PhD Michele L Shaffer

MD, PhD Olivier Baud

MD, PhD Thierry Lacaze-Masmonteil

MD, PhD Outi M Peltoniemi

MD Francesco Bonsante

MD Kristi L Watterberg

Effect of Prophylaxis for Early Adrenal Insufficiency Using Low-Dose Hydrocortisone in Very Preterm Infants: An Individual Patient Data Meta-Analysis

Objective To assess the effect of prophylaxis for early adrenal insufficiency using low-dose hydrocortisone on survival without bronchopulmonary dysplasia (BPD) in very preterm infants using an individual patient data meta-analysis.

Study design All existing randomized controlled trials testing the efficacy of the prophylaxis of early adrenal insufficiency using low-dose hydrocortisone on survival without BPD were considered for inclusion when data were available. The primary outcome was the binary variable survival without BPD at 36 weeks of postmenstrual age.

Results Among 5 eligible studies, 4 randomized controlled trials had individual patient data available (96% of participants identified; n = 982). Early low-dose hydrocortisone treatment for 10-15 days was associated with a significant increase in survival without BPD (OR, 1.45; 95% CI, 1.11-1.90; P = .007; I 2 = 0%), as well as with decreases in medical treatment for patent ductus arteriosus (OR, 0.72; 95% CI, 0.56-0.93; P = .01; I 2 = 0%) and death before discharge (OR, 0.70; 95% CI, 0.51-0.97; P = .03; I 2 = 0%). The therapy was associated with an increased risk of spontaneous gastrointestinal perforation (OR, 2.50; 95% CI, 1.33-4.69; P = .004; I 2 = 31.9%) when hydrocortisone was given in association with indomethacin exposure. The incidence of late-onset sepsis was increased in infants exposed to hydrocortisone (OR, 1.34; 95% CI, 1.02-1.75; P = .04; I 2 = 0%), but no adverse effects were reported for either death or 2-year neurodevelopmental outcomes as assessed in an aggregate meta-analysis.

Conclusions

This individual patient data meta-analysis showed that early low-dose hydrocortisone therapy is beneficial for survival without BPD in very preterm infants. (J Pediatr 2018;■■:■■-■■).

See related article, p •••

 Data reported since 1995 have supported the hypothesis that very preterm infants who develop BPD often have relative adrenal insufficiency during the first postnatal week, suggesting that early hydrocortisone replacement could be beneficial. 4,9,10 Among 11 randomized controlled trials (RCTs) that included hydrocortisone treatment initiated before 7 postnatal days in very preterm infants, 7 5 were designed specifically to test the efficacy of early prophylaxis of early adrenal insufficiency BPD Bronchopulmonary dysplasia NDI Neurodevelopmental impairment PDA

to improve survival without BPD. [START_REF] Watterberg | Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants[END_REF][START_REF] Watterberg | Prophylaxis of early adrenal insufficiency to prevent bronchopulmonary dysplasia: a multicenter trial[END_REF][START_REF] Peltoniemi | Pretreatment cortisol values may predict responses to hydrocortisone administration for the prevention of bronchopulmonary dysplasia in high-risk infants[END_REF][START_REF] Bonsante | Early low-dose hydrocortisone in very preterm infants: a randomized, placebo-controlled trial[END_REF][START_REF] Baud | Effect of early low-dose hydrocortisone on survival without bronchopulmonary dysplasia in extremely preterm infants (PREMILOC): a doubleblind, placebo-controlled, multicentre, randomised trial[END_REF] These trials extended hydrocortisone therapy beyond the first postnatal week and used a dose of 1-2 mg/kg/day, which has been shown to moderately but significantly increase serum cortisol concentrations in extremely preterm neonates compared with placebo. [START_REF] Watterberg | Prophylaxis of early adrenal insufficiency to prevent bronchopulmonary dysplasia: a multicenter trial[END_REF] Although 2-year follow-up data have been consistently reassuring, [START_REF] Doyle | Early (<8 days) systemic postnatal corticosteroids for prevention of bronchopulmonary dysplasia in preterm infants[END_REF][START_REF] Watterberg | Growth and neurodevelopmental outcomes after early lowdose hydrocortisone treatment in extremely low birth weight infants[END_REF][START_REF] Peltoniemi | Trial of early neonatal hydrocortisone: two-year follow-up[END_REF][START_REF] Baud | Association between early low-dose hydrocortisone therapy in extremely preterm neonates and neurodevelopmental outcomes at 2 years of age[END_REF][START_REF] Baud | Twoyear neurodevelopmental outcomes of extremely preterm infants treated with early hydrocortisone: treatment effect according to gestational age at birth[END_REF] the effect of early hydrocortisone treatment on survival without BPD and potential side effects remain unclear. Therefore, we undertook an individual patient data metaanalysis of clinical trials to examine the effect of prophylaxis of early adrenal insufficiency on these outcomes.

Methods

The PRISMA-IPD checklist of items requested to report a metaanalysis of individual patient data is available at www.jpeds.com (Supplement). In addition, some data items are available in the statistical analysis plan (Appendix; available at www.jpeds.com). Two meta-analyses identified published RCTs of early hydrocortisone therapy conducted before 2017. [START_REF] Doyle | Early (<8 days) systemic postnatal corticosteroids for prevention of bronchopulmonary dysplasia in preterm infants[END_REF][START_REF] Doyle | Postnatal hydrocortisone for preventing or treating bronchopulmonary dysplasia in preterm infants: a systematic review[END_REF] We also searched MEDLINE for the terms "hydrocortisone," "cortisone,""preterm infant,""randomized," and "human." The last search was done in July 2018. We did not identify any RCTs of early low-dose hydrocortisone therapy to prevent BPD other than those included in these 2 reports.

Individual Patient Data Acquisition, Data Processing, and Quality Assessment

The principal investigators of the 5 eligible trials agreed to share deidentified data to perform an individual patient data metaanalysis. Individual patient data from 1 pilot trial (n = 40) were no longer available [START_REF] Watterberg | Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants[END_REF] ; this individual patient data metaanalysis includes all data from the remaining 4 studies (n = 982). Because an individual patient data meta-analysis can improve the ability to address confounders or covariates of interest, we were able to account for individual patient-level factors that affected outcomes, which would not have been possible with an aggregate meta-analysis based on published data without the risk of drawing potentially incorrect conclusions owing to ecological fallacy with the application of meta-regression. In addition, meta-regression often has low power to detect relationships. [START_REF] Lambert | A comparison of summary patient-level covariates in meta-regression with individual patient data meta-analysis[END_REF][START_REF] Berlin | Individual patient-versus group-level data meta-regressions for the investigation of treatment effect modifiers: ecological bias rears its ugly head[END_REF][START_REF] Higgins | Controlling the risk of spurious findings from meta-regression[END_REF] Individual patient data also allowed for inclusion of data not previously reported and standardization of outcomes and exposures across studies. In addition, the cooperation of all authors of the original publications allowed for detailed data checking.

We created a common data dictionary and asked corresponding authors to identify which data elements were available, or to suggest alternative measures if data were not available. After receiving all authors' available data, we created a final data capture template to request the individual patient datasets from all authors to reduce the amount of postprocessing needed to harmonize the datasets. Any questions regarding the individual patient datasets were discussed with the authors and corrected.

The statistician created any necessary derived variables and checked the summary statistics against available published data.

All data summaries by study and treatment group were shared with the corresponding authors to check for discrepancies, and any identified errors were corrected.

Outcomes and Subgroups of Interest

The primary outcome of interest was the binary variable survival without BPD at 36 weeks postmenstrual age (PMA), and the primary predictor was receipt of early low-dose hydrocortisone treatment. Adjustment variables to be included in all models were birth weight, sex, gestational age, and antenatal steroid use. Predefined subgroups of interest included sex, histologic chorioamnionitis, gestational age strata (<26 or ≥26 weeks), and indomethacin treatment.

Secondary outcomes included days of ventilation, continuous positive airway pressure, and oxygen; supplemental oxygen at discharge; and medical or surgical treatment for patent ductus arteriosus (PDA). Prespecified potential adverse outcomes included pneumothorax, spontaneous gastrointestinal perforation, necrotizing enterocolitis, severe intraventricular hemorrhage, cystic periventricular leukomalacia, severe retinopathy of prematurity, late online sepsis (bacterial or fungal), and the effects of hydrocortisone on weight or head circumference at 36 weeks PMA and on the 2-year neurodevelopmental outcomes.

Statistical Analyses

An a priori statistical analysis plan (Appendix) was created to describe and prioritize the outcomes and analyses of interest, including subgroup and sensitivity analyses, before the analysis was begun. No multiple comparisons adjustments were used for subgroup analyses. We used a 1-step approach to individual patient data meta-analysis using generalizations of logistic regression models for binary outcomes and linear regression models for quantitative outcomes based on generalized linear mixed models with Kenward-Roger approximation of degrees of freedom. [START_REF] Kenward | Small sample inference for fixed effects from restricted maximum likelihood[END_REF][START_REF] Fitzmaurice | Applied longitudinal analysis[END_REF] Logistic regression models were summarized using ORs and associated 95% CIs. Linear regression models were summarized using mean differences and associated CIs. We attempted to account for clustering of patients within different studies by specifying a random intercept term, assuming that the baseline is drawn at random from a normal distribution. We first considered treatment a random effect, but then simplified the models to fixed treatment effects, which yielded similar findings in terms of magnitude, direction, and significance. Statistical heterogeneity was summarized as I2 value and associated P value. I 2 values were computed using the meta package in R (R Foundation for Statistical Computing, Vienna, Austria). We also conducted a traditional aggregate random-effects meta-analysis based on available published data for comparison of long-term developmental outcomes. [START_REF] Watterberg | Growth and neurodevelopmental outcomes after early lowdose hydrocortisone treatment in extremely low birth weight infants[END_REF][START_REF] Peltoniemi | Trial of early neonatal hydrocortisone: two-year follow-up[END_REF][START_REF] Baud | Association between early low-dose hydrocortisone therapy in extremely preterm neonates and neurodevelopmental outcomes at 2 years of age[END_REF] Statistical analyses were performed using SAS 9.4 (SAS Institute, Cary, North Carolina) for the individual patient data analysis and using Stata 12.0 (StataCorp, College Station, Texas) for the aggregate meta-analysis. Findings were considered significant at P <.05.

Results

Among 11 RCTs testing hydrocortisone early after birth in neonates (Table I; available at www.jpeds.com), 5 were specifically designed to test the efficacy of prophylaxis of early adrenal insufficiency to improve survival without BPD. [START_REF] Watterberg | Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants[END_REF][START_REF] Watterberg | Prophylaxis of early adrenal insufficiency to prevent bronchopulmonary dysplasia: a multicenter trial[END_REF][START_REF] Peltoniemi | Pretreatment cortisol values may predict responses to hydrocortisone administration for the prevention of bronchopulmonary dysplasia in high-risk infants[END_REF][START_REF] Bonsante | Early low-dose hydrocortisone in very preterm infants: a randomized, placebo-controlled trial[END_REF][START_REF] Baud | Effect of early low-dose hydrocortisone on survival without bronchopulmonary dysplasia in extremely preterm infants (PREMILOC): a doubleblind, placebo-controlled, multicentre, randomised trial[END_REF] The other 6 trials were excluded because of significant differences in study design, such as larger study group and use of hydrocortisone at higher doses [START_REF] Baden | A controlled trial of hydrocortisone therapy in infants with respiratory distress syndrome[END_REF] or for a different purpose, including refractory hemodynamic failure, [START_REF] Efird | A randomizedcontrolled trial of prophylactic hydrocortisone supplementation for the prevention of hypotension in extremely low birth weight infants[END_REF][START_REF] Ng | A doubleblind, randomized, controlled study of a "stress dose" of hydrocortisone for rescue treatment of refractory hypotension in preterm infants[END_REF][START_REF] Batton | Feasibility study of early blood pressure management in extremely preterm infants[END_REF][START_REF] Hochwald | Adding hydrocortisone as first line of inotropic treatment for hypotension in very low birth weight infants[END_REF] or in combination with another treatment. [START_REF] Baden | A controlled trial of hydrocortisone therapy in infants with respiratory distress syndrome[END_REF][START_REF] Biswas | Pulmonary effects of triiodothyronine (T3) and hydrocortisone (HC) supplementation in preterm infants less than 30 weeks gestation: results of the THORN trial: Thyroid Hormone Replacement in Neonates[END_REF] The PRISMA-IPD flow diagram is depicted in the Figure (available at www.jpeds.com).

Although this individual patient data meta-analysis was not prospectively planned, the studies were quite similar in hypothesis, design, and primary outcome. As shown in Table II, patient eligibility varied slightly across the studies, as did the dose and duration of hydrocortisone therapy; however, these differences were minor and the populations were generally comparable. Table III presents summary statistics by study and treatment group for all patient characteristics and primary and secondary outcomes of interest.

Primary Outcome

Table IV presents the results of the individual patient data metaanalysis for the primary outcome and its components. Table V (available at www.jpeds.com) provides heterogeneity estimates of individual patient data meta-analysis of all outcomes unadjusted. Heterogeneity was low in most of the outcomes, with the exception of a few respiratory support items for which not all trials had data available.

Treatment with early low-dose hydrocortisone was associated with greater odds of survival without BPD at 36 weeks PMA (unadjusted OR, 1.37; 95% CI, 1.07-1.76). Findings were similar after adjustment for sex, gestational age, and antenatal steroid use (aOR, 1.45; 95% CI, 1.11-1.90; I 2 = 0%). For the components of the primary outcome, receipt of hydrocortisone was associated with significantly lower odds of BPD (aOR, 0.73; 95% CI, 0.54-0.98; I 2 = 0%), but not with a significant decrease in death before 36 weeks PMA (aOR, 0.76; 95% CI, 0.54-1.07; I 2 = 0%). However, hydrocortisone treatment was associated with a significant decrease in death before discharge (aOR, 0.70; 95% CI, 0.51-0.97; I 2 = 0%).

Secondary Outcomes

Table IV also presents the results of the individual patient data meta-analysis for all secondary outcomes. Days of ventilation, continuous positive airway pressure, and oxygen were not significantly different between the hydrocortisone and placebo groups, and BPD severity was similar in the 2 groups. There was no significant difference in exposure to oxygen at discharge. There were significantly lower odds of any medical treatment for PDA (aOR, 0.72; 95% CI, 0.56-0.93; I 2 = 0%), including both indomethacin and ibuprofen; however, there was no difference in the odds of ligation.

Exposure to hydrocortisone was associated with a significant increase in spontaneous gastrointestinal perforation (aOR, 2.50; 95% CI, 1.33-4.69; I 2 = 31.9%). However, in the absence of indomethacin cotreatment, hydrocortisone was not associated with a significant increase in gastrointestinal perforation (Table VI). Exposure to hydrocortisone also was associated with significantly increased odds of late sepsis, both bacterial and fungal (aOR, 1.34; 95% CI, 1.02-1.75; I 2 = 0%). This observed difference was not associated with increased mortality or other in-hospital adverse outcomes, or with any detectable adverse effect on 2-year neurodevelopmental outcomes in the hydrocortisone-treated group. There were no significant differences between the hydrocortisone and placebo groups for any of the remaining adverse outcomes (Table IV) of

Table II. Characteristics of 4 included studies providing individual patient data

Characteristics Sources Watterberg et al, 2004 [START_REF] Watterberg | Prophylaxis of early adrenal insufficiency to prevent bronchopulmonary dysplasia: a multicenter trial[END_REF] Peltoniemi et al, 2005 [START_REF] Peltoniemi | Pretreatment cortisol values may predict responses to hydrocortisone administration for the prevention of bronchopulmonary dysplasia in high-risk infants[END_REF] Bonsante et al, 2007 [START_REF] Bonsante | Early low-dose hydrocortisone in very preterm infants: a randomized, placebo-controlled trial[END_REF] Baud et al, 2016 If an empty cell appears under inclusion criteria, the study did not have this criterion specified. *A subgroup of infants with birth weight of 1000-1250 g had the additional requirement of supplemental oxygen and mechanical ventilation beyond 24 h despite surfactant therapy.

Table III. Patient characteristics and outcomes of 4 included studies providing individual patient data

Source Watterberg et al, 2004 [START_REF] Watterberg | Prophylaxis of early adrenal insufficiency to prevent bronchopulmonary dysplasia: a multicenter trial[END_REF] Peltoniemi et al, 2005 [START_REF] Peltoniemi | Pretreatment cortisol values may predict responses to hydrocortisone administration for the prevention of bronchopulmonary dysplasia in high-risk infants[END_REF] Bonsante et al, 2007 [START_REF] Bonsante | Early low-dose hydrocortisone in very preterm infants: a randomized, placebo-controlled trial[END_REF] For any cell with missing data, the denominator is provided within the cell. An empty cell indicates that not of the data were not available.

pneumothorax, necrotizing enterocolitis, severe intraventricular hemorrhage, cystic periventricular leukomalacia, and severe retinopathy of prematurity. There also were no significant differences in weight or head circumference at 36 weeks PMA between the 2 groups.

Planned Subgroup Analyses

Table VI summarizes the effect of treatment group on the primary study outcome as well as on late-onset sepsis and mortality before discharge, by subgroup of interest (sex, gestational age, histologic chorioamnionitis, and indomethacin exposure) adjusted for sex, gestational age, and any antenatal steroids unless the adjustment factor is used to define the subgroup of interest. Although individual odds ratios vary somewhat among subgroups, the effect is generally consistent for improvement in survival without BPD at 36 weeks PMA. Of particular interest is that the effect was similar for boys and girls. The outcomes for the subgroup chorioamnionitis showed that the incidence of late-onset sepsis was significantly increased (OR, 1.91; 95% CI, 1.18-3.08), but at the same time the incidence of survival without BPD was increased (OR, 2.01; 95% CI, 1.19-3.39) and mortality before discharge was decreased (OR, 0.43; 95% CI, 0.23-0.82).

The results of the individual patient data meta-analysis were compared with aggregate meta-analysis based on the published data of the four studies included. For the aggregate metaanalysis of the four studies, there was no significant difference in the odds of survival without BPD at 36 weeks PMA (OR, 1.40; 95% CI, 0.98-2.00).

Long-Term Outcomes

Because of differences in assessment tools used (the Griffiths Developmental Scale, Bayley Scale of Infant Development, and revised Brunet-Lezine Scale), only cerebral palsy (CP) and NDI were compared in 709 of 785 (90%) and 706 of 785 (90%) children at age 2 years, respectively, using aggregate meta-analysis. These analyses were performed based on the available data collected from the 4 RCTs included in the individual patient data meta-analysis (Table VII; available at www.jpeds.com). [START_REF] Watterberg | Growth and neurodevelopmental outcomes after early lowdose hydrocortisone treatment in extremely low birth weight infants[END_REF][START_REF] Peltoniemi | Trial of early neonatal hydrocortisone: two-year follow-up[END_REF][START_REF] Baud | Association between early low-dose hydrocortisone therapy in extremely preterm neonates and neurodevelopmental outcomes at 2 years of age[END_REF] Hydrocortisone therapy did not show a significant benefit; however, the direction of effect consistently favored the hydrocortisone-treated group for NDI (OR, 0.76; 95% CI, 0.52-1.12). For CP, individual study results were mixed in direction, but overall there was no significant relationship between hydrocortisone exposure and CP (OR, 0.95; 95% CI, 0.56-1.60).

Discussion

In this study, an individual patient data meta-analysis of 4 published RCTs showed that early low-dose hydrocortisone treatment in very preterm infants was associated with significantly increased survival without BPD, as well as a decreased need for PDA treatment and reduced mortality before discharge. The fifth published study of this therapy, a 40-patient pilot RCT for which data were no longer available, also showed a benefit; therefore, its omission does not affect the conclusions of our analysis. [START_REF] Watterberg | Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants[END_REF] Other findings of note include a significant decrease in the incidence of treatment for PDA in infants treated with hydrocortisone. We have previously reported significantly lower cortisol concentrations in infants diagnosed with PDA, as well as in infants who subsequently develop BPD, 4,[START_REF] Watterberg | Evidence of early adrenal insufficiency in babies who develop bronchopulmonary dysplasia[END_REF][START_REF] Watterberg | Impaired glucocorticoid synthesis in premature infants developing chronic lung disease[END_REF] suggesting that adrenal insufficiency may be a contributing factor to the wellknown association of PDA with BPD. [START_REF] Schena | Association between hemodynamically significant patent ductus arteriosus and bronchopulmonary dysplasia[END_REF] In addition, in the absence of indomethacin exposure, hydrocortisone therapy did not have an effect on the incidence of spontaneous gastrointestinal perforation. Studies in which ibuprofen was used as treatment for PDA also reported no effect of hydrocortisone therapy on perforation. [START_REF] Bonsante | Early low-dose hydrocortisone in very preterm infants: a randomized, placebo-controlled trial[END_REF][START_REF] Baud | Effect of early low-dose hydrocortisone on survival without bronchopulmonary dysplasia in extremely preterm infants (PREMILOC): a doubleblind, placebo-controlled, multicentre, randomised trial[END_REF][START_REF] Peltoniemi | Trial of early neonatal hydrocortisone: two-year follow-up[END_REF] Our analysis confirms an association between early lowdose hydrocortisone exposure and late-onset sepsis; nonetheless, treatment was associated with a significantly improved survival without BPD at 36 weeks PMA and survival to discharge, with no adverse effects on neurodevelopmental outcomes at 2 years. [START_REF] Watterberg | Growth and neurodevelopmental outcomes after early lowdose hydrocortisone treatment in extremely low birth weight infants[END_REF][START_REF] Peltoniemi | Trial of early neonatal hydrocortisone: two-year follow-up[END_REF][START_REF] Baud | Association between early low-dose hydrocortisone therapy in extremely preterm neonates and neurodevelopmental outcomes at 2 years of age[END_REF][START_REF] Baud | Twoyear neurodevelopmental outcomes of extremely preterm infants treated with early hydrocortisone: treatment effect according to gestational age at birth[END_REF] Follow-up of a small number of children (n = 27) at age 5-7 years suggested a correlation between early hydrocortisone treatment and later neurocognitive impairment [START_REF] Peltoniemi | Randomised trial of early neonatal hydrocortisone demonstrates potential undesired effects on neurodevelopment at preschool age[END_REF] ; however, the 2-year outcomes of 694 children enrolled in all these studies showed no adverse neurodevelopmental effects and identified possible benefits. Longer-term follow-up of previous cohort studies also have been reassuring. [START_REF] Rademaker | Long-term effects of neonatal hydrocortisone treatment for chronic lung disease on the developing brain and heart[END_REF] Children in the PREMILOC study will be assessed at age 5-7 years. [START_REF] Baud | Association between early low-dose hydrocortisone therapy in extremely preterm neonates and neurodevelopmental outcomes at 2 years of age[END_REF] Planned subgroup analyses showed that hydrocortisone appears to be similarly efficacious for both boys and girls. The direction of effect was beneficial in both gestational age strata; however, effects were more pronounced in the infants born at ≥26 weeks of gestation, consistent with the increased fragility and resistance to therapies of the most immature infants. 2 In the presence of chorioamnionitis, the incidence of late-onset sepsis was increased with hydrocortisone therapy, but a benefit was still seen in the primary outcome and in survival to discharge. Chorioamnionitis is a risk factor for late-onset sepsis [START_REF] Gowda | Late-onset neonatal sepsis: a 10-year review from North Queensland, Australia[END_REF] ; treatment with hydrocortisone may affect the immune response, yet reduce inflammatory injury and thereby improve outcomes in exposed infants, because systemic inflammation has been shown to precede clinical symptoms of the early phase of BPD. [START_REF] Leroy | A time-based analysis of inflammation in infants at risk of bronchopulmonary dysplasia[END_REF] As always, subgroup analyses, even when preplanned, should be interpreted with caution.

Only 5 trials, including the 4 included in this analysis and that reported by Watterberg et al in 1999, [START_REF] Watterberg | Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants[END_REF] were specifically designed to assess the effect of early low-dose hydrocortisone as prophylaxis of early adrenal insufficiency in very preterm infants. Four other RCTs tested the effect of early hydrocortisone in hypotensive preterm infants, [START_REF] Efird | A randomizedcontrolled trial of prophylactic hydrocortisone supplementation for the prevention of hypotension in extremely low birth weight infants[END_REF][START_REF] Ng | A doubleblind, randomized, controlled study of a "stress dose" of hydrocortisone for rescue treatment of refractory hypotension in preterm infants[END_REF][START_REF] Batton | Feasibility study of early blood pressure management in extremely preterm infants[END_REF][START_REF] Hochwald | Adding hydrocortisone as first line of inotropic treatment for hypotension in very low birth weight infants[END_REF] including 1 trial testing hydrocortisone in association with dopamine [START_REF] Hochwald | Adding hydrocortisone as first line of inotropic treatment for hypotension in very low birth weight infants[END_REF] and 1 trial investigating the effect of early triiodothyronine therapy on mortality and respiratory morbidity that included lowdose hydrocortisone as an adjunct therapy. [START_REF] Biswas | Pulmonary effects of triiodothyronine (T3) and hydrocortisone (HC) supplementation in preterm infants less than 30 weeks gestation: results of the THORN trial: Thyroid Hormone Replacement in Neonates[END_REF] Those studies found no significant benefit in survival without BPD at 36 weeks; however, the shorter treatment periods in those studies might have affected their results. We have reported lower cortisol concentrations and a decreased response to ACTH stimulation continuing beyond the first postnatal week in infants who subsequently developed BPD. [START_REF] Watterberg | Evidence of early adrenal insufficiency in babies who develop bronchopulmonary dysplasia[END_REF][START_REF] Watterberg | Impaired glucocorticoid synthesis in premature infants developing chronic lung disease[END_REF][START_REF] Watterberg | Effect of dose on response to adrenocorticotropin in extremely low birth weight infants[END_REF] Strengths of this study include access to individual patient data for 982 patients, harmonization of data and outcome definitions across studies, and very close comparability of the study populations and the therapeutic intervention. Our results differ from those of an aggregate meta-analysis of the 4 studies showing no difference in the odds of survival without BPD at 36 weeks PMA, demonstrating that an individual patient data meta-analysis can improve the ability to address important confounders at the individual patient level.

Limitations of the study include the loss of 40 patients in the original pilot study [START_REF] Watterberg | Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants[END_REF] and loss of accuracy regarding the time of first dose. In addition, exposure to indomethacin as a confounding variable was not balanced across all studies.

In conclusion, this individual patient data meta-analysis shows that early, low-dose hydrocortisone therapy provides significant benefits in survival without BPD, PDA closure, and survival to discharge in very preterm infants. Increases in intestinal perforation and late-onset sepsis, 2 reported adverse effects of this hydrocortisone treatment, did not appear to negate the overall benefits of hydrocortisone in this population.

  [START_REF] Baud | Effect of early low-dose hydrocortisone on survival without bronchopulmonary dysplasia in extremely preterm infants (PREMILOC): a doubleblind, placebo-controlled, multicentre, randomised trial[END_REF]

	Country	US	Finland	Italy	France
	Funding source	National Institute of Child Health	Foundation for Pediatric	University of Bari	Public Hospitals of Paris
		and Human Development	Research
	Ethics Committee review	Yes	Yes	Yes	Yes
	Parental informed consent	Yes	Yes	Yes	Yes
	Loss to follow-up for primary	3/360 (<1)	None	None	2/523 (<1)
	outcome, n/N (%)
	Design	Double-blind RCT	Double-blind RCT	Double-blind RCT	Double-blind RCT
	Inclusion criteria
	Gestational age, wk		23 0/7 -29 6/7	24 0/7 -29 6/7	24 0/7 -27 6/7
	Birth weight, g	500-999 g	501-1250	500-1249
	Respiratory status	Need for mechanical ventilation	Need for mechanical	Need for mechanical ventilation
		at study entry	ventilation before 24 h	after rescue surfactant
			of life*
	Enrolled	Between 12 and 48 h postnatal	Before 36 h	Before 48 h	Before 24 h
		age
	Duration of exposure	1 mg/kg/d for 12 d, then	10 d tapered from 2.0 to
		0.5 mg/kg/d for 3 d	0.75 mg/kg/d

1 mg/kg/d divided into 2 doses/d for 9 d, then 0.5 mg/kg/d for 3 d 1 mg/kg/d divided into 2 doses/d for 7 d, then 0.5 mg/kg/d for 3 d

Table IV . Results of individual patient data meta-analysis of all outcomes adjusted for sex, gestational age, and any an- tenatal steroids

 IV

				OR or Mean	95% CI of OR or
	Outcomes	Hydrocortisone	Placebo	Difference	Mean Difference	P Value
	Survival without BPD at 36 wk PMA, n/N (%)	258/484 (53.3)	225/495 (45.5)	1.45	1.11-1.90	.007
	Death before 36 wk PMA, n/N (%)	79/484 (16.3)	98/495 (19.8)	0.76	0.54-1.07	.12
	BPD at 36 wk PMA, n/N (%)	147/405 (36.3)	172/397 (43.3)	0.73	0.54-0.98	.038
	Death before discharge, n/N (%)	85/485 (17.5)	112/497 (22.5)	0.70	0.51-0.97	.0327
	Weight at 36 wk PMA, g, mean	2036 (n = 390)	2061 (n = 379)	-24.11	-71.36 to 23.14	.32
	Head circumference at 36 wk, cm, mean	31.0 (n = 346)	30.9 (n = 337)	0.19	-0.05 to 0.42	.12
	Respiratory support
	Days of ventilation, mean	32.3 (n = 226)	31.7 (n = 221)	-0.63	-7.28 to 6.01	.85
	Days of CPAP, mean	17.2 (n = 47)	17.7 (n = 49)	-0.12	-5.78 to 5.55	.97
	Days of oxygen, mean	74.1 (n = 226)	75.2 (n = 221)	-2.17	-12.07 to 7.73	.67
	Oxygen at discharge, n/N (%)	73/401 (18.2)	75/386 (19.4)	0.92	0.64-1.33	.65
	Open-label steroid use, n/N (%)	195/485 (40.2)	211/497 (42.5)	0.90	0.70-1.17	.44
	Pneumothorax, n/N (%)	31/485 (6.39)	32/497 (6.44)	0.98	0.58-1.64	.93
	Insulin treatment, n/N (%)	204/485 (42.1)	194/497 (39.0)	1.12	0.86-1.45	.42
	Medical treatment for PDA (indomethacin or ibuprofen), n/N (%)	202/485 (41.7)	246/497 (49.5)	0.72	0.56-0.93	.012
	Any prophylactic indomethacin, n/N (%)	61/485 (12.6)	63/497 (12.7)	0.96	0.65-1.41	.83
	Surgical ligation, n/N (%)	68/485 (14.0)	83/497 (16.7)	0.80	0.56-1.14	.21
	Necrotizing enterocolitis, n/N (%)	27/485 (5.57)	29/497 (5.84)	0.95	0.55-1.63	.85
	Spontaneous gastrointestinal perforation	35/483 (7.25)	15/497 (3.02)	2.50	1.33-4.69	.004
	Late-onset sepsis (bacterial/fungal), n/N (%)	176/485 (36.3)	148/497 (29.8)	1.34	1.02-1.75	.0357
	Severe IVH (grade 3-4), n/N (%)	76/477 (15.9)	71/493 (14.4)	1.10	0.76-1.59	.60
	Cystic PVL, n/N (%)	22/447 (4.92)	25/459 (5.45)	0.89	0.49-1.60	.69
	Severe ROP, n/N (%)	50/458 (10.9)	54/467 (11.6)	0.92	0.59-1.45	.72

Table VI . Subgroup analyses of primary outcome and other selected outcomes adjusted for sex, gestational age, and any antenatal steroids unless the adjustment factor is used to define the subgroup

 VI

	Outcomes	OR	95% CI	P value
	Survival to 36 wk without BPD
	Male	1.40	0.97-2.02	.074
	Female	1.52	1.02-2.26	.038
	Gestational age <26 wk	1.38	0.91-2.09	.13
	Gestational age ≥26 wk	1.52	1.07-2.17	.020
	No chorioamnionitis	1.40	0.97-2.02	.074
	Chorioamnionitis	2.01	1.19-3.39	.009
	Death before discharge
	Male	0.73	0.47-1.14	.17
	Female	0.66	0.41-1.07	.094
	Gestational age <26 wk	0.87	0.58-1.32	.53
	Gestational age ≥26 wk	0.46	0.26-0.82	.008
	No chorioamnionitis	0.71	0.44-1.15	.16
	Chorioamnionitis	0.43	0.23-0.82	.010
	Late-onset sepsis
	Male	1.41	0.96-2.05	.076
	Female	1.23	0.83-1.83	.29
	Gestational age <26 wk	1.60	1.08-2.37	.019
	Gestational age ≥26 wk	1.14	0.78-1.65	.50
	No chorioamnionitis	1.06	0.72-1.55	.77
	Chorioamnionitis	1.91	1.18-3.08	.009
	Spontaneous gastrointestinal perforation
	No indomethacin	1.52	0.73-3.15	.26
	Indomethacin	9.37	2.02-43.49	.004

  ■

	■■ 2018						Volume ■■ ORIGINAL ARTICLES
				Peltoneimi et al,		Watterberg et al,	Baud et al,
				2005 2		2004 3	2016 4
	Survival without BPD	Y (clinical)		Y (clinical)		Y (clinical, physiological) Y (physiological)	Binary (Y/N clinical and
							Y/N physiological)
	Severity at 36 wk	FiO2 >30%/vent;		FiO2/CPAP/vent		FiO2/MAP/vent/CPAP	FiO2/MAP/vent/CPAP	Categorical (moderate,
		Home on O2					O2 <30% at 36 wk;
							severe, >30% or any
							positive pressure)
	Days of ventilation/CPAP/	Y/Y/Y		Y/Y/Y		Y/N/Y	N/N/N	Quantitative
	oxygen
	Oxygen at discharge	Y		Y		Y	N	Binary
	Pneumothorax/pulmonary	Y/N		Y/N		Y/Y	Y/N	Binary/binary
	interstitial emphysema
	PDA treatment	Y		Y (indomethacin/ibuprofen/	Y (indomethacin/ligation/	Y (iIbuprofen/ligation) Binary (any treatment Y/N)
				ligation/other)		other)
	Insulin treatment	Y		Y		Y	Y	Binary
	Necrotizing enterocolitis	Y		Y		Y (stage)	Y	Binary
	Gastrointestinal perforation/	Y/Y		Y/Y		Y/Y	Y (ibuprofen)	Binary/binary
	indomethacin
	Late-onset sepsis (bacterial	Y		Y		Y	Y	Binary/binary
	or fungal)
	IVH grade 3-4/PVL	Y/Y		Y/Y		Y/Y	Y severe/Y	Binary (severe Y/N)/binary
	ROP grade	Y		Y		Y	Y (severe)	Binary (severe Y/N)
	Death before discharge	Y		Y		Y	Y	Binary
	36 wk weight and head	Y z-scores		Y actual values		Y actual values; length	Y actual values	z-scores
	circumference					not available
	Open-label steroid use/type	Y (dex BPD		Y (+ inhaled)		Y (dex all)	Y (Y, inhaled)	Binary (use Y/N); binary
	of steroid use	hydrocortisone-BP)				(inhaled Y/N); binary
							(systemic Y/N)
	Any indomethacin exposure Inbuprofen prophylaxis	Indomethacin Rx		Prenatal or subsequent	Ibuprofen	Any exposure (Y/N)
		for PDA				indomethacin Rx
				Peltoneimi et al,		Watterberg et al,	Baud et al,
				2005 2		2004 3	2016 4
	Birth weight	Y	Y		Y		Y	Quantitative (g)
	Gestational age	Y	Y		Y		Y	Quantitative (wk)
	Sex	Y	Y		Y		Y	Binary
	Apgar 1-min score	Y	Y		Y		N	Quantitative
	Apgar 5-min score	Y	Y		Y		Y	Quantitative
	Race/ethnicity	Y	All white	Y		Y	Categorical: 1, white; 2, black;
	Antenatal steroids	Y (any/full)	Y (any/full/partial)	3, Asian; 4, other; 9, Submitted for publication May 17, 2018; last revision received Oct 1, 2018; unknown accepted Oct 3, 2018 Y (detailed) Y Binary (any vs none)
	Rupture of membranes	N	>24 h Y/N	Y (h)	Reprint requests: Olivier Baud, MD, PhD, Division of Neonatology, Department Y (>24 h) Binary (>24 h Y/N)
	Vaginal/Cesarean delivery Y	Y		Y	of Pediatrics, University Hospitals, Rue Willy-Donzé 6, 1205 Genève, Geneva, Y Binary
	Chorioamnionitis	Y (histologic)	Switzerland. E-mail: olivier.baud@hcuge.ch Y (histologic)/Y (clinical) Y (histologic detailed, clinical) Y (histologic/ clinical) Binary (histologic Y/N); binary
							(clinical Y/N)
	Preeclampsia	Y	Y		Y		Y	Binary
	Multiple birth	Y (individual random) Y (individual random)	Y (twin, random together)	Y (how random?)	Binary (Y/N)
	Inborn/outborn	Y (all inborn)	Y (all inborn)	Y		Y (inborn)	Binary
	Intubated at entry	Y (all)	Y (all)	Y (all)		Y (both)	Binary
	Age at entry	Y (at first dose)	Y (at first dose)	Y (at first dose)	Y (at first dose)	Quantitative, h
	CRIB score	Y	N		Y		N	Quantitative
	Blood pressure
	Daily	N	Y		Y		Y	Daily systolic
	Inotropic therapy at	Y	Y		Y		Y	Binary (any Y/N)
	study entry
	7.e1						Shaffer et al

CRIB, Clinical Risk Index for Babies.

THE JOURNAL OF PEDIATRICS • www.jpeds.com FLA 5.5.0 DTD ■ YMPD10344_proof ■ November 6, 2018 CPAP, continuous positive airway pressure; IVH, intraventricular hemorrhage; MAP, mean airway pressure; PVL, periventricular leukomalacia; ROP, retinopathy of prematurity.

7.e2

Effect of Prophylaxis for Early Adrenal Insufficiency Using Low-Dose Hydrocortisone in Very Preterm Infants: An Individual Patient Data Meta-Analysis FLA 5.5.0 DTD ■ YMPD10344_proof ■ November 6, 2018

Shaffer et alFLA 5.5.0 DTD ■ YMPD10344_proof ■ November 6, 2018

Effect of Prophylaxis for Early Adrenal Insufficiency Using Low-Dose Hydrocortisone in Very Preterm Infants: An Individual Patient Data Meta-Analysis FLA 5.5.0 DTD ■ YMPD10344_proof ■ November 6, 2018

FLA 5.5.0 DTD ■ YMPD10344_proof ■ November 6, 2018

Effect of Prophylaxis for Early Adrenal Insufficiency Using Low-Dose Hydrocortisone in Very Preterm Infants: An Individual Patient Data Meta-Analysis FLA 5.5.0 DTD ■ YMPD10344_proof ■ November

[START_REF] Doyle | Early (<8 days) postnatal corticosteroids for preventing chronic lung disease in preterm infants[END_REF] 2018

Shaffer et alFLA 5.5.0 DTD ■ YMPD10344_proof ■ November 6, 2018

Supported by a research grant from the French Ministry of Health and funded by the Département de la role in the data analysis and reporting. The authors declare no conflicts of interest.

Availability Scale Bonsante et al, 2007 1 Availability Scale Bonsante et al, 2007 1

Appendix

Brief Statistical Analysis Plan for Hydrocortisone Meta-Analysis

Overview. Five RCTs that tested low-dose hydrocortisone during the first postnatal days with the primary outcome of improving survival without BPD have been completed and published. Individual patient data are no longer available for the first trial of 40 patients. We will conduct an individual patient data meta-analysis of the remaining 4 trials. Although this individual patient data meta-analysis was not prospectively planned, the studies are quite similar in hypothesis, design, and primary outcome. Patient eligibility varies slightly across the studies, as do the dose and duration of hydrocortisone therapy; however, these differences are minor, and the populations generally overlap. Data Analysis. Descriptive data will be presented for each study when available, and overall, including the variables shown in Appendix Table I. We will take a 1-step approach to individual patient data meta-analysis using generalizations of logistic regression models for binary outcomes and linear regression models for quantitative outcomes based on generalized linear mixed models with Kenward-Rogers approximation of degrees of freedom. We will account for clustering of patients within different studies by specifying a random intercept term, assuming that the baseline is drawn at random from a normal distribution. We will consider treatment a random effect and adjustment factors random effects. We will assume different residual variances for each study because patient eligibility varies slightly from study to study, as well as dose and duration of hydrocortisone therapy. Simplifications of modeling assumptions, such as fixed effects in place of random effects and a common residual variance, will be considered if models fail to converge.

The primary outcome of interest is the binary variable survival without BPD at 36 weeks, and the primary predictor is receipt of hydrocortisone treatment. Primary and secondary outcomes of interest are summarized in Appendix Table II. Adjustment variables included in all models are birth weight, sex, gestational age, antenatal steroids, and age at first dose.

Similar subgroup analyses will be conducted for the following groups: boys/girls, histologic chorioamnionitis, gestational age strata, and receipt of indomethacin.

Appendix Table I. Descriptive variables to be summarized overall and by study

Variable

Appendix Table II. Primary and secondary outcomes

Outcomes

Table V. Heterogeneity estimates of individual patient data meta-analysis of all outcomes unadjusted

Table VII. Characteristics of the studies in the long-term outcomes meta-analysis

Characteristics

Watterberg et al, 2007 [START_REF] Watterberg | Growth and neurodevelopmental outcomes after early lowdose hydrocortisone treatment in extremely low birth weight infants[END_REF] Peltoniemi et al, 2009 [START_REF] Peltoniemi | Trial of early neonatal hydrocortisone: two-year follow-up[END_REF] Bonsante et al, 2007 follow-up study as reported in Peltoniemi et al, 2009 [START_REF] Peltoniemi | Trial of early neonatal hydrocortisone: two-year follow-up[END_REF] Baud et al, 2017


Frederic Denis
email: frederic.denis@chlcdijon.fr

Gérard Milleret

Thomas Wallenhorst

Maud Carpentier

Nathalie Rude

Benoit Trojak

LA SANTE BUCCO-DENTAIRE DES PATIENTS SCHIZOPHRENES : UNE ETUDE FRANÇAISE TRANSVERSALE MULTICENTRIQUE ORAL HEALTH IN SCHIZOPHRENIA PATIENTS: A FRENCH MULTICENTER CROSS-SECTIONAL STUDY

Objective:

The aim of this study was to assess the oral health status of a sample of patients with schizophrenia (PWS) in Côte d'Or region (France) and to determine the factors associated with dental status.

Material and Methods:

A multicenter cross-sectional descriptive study was performed. PWS were selected using a random stratified method. Dental status was investigated using the Decayed, Missing, or Filled Teeth (DMFT) index, based on 32 teeth. The Simplified Oral Hygiene Index (OHI-S), the Xerostomia Index (XI) and the Global Oral Health Assessment Index (GOHAI) for Oral Health related Quality of Life (OHrQoL) were recorded.

Results: Among the 302 persons identified, 109 were included (acceptance rate was 49.1%). Of these, 61.5% were men. The mean age was 46.8 years ±12.0 years. The majority (78%) had completed a secondary education (SE). The mean duration of mental illness (DMI) was 17.9±9.4 years, and 55.5% were taking drugs.

The mean DMFT was 16.6±8.1. There was a significant relationship between age, SE, oral health variables, medical conditions and OHrQoL.

Conclusions: PWS describe a poor OHrQoL. The dental and hygiene index scores are poor. We observed a lack of brushing and of dental visit for these persons and a gap in oral health between PWS and general population.

La santé bucco

-dentaire des patients schizophrènes: une étude française transversale multicentrique Objectif : Le but de cette étude était d'évaluer l'état de santé bucco-dentaire d'un échantillon de patients atteints de schizophrénie (PS) en Côte d'Or (France) et de préciser les facteurs associés à la santé bucco-dentaire. Matériel et méthodes : Une étude descriptive transversale multicentrique a été réalisée. Les PS ont été sélectionnés en utilisant une méthode stratifiée aléatoire. L'état de santé bucco-dentaire a été étudié en utilisant l'indice carieux (CAO) qui fait la somme des dents cariées, (C), absentes (O) et obturées (O) basé sur 32 dents, l'indice d'hygiène buccale simplifié (OHI-S), l'indice de xérostomie (XI) et un indice global d'évaluation de la qualité de vie lié à la santé bucco-dentaire, le Global Oral Health Assessment Index (GOHAI).

Résultats : Parmi les 302 personnes identifiées pour cette étude, 109 ont été incluses (taux d'acceptation de 49,1%). Parmi celles-ci, 61,5% étaient des hommes. L'âge moyen était de 46,8 ans ± 12,0 ans. La majorité des PS (78%) avaient terminé leurs études secondaires (ES).

La durée moyenne de la maladie mentale était de 17,9 ± 9,4 ans et 55,5% prenaient des médicaments.

Le CAO moyen était de 16,6 ± 8,1. Il y avait une association significative l'âge, les ES, les variables de santé bucco-dentaire, les conditions médicales et la qualité de vie liée à la santé bucco-dentaire.

Conclusions : Les PS ont une mauvaise qualité de vie liée à leur santé bucco-dentaire. Les scores de l'indice dentaire et d'hygiène sont de mauvaises qualités. Nous avons observé un manque d'hygiène bucco-dentaire et de fréquentation des dentistes par ces populations ainsi qu'un écart entre la santé bucco-dentaire des PS et la population générale.

Schizophrenia is a severe mental disorder that affects between 0.7% and 1% of the world's population, including 600,000 people in France [START_REF] Jablensky | Epidemiology of schizophrenia: the global burden of disease and disability[END_REF]. Patients with schizophrenia (PWS) have high mortality (their life expectancy is reduced by 20%) and morbidity [START_REF] Wildgust | The paradox of premature mortality in schizophrenia: new research questions[END_REF]. Among physical comorbidities in PWS, poor oral health contributes to the overall poor health of these patients [START_REF] Kisely | A systematic review and meta-analysis of the association between poor oral health and severe mental illness[END_REF]. At various degrees of intensity, the symptoms of schizophrenia lead to disturbances in the progression of thought, errors in contextual analysis and errors of logic. PWS often do not recognize their health needs and delay seeking advice or treatment [START_REF] Grisso | Comparison of standards for assessing patients' capacities to make treatment decisions[END_REF]. This issue occurs in all related physical disorders, but because of a lack of analysis due to this disease, PWS do not recognize their condition or make the right decisions to solve problems independently [START_REF] Grisso | Comparison of standards for assessing patients' capacities to make treatment decisions[END_REF]. Difficult relationships with professional caregivers (fear of mental illness, lack of training) and the health system in general (difficulties in access to private practices, environment, cost, etc.) are additional obstacles that contribute to deficient physical care [START_REF] Kisely | Inequitable access for mentally ill patients to some medically necessary procedures[END_REF].

One of the most visible elements of poor oral health in PWS is a large number of missing or decayed teeth (leading to pain, infection, masticatory and digestive problems) [START_REF] Rai | Systemic effect of oral disease[END_REF].

Dental caries and periodontal or infectious diseases and metabolic disturbances (diabetes, obesity, xerostomia) induced by antipsychotic treatments, poor diet and lifestyle behaviours (diet rich in sugars, use of psychoactive substances such as tobacco, and inadequate oral hygiene) all combine to lead to poor health [START_REF] Arnaiz | Oral health and the symptoms of schizophrenia[END_REF][START_REF] Thomas | Factors which influence the oral condition of chronic schizophrenia patients[END_REF].

Generally, negative symptoms, age, duration of mental illness, and low socioeconomic and cultural status are risk factors common to tooth decay [START_REF] Thomas | Factors which influence the oral condition of chronic schizophrenia patients[END_REF][START_REF] Gurbuz | Oral health and treatment needs of institutionalized chronic psychiatric patients in Istanbul, Turkey[END_REF][START_REF] Mccreadie | The dental health of people with schizophrenia[END_REF]. These factors are aggravated by stigmatization and discrimination, which is why PWS receive less attention than others in terms of their physical health [START_REF] Moore | Promoting physical health for people with schizophrenia by reducing disparities in medical and dental care[END_REF]. Poor oral health can also affect quality of life through the social and psychological impact of the deterioration in smile aesthetics on self-esteem and self-confidence [START_REF] Wey | The oral health of people with chronic schizophrenia: a neglected public health burden[END_REF]. In France, medical and dental care costs are partly covered for conservative and surgical dental care (70%), prosthetics and orthodontics treatment (30 to 50%) by national health insurance and complementary health insurance or by PUMa (Protection Universelle Maladie) or by CMUc (Couverture Médicale Universelle Complémentaire) for people with a low income level (below 8723€ per year in 2017). The PUMa and CMUc are free [START_REF]Le portail du service public de la Sécurité social[END_REF].

International data confirm that oral health is poor in people with psychiatric disorders.

Dental caries and periodontal measurement indexes are often twice the level found in the general population [START_REF] Thomas | Factors which influence the oral condition of chronic schizophrenia patients[END_REF][START_REF] Gurbuz | Oral health and treatment needs of institutionalized chronic psychiatric patients in Istanbul, Turkey[END_REF][START_REF] Mccreadie | The dental health of people with schizophrenia[END_REF]. These results, however, must be clarified. Indeed, in many studies [START_REF] Kisely | A systematic review and meta-analysis of the association between poor oral health and severe mental illness[END_REF][START_REF] Bertaud-Gounot | Oral health status and treatment needs among psychiatric inpatients in Rennes, France: a cross-sectional study[END_REF], schizophrenic populations are rarely differentiated from other populations (bipolar disease, major depression) even though they present with distinctive clinical features. The perception of Oral Health related Quality of Life (OHrQoL) was found to be dependent on the particular psychiatric diagnosis [START_REF] Bertaud-Gounot | Oral health status and treatment needs among psychiatric inpatients in Rennes, France: a cross-sectional study[END_REF]. Additionally, most studies have been conducted with in-patients (most marked by the disease) even though most psychiatric patients today live in the community and are managed in psychiatric outpatient units [START_REF] Kisely | A systematic review and meta-analysis of the association between poor oral health and severe mental illness[END_REF].

This population is at greater risk of dental disease, and data on this health problem need to be collected in France. Knowledge of the determinants of oral health in PWS is important to assess preventive oral health needs in this population.

The aim of this study was to assess demographic (age, sex), socioeconomic (education, residential area), lifestyle (tooth brushing frequency, dentist visit frequency and smoking habits), medical condition (number of antipsychotic treatments, illness duration, body mass index, diabetes) and dental condition (oral hygiene, dental caries, xerostomia, and OHrQoL) predictors of oral health of a sample of PWS.

METHODS

Design

Sample strategy and sample size calculation

From a population of 1,868 PWS in Côte d'Or recorded in the French medico-administrative database PMSI (Programme de Médicalisation des Système d'Information) of the three hospitals of Cote d'Or, PWS were selected using a random stratified method according to age, sex and hospital, with a probability proportional to the total number of persons in each stratum. Assuming an average standard deviation of 8 [START_REF] Bertaud-Gounot | Oral health status and treatment needs among psychiatric inpatients in Rennes, France: a cross-sectional study[END_REF] and a precision of 1.5, 112 subjects were required. Assuming that 20% of subjects could not be reached and a response rate of 50%, 300 subjects were required. Eligible patients were informed by letter and contacted by phone within 2 weeks for out-patients and by the medical staff for in-patients.

Patients were included if they were over 18 years of age with a diagnosis of schizophrenia (Diagnosis: F20-F29 PMSI) according to the International Classification of Diseases 10 th Revision: ICD-10 [START_REF][END_REF] and were psychically stable. The following patients were excluded: pregnant or breast-feeding women, persons not stabilized from a psychiatric viewpoint, persons experiencing an acute psychiatric episode, persons who could not understand or had poor understanding of French. We also excluded persons who had not updated their social rights to cover the cost of medical and dental care. All the procedures were part of standard care as patients had regular oral and physical examinations.

Study procedure

Oral examinations / Dental condition

A single investigator, a dentist, interviewed and clinically examined all the participants using portable dental equipment in the hospital nearest to their place of residence. Pre-packaged and disposable instruments were used. Rating was calibrated in comparison with repeated examinations of a separate pilot sample. Kappa scores of 0.9 for inter-rater agreement were achieved.

Caries experience was assessed using the dentinal (D3) level and the Decayed, Missing, or Filled Teeth (DMFT) index based on 32 teeth and was calculated using WHO (World Health Organization) criteria [START_REF]Oral health surveys: basic methods[END_REF]. Dental plaque and calculi were evaluated on six teeth using the Simplified Oral Hygiene Index (OHI-S). The OHI-S is the sum of the plaque index and the calculus index. The mean OHI-S was computed, and the scores were classified into four levels: Excellent (0); Good (0.1-1.2); Fair (1.3-3.0); Poor (3.1-6.0) [START_REF] Greene | The simplified oral hygiene index[END_REF].

Oral Health Assessment

Each participant was asked to complete the 'General Oral Health Assessment Index' (GOHAI) [START_REF] Atchison | Development of the geriatric oral health assessment index[END_REF] and the 'Xerostomia Inventory' (XI) [START_REF] Thomson | Shortening the xerostomia inventory[END_REF] questionnaires and answer other questions related to their socio-economic status (SES) (age, sex, level of education, residential area) and data relating to oral health (tooth brushing frequency, last dental visit).

The Geriatric Oral Health Assessment Index (GOHAI)

The GOHAI is a self-assessment oral health index. A French version has been validated with PWS [START_REF] Denis | Psychometric characteristics of the "General Oral Health Assessment Index (GOHAI)" in a French representative sample of patients with schizophrenia[END_REF]. The questionnaire consists of 12 questions that are phrased positively or negatively. The answer modalities are based on a Likert scale with scores ranging from 1 to 5. The GOHAI score is the sum of the answers to the 12 questions. According to Atchison and Dolan, a score of 57 to 60 is regarded as high and corresponds to a satisfactory oral health quality of life. A score from 51 to 56 is regarded as average, and a score of 50 or less is regarded as a low score, reflecting a poor oral health quality of life [START_REF] Atchison | Development of the geriatric oral health assessment index[END_REF].

The Xerostomia Inventory (XI)

The Xerostomia Inventory (XI) is a self-assessment questionnaire. It is an 11-item summated rating scale that combines the responses to 11 individual items into a single continuous scale score, which represents the severity of chronic xerostomia; higher scores represent more severe symptoms. Respondents are asked to choose one of five responses ('Never', scoring 1; 'Hardly ever', 2; 'Occasionally', 3; 'Fairly often', 4; and 'Very often', 5). Each individual's responses are scored and summed to give a single XI score [START_REF] Thomson | Shortening the xerostomia inventory[END_REF].

General health-related information

Demographic and medical variables were extracted from institutional medical records: age, sex, presence of diabetes, prescribed drugs at the time of the examination, duration of mental illness (DMI) [START_REF] Lesh | Cognitive control deficits in schizophrenia: mechanisms and meaning[END_REF] and Body Mass Index (BMI) [START_REF]Waist Circumference and Waist-Hip Ratio: Report of a WHO Expert Consultation[END_REF]. The BMI was stratified into four groups: underweight (BMI<18.5), normal weight (BMI=18.5-25.0), overweight (BMI=25.0-30.0), and obese (BMI>30.0) based on cut-offs for the European population [START_REF]Waist Circumference and Waist-Hip Ratio: Report of a WHO Expert Consultation[END_REF]. Additional variables, namely, the level of education, residential area, smoking habits, tooth brushing frequency, and last dental visit, were collected directly by questioning the patient. Psychiatric status was determined by a clinical assessment conducted by the treating healthcare team.

Data analysis

Categorical variables were presented as percentages and continuous variables as the means with standard deviations (SD). Associations between the DMFT index or its components (dependent variables) and socio-demographic, lifestyle, clinical, treatment and oral health assessments were first analysed using analysis of variance and Pearson's correlation tests.

Dental caries is an irreversible condition. Assessment of dental status by DMFT index to evaluate the state of oral health based on the presence of decayed teeth (D) which is expressed in a need for care and missing teeth (M) which justifies prosthetic rehabilitating.

The presence of filled teeth (F) reflected the state of care. Tooth decay is directly dependent on dental hygiene (OHI-S). Poor oral health can may also affect the quality of life, OHrQOL and oral health is inseparable from general health [START_REF] Moore | Promoting physical health for people with schizophrenia by reducing disparities in medical and dental care[END_REF].

A multiple linear regression model was used in subjects who had completed both the OHI-S to find correlates of the DMFT score. A stepwise procedure was used to optimize the model's fit. Variables which reached a significance level of p<0.2 in the univariate analysis were included as independent variables and excluded from the final model during the backward elimination process, according to the amount of variance explained. The final model incorporated the standardized β coefficients, which represent a change in the standard deviation of the dependent variable (DMFT score) resulting from a change of one standard deviation in the various independent variables. Age, sex and centre were systematically forced into the multivariable model. Collinearity was investigated using collinearity diagnostic procedures. The statistical significance level was set at p<0.05 for a two-sided test.

All statistical analyses were performed using SAS statistical software (version 9.1, SAS Institute, Cary, N.C.).

Ethics

This study was approved by the Committee for the Protection of Persons (CPP) I of Eastern France (registration number: 2014-A00358-39). After providing participants with a complete description of the study, written informed consent was obtained from each participant (or from their legal guardians for persons under guardianship).The study was registered on www.ClinicalTrials.gov under number NCT02167724.

RESULTS

Study sample

A study population of 302 persons was selected from the administrative databases. Of the randomized subjects, 22% (n=67) could not be reached (14 had died, 52 had moved but were still registered at their previous address, one did not have schizophrenia). Among those contacted (n=235), 113 refused to participate in the study, and 13 were unable to cooperate (Figure 1). The acceptance rate was thus 46.4% (n=109 subjects). Table 1 shows the sociodemographic, lifestyle, clinical, treatment and oral health assessments of the sample.

Demographics

Among the 109 examined, 61.5% were men and 38.5% were women. Age ranged from 25 to 75 years (mean age 46.8±12.0). The mean age of the non included patients was 48.0±14.3 (n=193). In this group 35.8% were women and 64.2% were men. No significant differences were found between the two groups in terms of age (p = 0.40) and sex (p = 0. 619). The proportions of the sample by sex, age and centre reflected the proportions of the overall study population by sex, age and centre.

Medical condition

The mean number of antipsychotic treatments was 1.92 ± 1.0 and that for other treatments was 2.3±2.2. The mean DMI was 17.9 years ±9.4 and was between (5-19 years) for 54 (50.5%) patients. Thirty-two (29.6%) had a BMI>30, 20.9% of men and 43.9% of women, and 12 patients had non-insulin-dependent diabetes (11.3%).

Dental condition

The DMFT was 16.6±8.1 with a mean of 2.0±2.8 decayed teeth, a mean of 7.7 missing teeth ±9.2 and a mean of 6.9±5.5 filled teeth (Table 2 indicates tooth decay index by age). Thirteen patients were totally edentate (11.9%), eleven (84.6%) had full dentures but only five (38.5%) of these patients reported being satisfied with their dentures. Among the 96 dentate patients, the OHI-S index was excellent for only 0.9%, good for 21.1%, fair for 44% and poor for 22%. The mean GOHAI was 42.2±7.7 with poor oral health quality of life for 91% of the sample, and the mean Xerostomia Inventory score was 22.8±7.9.

Factors associated with DMFT score

The results of the univariate and multivariate analyses are presented in Tables 3,4.

In the univariate analysis, the DMFT score was positively associated to age (Pearson ρ=0.5, p<0.0001), XI score (ρ=0.4, p<0.0001) and OHI-S score (ρ=0.4, p=0.002). It was negatively associated to the GOHAI score (ρ=-0.4, p<0.0001). The mean DMFT score was significantly higher in subjects with a low level of school education, polypharmacy, DMI ≥ 20 years and infrequent tooth brushing than in subjects without any of these characteristics.

There was no significant association between the mean DMFT score and gender, residence, last dental visit, smoking, BMI and diabetic status.

As the XI score correlated with the GOHAI score (ρ=-0.7, p<0.0001) and as 32% of XI scores were missing, we decided to exclude the XI score from the multivariate analyses.

In subjects who had completed the OHI-S, a low DMFT score remained associated with age (β=0.256, p<0.001), the OHI-S score (β=1.786, p=0.002) and the GOHAI score (β=-0.225, p=0.021), but not with education, tooth brushing habits, polypharmacy and DMI (Table 4).

DISCUSSION

The study highlights that OHrQOL of PWS was low (mean GOHAI was 42.2 ± 7.7). The dental and hygiene index score was poor and we observed a lack of brushing and of dental visit in this population.

Dental condition

There are very few data available in the literature on the general population in France, and available data are old (1997)(1998)(1999). DMFT in 35-44-year-olds was 14.5, and these patients had an average of 1.2 decayed teeth. The number of missing teeth was 3.0, and the number of filled teeth was 10.4 [START_REF] Hescot | Oral health in 35-44 year old adults in France[END_REF]. In comparison, DMFT in 35-44-year-olds in our study was similar (14.1 vs 14.5), whereas the average number of decayed (2.5 vs 1.2) and missing teeth (4 vs 3) was higher, and the average number of filled teeth was lower (10.4 vs 7.5). Significantly more PWS had general extractions than did the general population (96% vs. 80%) [START_REF] Mccreadie | The dental health of people with schizophrenia[END_REF]. The number of filled teeth may be a reflection of the treatments available to patients. In our population, although free dental treatment is available, there was a difference between the number of filled teeth in the general population in France and the number in PWS in Côte d'Or. Actually, the difference between PWS and the general French population might be even greater. Available data regarding oral health in the French population are old (1997 and 1999) [START_REF] Hescot | Oral health in 35-44 year old adults in France[END_REF]. As in many industrialized countries, oral health in the French population might have improved over time.

Compared to the literature on PWS (Table 5), mean DMFT (16.6±8.1) of the study group with mean age 46.8±12.0 was lower than that in developing middle-income countries as classified by the World Bank [START_REF]Country and Lending Groups[END_REF] such as Malaysia (20.5±9.9) with a mean age of 54.8±16.0 [START_REF] Wey | The oral health of people with chronic schizophrenia: a neglected public health burden[END_REF] or Iran (19.4±7.7) with a mean age of 38.8±10.4 [START_REF] Nikfarjam | Oral health status in three long term care units of schizophrenic patients in Chaharmahal-Bakhtiari province, Iran[END_REF] for hospitalized patients.

Compared to high-income countries such as France, we observed that DMFT in Spain was lower (13.5±7.2) with a mean age of 40±11.2 [START_REF] Arnaiz | Oral health and the symptoms of schizophrenia[END_REF] and in Sweden (10.4±6.2) with a mean age between 20 and 65 years old for outpatients [START_REF] Persson | Monitoring oral health and dental attendance in an outpatient psychiatric population[END_REF]. In the present study, DMFT in France was lower compared to hospitalized patients in Japan (18.8±7.0) with a mean age of 55.6±13.4 [START_REF] Tani | Dental conditions in inpatients with schizophrenia: a large-scale multi-site survey[END_REF] and Greece (23.3±8.3) with a mean age of 50.3±13.0 [START_REF] Thomas | Factors which influence the oral condition of chronic schizophrenia patients[END_REF]. In the study by Bertaud-Gounot et al. [START_REF] Bertaud-Gounot | Oral health status and treatment needs among psychiatric inpatients in Rennes, France: a cross-sectional study[END_REF] the DMFT was 15.8±8.8 with a mean age of 46.9±17.5 in France (Rennes) in a sample of 165 psychiatric in-patients (59 schizophrenic persons in the sample). In this psychiatric hospital, the somatic care ward includes dental care or dental consultations.

In France, some psychiatric hospitals only have a somatic care ward that includes dental care or dental consultations. In this country, the somatic care of patients undergoing psychiatric treatment remains heterogeneous, and care management modes differ from one department to another, and data are not available.

Socio-economic status

In accordance with a previous study [START_REF] Bertaud-Gounot | Oral health status and treatment needs among psychiatric inpatients in Rennes, France: a cross-sectional study[END_REF], we showed that age over 45 years, length of hospitalization over 5 years, disability and plaque index over 1 were significant contributors to having a high DMFT (over 16). In other studies, among the subjects with schizophrenia, DMFT was significantly related to independent variables such as age, education, and grade of disability [START_REF] Chu | The relationship between body mass index, the use of second-generation antipsychotics, and dental caries among hospitalized patients with schizophrenia[END_REF].

To our knowledge, this is the first time the OHrQOL has been explored in PWS using the GOHAI scale. In the present study PWS clearly express their poor OHrQOL in accordance with poor oral health. Whereas some paradoxical situations were described where patients with serious and persistent disabilities report that they experience a good or excellent quality of life [START_REF] Albrecht | The disability paradox: high quality of life against all odds[END_REF]. In older adults, high GOHAI scores were found not in accordance with the severity of the impact of oral diseases [START_REF] Wallace | Age-related differences in the symptomatic expression of major depression[END_REF]. In this situation, the objective dental status is disconnected from the subjective perception of oral health and quality of life.

Given the impact of oral health on overall health and quality of life, the impact of self-perceived oral health in PWS should be explored in the future with qualitative studies to better understand the oral health and dental care needs in this population [START_REF] Tang | Self-reported and interviewer-rated oral health in patients with schizophrenia, bipolar disorder, and major depressive disorder[END_REF].

Medical condition

Schizophrenic patients generally have life-long treatment for the symptoms of the disease and take more than two drugs daily to address the symptoms of schizophrenia [START_REF] Parsons | Weight effects associated with antipsychotics: a comprehensive database analysis[END_REF].

Concerning medical conditions, our study showed that 29.6% of the patients had a BMI >30. Generally, in a psychiatric population, 31% of men and 37% of women have a BMI >30 [START_REF] Allison | Antipsychotic-induced weight gain: a review of the literature[END_REF].

PWS have a higher rate of diabetes mellitus than the general population. It seems that the frequency of diabetes mellitus is higher in older schizophrenic patients [START_REF] Eltas | An assessment of periodontal health in patients with schizophrenia and taking antipsychotic medication[END_REF]. Many factors can contribute to glycaemic disturbances, such as negative symptoms, which increase with time, unbalanced diets, toxic habits and the consumption of antipsychotics [START_REF] Tang | Self-reported and interviewer-rated oral health in patients with schizophrenia, bipolar disorder, and major depressive disorder[END_REF]. But in our study we don't observed this relation (p=0.0555) probably in relation with the small size of your population. Diabetes is a risk factor likely to contribute to the development of periodontal disease. The control of periodontal infection could furthermore contribute to the control of diabetes. Periodontal disease could may also be a risk factor for cardiovascular diseases (ischemic heart disease), and could be associated with a high rate of mortality in PWS [START_REF] Desvarieux | Relationship between periodontal disease, tooth loss, and carotid artery plaque: the Oral Infections and Vascular Disease Epidemiology Study (INVEST)[END_REF].

Lifestyle

In their study, Mallet et al. [START_REF] Mallet | Cigarette smoking and schizophrenia: a specific clinical and therapeutic profile? Results from the FACE-Schizophrenia cohort[END_REF] observed that 53.7% of PWS were smokers in France which is higher that observed in the general population (34.1%) [START_REF] Guignard | La consommation de tabac en France et son évolution: résultats du Baromètre santé 2014[END_REF]. Patients with tobacco use present clinical specificities that may involve different physiological pathways, involving at least interactions between cholinergic-nicotinic system and dopaminergic system. In our study, we observed more missing teeth in PWS smoking population. Therefore, understanding smoking patterns in major psychiatric disorders is important to develop effective interventions for smoking cessation and reduction of harmful outcomes.

A lack of brushing and of dental visit is observed in our population. It should also be noted that PWS visit dentists much less frequently than the general population [START_REF] Nielsen | Determinants of poor dental care in patients with schizophrenia: a historical, prospective database study[END_REF][START_REF] Sayegh | Oral health status and dental treatment needs among non-institutionalized psychiatric patients[END_REF].

Strengths and limitations

To our knowledge, this is the first study to consider in-and out-patients with schizophrenia in France. However, regardless the sampling procedure used to obtain samples, cohorts are ultimately composed of volunteers who may differ from the target population and our results in Côte d'Or region cannot be generalized to all of France. This also highlighted the difficulties to include PWS in research protocols. Secondly, some limitations in the use of the PMSI database to identify PWS can be pointed out. Diagnostic codes F20-F29 of PMSI data bases are not accurate enough. Indeed they take into account heterogeneous schizophrenia symptoms and can induce an under-estimation of the schizophrenia population.

Conversely, considering that schizophrenic symptoms might not be subsequently confirmed (transient schizophrenic disorder) could induce a risk of over-estimation. In order to be more exhaustive, we should take in account chronic disease status, drugs and hospitalisations conditions (mean duration, number of hospitalizations...) The REDSIAM (Réseau données Sniiram) network suggested different combinations of selected criteria in order to identify adult schizophrenia in the SNIIRAM (Système national d'information inter-regimes de l'assurance maladie) data base [START_REF] Quantin | Study of algorithms to identify schizophrenia in the SNIIRAM database conducted by the REDSIAM network[END_REF]. Our study could introduce a national survey with the REDSIAM network. Thirdly, in this study, indicators for oral function (deficiencies in chewing, swallowing, speaking) were not considered even though they are important oral problems. Alcohol and drug consummation habits were not assessed either and could be explored in further studies.

CONCLUSIONS

This study highlighted that PWS describe a poor OHrQoL. We observed a poor dental and hygiene index score. PWS visited a dentist less frequently, and they brushed their teeth less often. Finally, our findings illustrated a gap in oral health between PWS and general population in France.

In practice, to improve this major public health issue in France, since 2016, a dental check-up is recommended for all new admissions to a psychiatric hospital.

La Chartreuse Hospital

Total sample of schizophrenic persons (n=220)

Dijon University Hospital

Total sample of schizophrenic persons (n=65) Iran [START_REF]Country and Lending Groups[END_REF] Japan [START_REF] Persson | Monitoring oral health and dental attendance in an outpatient psychiatric population[END_REF] Greece [START_REF] Thomas | Factors which influence the oral condition of chronic schizophrenia patients[END_REF] Spain [START_REF] Arnaiz | Oral health and the symptoms of schizophrenia[END_REF] Sweden [START_REF] Nikfarjam | Oral health status in three long term care units of schizophrenic patients in Chaharmahal-Bakhtiari province, Iran[END_REF] In and outpatient

Robert Morlevat Hospital

Hospitalized

A

  multicentre cross-sectional descriptive study was performed between March 2014 and January 2016 in a sample population of PWS recruited from out-and in-patient populations of the three psychiatric hospitals of the Côte d'Or region (Bourgogne). It has 530,000 inhabitants and three hospitals monitoring PWS [15]. The Chartreuse hospital manages persons from rural and urban areas, the Robert Morlevat hospital (Semur-en-Auxois) manages persons from rural areas and Dijon University Hospital manages persons from urban areas. These three hospitals in Côte d'Or include physical and dental consultation and care.

Table 1 .

 1 Characteristics of sample studied (n=109)

	Total sample of schizophrenic persons (n=17)

Table 2 .

 2 Tooth decay index (DMFT scores) by age

		Decayed m(sd)	Missing m(sd)*** Filled m(sd)*	DMFT m(sd)***
	Age (years)
	25-34	2.0 (2.6)	3.4 (7.3)	4.6 (4.2)	10.0 (8.4)
	35-44	2.5 (3.6)	4.0 (5.3)	7.5 (4.5)	14.1 (6.4)
	45-54	2.3 (2.9)	7.3 (7.4)	8.8 (5.5)	18.4 (7.3)
	55-64	1.6 (1.9)	13.0 (10.4)	6.9 (6.2)	21.5 (5.8)
	≥65	0.8 (1.5)	16.4 (13.0)	4.4 (7.0)	21.7 (9.3)
	Legend: Sd: standard deviation; DMFT: decayed-missing-filled teeth. *p<0.05 **p<0.01
	***p<0.001

Table 3 .

 3 Dental status (DMFT scores) by demographic, socioeconomic, medical, dental and behavioural characteristics of the sample.

		n	Decayed	Missing	Filled	DMFT
			m(sd)	m(sd)	m(sd)	m(sd)
	Sex
	Female	42	1.9 (3.2)	7.0 (9.1)
	Male	67	2.1 (2.5)	8.1 (9.3)
	Education
	Primary school or less	24	2.1 (3.3)	11.3 (10.3)*
	Secondary or higher	85	2.0 (2.8)	6.6 (8.6)
	Residence
	Out-patient	87	1.8 (2.5)
	In-patient	22	3.2 (3.9)
	Tooth brushing habits
	Never to Occasionally	29	3.2 (3.6)*
	Every day	69	1.5 (2.4)
	Last dental visit
	<6 months	57	2.2 (3.0)
	≥6 months	50	1.7 (2.4)
	Smoking
	No	48	1.5 (2.1)
	Yes	60	2.4 (3.2
	Treatment (n° of drugs)
	<4	48	1.6 (2.6)
	≥4	60	2.3 (2.9)
	Duration of illness
	<20 years	58	1.8 (2.4)
	≥20 years	49	2.3 (3.2)
	BMI
	<30	76	2.2 (3.0)
	≥30	32	1.7 (2.4)
	Diabetic
	No	94	2.1 (2.8)
	Yes	12	1.4 (3.0)
	OHI-S
	<3	68	1.3 (1.7)*
	≥3	28	4.2 (4.0)
	GOHAI
	<44	48	2.5 (3.3)
	≥44	53	1.7 (2.3)
	<51	92	2.2 (2.9)
	≥51	9	1.6 (2.7)
	XI
	<22	37	1.4 (2.0)
	≥22	37	2.4 (3.6)

Table 5 . International comparisons of DMFT scores

 5

	Year Country	Outpatient or	n	Average	DMFT
			Hospitalized		age m(sd)	m(sd )
	2016	France
	2015	Malaysia [12]
	2013
	2012
	1996
	2011
	2009

Acknowledgements

The authors thank the staff and patients of all the hospitals (La Chartreuse Psychiatric Centre, University Hospital of Dijon and Semur en Auxois Hospitals) who participated in the survey.

We thank Nicolas Abello, Sandrine Lombion and Valérie Bertaud for their assistance in writing the manuscript and Philip Bastable for his help with English language correction. We are grateful to the Regional Health Agency of Burgundy (Agence Régionale de Santé de Bourgogne) and the University Hospital of Dijon for the financial support given to the study.

We are awaiting the results of a cluster randomized controlled trial to assess the effectiveness of a therapeutic educational programme in oral health for PWS (www.ClinicalTrials.gov under number NCT02167724) [43] funded by the French Ministry of Health. This therapeutic educational programme is potentially a new care support strategy for improving the OHrQoL of PWS.

Funding source

This research project was funded by the Agence Régionale de Santé de Bourgogne (ARS).

Oral health in schizophrenia patients: a French multicenter cross-sectional study


Dr Soraya Mezouar
email: soraya.mezouar@univ-amu.fr

Victor Morel

Laury Leveille

Noémie Resseguier

Céline Chartier

Didier Raoult

Jean-Louis Mege

Joana Vitte

Progenitor mast cells and tryptase in Q fever

Keywords: Mast cells progenitors, tryptase, Q fever, Coxiella burnetii, flow cytometry

Q fever is an infectious disease due to Coxiella burnetii. Following a primaryinfection, C. burnetii may persist in some patients, leading to endocarditis and vascular infections. Mast cells (MCs), known for their role in allergic diseases, innate immunity and cardiac function, are produced by bone marrow, circulate as progenitors in the bloodstream and reach tissues for their maturation and activation. The latter may be estimated by measuring serum tryptase levels. We wondered if MC progenitors and tryptase were affected in Q fever. We showed a decrease in MC progenitor count in Q fever patients whereas serum tryptase levels were increased. Interestingly, counts MC progenitors and levels of serum tryptase were correlated in Q fever patients with valvular lesions including Q fever endocarditis. Taken together, our results show alterations of MC numbers and activity in Q fever patients, suggesting that MC are involved in Q fever pathophysiology.

Introduction

Q fever is an infectious disease due to the intracellular bacterium Coxiella burnetii.

Following primary-infection that is symptomatic in some patients, the infection may become persistent in specific contexts such as immunodeficiency, valvulopathy or vascular disease.

The manifestations of persistent Q fever consist of endocarditis and vascular infections [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF].

The evolution of Q fever is largely determined by anti-C. burnetii immune response [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF]. This latter consists of an inappropriate inflammatory response and decreased counts of lymphocytes [START_REF] Layez | Foxp3(+)CD4(+)CD25(+) regulatory T cells are increased in patients with Coxiella burnetii endocarditis[END_REF], monocytes [START_REF] Ka | Imbalance of circulating monocyte subsets and PD-1 dysregulation in Q fever endocarditis: the role of IL-10 in PD-1 modulation[END_REF], dendritic cells [START_REF] Gorvel | Intracellular bacteria interfere with dendritic cell functions: role of the type I interferon pathway[END_REF], and plasmacytoid dendritic cells in Q fever patients [START_REF] Ka | Coxiella burnetii Induces Inflammatory Interferon-Like Signature in Plasmacytoid Dendritic Cells: A New Feature of Immune Response in Q Fever[END_REF]. It is likely that other innate immune cells including mast cells (MCs) are involved in the pathophysiology of Q fever. MCs leave the bone marrow as progenitors, pass through the bloodstream and complete their maturation in target tissues [START_REF] Dahlin | Mast cell progenitors: Origin, development and migration to tissues[END_REF]. MCs are key players in both inflammatory and immune responses, in addition to their well-known role during immediate hypersensitivity reactions [START_REF] Urb | The Role of Mast Cells in the Defence against Pathogens[END_REF]. MCs also contribute to cardiac functions and are involved in cardiovascular diseases [START_REF] Bot | Mast Cells: Pivotal Players in Cardiovascular Diseases[END_REF]. Mast cell progenitors may be found in the bloodstream and are identified as CD34 + cells expressing CD117 (c-kit) in association with the high affinity immunoglobulin (Ig) E receptor (FcεRI) [START_REF] Dahlin | KIT signaling is dispensable for human mast cell progenitor development[END_REF]. During infection, MC progenitors are recruited to infected tissues where they mature and get activated, contributing to host defense mechanisms against microorganisms [START_REF] Anthony | Protective immune mechanisms in helminth infection[END_REF]. The activation status of tissue MCs can be estimated by the determination of serum baseline tryptase (sbT) [START_REF] Amin | The role of mast cells in allergic inflammation[END_REF]. C. burnetii has a strong tropism for tissues rich in mast cells (MCs) such as adipose tissue [START_REF] Bechah | Persistence of Coxiella burnetii, the agent of Q fever, in murine adipose tissue[END_REF], bone-marrow [START_REF] Cottalorda | Osteoarticular Infection due to Coxiella Burnetii in Children[END_REF][START_REF] Poujol | Recurrent osteitis and Coxiella burnetii: the relation to chronic recurrent multifocal osteomyelitis[END_REF] or lung [START_REF] Whittick | Necropsy Findings in a Case of Q Fever in Britain[END_REF]. However, the role of MCs in Q fever is unknown.

We wondered if MCs are involved in the pathophysiology of Q fever. The main purpose of this study was to investigate the MC progenitor population in Q fever patients.

Using flow cytometry, we report here that circulating MC progenitors were decreased whereas sbT was increased in Q fever patients compared to healthy donors. We also reported a correlation between the decrease of circulating MC progenitors and the increase in sbT in Q fever patients with valvular disease. Taken together, these results suggest a role of MCs in Q fever pathophysiology.

Materials and methods

Patients and controls

We included 23 healthy blood donors and 22 patients with Q fever. Q fever patients belonged to clinical subsets of acute (n = 10) and persistent Q fever (endocarditis group, n = 12). This study was conducted with the approval of the Ethics Committee of Aix-Marseille University and written consent of each patient. Patients consisted of 6 women and 16 men, median age 63, range 35 -89 years. Controls consisted of 11 women and 12 men, median age 41, range 24 -65 years. The diagnosis of acute and persistent Q fever was performed according to recently updated criteria [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF]. Briefly, patients with acute Q fever were diagnosed by the presence of fever and/or hepatitis and/or pneumonia with serological criteria ( IgG >200 and IgM>50 against C. burnetii phase II or seroconversion) [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF]. Persistent Q fever was diagnosed on the presence of hepatitis, endocarditis or vascular infection and IgG >800 against C. burnetii phase I. As additional controls we included 15 patients with acute, non-Q fever, native valve infective endocarditis. Patients consisted of 6 women and 9 men, median age 61, range 21 -81 years. The diagnosis of acute infective endocarditis was performed according to modified Duke criteria [START_REF] Li | Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis[END_REF]. Valvular surgery was performed in all patients as valvular repair or valvular replacement. A microbiological identification was obtained in all patients.

Staphylococcus aureus and S. lugdunensis were found in 4 and 1 patient respectively.

Enterococcus faecalis was found in 3 patients. Streptococcus anginosus, S. mitis and S. infarius was respectively found in 1 patient. The other microorganisms recovered were Gemella sanguinis, Haemophilius influenzae, Escherichia coli and Bartonella quintana.

Cell isolation

Blood was collected into EDTA tubes. Peripheral blood mononuclear cells (PBMCs) from healthy donors and patients were isolated after centrifugation through Ficoll cushion and suspended in RPMI 1640 containing 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Courtaboeuf, France) as previously described [START_REF] Ka | Imbalance of circulating monocyte subsets and PD-1 dysregulation in Q fever endocarditis: the role of IL-10 in PD-1 modulation[END_REF].

Serum baseline tryptase quantification

Serum baseline tryptase levels were measured with the immunofluorescent enzyme assay for αand β-tryptase isoforms (ImmunoCAP, Thermo Fisher, Uppsala, Sweden).

Flow cytometry

Mast cell progenitors were identified by staining with anti-CD34 (Beckman Coulter, Nyon, Switzerland), anti-CD117 (c-Kit receptor, CD117-APC, Beckman Coulter) and anti-IgE (FcεRI, anti-IgE-PE, Bühlmann, Schönenbuch, Switzerland) antibodies (Abs). Cytometry experiments were performed with a Canto II flow cytometer (Becton Dickinson, Le Pont de Claix, France). Fifty thousand events were acquired and analyzed with FACS Diva software (Becton Dickinson Bioscience).

Statistical analysis

Results were expressed as median and range. Statistical analysis was performed using the Mann-Whitney U test. Adjusted p-values were provided in order to take into account the multiplicity of comparisons within each analysis. The Benjamini and Hochberg method was used, thus controlling the false discovery rate. Statistical significance threshold was set at p < 0.05.

Results

Circulating MC progenitors were specifically decreased in Q fever patients

The expression of CD117, surface IgE and CD34 was assessed by flow cytometry to identify MC progenitors, as previously reported [START_REF] Dahlin | KIT signaling is dispensable for human mast cell progenitor development[END_REF]. We found that approximately 1% of total PBMCs were CD117 + /IgE + , and 0.003 % were CD34 + MC progenitors ranging from 0.001 % to 0.012 % of total PBMCs (Figure 1A). Then we wondered if this subset of circulating cells was modulated in Q fever. We found that the percentage of MC progenitors was significantly decreased in patients with Q fever (p = 0.001) (Figure 1B) whereas no differences were observed between acute and endocarditis group (p = 0.8836) (Figure 1C). In addition, the percentage of MC progenitors was not altered in patients with bacterial infection other than C. burnetii (p = 0.1515). These results showed that MC progenitors were specifically decreased in Q fever patients independently of their clinical form.

Serum baseline tryptase was specifically increased in patient infected with C. burnetii

We therefore investigated sbT in Q fever patients. As depicted in figure 2A, sbT was higher in Q fever patients than in controls (6.18 ± 2.87 µg/L versus 3.77 ± 1.85, p = 0.0448).

Interestingly, compared to controls this increase is specifically found in Q fever patients because other infections did not induce modification in sbT (Figure 2A). In addition, no differences were observed in acute and endocarditis groups (p = 0.4700). These results showed a specific increase of sbT in Q fever. Moreover, we found a correlation between the number of MC progenitors and sbT levels in one subgroup of Q fever patients, namely in Q fever patients with valvular lesions including Q fever endocarditis (R = 0.8240, p = 0.0034) (Figure 2B). These results suggested that this association was a marker of vascular involvement in Q fever.

Discussion

In this study, we measured the frequency of MC precursors in blood from healthy individuals and patients with Q fever. The identification was based on the assessment of the co-expression of IgE, CD117 and CD34. This approach is necessary because each maker is incompletely specific of MC progenitors. IgE expression identifies mostly MCs and basophils, but monocytes from atopic patients can also bear surface IgE [START_REF] Shin | The role of FcεRI expressed in dendritic cells and monocytes[END_REF]. Although CD117 is widely considered as a specific MC surface marker, it is also expressed by basophils, myeloid dendritic cells, TCRα/β + T cells, B cells and NK cells [START_REF] Escribano | Expression of the c-kit (CD117) molecule in normal and malignant hematopoiesis[END_REF]. We found that MC progenitors represent a minor population in blood from healthy individuals. This is in agreement with previous studies [START_REF] Dahlin | Mast cell progenitors: Origin, development and migration to tissues[END_REF][START_REF] Dahlin | KIT signaling is dispensable for human mast cell progenitor development[END_REF].

This study reported for the first time the decrease in circulating MC progenitors in Q fever patients. This result has to be related to previous reports in which we found a decrease in circulating lymphocytes [START_REF] Layez | Foxp3(+)CD4(+)CD25(+) regulatory T cells are increased in patients with Coxiella burnetii endocarditis[END_REF], monocytes [START_REF] Ka | Imbalance of circulating monocyte subsets and PD-1 dysregulation in Q fever endocarditis: the role of IL-10 in PD-1 modulation[END_REF], dendritic cells [START_REF] Gorvel | Intracellular bacteria interfere with dendritic cell functions: role of the type I interferon pathway[END_REF] and plasmacytoid dendritic cells counts [START_REF] Ka | Coxiella burnetii Induces Inflammatory Interferon-Like Signature in Plasmacytoid Dendritic Cells: A New Feature of Immune Response in Q Fever[END_REF]. While the decrease in circulating immune cells occurred mainly in Q fever patients with endocarditis, here we did not observe differences in the number of MC progenitors between acute and persistent Q fever. Decreased numbers of progenitors might be due to impaired bone marrow production, or increased recruitment of progenitors into C. burnetii-infected tissues. It has been reported that the numbers of MC increased in tissuespecific due to the maturation of MC progenitors in human diseases [START_REF] Dahlin | Mast cell progenitors: Origin, development and migration to tissues[END_REF][START_REF] Dahlin | CD11c+ cells are required for antigen-induced increase of mast cells in the lung[END_REF]. Alternatively, the decrease in MC progenitors may be due to their death. The interleukin (IL)-10 is known to induce MC apoptosis [START_REF] Bailey | Interleukin-10 induces apoptosis in developing mast cells and macrophages[END_REF] and is increased in persistent Q fever [START_REF] Meghari | Persistent Coxiella burnetii infection in mice overexpressing IL-10: an efficient model for chronic Q fever pathogenesis[END_REF].

The second major observation was the increase in serum tryptase in patients with Q fever independently of the clinical presentation. Circulating tryptase originates mainly from mature MCs and in minute amounts from MC-committed progenitors [START_REF] Ma | Direct development of functionally mature tryptase/chymase double-positive connective tissue-type mast cells from primate embryonic stem cells[END_REF]. Apart from acute MC degranulation, which was not relevant for the patients included in this study, an increase in tryptase levels has been reported in association with a poorer clinical condition or prognosis in cardiovascular diseases [START_REF] Pastorello | Serum tryptase detected during acute coronary syndrome is significantly related to the development of major adverse cardiovascular events after 2 years[END_REF]. Indeed, in our hands, the increase in tryptase levels was correlated in Q fever patients with valvular lesions including Q endocarditis. This association is reminiscent of the abundant presence of MCs in cardiovascular tissues. The cardiac tropism of MC and the variation of the sbT is well documented in particular in cardiovascular diseases such as aortic stenosis and myocardial infarction [START_REF] Filipiak | Tryptase levels in patients after acute coronary syndromes: the potential new marker of an unstable plaque?[END_REF][START_REF] Shi | Mast cells in human and experimental cardiometabolic diseases[END_REF].

In summary, we show that Q fever patients display less circulating MC progenitors but higher levels of sbT. The decrease in MC progenitors is correlated with sbT in Q fever patients with valvular lesions including endocarditis. These results suggest MCs take part to pathophysiology of Q fever.

Acknowledgments

We thank Dr. Christian Capo and Dr. Yassina Bechah for their help and advice during the preparation of the manuscript.

Formatting of funding sources

Soraya Mezouar was supported by a "Fondation pour la Recherche Médicale" (FRM) postdoctoral fellowship (SPF20151234951). This work was supported by the French Government under the "Investissements d'avenir" (Investments for the Future) program managed by the "Agence Nationale de la Recherche" (reference: Méditerranée Infection 10-IAHU-03).

Author contributions

S.M and J.V conceived and designed the experiments. S.M, V.M, C.C and L.L performed experiments and analyzed the data. N.R performed statistical analysis. S.M, D.R, J.L.M and J.V wrote the paper.

Declaration of interest

The authors declare no competing interests.

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