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1 Medical Microbiology
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11 a r t i c l e i n f o a b s t r a c t
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23 Vulvovaginal candidiasis (VVC) is characterized by an infec- ing, cracking, erythema and vulvar edema, leukorrhea and 30
24 tion of the genital mucosa by yeasts, which mainly the presence of whitish plaques on the vaginal mucosa
: Laboratrio de Micologia, Faculdade de Medicina, Universidade Federal do Rio Grande, Rio Grande, RS, Brazil.
E-mail: melissaxavierfurg@gmail.com (M.O. Xavier).
http://dx.doi.org/10.1016/j.bjm.2016.09.006
1517-8382/ 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Brandolt TM, et al. Prevalence of spp. in cervical-vaginal samples and the susceptibility of
isolates. (2016), http://dx.doi.org/10.1016/j.bjm.2016.09.006 BJM 156 16
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31 are common.1 VVC is considered the second most common under freezing at 20 C in saline with 30% glycerol for subse- 86
32 cause of genital infection in women of reproductive age, and quent susceptibility testing. 87
33 although it represents a problem of global importance in pub- The variables evaluated in the study were obtained by self- 88
34 lic health, its exact incidence is unknown.2,3 administered pre-coded questionnaire and included age, skin 89
35 Risk factors for vulvovaginitis include pregnancy, use color, educational level, marital status, family income, HIV 90
36 of oral contraceptives, genetic predisposition, and previous infection status, pregnancy, contraception and vaginal pH at 91
37 antibiotic therapy, among others. The incidence of infection, the time of sample collection. Signs and symptoms assessed 92
38 as well as the increase in colonization of the mucosa by the during the gynecological exam were used to consider women 93
39 yeast, is also higher in women with diabetes due to their as colonized or presenting VVC, the latter being those that had 94
40 higher glycogen levels and in those with HIV due to immune leukorrhea, pruritus, edema and vulvovaginal erythema asso- 95
41 suppression.4,5 ciated with the presence of whitish plaques in the mucosa and 96
42 Studies show that 7075% of all women of reproductive age the isolation of spp. in mycological cultivation. 97
43 develop at least one case of VVC during their lifetime.6,7 The The susceptibility analysis of clinical isolates was 98
44 recurrence rate is 4050%, and approximately 58% develop performed by broth microdilution using the M27-A2 standard 99
45 recurrent vulvovaginal candidiasis (RVVC), which is charac- protocol CLSI (2002).14 For the standardization of fungal inocu- 100
46 terized by four or more episodes of disease over a period of lum, isolates were subcultured on PDA for 24 h at 37 C, young 101
47 twelve months.68 colonies were homogenized in sterile saline, and the turbidity 102
48 The main etiologic agent is , accounting for was adjusted to 0.5 on the McFarland scale by spectrophoto- 103
49 7090% of VVC cases.7,8 Among the non- species metry (530 nm) to obtain a concentration of 15 106 CFU/mL. 104
50 is highlighted79 due to its prevalence and resistance Then, a dilution was performed at 1:100 and 1:20 in RPMI 1640, 105
52 An increasing number of spp. clinical isolates are inoculum concentration was confirmed using the Pour-plate 107
53 resistant to antifungal agents routinely used for the treat- technique. The susceptibility test was performed at concen- 108
54 ment of VVC. In addition, studies in different regions of Brazil trations of 64 !g/mL to 0.125 !g/mL for fluconazole and at 109
55 suggest that geographical factors interfere in the prevalence concentrations ranging from 16 !g/mL to 0.031 !g/mL for itra- 110
56 of species as well as in the sensitivity of isolates to conazole, miconazole and nystatin. 111
57 antifungals.1,1113 Considering this and the lack of data on The 96-well microplates were filled with 100 !L of the 112
58 this topic in Rio Grande do Sul, this study aimed to iden- antifungal concentrations and 100 !L of inoculum and were 113
59 tify the prevalence of spp. isolated from the vaginal incubated at 37 C for 48 h along with growth and sterility con- 114
60 mucosa of patients with and without vulvovaginitis treated trols. Each isolate was tested in duplicate, and the results were 115
61 at a university hospital in extreme southern Brazil, as well as visually evaluated by turbidity, comparing the fungal growth 116
62 the susceptibility of these isolates to four antifungals used in in the well with the growth control. The minimum inhibitory 117
zole was one that inhibited 80% of yeast growth, and the MIC 119
Materials and methods zole, the isolates were classified as S for sensitive, SDD for 122
64 The study included 263 patients attended on from April 2013 to by the cutoff point protocol M27-A2. For miconazole and nys- 124
65 October 2014 at the gynecology clinic of the University Hospi- tatin, the MIC50 and MIC90 were calculated corresponding to 125
66 tal of the Federal University of Rio Grande (FURG) Rio Grande the antifungal concentration capable of inhibiting the growth 126
67 do Sul Brazil. The women agreed to participate by signing the of 50% and 90%, respectively, of the isolates tested. After read- 127
68 Clarified Informed Consent form. The sample size, calculated ing the results of MIC, 10 !L of the solutions from the wells 128
69 using Epi Info 6.0 and considering a prevalence of 30%, a con- related to concentrations higher than and equal to the MIC 129
70 fidence level of 99%, maximum permissible error of 20% and were plated on a Petri dish containing agar SCl and were incu- 130
71 losses of 10%, was estimated to be a sample of 249 patients. bated at 37 C for 48 h to determine the fungicide minimum 131
73 vaginal mucosa was collected using a sterile brush, which The study was conducted according to ethical principles 133
74 was stored in sterile tubes containing PBS and kept refriger- and was approved by the Research Ethics Committee of the 134
75 ated until processing. The sample was sent to the Mycology Federal University of Rio Grande (65/2012). The results were 135
76 Laboratory of the Medicine Faculty from FURG, where it compiled by conducting descriptive analysis of the data and 136
77 was processed within 12 h. The cultures were processed chi-square tests for categorical variables using SPSS 19.0 137
78 in Sabouraud agar with chloramphenicol 0.01% (SCl) and software. -values below 0.05 were considered statistically sig- 138
85 agar (PDA) in the mycology collection at room temperature partners (64.5%), had white skin color (65.3%), were pregnant 141
Please cite this article in press as: Brandolt TM, et al. Prevalence of spp. in cervical-vaginal samples and the susceptibility of
isolates. (2016), http://dx.doi.org/10.1016/j.bjm.2016.09.006 BJM 156 16
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(chi-square test) in the development of vulvovaginal identified only in general, being classified as spp. 163
candidiasis (VVC) (n = 35). Regarding the variables analyzed, 82.4% ( = 28) of patients 164
Variables (%) VVC ( ) value diagnosed with VVC belonged to the group of women aged 30 165
years or younger ( = 0.047). For the other variables such as skin 166
71 (27.1) 10 0.833
color, marital status, educational level, family income, contra- 167
191 (72.9) 25
ception, pregnancy and HIV infection, there was no significant 168
158 (60.3) 26 0.069
104 (39.7) 09 difference between the group of women with and without VVC 169
(Table 1). The same occurred with the vaginal pH at the time of 170
0.542
collection, in which 133 patients had pH 4.5, but only 14.3% 171
White 160 (65.3) 20
Not white 85 (34.7) 13
( = 19) of them were diagnosed with VVC ( = 0.948). 172
142 (60.3%) and were HIV-negative (72.9%). The mean age was 28.5
143 years and ranged from 12 and 68 years, with more than 65% of Discussion
144 the women under 31 years of age ( = 176) and 54.7% ( = 135)
145 using hormonal contraception (Table 1). The present study provides data about spp. isola- 187
146 Of the 263 patients, spp. was isolated in the myco- tion from the vulvovaginal mucosa of women in southern Rio 188
147 logical culture from cervical-vaginal samples in 27% ( = 71). Grande do Sul, Brazil. We detected the yeast in 27% of the study 189
148 Of these, vulvovaginal candidiasis was diagnosed in 49.3% population, corresponding to a prevalence of approximately 190
149 ( = 35), resulting in a prevalence in the population of 13.3%. 13% both for colonization and for vulvovaginal candidiasis. 191
150 The remaining 50.7% ( = 36) that had no signs or symptoms Similar and lower rates of fungal isolation in women with and 192
151 of vulvovaginitis were classified as colonization cases. without vulvovaginitis have been described in other studies of 193
152 The predominant species was in both the southern region: 11 and 24.7% in patients in the state of 194
153 groups, corresponding to 62.9% ( = 22) of isolates originating Paran,1215 23.8% in patients in the state of Santa Catarina12 195
154 from the group of women colonized and 74.3% ( = 26) from the and 18.2% in the city of Santo ngelo-RS,16 the latter being 196
155 women with VVC. The other species identified colonizing the the only study held in Rio Grande do Sul. These differences 197
156 vaginal mucosa of healthy women were in 14.3% in the isolation of spp. from vaginal mucosa can be 198
157 ( = 5), followed by in 8.6% ( = 3) and explained by cultural habits of different regions, especially in 199
158 complex in 2.9% ( = 1). Among cases of VVC by . non- regard to hygiene practices, as this factor is directly related 200
159 ( = 5), 8.6% ( = 3) were due to , 2.9% ( = 1) were to self-contamination because the yeast belongs to the nor- 201
160 due to complex, and 2.9% ( = 1) were due to mal gastrointestinal tract microbiota.3,17,18 On the other hand, 202
Table 2 Results of the in vitro susceptibility test of Candida spp. from colonized and/or VVC patients against four
antifungal agents. S-sensitive, SDD-sensitive dose-dependent and R-resistant.
Species Fluconazole Itraconazole Miconazole Nystatin
( = 37) 12 (32.4) 4 (10.8) 21 (56.8) 8 (21.6) 9 (24.3) 20 (54.1) 0.0318.0 1.0/4.0 2.0>16 4.0/8.0
( = 6) 1 (16.7) 5 (83.3) 1 (16.7) 2 (33.3) 3 (50) 0.0310.250 0.031/0.125 4.08.0 4.0/8.0
complex ( = 2) 2 (100) 1 (50) 1 (50) 0.0310.125 0.031/0.125 8.0 8.0/8.0
( = 1) 1 (100) 1 (100) 1.0 1.0/1.0 8.0 8.0/8.0
( = 3) 3 (100) 3 (100) 0.031 0.031/0.031 2.04.0 2.0/4.0
spp. ( = 1) 1 (100) 1 (100) 0.5 0.5/0.5 2.0 2.0/2.0
Please cite this article in press as: Brandolt TM, et al. Prevalence of spp. in cervical-vaginal samples and the susceptibility of
isolates. (2016), http://dx.doi.org/10.1016/j.bjm.2016.09.006 BJM 156 16
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Table 3 Fungicide Minimum Concentration (FMC) results of the four antifungals tested against Candida spp. isolated
from colonized and/or VVC patients.
Species Fluconazole Itraconazole Miconazole Nystatin
203 similar studies in northeastern Brazil show fungal isolation in highlights the importance of laboratory tests for correct iden- 249
204 women with and without vulvovaginitis of up to 46%.1,17,19,20 It tification of the etiologic agent, although the clinical diagnosis 250
205 is assumed that other factors besides those mentioned above of the disease is easily performed and commonly applied.2,25 251
206 may explain the high prevalence of spp. in this region, Regarding the variables studied, the largest number of 252
207 such as weather conditions and social and environmental fac- patients with VVC was from the group of women younger than 253
208 tors that are favorable to the reproduction of yeast.20 31 years, being within the reproductive age group,3 which is 254
209 The rate of colonization of 13.7% observed in our study is considered a risk factor for VVC due to sexual activity.8 How- 255
210 consistent with the literature,3,9 which describes that ever, this difference in age between the group of women with 256
211 spp. is part of the genital microbiota of up to 30% VVC and the otherwise healthy group was not detected by 257
212 of healthy women.1,18 Whereas the disease occurs endoge- other authors.1,17,19,23 Similarly, use of hormonal contracep- 258
213 nously, is important to differentiate between the colonization tion or intrauterine devices, vaginal pH 4.5, pregnancy and 259
214 and infection of the vaginal mucosa.3,18 HIV infection, which are considered as risk factors for the 260
215 Ferrazza and co-workers12 found a VVC prevalence of 19.2% development of VVC3,5,20,26 were not significantly associated 261
216 in Santa Catarina and of 9.3% in Paran; these data are sim- with VVC infection in our study. This discrepancy may be due 262
217 ilar to the prevalence of VVC found in our study.12 However, to the small number of women with VVC in our study ( = 35), 263
218 it is below the 69% found by Holanda and co-workers17 in a which does not allow for a robust statistical analysis for risk 264
219 study conducted in Natal, the 47.9% found by Andrioli and co- factors for the disease. 265
220 workers1 in Bahia, the 42.7% described by S and co-workers20 According to our study as well as that of Rodrigues and 266
221 in Maranho and the 39.6% found by Dias and co-workers7 in co-workers23 the other variables such as marital status, educa- 267
222 Mato Grosso. Considering that this disease is opportunistic, tional level and skin color do not exert significant influence on 268
223 this discrepancy may be related to several factors related to the development of VVC. Moreover, lvares and co-workers4 269
224 the etiologic agent, the species distribution and/or virulence state that the vaginal microbiota of black women has a lower 270
225 of the strains, the immune status of the host, or the environ- incidence of bacterial species, and there would thus be a 271
226 ment, which influences the maintenance of temperature and decrease in natural defenses against fungal growth, predis- 272
227 humidity conducive to fungal proliferation.1,12,20,21 posing them to infection by spp.4,20 273
228 The differences in geographical location should be consid- studies have shown different resistance rates 274
229 ered among the epidemiologic factors that also interfere in the of spp. from the vaginal mucosa to the azole 275
230 prevalence of spp. isolated from vaginal mucosa.12 In drugs commonly used in the treatment and prophylaxis of 276
231 this study, was predominant, accounting for over VVC.7,8,10,11,13,22,24,27,28 Concerning fluconazole, the resistance 277
232 60% of the isolates both in colonized patients and in those rates range from 0.8% to 12.5%.7,10,11,24,28 These values are con- 278
233 with VVC. In fact, this species is the most pathogenic of the siderably lower than the results found in our study, in which 279
234 gender, being related to most cases of VVC described, and 42% of the isolates were resistant to fluconazole. However, 280
235 generally represents more than half of the isolates identified Dalazen and co-workers22 in a study conducted in Santa Cata- 281
236 in other studies.7,15,16,22 Ferraza and co-workers12 described rina, found 100% of fluconazole resistance. With respect to 282
237 the identification of this species in Santa Catarina in 100% itraconazole, resistance rates described in other studies range 283
238 and 72% of isolates from the asymptomatic and symptomatic from 1.9 to 43%,10,13,27,28 also lower than the 48% found in our 284
239 patients, respectively, and in 66.7% of patients with VVC in study. In addition to the high rate of resistance to flucona- 285
240 state of Paran. Similarly, Camargo and co-workers16 identi- zole and itraconazole observed, the FMC of more than 90% of 286
241 fied in more than 80% of the isolates from patients the isolates was greater than the maximum antifungal con- 287
242 with and without VVC in Santo ngelo-RS. centration tested. Therefore, there is a tendency to avoid the 288
243 Approximately 25% of cases of VVC were caused by prophylactic use of low doses of antifungals in gynecological 289
244 non- species in our study. Among these, , routines to prevent the emergence of drug-resistant isolates.2 290
245 described as the second most important species in cases Another group of azole drugs used for topical treatment 291
246 of VVC due to its frequency10,12,15,18,23 and greater resis- of VVC is miconazole.22 In our study, the MIC for this drug 292
247 tance to antifungals10,22,24 was found in 8.6% of symptomatic ranged from 0.031 to 8 !g/mL. These data were similar to those 293
248 patients and in 14.3% of colonized patients in our study, which found in a study by Choukri and co-workers29 in which the 294
Please cite this article in press as: Brandolt TM, et al. Prevalence of spp. in cervical-vaginal samples and the susceptibility of
isolates. (2016), http://dx.doi.org/10.1016/j.bjm.2016.09.006 BJM 156 16
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295 MIC to miconazole varied between 0.015 and 8 !g/mL and 8. Beikert FC, Le MT, Koeninger A, Technau K, Clad A. Recurrent 350
296 in a study by Richter and co-workers10 in which the varia- vulvovaginal candidosis: focus on the vulva. . 351
297 tion was between 0.007 and 4 !g/mL. Moreover, Dalazen and 2011;54:807810. 352
9. Colombo AL, Guimares T, Camargo LFA, et al. Brazilian 353
298 co-workers22 found a variation in the MIC of miconazole of
guidelines for the management of candidiasis a joint 354
299 between 0.097 and 100 !g/mL. The different genotypes of the meeting reporto of three medical societies: Sociedade 355
300 same species may be significantly different in their suscepti- Brasileira de Infectologia, Sociedade Paulista de Infectologia 356
301 bility to azoles.27 and Sociedade Brasileira de Medicina Tropical. 357
302 The polyenic agents are another group of drugs used for . 2013;17(3):283312. 358
303 the treatment of VVC; this group includes nystatin, which is 10. Richter SS, Galask RP, Messer SA, Hollis RJ, Diekema DJ, 359
304 the most used in Brazil and is freely available in the Unified Pfaller MA. Antifungal susceptibilities of species 360
causing vulvovaginitis and epidemiology of recurrent cases. 361
305 Health System (SUS) in Brazil.30 The MIC of nystatin found
. 2005;43:21552162. 362
306 in our study ranged from 2 to >16 !g/mL; however, in a study 11. Fan SR, e Liu XP. In vitro fluconazole and nystatin 363
307 by Choukri and co-workers,29 the MIC varied between 1 and susceptibility and clinical outcome in complicated 364
308 4 !g/mL, and in a study by Richter and co-workers,10 the vulvovaginal candidosis. . 2010;54:501505. 365
309 concentration ranged from 1 to 16 !g/mL. Knowledge of the 12. Ferrazza MHSH, Maluf MLF, Consolaro MEL, Shinobu CS, 366
310 patterns of susceptibility to typical drugs in isolates from dif- Svidzinski TIE, Batista MR. Caracterizaco de leveduras 367
isoladas da vagina e sua associaco com candidase 368
311 ferent regions will allow for the rationalization of the empirical
vulvovaginal em duas cidades do sul do Brasil. 369
312 use of antifungal agents, thus contributing to the control of the
. 2005;27(2):5863. 370
313 isolated resistance to drugs.2 13. Dota KFD, Consolaro MEL, Svidzinski TIE, Bruschi ML. 371
314 Our results will encourage the development of other stud- Antifungal activity of Brazilian propolis microparticles Q3 372
315 ies related to the resistance mechanisms of these yeasts and against yeasts isolated from vulvovaginal candidiasis. 373
316 also of clinical trials in the hospital to determine whether the . 2011. 374
317 high resistance to antifungals detected actually corre- 14. CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI). 375
376
318 sponds to therapeutic failure.
. 2nd ed; 377
319 According to this data discrepancies and the lack of epi- 2002. 378
320 demiological data in the state of Rio Grande do Sul, the present 15. Gunther LSA, Martins HPR, Gimenes F, Abreu ALP, Consolaro 379
321 study has been collaborating with local epidemiology, eluci- MEL, Svidzinski TIE. Prevalence of and 380
322 dating the etiological factors of VVC and warning of the high non- isolates from vaginal secretions: comparative 381
323 resistance rates found in spp. isolates evalu- evaluation of colonization, vaginal candidiasis and recurrent 382
Please cite this article in press as: Brandolt TM, et al. Prevalence of spp. in cervical-vaginal samples and the susceptibility of
isolates. (2016), http://dx.doi.org/10.1016/j.bjm.2016.09.006 BJM 156 16
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419 24. Oliveira PM, Mascarenhas RE, Lacroix C, et al. Candida candidiasis in China. . 432
420 species isolated from the vaginal mucosa of HIV-infected 2010;54(8):31263131. 433
421 women in Salvador, Bahia, Brazil. . 28. Nunes EB, Monteiro JCMS, Nunes NB, Paes ALV. Perfil de 434
422 2011;15(3):239244. sensibilidade do gnero a antifngicos em um 435
423 25. Rathod SD, Klausner JD, Krupp K, Reingold AL, Madhivanan hospital de referncia da Regio Norte do Brasil. 436
424 P. Epidemiologic features of vulvovaginal candidiasis among . 2011;2(4):2330. 437
425 reproductive-age women in India. . 29. Choukri F, Benderdouche M, Sednaoui P. susceptibility 438
426 2012:2012. profile of 200 recent clinical isolates of spp. to topical 439
427 26. Auler ME, Morreira D, Rodrigues FFO, et al. Biofilm formation antifungal treatments of vulvovaginal candidiasis, the 440
428 on intrauterine devices in patients with recurrent imidazoles and nystatin agents. . 2014;24:303307. 441
429 vulvovaginal candidiasis. . 2010;48:211216. 30. Martins HPR, Da Silva MC, Paiva LCF, Svidzinski TIE, 442
430 27. Ge SH, Wan Z, Li J, Xu J, Li RY, Bai FY. Correlation between Consolaro MEL. Efficacy of fluconazole and nystatin in the 443
431 azole susceptibilities, genotypes, and ERG11 Mutations in treatment of vaginal species. . 444
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Please cite this article in press as: Brandolt TM, et al. Prevalence of spp. in cervical-vaginal samples and the susceptibility of
isolates. (2016), http://dx.doi.org/10.1016/j.bjm.2016.09.006 BJM 156 16