT-Plus and QFT-GIT result (n=304) had similar demographic characteristics to the overall sample 10 .

Summary of demographic characteristics at enrolment.
Variable Participants Enrolled Participants enrolled with a QFT-Plus and QFT-GIT result
N 349 304
Age, years Median (IQR) 48 (45-52) 48 (44-52)
Sex Male 344 (98.6%) 300 (98.7%)
BCG scar No 84 (24.1%) 74 (24.3%)
Yes 253 (72.5%) 221 (72.7%)
Indeterminate 12 (3.4%) 9 (3.0%)
Country of birth South Africa 233 (66.8%) 204 (67.1%)
Lesotho 59 (16.9%) 50 (16.4%)
Mozambique 37 (10.6%) 32 (10.5%)
Other 20 (5.7%) 18 (5.9%)
Ethnicity Black/African 321 (92.0%) 279 (91.8%)
Hostel No 170 (48.7%) 155 (51.0%)
Hostel 100 (28.7%) 83 (27.3%)
Other mine house 79 (22.6%) 66 (21.7%)
Years worked underground Median (IQR) 24 (18-28) 24 (17.5-28)
Sleeping arrangement Alone 51 (14.6%) 46 (15.1%)
1 person 206 (59.0%) 182 (59.9%)
> 1 person 92 (26.4%) 76 (25.0%)
Occupation Unskilled 271 (77.7%) 236 (77.6%)

IQR, interquartile range; QFT-Plus, QuantiFERON-TB Gold-Plus; QFT-GIT, QuantiFERON-TB Gold.

 Comparison of QuantiFERON-TB Gold-Plus vs. Gold in-tube

Of the 304 who had QFT-Plus and QFT-GIT results, 214 (70.4%) had a positive QFT-Plus result, 205 (67.4%) were positive on both assays; 83 (27.3%) were negative on both assays, while 16 (5.3%) had discordant results (seven QFT-Plus negative/QFT-GIT positive; nine QFT-Plus positive/QFT-GIT negative) ( Table 2). Overall, the agreement between QFT-Plus and QFT-GIT was 94.7% (288/304) and Kappa was 0.87.

Comparison of QuantiFERON-TB Gold-Plus vs. QuantiFERON-TB Gold in-tube.
QFT-GIT N QFT-Plus QFT-Plus interferon-gamma concentration, among those positive on QFT- Plus
Positive Negative Positive result *: TB1 (%) Positive result *:TB2 (%) TB1-nil Median (IQR) TB2-nil Median (IQR) TB2 - TB1 median difference (IQR) TB2 - TB1 >0.6
Positive 212 205 7 198/205 a (96.6%) 200/205 b (97.6%) 3.06 (1.31, 7) 3.25 (1.53, 8.02) 0.00 (-0.16,0.39) 34/205 (16.6%)
Negative 92 9 83 4/9 c (44.4%) 5/9 d (55.6%) 0.35 (0.18, 0.53) 0.37 (0.28, 0.45) 0.02 (-0.23,0.30) 1/9 (11.1%)
Total 304 ^ 214 90 202/214 (94.4%) 205/214 (95.8%) 2.89 (1.18, 6.97) 2.95 (1.17, 7.79) 0.00 (-0.17,0.39) 35/214 (16.4%)

^ n=304 who have QFT-Plus and QFT-GIT. Percentage agreement 94.7% (95%CI: 91.6– 97.0%); Kappa 0.87.

a Of the 198, n=5 are positive on TB1 alone; b of the 200, n=7 are positive on TB2 alone; c of the four, all are positive on TB1 alone; d of the five, all are positive on TB2 alone.

e Of the 154, n=3 are positive on TB1 alone; f of the 157, n=6 are positive on TB2 alone; g of the three, all are positive on TB1 alone; h of the three, all are positive on TB2 alone.

* Defined as TB1-nil>0.35 or TB2-nil>0.35.

QFT-Plus, QuantiFERON-TB Gold-Plus; QFT-GIT, QuantiFERON-TB Gold in-tube; IQR interquartile range; CI confidence interval.

 Assessing the specific contribution of CD8+ T-cells

Of the 214 who had positive QFT-Plus results, 202 (94.4%) were positive on TB1, while 205 (95.8%) were positive on TB2 ( Table 2). The median (IQR) values for TB1 and TB2 among those who were QFT-Plus positive were 3.06 (1.31, 7.00) and 3.25 (1.53, 8.02), respectively. The median difference between the TB1 and TB2 was 0.00 (-0.17, 0.39). A >0.6 difference between TB2 and TB1 was observed in 16.4% (35/214) of those who were positive on QFT-Plus ( Table 2). Only BMI status was found to be associated with TB2-TB1 >0.6; unadjusted OR 1.94 (95% CI: 0.74–5.05) for BMI>30 kg/m 2 versus BMI between 18.5–24.9 ( Table 3).

Risk factors for having TB2-TB1>0.6 on QFT-plus.
Variable N TB2-TB1 >0.6 n (%) Univariable analysis
Crude OR 95% CI p-value *
Age group, years 0.85
<45 48 7 (14.6) 1
45–49 71 13 (18.3) 1.31 0.48 - 3.58
≥50 95 15 (15.8) 1.10 0.42 - 2.91
Gender 0.66
Male 210 34 (16.2) 1
Female 4 1 (25.0) 1.73 0.17 - 17.09
Ethnicity 0.20
Black/African 209 33 (15.8) 1
Other 5 2 (40.0) 3.56 0.57 - 22.11
Marital status 0.14
Married 193 29 (15.0) 1
Other 21 6 (28.6) 2.26 0.81 - 6.31
Country of birth 0.85
South Africa 141 25 (17.7) 1
Lesotho 39 5 (12.8) 0.68 0.24 - 1.92
Mozambique 23 3 (13.0) 0.70 0.19 - 2.52
Other 11 2 (18.2) 1.03 0.21 - 5.07
Occupational level 0.17
Unskilled 177 26 (14.7) 1
Skilled 37 9 (24.3) 1.87 0.79-4.41
Years worked underground 0.99
<20 61 10 (16.4) 1
20-29 112 18 (16.1) 0.98 0.42 - 2.27
≥30 41 7 (17.1) 1.05 0.36 - 3.03
Type of mine housing 0.31
Not staying in mine house 102 17 (16.7) 1
Hostel 68 8 (11.8) 0.67 0.27 - 1.64
Other mine housing 44 10 (22.7) 1.47 0.61 - 3.53
Sleeping arrangement 0.30
Alone 38 5 (13.2) 1
1 person 129 25 (19.4) 1.59 0.56 - 4.48
>1 person 47 5 (10.6) 0.79 0.21 - 2.94
BCG Scar 0.39
Yes/indeterminate ¥ 159 28 (17.6) 1
No 55 7 (12.7) 0.68 0.28-1.66
BMI, kg/m 2 0.03
18.5-24.9 47 7 (14.9) 1
25-29.9 92 9 (9.8) 0.62 0.22 - 1.78
≥30 75 19 (25.3) 1.94 0.74 - 5.05

* P-value from the likelihood ratio test; ¥, n=4 indeterminate.

QFT-Plus, QuantiFERON-TB Gold-Plus; OR, odds ratio; CI confidence interval.

 Discussion

In this study, conducted in South African goldmines, the overall agreement between QFT-Plus and QFT-GIT was high at 94.7%, suggesting that QFT-GIT may have similar performance to QFT-Plus; consistent with previous evaluation studies conducted in low TB incidence settings which showed similar diagnostic performance and high overall agreement between QFT-Plus and its predecessor 5, 8, 11– 15 . The high concordance (or low discordance) in our study may be because our study was in HIV negative adults among whom QFT-GIT would be expected to have relatively high sensitivity. A greater difference between the two tests might be expected in populations where QFT-GIT typically has poor sensitivity e.g. children and people with advanced HIV disease 11, 16– 18 . Overall, there were 16 discordant pairs (5.3%); seven were QFT-Plus negative/QFT-GIT positive and nine were QFT-Plus positive/QFT-GIT negative. It is interesting that only 56.3% were QFT-Plus positive/QFT-GIT negative and not higher. The discordancy rate found in our study was consistent with findings from Theel et al. and Moon et al. studies, which showed discordancy rates of 3.1% and 4.4%, respectively 5, 12 . However, of the five discordant pairs in the Theel et al. study, 60.0% (3/5) were QFT-Plus negative/QFT-GIT positive and 40.0% (2/5) were QFT-Plus positive/QFT-GIT negative; while in the Moon et al. study, 25.6% (11/43) were QFT-Plus negative/QFT-GIT positive and 74.4% (32/43) were QFT-Plus positive/QFT-GIT negative in the 43 discordant pairs. This is thought to be due to several factors, broadly classified as preanalytical, analytical, postanalytical, manufacturing, immunological, and interferon-gamma levels bordering on the binary 0.35 IU/ml cutoff for assay positivity 9, 19– 21 .

We observed a TB2−TB1 difference >0.6 among 35 (16.4%) individuals who had QFT-Plus positive results, associated with obesity. Barcellini et al. had also observed a similar TB2−TB1 difference >0.6 in a small proportion of TB contacts who had a positive QFT-Plus results 18 (15.1%); suggesting presence of Mtb-specific CD8+ T lymphocytes, which may be indicative of a higher antigenic burden 14, 22– 27 . The presence of Mtb-specific CD8+ T lymphocytes in latently infected miners may therefore be predictive of Mtb active replication and may be indicative of higher likelihood of disease progression 23 . In Barcellini et al. study, sleeping in the same room and the European origin were significantly associated with TB2−TB1 difference >0.6 (27). In this study, only obesity was associated with TB2−TB1 difference >0.6, suggesting a greater TB2 response among obese participants. However, multivariable analysis was not conducted, due to the small number of outcomes. A much bigger sample size may be needed to investigate relative prognostic value of the TB1 and TB2 antigen tubes and further explore the association between TB2−TB1 difference >0.6 and BMI.

Study limitations

This sub-analysis was based on a cross-sectional sample and did not include follow-up to confirm the LTBI status due to low number of individuals available to provide blood sample to conduct Mtb infection testing. The sample size of 304 was relatively small to make strong inferences. In addition, the study was conducted among HIV-negative goldminers who were most likely to have experienced prolonged and high exposure to Mtb. However, results may be generalizable as the performance of a diagnostic test is independent of setting and prevalence of infection. The lack of a gold-standard test for Mtb infection means that where results were discordant, we cannot know which (if either) was correct.

 Conclusion

Among HIV-negative goldminers in South Africa, QFT-Plus showed high agreement with QFT-GIT, suggesting similar performance. For most discordant results, interferon-gamma concentrations bordered on the binary cut-off for assay positivity.

 Data availability

LSHTM Data Compass: Data set for the comparison of the performance of QuantiFERON-TB Gold Plus with QuantiFERON-T Gold in-tube among highly TB exposed gold miners in South Africa. https://doi.org/10.17037/DATA.00001891 10 .

This project contains the following underlying data:

Hetu-dataset.txt

Hetu_data_codebook.html

Hetu_data_userguide.html

Due to ethical concerns, dataset access is restricted to ensure privacy and confidentiality of participant data. However, raw data is available upon request under a custom data sharing agreement and will require authorization from Principal Investigators (Professor Violet Chihota: VChihota@auruminstitute.org and Professor Katherine Fielding: Katherine.Fielding@lshtm.ac.uk). Once access is granted, the files will be made available on LSHTM Data Compass.

The data codebook and user guide are available under the terms of the Creative Commons Attribution 3.0 International license (CC-BY 3.0).

 Acknowledgements

The authors are thankful to the North West Department of Health and gold mining companies for the gracious endorsement for undertaking of this study. We also thank all the participants who participated in the HETU and the following lab and field researchers: Ithabeleng Morojele, Keolebogile Ntshamane, Tebogo Rampai, Ndumiso Sithole, Zamakhabako Mhlanga, Ntombomzi Motsoeneng, James Seseng, Samuel Tlhabakwane, Abel Qas, Kabelo Leshoro, Samantha Naicker, Martha Albani, Ken Clarke, Palesa Mosweu, Letlhogonolo Seabela, Nondumiso Langa.

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AIDS. 2008;22(17):2241–50. 18981763 10.1097/QAD.0b013e3283117f18 2743094 Rose MV Kimaro G Nissen TN : QuantiFERON ®-TB Gold In-Tube Performance for Diagnosing Active Tuberculosis in Children and Adults in a High Burden Setting. PLoS One. 2012;7(7):e37851. 22808002 10.1371/journal.pone.0037851 3395691 Santin M Muñoz L Rigau D : Interferon-γ Release Assays for the Diagnosis of Tuberculosis and Tuberculosis Infection in HIV-Infected Adults: A Systematic Review and Meta-Analysis. PLoS One. 2012;7(3):e32482. 22403663 10.1371/journal.pone.0032482 3293815 Whitworth WC Hamilton LR Goodwin DJ : Within-Subject Interlaboratory Variability of QuantiFERON-TB Gold In-Tube Tests. PLoS One. 2012;7(9):e43790. 22970142 10.1371/journal.pone.0043790 3435391 Detjen AK Loebenberg L Grewal HMS : Short-Term Reproducibility of a Commercial Interferon Gamma Release Assay. Clin Vaccine Immunol. 2009;16(8):1170–5. 19535542 10.1128/CVI.00168-09 2725540 Banaei N Gaur RL Pai M : Interferon Gamma Release Assays for Latent Tuberculosis: What Are the Sources of Variability? J Clin Microbiol. 2016;54(4):845–50. 26763969 10.1128/JCM.02803-15 4809912 Rozot V Vigano S Mazza‐Stalder J : Mycobacterium tuberculosis-specific CD8 + T cells are functionally and phenotypically different between latent infection and active disease. Eur J Immunol. 2013;43(6):1568–77. 23456989 10.1002/eji.201243262 6535091 Day CL Abrahams DA Lerumo L : Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load. J Immunol. 2011;187(5):2222–32. 21775682 10.4049/jimmunol.1101122 3159795 Lewinsohn DA Heinzel AS Gardner JM : Mycobacterium tuberculosis–specific CD8 + T Cells Preferentially Recognize Heavily Infected Cells. Am J Respir Crit Care Med. 2003;168(11):1346–52. 12969871 10.1164/rccm.200306-837OC Chiacchio T Petruccioli E Vanini V : Polyfunctional T-cells and effector memory phenotype are associated with active TB in HIV-infected patients. J Infect. 2014;69(6):533–45. 24975174 10.1016/j.jinf.2014.06.009 Lancioni C Nyendak M Kiguli S : CD8 + T Cells Provide an Immunologic Signature of Tuberculosis in Young Children. Am J Respir Crit Care Med. 2012;185(2):206–12. 22071329 10.1164/rccm.201107-1355OC 3297089 Barcellini L Borroni E Brown J : First independent evaluation of QuantiFERON-TB Plus performance. Eur Respir J. 2016;47(5):1587–90. 26869677 10.1183/13993003.02033-2015 10.21956/gatesopenres.15091.r34041 Reviewer response for version 3 Sarkar Sonali 1 Referee JIPMER, Puducherry, India

Competing interests: No competing interests were disclosed.

 7 8 2023 Copyright: © 2023 Sarkar S 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The authors have compared the agreement between QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-Tube Interferon Gamma Release Assays in South African gold miners with the premise that the results might vary from the multiple studies done in low burden settings.

The assumption that the agreement between the two tests can vary is a valid given the knowledge that the spectrum of the condition under study has some influence on sensitivity and specificity of the tests performed. Having said that, it is also noted that the artifactual variability that is introduced by excluding participants in whom the TB infection is difficult to diagnose, such as those with body mass index (BMI) <18.5, having serious medical conditions, HIV positive and current treatment for cancer, treatment with steroid tablets, inhalers or injections, overestimates the accuracy of the tests.(1) There are multiple studies reporting the agreement between the two tests in lower incidence setting. Results from repetition of a similar study in a high-burden setting was expected and does not contribute to the clinical decision of using the QFT-Plus in similar or other settings. The inputs required for justification for the use of QFT-Plus would come from directional hypothesis with minimal acceptable criteria set in calculation of sample size (2). An attempt at sample size calculation should have been done to justify the validity of the results.

Minor comments regarding the study are as follows.

The flowchart of the number of participants does not match with the numbers stated in the text.

Typographical error in line 3 of the results section under the demographic characteristics, work-face to be corrected as work-force.

 References

Leeflang MM, Rutjes AW, Reitsma JB, Hooft L, Bossuyt PM. Variation of a test's sensitivity and specificity with disease prevalence. CMAJ. 2013 Aug 6;185(11):E537-44. doi: 10.1503/cmaj.121286. Epub 2013 Jun 24. PMID: 23798453; PMCID: PMC3735771.

Korevaar, D.A., Gopalakrishna, G., Cohen, J.F. et al. Targeted test evaluation: a framework for designing diagnostic accuracy studies with clear study hypotheses. Diagn Progn Res 3, 22 (2019). https://doi.org/10.1186/s41512-019-0069-2

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

No

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Epidemiology of Tuberculosis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

References : Variation of a test's sensitivity and specificity with disease prevalence. CMAJ .2013;185(11) : 10.1503/cmaj.121286 E537-44 23798453 10.1503/cmaj.121286 : Targeted test evaluation: a framework for designing diagnostic accuracy studies with clear study hypotheses. Diagn Progn Res .2019;3: 10.1186/s41512-019-0069-2 22 31890896 10.1186/s41512-019-0069-2 10.21956/gatesopenres.15091.r34036 Reviewer response for version 3 Barcellini Lucia 1 Referee University of Milan, Milan, Italy

Competing interests: No competing interests were disclosed.

 2 8 2023 Copyright: © 2023 Barcellini L 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The study compared the performance of QuantiFERON-TB Gold-Plus (QFT-Plus) with the standard QFT-GIT assay in diagnosing latent tuberculosis infection in goldminers. QFT-Plus showed high agreement (94.7%) with QFT-GIT, suggesting similar performance. 

The microbiological procedure used in the study is reliable, and the inclusion of subclinical TB testing with a culture is a strong aspect.

However, the inclusion criteria have led to a significant reduction in the population included, and they are not entirely clear.

While some criteria are clear, such as excluding previous TB patients or those currently on prophylaxis, the rationale behind others is less transparent.

If the objective was to assess the agreement between the two tests, having a more diverse sample, including different age groups, individuals with low BMI, and those with comorbidities, could actually be advantageous.

Please, clarify the specific rationale behind the chosen criteria and /or discuss them as a limitation.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Microbiology: diagnostic test for TB

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.21956/gatesopenres.15091.r32392 Reviewer response for version 3 Hatherill Mark 1 Referee https://orcid.org/0000-0003-3491-1809 South African Tuberculosis Vaccine Initiative (SATVI), University of Cape Town, Cape Town, South Africa

Competing interests: No competing interests were disclosed.

 25 8 2022 Copyright: © 2022 Hatherill M 2022 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

This revision is satisfactory, I have no further comments.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

No source data required

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

NA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.21956/gatesopenres.14885.r31933 Reviewer response for version 2 Hatherill Mark 1 Referee https://orcid.org/0000-0003-3491-1809 South African Tuberculosis Vaccine Initiative (SATVI), University of Cape Town, Cape Town, South Africa

Competing interests: No competing interests were disclosed.

 6 6 2022 Copyright: © 2022 Hatherill M 2022 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The authors have revised statements that “ QFT-Plus showed similar performance to QFT-GIT” to state that QFT-Plus showed high agreement with QFT-GIT, suggesting similar performance, since performance of these tests to differentiate true Mtb infection was not measured.

However, in version 2 the abstract, introduction and methods still state incorrectly that the study compared performance of QFT-Plus with QFT-GIT. This needs to be corrected to reflect that agreement was tested, not performance.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

No source data required

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

tuberculosis vaccinology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.21956/gatesopenres.14396.r30700 Reviewer response for version 1 Hatherill Mark 1 Referee https://orcid.org/0000-0003-3491-1809 South African Tuberculosis Vaccine Initiative (SATVI), University of Cape Town, Cape Town, South Africa

Competing interests: No competing interests were disclosed.

 4 6 2021 Copyright: © 2021 Hatherill M 2021 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Ntshiqa et al. report a comparison of QuantiFERON-TB Gold Plus with QuantiFERON-TB Gold in-tube among highly TB exposed gold miners, showing that agreement was high (94%). 

Major comments:

The data analysed are from a subset of a larger parent study and the 304 participants available for direct comparison form only 1.8% of the 17,030 who consented to screening; and the study participants included in the analysis are all very highly exposed to Mtb over a long period. i.e. It is not clear if the study population is representative of other settings, or to what extent the results are generalisable.

Minor comments:

Abstract: Typo 33 years of age?

Study limitations: The statement that “it is unlikely that the LTBI status would change over a relatively short period of follow-up” does not justify the lack of confirmatory testing.

Discussion: No TST information is included in the title, abstract or discussion, but is included in the methods and main results. Suggest TST information is either included in the title and abstract, and concordance/discordance with the IGRA tests is interpreted and discussed further, or TST is removed entirely from this manuscript.

The observation that TB2-TB1 was associated with BMI deserves further discussion.

Conclusion (abstract and main text): Here and elsewhere - suggest statements that “QFT-Plus showed similar performance to QFT-GIT” should be revised to state that QFT-Plus showed high agreement with QFT-GIT, or qualified as per the Discussion “suggesting similar performance” since performance of these tests to differentiate true Mtb infection was not measured.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

No source data required

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

tuberculosis vaccinology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.