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.

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).

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.

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.

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.

[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.