Journal of Clinical Medicine Article Tobacco Smoking and Second-Hand Smoke Exposure Impact on Tuberculosis in Children Neus Altet 1,2,† , Irene Latorre 3, *,† , María Ángeles Jiménez-Fuentes 1 , Antoni Soriano-Arandes 1 , Raquel Villar-Hernández 3 , Celia Milà 1 , Pablo Rodríguez-Fernández 3,4 , Beatriz Muriel-Moreno 3 , Patricia Comella-del-Barrio 3 , Pere Godoy 5,6,7 , Joan-Pau Millet 2,6 , Maria Luiza de Souza-Galvão 1 , Carlos A. Jiménez-Ruiz 8 , Jose Domínguez 3 and on behalf of PII Smoking SEPAR Working Group ‡ 1 Unitat de Tuberculosi Vall d’Hebron-Drassanes, Hospital Universitari Vall d’Hebron, 08001 Barcelona, Spain; mneusagomez@gmail.com (N.A.); m.jimenez@vhebron.net (M.Á.J.-F.); asoriano@vhebron.net (A.S.-A.); cmila@vhebron.net (C.M.); msouza@vhebron.net (M.L.d.S.-G.) 2 Serveis Clínics, Unitat Clínica de Tractament Directament Observat de la Tuberculosi, 08022 Barcelona, Spain; jmillet@aspb.cat 3 Institut d’Investigació Germans Trias i Pujol, CIBER Enfermedades Respiratorias, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; rvillar@igtp.cat (R.V.-H.); pablobena20@outlook.es (P.R.-F.); bmoreno@igtp.cat (B.M.-M.); patricia.comella@gmail.com (P.C.-d.-B.); jadominguez@igtp.cat (J.D.) 4 Institut de Biotecnologia i Biomedicina, 08193 Cerdanyola del Vallès, Spain 5 Departament de Salut, Generalitat de Catalunya, 08028 Barcelona, Spain; pere.godoy@gencat.cat  6 CIBER Epidemiología y Salud Pública, 28029 Madrid, Spain  7 IRB-Lleida, Universitat de Lleida, 25198 Lleida, Spain Citation: Altet, N.; Latorre, I.; 8 Unidad de Tabaquismo de la Comunidad Autónoma de Madrid, 28015 Madrid, Spain; victorina@ctv.es Jiménez-Fuentes, M.Á.; * Correspondence: ilatorre@igtp.cat; Tel.: +34-93-033-0537 Soriano-Arandes, A.; † These authors contributed equally to this work. Villar-Hernández, R.; Milà, C.; ‡ PII Smoking SEPAR Working Group is provided in the Acknowledgments. Rodríguez-Fernández, P.; Muriel-Moreno, B.; Abstract: Little is known about whether second-hand smoke (SHS) exposure affects tuberculosis Comella-del-Barrio, P.; Godoy, P.; (TB). Here, we investigate the association of cigarette smoke exposure with active TB and latent TB et al. Tobacco Smoking and infection (LTBI) in children, analyzing Interferon-Gamma Release Assays’ (IGRAs) performance and Second-Hand Smoke Exposure cytokine immune responses. A total of 616 children from contact-tracing studies were included and Impact on Tuberculosis in Children. J. classified regarding their smoking habits [unexposed, SHS, or smokers]. Risk factors for positive Clin. Med. 2022, 11, 2000. IGRAs, LTBI, and active TB were defined. GM-CSF, IFN-γ, IL-2, IL-5, IL-10, IL-13, IL-22, IL-17, https://doi.org/10.3390/ TNF-α, IL-1RA and IP-10 cytokines were detected in a subgroup of patients. Being SHS exposed jcm11072000 was associated with a positive IGRA [aOR (95% CI): 8.7 (5.9–12.8)] and was a main factor related Academic Editor: Martin with LTBI [aOR (95% CI): 7.57 (4.79–11.94)] and active TB [aOR (95% CI): 3.40 (1.45–7.98)]. Moreover, John Dedicoat IGRAs’ sensitivity was reduced in active TB patients exposed to tobacco. IL-22, GM-CSF, IL-5, TNF-α, Received: 10 March 2022 IP-10, and IL-13 were less secreted in LTBI children exposed to SHS. In conclusion, SHS is associated Accepted: 31 March 2022 with LTBI and active TB in children. In addition, false-negative IGRAs obtained on active TB patients Published: 2 April 2022 exposed to SHS, together with the decrease of specific cytokines released, suggest that tobacco may alter the immune response. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- Keywords: Mycobacterium tuberculosis; cigarette smoking; passive smoking; child; immunology iations. 1. Introduction Copyright: © 2022 by the authors. Active smoking and second-hand smoke (SHS) exposure is a public health problem Licensee MDPI, Basel, Switzerland. associated with active tuberculosis (TB) and latent TB infection (LTBI), as well as disease This article is an open access article relapse and mortality [1]. In 2020, there were an estimated 9.9 million new TB cases distributed under the terms and worldwide, of which 11% were children (aged < 15 years). In addition, about 1.7 billion conditions of the Creative Commons people (23% of the world’s population) are estimated to have LTBI, being at risk of disease Attribution (CC BY) license (https:// progression during life [2]. Tobacco kills around 8 million people/year across the world. creativecommons.org/licenses/by/ 4.0/). From these deaths, around 1.2 million are due to SHS exposure in non-smokers [3,4]. J. Clin. Med. 2022, 11, 2000. https://doi.org/10.3390/jcm11072000 https://www.mdpi.com/journal/jcm J. Clin. Med. 2022, 11, 2000 2 of 12 Infants are especially susceptible to SHS exposure, being vulnerable to developing health problems and having a significant risk factor for TB progression [3]. A systematic review conducted by the WHO and the International Union Against Tuberculosis and Lung Diseases indicated that the association of LTBI and active TB with tobacco smoke (passive or active) was independent of alcohol abuse, socioeconomic status, and other variable confounders. Furthermore, it is estimated that smoking can increase the risk for TB disease more than two-and-a-half times [1]. Even though there is an absence of a gold standard test for LTBI diagnosis, Inter- feron (IFN)-gamma (γ) Release Assays (IGRAs) are more specific than tuberculin skin test (TST) for this purpose because they do not present a cross-reaction with the BCG vaccine or non-tuberculous mycobacteria. However, their performance in children can be differentially affected by age, especially in those aged < 5 years [5]. Therefore, gaining evidence in this field using IGRAs is required, especially in children who are exposed to SHS [6–9]. Tobacco can increase the risk of infections by impairing immune defense mech- anisms. Smoking promotes an inflammatory state leading to oxidative stress, mucosal in- flammation, and the production of pro-inflammatory cytokines [10,11]. Altogether, this inflammatory environment can lead to a negative effect on the production of specific cy- tokines that play a role against infections, such as those caused by Mycobacterium tuberculosis. It has been described that cigarette smoke decreases specific M. tuberculosis cytokines in vitro [12,13]. In addition, has been shown that smokers have a reduced number of cells secreting Th1 cytokines, and as a consequence, can be more susceptive to viral or mycobacterial infections [14]. Interestingly, tobacco can also alter the IFN-γ response de- tected by IGRAs, impairing their assay performance [15]. However, only a few studies have addressed this impact in individuals exposed to SHS [16]. Here, we investigate the influence of cigarette smoke exposure on children as a risk factor of LTBI and active TB disease, analyzing IGRAs’ diagnostic performance and assessing specific cytokine immune responses against infection. 2. Material and Methods 2.1. Study Design and Sample Collection This is a prospective study performed on children (<15 years old) who attended Uni- tat de Tuberculosi Vall d’Hebron-Drassanes (Barcelona, Spain) between September 2013 and September 2015. Patients were recruited from contact-tracing studies with an adult TB index case. Blood (<11 mL) was drawn from each patient for performing T-SPOT.TB (Oxford Immunotec, Abingdon, UK) and QuantiFERON-TB Gold In-Tube (QFN-G-IT, Qiagen, Düsseldorf, Germany) assays. The blood was sent to Institut d’Investigació Ger- mans Trias i Pujol (IGTP, Badalona, Spain) to be tested by IGRAs. This study was approved by the Ethical Committee of the Institut d’Investigació en Atenció Primària (IDIAP) Jordi Gol (protocol code P13/46). Informed consent was signed by the children’s parents or legal guardians before blood sampling. A questionnaire was collected from all patients detailing demographic information and clinical data, as well as index case data (diagnostic delay, bacteriology, and smoking condition) and degree of contact with index case (living together, days exposed, and daily contact hours). Exclu- sion criteria were HIV-positive, immunosuppression or past TB treatment for all patients enrolled in this study. 2.2. Study Population and Contact-Tracing Study Procedure A flow-chart detailing the contact-tracing study procedure and the final diagnosis of the children recruited is represented in Figure 1. Contact-tracing studies were conducted ac- cording to Spanish guidelines [17,18]. TST, IGRAs, and chest radiography were performed in the first visit during the contact-tracing study. If the radiography was abnormal, clinical and microbiological studies for active TB diagnosis were performed. In those exposed children with negative assays for tuberculosis diagnosis infection, a Window Period Pro- phylaxis (WPP, primary prophylaxis) was prescribed until a second phase of the study. J. Clin. Med. 2022, 11, x FOR PEER REVIEW 3 of 13 J. Clin. Med. 2022, 11, 2000 clinical and microbiological studies for active TB diagnosis were performed. In those 3 ofex- 12 posed children with negative assays for tuberculosis diagnosis infection, a Window Pe- riod Prophylaxis (WPP, primary prophylaxis) was prescribed until a second phase of the study. In this In this second second phase, phase, an IGRA an and/or IGRA and/or TST (only TST (only in those in those cases cases with initial with initial negative negative TST) TST) repeated were were repeated 8–12 weeks 8–12 weeks after theafter the initial initial visit. Radiological visit. Radiological imagesimages and studies and studies for activefor active TB were conducted if needed. Patient groups were classified as: TB were conducted if needed. Patient groups were classified as: (i) LTBI contacts with an (i) LTBI contacts with anTB active active case TB andcase and normal normal chest radiograph. chest radiograph. LTBI was LTBI was diagnosed diagnosed when awhen a positive positive result result was obtained with either of the two IGRAs (QFN-G-IT and/or was obtained with either of the two IGRAs (QFN-G-IT and/or T-SPOT.TB). (ii) Active T-SPOT.TB). (ii) Ac- TB tive TB cases cases with with a M. a M. tuberculosis tuberculosis positivepositive culture culture (sputum (sputum bacteriology bacteriology or gastric or gastric aspi- aspirate) or rate) or a probable TB based on clinical evaluation, compatible radiological a probable TB based on clinical evaluation, compatible radiological images (when chest images (when chest radiography radiography was doubtful, was doubtful, a thoracic a thoracic computerized computerized tomography tomography was performed), was performed), and and clinical response to treatment. Having a positive TST/IGRA clinical response to treatment. Having a positive TST/IGRA or being closely or being closely exposed exposed to an to an active active TB caseTBwas casealso wasused alsoasused as diagnostic diagnostic support.support. Lastly,Lastly, (iii) uninfected (iii) uninfected individu- individuals with als with negative negative TSTIGRA, TST and/or and/orand IGRA, and a chest a normal normal chest radiograph radiograph imaging.imaging. Figure 1. Figure 1. Flow-chart Flow-chart with with the the final final diagnosis diagnosis of of the the 616 616 children children included included in in the the study. study. Briefly, Briefly, children coming from contact-tracing studies were stratified according to their age (<5 years and children coming from contact-tracing studies were stratified according to their age (<5 years old old 5–14 years old) and classified as uninfected, LTBI, and active TB. A Window Period Prophylaxis and 5–14 years old) and classified as uninfected, LTBI, and active TB. A Window Period Prophylaxis (WPP, primary prophylaxis) was indicated in the first screening in those children with negative TST (WPP, primary prophylaxis) was indicated in the first screening in those children with negative TST and/or IGRAs. Then, after 8–12 weeks, a second screening was performed. In this second phase, TST and/or IGRAs.were and/or IGRAs Then,repeated. after 8–12 WPPweeks, wasa not second screening prescribed was performed. in some Innon-acceptance. cases due to this second phase, TST and/or IGRAs were repeated. WPP was not prescribed in some cases due to non-acceptance. Children were classified regarding their smoking habits: (i) tobacco unexposed; (ii) Children were classified regarding their smoking habits: (i) tobacco unexposed; SHS exposed; and (iii) smokers. Tobacco consumption was assessed by two independent (ii) SHS exposed; and (iii) smokers. Tobacco consumption was assessed by two independent interviews as described previously [6]. The pack-years ratio was calculated as (number of interviews as described previously [6]. The pack-years ratio was calculated as (number cigarettes consumed per day/20) × (number of years the person has smoked) [19]. SHS of cigarettes consumed per day/20) × (number of years the person has smoked) [19]. exposure was assessed in those children whose relatives currently smoked at home, esti- SHS exposure was assessed in those children whose relatives currently smoked at home, mating pack-years exposure for the year preceding examination of the child as a contact. estimating pack-years exposure for the year preceding examination of the child as a contact. 2.3. Tuberculin 2.3. Skin Test, Tuberculin Skin Test,T-SPOT.TB T-SPOT.TBandandQFN-G-IT QFN-G-IT TST was TST was performed performed using using 22 tuberculin tuberculin units units of of PPD PPD RT23 RT23 (Statens (Statens Serum Serum Institut, Institut, Copenhagen, Denmark) Copenhagen, Denmark)and andevaluated evaluatedwithin within 48–72 48–72 h by h by specialized specialized nurses nurses and and physi- physicians. cians. Indurations Indurations ≥ 5 mm≥ 5were mm were considered considered positive positive according according to thetoSpanish the Spanish Pneumology Pneumology and and Thoracic Surgery Society guidelines [17,18]. T-SPOT.TB and QFN-G-IT Thoracic Surgery Society guidelines [17,18]. T-SPOT.TB and QFN-G-IT were done and were done and interpreted interpreted according according to thetomanufacturer’s the manufacturer’s instructions. instructions. J. Clin. Med. 2022, 11, 2000 4 of 12 2.4. Cytokine Detection by Means of a Bead-Based Multiplex Assay Cytokine detection was performed on QFN-G-IT supernatants using a bead-based multiplex assay (Luminex 11-plex cytokine kit, R&D Systems, Minneapolis, MN, USA) and analyzed using Bioplex manager software (version 5.0, Bio-Rad, Hercules, CA, USA). Cytokines with a high relevance role on the immune response against M. tuberculosis were selected: granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, interleukin (IL)-2, IL-5, IL-10, IL-13, IL-22, IL-17, tumour necrosis factor (TNF)-alpha (α), IL-1RA and IFN-γ-induced protein (IP)-10. The specific response was measured on antigen-stimulated plasmas after the subtraction of cytokine unstimulated concentration. 2.5. Statistical Methods Comparisons between qualitative variables were done using the chi-square and Fisher’s exact tests. Risk factors for (i) positive IGRAs and for (ii) LTBI and active TB were defined using an adjusted Odds Ratio (aOR). Sensitivity, specificity, and predictive values (PVs) were calculated for TST, QFN-G-IT, and T-SPOT.TB. Data were analyzed using Epi Info 7.1.2 (www.cdc.gov/epiinfo/). Graphs were represented using GraphPad Prism version 4 (GraphPad Software, Inc., San Diego, CA, USA). Spot-forming cells (SFCs) counted in T-SPOT.TB were considered as an overall RD1 response (sum of SFCs in ESAT- 6 and CFP-10). Differences in cytokine levels between groups were assessed using the two-tailed Mann–Whitney U-test for pairwise comparisons. Differences were considered statistically significant when p-values were <0.05. 3. Results 3.1. Participant’s Characteristics and Final Diagnosis A total of 616 children from contact-tracing studies were included. A flow-chart indicating the final diagnosis of the children recruited is represented in Figure 1. Children were grouped according to their age (148 children aged < 5 years old and 468 children aged between 5–14 years old). LTBI and active TB were initially diagnosed in 8/148 (5.4%) and 14/148 (9.5%) of the children aged < 5; and in 121/468 (25.9%) and 30/468 (6.4%) of the children aged between 5–14 years, respectively. After the WPP, the total LTBI and active TB cases were 14/148 (9.5%) and 17/148 (11.5%) in children < 5 years; and 150/468 (32.1%) and 36/468 (7.7%) in 5–14 years children, respectively. Globally, active TB was higher in children < 5 years (11.5%) when compared with the group of children aged between 5–14 years old (7.7%), however, differences were not significant. 3.2. Risk Factors Associated with a Positive IGRA IGRAs’ performance was globally assessed according to smoking habits, showing that the QFN-G-IT or T-SPOT.TB positivity rate increased in children exposed to SHS or smokers (for QFN-G-IT: 12.0% vs. 47.5% vs. 67.6% in unexposed, SHS and smokers respectively; for T-SPOT.TB: 12.3% vs. 49.8% vs. 76.5% in unexposed, SHS and smokers respectively). On the contrary, the highest negative percentages were observed in unexposed children (for QFN-G- IT: 88.0% vs. 52.5% vs. 32.4% in unexposed, SHS and smokers respectively; for T-SPOT.TB: 87.7% vs. 50.2% vs. 23.5% in unexposed, SHS and smokers respectively) (Figure 2). Table 1 shows the main demographic characteristics and the possible risk factors associated with positive IGRAs (QFN-G-IT and/or T-SPOT.TB). Being exposed to SHS and/or being a smoker was significantly associated with having a positive IGRA [aOR and 95% confidence interval (CI): 8.7 (5.9–12.8) for SHS; and 25.6 (9.95–70.5) for smokers; p < 0.00001 for both conditions; Table 1]. This risk was also significantly higher when pack-years exposure increased [aOR (95% CI): 5.20 (3.4–8.0) for 1–15 pack-years exposure; and 23.8 (13.7–41.4) for >15 pack-years exposure; p < 0.00001]. Moreover, being a contact of a smoking index case was an important risk factor as well [aOR (95% CI): 2.47 (1.72–3.53); p < 0.00001]. J. Clin. Med. 2022, 11, 2000 5 of 12 J. Clin. Med. 2022, 11, x FOR PEER REVIEW 5 of 13 Figure 2. IGRA positive or negative results stratification according to the smoking habit. T- Figure 2. IGRA positive or negative results stratification according to the smoking habit. T- SPOT.TB and QFN-G-IT results in the 616 children recruited in the study, and stratification regard- SPOT.TB and ing their QFN-G-IT tobacco results exposure in the 616 (unexposed, children SHS, recruited in the study, and stratification regarding or smokers). their tobacco exposure (unexposed, SHS, or smokers). Table 1 shows the main demographic characteristics and the possible risk factors as- Table 1. Main sociated demographic with characteristics positive IGRAs (QFN-G-ITand risk factors and/or according T-SPOT.TB). Beingtoexposed positive to IGRA SHSresults (QFN-G-IT and/or T-SPOT.TB positive result) in contact-tracing studies of and/or being a smoker was significantly associated with having a positive IGRA [aOR and children included in the 95% study. confidence interval (CI): 8.7 (5.9–12.8) for SHS; and 25.6 (9.95–70.5) for smokers; p < 0.00001 for both conditions; Table 1]. This risk was also significantly higher when pack- years exposure increasedPositive QFN-G-IT and/or T-SPOT.TB [aOR (95% CI): 5.20 (3.4–8.0) for 1–15 pack-years exposure; and 0–4 Years:23.8 148 Contacts (13.7–41.4) for >15 pack-yearsYears: 5–14 468 Contacts exposure; p < 0.00001]. Moreover, 0–14 Years:a616 being Contacts contact of a Variables (Positive IGRAs, n = 31) (Positive IGRAs, n = 181) (Positive IGRAs, n = 212) smoking index case was an important risk factor as well [aOR (95% CI): 2.47 (1.72–3.53); p n (%) * OR (95% CI) p-Value n (%) * OR (95% CI) p-Value n (%) * OR (95% CI) p-Value < 0.00001]. Gender Female 18 (22.0) 1 79 (32.9) 1 97 (30.1) 1 Male 13 (19.7) 0.87Table 1. Main NS (0.4–1.9) demographic characteristics 102 (44.7) 1.65 (1.1–2.4) <0.001 according and risk factors 115 (39.1)to positive IGRA results<0.05 1.49 (1.06–2.1) BCG (QFN-G-IT and/or T-SPOT.TB positive result) in contact-tracing studies of children included in the No 23 (19.0) 1 study. 78 (33.8) 1 101 (28.7) 1 Yes 8 (29.6) 1.8 (0.7–4.6) NS 103 (43.5) 1.51 (1.0–2.2) <0.05 111 (42.0) 1.80 (1.3–2.5) <0.005 Immigrant No 22 (18.3) 1 Positive 77 (33.5)QFN-G-IT1and/or T-SPOT.TB 99 (28.3) 1 NS <0.05 <0.0005 Yes 9 (32.1) 0–42.1Years: (0.8–5.3)148 Contacts 5–14 Years: 104 (43.7) 468 Contacts 1.5 (1.1–2.2) 0–14 113 Years: 1.87 (42.5) 616(1.33–2.62) Contacts Smoking Habit Unexposed (Positive 1 IGRAs, n = 31) (Positive IGRAs, n = 181) (Positive 41 (12.9) IGRAs,1n = 212) SHS Variables 247 (37.5) (8.3) 6.6 (2.4–18.6) <0.00005 34 (14.6) 120 (59.7) 1 8.7 (5.31–14.1) <0.00001 144 (54.3) OR (95% 8.7 (5.9–12.8) <0.00001 Smoker † – n (%) * OR – (95% CI) p-Value – 27 n (%) * OR (79.4) 22.6(95% CI) p-Value (8.5–62.3) <0.00001 n (%) * 27 (79.4) p-Value<0.00001 25.6 (9.95–70.5) Pack-years exposed CI) None Gender 8 (9.4) 1 35 (14.8) 1 43 (13.4) 1 1 to 15 10 (24.4) 3.1 (1.01–9.7) <0.05 71 (50.3) 5.8 (3.6–9.56) <0.00001 81 (44.5) 5.20 (3.4–8.0) <0.00001 >15 Female 13 (61.9) 18 (22.0) 1 13.9 (4.01–50.4) <0.00001 7579 (32.9) 28.8 (14.9–55.8) (83.3) 1 <0.00001 97 (30.1) 88 (78.6) 1 (13.7–41.4) 23.8 <0.00001 Underweight NS <0.001 1.49 (1.06– <0.05 No Male 28 (20.3)13 (19.7) 0.87 1 (0.4–1.9) NS 102 165 (44.7) 1.65 (1.1–2.4) (38.8) 1 NS 115193 (39.1) (34.3) 1 NS Yes 3 (30.0) 1.68 (1.4–6.92) 16 (37.2) 0.93 (0.5–1.78) 19 (35.8) 2.1) 1.07 (0.6–1.93) IC drug-resistant BCG No 30 (21.4) 1 166 (38.4) 1 196 (34.3) 1 Yes No 1 (12.5)23 (19.0) 0.52 (0.06–4.4)1 NS 1578 (33.8) 1.14 (0.6-2.3) (41.7) 1 NS 101 16 (28.7) (36.4) 11.09 (0.6–2.07) NS Living Together NS <0.05 <0.005 No Yes 7 (11.5) 8 (29.6) 1.8 1 (0.7–4.6) 103 (43.5) 1.51 (1.0–2.2) 72 (26.5) 1 111 79 (42.0) 1.80 (1.3–2.5) (23.7) 1 YesImmigrant24 (27.6) 2.9 (1.2–7.35) <0.05 109 (55.6) 3.48 (2.4–5.14) <0.00001 133 (47.0) 2.85 (2.02–4.02) <0.00001 Days Exposed <50 No 22 (18.3) 1 19 (16.8) 1 77 (33.5) 115 (31.4) 11 99 (28.3) 134 (28.0) 1 1 <0.05 <0.00001 ≥50 12 (34.3) 2.6 (1.01–6.6) NS 66 (64.7) 5.83 (3.6–9.5) <0.05 78 (56.9) 1.87 (1.33– <0.0005<0.00001 3.4 (2.26–5.14) Yes Daily contact hours 9 (32.1) 2.1 (0.8–5.3) 104 (43.7) 1.5 (1.1–2.2) 113 (42.5) <6 8 (11.4) 1 74 (25.3) 1 59 (23.0) 2.62) 1 <0.01 <0.00001 <0.00001 Smoking ≥6 Habit 23 (29.5) 3.24 (1.3–4.83) 107 (61.1) 4.7 (3.1–6.96) 153 (42.5) 2.47 (1.72–3.53) IC Smoker No Unexposed 11 (14.1) 7 (8.3) 1 1 4834 (14.6) (27.0) 11 41 (12.9) 59 (23.0) 1 1 Yes 20 (28.6) 2.44 (1.07–5.4) <0.05 133 (45.9) 2.29 (1.53–3.43) <0.0001 153 (42.5) 2.47 (1.72–3.53) <0.00001 J. Clin. Med. 2022, 11, 2000 6 of 12 Table 1. Cont. Positive QFN-G-IT and/or T-SPOT.TB 0–4 Years: 148 Contacts 5–14 Years: 468 Contacts 0–14 Years: 616 Contacts Variables (Positive IGRAs, n = 31) (Positive IGRAs, n = 181) (Positive IGRAs, n = 212) n (%) * OR (95% CI) p-Value n (%) * OR (95% CI) p-Value n (%) * OR (95% CI) p-Value IC Diagnostic Delay <50 days 14 (17.7) 1 75 (32.1) 1 89 (28.4) 1 ≥50 days 17 (24.6) 1.52 (0.6–3.6) NS 106 (45.3) 1.76 (1.18–2.6) <0.005 123 (40.6) 1.72 (1.21–2.44) <0.005 IC AFB sputum grade 1 + (10–99/100 fields) 3 (5.9) 1 32 (23.9) 1 35 (18.9) 1 2 + (1–10/field) 15 (20.3) 4.1 (1.05–23.0) <0.05 95 (41.3) 2.24 (1.4–3.7) <0.05 110 (36.2) 2.4 (1.5–3.8) <0.0001 3 + (>10/field) 13 (56.5) 20.8 (4.3–127.2) <0.00001 54 (40.3) 3.44 (2.0–6.0) <0.00001 67 (52.3) 4.7 (2.8–7.8) <0.00001 * Percentages are calculated over the total amount of the given variable. † No children < 5 years were classified as smokers in this study. OR: Odds Ratio; CI: Confidence Interval; SHS: Second-hand smoke; IC: index case; AFB: acid-fast bacilli; NS: not statistically significant. 3.3. Risk Factors for LTBI and Active TB Being exposed to SHS and/or being a smoker was one of the main factors associated with LTBI, together with pack-years exposure to tobacco smoke. These factors were also associated with active TB, except for the smoker group, where no significant differences were observed. Risk factors associated with the latter group were difficult to establish due to the low number of children with active TB cataloged as smokers (n = 7). Acid-fast bacilli sputum grade of the index case, and having a daily contact of >6 h were other two risk factors associated with LTBI and active TB (Table 2). Table 2. LTBI and active TB risk factors in children included in the study. LTBI * Active TB Variable aOR (95% CI) p-Value aOR (95% CI) p-Value Gender (Male) No 1 1 Yes 1.73 (1.13–2.64) <0.05 1.64 (0.81–3.32) NS BCG No 1 1 Yes 0.65 (0.15–2.79) NS 1.68 (0.06–47.10) NS Age Group 0–4 years 1 1 5–15 years 2.59 (1.47–4.57) <0.05 0.46 (0.20–1.03) NS Immigrant No 1 1 Yes 2.25 (0.53–9.61) NS 0.42 (0.03–6.01) NS Smoking Habit Unexposed 1 1 SHS 7.57 (4.79–11.94) <0.00001 3.40 (1.45–7.98) <0.005 Smokers 21.71 (8.18–57.60) <0.00001 3.31 (0.80–13.76) NS Pack-years exposed None 1 1 1 to 5 3.54 (1.94–6.44) <0.00001 1.02 (0.20–5.19) NS 6 to 15 10.30 (5.60–18.97) <0.00001 3.52 (1.19–10.36) <0.05 >15 18.21 (9.80- 41.81) <0.00001 7.31 (2.61–20.49) <0.0005 Underweight No 1 1 Yes 1.09 (0.52–2.29) NS 3.64 (1.30–10.14) <0.05 IC AFB sputum grade 1 + (10–99/100 fields) 1 1 2 + (1–10/field) 2.83 (1.66–4.80) <0.0001 7.49 (1.65–34.02) <0.005 3 + (>10/field) 4.93 (2.61–9.33) <0.00001 24.80 (5.31–115.5) <0.00001 Daily contact hours <6 1 1 ≥6 4.00 (2.46–6.51) <0.00001 7.65 (3.17–18.5) <0.00001 Days Exposed ≥50 1 1 <50 0.97 (0.55–1.71) NS 1.18 (0.53–2.65) NS * LTBI was defined as having positive IGRAs (T-SPOT.TB and/or QFN-G-IT) and chest radiography without alterations. OR: Odds Ratio; CI: Confidence Interval; SHS: Second-hand smoke; IC: index case; AFB: acid-fast bacilli; NS: not statistically significant. 3.4. Sensitivity Values for Active TB According to Tobacco Smoke Exposure As shown in Table 3, IGRAs’ sensitivity decreased in children with active TB who were directly or indirectly exposed to tobacco smoke, being that the T-SPOT.TB is more J. Clin. Med. 2022, 11, 2000 7 of 12 sensitive than the QFN-G-IT. All these data may indicate that tobacco exposure can be associated with false-negative IGRA results when diagnosing active TB. The percentage of active TB cases was significantly higher in individuals exposed to SHS or smokers when compared with unexposed [3.1% (10/317) in unexposed children, vs. 13.6% (36/265; p < 0.000001) in SHS exposed, and vs. 20.6% (7/34; p < 0.001) in smokers]. Finally, TB disease prevalence increased based on tobacco smoke exposure, being higher in children exposed to SHS and/or being smokers [% disease prevalence (95% CI): 3.15 (1.52–5.72) in unexposed vs. 13.48 (9.63–18.17) in SHS exposed vs. 20.59 (8.70–37.9) in smokers]. Interestingly, the percentage of positive IGRAs’ results increased with tobacco exposure due to the high risk of infection. As a consequence, the specificity for diagnosing active TB in contact-tracing is reduced. Table 3. Sensitivity, specificity, and predictive values of TST, QFN-G-IT, and T-SPOT.TB according to direct and/or indirect tobacco smoke exposure in the 616 children recruited during contact-tracing studies. Tobacco Smoke TB Non TB Sensitivity Specificity PPV % NPV % Test Exposition Cases Cases (95% CI) (95% CI) (95% CI) (95% CI) Unexposed TST ≥ 5 mm 10 95 100 69.1 10 100 n = 317 TST < 5 mm 0 212 (69.1–100) (63.6–74.2) (8.2–11.1) QFN.G-IT pos 10 28 100 90.9 26.3 Disease Prevalence % 100 QFN.G-IT neg 0 279 (69.1–100) (87.1–93.9) (20.1–33.7) (95% CI) 3.15 (1.52–5.72) T-SPOT.TB pos 10 29 100 90.5 25.6 100 T-SPOT.TB neg 0 278 (69.1–100) (87.2–94.3) (12.6–39.7) SHS TST ≥ 5 mm 36 136 100 40.6 21 100 n = 265 TST < 5 mm 0 93 (90.3–100) (34.2–47.3) (19.2–22.8) QFN.G-IT pos 30 96 83.3 58.1 23.8 96 Disease Prevalence % QFN.G-IT neg 6 133 (67.2–93.6) (51.4–64.5) (20.2–27.8) (91.4–97.9) (95% CI) 13.48 (9.63–18.17) T-SPOT.TB pos 31 101 86.1 55.9 23 96.2 T-SPOT.TB neg 5 128 (74.8–97.4) (49.0–62.4) (20.1–27.2) (91.8–98.3) Smokers TST ≥ 5 mm 7 23 100 14.8 23 100 n = 34 TST < 5 mm 0 4 (59.0–100) (4.2–33.7) (20.6–26.3) QFN.G-IT pos 4 19 57.1 29.6 17.4 72.7 Disease Prevalence % QFN.G-IT neg 3 8 (18.4–90.1) (13.7–50.2) (9.6–29.5) (48.7–88.2) (95% CI) 20.59 (8.70–37.9) T-SPOT.TB pos 7 19 100 29.6 26.9 100 T-SPOT.TB neg 0 8 (59.0–100) (13.7–50.2) (22.4–32.0) SHS: Second-hand smoke; pos: Positive; neg: Negative; TB: tuberculosis; PPV: positive predictive value; NPV: negative predictive value; CI: Confidence Interval. 3.5. Cytokine Responses in Children Exposed to SHS We investigated whether SHS could affect cytokine responses (other than IFN-γ) against M. tuberculosis infection. For this purpose, cytokine responses were analyzed in a subgroup of unexposed and SHS exposed patients, classified as uninfected controls or LTBI. Tobacco unexposed children had a significantly higher secretion of IL-22, GM-CSF, and IL-5 when infected than when they were not (p < 0.05 for IL-22; and p < 0.01 for both GM-CSF and IL-5). In contrast, LTBI children exposed to SHS failed to secrete significantly more IL-22, GM-CSF, and IL-5 than those exposed to SHS who were uninfected. In addition, IL-5 levels were significantly higher in tobacco unexposed LTBI compared to those LTBI individuals exposed to SHS (p < 0.01). Although differences for TNF-α in unexposed and SHS exposed children were not significant when comparing controls and infected individuals, the level of this cytokine tended to be reduced in children exposed to SHS [median (pg/mL) and interquartile range (IQR): 174.8 (0–828.5) for LTBI individuals unexposed to tobacco versus 0 (0–173.7) for LTBI individuals exposed to SHS]. For IP-10 and IL-13, cytokine responses were significantly higher in LTBI individuals than in uninfected controls for both conditions (unexposed: p < 0.01 for both IP-10 and IL-13; and SHS exposed: p < 0.05 for IP-10 and individuals, the level of this cytokine tended to be reduced in children exposed to SHS [median (pg/mL) and interquartile range (IQR): 174.8 (0–828.5) for LTBI individuals un- J. Clin. Med. 2022, 11, 2000 exposed to tobacco versus 0 (0–173.7) for LTBI individuals exposed to SHS]. For IP-108 and of 12 IL-13, cytokine responses were significantly higher in LTBI individuals than in uninfected controls for both conditions (unexposed: p < 0.01 for both IP-10 and IL-13; and SHS ex- posed: p < 0.01p for < 0.05 for IP-10 IL-13) and3).p