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Research ArticleEvaluation ofCandidaColonization andSpecific Humoral Responses against Candida albicansin Patients with Atopic Dermatitis

Ghaffari Javad,1 Mehdi Taheri Sarvtin,2,3 Mohammad Taghi Hedayati,3,4

Zohreh Hajheydari,4,5 Jamshid Yazdani,6 and Tahereh Shokohi3,4

Department o Pediatrics, School o Medicine, Mazandaran University o Medical Sciences, Sari, IranDepartment o Medical Mycology and Parasitology, School o Medicine, Jirof University o Medical Sciences, Jirof, IranDepartment o Medical Mycology and Parasitology, School o Medicine, Mazandaran University o Medical Sciences, Sari, IranInvasive Fungi Research Center, Mazandaran University o Medical Sciences, Sari, IranDepartment o Dermatology, School o Medicine, Mazandaran University o Medical Sciences, Sari, IranDepartment o Statistics, School o Health, Mazandaran University o Medical Science, Sari, Iran

Correspondence should be addressed to ahereh Shokohi; shokohi.tahereh@gmail.com

Received December ; Revised March ; Accepted March

Academic Editor: Davinder Parsad

Copyright Ghaffari Javad et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Te aim of this study was to assess the candidal colonization and specic humoral responses against Candida albicansin patientswith atopic dermatitis. One hundred patients with atopic dermatitis and healthy individuals were enrolled in the study. Skin andoral specimens from all participants were cultured on CHROMagarCandidamedium. Isolated yeasts were identied by using thesequence of the D/D domain of the S rRNA gene. ELISA was used for detection of IgM, IgA, and IgG antibodies against C.albicans in sera of participants.Candidaspecies were isolated from the skin and oral cavity of % of the patients and % of thecontrols. Tere was no signicant difference betweenCandidacolonization in patients and controls ( >.).Candida albicanswas isolated from the skin and oral cavity of % of the patients and % of the controls ( .). Serum level of IgG was signicantly lower inpatients than in controls (

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with this disease should be assessed and controlled. So faronly two studies investigated Candida colonization in skinand oral cavity of patients with atopic dermatitis and haveprovided different results [, ]. Candida species can playan important role in the pathogenesis of atopic dermatitis

via stimulation of humoral immune system and reaction

with immunoglobulins []. Some researchers believe thatproduction of specic antibodies againstCandida albicansisassociated with increased severity of atopic dermatitis. Butall the studies only examined the production of IgE antibodyagainstCandida albicans in these patients [,]. Tereforethis study was designed to investigate the colonization ofCandida species on the skin and oral cavity and productionof IgM, IgG, and IgA antibodies againstCandida albicansinpatients with atopic dermatitis.

2. Materials and Methods

.. Patients. One hundred patients with atopic dermatitis

and healthy individuals as control group from January to March were enrolled in the study. Te patientsand controls lled out the consent form to participate inresearch and the study was approved by the ethical committeeof Mazandaran University of Medical Sciences, Sari, Iran.

Control subjects were selected from persons who werereferred for cosmetic problems. People who had diabetes andthose who had used broad spectrum antibiotics and steroidsas well as pregnant patients were excluded from the study. Inorder to assess clinical severity of the disease, the SCORAD(SCORing Atopic Dermatitis) index was calculated as eluci-dated by Kunz et al. in []. Based on this denition,clinical severity of atopic dermatitis was categorized to mild

(SCORAD index ).

.. Mycological Investigation. Te samples were collectedfrom oral cavity and skin by swab and scalpel, respectively.All of the samples were cultured on CHOROMagar Candidamedium (CHOROMagar Company, Paris, France). Te iso-lated species ofCandida were subcultured on Sabouraudsdextrose agar containing chloramphenicol (SC) and incu-bated at C for days.

.. Molecular Investigation. Te DNA of the isolated yeastswasextracted according to the procedureof Yamada et al. [].

Yeasts were identied to the species level using sequenceanalysis of the D/D domain of the S ribosomal RNAgene. For amplication of the D/D domain, the externalprimers NL- (-GCA A CAA AA GCG GAG GAAAAG-) and NL- (-GG CCG G C AAG ACGG-) [] were used. Te reactions were performed in anautomatic thermal cycler (C Termal Cycler, Bio-RadLaboratories) with initial denaturation at C for min; cycles at C for s, C for s, and C for s; nalextension at C for min. Te quality of PCR productswas determined by electrophoresis in % (w/v) agarose gelin x BE (mM ris-base, mM boric acid, and mMEDA) at V for h. GenRuler DNA ladder mix was

used as a marker. Sequencing of the puried PCR productswas performed using the primers NL-. Sequences werealigned to the S rRNA gene sequences obtained fromthe National Center for Biotechnology Information (NBCI)Genbank database (http://www.ncbi.nlm.nih.gov/) and yeastspecies were identied by searching databases using the

BLAS algorithm.

.. Detection o Anti-C. albicans Antibodies. Serum IgG,IgM, and IgA levels were measured with enzyme-linkedimmunosorbent assay (ELISA) test kits (Genesis Diagnostic,England) according to the manufacturers instructions.

.. Statistical Analysis. Numbers of individuals with yeastgrowth were compared with the Chi-Square test. Indepen-dent -test was used to compare the results of IgG and IgMassay in two groups. values less than . were consideredsignicant. Te results of IgA assay were analyzed by Kruskal-Wallis test and paired comparisons were performed by means

of the Mann-Whitney test. values less than . wereconsidered signicant. Te correlation between Candidacarriage and levels of antibodies against C. albicans withthe severity of disease (SCORAD index) were examinedby means of Chi-Square test and Pearsons correlation test,respectively. Logistic regression was used to control con-founding effects of age and sex.

3. Results

In this study, patients ( male and female; age mean12.1 11.5years) and controls ( male and female; agemean . . years) wereexamined. By logistic regressionanalysis, age and sex do not inuence Candidacolonizationand levels of antibodies ( > 0.05). Tirty-one percent ofpatients and % of controls were colonized by yeast species( = 0.243). Candida species were isolated from the oralcavity of (%) patients and (%) controls ( = 0.133).Candida species were isolated from the skin of (%) patientsand (%) of controls. wenty-nine percent of patients and% of controls were colonized by only one yeast species.wo percent of patients and % of controls were colonizedby two different yeast species.Candida albicansandCandida

glabratawere the most common yeast species isolated frompatients and control, respectively. Isolated yeast species arelisted inable . wenty-three percent of patients and % of

controls were colonized byCandida albicans ( = 0.006).SCORAD index wereobserved in %, %, and % of the patientswith atopic dermatitis, respectively. % of patients with milddisease, .% of patients with moderate disease, and %of patients with severe disease were colonized byCandidaspecies. IgG and IgM titers against Candida show normaldistribution in patients and control group. Te mean level ofIgMagainst C. albicans in patients andcontrols was. U/mLand . U/mL, respectively ( = 0.112). Te mean levelof IgG againstC. albicansin patients and controls was .and . U/mL, respectively ( < 0.001). IgA levels againstCandidashow abnormal distribution in patients and control

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: Yeast species isolated from patients with atopic dermatitis and controls.

Yeast species

Oral specimens Skin specimens

Patients(%)

Controls(%)

Patients(%)

Controls(%)

Candida albicans (.%) (%) (%) (%)

Candida glabrata (%) (.%) (%) (%)Debaryomyces hansenii (.%) (%) (%) (%)

Candida tropicalis (%) (.%) (%) (%)

Candida dubliniensis (.%) (%) (%) (%)

Candida parapsilosis (.%) (%) (%) (%)

Issatchenkia orientalis (.%) (.%) (%) (%)

otal (%) (%) (%) (%)

group. Te mean level of IgA against C. albicans in patientsandcontrols was. U/mL and. U/mL, respectively ( =0.022). Te Pearson correlation between levels of IgM, IgG,and IgA and severity of disease was ., ., and .,respectively ( > 0.05). Fourteen percent of patients and %of controls withCandidacolonization showed IgM level lessthan U/mL (Figure ). Fourteen percent of patients and %of controls withCandidacolonization had IgA level less than U/mL (Figure ). Te IgG levels of % of patients and %of controlswith Candida colonization were less than U/mL(Figure ).

4. Discussion

In the present study, there was no signicant differencebetween the rate of Candida species colonization in theoral cavity and skin of patients with atopic dermatitis andcontrols. Tis result is incompatible with Henseler andauschs study [] and is compatible with Leibovici et al.sstudy []. However, the differences in the results of thesestudies may be due to differences in race, age, and diseaseseverity of the study population. In thepresent studyalthoughthere was no signicant difference between the colonizationofCandida species in the patients and controls, impairedimmune systems of patients with atopic dermatitis can reactto the normal rate ofCandidaspecies colonization and alterthe course of the disease []. In the present study, Candidaalbicans colonization in the oral cavity of patients was sig-nicantly higher than controls.Candida albicansis the most

common and important species []. Protein compounds ofthis species such as proteins , , , , , and kDacan play an important role in the pathogenesis of atopicdermatitis via stimulation of immune system and reactionwith immunoglobulins []. Some researchers have reportedthat increased severity of atopic dermatitis is associated withthe production ofCandida albicans-specic antibodies [,,]. In Matsumura et al.s study [], level of IgE antibodyagainstCandida albicans in patients with atopic dermatitiswas signicantly higher than in controls. In Faergemannstudy, pediatric patients and patients with severe atopicdermatitis had higher level of IgE antibody against Candidaalbicans than those with mild disease and controls []. In

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0

10.0 10.1

20.0 20.1

30.0 30.1+

F : Distribution of serum level of IgM specic againstCandida albicans in patients with atopic dermatitis and control(mean of IgM in patients: 11.6 7.7and mean of IgM in controls:14.68 12.00).

Matsumura et al.s study [], % of patients with atopic

dermatitis have a high level of IgE antibody against Candidaalbicans. As noted above, most studies examined specic IgEto Candida albicans and its immunoglobulin-reactive proteinin patients with atopic dermatitis. Hence, in this study, theproduction of other classes of antibody to Candida albicansincluding IgG, IgA, and IgM was evaluated. In the presentstudy, there were no signicant differences between the levelsof IgM and IgA antibodies againstCandida albicansin serumof the patient and control groups, but level of specic IgGtoCandida albicanswas signicantly lower in patients thanin controls. IgG is the major immunoglobulin in normalhuman serum and is a key player in the humoral immuneresponse []. Te reductionin specic IgG production maybe

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Patients withoutCandida

species colonizationPatients with Candidaspecies colonization

Control without Candidaspecies colonization

Control with Candidaspecies colonization

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010.0 10.120.0 20.130.0 30.1+

F : Distribution of serum level ofIgA specic against Candidaalbicansin patients with atopic dermatitis and control (mean of IgAin patients: 13.18 12.83and mean of IgA in controls:13.8 4.74).

030.0 30.16 0. 0 6 0. 180.0 80.11 00 .0 1 00 .0 +

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Patients without Candidaspecies colonization

Patients with Candidaspecies colonization

Control without Candidaspecies colonizationControl with Candidaspecies colonization

F : Distribution ofserum level of IgG specic against Candidaalbicansin patients with atopic dermatitis and control (mean of IgGin patients:42.64 31.5and mean of IgG in controls: 90.03 40.21).

the etiology for the increased Candida albicanscolonizationin patients with atopic dermatitis. According to the surveywhich was conducted, so far the levels of specic antibodiesagainstCandida albicans have not been evaluated in serumof patients with atopic dermatitis. So it seems that thepresent study is the only study that has addressed this issue.

In our study, there were no signicant differences amongantibody levels, severity of illness, and intensity ofCandidacolonization. In present study, we applied logistic regressionto control confounding effects of age and sex. Terefore ageand gender had no effect on the results of this study.

Te results of this study showed that type ofCandida

colonization can change in patients with atopic dermatitis.Moreover, these patients have abnormalities in the produc-tion of antibodies againstCandida albicans that may have arole in the pathogenesis of atopic dermatitis.

Conflict of Interests

Te authors declare that they have no conict of interests.

Acknowledgments

Te authors are grateful to the Vice-Chancellor of Researchof Mazandaran University of Medical Sciences and for the

nancial support that they received.

References

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