Morphological and ultrastructural changes in fungal agents after LASER application.

BACKGROUND: Onychomycosis is a fungal nail infection of difficult treatment due to the fungal survival capacity and reduced number of effective therapies. The present study aimed to isolate fungal agents that cause onychomycosis in immunocompetent patients and evaluate how LASER treatments affect the growth and ultrastructure of isolates. METHODS: In total, 21 patients with positive direct microscopic examination (DME) for onychomycosis had nail samples collected for cultivation and phenotypic identification of microorganisms. From these patients, 12 underwent LASER treatment, divided in Group 1 (n = 5) treated with Nd: YAG 1,064 nm, and Group 2 (n = 7) treated with Nd: YAG 1,064 nm + Er: YAG 2,940 nm + topical isoconazole. Transmission Electron Microscopy (TEM) was performed to evaluate ultrastructural changes after treatment. RESULTS: DME, cultivation, and phenotypic identification showed that the most identified fungus was Trichophyton rubrum spp. After LASER therapy, sample cultivation showed alterations in the fungal morphology with reduction of hyphae, conidia, and reproductive structures. Alterations in fungal cell wall structure, cytoplasm density, and organelles were observed by TEM. CONCLUSION: LASER irradiation causes changes in the fungal cells, especially in the number of hyphae and the presence of conidia. In addition, it affects fungal growth and reproduction capacity, which interferes with their infection ability and virulence.

Emergence of cryptic species and clades of Meyerozyma guilliermondii species complex exhibiting limited in vitro susceptibility to antifungals in patients with candidemia.

Members of the Meyerozyma guilliermondii species complex are able to cause superficial and life-threatening systemic infections with low susceptibility to azoles and echinocandins. We tested 130 bloodstream M. guilliermondii complex isolates collected from eight Latin American medical centers over 18 years (period 1 = 2000-2008 and period 2 = 2009-2018) to investigate trends in species distribution and antifungal resistance. The isolates were identified by rDNA ITS region sequencing, and antifungal susceptibility tests were performed against fluconazole, voriconazole, anidulafungin, and amphotericin B using the CLSI microbroth method. M. guilliermondii sensu stricto (s.s.; n = 116) was the most prevalent species, followed by Meyerozyma caribbica (n = 12) and Meyerozyma carpophila (n = 2). Based on rDNA ITS identification, three clades within M. guilliermondii sensu stricto were characterized (clade 1 n = 94; clade 2 n = 19; and clade 3 n = 3). In the second period of study, we found a substantial increment in the isolation of M. caribbica (3.4% versus 13.8%; P = 0.06) and clade 2 M. guilliermondii s.s. exhibiting lower susceptibility to one or more triazoles. IMPORTANCE Yeast-invasive infections play a relevant role in human health, and there is a concern with the emergence of non-Candida pathogens causing disease worldwide. There is a lack of studies addressing the prevalence and antifungal susceptibility of different species within the M. guilliermondii complex that cause invasive infections. We evaluated 130 episodes of M. guilliermondii species complex candidemia documented in eight medical centers over 18 years. We detected the emergence of less common species within the Meyerozyma complex causing candidemia and described a new clade of M. guilliermondii with limited susceptibility to triazoles. These results support the relevance of continued global surveillance efforts to early detect, characterize, and report emergent fungal pathogens exhibiting limited susceptibility to antifungals.

Development of quantitative multiplex RT-qPCR one step assay for detection of hepatitis delta virus.

Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruses, usually with a more severe clinical outcome when compared to an HBV monoinfection. To date, the real prevalence of HDV infection is underestimated and detection methods are poorly available, especially in more endemic regions. Therefore, a one-step RT-qPCR method for quantification of HDV-RNA was developed. Biological samples were selected between 2017 and 2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada and underwent the test developed by this study and a second quantitative RT-qPCR assay. The slope of the initial quantitative assay was - 3.321 with an efficiency of 100.04% and amplification factor equal to 2. Analysis of the repeatability data revealed a Limit of Quantification of 5 copies/reaction and Limit of Detection (95%) of 2.83 copies per reaction. In the diagnostic sensitivity tests, there was an accuracy of 97.37% when compared to the reference test. This assay proved to be highly efficient and reproducible, making it a valuable tool to monitor hepatitis Delta patients and assess the risk of disease progression, as well as the effectiveness of treatment.

Candida species causing fungal keratitis: molecular identification, antifungal susceptibility, biofilm formation, and clinical aspects.

The study aimed to evaluate the clinical aspects, molecular identification, biofilm formation, and antifungal susceptibility profile of Candida species isolated from fungal keratitis. Thirteen Candida isolates from 13 patients diagnosed with Candida keratitis were retrieved and grown in pure culture. Species identification was performed by micromorphology analysis and ITS-rDNA sequencing. The broth microdilution method tested the minimum inhibitory concentration (MIC) of four antifungal drugs (fluconazole, amphotericin B, voriconazole, and anidulafungin). The biofilms were cultured and incubated with antifungal drugs for 24 h. The XTT reduction assay measured the biofilm activity. Biofilm MICs were calculated based on a 50% reduction in metabolic activity compared with the activity of the drug-free control. Among isolates, two were C. albicans, 10 were C. parapsilosis (sensu stricto), and one was C. orthopsilosis. All isolates were classified as susceptible or intermediate to all four antifungal drugs. Four isolates were very low biofilm producers (30%). Nine isolates were biofilm producers, and all biofilm samples were unsusceptible to all drugs tested. Previous ocular surgery was the most common underlying condition for fungal keratitis (84.6%), and C. parapsilosis was the most frequent Candida species (76.9%). Four patients (30.7%) needed keratoplasty, whereas two (15.3%) required evisceration. The biofilm formation ability of Candida isolates decreased antifungal susceptibility compared with planktonic cells. Despite in vitro antifungal susceptibility, almost half of the patients were unresponsive to clinical treatment and needed surgery.

Trends towards lower azole susceptibility among 200 Candida tropicalis bloodstream isolates from Brazilian medical centres.

OBJECTIVES: Candida tropicalis is one of the three most frequent species causing candidaemia in Latin America. Despite the high prevalence of C. tropicalis in candidaemia cases in Brazil, little is known about the trends in fluconazole susceptibility over time. The objective of this study was to evaluate temporal trends in azole resistance rates among C. tropicalis bloodstream isolates from patients treated in six Brazilian medical centres over a 12-year period. METHODS: We selected 200 C. tropicalis bloodstream isolates from six medical centres in Brazil collected between 2007 and 2018. Species identification was confirmed by MALDI-TOF/MS. Antifungal susceptibility testing for four antifungal agents was performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. RESULTS: Overall, rates of non-susceptibility were 4% and 3.5% to fluconazole and voriconazole, respectively. All isolates were susceptible to amphotericin B and only one isolate was resistant to echinocandins. CONCLUSION: Although we failed to demonstrate statistical differences in the rates of azole resistance documented during the period of analysis, trends towards lower susceptibility to fluconazole and voriconazole were shown.

Oral colonization by Candida spp. in liver transplant patients: Molecular identification and antifungal susceptibilityOral colonization by Candida spp. in liver transplant.

Candida species are commensal to normal oral microbiota; however, they can cause infections if immune functions are reduced. The aim of this study was to investigate oral colonization, identify species, and test the susceptibility profile to antifungals. A descriptive study included 97 liver transplant patients who attended the transplant center of a referral hospital in southern Brazil. Two oral swab collections were performed, with a 6-month gap between collections. The samples were identified by sequencing the internal transcribed spacer ITS region of the ribosomal DNA. The sensitivity test was performed with fluconazole, amphotericin B, and micafungin using a broth microdilution method recommended by Clinical and Laboratory Standards Institute document M27-A4. Eighty-two patients were investigated and 15 were excluded for presenting clinical infection. The identification of yeasts showed colonization in 66% and 61.9% in collections A and B, respectively. Candida albicans was the most prevalent species in both collections (n = 29/50 and n = 27/49, respectively). In 31 (62%) patients, the yeast species remained the same for 6 months, and in 19 (38%) the colonizing species was substituted. Thirty-two isolates from collection A were sensitive (S) to Fluconazole, 13 sensitive dose-dependent (SDD), and five resistant (R). In collection B, 32 were S, 12 SDD, and 5 R. For amphotericin B and micafungin, all isolates were sensitive. With knowledge of the species and identification of strains resistant to fluconazole, useful information can be alerts about the emergence of antifungal resistance strains. LAY SUMMARY: Study of great importance because it is the first investigation that identifies Candida in the oral cavity of liver transplant patients, allowing an understanding of epidemiology and contributing to the knowledge about strains resistant to fluconazole.

Genotyping Reveals High Clonal Diversity and Widespread Genotypes of Candida Causing Candidemia at Distant Geographical Areas.

The objectives of this study were to gain further insight on Candida genotype distribution and percentage of clustered isolates between hospitals and to identify potential clusters involving different hospitals and cities. We aim to genotype Candida spp. isolates causing candidemia in patients admitted to 16 hospitals in Spain, Italy, Denmark, and Brazil. Eight hundred and eighty-four isolates (Candida albicans, n = 534; C. parapsilosis, n = 282; and C. tropicalis, n = 68) were genotyped using species-specific microsatellite markers. CDC3, EF3, HIS3, CAI, CAIII, and CAVI were used for C. albicans, Ctrm1, Ctrm10, Ctrm12, Ctrm21, Ctrm24, and Ctrm28 for C. tropicalis, and CP1, CP4a, CP6, and B for C. parapsilosis. Genotypes were classified as singletons (genotype only found once) or clusters (same genotype infecting two or more patients). Clusters were defined as intra-hospital (involving patients admitted to a single hospital), intra-ward (involving patients admitted to the same hospital ward) or widespread (involving patients admitted to different hospitals). The percentage of clusters and the proportion of patients involved in clusters among species, genotypic diversity and distribution of genetic diversity were assessed. Seven hundred and twenty-three genotypes were detected, 78 (11%) being clusters, most of which (57.7%; n = 45/78) were intra-hospital clusters including intra-ward ones (42.2%; n = 19/45). The proportion of clusters was not statistically different between species, but the percentage of patients in clusters varied among hospitals. A number of genotypes (7.2%; 52/723) were widespread (found at different hospitals), comprising 66.7% (52/78) of clusters, and involved patients at hospitals in the same city (n = 21) or in different cities (n = 31). Only one C. parapsilosis cluster was a widespread genotype found in all four countries. Around 11% of C. albicans and C. parapsilosis isolates causing candidemia are clusters that may result from patient-to-patient transmission, widespread genotypes commonly found in unrelated patients, or insufficient microsatellite typing genetic discrimination.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†.

The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries-and the same number of singleton genotypes used as controls-were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters-particularly in the case of C. albicans-show significantly more biomass production and metabolic activity than singleton genotypes.

Revisiting Species Distribution and Antifungal Susceptibility of Candida Bloodstream Isolates from Latin American Medical Centers.

The epidemiology of candidemia varies geographically, and there is still scarce data on the epidemiology of candidemia in Latin America (LA). After extensive revision of medical literature, we found reliable and robust information on the microbiological aspects of candidemia in patients from 11 out of 21 medical centers from LA countries and 1 out of 20 from Caribbean countries/territories. Based on 40 papers attending our search strategy, we noted that C. albicans remains the most common species causing candidemia in our region, followed by C. parapsilosis and C. tropicalis. In Argentina, Brazil, and Colombia, a trend towards an increase in frequency of C. glabrata candidemia was observed. Although resistance rates to fluconazole is under 3%, there was a slight increase in the resistance rates to C. albicans, C. parapsilosis and C. tropicalis isolates. Echinocandin resistance has been reported in a few surveys, but no single study confirmed the resistant phenotype reported by using molecular methods. We highlight the importance of conducting continuous surveillance studies to identify new trends in terms of species distribution of Candida and antifungal resistance related to episodes of candidemia in LA. This information is critical for helping clinicians to prevent and control Candida bloodstream infections in their medical centers.

In vitro susceptibility of a large collection of Candida Strains against fluconazole and voriconazole by using the CLSI disk diffusion assay.

We evaluated all Candida spp. isolates obtained from patients admitted to two tertiary care hospitals between 1999 and 2003 in the city of São Paulo, Brazil. The in vitro activities of fluconazole (FCZ) and voriconazole were determined by the agar disk diffusion test using the Clinical and Laboratory Standards Institute M44-A guidelines. The inhibition zone diameters were read and interpreted automatically by the BIOMIC(®) image-analysis plate reader system. We tested a total of 4,625 strains, including 2,393 strains of C. albicans (51.7%), 658 of C. tropicalis (14.2%), 503 of C. glabrata (10.9%), 495 of C. parapsilosis (10.7%), 292 of C. rugosa (6.3%), 195 of C. guilliermondii (4.2%) and 89 of other Candida species (2.0%). Only 2.0% of the strains tested were classified as dose-dependent susceptible (DDS), and 5.8% of them were resistant to FCZ. The resistance or DDS to fluconazole was verified mainly among C. glabrata (7.8%), C. krusei (67.9%) and C. rugosa (65.1%). Voriconazole exhibited better activity in vitro than fluconazole, even in isolates fluconazole resistant. The resistance of fluconazole and voriconazole did not increase in the isolates of Candida spp. during the evaluated period.

Candidemia surveillance in Brazil: evidence for a geographical boundary defining an area exhibiting an abatement of infections by Candida albicans group 2 strains.

Prospective population surveillance has been conducted for candidemia in Brazil (A. L. Colombo, M. Nucci, B. J. Park, et al., J. Clin. Microbiol. 44:2816-2823, 2006). In the present study, a total of 63 isolates from 61 patients, representing 11 medical centers from nine geographic regions, were characterized by multilocus sequence typing (MLST). A total of 48 unique profiles or diploid sequence types (DSTs) were observed, with nine new sequence types (STs) and 32 new DSTs. There were no apparent correlations between center/region and DST patterns. Subtypes were compared to those in a known characterized reference set, including a large database of strains obtained worldwide. Significantly, only one C. albicans group 2 isolate was found in our collection, although isolates from this particular group are commonly found worldwide. These data, combined with information from other previously reported studies, establish a statistically significant diminishment of group 2 strains in Central and South America, including Mexico and portions of the Southwestern United States.

Multilocus sequence typing of sequential Candida albicans isolates from patients with persistent or recurrent fungemia.

Multilocus sequence typing (MLST) is a useful tool to explore the phylogenetics and epidemiology of Candida albicans isolates recovered from cases of invasive candidiasis. The goal of this study was to determine whether the same or different strains were responsible for persistent or recurrent fungemia through the use of MLST and ABC typing on sequential C. albicans isolates from the same patient. We applied both typing methods to 21 C. albicans strains recovered from 8 patients with persistent or recurrent candidemia. The isolates were collected during a multicenter surveillance study in four public tertiary care hospitals in Brazil. Persistent candidemia was defined as two or more blood cultures positive for C. albicans on 2 or more separate days. Recurrent candidemia was defined as an episode of candidemia occurring at least 1 month after the apparent complete resolution of an infectious episode caused by Candida species. We observed that, except for one patient, all strains from the first and second samples of the same patient showed the same MLST diploid sequence type (DST), ABC type and susceptibility profile to antifungals. Three distinct strains, well discriminated by MLST, were found in the seven samples collected sequentially over 10 days from one patient. The strains from the first four samples were indistinguishable, the fifth and sixth were also indistinguishable but different from the first four and seventh samples. Significantly, the seventh strain was the only C. albicans clade 2 isolate found in our total collection involving 61 patients, although clade 2 is commonly found worldwide. To the best of our knowledge, this is the first study describing the recovery of three distinct C. albicans strains in the same patient with a persistent blood stream infection within a short period of time.

Antifungal susceptibility of 1000 Candida bloodstream isolates to 5 antifungal drugs: results of a multicenter study conducted in São Paulo, Brazil, 1995-2003.

We evaluated all Candida sp. bloodstream isolates obtained from patients admitted to 4 tertiary care hospitals between 1995 and 2003 in the city of São Paulo, Brazil. Susceptibility to amphotericin B, 5-fluorocytosine, fluconazole (FCZ), itraconazole (ITZ), and voriconazole (VCZ) was determined using the Clinical Laboratory Standards Institute broth microdilution method. We tested a total of 1000 strains, including 400 strains of Candida albicans (40%), 243 of Candida tropicalis (24.3%), 238 of Candida parapsilosis (23.8%), 44 of C. glabrata (4.4%), 30 of Candida guilliermondii (3%), and 25 of Candida rugosa (2.5%). Only 1.9% of the strains tested were susceptible in a dose-dependent manner, and 0.2% of them were resistant to FCZ. Almost 100% of the strains were susceptible to VCZ. Despite that azole resistance was a rare finding, a trend toward increased resistance among C. rugosa strains to FCZ and ITZ was noted.

Epidemiology of candidemia in Brazil: a nationwide sentinel surveillance of candidemia in eleven medical centers.

Candidemia studies have documented geographic differences in rates and epidemiology, underscoring the need for surveillance to monitor trends. We conducted prospective candidemia surveillance in Brazil to assess the incidence, species distribution, frequency of antifungal resistance, and risk factors for fluconazole-resistant Candida species. Prospective laboratory-based surveillance was conducted from March 2003 to December 2004 in 11 medical centers located in 9 major Brazilian cities. A case of candidemia was defined as the isolation of Candida spp. from a blood culture. Incidence rates were calculated per 1,000 admissions and 1,000 patient-days. Antifungal susceptibility tests were performed by using the broth microdilution assay, according to the Clinical and Laboratory Standards Institute guidelines. We detected 712 cases, for an overall incidence of 2.49 cases per 1,000 admissions and 0.37 cases per 1,000 patient-days. The 30-day crude mortality was 54%. C. albicans was the most common species (40.9%), followed by C. tropicalis (20.9%) and C. parapsilosis (20.5%). Overall, decreased susceptibility to fluconazole occurred in 33 (5%) of incident isolates, 6 (1%) of which were resistant. There was a linear correlation between fluconazole and voriconazole MICs (r = 0.54 and P < 0.001 [Spearman's rho]). This is the largest multicenter candidemia study conducted in Latin America and shows the substantial morbidity and mortality of candidemia in Brazil. Antifungal resistance was rare, but correlation between fluconazole and voriconazole MICs suggests cross-resistance may occur.

Clinical and microbiological aspects of candidemia due to Candida parapsilosis in Brazilian tertiary care hospitals.

In order to characterize the epidemiology, microbiology and outcome of candidemia due to Candida parapsilosis, we examined a database of 282 episodes of candidemia prospectively collected from four tertiary care hospitals in São Paulo, Brazil between March 2002 and February 2003, and compared the characteristics of patients with candidemia due to C. parapsilosis (n=64) with those caused by Candida albicans (n=107). C. parapsilosis candidemia was associated with neutropenia (p=0.005), tunneled central venous catheter (p=0.005) and cancer chemotherapy (p=0.03). By multivariate analysis, candidemia due to C. parapsilosis was associated with the presence of a tunneled central venous catheter (relative risk 3.71, 95% confidence interval 1.28-10.70). Except for a single isolate of C. parapsilosis that exhibited MIC >1 microg/ml to amphotericin B, no resistance was observed in 166 isolates tested against fluconazole, itraconazole, 5-flucytosine and amphotericin B. The caspofungin MIC values of C. parapsilosis isolates were significantly higher than those exhibited by C. albicans isolates (p<0.001). The overall mortality of patients with candidemia due to C. parapsilosis was significantly lower (45% vs. 62%, p=0.03). The association between C. parapsilosis candidemia and a tunneled central venous catheter supports the idea that the main mode of acquisition of C. parapsilosis is from an external source.

Candida dubliniensis identification in Brazilian yeast stock collection.

We investigated the presence of Candida dubliniensis among isolates previously identified as Candida albicans and maintained in a yeast stock collection from 1994 to 2000. All isolates were serotyped and further evaluated for antifungal susceptibility profile. After doing a screening test for C. dubliniensis isolates based on the capability of colonies to grow at 42 C, its final identification was obtained by randomly amplified polymorphic DNA (RAPD) analysis using three different primers. A total of 46 out of 548 screened isolates did not exhibit growth at 42 C and were further genotyped by RAPD. Eleven isolates were identified as C. dubliniensis with RAPD analysis. Regarding serotypes, 81.5% of C. albicans and all C. dubliniensis isolates belonged to serotype A. Of note, 9 out of 11 C. dubliniensis isolates were obtained from patients with acquired immunodeficiency syndrome (Aids) and all of them were susceptible to azoles and amphotericin B. We found 17 (3%) C. albicans isolates that were dose-dependent susceptibility or resistant to azoles. In conclusion, we found a low rate of C. dubliniensis isolates among stock cultures of yeasts previously identified as C. albicans. Most of these isolates were recovered from oral samples of Aids patients and exhibited high susceptibility to amphotericin B and azoles. C. albicans serotype A susceptible to all antifungal drugs is the major phenotype found in our stock culture.

Species distribution and antifungal susceptibility profile of Candida spp. bloodstream isolates from Latin American hospitals.

From March 1999 to March 2000, we conducted a prospective multicenter study of candidemia involving five tertiary care hospitals from four countries in Latin America. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to the National Committee for Clinical Laboratory Standards microbroth assay method. During a 12 month-period we were able to collect a total of 103 bloodstream isolates of Candida spp. C. albicans was the most frequently isolated species accounting for 42% of all isolates. Non-albicans Candida species strains accounted for 58% of all episodes of candidemia and were mostly represented by C. tropicalis (24.2%) and C. parapsilosis (21.3%). It is noteworthy that we were able to identify two cases of C. lusitaniae from different institutions. In our casuistic, non-albicans Candida species isolates related to candidemic episodes were susceptible to fluconazole. Continuously surveillance programs are needed in order to identify possible changes in the species distribution and antifungal susceptibility patterns of yeasts that may occurs after increasing the use of azoles in Latin American hospitals.

Susceptibility of Malassezia pachydermatis to azole antifungal agents evaluated by a new broth microdilution method

Malassezia pachydermatis   is considered an opportunistic pathogen of the outer ear duct in dogs and cats. This yeast can also be found in the skin, rectum, anal sacks and vagina. Eighty-two samples of this yeast isolated from dogs with the symptoms of external otitis from the Porto Alegre region were tested for their susceptibility to antifungal agents using the Broth Microdilution Method. The testing antifungal agents were Ketoconazole, Fluconazole and Itraconazole. Experimental essays determined that Sabouraud dextrose broth supplemented with 1% Tween 80 was the most appropriate medium for culture, for a ten-fold dilutions for the inocula, and 48 hours as the interval of readings. The ranges of the Minimal Inhibitory Concentrations (MICs) for the 82 samples were (a) Ketoconazole, from 0.015 to 0.25 mg/mL (mean of 0.08 mg/mL), (b) Fluconazole, from 1 to 32 mg/mL (mean of 9.22 mg/mL), and (c) Itraconazole, from 0.007 to 0.125 mg/mL (mean of 0.05 mg/mL). The isolates of M. pachydermatis showed an excellent level of susceptibility to antifungal azole agents, with all strains being susceptible to Itraconazole, and with only 2.4 % and 3.7% being resistant to Fluconazole and Kketoconazole, respectively. The use of the broth microdilution method allows the assessment of the susceptibility of large numbers of samples from M. pachydermatis isolates to the most common antifungal agents

Susceptibility of Malassezia pachydermatis to azole antifungal agents evaluated by a new broth microdilution method

Malassezia pachydermatis   is considered an opportunistic pathogen of the outer ear duct in dogs and cats. This yeast can also be found in the skin, rectum, anal sacks and vagina. Eighty-two samples of this yeast isolated from dogs with the symptoms of external otitis from the Porto Alegre region were tested for their susceptibility to antifungal agents using the Broth Microdilution Method. The testing antifungal agents were Ketoconazole, Fluconazole and Itraconazole. Experimental essays determined that Sabouraud dextrose broth supplemented with 1% Tween 80 was the most appropriate medium for culture, for a ten-fold dilutions for the inocula, and 48 hours as the interval of readings. The ranges of the Minimal Inhibitory Concentrations (MICs) for the 82 samples were (a) Ketoconazole, from 0.015 to 0.25 mg/mL (mean of 0.08 mg/mL), (b) Fluconazole, from 1 to 32 mg/mL (mean of 9.22 mg/mL), and (c) Itraconazole, from 0.007 to 0.125 mg/mL (mean of 0.05 mg/mL). The isolates of M. pachydermatis showed an excellent level of susceptibility to antifungal azole agents, with all strains being susceptible to Itraconazole, and with only 2.4 % and 3.7% being resistant to Fluconazole and Kketoconazole, respectively. The use of the broth microdilution method allows the assessment of the susceptibility of large numbers of samples from M. pachydermatis isolates to the most common antifungal agents

Susceptibility of Malassezia pachydermatis to azole antifungal agents evaluated by a new broth microdilution method

Malassezia pachydermatis   is considered an opportunistic pathogen of the outer ear duct in dogs and cats. This yeast can also be found in the skin, rectum, anal sacks and vagina. Eighty-two samples of this yeast isolated from dogs with the symptoms of external otitis from the Porto Alegre region were tested for their susceptibility to antifungal agents using the Broth Microdilution Method. The testing antifungal agents were Ketoconazole, Fluconazole and Itraconazole. Experimental essays determined that Sabouraud dextrose broth supplemented with 1% Tween 80 was the most appropriate medium for culture, for a ten-fold dilutions for the inocula, and 48 hours as the interval of readings. The ranges of the Minimal Inhibitory Concentrations (MICs) for the 82 samples were (a) Ketoconazole, from 0.015 to 0.25 mg/mL (mean of 0.08 mg/mL), (b) Fluconazole, from 1 to 32 mg/mL (mean of 9.22 mg/mL), and (c) Itraconazole, from 0.007 to 0.125 mg/mL (mean of 0.05 mg/mL). The isolates of M. pachydermatis showed an excellent level of susceptibility to antifungal azole agents, with all strains being susceptible to Itraconazole, and with only 2.4 % and 3.7% being resistant to Fluconazole and Kketoconazole, respectively. The use of the broth microdilution method allows the assessment of the susceptibility of large numbers of samples from M. pachydermatis isolates to the most common antifungal agents