First isolation of Cryptococcus neoformans genotype VNI MAT-alpha from wood inside hollow trunks of Hymenaea courbaril.

Cryptococcal infection is transmitted by the inhalation of Cryptococcus spp. propagules. Information about the Cryptococcus species inhabiting plants might be clinically relevant due to the epidemiological role of these habitats as possible sources of human infection. The aim of this study was to increase the knowledge about the environmental occurrence of cryptococcosis agents. Hollow tree vegetal debris of nine plant species was sampled quarterly over a 12-month period. Melanized colonies were screened for Cryptococcus neoformans and C. gattii by biochemical tests, followed by URA5-RFLP molecular analysis, M13 fingerprinting assays, and mating-typing with the specific a and α primers. The susceptibility to fluconazole of all of the confirmed species colonies was determined using the AFST-EUCAST broth dilution method. We found that the typical Brazilian flora tree Hymenaea courbaril yielded a high cryptococcal burden (median, 10(2) CFU/g) during the summer, autumn and winter seasons. C. neoformans VNI molecular type MAT alpha was identified in all of the samples. The fingerprinting analyses showed great molecular variability with no correlation with the susceptibility profile to fluconazole (MIC range 4 to ≥64 mg/l). To our knowledge, this study is the first describing the association between C. neoformans and Hymenaea courbaril. These observations extend the known geographic distribution of and substantiate a new urban environmental niche for C. neoformans and also emphasize the genetic diversity of the environmental C. neoformans VNI molecular type isolates.

First isolation of cryptococcus neoformans genotype VNI MAT-alpha from wood hollow trunks of hymenaea courbaril

Cryptococcal infection is transmitted by the inhalation of Cryptococcus spp. propagules. Information about the Cryptococcus species inhabiting plants might be clinically relevant due to the epidemiological role of these habitats as possible sources of human infection. The aim of this study was to increase the knowledge about the environmental occurrence of cryptococcosis agents. Hollow tree vegetal debris of nine plant species was sampled quarterly over a 12-month period. Melanized colonies were screened for Cryptococcus neoformans and C. gattii by biochemical tests, followed by URA5-RFLP molecular analysis, M13 fingerprinting assays, and mating-typing with the specific a and α primers. The susceptibility to fluconazole of all of the confirmed species colonies was determined using the AFST-EUCAST broth dilution method. We found that the typical Brazilian flora tree Hymenaea courbaril yielded a high cryptococcal burden (median, 10(2) CFU/g) during the summer, autumn and winter seasons. C. neoformans VNI molecular type MAT alpha was identified in all of the samples. The fingerprinting analyses showed great molecular variability with no correlation with the susceptibility profile to fluconazole (MIC range 4 to ≥64 mg/l). To our knowledge, this study is the first describing the association between C. neoformans and Hymenaea courbaril. These observations extend the known geographic distribution of and substantiate a new urban environmental niche for C. neoformans and also emphasize the genetic diversity of the environmental C. neoformans VNI molecular type isolates.

MDGC-MS analysis of essential oils from Protium heptaphyllum (Aubl. ) and their antifungal activity against Candida specie / Análise MDGC-MS de óleos essenciais de Protium heptaphyllum (Aubl. ) e sua atividade antifúngica contra espécies de Candida

ABSTRACT Protium heptaphyllum is found in the Amazon region, and in various Brazilian states and South American countries. Also Known as almecega, it produces an oil resin used in traditional medicine as analgesic, anti-inflammatory, cicatrizant and expectorant, it is rich in pentacyclic triterpenes and essential oil. The main objective of this study was to analyze the chemical composition of P. heptaphyllumresin (OEPh) over different extraction times and to evaluate their antifungal activity against Candida species, obtained from gardeners with onychomycosis, using the disk diffusion method. The OEPh was obtained by hydrodistillation and analyzed by Multidimensional Gas Chromatography coupled with Mass Spectrometry (MDGC / MS). Candida species were obtained from lesions on the nails of horticulturist from a community garden in the city of Teresina, Piauí, Brazil. The antifungal activity in concentrations of 1000 µg/L, 500 µg/L and 250 µg/L, PROTOCOL M44-A2 (CLSI 2009) OEPh was tested. The main constituents identified were: l-limonene, α-terpineol, p-cineol, o-cymene and α-phellandrene, however, its composition varies significantly with extraction time. All species, except C. rugosa, were inhibited with halo (≥ 14 mm) at 1000 μg / L. C. krusei is naturally resistant to the drug fluconazole, but when tested with OEPh the clinical species (case 9) demonstrated sensitivity in three dilutions (halo ≤ 10 ≥ 14) and the standard strain was inhibited at concentration of 1000 μg/Lg / L (halo 14mm). A similar situation also occurred with the standard strain of C. parapsilosis (halo ≥ 11mm). OEPh has considerable antifungal activity, which merits further investigation for alternative clinical applications, since this species is widely distributed in our community, and it presents good yields, and also has important therapeutic applications.

RESUMO Protium heptaphyllum é encontrada na região amazônica, em vários estados do Brasil e países da América do Sul. Conhecida como almecega produz uma resina oleosa usada na medicina popular como analgésica, antiinflamatória, cicatrizante e expectorante, é rica em triterpenos pentaciclicos e óleo essencial. O objetivo principal do presente trabalho foi analisar a composição química do óleo essencial da resina P. heptaphyllum (OEPh) em diferentes tempo de extração e avaliarsuaatividade antifúngica contra espécies de Candida, isoladas de horticultores com onicomicoses, por método de disco-difusão. O OEPh foi obtido por hidrodestilação, analisado por Cromatografia Gasosa Multidimensinal Acoplada a Espectrometria de Massas (MDGC/MS). As espécies de Candida foram obtidas de lesões nas unhas de horticultores de uma horta comunitária na cidade de Teresina, Piauí, Brasil. Testou-se a atividade antifúngica do OEPhnas concentrações de 1000 μg/L, 500 μg/L e 250 μg/L, protocolo M44-A2 (CLSI 2009). Os principais constituintes identificados foram l- limoneno, α-terpineol, p-cineol, o-cimeno e α-felandreno, entretanto, sua composição varia significativamente em decorrência do tempo de extração. Todas as espécies, exceto a C. rugosa, foram inibidas com halo ( Χ ≥ 14 mm) na concentração de 1000 μg/L. C. krusei é naturalmente resistente ao fármaco fluconazol, mas quando testado com OEPh,a espécie clínico (caso 9) demonstrou sensibilidade nas três diluições (halo Χ ≤ 10 ≥ 14) e a cepa padrão foi inibida na concentração de 1000 μg/L (halo Χ 14mm). Fato semelhante também ocorreu com a cepa padrão de C. parapsilosis (halo Χ ≥ 11mm). O OEPh possui atividade antifúngica considerável, merecendo uma investigação mais aprofundada para aplicações clínicas alternativas, uma vez que esta espécie é amplamente distribuída em nossa comunidade, apresenta bom rendimento e, ainda, aplicações terapêuticas importantes.

Species distribution and antifungal susceptibility profile of Candida isolates from bloodstream infections in Lima, Peru.

Yeast identification and in vitro susceptibility testing provide helpful information for appropriate administration of antifungal treatments; however, few reports from the Latin American region have been published. The aim of this study was to identify the species present in isolates from bloodstream infections diagnosed in nine hospitals in Lima, Peru and to determine their in vitro susceptibility to four antifungal drugs. We tested and identified 153 isolates collected between October 2009 and August 2011 using standard methods. PCR and PCR-RFLP assays were performed to distinguish Candida albicans from Candida dubliniensis and to identify species of the Candida parapsilosis and Candida glabrata complexes. Antifungal susceptibility testing for fluconazole, anidulafungin and voriconazole was performed using the CSLI M27-A3 method, and amphotericin B susceptibility was determined using the Etest method. The most frequently isolated species were: C. albicans (61; 39.9 %), C. parapsilosis (43; 28.1 %), C. tropicalis (36; 23.5%) and C. glabrata (8; 5.2 %). The overall susceptibility rates were 98.0 %, 98.7 %, 98.0 % and 97.4 % for amphotericin B, fluconazole, voriconazole and anidulafungin, respectively. No isolate was resistant to more than one drug. These results showed that the rate of resistance to four antifungal drugs was low among Candida bloodstream isolates in Lima, Peru.

Interlaboratory variability of Caspofungin MICs for Candida spp. Using CLSI and EUCAST methods: should the clinical laboratory be testing this agent?

Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 µg/ml for C. albicans and C. tropicalis, 0.031 to 0.5 µg/ml for C. glabrata, and 0.063 to 1 µg/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.

Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species.

Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods.

Prevalence and antifungal susceptibility of Candida parapsilosis complex isolates collected from oral cavities of HIV-infected individuals.

At present, few data are available on the prevalence and antifungal susceptibility of Candida parapsilosis complex isolates from HIV-infected individuals. The C. parapsilosis complex comprises three species, C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis. Fifteen of 318 Candida isolates were identified as members of the C. parapsilosis complex by PCR and restriction fragment length polymorphism (RFLP). The prevalence of C. parapsilosis complex isolates was 4.7 %, 2.2 % being identified as C. parapsilosis sensu stricto and 2.5 % as C. metapsilosis, while no C. orthopsilosis was isolated. This is believed to be the first study that has identified isolates of C. metapsilosis obtained from the oral cavity of HIV-infected individuals. Antifungal susceptibility tests indicated that all the isolates were susceptible to amphotericin B (AMB), fluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), voriconazole (VRC) and caspofungin (CASPO). Although isolates of C. parapsilosis sensu stricto and C. metapsilosis were susceptible to FLC, isolates of C. metapsilosis showed a tendency for higher MICs (≥1.0 µg ml(-1)). Based upon the frequency of candidiasis and the fact that certain isolates of the C. parapsilosis complex respond differently to FLC therapy, our data may be of therapeutic relevance with respect to susceptibility and potential resistance to specific antifungal agents. Our data suggest that C. metapsilosis can be a human commensal; its importance as a pathogen has yet to be confirmed.

Ten-year study of species distribution and antifungal susceptibilities of Candida bloodstream isolates at a Brazilian tertiary hospital.

To describe the incidence and susceptibility profile of Candida bloodstream infections in a tertiary-care hospital, we performed a retrospective observational study from 1998 to 2007. Comorbidities and risk factors were compiled from all cases. In vitro susceptibility testing to fluconazole, itraconazole, voriconazole, and amphotericin B was performed for 100 isolates, and caspofungin was tested for C. parapsilosis complex. In a ten-year evaluation of candidemias, 44 % were caused by C. albicans, and species of the C. parapsilosis complex were the second most frequent agents (37 %). Other species presented lower incidences (C. tropicalis, 13 %, C. glabrata, 5 %, and C. krusei, 1 %). Neither C. dubliniensis nor C. metapsilosis were observed in this study. C. orthopsilosis (3 %) and C. parapsilosis stricto sensu (34 %) were also found. Species distribution was independent of catheterization, mechanical ventilation, or previous use of antifungals or corticoids. Parenteral nutrition administration was strongly related to C. glabrata infection, and the highest mortality (80 %) was observed in patients infected by this species. All C. albicans isolates showed high susceptibility to all tested drugs. However, two C. parapsilosis stricto sensu isolates presented high minimum inhibitory concentration (MIC) (4 mg/L each) to fluconazole, and one exhibited voriconazole MIC of 0.25 mg/L, highlighting the cross-resistance to these azoles. All isolates of C. tropicalis and C. glabrata showed no resistance to any drug tested. No difference was noted between C. parapsilosis and C. orthopsilosis susceptibilities to caspofungin. Our results suggest that resistance to amphotericin B, fluconazole, voriconazole, itraconazole, and caspofungin in Brazilian Candida bloodstream isolates is still uncommon.

Wild-type MIC distributions and epidemiological cutoff values for amphotericin B, flucytosine, and itraconazole and Candida spp. as determined by CLSI broth microdilution.

Clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs) have been established for several Candida spp. and the newer triazoles and echinocandins but are not yet available for older antifungal agents, such as amphotericin B, flucytosine, or itraconazole. We determined species-specific ECVs for amphotericin B (AMB), flucytosine (FC) and itraconazole (ITR) for eight Candida spp. (30,221 strains) using isolates from 16 different laboratories in Brazil, Canada, Europe, and the United States, all tested by the CLSI reference microdilution method. The calculated 24- and 48-h ECVs expressed in µg/ml (and the percentages of isolates that had MICs less than or equal to the ECV) for AMB, FC, and ITR, respectively, were 2 (99.8)/2 (99.2), 0.5 (94.2)/1 (91.4), and 0.12 (95.0)/0.12 (92.9) for C. albicans; 2 (99.6)/2 (98.7), 0.5 (98.0)/0.5 (97.5), and 2 (95.2)/4 (93.5) for C. glabrata; 2 (99.7)/2 (97.3), 0.5 (98.7)/0.5 (97.8), and 05. (99.7)/0.5 (98.5) for C. parapsilosis; 2 (99.8)/2 (99.2), 0.5 (93.0)/1 (90.5), and 0.5 (97.8)/0.5 (93.9) for C. tropicalis; 2 (99.3)/4 (100.0), 32 (99.4)/32 (99.3), and 1 (99.0)/2 (100.0) for C. krusei; 2 (100.0)/4 (100.0), 0.5 (95.3)/1 (92.9), and 0.5 (95.8)/0.5 (98.1) for C. lusitaniae; -/2 (100.0), 0.5 (98.8)/0.5 (97.7), and 0.25 (97.6)/0.25 (96.9) for C. dubliniensis; and 2 (100.0)/2 (100.0), 1 (92.7)/-, and 1 (100.0)/2 (100.0) for C. guilliermondii. In the absence of species-specific CBP values, these wild-type (WT) MIC distributions and ECVs will be useful for monitoring the emergence of reduced susceptibility to these well-established antifungal agents.

Mutants with heteroresistance to amphotericin B and fluconazole in Candida

Several studies have reported the occurrence of infections caused by Candida yeasts as well as the increasing prevalence of non albicans species. The aim of the present work is focused on the obtaining of heteroresistance to amphotericin B and fluconazole in Candida species using two distinct methodologies: selection and induction. Resistant samples were obtained by selective pressure using a medium with fluconazole for growth, followed by growth in a medium with amphotericin B. The selective pressure was also created beginning with growth in amphotericin B medium followed by growth in fluconazole medium. Concomitantly, samples were submitted to the induction of resistance through cultivation in increasing concentrations of fluconazole, followed by cultivation in increasing concentrations of amphotericin B. Subsequently, the induction began with amphotericin B followed by fluconazole. Three samples resistant to fluconazole and amphotericin B were obtained, two by induction (C. glabrata and C. tropicalis) and one by selection (C. tropicalis). Both C. tropicalis originated from the same wild sample. After successive transfers for drug free medium, only the sample obtained by selection was able to maintain the resistance phenotype. These results suggest that the phenotype of heteroresitance to fluconazole and amphotericin B can be produced by two methodologies: selection and induction.

Mutants with heteroresistance to amphotericin B and fluconazole in Candida.

Several studies have reported the occurrence of infections caused by Candida yeasts as well as the increasing prevalence of non albicans species. The aim of the present work is focused on the obtaining of heteroresistance to amphotericin B and fluconazole in Candida species using two distinct methodologies: selection and induction. Resistant samples were obtained by selective pressure using a medium with fluconazole for growth, followed by growth in a medium with amphotericin B. The selective pressure was also created beginning with growth in amphotericin B medium followed by growth in fluconazole medium. Concomitantly, samples were submitted to the induction of resistance through cultivation in increasing concentrations of fluconazole, followed by cultivation in increasing concentrations of amphotericin B. Subsequently, the induction began with amphotericin B followed by fluconazole. Three samples resistant to fluconazole and amphotericin B were obtained, two by induction (C. glabrata and C. tropicalis) and one by selection (C. tropicalis). Both C. tropicalis originated from the same wild sample. After successive transfers for drug free medium, only the sample obtained by selection was able to maintain the resistance phenotype. These results suggest that the phenotype of heteroresitance to fluconazole and amphotericin B can be produced by two methodologies: selection and induction.

Mutants with heteroresistance to amphotericin B and fluconazole in Candida

Several studies have reported the occurrence of infections caused by Candida yeasts as well as the increasing prevalence of non albicans species. The aim of the present work is focused on the obtaining of heteroresistance to amphotericin B and fluconazole in Candida species using two distinct methodologies: selection and induction. Resistant samples were obtained by selective pressure using a medium with fluconazole for growth, followed by growth in a medium with amphotericin B. The selective pressure was also created beginning with growth in amphotericin B medium followed by growth in fluconazole medium. Concomitantly, samples were submitted to the induction of resistance through cultivation in increasing concentrations of fluconazole, followed by cultivation in increasing concentrations of amphotericin B. Subsequently, the induction began with amphotericin B followed by fluconazole. Three samples resistant to fluconazole and amphotericin B were obtained, two by induction (C. glabrata and C. tropicalis) and one by selection (C. tropicalis). Both C. tropicalis originated from the same wild sample. After successive transfers for drug free medium, only the sample obtained by selection was able to maintain the resistance phenotype. These results suggest that the phenotype of heteroresitance to fluconazole and amphotericin B can be produced by two methodologies: selection and induction.

Correlation between CLSI, EUCAST and Etest methodologies for amphotericin B and fluconazole antifungal susceptibility testing of Candida spp. clinical isolates.

This study analyzed the correlation between the results obtained through two microdilution methods: Clinical and Laboratory Standard Institute (CLSI) (M27-A2) and European Committee on Antibiotic Susceptibility Testing (EUCAST) (document E. Dis. 7.1) and an agar base method Etest for determining minimmun inhibitory concentration (MIC) for amphotericin B and fluconazole against 30 clinical isolates of Candida spp. The agreement between Etest, CLSI and EUCAST MICs within +/- 2 log2 dilutions was higher for amphotericin B than for fluconazole However, Pearson correlation demonstrated a greater agreement for fluconazole. The categorical agreement between MICs provided by the Etest/ CLSI and Etest/EUCAST methodologies was high for both amphotericin B (100%) and fluconazole (> or = 96.66%). This study demonstrated the adequacy of Etest method using Mueller Hinton agar to evaluate amphotericin B and fluconazole susceptibility of clinical isolates of Candida spp.

Clinical and epidemiological study in an AIDS patient with Microsporum gypseum infection.

A 37 years old homosexual male with AIDS was diagnosed as having Tinea cruris and Tinea corporis. The patient was a garden designer and therefore he used to handle soil very often. Microsporum gypseum was identified on cultures from skin-scrapings and biopsy material taken from different cutaneous lesion. The same species was isolated from samples of soil the patient used to work with. The clinical history of the patient and its epidemiological aspects were deeply studied by the authors. We stress the value of identifying possible sources of dermatophytic infections in order to give advice to patients.