PLOS ONE RESEARCH ARTICLE Serum cotinine cut-points for secondhand smoke exposure assessment in children under 5 years: A systemic review Nerea Mourino ID1, Alberto Ruano-Raviña1,2, Leonor Varela Lema ID1*, Esteve Fernández3,4,5,6, Marı́a José López2,7,8, Marı́a Isolina Santiago-Pérez9, Julia Rey- Brandariz1, Alexandra Giraldo-Osorio1,10,11, Mónica Pérez-Rı́os1,2 1 Department of Preventive Medicine and Public Health, University of Santiago de Compostela, Santiago de Compostela, Spain, 2 CIBER Epidemiology and Public Health, CIBERESP, Madrid, Spain, 3 Tobacco Control Unit, WHO Collaborating Center for Tobacco Control, Institut Català d’Oncologia (ICO), Badalona, Spain, 4 Tobacco Control Research Group, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), a1111111111 Barcelona, Spain, 5 School of Medicine and Health Sciences, Universitat de Barcelona, Barcelona, Spain, 6 CIBER of Respiratory Diseases (CIBERES), Madrid, Spain, 7 Servicio de Evaluación y Métodos de a1111111111 Intervención, Agència de Salut Pública de Barcelona, Barcelona, Spain, 8 Institut d’Investigació Biomèdica a1111111111 de Sant Pau (IIB Sant Pau), Barcelona, Spain, 9 Epidemiology Unit, Galician Directorate for Public Health, a1111111111 Santiago de Compostela, Spain, 10 Departamento de Salud Pública, Grupo de investigación Promoción de a1111111111 la Salud y Prevención de la Enfermedad (GIPSPE), Universidad de Caldas, Manizales, Colombia, 11 Fundación Carolina, Madrid, Spain * leonor.varela@usc.es OPEN ACCESS Citation: Mourino N, Ruano-Raviña A, Varela Lema Abstract L, Fernández E, López MJ, Santiago-Pérez MI, et al. (2022) Serum cotinine cut-points for secondhand smoke exposure assessment in children under 5 years: A systemic review. PLoS ONE 17(5): Background e0267319. https://doi.org/10.1371/journal. Serum cotinine has become the most widely used biomarker of secondhand smoke expo- pone.0267319 sure (SHS) over time in all ages. The aim of this study was to review the serum cotinine cut- Editor: Pal Bela Szecsi, Holbaek Sygehus, points used to classify children under 5 years as exposed to SHS. DENMARK Received: August 9, 2021 Methods Accepted: April 6, 2022 A systematic review performed in the Pubmed (MEDLINE) and EMBASE databases up to Published: May 5, 2022 April 2021 was conducted using as key words "serum cotinine", “tobacco smoke pollution” Copyright: © 2022 Mourino et al. This is an open (MeSH), "secondhand smoke", "environmental tobacco smoke" and “tobacco smoke expo- access article distributed under the terms of the sure”. Papers which assessed SHS exposure among children younger than 5 years old Creative Commons Attribution License, which permits unrestricted use, distribution, and were included. The PRISMA 2020 guidelines were followed. Analysis was pre-registered in reproduction in any medium, provided the original PROSPERO (registration number: CRD42021251263). author and source are credited. Data Availability Statement: All relevant data are Results within the manuscript. No datasets were generated or analyzed during the current study. 247 articles were identified and 51 fulfilled inclusion criteria. The selected studies were pub- Funding: The authors received no specific funding lished between 1985–2020. Most of them included adolescents and adults. Only three for this work. assessed postnatal exposure exclusively among children under 5 years. None of the Competing interests: The authors have declared selected studies proposed age-specific cut-points for children < 5 years old. Cut-point val- that no competing interests exist. ues to assess SHS exposure ranged from 0.015 to 100 ng/ml. The most commonly used PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 1 / 17 PLOS ONE Cotinine cut-points in children under 5 years cut-point was 0.05 ng/ml, derived from the assay limit of detection used by the National Health and Nutrition Examination Survey (NHANES). Conclusions No studies have calculated serum cotinine age-specific cut-points to ascertained SHS expo- sure among children under 5 years old. Children’s age-specific cut-points are warranted for health research and public health purposes aimed at accurately estimating the prevalence of SHS exposure and attributable burden of disease to such exposure, and at reinforcing 100% smoke-free policies worldwide, both in homes, private vehicles and public places. Introduction The assessment of the involuntary smoking or exposure to secondhand smoke (SHS) in chil- dren during pregnancy and early childhood is essential for public health and research pur- poses. Estimating the children at-risk from environmental tobacco can have important public health implications in terms of determining the population attributable burden of disease and evaluating the impact of the information, cessation interventions and prohibition laws on this target group. SHS is defined as tobacco smoke produced by an active smoker from exhaled mainstream smoke together with sidestream smoke. Sidestream smoke is a combination of smoke from the smoldering tobacco product between and during puffs and smoke compo- nents which diffuse through cigarette paper [1,2]. Pediatric population is especially vulnerable to the effects of SHS due to their narrower bronchi, faster respiratory rate and immature immune system [3]. Prejudicial effects derived from SHS exposure on children’s health have been documented since the 1970s, including but not limited to the following: increased risk of sudden infant death syndrome, acute respiratory symptoms (cough, phlegm, wheeze and breathlessness) and ear infections [1]. In 2004, 165,071 deaths among children younger than 5 years were attributed worldwide to SHS exposed [4]. However, these estimations were considered to be prone to bias because exposure data was mainly derived from surveys. Parental self-report questionnaires have been widely used to assess both prenatal and postnatal SHS exposure [5,6] but frequently provide underestimations due to parents’ reluctance to disclose their smoking status and underreporting of children’s SHS exposure, aswell as the recall bias or lack of knowledge of exposure [6,7]. This highlights the need for objective methods based on the determination of biomarkers to provide reliable assessment of SHS exposure [5]. Cotinine, the major metabolite of nicotine, is considered the best biomarker for assessing recent SHS exposure due to its high specificity and sensitivity, as well as its relatively prolonged half-life which ranges from 16 to 20 hours in children [6,8]. Cotinine can be collected from various biological samples such as blood (serum/plasma), urine, saliva, hair, meconium and maternal milk, and quantified through several analytical techniques [6,8–10]. Although the collection of serum or plasma cotinine is more invasive than that from other samples, it is not influenced by renal function, flow rate, and urinary pH and it does not require urine dilution adjustments; over time, serum cotinine has become the most widely used biomarker of SHS exposure among children [1,6]. Serum cotinine allows for differentiating active smokers from passive and non-smokers. Nonetheless, the determination of SHS exposure is rather challenging as currently there is no standardized consensus regarding the optimal cut-point to be used to classify SHS exposure PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 2 / 17 PLOS ONE Cotinine cut-points in children under 5 years among young children. Some prior studies, based on NHANES data for older children and adolescents, proposed the serum cotinine cut-point of 0.05 ng/ml which derives from the assay limit of detection (LOD) [8,11]. This cut- point maximises sensitivity to avoid missing positive cases, but it should be acknowledged that a trade-off always exists between sensitivity and spec- ificity, and cotinine metabolism and clearance can vary by sex, race and age [1,5]. Considering that children´s physiology is quite different within the 0–5 years old age range, age-specific cut-points might be useful for different studies to accurately classify nonsmoking children as exposed or unexposed to tobacco smoke in a standardized manner [5]. As far as we are aware of, there are no reviews focused on the serum cotinine cut-points used to classify SHS exposure in the pediatric population. The aim of this study is to review serum cotinine cut-points used so far to assess SHS expo- sure in children under 5 years and assess the changes on the cut-point values over time and across different countries. Material and methods Literature search We performed a systematic review in PubMed (MEDLINE) and EMBASE databases following the PRISMA 2020 guidelines [12]. Search included papers published until April 2021 and “serum cotinine”, “Tobacco smoke pollution” (MeSH), “secondhand smoke” and “environ- mental tobacco smoke” were used as key words. Search strategy underwent peer review by three knowledgeable reviewers. Age filters were set (newborns-infant-preschool child). No reports, communications to congresses or simulation essays were included. The bibliographic search was limited to English, Spanish, French and German. Analysis was pre-registered in PROSPERO (registration number: CRD42021251263). Inclusion/Exclusion criteria To be selected for this review, articles had to assess pre-natal SHS exposure in newborns and/ or post-natal SHS exposure in children younger than 5 years by using serum cotinine assays in addition to or in lieu of parental self-report. We included investigations with mother-child pairs and those grouping children under 5 with older ones, adolescents and adults. Among the latter, when data was stratified by age groups, we selected that including children younger than 5 years. On the other hand, we excluded studies with serum cotinine obtained just from moth- ers; without cut-point values to distinguish between exposed and unexposed children, and those measuring cotinine from other biological fluids as urine, saliva, hair, meconium or maternal milk. In the case of longitudinal studies, we only considered the information corre- sponding to the period of time in which SHS exposure was ascertained accompanied with serum cotinine in a sample including children under 5 years. Selection of articles and evidence synthesis After excluding duplicate articles from the search, the title and abstract of the records were screened individually by two independent reviewers to ascertain that they met eligibility cri- teria. Full text of the eligible papers was obtained for potentially relevant articles. Bibliogra- phy from the selected papers was revised to manually complete the initial search. Thereafter, data from those meeting eligibility criteria were extracted and discrepancies in its interpreta- tion were solved by consensus. A modified Newcastle-Ottawa scale was used to assess appro- priateness of the representativeness of the sample, participation rate, ascertainment of the exposure, and the adequacy of the method used for the selection of the cut-points. Score PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 3 / 17 PLOS ONE Cotinine cut-points in children under 5 years disagreements were resolved by consensus and a final agreed-upon rating was assigned to each study. The data extracted for analysis were: study characteristics (author, publication year, design, period of the study, use of questionnaire in conjunction with cotinine (yes/no) and country); population characteristics (sample size, age and race); information regarding the analytical technique used to quantify serum cotinine (radioimmunoassay (RIA), enzyme-linked immu- nosorbent essay (ELISA), gas chromatography (GC), gas-chromatography mass spectrometry (GC/MS), gas-liquid chromatographic procedure with nitrogen-phosphorus-specific detector (GLC/NPD) and isotope dilution liquid chromatography tandem mass spectrometry (LC-MS)); serum cotinine cut-point values to classify SHS exposure in children and method for the selection of the cut-points. Results A total of 247 articles were obtained and 51 of them fulfilled the inclusion criteria. The PRISMA flow diagram of the included studies is displayed in Fig 1. Fig 1. PRISMA 2020 flow diagram for studies selection (search until April 2021). https://doi.org/10.1371/journal.pone.0267319.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 4 / 17 PLOS ONE Cotinine cut-points in children under 5 years Fig 2. Study and population characteristics (design, country; age); analytical technique for serum cotinine quantification (isotope dilution-liquid chromatography-tandem mass spectrometry (LC/MS), radioimmunoassay (RIA), gas chromatography (GC), gas-chromatography mass spectrometry (GC/MS), gas-liquid chromatographic procedure with nitrogen-phosphorus-specific detector (GLC/NPD) and enzyme-linked immunosorbent essay (ELISA)); cut-point values range (ng/ml) and methods for cut-point identification (assay limit of detection (LOD), previous studies or other (separation point in the bimodal distribution, median cotinine levels of the cohort, receiver operating characteristic (ROC) curves or percentiles)). https://doi.org/10.1371/journal.pone.0267319.g002 Study and population characteristics The 51 papers included were published between 1985 and 2020. They included one case-con- trol study [13], 13 cohort studies [14–26] and 37 [27–63] cross-sectional studies from which, one included a matched case-control substudy [53]; 26 studies obtained data from different cycles of the National Health and Nutrition Examination Survey (NHANES). When applying the modified Newcastle-Ottawa scale, two cohort studies [17,19] and one cross-sectional study [36], which used data from the NHANES, were judged to be of high quality, and thirty six studies were judged to be of low quality. Studies were mainly conducted in the USA (34 papers) and Europe (15 studies) (Fig 2). Thirty-three out of 51 papers measured postnatal exposure to SHS with serum cotinine; 17 prenatal exposure with newborns’cord serum cotinine and one both pre- and post-natal expo- sure [19]. 38 studies ascertained exposure by using both serum cotinine and questionnaires (Table 1). Considering those studies assessing prenatal exposure, sample sizes ranged from 8 to 1,323 newborns. Among the 33 studies assessing postnatal exposure, 27 papers grouped children under 5 years with older/adolescents (aged 12–19) and 3 papers, also with adults (aged � 20). The remaining 3, measured postnatal SHS exposure exclusively in children under 5 years with sample sizes ranging from 132 to 504 children [14,56,59] (Table 1 and Fig 2). Analytical technique, cut-point value/s, and method for cut-point identification Isotope dilution liquid chromatography tandem mass spectrometry (LC-MS) was used in 35 of the selected papers followed by RIA in 7, GC/GC-MS/ GLC/NPD in 7 and ELISA in 2 (Fig 2). PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 5 / 17 PLOS ONE Cotinine cut-points in children under 5 years Table 1. Study and population characteristics; analytical technique for serum cotinine quantification; cotinine cut-points (CP) and method for their selection. STUDY CHARACTERISTICS POPULATION CHARACTERISTICS ANALYTICAL TECHNIQUE, CP, METHOD FOR THE SELECTION OF THE CP AND REFERENCE NUMBER Author, yr Design and Questionnaire Country Sample size Age Race Analytical CP Method Reference period (yes/no) technique (ng/ ml) Luck et al, Cross-sectional, Yes Germany 8 Newborns N/A GLC/NPD 5 Assay LOD 27 1985 [27] N/A Ahlsten et al, Cross-sectional, Yes Sweden 39 Newborns N/A GC 1 Assay LOD 28 1989 [28] N/A Etzel et al, Cohort study, No USA 132 4 months-3 White, black or RIA 2.5 ROC curves 14 1992 [14] 1964–1983 yr mixed race Bardy et al, Cross-sectional, Yes Finland 1,237 Newborns N/A GC/MS 6 Assay LOD 31 1993 [31] 1991 CDC,1993 [29] Cross-sectional Yes USA 800† 4–91 yr N/A LC/MS 10/15 Authors selected the CP 29 (NHANES, value from a separation 1988–1991) point in the bimodal distribution of SC in smokers and nonsmokers Bardy et al, Cross-sectional, Yes Finland 1,323 Newborns N/A GC/MS 35 Assay LOD 30 1994 [30] 1991 Martinez et al, Cohort Study, Yes USA 175 Newborns Hispanic or other RIA 1 Assay LOD 15 1994 [15] 1980–1984 non-Anglo Ruhle et al, Cross-sectional, Yes Germany 75 Newborns N/A RIA 15 Authors selected the CP 32 1995 [32] N/A value based on data from a previous study Pirkle et al, Cross-sectional Yes USA 1,793‡ 4–11 yr Non-Hispanic LC/MS 10/15 Authors selected the CP 33 1996 [33] (NHANES, white, non- value from a separation 1988–1991) Hispanic black, point in the bimodal Mexican American distribution of SC in or other smokers and nonsmokers Nafstad et al, Cohort study, Yes Norway 202 Newborns N/A RIA 14 Authors selected the CP 16 1996 [16] 1992–1993 value based on data from a previous study Bearer et al, Case-control, Yes USA 70 Newborns Non-Hispanic GC/MS 1 Assay LOD 13 1997 [13] 1991–1992 white, non- Hispanic black or other (Hispanic and Asian) Pichini et al, Cohort Study, Yes Spain 429 Newborns N/A RIA 1,78 ROC curves 17 2000 [17] 1997–1998 Mannino et al, Cross-sectional Yes USA 1,533 4–6 yr Non-Hispanic LC/MS 0.05 Assay LOD 34 2001 [34] (NHANES, white, non- 1988–1994) Hispanic black or Mexican American Strauss, 2001 Cross-sectional Yes USA 2,968‡ 4–18 yr N/A LC/MS 2 Author selected the CP 35 [35] (NHANES, value based on data 1988–1991) from a previous study Mannino et al, Cross-sectional Yes USA 5,653‡ 4–16 yr Non-Hispanic LC/MS 0.05 Assay LOD 36 2001 [36] (NHANES, white, non- 1989–1994) Hispanic black or Mexican American Lieu et al, 2002 Cross-sectional Yes USA 1,825‡ 4–12 yr Non-Hispanic LC/MS 10/20 Authors selected the CP 37 [37] (NHANES, white, non- value based on data 1989–1994) Hispanic black or from a previous study Mexican American (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 6 / 17 PLOS ONE Cotinine cut-points in children under 5 years Table 1. (Continued) STUDY CHARACTERISTICS POPULATION CHARACTERISTICS ANALYTICAL TECHNIQUE, CP, METHOD FOR THE SELECTION OF THE CP AND REFERENCE NUMBER Author, yr Design and Questionnaire Country Sample size Age Race Analytical CP Method Reference period (yes/no) technique (ng/ ml) Mannino et al, Cross-sectional No USA 523‡ 4–16 yr Non-Hispanic LC/MS 0.05 Assay LOD 38 2002 [38] (NHANES, white, non- 1989–1994) Hispanic black or Mexican American Rubin et al, Cross-sectional Yes USA 6,153‡ 4–16 yr N/A LC/MS 2.9 Authors selected both 39 2004 [39] (NHANES, CP values based on data 1989–1994) from a previous study Aligne et al, Cross-sectional No USA 3,531‡ 4–11 yr Non-Hispanic LC/MS 0.05 Assay LOD 40 2003 [40] (NHANES, white, non- 1989–1994) Hispanic black, Mexican American or other Wilkinson Cross-sectional Yes USA 2,516‡ 4–16 yr Non-Hispanic LC/MS 0.05 Assay LOD 41 et al,2006 [41] (NHANES, white, non- 1989–1991) Hispanic black, Mexican American or other Pirkle et al, Cross-sectional Yes USA 1988–91: 4–11 yr Non-Hispanic LC/MS 0.05 Assay LOD 42 2006 [42] (NHANES, 1,839‡ white, non- 0.015 1989–2002) 1991–94: Hispanic black, 2,090‡ Mexican American 1999–2000: or other 1,065‡ 2001–2002: 1,278‡ Franchini et al, Cross-sectional, Yes Italy 979 Newborns Italian or foreign GC/MS 1 Authors selected the CP 43 2008 [43] 2004–2005 value based on data from previous studies Puig et al, 2008 Cohort Study, Yes Spain 487 Newborns Spanish or non- RIA 1 Authos selected the CP 18 [18] 1996–1998. Spanish value based on data from a previous study Max et al, 2009 Cross-sectional Yes USA 1999–2000: 3–11 yr Non-Hispanic LC/MS 0.05 Assay LOD 44 [44] (NHANES, 1,179‡ white, non- 0.015 1999–2006) 2001–2002: Hispanic black, 1,423‡ Mexican American, 2003–2004: other Hispanic or 1,265‡ other race/ethnicity 2005–2006: 1,300‡ Dixon et al, Cross-sectional Yes USA 829‡ 1–5 yr Non-Hispanic LC/MS 0.05 Assay LOD 45 2009 [29] (NHANES, white, non- 0.015 1999–2004) Hispanic black, Hispanic or other CDC (Vital Cross-sectional Yes USA N/A 3–11 yr Non-Hispanic LC/MS 0.05 Assay LOD 46 signs), 2010 (NHANES, white, non- [46] 1999–2008) Hispanic black, Mexican American or other Dove et al, Cross-sectional Yes USA 1,582‡ 3–5 yr Non-Hispanic LC/MS 0.05 Assay LOD 47 2010 [47] (NHANES, white, non- 1999–2006) Hispanic black, Mexican American or other (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 7 / 17 PLOS ONE Cotinine cut-points in children under 5 years Table 1. (Continued) STUDY CHARACTERISTICS POPULATION CHARACTERISTICS ANALYTICAL TECHNIQUE, CP, METHOD FOR THE SELECTION OF THE CP AND REFERENCE NUMBER Author, yr Design and Questionnaire Country Sample size Age Race Analytical CP Method Reference period (yes/no) technique (ng/ ml) Vesper et al, Cross-sectional No USA N/A 3–11 yr Non-Hispanic LC/MS 10 Authors selected the CP 48 2010 [48] (NHANES, white, non- value based on data 2003–2004) Hispanic black or from a previous study Mexican American Xu et al, 2010 Cross-sectional No USA 4,508‡ 4–15 yr Non-Hispanic LC/MS 0.035 Authors selected CP 49 [49] (NHANES, white, non- values based on the 2001–2002) Hispanic black, 33rd and 67th Mexican American percentiles of the SC or other concentrations Preston et al, Cross-sectional, Yes USA 30 Newborns Asian, Caucasian or LC/MS 1 Authors selected the CP 50 2010 [50] 2004–2005 African American value based on data from a previous study Sharief et al, Cross-sectional No USA 3,136† 1–21 yr Non-Hispanic LC/MS 2.9 Authors selected the CP 51 2011 [51] (NHANES, white, non- value based on data 2005–2006) Hispanic black, from a previous study Mexican American or other Spanier et al, Cohort Study, Yes USA Newborns: Newborns- Non-Hispanic LC/MS 0.015 Assay LOD 19 2011 [19] 2003–2006 273 1 2 yr White, non- yr: 275 2 yr: Hispanic Black or 206 other Cardwell et al, Cross-sectional, No USA 220‡ 1–16 yr N/A LC/MS 0.9 ROC curves; 0.9 ng/ml 52 2012 [52] N/A 0.6 for children aged < 12 yr; 0.6 ng/ml for those aged �12 yr. Dempsey et al, Cross-sectional Yes USA 274‡ 8 months- Latino, African LC/MS 0.05 Assay LOD 53 2012 [53] with a matched 17 yr American, Asian, case control (70% White non- substudy, 2009– were < 3 Hispanic or other 2010 yr) Florath et al, Cohort Study, Yes Germany 972 Newborns German or non- LC/MS 14 Authors selected the CP 20 2014 [20] 2000–2001 German value based on data from previous studies Wang et al, Cohort study Yes China 14 Newborns N/A LC/MS 0,12 Authors selected the CP 21 2013 [21] value based on the median cotinine levels of the entire cohort Andersen et al, Cross-sectional, No Denmark 133 Newborns N/A Immulite 5 Authors selected the CP 54 2013 [54] 1988–1990 2000 values based on data from a previous study Kit et al, 2013 Cross-sectional Yes USA 1988–1994: 4–11 yr Non-Hispanic LC/MS 0.05 Assay LOD 55 [55] (NHANES, 248‡ White, non- 1988–2010) 1999–2004: Hispanic Black, 336‡ Mexican American 2005–2010: or other 392‡ Howrylak et al, Cohort Study, Yes USA 619 1–16 yr White, African LC/MS 0.1 Assay LOD 22 2014 [22] 2010–2011 American, Multiracial or other Pino et al, Cross-sectional, No Chile 504 1 yr N/A RIA 100 Assay LOD (from a 56 2004 [56] 1995–1997 study conducted in 1987) (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 8 / 17 PLOS ONE Cotinine cut-points in children under 5 years Table 1. (Continued) STUDY CHARACTERISTICS POPULATION CHARACTERISTICS ANALYTICAL TECHNIQUE, CP, METHOD FOR THE SELECTION OF THE CP AND REFERENCE NUMBER Author, yr Design and Questionnaire Country Sample size Age Race Analytical CP Method Reference period (yes/no) technique (ng/ ml) Mason et al, Cross-sectional No USA N/A† � 3 yr N/A LC/MS 0.05 Assay LOD 57 2015 [57] (NHANES, 0.015 2007–2010) West et al, Cohort Study, Yes Finland 1,578‡ 3–18 yr N/A GC 3 Authors selected the CP 23 2015 [23] 1980–2014 value based on data from a previous study Merianos et al, Cross-sectional No USA 2,707‡ 3–11 yr Non Hispanic LC/MS 0.05 Assay LOD 58 2017 [58] (NHANES, White, non 2009–2012) Hispanic-Black, Hispanic, other races/multiracial Yilmaz et al, Cross-sectional, No Turkey 150 1–3 yr N/A ELISA 3 Authors selected the CP 59 2018 [59] 2012–2013 value based on data from a previous study Shenassa et al, Cross-sectional Yes USA 2,679‡ 3–5 yr Non-Hispanic LC/MS 10 Authors used CP value 60 2017 [60] (NHANES, White, non- based on data from a 1999–2012) Hispanic Black, previous study Mexican American or other Hedengran Cohort study, Yes Denmark 263 Newborns N/A LC/MS 0.2 Authors selected CP 24 et al, 2018 [24] 2003–2004 value based on data from a previous study Nwosu et al, Cross-sectional No USA 1,013� 3–9 yr Non-Hispanic LC/MS 0.05 Assay LOD 61 2018 [61] (NHANES, White, Mexican 2009–2010) American, Other Hispanics, African American or other Chelchowska Cohort study, Yes Poland 80 Newborns Caucasian ELISA 13.7 Authors selected the CP 25 et al, 2019 [25] 2013–2015 value based on data from a previous study Brody et al, Cross-sectional Yes USA 2,833‡ 3–11 yr Non-Hispanic LC/MS 0.05 Assay LOD 62 2019 [62] (NHANES, White, non- 2013–2016) Hispanic Black, non-Hispanic Asian or Hispanic Biren et al, Cross-sectional Yes USA 257 3–5 yr Non-Hispanic LC/MS 0.05 Assay LOD 63 2020 [63] (NHANES, white, non 2015–2016) Hispanic-black, Mexican- American, other Hispanic or other/ multirace. Rovio et al, Cohort study, Yes Findland 1,504‡ 3–18 yr N/A LC/MS 3 Authors used CP value 26 2020 [26] 1980–2011 based on data from a previous study Abreviations: N/A, not available; GLC/NPD, gas-liquid chromatographic procedure with nitrogen-phosphorus-specific detector; LOD, limit of detection; GC, gas chromatography; USA, United States of America; yr, year; RIA, radioimmunoassay; ROC, receiver operating characteristic; GC/MS, Gas- chromatography mass spectrometry; NHANES, National Health and Nutrition Examination Survey; LC/MS, isotope dilution-liquid chromatography-tandem mass spectrometry; CP, cut- point; SC, serum cotinine; SHS, secondhand smoke; ELISA, Enzyme-linked immunosorbent essay. † Sample sizes includes children (3–11 yr), adolescents (12–19 yr) and adults (> 19 yr). ‡ Sample size includes children aged 5 years and older. https://doi.org/10.1371/journal.pone.0267319.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 9 / 17 PLOS ONE Cotinine cut-points in children under 5 years A total of 26 studies mentioned the assay LOD threshold as cut-point (Fig 2). In order to ascer- tain SHS exposure among children, cut-point values ranged between 0.015–35 ng/ml, except- ing in one study that used 100 ng/ml; the most commonly used threshold was 0.05 ng/ml (17 out of 51) (Table 1). Eighteen articles selected the serum cotinine cut-point value/s based on previous studies (Fig 2). The lowest value was mentioned in North American studies and the highest, in a Chilean study [56]. One study calculated age-specific cut-point with receiver operating characteristic (ROC) curves [52]: 0.9 ng/ml (< 12 years) vs. 0.6 ng/ml (=/> 12 years). However, none of the selected articles proposed sex, race or age-specific serum cotinine cut-points in children under 5 years. Discussion As summarized in Table 1, the cut-points to classify SHS exposure varied widely between stud- ies. None of these studies have proposed age-specific cut-points to classify SHS exposure in children under 5 years. Only one study calculated two age-specific cut-points setting the refer- ence age at 12. Commonly, SHS exposure thresholds were established attending to the assay LOD or based on previously defined adolescent and adult cut-points without accounting for population characteristics, analytical technique or prevalence of SHS exposure in the country of the study. Several studies have concluded that cotinine metabolism is influenced by sex, race and age after having observed higher cotinine concentrations among children, females and non-His- panic Blacks [42,44,64]. With respect to age, young children seem to have higher serum cotin- ine concentrations than older/adolescents and nonsmokers adults at similar exposures to SHS and this could be due to differences in cotinine metabolism and clearance. However, young children could also be more exposed to SHS due to faster respiratory rate, inability to distract themselves or breastfeeding by smoking mothers [42,64–66]. The NHANES, which has mea- sured SHS exposure since 1988 by using questionnaires and serum cotinine, found higher cotinine concentrations among children aged 3–11 years compared to adolescents aged 12–19 years and adults [42,67]. In our systematic review, we have observed that serum cotinine cut-point values to charac- terize children’s exposure to SHS have varied remarkably since 1985 and across the countries, being the highest value mentioned in a Chilean study conducted in 1990s, more than 6,600 times that of the lowest used in US studies, 100 versus 0.015 ng/ml. European studies used val- ues between 0.2–35 ng/ml. We only found three studies assessing postnatal exposure exclu- sively among children under 5 years, between 1–3 years, with disparate cut-points ranging from 2.5 ng/ml to 100 ng/ml. We have observed that some of the studies that classified pre- or postnatal SHS exposure selected the threshold used to distinguish between smoker and non smoker (ranging from 3 to 20 ng/ml) and thus, being liable to have underestimate exposure on this target group. The only paper included in our review, which measured both pre- and postnatal exposure to SHS with serum cotinine, used the same cut-point for newborns and children aged 1 and 2 years [19]. The most widely used cut-point for the classification of prenatal SHS exposure, via new- borns’ cord serum cotinine, was 1 ng/ml; One study obtained a cut-point of 1.78 ng/ml from the ROC curves to discriminate newborns from nonexposed and exposed nonsmokers with a sensitiviy and specificity of 60% [17]; this value exceeds some of those used to classify exposure among young children. However, taking into consideration the transfer of serum cotinine to fetus across the placenta and the higher metabolism and faster clearance of cotinine in preg- nant women, it can not be disregarded that newborns may have lower cotinine concentrations than young children for the same exposure levels. Nonetheless, it should be acknowledged that PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 10 / 17 PLOS ONE Cotinine cut-points in children under 5 years the characterization of SHS prenatal exposure from cord serum cotinine samples from the newborn could be imprecise; taking into account that newborn refers to the moment of deliv- ery and that the measured cotinine concentrations might cover the period of time in which women may be hospitalised [24,43]. In general terms, a decrease in the serum cotinine cut-point values has been observed over time and this could be partly explained by the better LOD threshold of the analytical tech- niques. Beginning in 2001–2002, the LC/MS assay LOD used to determine serum cotinine concentrations in the samples collected in NHANES was lowered in some studies from 0.05 to 0.015 ng/ml as a result of the introduction of a more sensitive mass spectrometer [42]. These two have been the LOD derived cut-points most widely used over time to classify exposure to SHS in the USA. Of note, it should be acknowledged that serum cotinine, like other biomark- ers, has an important limitation, being that it does not provide information on the route and source of exposure. Although the main health risks associated with SHS are related to inhala- tion of fine particulate and gases from sidestream smoke [2,68], values around 0.015 ng/ml could reflect not only transient and accidental SHS exposure (sometimes unnoticed by chil- dren’s caregivers), but also third-hand smoke exposure (THS) or residual tobacco smoke (such as involuntary inhalation, ingestion or cutaneous absorption of nicotine particles from SHS deposited on surfaces, clothing or furniture which remain there long after smoke is gone) or exposure from other sources such as food. Thus, a child ingesting nicotine laden dust through crawling or hand-to-mouth activity (THS exposure) may have high serum cotinine from ingesting that dust, but could have experienced very little exposure to SHS. Previous studies reported that certain foodstuffs such as potatoes, tomatoes, eggplant, cauliflower, green pep- pers and black tea have measurable levels of nicotine [69,70]. Whilst food consumption levels of dietary nicotine are insignificant compared with moderate SHS exposure, the consumption of high quantities might contribute to low-level elevations in serum cotinine (e.g. 80 g of egg- plant is equivalent to approximately 0.01 ng/ml of serum cotinine) [8]. Further studies in chil- dren could verify this hypothesis using detailed dietary information and sensitive cotinine biomarkers; this information could be useful to propose high level cut-points that could allow for a better characterization of the SHS exposure in children, differentiating significant expo- sure from minor or incidental exposure. The lowering of the thresholds also results from the acknowledgement that no safe levels of SHS exposure can be established. Some prior investigators have emphasized that even very low levels of SHS exposure (serum cotinine concentrations < 0.1 ng/mL) can cause adverse effects on cognitive outcomes among children [71] and this could be also attributed to the higher lead concentrations found in children exposed to SHS compared to those unexposed [72]. In the USA, as a result of combined tobacco control policies, prevalence of tobacco consumption and SHS exposure have declined during the last 3 decades. Consequently, serum cotinine cut- points have become lower to be able to avoid misclassifying any SHS even at low level or acci- dental exposure [73]. Whilst these revised lower cut-points could be recommended from a health perspective, they could lead to an overestimation of the population exposure to SHS because as already mentioned, there is always a trade off between sensitivity and specificity. In this case, this lowering of the cut-point can lead to a higher false positive rate. This could impact on the calculations of SHS exposure in a given population and in term, on the esti- mated attributable burden of disease. The lack of consensus with respect to the most appropri- ate cut-point has also important implications with regards to the comparison of data and trend analysis. This review has some limitations. First, we only used PubMed and EMBASE databases but we do not expect to have missed any relevant papers because we have expanded the search by including articles from the bibliography of those papers selected. Second, when limiting the PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 11 / 17 PLOS ONE Cotinine cut-points in children under 5 years search to English, Spanish, French and German, papers published in other languages might have been excluded. To the best of our knowledge, 1 paper published in Polish was excluded. Third, period of study was not especified in 4 cross-sectional studies [27,28,32,52]. Another limitation is the low sample size of some investigations with 3 articles without specification of the participants’ sample size [46,48,57]. Finally, 36 out of 51 studies were judged to be of low quality when applying the modified Newcastle-Ottawa scale, and the majority of the included studies grouped children under 5 years with older and adolescents, being this one the main limitation. As the main strength, this is the first review on the serum cotinine cut-points used across different countries to assess SHS exposure in children. Prior investigations have identified the cotinine cut-point values used over time to discriminate between smokers and nonsmok- ers. The 2006 Surgeon General´s report mentioned the need to review the cut-points used when assessing tobacco consumption and SHS exposure among pregnant women consider- ing the higher metabolic clearance rates and therefore, the shorter half-life of cotinine during pregnancy [1]. For those studies which found discrepancies between self-report and serum cotinine, most assumed that parents did not accurately report their child´s SHS exposure [15,18,53]. One study considered the possibility of misclassification due to the inadequacy of the cut-point [40]. A recent cohort study assessing SHS exposure among children younger than 5 years, observed that, compared to the assay LOD derived cut-point of 0.015 ng/ml, when using new age-specific cut-points, all of them higher than the mentioned assay thresh- old, concordance between maternal self-report and serum cotinine improved; actually, the percentage of children reclassified as SHS exposed with cotinine disminished in approxi- mately fifty percentage points when using the age-specific cut-points obtained with ROC curves [74]. It is necessary to raise public awareness of the need to adopt 100% smoke-free policies worldwide aimed at avoiding the long-term adverse effects resulting from early exposures to tobacco smoke. SHS exposure has been associated with cognitive impairment and chronic diseases (such as respiratory and cardiovascular diseases) which begin early in childhood and cause premature disability, death and high healthcare costs. Thus, adequate classifica- tion of SHS exposure is warranted to accurately estimate the prevalence of SHS exposure and the attributable burden of disease to such exposure. Biomarker assessment of SHS pro- vides an advantage over questionnaire assessment with respect to their accuracy; however, valid non-biased cotinine cut-points are needed. Considering that young children are espe- cially vulnerable to the health consequences derived from SHS exposure, and the develop- mental changes in child behaviour, anatomy and physiology during the first years of life, further well-designed studies would be recommended to propose age-specific serum cotinine cut-points or multiple cut-point values which maximises both sensitivity and speci- ficity to minimise misclassification either as exposed or unexposed based on cotinine concentrations. Conclusions No studies have calculated serum cotinine age-specific cut-points to ascertained SHS exposure among children under 5 years old. The adverse health consequences derived from any level of SHS exposure, especially from the sidestream smoke, and the developmental changes in child behaviour, anatomy and physiology during the first years of life, support the need for age-spe- cific cut-points for health research and public health purposes aimed at accurately estimating SHS exposure and attributable burden of disease to such exposure, and at reinforcing 100% smoke-free policies worldwide, both in homes, private vehicles and public places. PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 12 / 17 PLOS ONE Cotinine cut-points in children under 5 years Supporting information S1 Checklist. PRISMA checklist. (DOC) Author Contributions Conceptualization: Nerea Mourino, Alberto Ruano-Raviña, Leonor Varela Lema, Mónica Pérez-Rı́os. Data curation: Nerea Mourino, Alberto Ruano-Raviña, Leonor Varela Lema, Mónica Pérez- Rı́os. Formal analysis: Alberto Ruano-Raviña, Leonor Varela Lema. Investigation: Nerea Mourino. Methodology: Nerea Mourino, Alberto Ruano-Raviña, Leonor Varela Lema, Esteve Fernán- dez, Marı́a José López, Marı́a Isolina Santiago-Pérez, Mónica Pérez-Rı́os. Supervision: Alberto Ruano-Raviña, Leonor Varela Lema, Esteve Fernández, Marı́a José López, Marı́a Isolina Santiago-Pérez, Mónica Pérez-Rı́os. Validation: Nerea Mourino, Alberto Ruano-Raviña, Leonor Varela Lema, Esteve Fernández, Marı́a José López, Marı́a Isolina Santiago-Pérez, Julia Rey-Brandariz, Alexandra Giraldo- Osorio, Mónica Pérez-Rı́os. Visualization: Nerea Mourino, Marı́a Isolina Santiago-Pérez. Writing – original draft: Nerea Mourino, Alberto Ruano-Raviña, Leonor Varela Lema. Writing – review & editing: Nerea Mourino, Alberto Ruano-Raviña, Leonor Varela Lema, Esteve Fernández, Marı́a José López, Marı́a Isolina Santiago-Pérez, Julia Rey-Brandariz, Alexandra Giraldo-Osorio, Mónica Pérez-Rı́os. References 1. U.S. Department of Health and Human Services. The Health Consequences of Involuntary Exposure to Tobacco Smoke: A Report of the Surgeon General. Atlanta: U.S. Department of Health and Human Ser- vices, Centers for Disease Control and Prevention, Coordinating Center for Health Promotion, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. 2006. 2. Jaakkola MS, Jaakkola JJ. Assessment of exposure to environmental tobacco smoke. Eur Respir J. 1997; 10(10):2384–97. https://doi.org/10.1183/09031936.97.10102384 PMID: 9387970 3. Hang B, Wang P, Zhao Y, Sarker A, Chenna A, Xia Y, et al. Adverse Health Effects of Thirdhand Smoke: From Cell to Animal Models. Int J Mol Sci. 2017; 18(5): 932. https://doi.org/10.3390/ ijms18050932 PMID: 28452951 4. Öberg M, Jaakkola MS, Woodward A, Peruga A, Prüss-Ustün A. Worldwide burden of disease from exposure to second-hand smoke: a retrospective analysis of data from 192 countries. The Lancet. 2011; 377(9760):139–46. https://doi.org/10.1016/S0140-6736(10)61388-8 PMID: 21112082 5. Kim S. Overview of Cotinine Cutoff Values for Smoking Status Classification. Int J Environ Res Public Health. 2016; 13(12). https://doi.org/10.3390/ijerph13121236 PMID: 27983665 6. Florescu A, Ferrence R, Einarson T, Selby P, Soldin O, Koren G. Methods for quantification of exposure to cigarette smoking and environmental tobacco smoke: focus on developmental toxicology. Ther Drug Monit. 2009; 31(1):14–30. https://doi.org/10.1097/FTD.0b013e3181957a3b PMID: 19125149 7. Aurrekoetxea JJ, Murcia M, Rebagliato M, Guxens M, Fernández-Somoano A, López MJ, et al. Sec- ond-hand smoke exposure in 4-year-old children in Spain: Sources, associated factors and urinary cotinine. Environ Res. 2016; 145:116–25. https://doi.org/10.1016/j.envres.2015.11.028 PMID: 26656512 8. Benowitz NL. Cotinine as a biomarker of environmental tobacco smoke exposure. Epidemiol Rev. 1996; 18(2):188–204. https://doi.org/10.1093/oxfordjournals.epirev.a017925 PMID: 9021312 PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 13 / 17 PLOS ONE Cotinine cut-points in children under 5 years 9. Braun JM, Kalloo G, Chen A, Dietrich KN, Liddy-Hicks S, Morgan S, et al. Cohort Profile: The Health Outcomes and Measures of the Environment (HOME) study. Int J Epidemiol. 2017; 46(1):24. https:// doi.org/10.1093/ije/dyw006 PMID: 27006352 10. Luck W, Nau H. Exposure of the fetus, neonate, and nursed infant to nicotine and cotinine from maternal smoking. N Engl J Med. 1984; 311(10):672. https://doi.org/10.1056/NEJM198409063111014 PMID: 6472348 11. Levy RV, Brathwaite KE, Sarathy H, Reidy K, Kaskel FJ, Melamed ML. Analysis of Active and Passive Tobacco Exposures and Blood Pressure in US Children and Adolescents. JAMA Netw Open. 2021; 4(2). https://doi.org/10.1001/jamanetworkopen.2020.37936 PMID: 33620445 12. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. Syst Rev. 2021; 10(1):89. https://doi. org/10.1186/s13643-021-01626-4 PMID: 33781348 13. Bearer C, Emerson RK, O’Riordan MA, Roitman E, Shackleton C. Maternal tobacco smoke exposure and persistent pulmonary hypertension of the newborn. Environ Health Perspect. 1997; 105(2):202–6. https://doi.org/10.1289/ehp.97105202 PMID: 9105795 14. Etzel RA, Pattishall EN, Haley NJ, Fletcher RH, Henderson FW. Passive smoking and middle ear effu- sion among children in day care. Pediatrics. 1992; 90(2 Pt 1):228–32. PMID: 1641287 15. Martinez FD, Wright AL, Taussig LM. The effect of paternal smoking on the birthweight of newborns whose mothers did not smoke. Group Health Medical Associates. Am J Public Health. 1994; 84(9):1489–91. https://doi.org/10.2105/ajph.84.9.1489 PMID: 8092378 16. Nafstad P, Kongerud J, Botten G, Urdal P, Silsand T, Pedersen BS, et al. Fetal exposure to tobacco smoke products: a comparison between self-reported maternal smoking and concentrations of cotinine and thiocyanate in cord serum. Acta Obstet Gynecol Scand. 1996; 75(10):902–7. https://doi.org/10. 3109/00016349609055025 PMID: 9003090 17. Pichini S, Basagaña XB, Pacifici R, Garcia O, Puig C, Vall O, et al. Cord serum cotinine as a biomarker of fetal exposure to cigarette smoke at the end of pregnancy. Environ Health Perspect. 2000; 108(11):1079–83. https://doi.org/10.1289/ehp.001081079 PMID: 11102300 18. Puig C, Garcia-Algar O, Monleon T, Pacifici R, Zuccaro P, Sunyer J, et al. A longitudinal study of envi- ronmental tobacco smoke exposure in children: parental self reports versus age dependent biomarkers. BMC Public Health. 2008; 8:47. https://doi.org/10.1186/1471-2458-8-47 PMID: 18254964 19. Spanier AJ, Kahn RS, Xu Y, Hornung R, Lanphear BP. Comparison of biomarkers and parent report of tobacco exposure to predict wheeze. J Pediatr. 2011; 159(5):776–82. https://doi.org/10.1016/j.jpeds. 2011.04.025 PMID: 21645908 20. Florath I, Kohler M, Weck MN, Brandt S, Rothenbacher D, Schöttker B, et al. Association of pre- and post-natal parental smoking with offspring body mass index: an 8-year follow-up of a birth cohort. Pediatr Obes. 2014; 9(2):121–34. https://doi.org/10.1111/j.2047-6310.2012.00146.x PMID: 23420701 21. Wang I-J, Chen S-L, Lu T-P, Chuang EY, Chen P-C. Prenatal smoke exposure, DNA methylation, and childhood atopic dermatitis. Clin Exp Allergy. 2013; 43(5):535–43. https://doi.org/10.1111/cea.12108 PMID: 23600544 22. Howrylak JA, Spanier AJ, Huang B, Peake RWA, Kellogg MD, Sauers H, et al. Cotinine in children admitted for asthma and readmission. Pediatrics. 2014; 133(2):e355–362. https://doi.org/10.1542/ peds.2013-2422 PMID: 24446438 23. West HW, Juonala M, Gall SL, Kähönen M, Laitinen T, Taittonen L, et al. Exposure to parental smoking in childhood is associated with increased risk of carotid atherosclerotic plaque in adulthood: the Cardio- vascular Risk in Young Finns Study. Circulation. 2015; 131(14):1239–46. https://doi.org/10.1161/ CIRCULATIONAHA.114.013485 PMID: 25802269 24. Hedengran KK, Andersen MR, Szecsi PB, Lindh C, Uldbjerg N, Stender S. Environmental tobacco smoke exposure during pregnancy has limited effect on infant birthweight and umbilical vein endothelial nitric oxide synthase. Acta Obstet Gynecol Scand. 2018; 97(11):1309–16. https://doi.org/10.1111/aogs. 13419 PMID: 29969842 25. Chełchowska M, Maciejewski TM, Mazur J, Gajewska J, Zasimovich A, Ołtarzewski M, et al. Active Tobacco Smoke Exposure in Utero and Concentrations of Hepcidin and Selected Iron Parameters in Newborns. Int J Environ Res Public Health. 2019; 16(11). https://doi.org/10.3390/ijerph16111996 PMID: 31195607 26. Rovio SP, Pihlman J, Pahkala K, Juonala M, Magnussen CG, Pitkänen N, et al. Childhood Exposure to Parental Smoking and Midlife Cognitive Function. Am J Epidemiol. 2020; 189(11):1280–91. https://doi. org/10.1093/aje/kwaa052 PMID: 32242223 27. Luck W, Nau H. Nicotine and cotinine concentrations in serum and urine of infants exposed via passive smoking or milk from smoking mothers. J Pediatr. 1985; 107(5):816–20. https://doi.org/10.1016/s0022- 3476(85)80427-3 PMID: 4056988 PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 14 / 17 PLOS ONE Cotinine cut-points in children under 5 years 28. Ahlsten G, Tuvemo T, Gebre-Medhin M. Selected trace elements and proteins in serum of apparently healthy newborn infants of mothers who smoked during pregnancy. Acta Paediatr Scand. 1989; 78(5):671–6. https://doi.org/10.1111/j.1651-2227.1989.tb11124.x PMID: 2596272 29. Centers for Disease Control and Prevention (CDC). Preliminary data: exposure of persons aged > or = 4 years to tobacco smoke—United States, 1988–1991. MMWR Morb Mortal Wkly Rep. 1993; 42(2):37– 9. PMID: 8419790 30. Bardy AH, Seppälä T, Lillsunde P, Koskela P, Gref CG. Objectively measured tobacco exposure among pregnant women in Finland in 1986 and 1990. Acta Obstet Gynecol Scand. 1994; 73(1):30–4. https://doi.org/10.3109/00016349409013389 PMID: 8304021 31. Bardy AH, Seppälä T, Lillsunde P, Kataja JM, Koskela P, Pikkarainen J, et al. Objectively measured tobacco exposure during pregnancy: neonatal effects and relation to maternal smoking. J Obstet Gynaecol. 1993; 100(8):721–6. https://doi.org/10.1111/j.1471-0528.1993.tb14262.x PMID: 8399009 32. Rühle W, Graf von Ballestrem CL, Pult HM, Gnirs J. [Correlation of cotinine level in amniotic fluid, umbil- ical artery blood and maternal blood]. Geburtshilfe Frauenheilkd. 1995; 55(3):156–9. https://doi.org/10. 1055/s-2007-1022795 PMID: 7665063 33. Pirkle JL, Flegal KM, Bernert JT, Brody DJ, Etzel RA, Maurer KR. Exposure of the US population to environmental tobacco smoke: the Third National Health and Nutrition Examination Survey, 1988 to 1991. JAMA. 1996; 275(16):1233–40. PMID: 8601954 34. Mannino DM, Moorman JE, Kingsley B, Rose D, Repace J. Health effects related to environmental tobacco smoke exposure in children in the United States: data from the Third National Health and Nutri- tion Examination Survey. Arch Pediatr Adolesc Med. 2001; 155(1):36–41. https://doi.org/10.1001/ archpedi.155.1.36 PMID: 11177060 35. Strauss RS. Environmental tobacco smoke and serum vitamin C levels in children. Pediatrics. 2001; 107(3):540–2. https://doi.org/10.1542/peds.107.3.540 PMID: 11230596 36. Mannino DM, Caraballo R, Benowitz N, Repace J. Predictors of cotinine levels in US children: data from the Third National Health and Nutrition Examination Survey. Chest. 2001; 120(3):718–24. https://doi. org/10.1378/chest.120.3.718 PMID: 11555498 37. Lieu JEC, Feinstein AR. Effect of gestational and passive smoke exposure on ear infections in children. Arch Pediatr Adolesc Med. 2002; 156(2):147–54. https://doi.org/10.1001/archpedi.156.2.147 PMID: 11814376 38. Mannino DM, Homa DM, Redd SC. Involuntary smoking and asthma severity in children: data from the Third National Health and Nutrition Examination Survey. Chest. 2002; 122(2):409–15. https://doi.org/ 10.1378/chest.122.2.409 PMID: 12171810 39. Rubin RN, Navon L, Cassano PA. Relationship of serum antioxidants to asthma prevalence in youth. Am J Respir Crit Care Med. 2004; 169(3):393–8. https://doi.org/10.1164/rccm.200301-055OC PMID: 14630617 40. Aligne CA, Moss ME, Auinger P, Weitzman M. Association of pediatric dental caries with passive smok- ing. JAMA. 2003; 289(10):1258–64. https://doi.org/10.1001/jama.289.10.1258 PMID: 12633187 41. Wilkinson JD, Arheart KL, Lee DJ. Accuracy of parental reporting of secondhand smoke exposure: The National Health and Nutrition Examination Survey III. Nicotine Tob Res. 2006; 8(4):591–7. https://doi. org/10.1080/14622200600790021 PMID: 16920657 42. Pirkle JL, Bernert JT, Caudill SP, Sosnoff CS, Pechacek TF. Trends in the exposure of nonsmokers in the U.S. population to secondhand smoke: 1988–2002. Environ Health Perspect. 2006; 114(6):853–8. https://doi.org/10.1289/ehp.8850 PMID: 16759984 43. Franchini M, Caruso C, Perico A, Pacifici R, Monleon T, Garcia-Algar O, et al. Assessment of foetal exposure to cigarette smoke after recent implementations of smoke-free policy in Italy. Acta Paediatr. 2008; 97(5):546–50. https://doi.org/10.1111/j.1651-2227.2008.00762.x PMID: 18394097 44. Max W, Sung H-Y, Shi Y. Who is exposed to secondhand smoke? Self-reported and serum cotinine measured exposure in the U.S., 1999–2006. Int J Environ Res Public Health. 2009; 6(5):1633–48. https://doi.org/10.3390/ijerph6051633 PMID: 19543411 45. Dixon SL, Gaitens JM, Jacobs DE, Strauss W, Nagaraja J, Pivetz T, et al. Exposure of U.S. children to residential dust lead, 1999–2004: II. The contribution of lead-contaminated dust to children’s blood lead levels. Environ Health Perspect. 2009; 117(3):468–74. https://doi.org/10.1289/ehp.11918 PMID: 19337524 46. Centers for Disease Control and Prevention (CDC). Vital signs: nonsmokers’ exposure to secondhand smoke—United States, 1999–2008. MMWR Morb Mortal Wkly Rep. 2010; 59(35):1141–6. PMID: 20829748 PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 15 / 17 PLOS ONE Cotinine cut-points in children under 5 years 47. Dove MS, Dockery DW, Connolly GN. Smoke-free air laws and secondhand smoke exposure among nonsmoking youth. Pediatrics. 2010; 126(1):80–7. https://doi.org/10.1542/peds.2009-3462 PMID: 20530079 48. Vesper HW, Caudill SP, Osterloh JD, Meyers T, Scott D, Myers GL. Exposure of the U.S. population to acrylamide in the National Health and Nutrition Examination Survey 2003–2004. Environ Health Per- spect. 2010; 118(2):278–83. https://doi.org/10.1289/ehp.0901021 PMID: 20123601 49. Xu X, Cook RL, Ilacqua VA, Kan H, Talbott EO. Racial differences in the effects of postnatal environ- mental tobacco smoke on neurodevelopment. Pediatrics. 2010; 126(4):705–11. https://doi.org/10. 1542/peds.2009-3589 PMID: 20855396 50. Guerrero-Preston R, Goldman LR, Brebi-Mieville P, Ili-Gangas C, Lebron C, Witter FR, et al. Global DNA hypomethylation is associated with in utero exposure to cotinine and perfluorinated alkyl com- pounds. Epigenetics. 2010; 5(6):539–46. https://doi.org/10.4161/epi.5.6.12378 PMID: 20523118 51. Sharief S, Jariwala S, Kumar J, Muntner P, Melamed ML. Vitamin D levels and food and environmental allergies in the United States: results from the National Health and Nutrition Examination Survey 2005– 2006. J Allergy Clin Immunol. 2011; 127(5):1195–202. https://doi.org/10.1016/j.jaci.2011.01.017 PMID: 21329969 52. Cardwell K, Pan Z, Boucher R, Zuk J, Friesen RH. Screening by pulse CO-oximetry for environmental tobacco smoke exposure in preanesthetic children. Paediatr Anaesth. 2012; 22(9):859–64. https://doi. org/10.1111/j.1460-9592.2012.03867.x PMID: 22587734 53. Dempsey DA, Meyers MJ, Oh SS, Nguyen EA, Fuentes-Afflick E, Wu AHB, et al. Determination of tobacco smoke exposure by plasma cotinine levels in infants and children attending urban public hospi- tal clinics. Arch Pediatr Adolesc Med. 2012; 166(9):851–6. https://doi.org/10.1001/archpediatrics.2012. 170 PMID: 22566513 54. Andersen SL, Nøhr SB, Wu CS, Olsen J, Pedersen KM, Laurberg P. Thyroglobulin in smoking mothers and their newborns at delivery suggests autoregulation of placental iodide transport overcoming thiocy- anate inhibition. Eur J Endocrinol. 2013; 168(5):723–31. https://doi.org/10.1530/EJE-12-0759 PMID: 23444413 55. Kit BK, Simon AE, Brody DJ, Akinbami LJ. US prevalence and trends in tobacco smoke exposure among children and adolescents with asthma. Pediatrics. 2013; 131(3):407–14. https://doi.org/10.1542/ peds.2012-2328 PMID: 23400612 56. Pino P, Walter T, Oyarzún MJ, Burden MJ, Lozoff B. Rapid drop in infant blood lead levels during the transition to unleaded gasoline use in Santiago, Chile. Arch Environ Health. 2004; 59(4):182–7. https:// doi.org/10.3200/AEOH.59.4.182-187 PMID: 16189990 57. Mason J, Wheeler W, Brown MJ. The economic burden of exposure to secondhand smoke for child and adult never smokers residing in U.S. public housing. Public Health Rep. 2015; 130(3):230–44. https:// doi.org/10.1177/003335491513000310 PMID: 25931627 58. Merianos AL, Jandarov RA, Mahabee-Gittens EM. Secondhand Smoke Exposure and Pediatric Health- care Visits and Hospitalizations. Am J Prev Med. 2017; 53(4):441–8. https://doi.org/10.1016/j.amepre. 2017.03.020 PMID: 28532658 59. Yilmaz O, Turkeli A, Onur E, Bilge S, Yuksel H. Secondhand tobacco smoke and severity in wheezing children: Nasal oxidant stress and inflammation. J Asthma. 2018; 55(5):477–82. https://doi.org/10. 1080/02770903.2017.1350970 PMID: 28881145 60. Shenassa ED, Rossen LM, Cohen J, Morello-Frosch R, Payne-Sturges DC. Income Inequality and US Children’s Secondhand Smoke Exposure: Distinct Associations by Race-Ethnicity. Nicotine Tob Res. 2017; 19(11):1292–9. https://doi.org/10.1093/ntr/ntw293 PMID: 27811157 61. Nwosu BU, Kum-Nji P. Tobacco smoke exposure is an independent predictor of vitamin D deficiency in US children. PLoS ONE. 2018; 13(10):e0205342. https://doi.org/10.1371/journal.pone.0205342 PMID: 30296288 62. Brody DJ, Lu Z, Tsai J. Secondhand Smoke Exposure Among Nonsmoking Youth: United States, 2013–2016. NCHS Data Brief. 2019;(348):1–8. PMID: 31442196 63. Biren C, Zhang L, Bhandari D, Blount BC, De Jesús VR. Isoprene Exposure in the United States Based on Urinary IPM3: NHANES 2015–2016. Environ Sci Technol. 2020; 54(4):2370–8. https://doi.org/10. 1021/acs.est.9b06587 PMID: 31961658 64. Benowitz NL, Hukkanen J, Jacob P. Nicotine chemistry, metabolism, kinetics and biomarkers. Handb Exp Pharmacol. 2009;(192):29–60. https://doi.org/10.1007/978-3-540-69248-5_2 PMID: 19184645 65. Kehl D, Thyrian JR, Lüdemann J, Nauck M, John U. A descriptive analysis of relations between parents’ self-reported smoking behavior and infants’ daily exposure to environmental tobacco smoke. BMC Pub- lic Health. 2010; 10:424. https://doi.org/10.1186/1471-2458-10-424 PMID: 20642817 PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 16 / 17 PLOS ONE Cotinine cut-points in children under 5 years 66. Dempsey D, Jacob P, Benowitz NL. Nicotine metabolism and elimination kinetics in newborns. Clin Pharmacol Ther. 2000; 67(5):458–65. https://doi.org/10.1067/mcp.2000.106129 PMID: 10824624 67. Homa DM, Neff LJ, King BA, Caraballo RS, Bunnell RE, Babb SD, et al. Vital signs: disparities in non- smokers’ exposure to secondhand smoke—United States, 1999–2012. MMWR Morb Mortal Wkly Rep. 2015; 64(4):103–8. PMID: 25654612 68. Schick S, Glantz S. Philip Morris toxicological experiments with fresh sidestream smoke: more toxic than mainstream smoke. Tob Control. 2005; 14(6):396–404. https://doi.org/10.1136/tc.2005.011288 PMID: 16319363 69. Domino EF, Hornbach E, Demana T. The nicotine content of common vegetables. N Engl J Med. 1993; 329(6):437. https://doi.org/10.1056/NEJM199308053290619 PMID: 8326992 70. Davis RA, Stiles MF, deBethizy JD, Reynolds JH. Dietary nicotine: a source of urinary cotinine. Food Chem Toxicol. 1991; 29(12):821–7. https://doi.org/10.1016/0278-6915(91)90109-k PMID: 1765327 71. Yolton K, Dietrich K, Auinger P, Lanphear BP, Hornung R. Exposure to environmental tobacco smoke and cognitive abilities among U.S. children and adolescents. Environ Health Perspect. 2005; 113 (1):98–103. https://doi.org/10.1289/ehp.7210 PMID: 15626655 72. Apostolou A, Garcia-Esquinas E, Fadrowski JJ, McLain P, Weaver VM, Navas-Acien A. Secondhand tobacco smoke: a source of lead exposure in US children and adolescents. Am J Public Health. 2012; 102(4):714–22. https://doi.org/10.2105/AJPH.2011.300161 PMID: 21852639 73. Jarvis MJ, Fidler J, Mindell J, Feyerabend C, West R. Assessing smoking status in children, adoles- cents and adults: cotinine cut-points revisited. Addiction. 2008; 103(9):1553–61. https://doi.org/10. 1111/j.1360-0443.2008.02297.x PMID: 18783507 74. Mourino N, Pérez-Rı́os M, Santiago-Pérez MI, Lanphear B, Yolton K, Braun JM. Secondhand tobacco smoke exposure among children under 5 years old: questionnaires versus cotinine biomarkers: a cohort study. BMJ Open. 2021; 11(6):e044829. https://doi.org/10.1136/bmjopen-2020-044829 PMID: 34183339 PLOS ONE | https://doi.org/10.1371/journal.pone.0267319 May 5, 2022 17 / 17