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Host, technical, and environmental factors affecting QuantiFERON-TB Gold In-Tube performance in children below 5 years of age

Interferon-gamma release assays performance can be impaired by host-related, technical and environmental factors, but data in young children are limited. We performed a cross-sectional study of children < 5 years-of-age at risk of tuberculosis (TB), using QuantiFERON-TB Gold In-Tube (QFT-GIT) assays. The impact of the following was evaluated: (i) host-related [age; hematological parameters; erythrocyte sedimentation rate (ESR); C-reactive protein (CRP); and tobacco smoke exposure (TSE) based on serum cotinine concentrations], (ii) technical (pre-analytical delay) and (iii) environmental factors (annual season; monthly temperatures). Of 204 children, 35 (17.2%) were diagnosed with latent TB infection or TB disease. QFT-GIT results were indeterminate in 14 (6.9%) patients. In multivariate analysis, younger age and higher ESR were associated with lower positive control responses (beta: 0.247, p = 0.002 and − 0.204, p = 0.007, respectively), and increasing age was associated with lower rates of indeterminate QFT-GIT results [OR (95% CI) 0.948 (0.903–0.996) per month, p = 0.035]. In children with positive QFT-GIT results, average monthly temperatures correlated with antigen responses (r = 0.453, p = 0.020); also, antigen responses were lower in winter than in other seasons (p = 0.027). Serum cotinine concentrations determined in a subgroup of patients (n = 41) indicated TSE in 36 (88%), positive control responses being lower in children with TSE (p = 0.034). In children < 5 years-of-age, young age, elevated ESR, temperature, annual season and TSE can affect the performance of QFT-GIT assays.

In adults, cigarette smoking has been associated with a reduction in IFN-γ production and higher rates of false-negative and indeterminate QFT-GIT results 20,21 . However, to date, the impact of tobacco smoke exposure (TSE) on IGRA results in children has not been investigated. Cotinine, a major metabolite of nicotine, has been used to assess tobacco use and environmental exposure both in active and passive smokers 22,23 . The half-life of cotinine in plasma is longer than that of nicotine. The former is estimated to be around 16 h in adults, but longer in neonates, infants, and children, thereby reflecting exposure over the preceding days [24][25][26] .
This study aimed to investigate the impact of a range of host-related factors on QFT-GIT assay performance specifically in children below 5 years of age in routine clinical practice in a low TB prevalence setting. Secondary objectives included the evaluation of other technical and environmental variables, including TSE for the first time, as well as the prevalence and risk factors of indeterminate QFT-GIT results.
In children with positive QFT-GIT results, background-corrected IFN-γ concentrations after TB antigenstimulation showed no significant correlation with age, hematological variables including WBC ratios, CRP concentration, ESR values, cotinine levels or pre-analytical delay. However, there was a positive correlation between average monthly temperatures (r = 0.453, p = 0.020) and background-corrected IFN-γ concentrations ( Supplementary Fig. S3). Also, in patients with positive QFT-GIT results, median background-corrected IFN-γ concentrations differed significantly between the seasons during which the tests were performed (winter: 1.41 [1.15-8.56 Fig. S4).

Effect of factors studied on QFT-GIT positive control responses and indeterminate result rates.
In the entire cohort, background-corrected IFN-γ concentrations following mitogen stimulation showed weak positive correlations with age (r = 0.231, p = 0.001), Hb level (r = 0.205, p = 0.005), and eosinophil count (r = 0.157, p = 0.035), weak inverse correlations with ESR (r = − 0.163, p = 0.040) and CRP concentrations (r = − 0.257, p = 0.001) and no associations with the rest of variables. In multivariate linear regression analysis that included age, eosinophil counts, CRP and ESR values, only age and ESR values remained significantly associated with background-corrected mitogen-induced IFN-γ concentrations (Fig. 1).
In bivariate analysis, age, AMC, MLR, and CRP values differed significantly between children with determinate and those with indeterminate QFT-GIT results (Table 1). Pre-analytical delay and the median temperature of the month did not have an impact on mitogen responses or the rate of indeterminate results (data not shown), and the rate of indeterminate results did not differ significantly between the seasons of the year (winter 5.1%, spring 5.8%, summer 7.7% and autumn 10.8%; p = 0.715). In binary logistic regression analysis including age, MLR, and CRP, only increasing age remained independently associated with a lower rate of indeterminate QFT-  Table S5).

Discussion
To our knowledge, this is one of the largest studies of young children at risk of TB to analyze the impact of a broad range of several host-related, technical, and environmental factors on the performance of IGRAs in a highincome, low TB prevalence setting, and the first exploratory study investigating the impact of TSE on IGRAs in children below 5 years of age at risk of TB.
The proportion of indeterminate results in our cohort (6.9%) was consistent with the proportions reported by other pediatric studies 3,17,27 . In line with other studies reporting that the large majority of indeterminate results in children are due to insufficient positive control responses rather than failed nil controls, we found that all indeterminate results in our study were due to the former. Lower age was significantly associated with indeterminate QFT-GIT results in logistic regression analysis, and multivariate linear regression analysis showed a significant correlation between lower age and lower background-corrected mitogen-induced IFN-γ concentrations. www.nature.com/scientificreports/ These findings are in concordance with previous studies that have shown that indeterminate IGRA results are significantly more common in young children than in adolescents or middle-aged adults 3,17,27,28 . Similar to our observations, other pediatric studies also found that there is a positive correlation between age and the magnitude of positive control responses, likely reflecting evolving immune maturation over the first few years of life 27,28 . However, in contrast to positive control responses, TB antigen-induced IFN-γ responses appear to be age-independent 3 . These data suggest that in order to reduce the rate of indeterminate assay results in infants and toddlers, either age-specific cut-offs for positive control responses or alternative positive control stimulants should be used 3,27 .
Comparisons between children with determinate and those with indeterminate results using bivariate analysis showed that the latter had higher AMCs, MLRs, and CRP values, indicating that there may be an association between systemic inflammation and indeterminate test results. However, inflammatory parameters were within the normal range or only mildly elevated in most patients in our cohort and only the association between ESR values and the magnitude of mitogen responses in QFT-GIT remained significant in multivariate analysis. This aligns with recent studies showing that inflammation impairs IGRAs performance, both in healhy children and in those affected with immune-mediated inflammatory disorders [29][30][31] .
Previous data suggest that indeterminate IGRA results are more common during colder months (i.e. during autumn and winter) than in warmer months (i.e. during spring and summer) 19 , which we did not observe, potentially due to the limited size of our cohort. In contrast to the aforementioned study 19 , all IGRA samples were taken and processed within the same building, thereby limiting the potential impact of environmental temperatures, which have been shown to impact on QFT-GIT assay performance 18 . However, we found that TB antigeninduced IFN-γ responses were lower during winter than any of the other seasons. Given our study set-up it is very unlikely that environmental temperatures are the cause of this observation. A more likely explanation lies in seasonal variations in the functioning of the human immune system, which have been reported previously 32 .
The observed TSE was surprisingly high, especially considering that children are particularly susceptible to adverse consequences related to SHS exposure, including an increased risk of lower respiratory infections and severe asthma 23 . The information regarding SHS exposure in infants is currently scarce. In a study conducted in San Francisco in which 70% of the participants were < 3 years of age, more than 55% showed evidence of smoke exposure, based on plasma cotinine measurements 33 . Smoking is also an independent risk factor for TB infection, progression of LTBI to TB disease, and death from TB 34,35 . There is some evidence to suggest that smoke exposure impacts adversely on inflammatory and immune responses, including IFN-γ mediated signaling, which may impair QFT-GIT performance in adult smokers 21,36 . In our cohort higher cotinine concentrations were associated with lower IFN-γ responses after mitogen stimulation, although the rate of indeterminate results did not vary between the TSE categories used in this study. However, it is possible that the number of patients in whom cotinine levels were determined in our study was too small to detect relatively subtle, but potentially relevant differences.
Our study has a number of limitations, including the limited number of children diagnosed with LTBI or TB, and the lack of inclusion of other clinical (i.e. nutritional status or atopic background) and biochemical (i.e. ferritin, albumin or vitamin D levels) parameters that have been reported to potentially interfere with QFT-GIT performance. Furthermore, we did not collect any parental reports regarding smoking and their child's SHS exposure; however, patient/parental reporting is deemed to be less accurate than biochemical testing 33 . Also, the design of the study prevents us from determining the impact of delays between blood draw and incubation of the samples 37,38 , as all blood samples were incubated in less than 90 min after phlebotomy.
In conclusion, in this cohort of previously healthy children below 5 years of age at risk of TB in a low prevalence setting, the rate of indeterminate QFT-GIT results was far higher than that reported by most adult studies, but consistent with other pediatric studies. Our observations show that indeterminate results are associated with   TST and IGRA testing. TSTs were performed by intradermal injection of 0.1 ml (2TU) of purified protein derivative (Tuberkulin PPD RT23 SSI, Statens Serum Institut; Copenhagen, Denmark), with results being read after 48-72 h. Following current national Spanish guidelines, the cut-offs for a positive TST result were ≥ 5 mm of induration in children assessed for clinically and/or radiologically suspected TB disease and those assessed following TB contact, and ≥ 10 mm in children undergoing new-entrant screening, irrespective of BCG vaccination status 6,39 . QFT-GIT (Cellestis/Qiagen; Carnegie, Australia) assays were performed and interpreted according to the manufacturer's instructions 40 . Blood samples were collected by a trained pediatric phlebotomist or by a pediatric nurse. Following shaking to ensure complete mixing of the blood sample with the tube additives, tubes were transported to an on-site, fully-accredited routine diagnostic laboratory in less than 90 min and were incubated immediately at 37 °C for 16 to 24 h. Then, tubes were centrifuged and plasma was removed and stored at 2-8 °C until the analysis was performed (every 1-2 weeks) measuring the amount of IFN-γ (in IU/mL) by enzyme-linked immunosorbent assay, after having brought the samples to room temperature (22 °C ± 5 °C). Briefly, a test was considered positive if the background-corrected IFN-γ response (i.e. concentration in TB-antigen tube minus concentration in the nil control tube) was ≥ 0.35 IU/mL and > 25% of the nil value. An indeterminate result was defined as insufficient IFN-γ response in the positive control tube (concentration < 0.5 IU/mL) and/or an IFN-γ concentration > 8.0 IU/mL in the nil control tube.
In a subset of patients for whom frozen serum samples taken at the initial assessment were available, serum cotinine concentrations were measured with the Cotinine Direct ® ELISA Kit (EIA-5496; DRG International Inc.; Springfield, NJ). The established range of serum cotinine to define secondhand smoke exposure used in most publications is 0.05-10 ng/mL 46 . In the present study TSE was classified according to serum cotinine concentrations as follows: (1) 'no/minimal TSE': < 0.05 ng/mL; (2) 'low TSE': 0.05-2.99 ng/mL; and (3) 'high TSE': ≥ 3 ng/mL 47 .
Statistical analyses were carried out using SPSS version 21.0 (IBM Corp., Armonk, NY, USA). Categorical variables are reported as proportions, and continuous variables as medians with interquartile ranges (IQRs). In bivariate analysis, patients with determinate (i.e. positive or negative) and those with indeterminate QFT-GIT results were compared by means of chi-square tests and Fisher's exact tests for qualitative variables, and Mann-Whitney U and Kruskal-Wallis tests for quantitative variables. Potential correlations between variables were assessed with Spearman's Rho tests. Variables with p-values < 0.10 without collinearity were included in subsequent binary logistic regression and multivariate linear regression analysis. Cotinine was excluded from these particular analyses, as results were only available in a subset of the study population. Statistical significance was defined as a p-value < 0.05.

Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.