Higher parental occupational social contact is associated with a reduced risk of incident pediatric type 1 diabetes : Mediation through molecular enteroviral indices

RESEARCH ARTICLE Higher parental occupational social contact is associated with a reduced risk of incident pediatric type 1 diabetes: Mediation through molecular enteroviral indices Anne-Louise Ponsonby 1,2 *, Angela Pezic 1 , Fergus J. Cameron 1 , Christine Rodda 1,3 , Andrew S. Kemp 1 , John B. Carlin 1 , Heikki Hyoty 4 , Amirbabak Sioofy-Khojine 4 , Terence Dwyer 1,5 , Justine A. Ellis 1,6‡ , Maria E. Craig 7,8‡ 1Murdoch Children’s Research Institute, Royal Children’s Hospital, University of Melbourne, Flemington Rd, Parkville, Victoria, Australia, 2National Centre for Epidemiology, Australian National University, Canberra, Australia, 3Western Centre for Health Research and Education, Sunshine Hospital, St Albans, Victoria, Australia, 4School of Medicine, Virology, University of Tampere, La ¨a ¨ka ¨rinkatu, Finland, 5The George Institute for Global Health, Oxford Martin School, University of Oxford, Oxford, United Kingdom, 6Centre for Social and Early Emotional Development, Faculty of Health, Deakin University, Burwood, Victoria, Australia, 7School of Women’s and Children’s Health, University of New South Wales, New South Wales, Australia, 8Discipline of Child and Adolescent Health, University of Sydney, New South Wales, Australia ‡ These authors are equal last authors on this work. *[email protected] Abstract We aimed to examine the association between parental occupational social contact and hygiene factors on type 1 diabetes (T1D) risk and possible mediation of these effects through child enteroviral infection. We interviewed 333 incident T1D cases and 660 controls from 2008–2011 in Melbourne, Australia. Enteroviral indices (ribonucleic acid by reverse transcription polymerase chain reaction and Coxsackie B virus antibody levels) in peripheral blood were measured in nested case control samples. Parent occupational social contact was assessed by the number of well or sick children, adults or animals contacted daily through work. Higher parental occupational social contact was strongly associated with reduced T1D risk with evidence of dose response (contact with the well or sick score, Adjusted odds ratio (AOR) per category: 0.73 (95% Confidence Interval (CI): 0.66, 0.81); P<0.001 or AOR 0.63 (95% CI: 0.53, 0.75); P<0.001) respectively). Nine of the ten parental social contact indices, were significant mediated through one or more enteroviral indices. The strength of association between enterovirus presence and T1D onset increased with child age (1.2 fold increase per year; P = 0.05). Lower child hand hygiene enhanced the adverse effect of low parental occupational contact with the sick; Synergy Index 5.16 (95% CI: 3.61, 7.36). The interaction between hand washing and parental occupational contact is more consistent with protection against parental enteroviral shedding than the sharing of a protective infectious agent or microbiome. PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 1 / 16 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Ponsonby A-L, Pezic A, Cameron FJ, Rodda C, Kemp AS, Carlin JB, et al. (2018) Higher parental occupational social contact is associated with a reduced risk of incident pediatric type 1 diabetes: Mediation through molecular enteroviral indices. PLoS ONE 13(4): e0193992. https://doi. org/10.1371/journal.pone.0193992 Editor: Marc S. Horwitz, University of British Columbia, CANADA Received: September 13, 2017 Accepted: February 22, 2018 Published: April 17, 2018 Copyright: ©2018 Ponsonby et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data are from the Early Environment and Type 1 Diabetes Study. Data requests should be directed to the Royal Children’s Hospital Human Research Ethics Committee, 50 Flemington Road, Parkville, Victoria 3052, Australia, [email protected]. Funding: Funding was obtained from the National Health and Medical Research Council of Australia, the Australian Research Council, Arthritis Australia, Diabetes Australia, Rebecca L Cooper Medical Introduction The incidence of paediatric type 1 diabetes mellitus (T1D) has increased over time [1]. This autoimmune disease has a first stage of preclinical autoimmunity and a second stage of clinical onset [2]. Meta-analysis indicates the presence of enterovirus (EV) by polymerase chain reaction (PCR) in peripheral blood is associated with a summary odds ratio of 9.8 (95% Confidence Interval (CI): 5.5, 17.4) for clinical TID onset [3]. EV is also markedly more commonly detected among the peripheral blood of family members (63% of parents; 60% of siblings) of incident T1D cases compared to only 3% and 0% of non-family child and adult controls respectively [4]. EV genome can be eliminated relatively quickly from peripheral blood [5]. In contrast, EV genome may be present in host gut mucosa and pancreatic islets for many years, leading to persistent disease with viral shedding [6] [7]. Prolonged EV elimination in faeces has been postulated to be responsible for T1D clustering among sibsets [4]. The role of EV infection in T1D is complex. T1D incidence has particularly increased in modern populations where EV is less prevalent [8]. Two possible mechanisms include:- (i) that in such populations EV is acquired at a later age which leads to adverse consequences and/or (ii) that the infectious contact load is reduced in such populations, leading to reduced ‘herd immunity’ (partly due to lack of maternal enterovirus antibodies in new-borns) and adverse immune consequences upon EV exposure. EV infection during the first year of life has been associated with a reduced risk of T1D onset [9]. However, to date, no study has demonstrated that the adverse effect of EV on T1D onset significantly increases with increasing age. The second mechanism has been difficult to investigate for T1D but occupational social contact (daily contact with a number of children, adults or animals through work) has been used as a proxy for investigating herd immunity issues for other diseases [10] [11]. High social contact occupations are associated with a greater infection rates [12] and reboosting of established immune responses against pathogens [13]. Such re-exposure is particularly valuable for short term host immune responses and/or persistent infections [14]. EV may meet this criteria [6,7] and Varicella-zoster virus does:- higher adult occupational social mixing or contact with children is associated with a reduced risk of herpes zoster in adulthood, likely mediated through boosted humoral immunity against latent Varicella-zoster virus [15]. High paternal occupational social contact is associated with maternal primary cytomegalovirus infection during pregnancy [16]. Apart from one small study [17], parental social contact has never been systematically evaluated for T1D. The purpose of this report was to evaluate (i) whether higher parental occupational social contact with well and sick adults, children and animals was associated with a reduced risk of T1D, (ii) whether any such effect was modified by child hand hygiene before meals, and (iii) the extent that any apparent beneficial effect of higher parental occupational social contact on child T1D onset is mediated through altered EV indices at T1D onset. We also consider these findings in the context of age of T1D onset. Table 1. Characteristics of children in the early environment and type 1 diabetes prevention project. Factor Cases Mean (SD) or % (n/N) Controls Mean (SD) or % (n/N) P-value Age at recruitment (years) 8.4 (3.6) 6.5 (3.5) <0.001 Maternal age at child’s birth (years) 30.5 (4.8) 29.4 (5.5) 0.001 Child ever breastfed (yes) 85.9 (279/325) 80.4 (510/634) 0.038 Male sex 50.8 (169/333) 59.6 (393/660) 0.008 Family history of insulin dependent diabetes mellitus 15.8 (50/316) 12.8 (77/600) 0.213 Caucasian 91.5 (300/328) 78.8 (402/510) <0.001 https://doi.org/10.1371/journal.pone.0193992.t001 Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 2 / 16 Research Foundation, L.E.W. Carty Charitable Fund, ANZ Medical Research and Technologies in Victoria Fund, Equity Trustees Lynne Quayle Charitable Trust Fund, the Australian Academy of Science, The Victorian State Government Operational Infrastructure Support Program, and the Murdoch Childrens Research Institute Internal Grant Scheme. JAE was supported by an Australian Research Council Future Fellowship. ALP was supported by a National Health and Medical Research Council Senior Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Methods Cases Participants with incident T1D were recruited between March 2008 and March 2011 at the Royal Children’s Hospital and Monash Medical Centre, Melbourne, Australia [18]. Inclusion criteria were participants with newly diagnosed T1D aged 1 to 14 years inclusive (Table 1). Controls Controls were recruited between January 2008 and July 2012 from the Royal Children’s Hospital day surgery unit which they attended for a minor surgical procedure. The healthy control children were aged 14 years or under and born in the state of Victoria, Australia. A range of minor reasons for surgery were targeted for inclusion [19]. These controls were recruited as part of the larger paediatric autoimmune disease platform. Cases and controls with a major congenital abnormality or an illness that would forgo usual school attendance in the year prior to recruitment were excluded from study interview which involved parental questionnaire and clinical examination. A comprehensive questionnaire which included infection, demographic, lifestyle and environmental history over the child’s life course was obtained at a single interview. This included ancestry by grandparents’ racial origin, child sun exposure [20] and current child hygiene practices [21]Current weekday parental occupational social exposure to children, adults or animals and whether these groups were sick or well was recorded (S1 Table), following the approach of Thomas et al [10]. This approach was chosen as it provides quantitative responses, allowing dose response trends to be better evaluated. Birth dates of all participating children and their siblings were used to provide sibling number and inter-sibling interval. Composite scores for contact with sick people or well people and animals were constructed as outlined in Table 2. Ethical approval was obtained from the Royal Children’s Hospital and the Monash Medical Centre Human Research Ethics Committees. Written consent was obtained from parents and assent from children aged 12 years and over. Blood samples For T1D cases, serum samples were obtained at time of admission and blood samples were obtained again at interview (median sampling time after initial admission to blood draw, 0.4 (IQR = 0.1 to 6.6) weeks). Control children provided a venous blood sample collected at insertion of the peripheral line for day surgery. Case and control blood samples were separated into heparinized plasma and peripheral blood mononuclear cells. Plasma was stored in 1 ml aliquots in a -80˚C facility. Enteroviral indices To allow direct matching by sex and within a year of age, nested case control samples were randomly selected for viral studies. Case admission serum and control plasma samples were tested for detectable EV ribonucleic acid by one step quantitative real time reverse transcription polymerase chain reaction with SYBR green dye using the LightCycler RNA Amplification Kit SYBR Green I (Product No. 12015137001, Roche applied systems, USA) [22] on the LightCycler 2.0 Instrument (Roche Diagnostics, USA) at the South Eastern Area Laboratory Services at Prince of Wales Hospital, as previously described [22]. Multiplex real time-polymerase chain reaction (PCR) for EV, herpes simplex 1, Epstein-Barr virus, Varicella-zoster virus and cytomegalovirus detection was also conducted [23]. Neutralizing antibodies were measured against Coxsackie B1 virus (CVB) (American Type Culture Collection prototype strain) with a Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 3 / 16 Table 2. Higher parent occupational microbial contact is associated with a reduced risk of type 1 diabetes onset: Ten measures and two composite indices. Category Cases, % (n/N) Controls, % (n/N) AOR95% CIP-value AOR† 95% CI† P-value Contact with well adults, mother Not at all 14.7% (45/307) 4.8% (26/540) Ref Ref n = <10 34.5% (106/307) 40.7% (220/540) 0.25 0.15, 0.44 <0.001 0.11 0.05, 0.25 <0.001 n = 10 <30 29.6% (91/307) 28.9% (156/540) 0.27 0.15, 0.49 <0.001 0.10 0.04, 0.23 <0.001 n = 30 or more 21.2% (65/307) 25.6% (138/540) 0.20 0.11, 0.37 <0.001 0.09 0.04, 0.22 <0.001 Test of trend <0.001 <0.001 Contact with well adults, father Not at all 3.3% (10/307) 1.5% (7/477) Ref Ref n = <10 21.5% (66/307) 23.7% (113/477) 0.35 0.12, 1.01 0.05 0.17 0.04, 0.73 0.02 n = 10 <30 45.9% (141/307) 35.4% (169/477) 0.54 0.19, 1.52 0.24 0.26 0.06, 1.11 0.07 n = 30 or more 29.3% (90/307) 39.4% (188/477) 0.29 0.10, 0.83 0.02 0.15 0.04, 0.65 0.01 Test of trend 0.06 0.01 Contact with well children, mother Not at all 30.4% (93/306) 10.9% (57/521) Ref Ref n = <10 42.2% (129/306) 55.1% (287/521) 0.24 0.16, 0.36 <0.001 0.14 0.08, 0.25 <0.001 n = 10 <30 18.3% (56/306) 19.4% (101/521) 0.31 0.19, 0.50 <0.001 0.17 0.09, 0.31 <0.001 n = 30 or more 9.2% (28/306) 14.6% (76/521) 0.16 0.09, 0.28 <0.001 0.09 0.05, 0.19 <0.001 Test of trend <0.001 <0.001 Contact with well children, father Not at all 39.7% (121/305) 26.6% (119/448) Ref Ref n = <10 50.2% (153/305) 60.5% (271/448) 0.52 0.37, 0.72 <0.001 0.43 0.29, 0.63 <0.001 n = 10 <30 7.5% (23/305) 6.7% (30/448) 0.74 0.40, 1.38 0.35 0.70 0.33, 1.49 0.36 n = 30 or more 2.6% (8/305) 6.3% (28/448) 0.25 0.11, 0.58 0.001 0.21 0.08, 0.54 0.001 Test of trend <0.001 <0.001 Contact with well animals, mother Not at all 56.7% (174/307) 39.7% (204/514) Ref Ref n = <10 40.1% (123/307) 58.6% (301/514) 0.44 0.33, 0.60 <0.001 0.33 0.23, 0.48 <0.001 n = 10 or more 3.3% (10/307) 1.8% (9/514) 1.12 0.43, 2.88 0.82 0.60 0.23, 1.57 0.30 Test of trend <0.001 <0.001 Contact with well animals, father Not at all 49.0% (150/306) 44.1% (198/449) Ref Ref n = <10 48.0% (147/306) 52.8% (237/449) 0.73 0.54, 0.99 0.05 0.60 0.42, 0.86 0.006 n = 10 or more 2.9% (9/306) 3.1% (14/449) 0.92 0.38, 2.24 0.85 0.84 0.31, 2.23 0.72 Test of trend 0.09 0.02 Any contact with sick adults, mother No 83.0% (253/305) 75.6% (380/503) Ref Ref Yes 17.1% (52/305) 24.5% (123/503) 0.58 0.40, 0.84 0.004 0.51 0.34, 0.78 0.002 Any contact with sick adults, father No 89.6% (268/299) 79.6% (356/447) Ref Ref Yes 10.4% (31/299) 20.4% (91/447) 0.43 0.27, 0.68 <0.001 0.33 0.20, 0.55 <0.001 Any contact with sick children, mother No 88.0% (270/307) 76.8% (381/496) Ref Ref Yes 12.1% (37/307) 23.2% (115/496) 0.43 0.28, 0.65 <0.001 0.36 0.23, 0.56 <0.001 Any contact with sick children, father No 95.9% (282/294) 87.6% (381/435) Ref Ref Yes 4.1% (12/294) 12.4% (54/435) 0.25 0.13, 0.49 <0.001 0.15 0.07, 0.33 <0.001 Composite any contact with well adults, children or animals, mother and father (occupational well score) ‡ (Continued) Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 4 / 16 plaque neutralization assay at the Department of Virology, University of Tampere, Finland [24]. The plasma sample was first mixed with 100 plaque-forming units of the virus and incubated for 1 h at 37˚C followed by overnight incubation at room temperature. This mixture was then transferred to a monolayer of green monkey kidney cells on six-well plastic plates (Nunclon, ThermoFisher Scientific, product No. 140685) in plaque assay medium containing minimal essential medium supplemented with 1% FBS, 40 U/mL penicillin-streptomycin, 0.0023% glucose, 1 X L-glutamine, 1.5 mmol/L MgCl2, and 1.5 mmol/L carboxymethyl cellulose (HEPES). The number of virus-generated plaques was counted manually after 48 h of incubation at 37˚C. All test runs included both virus-positive and virus-negative control wells. The final dilutions of plasma in the assay were 1/4 and 1/16, and the sample was judged seropositive if either of these dilutions inhibited at least 75% of the plaques. The range of inhibition was 0 to 100%. Detection of neutralizing antibodies in plasma in such titers has been shown to be a reliable marker of past infection [25]. We examined the case serum samples at first presentation because timing of EV infection in relation to T1D disease course is very important [26]. Although serum and plasma samples provide comparable Immunoglobulin G (IgG) measures be enzyme-linked immunosorbent assay [27,28] and very similar for viral PCR measures [29], some studies have had lower viral detection levels in serum than plasma [30,31]. Thus, the excess proportion of cases with detectable EV to controls could possibly be even a little higher than reported. Statistical methods Characteristics of the cases and controls are presented as mean (standard deviation) or percentages. Sibling birthdates were used to obtain the number of age-specific siblings. SiblingTable 2. (Continued) Category Cases, % (n/N) Controls, % (n/N) AOR95% CIP-value AOR† 95% CI† P-value Not at all 2.5% (8/325) 1.4% (8/562) Ref Ref Category 1 8.0% (26/325) 4.5% (25/562) 1.03 0.33, 3.29 0.95 2.92 0.51, 16.86 0.23 Category 2 12.9% (42/325) 13.9% (78/562) 0.53 0.18, 1.56 0.25 0.59 0.14, 2.53 0.48 Category 3 24.9% (81/325) 15.8% (89/562) 0.72 0.25, 2.08 0.55 0.40 0.10, 1.63 0.20 Category 4 14.2% (46/325) 21.2% (119/562) 0.38 0.13, 1.09 0.07 0.20 0.05, 0.83 0.03 Category 5 7.4% (24/325) 7.8% (44/562) 0.46 0.15, 1.42 0.18 0.17 0.04, 0.75 0.02 Category 6 30.2% (98/325) 35.4% (199/562) 0.43 0.15, 1.22 0.11 0.20 0.05, 0.78 0.02 Test of trend 0.002 <0.001 Composite any contact with sick adults or children, mother and father (occupational sick score) § Not at all 76.5% (244/319) 62.8% (329/524) Ref Ref Category 1 11.6% (37/319) 15.7% (82/524) 0.55 0.35, 0.85 0.007 0.53 0.32, 0.87 0.01 Category 2 7.5% (24/319) 12.8% (67/524) 0.46 0.28, 0.77 0.003 0.38 0.22, 0.68 0.001 Category 3 2.8% (9/319) 3.2% (17/524) 0.69 0.30, 1.62 0.40 0.45 0.19, 1.09 0.08 Category 4 1.6% (5/319) 5.5% (29/524) 0.20 0.08, 0.54 0.001 0.11 0.03, 0.36 <0.001 Test of trend <0.001 <0.001 Ref =Reference category. n = number of person-specific or animal-specific contacts. Adjusted for age and sex only † Adjusted for age, sex, family history of insulin dependent diabetes mellitus, time spent in sun during last winter weekdays, ever breastfed, maternal age at birth, SEIFA disadvantage index and Caucasian ancestry ‡ Occupational well score is a summation of individual occupational well categories. 0 = no exposure in all the six categories, 2 = some exposure in 2 of the 6 categories etc. § Occupational sick score is a summation of individual occupational sick categories. 0 = no exposure in all the four categories, 2 = some exposure in 2 of the 4 categories etc. https://doi.org/10.1371/journal.pone.0193992.t002 Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 5 / 16 years, defined as the total number of years a child had been exposed to any siblings, regardless of sibling age (up to age 18), was calculated for the subject at time of interview [19] and reconstructed for past ages of the subjects, for example, at age 2 years. Multivariable logistic regression was used to examine case-control differences. Adjusted odds ratios (AOR) and 95% CI are reported. All AORs were adjusted for age at recruitment and sex, and then additionally for other factors such as family history of T1D, Caucasian ancestry, ever breastfed, maternal age at birth, skin type, low sun exposure in past winter on weekends and the socioeconomic indexes for areas disadvantage index [32]. These factors were included as covariates as they potentially confounded the association between parental occupational social contact and T1D onset. Tests for trend with categorical covariates were undertaken by using a single predictor taking category rank scores, based on the Wald test. To assess interaction on the multiplicative scale, we added product terms to the logistic models. To assess interaction on the additive scale, we focussed on the Synergy Index as this allows confounding to be considered also [33]. Mediation analysis was undertaken to determine if EV infection was a likely intermediate factor in a causal pathway between the selected proxy microbial exposures (parental occupational social contact, child attending day care etc.) and T1D onset [34]. We followed the methods of VanderWeele [35] and assessed two EV indices:- EV presence by PCR and antibodies against EV serotypes previously linked with T1D as EV infection biomarkers. Multiple linear regression was used to assess the influence of environmental factors on age of onset in completed years, after first accounting for constitutional factors of ancestry, sex and parental history of T1D. The interaction between age of onset, EV presence and T1D risk was assessed by adding a product term and assessing the reduction in deviance using the log likelihood ratio test. Alternative models allowing age to be categorized in a non-linear form were also developed and compared using the log likelihood ratio test. The common odds ratio test was also used to assess whether the association between the EV indices and T1D onset varied by age in the age and sex matched analyses [36]. We conducted an additional analysis aimed to recalculate the main study findings using an estimation method to better reflect all Victorian births. Cases born outside Victoria were excluded. For controls, inverse probability weighting was used to re-weight the available controls to better reflect the entire Victorian paediatric population. Weights were calculated as the inverse of the probability of the controls being selected for the study [37] compared to 99.9% of live births in the same birth year, available from the Victorian Perinatal Data Collection Unit. Probability of selection was modelled using month of birth, birth weight, gestational age at birth, maternal marital status, mode of delivery, maternal age and SEIFA disadvantage index. We used Stata 14.1 software (StataCorp, College Station, TX) for all analyses [38]. Results 333 cases (83% of incident cases) and 660 controls (a participation rate of 82%) were involved. Table 1 shows that the cases had a mean age of 8.4 (SD 3.6) years and 50.8% were male. The control mean age was 6.5 (SD 3.5) years and 59.6% were male. In our setting, day care was associated with high child contact, with 86% (102/118) of control children at day care being exposed to 11 or more children in the same room. The inverse association between parental occupational social contact and T1D onset Higher parental occupational social contact was strongly associated with reduced T1D risk (Table 2). Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 6 / 16 The magnitude of these inverse associations were high, with evidence of dose response. The inverse associations were consistently evident across all ten exposure categories: Higher parental occupational social contact was strongly associated with reduced T1D risk with evidence of dose response (AOR per category of increasing contact with the well or sick score AOR (0.73 (95% CI: 0.66, 0.81); P<0.001 or AOR 0.63 (95% CI: 0.53, 0.75); P<0.001) respectively. The magnitude of effect was substantial. For example, the highest category of composite well or sick parental occupational score was associated with a more than five-fold or ten-fold reduction in T1D risk, respectively, compared to no exposure. The association between child hand hygiene, day care and other factors and T1D onset Better hand hygiene before meals was also strongly associated with reduced T1D risk, with evidence of dose response (Table 3). Day care was also associated with reduced T1D risk. (Table 3). Of interest, better child hand hygiene, as reported in Fig 1, was associated with the child having less colds or flu (AOR 0.64, P = 0.003),but not significantly less gastroenteritis (AOR 0.83, P = 0.16) over the past year. Poorer child hygiene before meals exacerbates the association between low parent occupational contact and a higher risk of type 1 diabetes onset The association between parental occupational social contact and T1D differed by child hand hygiene standards. Fig 1 shows the lowest risk was for those with high parental occupational social contact and also high hand hygiene and that the combined risk of both low parental occupational social contact and also low hand hygiene before meals was greater than expected. For parental occupational well score (0–1) and poor child hand hygiene before meals (never, occasionally), the Synergy Index was 1.22 (95% CI: 0.39, 3.87) and parental occupational sick score (none) and poor child hand hygiene before meals (never, occasionally), the Synergy Index was 5.16 (95% CI: 3.61, 7.36) (Fig 1). Table 3. The association between child hygiene, day care and recent illness and type 1 diabetes onset. Cases, % (n/N) Controls, % (n/N) AOR95% CIP-value AOR† 95% CI† P-value Hand washing before meals frequency Never 3.0% (10/330) 1.3% (8/635) Ref Ref Occasionally 17.6% (58/330) 10.2% (65/635) 0.74 0.26, 2.09 0.57 0.83 0.22, 3.09 0.78 Sometimes 23.0% (76/330) 16.5% (105/635) 0.59 0.21, 1.63 0.31 0.62 0.17, 2.26 0.47 Usually 35.5% (117/330) 36.7% (233/635) 0.46 0.17, 1.24 0.13 0.56 0.16, 1.98 0.37 Always 20.9% (69/330) 35.3% (224/635) 0.28 0.10, 0.76 0.01 0.37 0.10, 1.32 0.12 Test of trend <0.001 0.002 Child attended day care in the past year No 87.8 (280/319) 79.7 (463/581) Ref Ref Yes 12.2 (39/319) 20.3 (118/581) 0.64 0.43, 0.96 0.03 0.52 0.33, 0.81 0.004 Flu or cold in the past 12 months No 27.2 (88/323) 48.9 (278/569) Ref Ref Yes 72.8 (235/323) 51.1 (291/569) 2.70 1.98, 3.68 <0.001 2.58 1.80, 3.71 <0.001 Ref = Reference category Adjusted for age and sex only † Adjusted for age, sex, family history of insulin dependent diabetes mellitus, time spent in sun during last winter weekdays, ever breastfed, maternal age at birth, SEIFA disadvantage index and Caucasian ancestry https://doi.org/10.1371/journal.pone.0193992.t003 Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 7 / 16 Enterovirus indices. EV was detected by PCR more commonly among T1D cases than controls, with an adjusted odds ratio of 5.61 (95% CI: 3.16, 9.98). T1D cases had higher EV IgG levels (Table 4). The correlation between detectable EV and EV seropositivity was r = -0.01; P =0. 89 and r = 0.03, P =0.57 for cases and controls respectively. Among controls, day care attendance associated with a five-fold increase in the likelihood of EV presence by PCR (AOR 5.30 (95% CI: 1.27, 22.10); P =0.02). Further, day care attendance was associated with EV seropositivity (AOR 2.72 (95% CI: 1.07, 6.95); P =0.04). Mediation analyses—the association between higher parental occupational well or sick contact on type 1 diabetes is mediated through altered enteroviral indices in the child Restricting to the nested viral study, parental occupational well or sick contact was again strongly associated with reduced T1D risk. For mediation, highly consistent patterns were seen across the ten exposure categories (Table 5). Fig 1. Combined exposure to low occupational sick score and low hand washing is associated with greater risk of type 1 diabetes onset: Evidence of interaction. The combined exposure to low occupational sick score (0–2 vs. rest) and low hand washing (never, occasionally) had an odds ratio of 3.86 (95% CI: 2.08, 7.16); among those with a low occupational sick score and high hand washing the odds ratio was 1.74 (95% CI: 0.89, 3.41); among those with low hand washing and a high occupational sick score the odds ratio was 1.67 (95% CI: 0.86, 3.24) compared to the lowest risk category associated with both high occupational sick score and high hand washing (AOR 1.00 (reference)). The Synergy Index is 5.16 (95% CI: 3.61, 7.36) with a Relative Excess Risk due to Interaction of 37.46 (95% CI: 13.96, 60.95) and Attributable Proportion of 0.79 (95% CI: 0.72, 0.86). The AOR multi is 1.19, P = 0.03. All odds ratio adjusted for age and sex. Thus, the interaction is evident on the additive and multiplicative scale. https://doi.org/10.1371/journal.pone.0193992.g001 Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 8 / 16 Five of the six indicators of parental occupational well contact were demonstrated to be significantly mediated through EV PCR presence and/or also EV seropositivity. The mediated fractions were not large in magnitude but the 95% confidence intervals excluded a zero mediation value. Similarly, all four indicators of parental occupational sick contact were demonstrated to be significantly mediated through either EV PCR presence and/or EV seropositivity. A greater portion of mediated effect was accounted for by variation in EV presence than EV seropositivity. However, there was an anomalous finding where inverse association between maternal contact sick adults was not partly accounted for by mediation in EV presence, rather the reverse. However, this factor did appear to be partly mediated through EV seropositivity. The associations between child hand hygiene or day care and T1D onset were not demonstrated to be directly mediated through variation in EV presence or EV seropositivity. There was no interaction between parental occupational contact, EV infection and T1D. Age of onset of type 1 diabetes Day care attendance was associated with a younger age of T1D onset (mean difference, 2.49 (95% CI: 1.29, 3.69) years) and also associated with younger age among controls (mean difference, 1.15 (95% CI: 0.37, 1.94 years). Increasing composite score for parental occupational well or sick contact and child hand hygiene were not associated with age of T1D onset. We examined effect modification by child age. EV infection was associated with a moderate risk of T1D onset for children aged 1–6 years (matched OR 1.48, P =0.002) but a higher risk for children aged 7–15 years (matched OR 6.00, P =0.097); common odds ratio test P =0.98. When examining the linear influence of child age on the magnitude of association between EV presence and T1D onset, for every year beyond age 1, the risk associated with EV presence increased 1.2 fold; P =0.05 (Fig 2). There was no evidence a non-linear model provided a better fit to the data (P =0.97). There was no difference in effect by child age on the magnitude of association between EV seropositivity, parental occupational social contact, child hand hygiene or day care attendance and T1D onset. Sensitivity analyses We reconducted the analyses for Victorian-born cases with controls weighted to all Victorian live births. The findings were not materially altered. For example, higher parental occupational social contact composite score-T1D onset (composite well score; AOR 0.71 (95% CI: 0.63, 0.80) per category, composite sick score; AOR 0.61 (95% CI: 0.50, 0.73) per category. EV PCR was strongly associated with T1D onset (AOR 5.31 (95% CI: 3.02, 9.35). Again, the mediation analysis demonstrated that a significant proportion of the association between parental occupational well or sick contact was mediated through EV presence and also EV seropositivity, with a lesser magnitude for the later mediation pathway. Discussion Higher parental occupational social contact is strongly associated with a reduction in child T1D risk with consistent dose response trends. The association is mediated partly through a reduction in EV presence in the peripheral blood of the child at T1D onset and, to a lesser extent, associated EV seropositivity. At diagnosis, T1D cases were more likely to have detectable EV in their peripheral blood and elevated EV seropositivity, indicating greater past exposure to EV, than controls. The observation that this apparent protective association for parental occupational social contact is enhanced by high child hand hygiene before meals is consistent with several Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 9 / 16 35. VanderWeele TJ. Mediation Analysis: A Practitioner’s Guide. Annu Rev Public Health. 2016; 37:17–32. https://doi.org/10.1146/annurev-publhealth-032315-021402 PMID: 26653405. 36. He XX, Jin SG. Interaction and its solution in individual matching case-control study. Biomedical and environmental sciences: BES. 2003; 16(1):40–6. PMID: 12747006. 37. Little RJA, Rubin DB. Statistical analysis with missing data. 2 ed. New York: Wiley; 2002. 38. StataCorp. 2015. Stata Statistical Software: Release 14. 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Statistics: Biomedicine must look beyond P values. Nature. 2014; 507 (7491):169. https://doi.org/10.1038/507169b PMID: 24622193. 44. Kaila B, Taback SP. The effect of day care exposure on the risk of developing type 1 diabetes: a metaanalysis of case-control studies. Diabetes care. 2001; 24(8):1353–8. PMID: 11473069. 45. Krogvold L, Edwin B, Buanes T, Frisk G, Skog O, Anagandula M, et al. Detection of a low-grade enteroviral infection in the islets of langerhans of living patients newly diagnosed with type 1 diabetes. Diabetes. 2015; 64(5):1682–7. https://doi.org/10.2337/db14-1370 PMID: 25422108. Parental occupational contact, enterovirus and type 1 diabetes PLOS ONE | https://doi.org/10.1371/journal.pone.0193992 April 17, 2018 16 / 16