Oral yeast carriage in HIV-infected and non-infected populations in Rosario, Argentina

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1 Original article Oral yeast carriage in HIV-infected and non-infected populations in Rosario, Argentina A. G. Luque, 1 M. S. Biasoli, 1 M. E. Tosello, 1 A. Binolfi, 1 S. Lupo 2 and H. M. Magaró 1 1 CEREMIC, Fac. Cs. Bioquıḿicas y Farmace uticas, Rosario and 2 Primera Ca tedra de Clıńica Me dica Fac. Cs. Me dicas, Universidad Nacional de Rosario, Rosario, Argentina Summary The objectives of the present study were: (i) to assess the frequency of oral colonisation by Candida species in HIV-positive patients and to compare it with a population of HIVnegative individuals, (ii) to determine the prevalence of C. dubliniensis in both populations and (iii) to determine the susceptibility of C. dubliniensis and other Candida species isolated from HIV-positive patients to the most commonly used antifungal agents. Oral samples were obtained from 101 HIV-positive and 108 HIV-negative subjects. For yeast identification, we used morphology in cornmeal agar, the API 20C Aux, growth at 45 C, D-xylose assimilation, morphology in sunflower seed agar and PCR. The frequency of isolation of Candida in HIV-positive patients was: C. albicans, 60.7%; C. dubliniensis, 20.2%; C. glabrata, 5.6%; C. krusei, 5.6%; C. tropicalis, 4.5%; others, <5%. The frequency of isolation of Candida in HIV-negative patients was: C. albicans, 73.9%; C. tropicalis, 15.5%; C. dubliniensis, 2.1%; C. glabrata, 2.1%; C. parapsilosis, 2.1%; others, <5%. The oral colonisation by yeast in the HIV-positive patients was higher than that in the HIV-negative subjects. The susceptibilities of 42 Candida isolates to three antifungal agents were determined. All isolates of C. dubliniensis were susceptible to fluconazole, although several individuals had been previously treated with this drug. Out of the 42 Candida isolates, 10 presented resistance to fluconazole and 10 to itraconazole. The presence of Candida species, resistant to commonly used antifungal agents, represents a potential risk in immunocompromised patients. Key words: Antifungal susceptibility, Candida, HIV, oral carriage. Introduction Candida species, along with other micro-organisms, are usually found in the normal flora of the human oral mucosa. These species are potential sources of oral candidiasis as well as other more serious forms of the disease, such as oesophageal and systemic candidiasis. 1 As a consequence, in most of the patients, these diseases arise from the endogenous pool (oral or digestive) of the same patient. In the general population, the prevalence of oral colonisation by yeast is nearly 35%. 2 There are Correspondence: Alicia Graciela Luque, CEREMIC, Fac.Cs. Bioquímicas y Farmacéuticas, Pte Quintana 3356, 2000 Rosario, Argentina. Tel.: Fax: agluquear@yahoo.com.ar Accepted for publication 7 April 2008 several factors that favour the oral colonisation by Candida, such as hospitalisation, several therapies and eating habits among others. 1 Candida albicans is the most frequently isolated species as coloniser and pathogen of the oral mucosa. However, the isolation of other species, such as Candida tropicalis, Candida krusei, Candida parapsilosis and Candida glabrata, has increased. 3 5 In the past years, Candida dubliniensis has been recovered from immunocompromised patients as a source of oral candidiasis. 6 8 AIDS, a disease caused by HIV, is characterised by a decrease of cellular immunity because of a progressive loss of CD4 + T lymphocytes. This decrease leads to multiple opportunistic infections such as oral, oesophageal, tracheal and pulmonary candidiasis. 4,9,10 Many of HIV-infected patients have received multiple antifungal treatments for candidiasis or other opportunistic mycoses, and in the last years, cases of oral Journal compilation Ó 2008 Blackwell Publishing Ltd Mycoses 52, doi: /j x

2 A. G. Luque et al. candidiasis unresponsive to azoles were observed. 11 Development of resistance to fluconazole in immunocompromised patients has become an issue of growing concern and is usually correlated to the degree of immunosuppression and the total dose of drug used in long-term antifungal treatments. 12,13 There are several reports of the prevalence of Candida species in patients with fungal infections in Argentina However, there has been only one study about oral carriage of Candida species carried out by Negroni et al. [23] on population of students. A systematic study of oral colonisation by yeast in HIV-positive patients has not been performed in Argentina so far. The objectives of the present study were: (i) to assess the frequency of oral colonisation by Candida species in HIV-positive patients and to compare it with a population of HIV-negative individuals, (ii) to determine the prevalence of C. dubliniensis in both populations and (iii) to determine the susceptibility of C. dubliniensis and other Candida species isolated from HIV-positive patients to the most commonly used antifungal agents. Materials and methods Study population One hundred and one HIV-positive patients took part in this study. The individuals were recruited from both inpatients and outpatients from the Hospital Provincial del Centenario in Rosario, Santa Fe, in 2001 and The oral mucosa was clinically examined by a physician and the patients with clinical signs and symptoms of candidiasis (pseudomembranous and erythematous forms) were excluded from the study. The data obtained from the clinical history of each patient were the following: previous antifungal treatment, CD4 and CD8 counts and treatment with highly active antiretroviral therapy (HAART). One hundred and eight healthy university students of the Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, HIV negative (control group), without oral mucosa lesions and without recent treatments with antibiotics and antifungal agents were also studied. In this study, the procedures used were in accordance with the Helsinki Declaration (1964, amended in 1975 and 1983) of the World Medical Association. Sample collection and isolation of yeast The samples were obtained by swabbing the oral cavities with a sterile cotton swab and then suspended and shaken in 1 ml of sterile physiological solution. This solution was used for direct microscopic examinations, performed with lactophenol cotton blue. Two hundred and fifty microlitres of this saline solution was inoculated in agar slats of Sabouraud dextrose agar (SDA) with and without chloramphenicol (Britania, Buenos Aires, Argentina). The tubes were incubated at 28 C for a week. Identification of isolates All yeast colonies growing on each SDA tube were suspended in physiological solution and 10 ll of the suspension was used to inoculate plates of CHROMagar Candida media (CHROMagar Company, Paris, France) which were incubated at 37 C for 72 h. A presumptive identification of Candida isolates was attempted on the basis of the characteristic colour of the colonies appearing on CHROMagar Candida. When green colonies grew, which is characteristic of both C. albicans and C. dubliniensis, a single colony was selected from each plate. The following tests were performed in order to differentiate both species: growth at 45 C on potato dextrose agar (PDA), 8 D-xylose assimilation, the API 20C Aux system (BioMerieux, Marcy LÕEtoile, France), morphology in sunflower seed agar (SSA) 24 and PCR using primers that hybridise to the class IV intron of the ACT1 gene 25 according to the scheme of identification of Binolfi et al. [26]. Colonies of different colours other than green, which correspond to other yeast species, on CHROMagar Candida were isolated and identified using the carbohydrate assimilation and fermentation tests and the API 20C Aux system. Antifungal susceptibility testing All the C. dubliniensis (18), isolated from HIV-positive patients, were investigated for their in vitro susceptibility to amphotericin B, fluconazole and itraconazole. Also, 16 C. albicans, four C. glabrata, two C. tropicalis and two C. krusei isolated from the HIV-positive patients treated and not previously treated with antifungal agents were selected to detect the susceptibility to amphotericin B, fluconazole and itraconazole. The following drug concentration ranges were used: amphotericin B ( mg ml )1 ), fluconazole ( mg ml )1 ) and itraconazole ( mg ml )1 ). The minimal inhibitory concentrations (MICs) were determined in RPMI-1640 with 2% glucose, using reference method for broth dilution antifungal susceptibility testing of yeast according to the M 27-A 2 Document of the Clinical and Laboratory Standards Institute (CLSI), previously named National Committee 54 Journal compilation Ó 2008 Blackwell Publishing Ltd Mycoses 52, 53 59

3 Oral yeast carriage in Argentina for Clinical Laboratory Standards (NCCLS). 27 The results were visually recorded after incubation at 35 C for 24 and 48 h. Candida parapsilosis ATCC and C. krusei ATCC 6258 were used as the reference strains. The MICs were determined as the lowest concentration of the drug inhibiting the 100% of the growth (for amphotericin B) and at least 80% of the growth (for azoles). The isolates were tested in duplicate. The breakpoints used as interpretive guidelines for in vitro susceptibility testing of Candida species were based on the M 27-A 2 Document of the CLSI. 27 Statistical analysis Data were analysed using the chi-squared independence test and the Fisher exact test considering a significance level of P = Results Out of the 101 HIV-positive patients studied, 67 were male and 34 were female, with a mean age of 35.2 years (range between 17 and 64 years). The CD4 counts (mm 3 ) were <100 in 21%, in 21%, > in 21% and from 400 to 1000 in 37% of patients. The CD8 counts (mm 3 ) were in 55% and >1000 in 45% of patients. Eighty-nine per cent of them were treated with HAART that included: indinavir, nelfinavir, lamivudine and abacavir. Twentyfour patients had been previously treated with some of the following antifungal agents: fluconazole, itraconazole, ketoconazole and nystatin. In the control group, 70 were male and 38 were female, with a mean age of 30.3 years (range between 22 and 52 years). Out of 101 HIV-positive patients investigated, 73 (72.3%) were positive for oral carriage of yeast. Within the control group, 43 (39.8%) showed yeast colonisation. The chi-squared independence test (v 2 = 22.27; P = ) shows that there is a significant association between the colonisation by yeast and the HIV-positive status. HAART-treated patients showed a higher yeast colonisation frequency (89%) than non-treated ones (50%) (Fisher exact test, P < 0.05). The patients with CD4 counts <200 presented 94% of yeast colonisation, whereas the individuals with CD4 counts >200 showed 80% of yeast colonisation. A statistical analysis of results has shown no association between CD4 counts and yeast colonisation (P = 0.374). HAART-treated patients had 33% of resistant yeast to some of studied antifungal agents and non-treated patients presented 45% of resistant yeast. The patients with CD4 counts <200 presented 29% of resistant yeast and the patients with CD4 counts >200 showed 40% of resistant yeast. According to our data, there was no association between susceptibility to antifungal agents and HAART therapy (P = 0.687) or CD4 counts (P = 0.545). There was no significant association between antiretroviral therapy and C. albicans isolated from HIV-positive patients (P = 0.440). The relationship between yeast oral carriage and previous antifungal treatment was analysed and no statistical association (P = 0.210) was found. The frequency of isolation of Candida in HIV-positive patients was: C. albicans, 60.7%; C. dubliniensis, 20.2%; C. glabrata, 5.6%; C. krusei, 5.6%; C. tropicalis, 4.5%; others, <5%. The frequency of isolation of Candida in HIV-negative patients was: C. albicans, 73.9%; C. tropicalis, 15.5%; C. dubliniensis, 2.1%; C. glabrata, 2.1%; C. parapsilosis, 2.1%; others, <5%. Table 1 shows the yeast distribution in 101 HIVpositive subjects and 108 HIV-negative subjects. Candida albicans was the most common isolate from both HIV-positive and HIV-negative subjects while C. dubliniensis was the second most common isolate for HIV-positive patients but not among HIV-negative subjects. Table 1 Analysis of yeast distribution in HIV-positive subjects and HIV-negative subjects Yeast isolates Number of HIV-positive subjects (n = 73) (%) 1 Number of HIV-negative subjects (n = 43) (%) 1 C. albicans 43 (58.9) 32 (74.4) C. dubliniensis 13 (17.8) 1 (2.3) C. krusei 1 (1.4) (0) C. parapsilosis (0) 1 (2.3) C. tropicalis 1 (1.4) 5 (11.6) R. graminis 1 (1.4) (0) Rhodotorula spp. (0) 1 (2.3) C. albicans + C. dubliniensis 2 (2.7) (0) C. albicans + C. glabrata 4 (5.5) (0) C. albicans + C. tropicalis 3 (4.1) 2 (4.6) C. albicans + C. krusei 1 (1.4) (0) C. albicans + P. ohmeri 1 (1.4) (0) C. dubliniensis + C. krusei 1 (1.4) (0) C. glabrata + C. milleri (0) 1 (2.3) C. dubliniensis + C. krusei + C. 1 (1.4) (0) parapsilosis C. dubliniensis + C. krusei + C. glabrata 1 (1.4) (0) C., Candida; R., Rhodotorula; P., Pichia. 1 Frequency of isolation. Journal compilation Ó 2008 Blackwell Publishing Ltd Mycoses 52,

4 A. G. Luque et al. Table 2 Susceptibility to amphotericin B, fluconazole and itraconazole of 42 Candida species clinical isolates Isolate Drug Amphotericin B Fluconazole Itraconazole C. albicans (S) 0.03 (S) C. albicans (S) 0.25 (SDD) C. albicans (S) 0.25 (SDD) C. albicans (S) 0.06 (S) C. albicans (S) 0.25 (SDD) C. albicans (SDD) 0.5 (SDD) C. albicans (S) >16 (R) C. albicans (S) 0.25 (SDD) C. albicans 69 1, (R) 8 (R) C. albicans (S) 0.06 (S) C. albicans (S) 0.06 (S) C. albicans (S) 0.03 (S) C. albicans (R) 8 (R) C. albicans >128 (R) >16 (R) C. albicans (R) >16 (R) C. albicans (S) 0.06 (S) C. dubliniensis (S) 0.03 (S) C. dubliniensis (S) 1 (R) C. dubliniensis (S) 0.03 (S) C. dubliniensis (S) 0.03 (S) C. dubliniensis (S) 0.06 (S) C. dubliniensis (S) 0.06 (S) C. dubliniensis (S) 0.13 (S) C. dubliniensis (S) 0.5 (SDD) C. dubliniensis (S) 0.06 (S) C. dubliniensis (S) 0.03 (S) C. dubliniensis (S) 0.06 (S) C. dubliniensis (S) 0.06 (S) C. dubliniensis (S) 0.03 (S) C. dubliniensis (S) 0.06 (S) C. dubliniensis (S) 0.03 (S) C. dubliniensis (S) 0.13 (S) C. dubliniensis (S) 0.03 (S) C. dubliniensis (S) 0.06 (S) C. glabrata (S) 0.13 (S) C. glabrata 69 1, (R) 8 (R) C. glabrata (R) >16 (R) C. glabrata >128 (R) >16 (R) C. tropicalis (SDD) 1 (R) C. tropicalis (R) 8 (R) C. krusei (R) 0.5 (SDD) C. krusei (R) 0.5 (SDD) Minimal inhibitory concentration (mg l )1 ) was determined by the NCCLS reference method. Results have been read after 48 h of incubation at 35 C. S, susceptible; R, resistant; SDD, susceptible dose dependent. 1 Patients previously treated with fluconazole. 2 Patients previously treated with ketoconazole. 3 Patients previously treated with itraconazole. In Table 2, the MIC of 42 clinical isolates is listed. Amphotericin B, fluconazole and itraconazole MICs for the quality control strains were within the accepted limits. The MIC data were recorded at 48 h of incubation and, in general, there were no differences with the results obtained at 24 h. The growth of the C. dubliniensis isolates was very poor at 24 h of incubation, which made it impossible to get the readings within this period of time. Candida albicans 10 and C. albicans 22 isolates were read at 24 h because of a trailing effect. Six isolates of C. albicans, 16 isolates of C. dubliniensis and one of C. glabrata isolates were shown to be susceptible to all the three antifungal agents tested. Out of the 42 Candida isolates, 10 presented resistance to fluconazole and 10 to itraconazole. Discussion Two different populations of individuals were studied, an HIV-infected one and an HIV-negative one (control group). The prevalence of colonisation by yeast in the oral cavities of the HIV-positive patients was considerably higher than in the control group as it was previously reported. 4,5 In the HIV-infected subjects, the oral carriage prevalence of yeast (72.3%) is high, but lower than that (93%) reported by Felix & Wray. 28 In HIV-negative subjects, the prevalence of yeast in our results (39.8%) was similar to that obtained by Schoofs et al. [29], who found an oral yeast carriage rate of 38.8% in healthy individuals. The isolation of more than one yeast species in the oral cavities of both, HIV-positive and HIV-negative individuals has been reported. 5,30 Sixteen HIV-positive patients were found to carry more than one yeast species in their oral cavities, 14 carried two different Candida species and two had three different species. In the control group, this feature was observed in three subjects, all of them with two different Candida species. The presence of more than one species of Candida in the oral mucosa may predispose to recurrent candidiasis, mainly with species resistant to azole drugs, such as C. glabrata and C. krusei. 9 The frequency of Candida species isolated from the control group was similar to that obtained in previous reports, C. albicans being the most frequently isolated, followed by C. tropicalis. 31 Candida krusei isolates, inherently less susceptible to fluconazole, were only recovered from the HIV-positive patients. Only one isolate of C. dubliniensis was recovered from the oral mucosa of HIV-negative individuals representing a prevalence of 0.93%. The percentage of HIVpositive patients carrying C. dubliniensis was 17.8%. Although these results were similar to the ones obtained in the other studies, 7,30,32 34 the frequency of C. dubliniensis is relatively high, fundamentally if we 56 Journal compilation Ó 2008 Blackwell Publishing Ltd Mycoses 52, 53 59

5 Oral yeast carriage in Argentina consided that the studied patients did not present lesions in the oral mucosa. The relationship between C. dubliniensis and HIV-positive patients remains to be elucidated. Although the advent of HAART therapy has produced a decrease in the incidence of opportunistic infections in HIV-positive individuals, 9 in our work we studied oral yeast carriage and found that the patients treated with HAART therapy presented higher frequency of Candida colonisation than the non-treated ones. This could be due to the low number of nontreated patients in comparison with treated ones. As in Campisi et al. [35] studies, no association between yeast carriage in the oral cavity and CD4 + -cell counts of HIV-positive subjects was found. Although Rodrigues Costa et al. [10] reported that fluconazole resistance was relatively high among patients treated with HAART, no association between susceptibility to antifungal agents and antiretroviral therapy was found in our study. Among the C. albicans isolates, four were resistant to fluconazole and five to itraconazole. All C. albicans isolates that presented in vitro resistance to itraconazole were recovered from patients who had previously received this antifungal agent. This feature could be due to a secondary resistance produced by exposure to the drug. Out of patients previously treated with fluconazole, four Candida isolates were isolated (three C. albicans and one C. glabrata). All presented fluconazole and itraconazole resistance. These findings suggest the possibility of a cross-resistance phenomenon between both antifungal agents. Active efflux and alteration in drug target are considered to be the most important mechanisms of azole resistance. In two patients who belonged to the group showing isolation of more than one Candida species, both of the species isolated were sensitive to fluconazole and itraconazole (C. albicans + C. glabrata and C. albicans + C. dubliniensis). In a patient previously treated with fluconazole and itraconazole, the C. albicans and C. glabrata isolates recovered were resistant to these antifungals. All C. glabrata isolates, resistant to fluconazole, were also resistant to itraconazole. Two C. krusei isolates resistant to fluconazole were susceptible dose dependent (SDD) to itraconazole. 27 One C. tropicalis strain tested was resistant to itraconazole and to fluconazole and the other C. tropicalis was resistant to itraconazole and SDD to fluconazole. In our study, the in vitro susceptibility of C. glabrata, C. krusei and C. tropicalis to antifungal agents was tested in a low number of isolates, but these showed a high degree of resistance to imidazolic compounds as previously described by other authors. 36 All isolates of C. dubliniensis were susceptible to fluconazole, although several individuals had been previously treated with this drug. The 24 and 78 C. dubliniensis isolates showed a decreased susceptibility to fluconazole and resistance and SDD to itraconazole respectively. The results are in agreement with previous findings of other authors in which C. dubliniensis isolates are sensitive to most antifungal agents. In several studies, the percentage of isolates susceptible to fluconazole varied between 74% and 97%. 37 The resistance profile encountered in C. dubliniensis was, as observed in previous works, different to that observed in C. albicans. 38 In this report, the finding of a high number of C. albicans isolates resistant to fluconazole and itraconazole should be highlighted. Although the breakpoint concentrations for amphotericin B are not clearly defined, a strain is considered to be resistant when it presents an MIC 2mgl )1. 39 According to this criterion, 19% of the isolates were found to be resistant to amphotericin B and this feature is difficult to analyse from a clinical point of view. Out of eight patients in which Candida isolates resistant to amphotericin B were recovered, four had been previously treated with fluconazole. These results agree with previous reports on the existence of strains resistant to amphotericin B which had previously been in contact with azoles. The azoles affect the cellular membrane and this could be a factor in a selection of mutants. 36 It has also been established that the resistant isolates are less virulent (or not virulent at all) in infection experimental models. Most of the resistant isolates to amphotericin B were recovered from immunocopromised individuals and this could be related to effects on cellular membrane of yeast, in relation to the multiple treatments received for these patients. 40 In general, resistance to amphotericin B because of previous exposure to this drug has not been detected. Pfaller et al. [41] described different proportion of resistance to amphotericin B according to Candida strains considered, with 5% of C. albicans, 59% of C. glabrata and 100% to C. krusei resistant to amphotericin B. In patients with fungaemia for C. glabrata and C. krusei, high doses of amphotericin B need to be used. In our study, a greater proportion of resistance in C. glabrata, C. krusei and C. tropicalis isolates compared with C. albicans was detected. The presence of Candida species, resistant to most commonly used antifungal agents, in the oral mucosa represents a potential risk in the generation of serious infections in immunocompromised patients. The high Journal compilation Ó 2008 Blackwell Publishing Ltd Mycoses 52,

6 A. G. Luque et al. percentage of resistant Candida species to the antifungal agents in the present study could be due to the fact that many patients had received previous treatments with these drugs. Therefore, the azole prophylaxis could be considered only in special cases. Oral colonisation with Candida seems to vary widely depending on global location and this can be critical in determining treatment strategies for oral and systemic disease. We present data from a previously unstudied population in Argentina. In conclusion, the oral colonisation by yeast in the HIV-positive patients was higher than in the HIVnegative subjects. The percentage of HIV-positive patients carrying C. dubliniensis (17.8%) was high. In HIV-positive patients, there was a significant increase in azole-resistant Candida species associated with previous antimycotic treatment. All isolates of C. dubliniensis were susceptible to fluconazole. Oral colonisation by isolates resistant to the most commonly used antifungal agents could represent a serious therapeutic problem among immunocopromised individuals. Acknowledgements We thank Licenciada Liliana Racca for her assistance in the statistical interpretation of the data; Marcela Culasso, María Robson and Mariana de Sanctis for their valuable assistance with the English language. References 1 Urizzar JMA. Candidiasis orales. Rev Iberoam Micol 2002; 19: Chave JP, Durussel C, Glauser MP, Bille J. Asymtomatic oral yeast carriage in HIV-infected patients: frequency and fluconazole susceptibility profile. Clin Microbiol Infect 1996; 18: Davies AN, Brailsford S, Broadley K, Beighton D. Oral yeast carriage in patients with advanced cancer. Oral Microbiol Immunol 2002; 17: Gugnami HC, Becker K, Fegeler W et al. Oropharyngeal carriage of Candida species in HIV-infected patients in India. Mycoses 2003; 46: Jabra-Rizk MA, Falkler WA, Enwonwu CO, Onwujekwe DI, Merz WG, Meiller TF. Prevalence of yeast among children in Nigeria and the United States. Oral Microbiol Immunol 2001; 16: Gutiérrez J, Morales P, Gonzáles MA, Quindós G. Candida dubliniensis, a new fungal pathogen. J Basic Microbiol 2002; 42: Kirkpatrick WR, Revankar SG, McAtee RK et al. Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus infected patients in North America by primary CHROMagar Candida screening and susceptibility testing of isolates. J Clín Microbiol 1998; 36: Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Coleman DC. Candida dubliniensis sp nov: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV infected individuals. Microbiology 1995; 141: Laet SantÕAna P, Milan EP, Martinez R et al. Multicenter brazilian study of oral Candida species isolated from AIDS patients. Mem Inst Oswaldo Cruz 2002; 97: Rodrigues Costa C, Aquino Lemos J, Sena Passos X et al. Species distribution and antifungal susceptibility profile of oral Candida isolates from HIV-infected patients in the antiretroviral therapy era. Mycopathologia 2006; 162: Saag MS, Fessel WJ, Kaufman CA et al. Treatment of fluconazole-refractory oropharyngeal candidiasis with itraconazole oral solution in HIV-positive patients. AIDS Res Hum Retroviruses 1999; 15: Redding SW, Zellars RC, Kirkpatrick WR et al. Epidemiology of oropharyngeal Candida colonization and infection in patient receiving radiation for head and neck cancer. J Clin Microbiol 1999; 37: Sobel JD, Ohmit SE, Schuman P et al. The evolution of Candida species and fluconazole susceptibility among oral and vaginal isolates recovered from human immunodeficiency virus (HIV)-seropositive and at-risk HIV-seronegative women. J Infect Dis 2001; 183: Garcia Heredia M, Garcia SD, Copolillo EF et al. Prevalence of vaginal candidiasis in pregnant women. Identification of yeasts and susceptibility to antifungal agents. Rev Argent Microbiol 2006; 38: Rodero L, Davel G, Soria M et al. Multicenter study of fungemia due to yeasts in Argentina. Rev Argent Microbiol 2005; 37: Buscemi L, Arechavala A, Negroni R. Study of acute vulvovaginitis in sexually active adult women, with special reference to candidosis, in patients of the Francisco J. Muniz Infectious Diseases Hospital. Rev Iberoam Micol 2004; 21: Mujica MT, Finquelievich JL, Jewtuchowicz V, Iovannitti CA. Prevalence of Candida albicans and Candida non-albicans in clinical samples during Rev Argent Microbiol 2004; 36: Giusiano GE, Mangiaterra M, Rojas F, Gomez V. Yeasts species distribution in neonatal intensive care units in northeast Argentina. Mycoses 2004; 47: Cuenca-Estrella M, Rodero L, Garcia-Effron G, Rodriguez- Tudela JL. Antifungal susceptibilities of Candida spp. isolated from blood in Spain and Argentina, J Antimicrob Chemother 2002; 49: Saporiti AM, Gomez D, Levalle S et al. Vaginal candidiasis: etiology and sensitivity profile to antifungal agents in clinical use. Rev Argent Microbiol 2001; 33: Journal compilation Ó 2008 Blackwell Publishing Ltd Mycoses 52, 53 59

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