Abst ract
Submitted: August 24, 2016 0RGL¿FDWLRQ1RYHPEHU Accepted: December 29, 2016
I nvest igat ions of t he prevalence
and virulence of
Candida albicans
in
periodont al and endodont ic lesions in
diabet ic and norm oglycem ic pat ient s
Pulpal and periodont al t issues have sim ilar m icrobiot a t hat allows cross-cont am inat ion bet ween t he pulp and periodont al t issues. Obj ect ive: The aim of t his st udy was t o invest igat e t he prevalence of isolat ed Candida albicans
from periodont al endodont ic lesions in diabet ic and norm oglycem ic pat ient s, and t he fungi's virulence in different at m ospheric condit ions. Mat erial and Met hods: A case- cont rol st udy was conduct ed on 15 pat ient s wit h t ype 2 diabetes m ellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Sam ples of root canals and periodont al pocket s were plat ed on &+520DJDUIRUODWHULGHQWL¿FDWLRQE\SRO\PHUDVHFKDLQUHDFWLRQ3&5DQG virulence t est . Result s: C. albicansZDVLGHQWL¿HGLQDQGRIWKH
60 sam ples collected from diabetic and norm oglycem ic patients, respectively. Of t he 30 sam ples collect ed from periodont al pocket s, 13 showed a posit ive cult ure for C. albicans, wit h 77% belonging t o G1 and 23% t o G2. Of t he 11
posit ive sam ples from root canals, 82% were from G1 and 18% from G2. 3URGXFWLRQRISURWHLQDVHSUHVHQWHGDSUHFLSLWDWLRQ]RQH3]RILQ G1 and 72% in G2, in redox and negative ( Pz= 1) , under anaerobic conditions in bot h groups. Hydrophobicit y of t he st rains from G1 indicat ed 16.4% wit h low, 19.3% wit h m oderat e, and 64.3% wit h high hydrophobicit y in redox. I n G2, 42.2% had low, 39.8% had m oderat e, 18% had high hydrophobicit y in redox. I n anaerobic condit ions, G1 showed 15.2% wit h low, 12.8% wit h m oderat e, and 72% wit h high hydrophobicit y; in G2, 33.6% had low, 28.8% had m oderat e, and 37.6% had high hydrophobicit y. There was st at ist ical GLIIHUHQFHLQWKHQXPEHURISRVLWLYHFXOWXUHVEHWZHHQ*DQG*S wit h predom inance in G1. There was st at ist ical difference for all virulence fact ors, except hem olysis ( p= 0.001) . Conclusions: Candida albicans was
isolat ed m ore frequent ly and had higher virulence in diabet ic pat ient s.
Ke yw or ds: I nfect ion. Candida albicans. Diabet es m ellit us.
Cinthya Cristina GOMES1 Ludmila Silva GUIMARÃES2 Larissa Christina Costa PINTO2 Gabriela Alessandra da Cruz Galhardo CAMARGO1 Maria Isabel Bastos VALENTE1 Maria Inêz de Moura SARQUIS3
http://dx.doi.org/10.1590/1678-7757-2016-0432
1Universidade Federal Fluminense, Faculdade de Odontologia, Departamento de Formação (VSHFt¿FD1RYD)ULEXUJR5-%UDVLO
2Universidade Federal Fluminense, Faculdade de Odontologia, Programa de Pós-graduação em Odontologia, Nova Friburgo, RJ, Brasil.
3Instituto Osvaldo Cruz, Departamento de Micologia, Laboratório de Taxonomia, Bioquímica e Prospecção de Fungos, Rio de Janeiro, RJ, Brasil.
Corresponding address: Cinthya Cristina Gomes
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I nt roduct ion
Com bined per iodont al/ endodont ic lesions have been charact erized as infect ious processes t hat occur sim ult aneously in t he pulp and t he periodont al t issue of t he sam e t oot h1.
Dental pulp and periodontal tissues have sim ilarities regarding m icrobiota and fungi infections in root canals and advanced periodont it is7,9. I n t he oral cavit y, fungi
can be isolated from m ucosal surfaces, however, it can
DOVREHIRXQGLQELR¿OPURRWFDQDOLQIHFWLRQVSHUL
im plant lesions, and periodont al pocket s24. Candida
albicansLVDFRPPRQO\LGHQWL¿HGVSHFLHVRIIXQJLDQG
has higher prevalence in saliva and in the root canal6. I t
is considered an opport unist m icroorganism found not only in teeth with pulp necrosis and apical periodontitis8,
but also in periodont al pocket s, suggest ing t hat it m ay be involved in t he pat hogenesis of periodont al disease5. Candida in the oral cavities could facilitate the
increase of pat hogenic m icroorganism s DVLWPRGL¿HV
t he host defense m echanism . There is a correlat ion bet ween increased incidence of infect ions caused by fungi and im m unocom prom ised pat ient s, including pat ient s wit h hem at ological diseases, AI DS, diabet es m ellit us, broad spect rum ant ibiot ics users, and t hose t hat use st eroids in high doses5,8,24.
Diabet es m ellit us is a chronic disease t hat m ay reduce resist ance t o m icrobial infect ion of t issues and decrease t issue repair capacit y27. I t is bot h an
endocr ine and a m et abolic dy sfunct ion inv olv ing t h e con t r ol of blood glu cose lev els, r esu lt in g in hyperglycem ia. Chronic hyperglycem ia m ay t rigger GH¿FLHQFLHVLQWKHLPPXQHV\VWHPLQFUHDVLQJWKHULVN of infect ions, including in t he oral cavit y14,15.
An associat ion bet ween diabet es and periodont al d i se a se h a s b e e n w e l l d o cu m e n t e d a n d p o o r glycem ic cont rol m ay lead t o increased severit y of periodont it is14. A bidirect ional relat ionship bet ween
diabet es m ellit us and periodont al diseases has been discussed. I t is est im at ed t hat pat ient s wit h poor glycem ic control are three tim es m ore likely to develop periodont al dest ruct ion com pared t o norm oglycem ic individuals14. However, t he lit erat ure on pat hogenesis,
progression, and endodont ic t reat m ent of pat hologies in diabet ic pat ient s is ext rem ely sparse7.
Candida species possess m ult iple virulence fact ors
t hat help in t he invasion of t he host t issue and evasion of host defence m echanism s. Such fact ors include hydrolit ic enzym es product ion, hydrophobic
interaction, hem olytic activity. The hydrolytic enzym es in clu d e secr et ed asp ar t y l p r ot ein ase ( SAP) an d phospholipase t hat cause t he inhibit ion of fagocit osis p r o cesses b y d eg r a d i n g i m m u n o g l o b u l i n s a n d extracellular m atrix proteins18. Hydrophobic interaction
play an im port ant role in t he adherence of pat hogenic m icroorganism s t o host cells by facilit at ing cont act bet w een t he parasit e and host cell1 9. Hem oly sin
act ivity, followed by iron acquisit ion, is considered an im port ant virulence at t ribut e of Candida species. I ron
is used by t he fungus for m et abolism , growt h and for facilitating hyphal invasion during host infection17. The
hem olysin act ivit y of C. albicans in diabet ic pat ient s m ay be direct ly or indirect ly increased in t he presence of high glucose concent rat ion in t he blood25.
Th e aim of t h is st u d y w as t o assess t h e p r e v a l e n ce o f Ca n d i d a a l b i c a n s i so l a t e d f r o m p er iod on t al en d od on t ic lesion s of d iab et ic an d nondiabet ic pat ient s. Addit ionally, som e vir ulence fact or s of Can dida albican s w er e evaluat ed, such as act iv it y of t he pr ot einase, phospholipase, and hem olysin, and hydrophobicit y in a reduced oxygen at m osphere or in anaerobic condit ions.
Mat erial and m et hods
This research was approved and is in accordance w it h t he Et hical Com m it t ee for Resear ch t hr ough t he docum ent CEP num ber 125.285, CAAE num ber
DQG&OLQLFDO7ULDOVLGHQWL¿FDWLRQ
NCT0 2 8 6 7 2 5 4 . All pr ocedu r es per for m ed in t h is st udy w er e in accor dance w it h t he 1964 Helsinki declarat ion. Addit ional inform ed consent was signed by all part icipant s for whom ident ifying inform at ion is included in t his paper. This case- cont rol st udy was conduct ed on t hirt y pat ient s ( 15 pat ient s wit h t ype 2 diabet es m ellit us and 15 norm oglycem ics) w it h clinical and radiographic diagnosis of a periodont al endodont ic lesion. To assess t he glycem ic cont rol of t he pat ient s, plasm a glucose level and glycosylat ed hem oglobin concent rat ions ( HbA1c) were m easured.
Plasm a glucose levels of each pat ient at t he m om ent of sam pling was not estim ated. The diabetes diagnosis was perform ed according t o t he Guidelines of The Am erican Diabet es Associat ion, which recom m ends t he m easurem ent of plasm a glucose and glycosylat ed hem oglobin level concent rat ions ( HbA1c)2.
t o verify t he clinical periodont al param et ers ( plaque in dex , pr obin g bleedin g, pr obin g dept h , clin ical at t achm ent level, and gingival recession) and pulp sensit ivit y t est ing. The st udy included pat ient s who present ed periodont al pocket s involving t he apical region of t he t oot h and pulp necrosis ( periodont al en dodon t ic lesion ) . Ex clu sion cr it er ia w er e: u se of ant ibiot ics in t he last six m ont hs, pr egnancy, sm oking, other system ic diseases, clinical signs of oral candidiasis, and pat ient s who did not sign a free and
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Sam ple collect ion
The teeth were isolated with a rubber dam isolation. The crown and surrounding structures were disinfected wit h 5.25% sodium hypochlorit e, and neut ralized wit h 5% sodium t hiosulfat e. The st erilit y of t he ext ernal surfaces of t he crown was checked by t aking a swab sam ple from t he crown surface and st reaking it on blood agar plat es, which were t hen incubat ed bot h aerobically and anaerobically. The preparat ion of t he access cavit y was m ade using a st erile diam ond drill speed, without the use of water spray, but with m anual irrigat ion wit h st erile saline. Before ent ering t he pulp cham ber, t he access cavit y was again disinfect ed according to the protocol described above. The sterility of the internal surface of the access cavity was checked as pr ev iously descr ibed, and all pr ocedur es w er e perform ed asept ically. Sam pling of t he periodont al pocket was m ade at t he deepest sit e t hrough t hree st erile paper point s, insert ed one by one for one m inut e. Prior t o periodont al sam pling, all part icipant s were request ed t o rinse t heir m out h wit h 10 m l of 0.12% chlorhexidine gluconat e solut ion ( Periogard®
– Colgat e- Palm olive Com pany, São Paulo, SP, Brazil) , and t he local area was dried and isolat ed wit h cot t on rolls. Mat erial collect ed from t he periodont al pocket and t he root canal was inoculat ed in Eppendorf t ubes
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Subsequent ly, sam ples from each sit e were dilut ed and plat ed on t he m edium Saboraund Dext rose Agar plu s Ch lor am ph en icol an d ch r om ogen ic m ediu m ( CHROMagar Candida®, BioMerieux, Rio de Janeiro,
5-%UD]LODQGLQFXEDWHGDW&IRUKLQDUHGXFHG
oxygen at m osphere ( 10% CO2 and 90% air) . The green colonies t hat grew on CHROMagar plat es were
FKRVHQDWUDQGRPDQGVWRUHGLQJO\FHURODW&IRU ODWHULGHQWL¿FDWLRQE\3&5
Molecular charact erizat ion of t he species
Fifty m l of Sabouraud m edium were inoculated with FRQLGLDDQGLQFXEDWHGIRUKDW&0\FHOLXPZDV t ransferred t o a 50 m l polypropylene screw- capped bot t le cont aining six glass beads ( 4 m m in diam et er) . The t ube was im m ersed in liquid nit rogen for 10 s and st irred vigorously wit h a vort ex m ixer for 20 s. Then, 2 m l of DNA ext ract ion buffer and 0.5 m g per m l prot einase K ( Sigm a Chem ical Co., St . Louis, MO, USA) were added, and t he m ixt ure was allowed t o thaw. For the next step, the m ixture was incubated for KDW&3URWHLQDVH.ZDVLQDFWLYDWHGE\KHDWLQJ WKHPL[WXUHDW&IRUPLQ$YROXPHRIPO of t he m ixt ure was t ransferred t o a 1.5 m l m icrofuge t ube and an equal volum e of phenol- chloroform ( 1: 1) was added. The m ixt ure was cent rifuged at 10.000x JIRUPLQDW&6XSHUQDWDQWZDVWUDQVIHUUHGWR a new t ube and t he sam e procedure was repeat ed using chloroform isoam yl alcohol ( 24: 1) . DNA was SUHFLSLWDWHGZLWKYROXPHVRIHWKDQRODW&DQG FHQWULIXJHG DW [ J IRU PLQ DW & 7KH pellet was allowed t o dry. Aft er washing wit h 70% HWKDQRO DW & WKH H[WUDFWHG '1$ ZDV GLVVROYHG in 50 NjOof dist illed wat er and 5 NjOof suspension and was used for PCR.
Polym erase chain react ion ( PCR)
PCR react ions were st andardized for each prim er, using genom ic DNA st rains as a posit ive cont rol. PCR react ion wit h DNA was perform ed for each universal prim er t o verify t he presence of DNA as C. albicans
(Forward: 5 ‘-ACT GCT CAA ACC ATC TCT GG- 3 ‘ and Reverse: 5 ‘ - CAC AAG GCA AAT GAA GGA AT- 3; wit h fragm ent size of 472 bp)19,and st erile dist illed wat er
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perform ed in T100 Therm al Cycler (PowerPac Basic,
Bio- Rad Laborat ories LTDA, Sant o Am aro, SP, Brazil) , program m ed according t o t he following st eps: pre-GHQDWXUDWLRQ&PLQF\FOHVGHQDWXUDWLRQ &VDQQHDOLQJ7DR&FDOFXODWHGVDQG H[WHQVLRQ & PLQ V )LQDO H[WHQVLRQ ZDV
SHUIRUPHGDW&IRUPLQ20.
(OHFWURSKRUHVLVRIDPSOL¿FDWLRQSURGXFW
$IWHUDPSOL¿FDWLRQDQDQDOLTXRWRINjO race m arker
wat er] , and t he t ot al volum e was applied t o a channel of t he gel. An aliquot of 10 NjO of PCR react ion product was added t o t he DNA 5 NjO at a t im e separat ely from t he running dye. Dilut ions were applied on different channels and subj ect ed t o elect rophoresis ( PowerPac Basic, Bio- Rad Laborat ories LTDA, Sant o Am aro, SP, Brazil) in agarose gel at 1.5% ( w/ v) in TAE 1X buffer ( Tris-acet at e, 40 m M EDTA 1 m M, pH 8.0) at 90 V for 90 m inut es. The gel was subsequent ly t reat ed wit h ethidium brom ide at 0.5 NjO/ m l and DNA was visualized wit h t he aid of an ult raviolet t ransillum inat or ( UV) and phot ographed wit h t he aid of an aut om at ic det ect ion and im age analysis syst em (Gel Doc XR + Syst em wit h I m ageLab Soft ware, Bio- Rad Laborat ories LTDA, Sant o Am aro, SP, Brazil) .
Pr o t e i n a se a n d p h o sp h o l i p a se a ct i v i t y
det erm inat ion
All isolat es of C. albicans were t est ed in t riplicat e, for three separate experim ents, to verify the enzym atic activity of proteinases ( SAPS) and phospholipases16,28.
The pr ot einase cult ur e m edium was BSA ( bov ine serum album in) and the phospholipase culture m edium consist ed of enriched egg yolk. Plat es were incubat ed DW & LQ D UHGXFHG R[\JHQ DWPRVSKHUH RU LQ anaerobic conditions for 48 h to exam ine the proteases and for 72 h t o exam ine phospholipases. Enzym at ic act ivit y was det erm ined by t he form at ion of a halo around t he colony of yeast . The halo was m easured in t erm s of t he relat ionship bet ween t he diam et er of t he colony and t he overall diam et er of t he colony plus t he precipit at ion zone ( Pz) , according t o t he m et hod described by Price, Wilkinson and Gent ry16 ( 1982) .
Det erm inat ion of hem olysin act ivity
Hem olysin activity was evaluated for all the isolates of C. albicans using blood agar m edium25. Sam ples
ZHUHJURZQRQ6'$IRUKDQGLQFXEDWHGDW& in eit her a reduced oxygen at m osphere or anaerobic condit ions for 48 and 72 h, respect ively. An addit ional 10 NjO of saline wit hout yeast was placed on t he sam e plat e. The r efer ence st rain of C. alb ican s ( ATCC 90.028) was used as a posit ive cont rol. I n addit ion, st andar d st rains of St aph y lococcu s au r eu s ( ATCC
10.832) and St rept ococcus m ut ans ( UA159) , which repect ively induce hem olysis bet a and alpha, were used as a posit ive cont rol. Gam m a- hem olyt ic sam ples produce no halo, alpha- hem olyt ic sam ples produce a green halo, and bet a- hem olyt ic sam ples produce a yellow halo. Assays were also conduct ed in t riplicat e.
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This assay was perform ed according t o Rodrigues, et al.19 ( 1999) for all isolat es of C. albicans, in t wo independent ex per im ent s. Fift y m l of Sabouraud dex t r ose br ot h ( SDB, Disco Laborat or ies Det r oit , MI , USA) w er e in ocu lat ed w it h y east cells an d LQFXEDWHGRYHUQLJKWDW&LQDUHGXFHGR[\JHQ environm ent or in anaerobic condit ions. Yeast cells were harvest ed and washed t wice in phosphat e buffer ( pH 8.0) . Yeast slur r y was pr epar ed in t he sam e buffer t o reach an opt ical densit y [ ( A0) 0.4 - 0.6 at 600 nm ] , and 150 NjO of hexadecane was added t o 3 m l of t his yeast suspension. Aft er 10 m in incubat ion DW&WXEHVZHUHDJLWDWHGE\YRUWH[LQJWZLFHIRU 30 seconds. Aft er allowing phase separat ion for 30 m in, t he opt ical densit y of t he lower aqueous phase ( A1) was m easured and com pared wit h t he opt ical densit y obt ained before t he m ixing st ep ( A0) . The percent age of cells in t he hexadecane layer ( adherent cells) was used t o est im at e t he hydrophobicit y, using t he following form ula: % H= A0-A1/ A0 x 100% .
St at ist ical analysis
Dat a dist r ibut ions w er e ex pr essed as m eans, st andard deviat ions ( SD) , ranges and percent ages, as appropriat e. The result s obt ained for t he t est ed v ir ulence fact or s w er e analy zed accor ding t o t he atm ospheric condition, using Chi- square and Wilcoxon nonparam et r ic t est s. Differ ences w er e consider ed VWDWLVWLFDOO\ VLJQL¿FDQW ZKHQ S $OO GDWD ZHUH analy zed using t he St at ist ical Pack age for Social Sciences ( SPSS, Chicago, I L) version 16.0.
Result s
Diabet ic and cont rol groups were hom ogenous. Mean an d st an d ar d d ev iat ion w er e ob t ain ed t o est ablish com parisons bet ween groups ( Table 1) .
Of t he 60 sam ples collect ed ( Figures 1 and 2)
– 30 fr om per iodont al pocket s and 30 fr om r oot canals – 24 ( 40% ) showed a posit ive cult ure for C. albicans. Ninet een of t hese 24 were from pat ient s
ZLWKGLDEHWHVPHOOLWXVDQG¿YHIURPQRUPRJO\FHPLFV
sam ples from root canals, 9 were from pat ient s wit h diabet es m ellit us ( 82% ) and 2 from norm oglycem ic pat ient s ( 18% ) .
C. alb ican s st rains w er e found m or e oft en in diabet ic pat ient s. There was a st at ist ical difference for t he num ber of st rains isolat ed from periodont al pockets and root canals of diabetic and norm oglycem ic SDWLHQWVZKHQDQDO\]HGE\&KLVTXDUHWHVWS
St r ain s of C. a l b i ca n s sh ow ed an in cr eased virulence in diabet ic pat ient s ( Table 2) , dem onst rat ing D VWDWLVWLFDOO\ VLJQL¿FDQW GLIIHUHQFH S DIWHU applying t he Wilcoxon t est . Hem olysin act ivit y was posit ive for bot h groups in bot h at m ospheres, but was
QRWVLJQL¿FDQWO\GLIIHUHQWDPRQJWHVWJURXSVLQERWK at m ospheres.
3URGXFWLRQ RI SURWHLQDVH SUHVHQWHG 3] RI 100% in G1 and 72% in G2 in redox and negat ive ( Pz= 1) , under anaerobic condit ions in bot h groups. Phospholipase act ivit y was higher in sam ples from G1 in redox and anaerobic condit ions. Hydrophobicit y of t he st rains of G1 indicat ed 16.4% wit h low, 19.3% wit h m oderat e and 64.3% wit h high hydrophobicit y in redox. I n G2, 42.2% showed low, 39.8% m oderat e, 18% high hydrophobicity in redox. Anaerobically, G1 present ed 15.2% wit h low, 12.8% wit h m oderat e and 72% wit h high hydrophobicit y; G2, showed 33.6%
Diabetics (n=15) Controls (n=15)
Min Max Mean SD Min Max Mean SD
age 38 69 52 10.23 39 70 53.3 12.12
PD 4 14 8.2 3 4 14 8.3 3.2
CAL 9 18 13.5 3.34 7 24 12.9 4.44
HBA1C 6.2 11.3 8.5 1.78 5 5 5 0
GLIC_L 27 295.8 190.3 71.64 74 90 84.5 5.01
Diabetics (n=15) Controls (n=15)
n (%) n (%)
Sex male 6 (40) male 5(33.3)
female 9 (60) female 10 (66.7)
PI
no 0 no 1 (6.7)
yes 15 (100) yes 14 (93.3)
GI
no 1 (6.7) no 2 (13.3)
yes 14 (93.3) yes 13 (86.7)
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Table 1- Main characteristics of the subjects studied
Figure 1- Radiographic image of tooth with periodontal
w it h low, 28. 8% w it h m oderat e and 37. 6% w it h high hydrophobicit y. There was st at ist ical difference in t he num ber of posit ive cult ures bet ween G1 and * S ZLWK SUHGRPLQDQFH LQ * 7KHUH ZDV st at ist ical difference for all virulence fact ors, except hem olysis ( p= 0.001) .
Discussion
Fungi are considered norm al inhabitants of the oral cavit y, however, it m ay cause diseases when local or syst em ic fact ors such as diabet es m ellit us predispose the individual. Prem kum ar, Ram ani and Chandrasekar15
( 2014) showed t hat pat ient s wit h diabet es m ellit us
KDYHDVLJQL¿FDQWO\LQFUHDVHGSUHYDOHQFHRIC. albicans
st rains in t he oral cavit y. The prevalence rat e of t he
Candida species in diabet ics was 87.5% , whereas in
the control subj ects it was 50% ( collected by oral rinse m ethod) . I n the present study, 40% showed a positive cult ure for C. albicans, 79.2% were from pat ient s wit h diabet es m ellit us, and 20.8% from norm oglycem ics. This discrepancy m ay be explained by t he fact t hat t he sam ples in t his st udy were collect ed from periodont al pocket sand root canals.
Can d i d a al b i can s is t h e f u n g al sp ecies m ost com m on ly d et ect ed in t h e or al cav it y, b ot h in healt hy individuals and in individuals wit h syst em ic involvem ent23. I t is also t he m ost oft en isolat ed fungal
species in chronic periodont it is19,21 and infect ed root
canals22,23. Kum ar, Muralidhar and Banerj ee11 ( 2015) isolat ed C. albicans in 21% of sam ples from pat ient s with periodontal disease. Urzua, et al.26 (2008) isolated
t he fungus in 69.2% of periodont al sit es of chronic periodont it is. I n t he present st udy, C. albicans was
isolat ed in 43.3% of t he sam ples collect ed from t he periodont al pocket s. Different result s could be relat ed t o eit her different collect ion sit es or syst em ic fact ors, as these sam ples were com posed by both diabetic and norm oglycem ic pat ient s.
This higher incidence of C. albicans in diabet ic pat ient s m ay be explained by t he alt erat ion of t he QRUPDORUDOÀRUDLQUHVSRQVHWRHQGRFULQHGLVRUGHUV of diabet es m ellit us. Moreover, it m ay be at t ribut ed t o incr eased adhesion of fungi in epit helial cells facilit at ed by increased glucose cont ent in saliva, genet ic suscept ibilit y t o infect ion, changes in defense m echanism s, and changes in local fact ors, including disadvant aged blood support1.
However, Brem enKam p, et al.4 ( 2011) showed no
VLJQL¿FDQWGLIIHUHQFHLQWKHSUHYDOHQFHRIC. albicans
bet ween diabet ic and norm oglycem ic pat ient s. This cont radict ion wit h our result s can be at t ribut ed t o t he fact t hat pat ient s included in t he st udy cit ed above beared glycat ed haem oglobin levels lower or equal t o 9% , whereas in t he present st udy pat ient s present ed glycat ed hem oglobin levels ranging from 6.2% t o 11.3% . Blood glucose levels have a posit ive
Virulence of Candida albicans strains Diabetic (G1) Normoglycemic (G2)
Redox Anaerobic Redox Anaerobic Negative (Pz=1) 0 100% Negative (Pz=1) 2% 100% PROTEINASE Positive (Pz=1 - 0.63) 0 0 Positive (Pz=1 - 0.63) 6% 0
Strongly Positive 100% 0 (Pz<0.63)
Strongly Positive 72% 0 (Pz<0.63)
Redox Anaerobic Redox Anaerobic Negative (Pz=1) 18.5% 51.2% Negative (Pz=1) 0.3% 100% PHOSPHOLIPASE Positive (Pz=1 - 0.63) 28.3% 15.3% Positive (Pz=1 - 0.63) 6.4% 0
Strongly Positive 53.2% 33.5%
(Pz<0.63) Strongly Positive 23.3% 0(Pz<0.63) Redox Anaerobic Redox Anaerobic Alpha 84.8% 80.4% Alpha 81% 70.3% HEMOLYSIN Beta 15.2% 19.6% Beta 19% 29.7%
relat ionship wit h increasing t ransport rat e and cell densit y in diabet ic pat ient s3.
Virulence fact ors of t he C. albicans species m ay be
involved in t he pat hogenesis of various oral diseases. However, few st udies have invest igat ed it s role in host colonizat ion and developm ent of infect ions10.
I n this study, it was observed that the fungi present in diabet ic pat ient s had prot einase, phospholipase, and hem olysin activity, and cell surface hydrophobicity in bot h t est ed environm ent s. This is in line wit h t he literature on this fungi’s adaptive survival conditions12.
I n the present investigation, proteinase expression was det ect ed in 100% of t he st rains of C. albicans
when incubat ed in a redox at m osphere. These result s DUHVLPLODUWRWKH¿QGLQJVRI6DUGLHWDO21 ( 2012), who
found a high activity of proteinase in patients with type 2 diabet es m ellit us.
Phospholipase act ivit y on C. albicans st rains not
only det er m ine com m ensal colonizat ion, but also provide pot ent ial pat hogenic yeast s11. St udies have
r epor t ed t hat 30 - 100% of t he oral C. albican s
isolates produce phospholipases, with varying degrees of enzym e act ivity25. I n t his st udy, t his enzym e was
det ect ed in 53.2% of Candida albicans st rains when incubat ed in a r educed ox y gen at m ospher e. The anaerobic condit ion showed 33.5% act ivit y.
2XU DQDO\VLV VKRZHG QR VWDWLVWLFDOO\ VLJQL¿FDQW difference for t he hem olysin act ivit y in diabet ic and norm oglycem ic pat ient s in bot h t est ed at m ospheres.
Manns, Mosser and Buckley13 ( 1994) det erm ined t he
conditions under which C. albicans can show hem olytic activity and found that hem olysis is not observed when glucose is not available in t he cult ure m edium .
The cell sur face hydr ophobicit y was evaluat ed u n der an aer obic an d r edox con dit ion s, w it h t h e result s indicat ing great er hydrophobicit y in anaerobic condit ions. The result s of t his st udy indicat ed t hat 72% of t he isolat es were highly hydrophobic under anaerobic at m ospheres and 64.3% under t he redox at m osp h er es. Hy d r op h ob ic in t er act ion s m ay b e im port ant in t he t issue invasion of t he m ycelia phase of yeast cells21.
2XU¿QGLQJVIROORZWKHVDPHWUHQGRIWKHDXWKRUV
Siqueira and Sen23 ( 2004) , and Gom es, et al.8 ( 2010) ,
w h o inv est igat ed t h e im por t an ce of filam en t ou s fungi in root canals of t eet h wit h pulp necrosis and periradicular lesions, which m ay part icipat e in t he et iology of periradicular diseases.
The durat ion of diabet es and qualit y of m et abolic
cont rol are relat ed t o t he developm ent of diabet ic com plicat ions. Poorly cont rolled diabet es has been associat ed wit h high incidence of Candida infect ions DQGVXFKVLWXDWLRQPD\VXJJHVWGLI¿FXOW\LQWUHDWPHQW4.
C. albicans seem s t o be present m ore frequent ly in t he root canals of obt urat ed t eet h in which t reat m ent has failed22.
Conclusion
The prevalence of Candida albicans was higher in pat ient s wit h diabet es m ellit us in sam ples collect ed from periodont al pocket s and root canals. St rains of Candida albicans showed increased virulence in diabetic patients com pared to norm oglycem ic patients. The pr oduct ion of pr ot einase and phospholipase activity were higher in diabetic patients when analyzed in redox. Hydrophobicit y of t he st rains were higher in diabetic patients under anaerobic conditions. However, hem olyt ic act ivit y was posit ive for bot h groups in bot h at m ospheres.
Acknowledgem ent s
7KHDXWKRUVGHQ\DQ\FRQÀLFWRILQWHUHVWUHODWHGWR this study. We thank FAPERJ – Rio de Janeiro Research )RXQGDWLRQ5-%UD]LOIRU¿QDQFLDOVXSSRUW3URFHVV num ber E.26/ 111.809/ 2012) .
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