Accepted Manuscript

Title: Vitamin D levels and effects of vitamin D replacementin children with Periodic Fever, Aphthous stomatitis,Pharyngitis, and cervical Adenitis (PFAPA) syndrome

Author: Stefano Stagi Federico Bertini Donato RiganteFernanda Falcini

PII: S0165-5876(14)00158-XDOI: http://dx.doi.org/doi:10.1016/j.ijporl.2014.03.026Reference: PEDOT 7056

To appear in: International Journal of Pediatric Otorhinolaryngology

Received date: 7-12-2013Revised date: 20-3-2014Accepted date: 21-3-2014

Please cite this article as: S. Stagi, F. Bertini, D. Rigante, F. Falcini, Vitamin D levels andeffects of vitamin D replacement in children with Periodic Fever, Aphthous stomatitis,Pharyngitis, and cervical Adenitis (PFAPA) syndrome, International Journal ofPediatric Otorhinolaryngology (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.026

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Stefano Stagi, Federico Bertini2, Donato Rigante

3, and Fernanda Falcini

2

Health’s Sciences Department, University of Florence, Anna Meyer Children’s University Hospital,

Florence, Italy; 2Department of BioMedicine, Section of Rheumatology, Transition Clinic,

University of Florence, Florence, Italy; 3

Institute of Pediatrics, Università Cattolica Sacro Cuore,

Rome, Italy.

Short running title: Vitamin D deficiency and treatment in PFAPA

Address all correspondence and requests for reprints to: dr. Stefano Stagi, Paediatric Endocrinology

Unit, Health’s Sciences Department, Anna Meyer Children’s University Hospital, viale Pieraccini

24, 50139, Florence, Italy. Tel. + 39-055-5662426, Fax +39-055-4221012, E-Mail:

stefano.stagi@yahoo.it

*Title Page

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Vitamin D levels and effects of vitamin D replacement in children with Periodic 1

Fever, Aphthous stomatitis, Pharyngitis, and cervical Adenitis (PFAPA) 2

syndrome 3

4

Stefano Stagi, Federico Bertini2, Donato Rigante

3, Fernanda Falcini

2 5

6

Health’s Sciences Department, University of Florence, Anna Meyer Children’s University Hospital, 7

Florence, Italy; 2Department of BioMedicine, Section of Rheumatology, Transition Clinic, 8

University of Florence, Florence, Italy; 3

Institute of Pediatrics, Università Cattolica Sacro Cuore, 9

Rome, Italy. 10

11

Short running title: Vitamin D deficiency and treatment in PFAPA 12

13

Key Indexing terms: 14

25-hydroxyvitamin D, periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis, PFAPA 15

syndrome, child. 16

17

Address all correspondence and requests for reprints to: dr. Stefano Stagi, Paediatric Endocrinology 18

Unit, Health’s Sciences Department, Anna Meyer Children’s University Hospital, viale Pieraccini 19

24, 50139, Florence, Italy. Tel. + 39-055-5662426, Fax +39-055-4221012, E-Mail: 20

stefano.stagi@yahoo.it 21

*Manuscript

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Abstract 1

Background. The periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) 2

syndrome is an autoinflammatory disease characterized by regularly recurrent fever episodes due to 3

seemingly unprovoked inflammation. 4

Objective. To assess serum 25-hydroxyvitamin D 25(OH)D concentrations in children with 5

PFAPA syndrome and evaluate longitudinally the effect of wintertime vitamin D supplementation 6

on the disease course. 7

Study design. We have evaluated 25 Italian patients (19 males, 6 females, aged 2.4-5.3 years), 8

fulfilling the Euro-Fever PFAPA criteria. For each patient, we recorded demographic and 9

anthropometric data, clinical manifestations, serum calcium, phosphate, and 25(OH)D. After 400 IU 10

vitamin D supplementation during wintertime, clinical and auxological characteristics, calcium, 11

phosphate, and 25(OH)D levels were re-evaluated. Data were compared with a sex- and age-12

matched control group. 13

Results. PFAPA patients showed reduced 25(OH)D levels than controls (p<0.0001). Regarding the 14

effect of seasons on vitamin D, winter 25(OH)D levels were significantly reduced than summer 15

ones (p<0.005). Moreover, these levels were significantly lower than in healthy controls (p<0.005), 16

and correlated with both fever episodes (p<0.005) and C-reactive protein values (p<0.005). 17

After vitamin D supplementation, PFAPA patients showed a significantly decreased number of 18

febrile episodes and modification of their characteristics (mean duration of fever episodes, p<0.05; 19

number of febrile episodes per year p<0.005). 20

Conclusions. Deficient and insufficient vitamin D serum levels were found in most children with 21

PFAPA syndrome, and hypovitaminosis D might be a significant risk factor for PFAPA flares. 22

However, vitamin D supplementation seems to significantly reduce the typical PFAPA episodes 23

and their duration, supporting the role of vitamin D as an immune-regulatory factor in this 24

syndrome. 25

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1

Introduction 2

The periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome, first 3

described as a clinical entity in 1987 [1], belongs to the group of autoinflammatory diseases [2]. 4

This condition is an idiopathic chronic disease of non-hereditary origin, primarily affecting pre-5

school children, characterized by regularly recurrent fever episodes, lasting for 3-6 days with a 6

remarkable clockwork periodicity of 3-8 weeks in approximately 50% of patients, due to seemingly 7

unprovoked inflammation [2]. By definition, a constellation of cardinal signs (pharyngitis, cervical 8

adenopathy, aphthous stomatitis) and systemic symptoms (headache, abdominal pain, vomiting, 9

malaise, skin rash, etc.) appear in each PFAPA inflammatory attack, which is typically observed in 10

less than 5 year-children who have no sign of respiratory tract infections [3]. At present, no specific 11

biomarkers are available to help the diagnostic evaluation [4]. As in other periodic fever syndromes, 12

there is a marked increase in C-reactive protein (CRP), other acute phase reactants, and white blood 13

cell count [5], which are indicative of a prominent systemic inflammatory response. In children with 14

PFAPA syndrome, these indicators return to normal levels when symptoms subside [5, 6]. 15

Therefore, in order to corroborate the diagnosis, it is necessary to rule out the monogenic periodic 16

fever syndromes that may show overlapping features with PFAPA syndrome [6]. 17

Vitamin D plays an important role in calcium balance, which is largely a function of calcium 18

dietary intake, intestinal absorption, renal excretion, and bone remodeling [7, 8]. More recently, 19

vitamin D has gained attention for a plethora of other crucial benefits, and emerging evidence 20

suggests that vitamin D plays a key-role in immunity regulation [8]. In fact, vitamin D receptors are 21

found on several immune cells, and vitamin D metabolites seem to modulate T cell proliferation and 22

dendritic cell function [9, 10]. Hence, vitamin D deficiency may represent a risk factor for the 23

development of autoimmune and other chronic diseases [8, 11], because of its potential role as an 24

immune-regulatory agent. 25

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Serum 25(OH)D is the most commonly used indicator of vitamin D status, reflecting dietary intake 1

from vitamin D2 (ergocalciferol) and cutaneous synthesis of vitamin D3 (cholecalciferol) [12]. 2

Vitamin D status in PFAPA children has been evaluated in only one study, which did not consider 3

the effects of vitamin D supplementation [13]. The purpose of our study was to assess serum 4

25(OH)D in children with PFAPA syndrome and evaluate longitudinally the effect of wintertime 5

vitamin D supplementation on the disease course. 6

7

Patients and methods 8

We have consecutively evaluated 25 Italian children (19 males, 6 females, mean age 3.6 ± 0.9 9

years, range 2.4-5.3 years), after having excluded other causes of recurrent fever. All patients 10

attended the Paediatric Units of different Tuscany Hospitals (Paediatric Unit of Villamarina 11

Hospital, Piombino, Livorno; Paediatric Unit of Mugello’s Hospital, Borgo san Lorenzo, Florence; 12

Rheumatology Section, Transition Clinic, University of Florence, Florence; and Health’s Sciences 13

Department, Anna Meyer Children’s University Hospital, Florence, Italy) between September 2009 14

and August 2013. It was verified that all patients fulfilled the Euro-Fever PFAPA criteria: 1) daily 15

fever of at least 38.5 °C (axillar) for two to seven days, periodically recurring for a minimum of six 16

months; 2) pharyngitis, cervical adenitis and/or oral aphthosis; 3) exclusion of other causes of 17

recurrent fever (by clinical or laboratory assessment, depending on the individual history); 4) 18

exclusion of infections, immunodeficiency, cyclic neutropenia, and monogenic autoinflammatory 19

disorders; 5) full recovery between episodes and normal linear growth [14]. The exclusion of 20

monogenic hereditary periodic fever syndromes was based on the patient’s ethnicity, medical 21

history, infectious or immunological investigations, and genetic analysis negative for MEFV and 22

MVK mutations. For each patient, we recorded longitudinally demographic and anthropometric 23

data, duration of fever episodes, duration of intervals between fever episodes, laboratory results, 24

and clinical manifestations. In all patients serum calcium, phosphate, and 25(OH)D were evaluated 25

during a period of wellness. Although solar winter comprises the period from November to 26

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February, for this study winter was defined as the period November-May and summer as June-1

October. As previously reported, 25(OH)D values were categorized as being desirable (or 2

“sufficient”) when at least 30 to 40 ng/mL (75 to 100 nmol/L), “insufficient” between 20 and 30 3

ng/mL (50 and 75 nmol/L), and “deficient” when <20 ng/mL [8]. Exclusion criteria from the study 4

were primary hyperparathyroidism, skeletal dysplasia, severe obesity, malabsorption disorders, 5

endocrinological and renal diseases or if patients had already received vitamin D3 as a treatment for 6

another underlying disease at the time of the start of our study. 7

Vitamin D supplementation (400 IU of vitamin D3/day from November to May) was established for 8

all patients, and clinical/auxological characteristics, serum calcium, phosphate, and 25(OH)D levels 9

were re-evaluated in the same children (mean age 4.8 ± 0.9 years, range 3.7-6.3 years). 10

Healthy controls were recruited among children that were hospitalized for minor surgery (113 11

healthy age- and sex-matched subjects: 74 males, 39 females, mean age 4.9±1.3 years, range 2.1-8.3 12

years). Our Ethics Committee approved the study protocol, conducted in accordance with the 13

Declaration of Helsinki guidelines. All parents’ subjects and/or their guardian signed an informed 14

consent. 15

16

Laboratory methods 17

Blood samples were obtained from each participant to the study after an overnight fast. Serum 18

concentrations of calcium and phosphate were determined following routine biochemical laboratory 19

protocols. Blood cell count was determined using an ADVIA cell counter. CRP was also evaluated. 20

Serum 25(OH)D was determined by chemiluminescence enzyme-labeled immunometric assay 21

using an IMMULITE 2000 Systems analyzer (Siemens, Gwynedd, UK). The intra- and inter-assays 22

CVs were <5% and <8%, respectively). 23

24

Statistical analysis 25

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The characteristics of the study population were described through frequency distributions for 1

categorical variables and through means and standard deviations (SD), medians, and range for 2

continuous variables. The crude association of vitamin D status and the other categorical variables 3

was assessed through chi-square test and Fisher’s exact test. The crude association of vitamin D 4

status and continuous variables was assessed through Wilcoxon’s rank-sum test and Kruskal-Wallis 5

test, since all continuous variables resulted not normally distributed according to Shapiro-Wilk’s 6

test. All p-values <0.05 were considered statistically significant. Multivariate logistic regression 7

analyses were performed to investigate factors associated with a possible insufficient vitamin D 8

status. The association between 25(OH)D concentration and each variable was expressed in terms 9

of 95% confidence interval (95% CI). 10

11

Results 12

No statistically significant differences were found between patients with PFAPA syndrome and the 13

control group with regards to height (-0.2±0.7 vs 0.1±0.9, p=NS), and body mass index (BMI) SD 14

(0.9±1.3 vs 0.6±1.1, p=NS). The median age at onset of symptoms was 30 months (range: 17-58 15

months). The mean duration of fever episodes was 4.3 days (95% CI: 3.5-5.3), and the mean 16

duration of intervals between fever episodes was 25.3 days (95% CI: 20.4-30.1). The number of 17

fever episodes per year was 8.9±2.0. Seasonal variation in the frequency of fever episodes was seen 18

in 56.0% (14/25) of patients. As regards 25(OH)D levels, all patients with PFAPA syndrome did 19

not reveal to have sufficient levels: namely 5 patients (20%) displayed insufficient levels (between 20

20 and 30 ng/mL), and 20 (80%) showed deficient levels (<20 ng/mL). In addition, at the cross-21

sectional evaluation PFAPA patients showed lower 25(OH)D levels than controls (16.5±7.3 ng/mL 22

vs 29.8±8.4, p<0.0001) (Figure 1a). Levels of 25(OH)D were not different between males (16.0±8.5 23

ng/mL) and females (17.0±5.5, p=NS). Moreover, PFAPA patients showed normal levels of total 24

calcium (2.4±0.2 vs 2.5±0.1 mmol/L; p=NS) and phosphate (2.1±0.1 vs 2.1±0.2 mmol/L, p=NS), 25

compared to controls. Regarding the effect of the different seasons on 25(OH)D status, in winter 26

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25(OH)D levels in PFAPA patients were significantly reduced (10.6±3.0 ng/mL) in comparison 1

with levels in summer (21.2±6.1 ng/mL; p<0.005). These values were always significantly lower 2

than those observed in controls (winter: 22.1±5.4 ng/mL, p<0.005; summer: 36.4±7.3 ng/mL, 3

p<0.005) (Figure 2a). Analyzing the correlation among 25(OH)D levels and age, sex, seasons, 4

episodes of fever (number), CRP, calcium, phosphate, BMI, and height, we found that 25(OH)D 5

levels were inversely correlated with fever episodes (r=-0.42, p<0.005), and directly with CRP 6

values (r=0.39, p<0.005). After vitamin D supplementation (for a mean period of 7.4±1,1 months, 7

range 5.9-9.4 months), the longitudinal evaluation (after a mean follow-up of 12.3 months, range 8

10.1-14.3 months) showed that 9 PFAPA patients (36.0%) reached sufficient vitamin D levels, 9

whereas 14 (56.0%) still had insufficient levels, but only 2 (8.0%) had deficient levels. The 10

improved status of 25(OH)D in PFAPA subjects was significant, though without differences in 11

comparison with controls (26.8±5.1 vs 29.1±8.0 ng/mL, p=NS) (Figure 1b). 12

After vitamin D supplementation we also noted a significant modification of PFAPA febrile 13

episodes: the mean duration of episodes became of 2.3 days (95% CI: 1.9-3.1; p<0.05), and 9 14

patients had a significantly decreased number of febrile episodes per year (their mean number of 15

febrile attacks per year was reduced to 2.9±2.1, p<0.005) (Figure 3). PFAPA patients confirmed to 16

have normal levels of total calcium (2.5±0.2 vs 2.5±0.2 mmol/L; p=NS) and phosphate (2.0±0.1 vs 17

2.0±0.2 mmol/L, p=NS) in comparison with controls. As regards the effect of the different seasons 18

on 25(OH)D status, in winter 25(OH)D levels in PFAPA patients were significantly reduced 19

(22.6±3.4 ng/mL) than summer values (30.4±2.9 ng/mL; p<0.005). These values were not 20

significantly lower than those observed in controls (winter: 21.8±5.9 ng/mL, p=NS; summer: 21

35.8±7.1 ng/mL, p=NS) (Figure 2b). Evaluating the correlation among 25(OH)D levels and age, 22

sex, seasons, episodes of fever (number), CRP, calcium, phosphate, BMI, and height, we did not 23

observe any relationship between 25(OH)D and febrile episodes. 24

25

Discussion 26

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Our data confirm the results of Mahamid et al. who discovered significantly decreased 25(OH)D 1

levels in children with PFAPA syndrome, compared with a control group, showing a correlation 2

between 25(OH)D levels and CRP, and hypothesizing that vitamin D deficiency might be a 3

significant risk factor for PFAPA febrile episode recurrence [13]. Indeed, our patients with PFAPA 4

syndrome displayed reduced serum 25(OH)D levels, apparently related with the number of febrile 5

episodes and CRP, disclosing a possible increased consumption of 25(OH)D in PFAPA patients. To 6

the best of our knowledge, for the first time, our study provides data about the response to vitamin 7

D supplementation in PFAPA patients, which led to the reduction of number of PFAPA episodes, 8

highlighting the possible immune-regulatory effect of vitamin D, and suggesting a potential 9

therapeutic role of vitamin D in these patients. Vitamin D biology has revealed its multi-functional 10

role in many immune-mediated diseases, such as multiple sclerosis, type 1 diabetes mellitus, and 11

systemic lupus erythematosus [15]. Both the innate and adaptive immune system are impacted by 12

the active metabolite of vitamin D [16]. In PFAPA patients, while the exogenous or endogenous 13

antigens causing this disorder remain unidentified, a decrease in CD4+/CD25+ lymphocytes, which 14

regulate natural immune responses to prevent autoimmunity, has been reported [17]. Some data 15

show that an abnormal innate immune response might be the initial step in PFAPA syndrome, and a 16

subsequent adaptive response with activation and redistribution of T cells might explain the peculiar 17

periodicity of the disease [18]. Vitamin D metabolizing enzymes and vitamin D receptors (VDR) 18

are present in many cell types, including various immune cells such as antigen-presenting-cells, T 19

cells, B cells and monocytes. In vitro data show that vitamin D also promotes a more tolerogenic 20

immunological status [19]. It is interesting to note that VDR agonists powerfully control innate and 21

adaptive immune system, exerting a significant suppression of inflammatory processes, switching 22

the immune response from T helper 1 to T helper 2 dominance and counteracting the self-enhancing 23

inflammatory loop between immune and resident cells, especially by cytokine release impairment 24

[20]. However, for an inflammatory disease such PFAPA syndrome, firm conclusions regarding 25

cause and effect of vitamin D cannot be based on epidemiological association studies, and 26

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prospective long-term studies, including large intervention trials, are needed to increase our 1

knowledge in the field of vitamin D properties. 2

3

Conclusions 4

In conclusion, deficient and insufficient vitamin D serum levels have been found in most children 5

with PFAPA syndrome. Hypovitaminosis D can be a significant risk factor for PFAPA episode 6

recurrence. In addition, vitamin D supplementation seems significantly to reduce PFAPA febrile 7

flares and their duration, proving the relevance of vitamin D as a promoter of T cell regulation in 8

this syndrome. 9

10

References 11

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and aphthous stomatitis. J Pediatr 1987; 110: 43-46. 13

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658-659. 17

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vitamin D. Physiol Rev 1998; 78: 1193-1231. 7

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therapeutic considerations. Ann Rheum Dis 2007; 66: 1137-1142. 9

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12. Heaney RP. Lessons for nutritional science from vitamin D. Am J Clin Nutr 1999; 69: 825-12

826. 13

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Pediatr Otorhinolaryngol 2013; 77: 362-364. 15

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registry on autoinflammatory diseases: the Eurofever experience. Ann Rheum Dis 2012; 71: 17

1177-1182. 18

15. Yang CY, Leung PS, Adamopoulos IE, Gershwin ME. The implication of vitamin D and 19

autoimmunity: a comprehensive review. Clin Rev Allergy Immunol 2013; 45: 217-226. 20

16. Bikle DD. Vitamin D regulation of immune function. Vitam Horm 2011; 86: 1-21. 21

17. Stojanov S, Lapidus S, Chitkara P, Feder H, Salazar JC, Fleisher TA, et al. Periodic fever, 22

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18. Førsvoll J, Kristoffersen EK, Øymar K. Incidence, clinical characteristics and outcome in 1

Norwegian children with periodic fever, aphthous stomatitis, pharyngitis and cervical 2

adenitis syndrome; a population-based study. Acta Paediatr 2013; 102: 187-192. 3

19. Prietl B, Treiber G, Pieber TR, Amrein K. Vitamin D and immune function. Nutrients 2013; 4

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20. Scolletta S, Colletti M, Di Luigi L, Crescioli C. Vitamin D receptor agonists target 6

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2013: 876319. 8

9

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Legends 1

Fig. 1. 25(OH) vitamin D levels (ng/mL) at trasversal (1a) and longitudinal (1b) evaluation in 2

patients with Periodic Fever, Aphthous stomatitis, Pharyngitis, and cervical Adenitis (PFAPA) 3

syndrome and healthy controls. * p<0.05; ** p<0.005; *** p<0.005. 4

5

Fig. 2. 25(OH) vitamin D levels (ng/mL) at trasversal (2a) and longitudinal (2b) evaluation in 6

patients with Periodic Fever, Aphthous stomatitis, Pharyngitis, and cervical Adenitis (PFAPA) 7

syndrome and healthy controls evaluated during winter and summer. PFAPA vs controls: * p<0.05; 8

** p<0.005; *** p<0.005. PFAPA vs PFAPA and controls vs controls: ° p<0.05; °° p<0.005; °°° 9

p<0.005. 10

11

Fig. 3. Number of fever episodes/year and duration of fever (days) before and after vitamin D 12

supplementation. * p<0.05; ** p<0.005; *** p<0.005. 13

14

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Figure(s)

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Vit

am

in D

(n

g/m

L)

4,5

4,0

3,5

3,0

2,5

2,0

1,5

95

% C

I

Patients and controls

2b 0

10

20

30

40

50

60

Winter Summer

Controls PFAPA Controls PFAPA

°°°

°°

Page 18 of 18

Accep

ted

Man

uscr

ipt

Nu

mb

er o

f fe

ver

ep

iso

des

/yea

r

4,5

4,0

3,5

3,0

2,5

2,0

1,5

95

% C

I

Patients

3 0

2.0

4.0

6.0

8.0

10.0

12.0

Follow-up Ist evaluation Follow-up Ist evaluation

***

*

Du

rati

on

of

fev

er (

da

ys)

0

2.0

4.0

6.0

8.0

10.0

12.0