Recurrent aphthous stomatitis is the most common inflammatory disease of the oral mucosa. It is characterized by painful round ulcers with pseudomembranous center and erythematous borders [1,2]. The prevalence of the disease is 0.5% to 75% all over the world. Recurrent aphthous stomatitis is more common in women than in men. The ulcers usually begin during the second decade of life [1]. Recurrent aphthous stomatitis is classified into three clinical types; minor, major, and herpetiform ulcers [2]. Most of the patients (>85%) with recurrent aphthous stomatitis present with minor ulcers that have a diameter less than 1 cm [2]. Major ulcers (Sutton’s disease) have a diameter greater than 1 cm and they can persist for weeks [2,3]. Herpetiform type presents as multiple, small ulcers with 1-2 mm in size, that may heal with scarring [1].

The etiopathogenesis of recurrent aphthous stomatitis remains unknown. However, several predisposing factors have been associated with the disease. Genetic predisposition, iron, vitamin B₁₂ and folic acid deficiencies, nonsteroidal anti-inflammatory drugs, angiotensin converting enzyme inhibitors, gluten sensitive enteropathy, inflammatory bowel disease, stress, trauma to oral mucosa like dental procedures, Helicobacter pylori and viral infections have been implicated in the development of recurrent aphthous stomatitis [4].

The differential diagnosis should include chickenpox, erythema multiforme, erosive lichen planus, Riga-Fede disease, pemphigus vulgaris and pemphigoid [5]. Treatment of recurrent aphthous stomatitis can be difficult particularly in severe cases [6]. It is crucial to rule out an underlying systemic disorder in the management of disease. However, most of the patients are healthy except for painful oral ulcers [7]. Topical therapies include chlorhexidine rinse, antibiotics like doxycycline and minocyclin, diclofenac, benzocaine, lidocaine and corticosteroids. Furthermore, penicillin G, rifampin, dapsone, zinc, pentoxifylline, thalidomide, colchicine and ascorbic acid have been used in the management of recurrent aphthous stomatitis [6].

The study included 20 patients with recurrent aphthous stomatitis and 20 healthy individuals within the control group. Medical records of the participants were reviewed retrospectively between January 2018 and September 2018. Laboratory tests including complete blood count, serum levels of ferritin, folate, vitamin B₁₂, zinc, 25-hydroxyvitamin D (25(OH)D), herpes simplex virus type-1 (HSV1) IgG and herpes simplex virus type-2 (HSV2) IgG were evaluated in each participant. The exclusion criteria were pregnancy, immunosuppression, malignancy, inflammatory bowel disease, diet restrictions, vitamin and mineral supplements.

Data were represented as mean ± standard deviation or median for quantitative variables; counts and percentage for categorical variables. Differences between two groups were tested with Mann Whitney U test for continuous variables and chi-square or Fisher exact tests as appropriate for categorical variables. The data were analyzed using SPSS 20.0 Statistical Package Program.

The study included 20 patients (15 female, 5 male) with recurrent aphthous stomatitis and 20 healthy individuals (14 female, 6 male) within the control group. The mean age of the patients was 34±12.3 (range: 18-58). The mean age of the healthy individuals was 33.9±13.4 (range: 18-60) (p=1). The mean disease duration was 24.6±32.9 months (range: 1-120 months). Three (15%) patients had at least one first-degree family member who suffered from recurrent aphthous stomatitis.

The mean hemoglobin level of the patients and healthy individuals were 13.6±1.5 g/dL and 14.3±1.2 g/dL, respectively (p=0.1) (normal range: 11.7-17 g/dL). Two (10%) patients had decreased serum hemoglobin levels, while all the healthy individuals had normal serum hemoglobin levels (p=0.4). The mean white blood cell count of the patients and healthy individuals were 7.3±1.6 10³/µL and 6.7±1.3 10³/µL, respectively (p=0.3) (normal range: 4.2-10.2 103/µL). All the participants had normal serum white blood cell count. The mean platelet count of the patients and healthy individuals were 255.4±68.5 10³/µL and 252.4±54.5 10³/µL, respectively (p=0.7) (normal range: 142-450 10³/µL). One (5%) patient had low platelet count, while all the healthy individuals had normal platelet counts (p=1). The mean platelet volume of the patients and healthy individuals were 7.888±1.4 fL and 7.913±0.9 fL, respectively (p=0.7) (normal range: 6.4-11 fL). Three (15%) patients and one (5%) healthy individual had low mean platelet volume. Increased mean platelet volume was observed only in one (5%) patient (p=0.3).

The mean serum ferritin level of the patients and healthy individuals were 26.5±25.5 ng/mL and 42±30 ng/mL, respectively (p=0.04) (normal range: 10-204 ng/mL). Low serum ferritin levels were observed in 6 (30%) patients and 2 (10%) healthy individuals (p=0.2) (Fig. 1). The mean serum folate level of the patients and healthy individuals were 5.7±1.8 µg/L and 6.6±2.2 µg/L, respectively (p=0.1) (normal range: 3.1-20 µg/L). All the participants had normal serum folate levels. The mean serum vitamin B₁₂ level of the patients and healthy individuals were 343.7±171.9 pg/mL and 281.6±93.9 pg/ mL, respectively (p=0.2) (normal range: 190-880 pg/mL). Four (20%) patients had low serum vitamin B₁₂ levels, while 3 (15%) healthy individuals had low serum vitamin B₁₂ levels (p=1). The mean serum zinc level of the patients and healthy individuals were 78.8±14.4 µg/dL and 85.3±15.9 µg/L, respectively (p=0.1) (normal range: 60-150 µg/dL). All the participants had normal serum zinc levels. The mean serum 25(OH)D level of the patients and healthy individuals were 13.6±6.5 ng/mL and 20.9±10 ng/mL, respectively (p=0.01) (normal range: 20-60 ng/mL). Fourteen (70%) patients had decreased serum 25(OH)D levels, while 9 (45%) healthy individuals had decreased serum 25(OH)D levels (p=0.2) (Fig. 2).

The mean serum HSV1 IgG level of the patients and healthy individuals were 52.6±72.4 RU/mL and 80.8±83.9 RU/mL, respectively (p=0.4) (range: negative<16, positive>22 RU/mL). Positive serum HSV1 IgG levels were observed in eleven (55%) patients and in 10 (50%) healthy individuals (p=1). The mean serum HSV2 IgG level of the patients and healthy individuals were 10.5±34.3 RU/mL and 2.6±2.9 RU/mL, respectively (p=0.4) (range: negative<16, positive>22 RU/mL). Positive serum HSV2 IgG level was observed in one (5%) patient. None of the healthy individuals had positive serum HSV2 IgG level (p=1).

Despite being the most common ulcerative disease of the oral mucosa, the etiology of recurrent aphthous stomatitis has not been clearly identified yet. Therefore, current treatment options are usually aim to reduce symptoms [8]. Impaired mucosal barrier function, stress, oral microbial flora including Streptococcus sanguinis and Streptococcus mitis, Helicobacter pylori infection, diet, allergies, vitamin and mineral deficiencies, hematological parameters like ferritin, hemoglobin and hematocrit have been associated with recurrent aphthous stomatitis. Moreover, high levels of erythrocyte sedimentation rate, mean platelet volume, white blood cells, neutrophils and homocysteine have been reported in patients with recurrent aphthous stomatitis [8].

Sun et al. investigated hemoglobin, iron, vitamin B₁₂, folic acid, and homocysteine concentrations in patients with recurrent aphthous stomatitis and in healthy controls. Elevated homocysteine levels and decreased levels of hemoglobin, iron, vitamin B₁₂ and folic acid were significantly more common in patients than in control group [9]. Compilato et al. evaluated hematological deficiencies in 32 patients with recurrent aphthous stomatitis and in 29 healthy controls. Serum iron, folic acid and vitamin B₁₂ deficiencies were significantly more common in patient group than in healthy individuals [10]. Chen et al. reported high rates of hemoglobin, ferritin, folic acid and vitamin B₁₂ deficiencies in patients with recurrent aphthous stomatitis [11]. Similarly, Lopez-Jornet et al. reported that iron, folic acid, and vitamin B₁₂ deficiencies were more frequent in patients with recurrent aphthous stomatitis compared to healthy controls [12].

Within this study, the mean serum hemoglobin level was lower in patients (13.6±1.5 g/dL) compared to healthy individuals (14.3±1.2 g/dL). However, no statistically significant difference has been observed in hemoglobin levels between two groups (p=0.1). White blood cell count, platelet count and mean platelet volume were similar in patients with recurrent aphthous stomatitis and healthy individuals within the control group. The mean serum ferritin level was significantly lower in patients (26.5±25.5 ng/mL) compared to healthy individuals (42±30 ng/mL) (p=0.04). The mean serum folate level was lower in patients (5.7±1.8 µg/L) than in control group (6.6±2.2 µg/L). However, no statistically significant difference has been observed in folate levels between two groups (p=0.1). In contrast, mean serum vitamin B₁₂ levels were higher in patients (343.7±171.9 pg/mL) compared to healthy individuals (281.6±93.9 pg/ mL). However, no statistically significant difference has been observed in vitamin B₁₂ levels between two groups (p=0.2).

Association between serum zinc concentration and recurrent aphthous stomatitis remains controversial. Ślebioda et al. detected no significant difference in serum zinc levels between patients with recurrent aphthous stomatitis and healthy controls, while Ozler reported a distinct association between zinc deficiency and recurrent aphthous stomatitis [13,14].

Within this study, serum zinc levels of the participants were all in normal limits. Patients with recurrent aphthous stomatitis had lower serum zinc levels (78.8±14.4 µg/dL) compared to healthy individuals (85.3±15.9 µg/L). However, no statistically significant difference has been observed in zinc levels between two groups (p=0.1).

Low serum vitamin D level has been implicated as a facilitating factor in the development of recurrent aphthous stomatitis. Oztekin et al. reported significantly lower serum vitamin D levels in patients with recurrent aphthous stomatitis compared to healthy individuals [15]. In contrast, Krawiecka et al. reported similar serum vitamin D levels in patients with recurrent aphthous stomatitis and healthy controls [16].

Within this study, the mean serum 25(OH)D level was significantly lower in patients (13.6±6.5 ng/mL) than in control group (20.9±10 ng/mL) (p=0.01). Vitamin D deficiency was detected in 70% of the patients with recurrent aphthous stomatitis.

It has been suggested that the human herpesviruses (HHVs) may play role in the development of recurrent aphthous stomatitis. However, Brice et al. examined the members of the HHV family including HSV1, HSV2, varicella zoster virus, Epstein-Barr virus, cytomegalovirus, HHV-6, HHV-7 and HHV-8, in the oral mucosa of patients with recurrent aphthous stomatitis and control subjects. The frequency of detection of HHV DNA was not greater in the mucosa of patients than in controls [17]. In addition, Seoudi et al. reported no significant difference between HSV1 IgG and HSV2 IgG levels in patients with Behçet’s disease, recurrent aphthous stomatitis and healthy controls [18].

Within this study, a positive HSV1 IgG result was detected in 55% of the patients with recurrent aphthous stomatitis and in 50% of healthy individuals, whereas only one patient was HSV2 IgG positive. The results indicate no correlation between HSV and recurrent aphthous stomatitis.

In conclusion, this study revealed no association between recurrent aphthous stomatitis and mean platelet volume, white blood cell count, HSV1, HSV2, serum levels of hemoglobin, folate, vitamin B₁₂ and zinc. However, decreased serum levels of ferritin and 25(OH)D were more prevalent in patients with recurrent aphthous stomatitis compared to healthy individuals. Therefore, we suggest that serum ferritin and 25(OH)D levels should be evaluated in all patients with recurrent aphthous stomatitis. Replacement therapies may be helpful in the management of the disease in cases with iron or vitamin D deficiency.