Comparison between MALDI-TOF MS and MicroScan in the identification of emerging and multidrug resistant yeasts in a fourth-level hospital in Bogotá, Colombia.

BACKGROUND: The introduction of MALDI-TOF MS in the clinical microbiology laboratory has modified the approaches for the identification of fungi. Thanks to this tool, it is possible to identify cryptic species, which possess critical susceptibility patterns. Clinical strains were identified using the MicroScan and MALDI-TOF MS systems. Discrepant results from both methods were investigated using ITS rDNA barcoding. Finally, these isolates were also tested for in vitro susceptibility. RESULTS: The percentage of agreement between both methods to 498 yeast isolates was of 93.6% (32 discrepant isolates). The concordance of ITS sequencing with MALDI-TOF MS was higher (99%) than that of MicroScan (94%). Several of these discordant yeasts displayed high MICs for antifungal agents. CONCLUSIONS: Our study highlights the need of the MS and molecular approaches such as MALDI-TOF MS and ITS rDNA barcoding for the correct identification of emerging or cryptic yeast species; besides, some of these could be multidrug resistant. This work was the first experience in the implementation of the MALDI-TOF MS technology in Colombia. We found the first uncommon yeasts including Candida auris and we could identify Trichosporon faecalis. Our work highlights a clear necessity of an accurate yeast identification as a much more pertinent technique than the susceptibility profiles, because the most unusual yeasts exhibit resistance profiles to the few available antifungals.

[Evaluation of Fusarium spp. pathogenicity in plant and murine models]. / Evaluación de la capacidad patogénica de Fusarium spp. en modelos vegetal y murino.

The genus Fusarium is widely recognized for its phytopathogenic capacity. However, it has been reported as an opportunistic pathogen in immunocompetent and immunocompromised patients. Thus, it can be considered a microorganism of interest in pathogenicity studies on different hosts. Therefore, this work evaluated the pathogenicity of Fusarium spp. isolates from different origins in plants and animals (murine hosts). Twelve isolates of Fusarium spp. from plants, animal superficial mycoses, and human superficial and systemic mycoses were inoculated in tomato, passion fruit and carnation plants, and in immunocompetent and immunosuppressed BALB/c mice. Pathogenicity tests in plants did not show all the symptoms associated with vascular wilt in the three plant models; however, colonization and necrosis of the vascular bundles, regardless of the species and origin of the isolates, showed the infective potential of Fusarium spp. in different plant species. Moreover, the pathogenicity tests in the murine model revealed behavioral changes. It was noteworthy that only five isolates (different origin and species) caused mortality. Additionally, it was observed that all isolates infected and colonized different organs, regardless of the species and origin of the isolates or host immune status. In contrast, the superficial inoculation test showed no evidence of epidermal injury or colonization. The observed results in plant and murine models suggest the pathogenic potential of Fusarium spp. isolates in different types of hosts. However, further studies on pathogenicity are needed to confirm the multihost capacity of this genus.

Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum.

The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds) were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

In-house protocol and performance of MALDI-TOF MS in the early diagnosis of bloodstream infections in a fourth-level hospital in Colombia: Jumping to full use of this technology.

BACKGROUND: Bloodstream infections (BSIs) are a major cause of mortality in hospitalized patients. Rapid diagnosis is crucial because any delay in the antimicrobial treatment is associated with an increase in adverse patient outcomes. The application of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology directly to blood cultures permits earlier identification of BSIs and facilitates treatment management. METHODS: A total of 470 positive blood cultures from patient samples were analyzed using Standard Aerobic/F and Anaerobic/F blood culture media. Isolates were identified using conventional identification methods and by the direct method using the MALDI-TOF MS system. RESULTS: In 470 blood cultures, the direct method showed good identification results (420/470, 89%); specifically, accurate species and genus identification in 283/470 (60%), and only correct genus identification in 137/470 (29%). The direct protocol had better performance for Gram-negative compared to Gram-positive bacteria (97% vs 76%) and was unable to identify the positive blood cultures for both yeasts and some bacteria, mostly Gram-positive (50/470). CONCLUSIONS: The protocol used here gave good and reliable results, being available up to 24 h earlier, while also leading to better use of MALDI-TOF.

First report of sporadic cases of Candida auris in Colombia.

BACKGROUND: Candida auris is a recently reported Candida species that is phenotypically similar to Candida haemulonii and related to hospital outbreaks. This organism can be misidentified as Candida haemulonii, Candida famata, Candida catenulata, or Rhodotorula glutinis by phenotypic approaches. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and DNA sequence analysis using internal transcribed spacer rDNA bar-coding provide an accurate identification. CASE REPORTS: Three cases of C. auris infection in patients with risk factors for fungal infection (one admitted to the intensive care unit, one with lymphoma, and one with HIV; all three with previous antibiotic use) are reported; these infections were not epidemiologically related. Yeast isolates were recovered from blood, ocular secretion, and bronchoalveolar lavage and were misidentified as C. catenulata and Candida albicans by the phenotypic MicroScan method. The isolates were confirmed to be C. auris by means of MALDI-TOF MS and DNA sequence analysis. Antifungal susceptibility testing was performed on these C. auris isolates, which exhibited high minimum inhibitory concentrations to triazoles and amphotericin B. One patient survived and the other two died. Only one of these deaths was related to fungemia. CONCLUSIONS: C. auris is an emerging and opportunistic multidrug-resistant human pathogen. It is necessary to strengthen measures to achieve an accurate and quick identification and also to avoid its dissemination. This will require improvements in health and infection control measures, as well as the promotion of antifungal stewardship in healthcare facilities.

[Results of nine years of the clinical and epidemiological survey on cryptococcosis in Colombia, 1997-2005]. / Estudio clínico y epidemiológico de la criptococosis en Colombia: resultados de nueve años de la encuesta nacional, 1997-2005.

INTRODUCTION: A national survey on cryptococcosis has been conducted in Colombia since 1997. The survey data recorded over a 9-year period, 1997 to 2005, was summarized. MATERIALS AND METHODS: The format provided by the European Confederation of Medical Mycology was adapted with the correspondent permission. RESULTS: Over the 9 year period, 931 surveys were received from 76 centers. The associated disease syndromes were as follows: 891 (95.7%) were neurocryptococosis cases, 27 (2.9%) pulmonary disease, 5 (0.5%) cutaneous lesions, 2 (0.2%) ganglionar forms, 2 (0.2%) oropharyngeal lesions and one case (0.1%) each from peritonitis, liver lesion, cellulitis and urinary tract infection. Demographic data indicated 82.7% of the subjects were males, and 59.4% were between 20-39 years old; 25 children less than 16 years old were reported. The prevalent risk factor was HIV infection (78.1%). The mean annual incidence rate of cryptococcosis in the general population was 2.4 X 106 inhabitants, but in AIDS patients the rate rose to one in 3 X 103. The most frequent clinical features were headache (85.2%), nausea and vomiting (59.1%), fever (59.0%), mental changes (46.2%), meningeal signs (33.4%), cough (23.6%) and visual alterations or loss of vision (20.9%). Laboratory data showed that direct examination of cerebrospinal fluid (CSF) was positive in 92.8% cases and Cryptococcus was recovered in 90.3% of the cases. Cryptococcal antigen reactivity was 98.9% in CSF and 93.7% in serum samples. From 788 isolates submitted, 95.9% were C. neoformans var. grubii serotype A, 0.3% var. neoformans serotype D, 3.3% C. gattii serotype B and 0.5% C. gattii serotype C. The majority of patients were treated initially with amphotericin B. CONCLUSION: Cryptococcosis incidence has increased dramatically in Colombia with the AIDS pandemic and it can be considered as a sentinel marker for HIV infection.

[In vitro antifungal activity of azoles and amphotericin B against Malassezia furfur by the CLSI M27-A3 microdilution and Etest® methods]. / Actividad antifúngica in vitro de azoles y anfotericina B frente a Malassezia furfur por el método de microdilución M27-A3 del CLSI y Etest®.

BACKGROUND: Malassezia furfur is a human skin commensal yeast that can cause skin and opportunistic systemic infections. Given its lipid dependant status, the reference methods established by the Clinical and Laboratory Standards Institute (CLSI) to evaluate antifungal susceptibility in yeasts are not applicable. AIMS: To evaluate the in vitro susceptibility of M. furfur isolates from infections in humans to antifungals of clinical use. METHODS: The susceptibility profile to amphotericin B, itraconazole, ketoconazole and voriconazole of 20 isolates of M. furfur, using the broth microdilution method (CLSI M27-A3) and Etest®, was evaluated. RESULTS: Itraconazole and voriconazole had the highest antifungal activity against the isolates tested. The essential agreement between the two methods for azoles antifungal activity was in the region of 60-85% and the categorical agreement was around 70-80%, while the essential and categorical agreement for amphotericin B was 10%. CONCLUSIONS: The azoles were the compounds that showed the highest antifungal activity against M. furfur, as determined by the two techniques used; however more studies need to be performed to support that Etest® is a reliable method before its implementation as a routine clinical laboratory test.

Identification of Malassezia species as part of normal skin and ear canal microbiota in horses / Identificación de especies de Malassezia como microbiota normal en piel y canal auditivo externo de equinos / Identificação de espécies de Malassezia como microbiota normal em pele e canal auditivo externo de cavalos

Abstract Background: The yeasts of the genus Malassezia are considered part of the normal skin microbiota in humans and animals. In horses, several species of the genus Malassezia have been reported in different areas of the skin and ear canal. Objective: Isolate, characterize and identify the different species belonging to the genus Malassezia isolated from the ear canal and skin of equine patients with no dermatological lesions that were referred to the large animal clinic of veterinary teaching hospital at the National University of Colombia. Methods: 22 horses were evaluated and sampled. Eighty-two samples were obtained by swabbing either the ear canals (left and right), skin areas of prepuce, mammary gland and inguinal region. The samples were examined by cytological evaluation and were cultured on modified Dixon's agar and phenotypic and molecular identification were performed for yeast colonies. Results: Fourteen yeast isolates were obtained from the 82 samples. Biochemical identification determined that 50% (n=7) were Malassezia spp., 35.7% (n=5) were identified as Candida spp. and 14.3% (n=2) as Cryptococcus spp.. Using molecular tests, the Malassezia species were M. slooffiae (28.6%) and M.nana (57.1%); only one isolate was classified as Trichosporo asahii. Conclusion: M.nana and M. slooffiae were identified as part of the normal ear canal and skin microbiota in the evaluated horses. The observed prevalence of Malassezia spp. was 18.2% (n=4/22) in this study sample.

Resumen Antecedentes: Las levaduras del género Malassezia hacen parte de la microbiota normal cutánea de humanos y animales. En equinos se han reportado diferentes especies de Malassezia aisladas de varias regiones de piel y canal auditivo externo. Objetivo: Aislar, caracterizar e identificar las especies del género Malassezia spp. a partir de canal auditivo externo y piel de equinos sin lesiones dermatológicas, remitidos a la Clínica de Grandes Animales de la Facultad de Medicina Veterinaria y de Zootecnia de la Universidad Nacional de Colombia. Metodología: Se evaluaron 22 equinos, a partir de los cuales se obtuvieron 82 muestras entre hisopados de canal auditivo externo (izquierdo y derecho) y diferentes regiones de piel (prepucio, glándula mamaria e ingle). Las muestras fueron procesadas mediante examen directo y cultivo en agar Dixon modificado. A partir de los aislamientos en los que se observaron colonias morfológicamente compatibles con Malassezia spp. se realizó la identificación fenotípica y molecular. Resultados: De las 82 muestras procesadas se obtuvieron 14 aislamientos de levaduras, de las cuales mediante identificación bioquímica el 50% (n=7) correspondió a Malassezia spp., el 35,7% (n=5) a Candida spp., y el 14,3% (n=2) a Cryptococcus spp. Luego mediante pruebas moleculares se identificaron las especies del género Malassezia como: M. slooffiae (28,6%) y M . nana (57,1%); y un aislamiento correspondió a Trichosporon asahii. Conclusión: Se logró identificar las especies M.nana y M. slooffiae como microbiota normal de la piel y el canal auditivo en los equinos evaluados. La prevalencia de Malassezia spp. para la población evaluada fue de 18,2% (n=4/22).

Resumo Antecedentes: As leveduras do gênero Malassezia fazem parte da microbiota cutânea normal de humanos e animais. Em cavalos, diferentes espécies de Malassezia isoladas de várias regiões da pele e do canal auditivo externo foram reproduzidas. Objetivo: Isolar, caracterizar e identificar as espécies do gênero Malassezia spp. do canal auditivo externo e pele eqüinos sem lesões cutâneas, referiu-se à Clínica de Grandes Animais da Faculdade de Medicina Veterinária e Zootecnia da Universidade Nacional da Colômbia. Métodos: 22 equinos foram avaliadas a partir dos quais 82 amostras a partir de esfregaços do canal auditivo externo (esquerda e direita) e diferentes regiões da pele (prepúcio, glândula mamaria e virilha) foram obtidos. As amostras foram processadas por exame direto e cultura em ágar Dixon modificado. Dos isolados nos quais as colônias foram observadas morfologicamente compatíveis com Malassezia spp. identificação fenotípica e molecular foi realizada. Resultados: Das 82 amostras processadas 14 isolados de levedura, que foram obtidos por identificação bioquímica de 50% (n=7) correspondia a Malassezia spp., 35,7% (n=5) a Candida spp., e 14,3% (n=2) para Cryptococcus spp.. Em seguida, usando o teste molecular espécie Malassezia foram identificadas como M. slooffiae (28,6%) e M . nana (57,1%); e um isolamento correspondia a Trichosporon asahii. Conclusão: As espécies M.nana e M. slooffiae foram identificadas como microbiota de pele normal e do canal auditivo nos equídeos avaliados. A prevalência de Malassezia spp. para a população avaliada foi 18,2% (n=4/22).

Genotyping of Malassezia pachydermatis disclosed genetic variation in isolates from dogs in Colombia / Genotipagem de Malassezia pachydermatis revelou variação genética em isolados de cães na Colômbia

Malassezia pachydermatis is a lipophilic and lipid-dependent yeast mostly isolated from animals' skin; hence, it is regarded as a zoophilic species causing otitis externa in dogs. Aspects associated with its epidemiology and pathogenicity is a matter of interest. This study aimed to conduct a molecular characterization of 43 isolates of M. pachydermatis obtained from dogs with otitis externa. For this purpose, the 5.8S internal transcribed spacer 2 (ITS2) and D1/D2 26S rRNA regions were amplified, sequenced and analyzed using restriction fragment length polymorphism (RFLP) with AluI, CfoI, and BstF5I endonucleases. Phylogenetic analyses revealed that these isolates grouped with the sequence types I, IV and V, previously proposed for M. pachydermatis. Interestingly, we found a new polymorphic RFLP pattern using BstF5I, these isolates were associated with the sequence types IV and V, nevertheless an association between polymorphic RFLP patterns, and fosfolipase activity or canine population data was not observed. These findings underline the genetic diversity of M. pachydermatis and provide new insights about the epidemiology of this species in the analyzed population.(AU)

Malassezia pachydermatis é uma levedura lipofílica e dependente de lipídios, principalmente da pele de animais. Sendo, por essa razão, considerada uma espécie zoofílica e causadora de otite externa em cães. Neste sentido, aspectos associados à sua epidemiologia e patogenicidade constituem um tema de interesse científico. O objetivo deste estudo foi realizar a caracterização molecular de 43 isolados de M. pachydermatis obtidos a partir de cães com otite externa. Para esta propósito, foram amplificadas, sequenciadas e analisadas com enzimas de restrição as regiões do gene 5.8S, do espaçador interno transcrito 2 (ITS2) e D1/D2 do 26S do rRNA pelo método RFLP, com as endonucleases AluI, CfOI e BstF5I. Análises filogenéticas revelaram que os isolados se agruparam com as sequências tipo I, IV e V de M. pachydermatis como já descrito anteriormente. De maneira interessante, se observou um novo RFLP polimórfico utilizando BstF5I. Os isolados que mostraram esse padrão foram associados com os padrões IV e V. No entanto, não foi observada associação entre padrões polimórficos de RFLP e atividade de fosfolipase ou dados da população canina. Estes resultados demonstram a diversidade genética de M. pachydermatis e fornecem novas perspectivas sobre a epidemiologia destas espécies na população analisada.(AU)

Evaluación de la capacidad patogénica de Fusarium spp. en modelos vegetal y murino / Evaluation of Fusarium spp. pathogenicity in plant and murine models

El género Fusarium es ampliamente conocido por su capacidad fitopatógena, típicamente asociada al marchitamiento vascular. Sin embargo, se ha reportado como un patógeno oportunista en pacientes inmunocompetentes e inmunocomprometidos, por lo que puede ser considerado como un microorganismo de interés en estudios de patogenicidad en diferentes hospederos. Este trabajo evaluó la capacidad patogénica de aislamientos de Fusarium spp. de diferentes orígenes en hospederos vegetales y en un hospedero animal (modelo murino). Doce aislamientos de Fusarium spp. de origen vegetal, animal superficial, humano superficial y humano sistémico fueron inoculados en plantas de tomate, gulupa y clavel, y en ratones BALB/c, inmunocompetentes e inmunosuprimidos. Las pruebas de patogenicidad en plantas no mostraron todos los síntomas asociados al marchitamiento vascular en los tres modelos vegetales, pero la colonización y la necrosis de los haces vasculares observada en todos los casos, independientemente de la especie de Fusarium y el origen del aislamiento, demostró el potencial infeccioso de Fusarium spp. en las diferentes especies de plantas. Por otro lado, las pruebas de patogenicidad en el modelo murino evidenciaron alteraciones del comportamiento. Asimismo, se observó en el modelo murino que todos los aislamientos infectaron y colonizaron diferentes órganos, independientemente de su origen, de la especie o del estado inmunitario del hospedero, pero solamente cinco (de diferente origen y correspondientes a diferentes especies) generaron mortalidad. En contraste, la prueba de inoculación superficial no evidenció lesiones ni colonización. Los resultados observados indican el potencial papel patogénico de los aislamientos de Fusarium spp. en los diferentes tipos de hospederos. Sin embargo, es necesario profundizar en estudios de factores de patogenicidad que expliquen la capacidad de este género para colonizar múltiples hospederos.

The genus Fusarium is widely recognized for its phytopathogenic capacity. However, it has been reported as an opportunistic pathogen in immunocompetent and immunocompromised patients. Thus, it can be considered a microorganism of interest in pathogenicity studies on different hosts. Therefore, this work evaluated the pathogenicity of Fusarium spp. isolates from different origins in plants and animals (murine hosts). Twelve isolates of Fusarium spp. from plants, animal superficial mycoses, and human superficial and systemic mycoses were inoculated in tomato, passion fruit and carnation plants, and in immunocompetent and immunosuppressed BALB/c mice. Pathogenicity tests in plants did not show all the symptoms associated with vascular wilt in the three plant models; however, colonization and necrosis of the vascular bundles, regardless of the species and origin of the isolates, showed the infective potential of Fusarium spp. in different plant species. Moreover, the pathogenicity tests in the murine model revealed behavioral changes. It was noteworthy that only five isolates (different origin and species) caused mortality. Additionally, it was observed that all isolates infected and colonized different organs, regardless of the species and origin of the isolates or host immune status. In contrast, the superficial inoculation test showed no evidence of epidermal injury or colonization. The observed results in plant and murine models suggest the pathogenic potential of Fusarium spp. isolates in different types of hosts. However, further studies on pathogenicity are needed to confirm the multihost capacity of this genus.

Actividad antifúngica in vitro de azoles y anfotericina B frente a Malassezia furfur por el método de microdilución M27-A3 del CLSI y Etest® / In vitro antifungal activity of azoles and amphotericin B against Malassezia furfur by the CLSI M27-A3 microdilution and Etest® methods

Antecedentes. Malassezia furfur es una levadura comensal de la piel del ser humano que ha sido asociada con la presencia de algunas entidades dermatológicas e infecciones sistémicas oportunistas. Por su condición dependiente de lípidos, los métodos de referencia establecidos para las levaduras por el Clinical and Laboratory Standards Institute (CLSI) para evaluar la sensibilidad antifúngica no son aplicables. Objetivos. Evaluar la sensibilidad in vitro de aislamientos de M. furfur asociados a procesos patológicos en el ser humano frente a antifúngicos de uso clínico. Métodos. Se evaluó el perfil de sensibilidad a la anfotericina B, el itraconazol, el ketoconazol y el voriconazol de 20 aislamientos de M. furfur mediante el método de microdilución en caldo (CLSI M27-A3) y Etest®. Resultados. El itraconazol y el voriconazol presentaron la mayor actividad antifúngica frente a los aislamientos evaluados. El acuerdo esencial entre los dos métodos usados para evaluar la actividad antifúngica de los azoles estuvo en el 60-85%, y el acuerdo categórico en el 70-80%; para la anfotericina B tanto el acuerdo esencial como el categórico fueron del 10%. Conclusiones. De acuerdo con los dos métodos evaluados los azoles fueron los compuestos que presentaron la mayor actividad antifúngica frente a M. furfur; sin embargo, es necesario realizar más estudios que permitan afirmar que Etest® es un método confiable para ser implementado en la rutina del laboratorio clínico (AU)

Background. Malassezia furfur is a human skin commensal yeast that can cause skin and opportunistic systemic infections. Given its lipid dependant status, the reference methods established by the Clinical and Laboratory Standards Institute (CLSI) to evaluate antifungal susceptibility in yeasts are not applicable. Aims. To evaluate the in vitro susceptibility of M. furfur isolates from infections in humans to antifungals of clinical use. Methods. The susceptibility profile to amphotericin B, itraconazole, ketoconazole and voriconazole of 20 isolates of M. furfur, using the broth microdilution method (CLSI M27-A3) and Etest®, was evaluated. Results. Itraconazole and voriconazole had the highest antifungal activity against the isolates tested. The essential agreement between the two methods for azoles antifungal activity was in the region of 60-85% and the categorical agreement was around 70-80%, while the essential and categorical agreement for amphotericin B was 10%. Conclusions. The azoles were the compounds that showed the highest antifungal activity against M. furfur, as determined by the two techniques used; however more studies need to be performed to support that Etest® is a reliable method before its implementation as a routine clinical laboratory test (AU)

Histoplasmosis: results of the Colombian national survey, 1992-2008.

INTRODUCTION: Histoplasmosis, a fungal disorder characterized by a wide spectrum of manifestations that range from subclinical infections to disseminated processes, affects both immunocompetent and immunosuppressed individuals. Histoplasmosis is not a reportable disease in Colombia and consequently, a survey was designed to collect histoplasmosis cases diagnosed in the country. OBJECTIVE: The aim of this work was to analyze the data collected from 1992 to 2008. Materials and methods. The survey included demographic data, risk factors, clinical manifestations, imaging data, diagnostic methods and antifungal treatment. Patients were grouped according to risk factors and comparisons of the various findings were done. RESULTS: A total of 434 surveys were gathered from 20 of the country's Departments. Most patients (96.1%) were adults, 77% were males with a mean age of 38.4 years. Only 3.9% were children less than 15 years of age. In the adult population, AIDS was reported in 70.5% of the cases; additionally, in 7.0% patients other immunosuppressive conditions were informed. The most frequent clinical manifestations were fever (76.1%), cough (54.8%) and constitutional symptoms (56.8%). X rays abnormalities were represented mainly by infiltrates (65.9%) and nodules (17.1%). Diagnosis was made by microscopic observation of H. capsulatum in 49.6% patients, by culture in 58.0% and by serological test in 14.6% cases. Antifungal use was recorded in 52.5% cases. CONCLUSIONS: Histoplasmosis is frequent in Colombia, especially in certain risk factor groups such as the HIV-infected population. Data collected from this large number of cases has allowed valid comparisons on various aspects of histoplasmosis in Colombia.

Phenotypic characterization of canine Malassezia spp., isolates / Caracterización fenotípica de aislamientos de Malassezia spp., de origen canino

ABSTRACT Objective . To characterize and identify yeasts of the genus Malassezia by phenotypic features. Materials and methods. First, the macroscopic and microscopic morphological characteristics were described. In addition we performed biochemical and physiological assays as Tweens and Cremophor, including more. Results. Our results evidenced of 105 isolates obtained from dogs diagnosed with external otitis, it was possible to identify two distinct species from 46 isolates within the Malassezia genus: 36.19% (n=38) were identified as M. pachydermatis and 7.62% (n=8) as M. furfur. According to phenotypic patterns the remaining 56.19% (n=59) were reported as Malassezia spp., possibly corresponding to M. furfur and/or M. pachydermatis. Conclusions. Results emphasize the necessity to characterize according to species. It is not feasible to define Malassezia by species based on morphological, biochemical, and physiological findings. Therefore, molecular genotyping should be performed to identify markers allowing a more precise isolate identification. This would broaden our epidemiological knowledge regarding different species involved in canine otitis pathologies.

RESUMEN Objetivo . Caracterizar e identificar levaduras del género Malassezia, mediante características fenotípicas. Materiales y métodos . Inicialmente se describieron las características morfológicas macroscópicas y microscópicas, adicionalmente se realizaron pruebas bioquímicas y fisiológicas como Tween y Cremophor, entre otras. Resultados . De 105 aislamientos de caninos diagnosticados previamente con otitis, 46 fueron caracterizados hasta especie, así: El 36.19% (n=38) correspondió a M. pachydermatis, el 7.2% (n=8) a M. furfur; y 56.19% (n=59) restante fueron reportados como Malassezia spp., debido a los patrones fenotípicos atípicos que presentaron, y que podrían corresponder a variantes de M. furfur y/o M. pachydermatis. Conclusión. Estos resultados enfatizan la necesidad de hacer una caracterización a nivel de especie y/o genotipos mediante marcadores moleculares que permitan una identificación más precisa de los aislamientos. Con el presente estudio, se contribuye al conocimiento de las diferentes especies involucradas en patologías óticas en caninos.

Concordance between otic cytology and culture in diagnosis of external otitis canine by Malassezia spp / Concordancia entre citología ótica y cultivo en el diagnóstico de otitis externa canina por Malassezia spp

Objective. To determine the correlation between microbiological culture and otic cytology for diagnoses of external otitis by Malassezia in dogs. Materials and Methods. 158 ear swabs of dogs with clinical diagnosis of external otitis were analyzed by cytology, mycological culture and metabolic tests. Results. Were obtained a positive results by cytology of 62% and 75.3% by culture. The 31.1% of isolates were identified as M. pachydermatis, 12.6% as M. furfur and 56.3% were classified as Malassezia spp., because was not possible to define the species. We found a positive concordance between cytology and culture for Malassezia spp., of 0.76 with a kappa index of 0.448 (95% CI 0.30 to 0.60) which represents a moderate strength of concordance between the two techniques, without regard the identified species. Conclusions. The use of a diagnostic test is not enough to establish the participation of Malassezia spp., as a causal disease agent.

Objetivo. Determinar la concordancia entre el cultivo microbiológico y la citología ótica para el diagnóstico de otitis externa causada por Malassezia spp. en caninos. Materiales y Métodos. Se analizaron 158 muestras de hisopados de caninos con diagnóstico clínico de otitis externa, todas las muestras fueron analizadas mediante citología, cultivo micológico y pruebas metabólicas. Resultados. Se obtuvo una positividad mediante citología del 62% y por cultivo del 75.3%. El 31.1% de los aislamientos fueron identificados como M. pachydermatis, el 12.6% como M. furfur y un 56.3% se clasificó como Malassezia spp., dado que bioquímicamente no fue posible hallar su especie. Se determinó una concordancia observada entre técnicas positivas para Malassezia spp. de 0.76 con índice Kappa de 0.448 IC 95% (0.30 - 0.60) lo que representa una fuerza de concordancia moderada entre las dos técnicas, sin tener en cuenta la especie identificada. Conclusiones. El uso de una prueba diagnóstica no es suficiente para establecer la participación de Malassezia pachydermatis como agente causal de enfermedad.

Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum

The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds) were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

Histoplasmosis en Colombia: resultados de la encuesta nacional, 1992-2008 / Histoplasmosis: results of the Colombian National Survey, 1992-2008

Introducción. La histoplasmosis está caracterizada por variadas manifestaciones que van desde la afección subclínica a la enfermedad diseminada, y suele presentarse tanto en huéspedes inmunocompetentes como inmunosuprimidos. Como la enfermedad no es de notificación obligatoria en Colombia, se diseñó una encuesta para recolectar información de los casos diagnosticados en el país. Objetivo. El objetivo de este trabajo fue analizar los datos recolectados desde 1992 hasta 2008. Materiales y métodos. La encuesta incluyó datos demográficos, factores de riesgo, manifestaciones clínicas, estudios de imágenes, métodos diagnósticos y tratamiento antifúngico. Los pacientes se agruparon de acuerdo con los factores de riesgo y se compararon los correspondientes hallazgos.Resultados. Se examinaron 434 encuestas provenientes de 20 de los departamentos colombianos. La mayoría (96,1 %) correspondían a adultos, 77 % eran hombres con edad promedio de 38,4 años, y sólo 3,9 % eran niños o adolescentes. En los adultos, 70,5 % tenían sida y 7 % presentaban otra inmunosupresión. Las manifestaciones predominantes fueron fiebre (76,1 %), tos (54,8 %) y síntomas constitucionales (56,8 %). En las radiografías, las anormalidades fueron principalmente infiltrados (65,9 %) y nódulos (17,1 %). El diagnóstico se estableció por observación microscópica de H. capsulatum en 49,6 % casos, por cultivo en 58 % y por pruebas serológicas en 14,6 %. El tratamiento se informó sólo en 52,5 % casos. Conclusiones. Se demostró que la histoplasmosis es frecuente en Colombia, especialmente en grupos de riesgo como son los pacientes infectados con el VIH. El análisis de este número relevante de pacientes permitió establecer comparaciones válidas sobre aspectos de la histoplasmosis en nuestro país.

Introduction. Histoplasmosis, a fungal disorder characterized by a wide spectrum of manifestations that range from subclinical infections to disseminated processes, affects both immunocompetent and immunosuppressed individuals. Histoplasmosis is not a reportable disease in Colombia and consequently, a survey was designed to collect histoplasmosis cases diagnosed in the country. Objective. The aim of this work was to analyze the data collected from 1992 to 2008. Materials and methods. The survey included demographic data, risk factors, clinical manifestations, imaging data, diagnostic methods and antifungal treatment. Patients were grouped according to risk factors and comparisons of the various findings were done. Results. A total of 434 surveys were gathered from 20 of the country’s Departments. Most patients (96.1%) were adults, 77% were males with a mean age of 38.4 years. Only 3.9% were children less than 15 years of age. In the adult population, AIDS was reported in 70.5% of the cases; additionally, in 7.0% patients other immunosuppressive conditions were informed. The most frequent clinical manifestations were fever (76.1%), cough (54.8%) and constitutional symptoms (56.8%). X rays abnormalities were represented mainly by infiltrates (65.9%) and nodules (17.1%). Diagnosis was made by microscopic observation of H. capsulatum in 49.6% patients, by culture in 58.0% and by serological test in 14.6% cases. Antifungal use was recorded in 52.5% cases. Conclusions. Histoplasmosis is frequent in Colombia, especially in certain risk factor groups such as the HIV-infected population. Data collected from this large number of cases has allowed valid comparisons on various aspects of histoplasmosis in Colombia.

Análisis retrospectivo (2009-2010) de las alteraciones dermatológicas, óticas y oftalmológicas con diagnóstico clínico presuntivo de micosis en caninos y felinos / Análise retrospectiva (2009-2010) das alterações dermatológicas, óticas e oftalmológicas com diagnóstico clínico presuntivo de micose em cães e gatos / A retrospective analysis (2009-2010) of the dermatological, ear and ophtalmological alterations with presumptive clinical diagnosis of mycosis in canines and felines

Objetivo. Determinar la prevalencia de las alteraciones dermatológicas, óticas y oftalmológicas clínicamente presuntivas de ser causadas por hongos y analizar demográficamente su presentación. Materiales y métodos. Se realizó un estudio retrospectivo en una clínica para pequeños animales en Bogotá; se revisaron historias clínicas de pacientes que consultaron por alteraciones dermatológicas, óticas y oftalmológicas durante 2009 y 2010. Resultados. En el período evaluado la prevalencia de las alteraciones dermatológicas en caninos fue de 7,8%, las óticas de 4,2% y las oftalmológicas de 3,3%. Dentro de las alteraciones dermatológicas, la sospecha etiológica principal por diagnóstico clínico fueron los dermatofitos con 86,9%, en las alteraciones óticas fue Malassezia con 86,5% y para las oftalmológicas fue Candida con 83,3%. El análisis estadístico de variables no mostró asociación con los síntomas ni con los agentes etiológicos diagnosticados clínicamente; sin embargo, la evaluación categorizada evidenció asociaciones entre razas y edades con las etiologías por diagnóstico clínico. El diagnóstico de las etiologías micóticas se estableció en la mayoría de los casos por hallazgos clínicos y con menor frecuencia por raspado de piel y citología ótica; en ninguno de los casos se realizó cultivo micológico. La población felina no presentó alteraciones dermatológicas, óticas y oftalmológicas clínicamente compatibles con etiología fúngica. Conclusión. En caninos, la prevalencia de las alteraciones dermatológicas clínicamente compatibles con dermatofitos fue de 86,9%, los procesos óticos asociados con Malassezia de 86,5% y los oftalmológicos sospechosos de Candida con 83,3%. Los diagnósticos definitivos se establecieron por hallazgos clínicos sin la realización de metodologías diagnósticas estándar como el cultivo...

A retrospective analysis (2009-2010) of the dermatological, ear and ophtalmological alterations with presumptive clinical diagnosis of mycosis in canines and felines. Objective. To determine the prevalence of dermatological, ear and ophthalmological alterations clinically presumptive to be caused by fungi and to analyze their occurrence demographically. Materials and methods. We carried out a retrospective study in a clinic in Bogota for small animals. We reviewed the medical histories of patients who requested medical appointments due to dermatological, ear and ophtalmological alterations in 2009 and 2010. Results. In the assessed period, the prevalence of dermatological alterations in dogs was of 7.8%, 4.2% of ear alterations and of 3.3% of ophtalmological alterations. The main etiological suspects through clinical diagnosis were: dermatophytes with an incidence of 86.9% among dermatological alterations, Malassezia with an 86.5% of incidence in ear alterations, and Candida with 83.3% of incidence in ophtalmological alterations. Statistical analysis of variables showed no association with symptoms or clinically diagnosed etiological agents, but the categorized evaluation showed associations of races and ages with the etiologies through clinical diagnosis. The diagnosis of fungal etiologies was accomplished in most cases by clinical findings and less frequently by skin scrapings and ear cytology; in none of the cases a mycological culture was done. The feline population showed no dermatological, ear or ophtalmological alterations compatible with a fungal etiology. Conclusion. In canines, the prevalence of dermatological alterations clinically compatible with dermatophytes was of 86.9%; ear alterations associated with Malassezia were of 86.5%; and ophtalmological alterations Candida-suspected were 83.3%. Final diagnoses were accomplished with the help of clinical findings without following standard diagnostic methodologies such as cultivation...

Análise retrospectiva (2009-2010) das alterações dermatológicas, óticas e oftalmológicas com diagnóstico clínico presuntivo de micose em cães e gatos. Objetivo. Determinar a prevalência das alterações dérmicas, óticas e oftálmicas clinicamente presuntivo de serem causadas por fungos e analisar demograficamente sua apresentação. Materiais e métodos. Foi realizado um estudo retrospectivo numa clínica para pequenos animais em Bogotá; foram revisadas as histórias clínicas de pacientes com alterações dérmicas, óticas e oftalmicas em 2009 e 2010. Resultados. Durante o período de avaliação a prevalência das alterações dérmicas em cães foi de 7,8%, as óticas de 4,2% e as oftálmicas de 3,3%. Dentro das alterações dérmicas, a principal suspeita etiológica por diagnóstico clínico foram os dermatófitos com 86,9%, nas alterações óticas foi Malassezia com 86,5% e para as oftálmicas foi Candida com 83,3%. A análise estatística das variáveis não apresentou associação com os sintomas ou com os agentes etiológicos diagnosticados clínicamente; embora, a avaliação categorizada evidenciou associações entre raças e idades com as etiologias por diagnóstico clínico. O diagnóstico das etiologias micóticas foi estabelecido na maioria dos casos pelos achados clínicos e, menos freqüentemente por raspados de pele e citologia ótica, em nenhum dos casos foi realizada uma cultura micótica. A população felina não apresentou alterações dérmicas, óticas e oftálmicas clinicamente compatíveis com etiologia fúngica. Conclusão. Em cães, a prevalência das alterações dérmicas clinicamente compatíveis com dermatófitos foi de 86,9%, os processos óticos associados com Malassezia foi de 86,5% e os oftálmicos suspeitos de Candida foi de 83,3%. Os diagnósticos definitivos foram estabelecidos pelos achados clínicos sem a realização de métodos diagnósticos padroes, como o cultivo...

Estudio clínico y epidemiológico de la criptococosis en Colombia: resultados de nueve años de la encuesta nacional, 1997-2005 / Results of nine years of the clinical and epidemiological survey on cryptococcosis in Colombia, 1997-2005

Introducción. Con el fin de conocer la situación de la criptococosis en Colombia se ha llevado a cabo una encuesta nacional. El presente estudio analiza la información recogida de 1997 a 2005. Materiales y métodos. Se utilizó el formato de encuesta de la Confederación Europea de Micología Médica. Resultados. Se recibieron 931 encuestas de 76 centros. Se identificaron 891 (95,7 por ciento) casos de neurocriptococosis, 27 (2,9 por ciento) de formas pulmonares, cinco (0,5 por ciento) de lesiones cutáneas, dos (0,2 por ciento) de ganglionares, dos (0,2 por ciento) de úlceras orofaríngeas y un caso (0,1 por ciento) de cada una de las siguientes formas clínicas: peritonitis, lesión hepática, celulitis de los miembros inferiores e infección de las vías urinarias. El 82,7 por ciento fueron hombres con predominio de los adultos jóvenes. Sin embargo, se reportaron 25 (2,7 por ciento) niños. La infección por el virus de la inmunodeficiencia humana se encontró en el 78,1 por ciento de los casos. La incidencia promedio anual de criptococosis en la población general fue de 2,4 casos x106, pero en los pacientes con sida se elevó a 3,0 casos x 103. Las manifestaciones clínicas más frecuentes fueron: cefalea (85,2 por ciento), náuseas y vómito (59,1 por ciento), fiebre (59 por ciento), cambios mentales (46,2 por ciento) y signos meníngeos (33,4 por ciento). El examen directo de líquido cefalorraquídeo fue positivo en el 92,8 por ciento de los casos y el hongo se recuperó en el 90,3 por ciento. El antígeno capsular tuvo una positividad del 98,9 por ciento en el líquido cefalorraquídeo y del 93,7 por ciento en el suero. De 788 aislamientos remitidos, 95,9 por ciento fueron Cryptococcus neoformans var. grubii serotipo A, 0,3 por ciento, var. neoformans serotipo D, 3,3 por ciento, C. gattii serotipo B y 0,5 por ciento, C. gattii serotipo C. La mayoría de los pacientes fueron tratados inicialmente con anfotericina B. Conclusión: La criptococosis es una enfermedad cuya incidencia a...

Introduction. A national survey on cryptococcosis has been conducted in Colombia since 1997. The survey data recorded over a 9-year period, 1997 to 2005, was summarized. Materials and methods. The format provided by the European Confederation of Medical Mycology was adapted with the correspondent permission. Results. Over the 9 year period, 931 surveys were received from 76 centers. The associated disease syndromes were as follows: 891 (95.7%) were neurocryptococosis cases, 27 (2.9%) pulmonary disease, 5 (0.5%) cutaneous lesions, 2 (0.2%) ganglionar forms, 2 (0.2%) oropharyngeal lesions and one case (0.1%) each from peritonitis, liver lesion, cellulitis and urinary tract infection. Demographic data indicated 82.7% of the subjects were males, and 59.4% were between 20-39 years old; 25 children less than 16 years old were reported. The prevalent risk factor was HIV infection (78.1%). The mean annual incidence rate of cryptococcosis in the general population was 2.4 X 106 inhabitants, but in AIDS patients the rate rose to one in 3 X 103. The most frequent clinical features were headache (85.2%), nausea and vomiting (59.1%), fever (59.0%), mental changes (46.2%), meningeal signs (33.4%), cough (23.6%) and visual alterations or loss of vision (20.9%). Laboratory data showed that direct examination of cerebrospinal fluid (CSF) was positive in 92.8% cases and Cryptococcus was recovered in 90.3% of the cases. Cryptococcal antigen reactivity was 98.9% in CSF and 93.7% in serum samples. From 788 isolates submitted, 95.9% were C. neoformans var. grubii serotype A, 0.3% var. neoformans serotype D, 3.3% C. gattii serotype B and 0.5% C. gattii serotype C. The majority of patients were treated initially with amphotericin B. Conclusion. Cryptococcosis incidence has increased dramatically in Colombia with the AIDS pandemic and it can be considered as a sentinel marker for HIV infection.