Testes bioquímicos para identificação de leveduras na medicina veterinária - revisão de literatura / Biochemical tests to yeast identification in veterinary medicine - literature review / Pruebas bioquímicas para identificación de levaduras en la medicina veterinaria - revisión de literatura

As leveduras são fungos de importância à medicina veterinária por causarem doenças infecciosas em diferentes hospedeiros animais. A presente revisão de literatura teve como objetivo relatar os principais testes bioquímicos capazes de auxiliar na identificação de fungos leveduriformes de interesse veterinário e zoonótico. Para o levantamento bibliográfico, foram consideradas 48 publicações científicas selecionadas na área e indexadas nas principais bases de dados, entre os anos de 1988 e 2020. Como resultados, observou-se que oito provas são as mais empregadas na rotina micológica. Devido à baixa variabilidade morfológica das espécies leveduriformes, testes bioquímicos complementares são fundamentais na rotina laboratorial. A análise do perfil bioquímico de leveduras contribui na determinação taxonômica dos fungos a partir de reações químicas, visto que o metabolismo varia de acordo com a espécie, resultando em metabólitos distintos, os quais podem ser avaliados por diferentes provas. Conclui-se que a identificação fenotípica das leveduras é imprescindível no diagnóstico, prognóstico, tratamento e controle de doenças fúngicas e contribui para a manutenção da saúde animal.(AU)

Yeasts are fungi of importance to veterinary medicine because they cause infectious diseases in different animal hosts. This literature review aimed to report the main biochemical tests capable of assisting in the identification of yeast-like fungi of veterinary and zoonotic interest. For the bibliographical survey, 48 selected scientific publications in the area and indexed in the main databases, between the years 1988 and 2020, were considered. As a result, it was observed that eight tests are the most used in the mycological routine. Due to the low morphological variability of yeast species, complementary biochemical tests are fundamental in the laboratory routine. The analysis of the biochemical profile of yeast contributes to the taxonomic determination of fungi based on chemical reactions, since the metabolism varies according to the species, resulting in different metabolites, which can be evaluated by different tests. It is concluded that the phenotypic identification of yeasts is essential in the diagnosis, prognosis, treatment and control of fungal diseases and contributes to the maintenance of animal health.(AU)

Las levaduras son hongos de importancia para la medicina veterinaria porque causan enfermedades infecciosas en diferentes animales huéspedes. Esta revisión de la literatura tuvo como objetivo informar las principales pruebas bioquímicas capaces de ayudar en la identificación de hongos tipo levadura de interés veterinario y zoonótico. Para el levantamiento bibliográfico se consideraron 48 publicaciones científicas seleccionadas en el área e indexadas en las principales bases de datos, entre los años 1988 y 2020. Como resultado se observó que ocho pruebas son las más utilizadas en la rutina micológica. Debido a la baja variabilidad morfológica de las especies de levaduras, las pruebas bioquímicas complementarias son fundamentales en la rutina del laboratorio. El análisis del perfil bioquímico de la levadura contribuye a la determinación taxonómica de los hongos en base a reacciones químicas, ya que el metabolismo varía según la especie, dando como resultado diferentes metabolitos, los cuales pueden ser evaluados mediante diferentes pruebas. Se concluye que la identificación fenotípica de levaduras es fundamental en el diagnóstico, pronóstico, tratamiento y control de enfermedades fúngicas y contribuye al mantenimiento de la salud animal.(AU)

Progress in protein extraction methods for fungal identification with MALDI-TOF MS / 中华微生物学和免疫学杂志

With the development of techniques for rapid microbial identification, MALDI-TOF MS has become an important tool for clinical identification of fungi. Problems such as the applicability and standardization of protein extraction methods have hindered the development of MALDI-TOF MS technology in the fungal field. This paper analyzed the complex structure of fungal cell walls, introduced the protein extraction methods recommended by MALDI-TOF MS commercial mass spectrometry systems, discussed the protein extraction methods for the identification of various genera of yeast-like fungi and filamentous fungi by MALDI-TOF MS, such as direct smear method, formic acid acetonitrile extraction method and magnetic bead grinding method, and summarized the current status and drawbacks of protein extraction methods in fungal identification by MALDI-TOF MS with a view to providing theoretical reference for subsequent research.

An Enzymolysis-Assisted Agrobacterium tumefaciens-Mediated Transformation Method for the Yeast-Like Cells of Tremella fuciformis

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

Differential diagnosis in bloodstream infection with Trichosporon asahii and Geotrichum capitatum / 中国感染控制杂志

Objective To provide reference for establishing diagnosis and differential diagnosis methods of rare yeast-like fungal bloodstream infection for clinical microbiology laboratory.Methods Trichosporon asahii (T.asa-hii)and Geotrichum capitatum (G.capitatum)bloodstream infection was diagnosed and differentially diagnosed through clinical data analysis,morphological examination,biochemical reactions,and molecular biology technology. Results Two types of yeast-like fungal bloodstream infections in case 1 and case 2 both occurred in leukemia agranulocytosis phase after chemotherapy,such infections were serious and highly similar.The cultivated colonies on blood agar plates of case 1 and case 2 were performed gram stain and microscopic examination.Hyphae,arthro-spores and microconidia were visible in the former,thickness of hyphae branches and length of arthrospores were different,most presented rectangular and barrel shape;the latter can be seen hyphae with transparent septum bro-ken up into arthrospores,presented rectangular shape,did not produce blastoconidia.Identification with API 20C AUX showed that they were T.asahii and G.capitatum.The PCR product sequences were compared with NCBI, suggesting that T.asahii and G.capitatum were at sexual stage.Conclusion Comprehensive application of a varie-ty of technical methods is helpful for improving the diagnosis accuracy of bloodstream infection with yeast-like fungi, identifying Trichosporon and Geotrichum to the species level may help physicians to understand such rare fungal in-fection,choose antifungal agents rationally,and improve clinical prognosis.

Candidiasis bucal en pacientes con diabetes mellitus / Oral Candidiasis in patients with diabetes mellitus

Se realizó un estudio descriptivo y transversal de 75 pacientes con diabetes mellitus, quienes presentaron candidiasis bucal, tratados en la consulta estomatológica del Policlínico de Especialidades del Hospital Provincial Docente Clinicoquirúrgico "Saturnino Lora Torres" de Santiago de Cuba, desde marzo de 2013 hasta igual mes de 2015, a fin de evaluar los resultados del diagnóstico clínico e histopatológico de la citada afección. Las muestras para biopsias se fijaron y procesaron con la técnica clásica de inclusión en parafina; se colorearon con una tinción especial de ácido peryódico de Schiff, lo cual confirmó la existencia de cambios celulares. Predominaron el sexo masculino, el grupo etario de 60 años y más; la ausencia de manifestaciones clínicas, el paladar duro y la comisura labial como los sitios de mayor prevalencia y la candidiasis eritematosa como la forma clínica más común. La presencia de hifas patógenas y células levaduriformes fueron los hallazgos histopatológicos más sobresalientes.

A descriptive and cross-sectional study of 75 patients with diabetes mellitus who presented oral candidiasis, treated in the stomatological service of the Specialties Polyclinic of "Saturnino Lora Torres" Teaching Provincial Clinical Surgical Hospital in Santiago de Cuba was carried out, from March, 2013 to the same month in 2015, in order to evaluate the results of the clinical and histopathologic diagnosis of the mentioned affection. The samples for biopsies were fixed and processed with the classic technique of inclusion in paraffin; they were colored with a special stain of Schiff periodic acid, which confirmed the existence of cellular changes. There was a prevalence of male sex, the 60 years and over age group; the absence of clinical symptoms, the hard palate and the corner of the mouth as the sites of greater prevalence and the erythematous candidiasis as the most common clinical form. The presence of pathogen hyphae and yeast-like cells were the most outstanding histopathologic findings.

Onicomicosis: epidemiología, agentes causales y evaluación de los métodos diagnósticos de laboratorio / Onychomycoses: epidemiology, causative agents and assessment of diagnostic laboratory methods

Desde marzo de 2007 hasta marzo de 2011 se estudiaron prospectivamente 414 pacientes con onicodistrofias en un laboratorio privado de Esquel. La prevalencia de onicomicosis de pie fue del 78 %; la de mano, del 58 %. Los principales agentes etiológicos fueron Trichophyton rubrum, Candida spp. y Trichophyton mentagrophytes. El desarrollo de dermatofitos prevaleció en las onicopatías de pie y el de Candida spp. en las de uñas de mano (ambos, p < 0,05). En las onicomicosis candidiásicas predominaron especies diferentes a Candida albicans. Las onicomicosis fueron más frecuentes en los hombres que en las mujeres. A su vez, en los hombres hubo más aislamientos de T. rubrum en pies (p < 0,05) y mayor proporción de exámenes directos (ED) y cultivos positivos (ambos, p < 0,05). La correlación entre los resultados del ED y del cultivo fue del 68 %. El rédito de ambos métodos se asoció a un mayor tamaño de la lesión ungueal. El ED fue más efectivo en onicodistrofias que superaban los 5 años de evolución. La positividad del cultivo fue independiente de la cronicidad de la onicodistrofia.

Since March 2007 to March 2011, 414 patients with onychopathies were prospectively analyzed. Prevalence of the toenail and fingernail mycoses was 78 % and 58 %, respectively. The major etiological agents were Trichophyton rubrum, Candida spp. and Trichophyton mentagrophytes. Dermatophytes were more frequently cultured from toenails, whereas Candida spp. from fingernails (both, p < 0.05). In candidal onychomycosis, species different from C. albicans were prevalent. A higher prevalence of toenail and fingernail mycoses, a predominance of T. rubrum in toenails (p < 0.05), and greater positivity in the direct examination (DE) and in culture (both, p < 0.05) were more frequently observed in men than in women. The correlation between DE and culture was 68 %. DE and culture yields were associated with a greater size lesion. DE was more effective in onycodystrophies with duration of more than 5 years. Culture positivity was independent of nail affection chronicity.

Unusual morphologies of Cryptococcus spp. in tissue specimens: report of 10 cases / Histopatologia, sorologia e cultivo no diagnóstico da criptococose

Ten cases of cryptococcosis due to unusual microscopic forms of Cryptococcus sp. observed over a twenty-eight year period (1981-2009) are presented. The most important clinicopathological and laboratory data are tabulated. The uncommon forms of cryptococcal cells given are: structures resembling germ tube (one case), chains of budding yeasts (one case), pseudohyphae (two cases) and nonencapsulated yeast-like organisms (eight cases). The diagnosis was based on the histopathological findings. The causative organism was isolated and identified in seven cases; five were due to C. neoformans, and two to C. gattii. In addition, the importance of using staining histochemical techniques - Grocott's silver stain (GMS), Mayer's mucicarmine stain (MM) and Fontana-Masson stain (FM) - in the diagnosis of cryptococcosis is argued.

A criptococose é a mais comum infecção fúngica oportunística observada em pacientes com síndrome da imunodeficiência adquirida (AIDS). Relatamos 13 casos da infecção baseados no diagnóstico histopatológico, sorológico e cultivo. Foram analisadas: a epidemiologia, as técnicas histoquímicas básicas de hematoxilina-eosina (HE) e coloração pela prata (GMS), bem como as técnicas histoquímicas especiais de mucicarmim de Mayer (MM) e Fontana-Masson (FM), o teste do antígeno criptocóccico (CrAg) e o isolamento em cultivos em ágar-Sabouraud (SAB), ágar infusão de cérebro-coração (BHI) e meio com canavanina azul de bromotimol (CGB). Em quatro casos, resultados tintoriais insatisfatórios pela coloração de MM associados a títulos negativos pelo teste do CrAg, a coloração de FM confirmou a infecção pelo Cryptococcus deficiente de cápsula. Oito isolados foram identificados: seis casos apresentaram a infecção por Cryptococcus neoformans e dois casos apresentaram a infecção por Cryptococcus gattii.

Mastite micótica em ruminantes causada por leveduras / Mycotic mastitis in ruminants caused by yeasts

A ocorrência de casos esporádicos de mastite causados por microrganismos de origem ambiental pode ser considerada como emergente e as leveduras, os fungos leveduriformes e os filamentosos são alguns dos principais agentes envolvidos. Em relação à mastite micótica, as leveduras são freqüentemente causas de infecções da glândula mamária em animais produtores de leite. A maior parte dos casos ocorre sob a forma de surtos localizados e/ou após tratamento com antimicrobianos. Os principais gêneros envolvidos são Candida e Cryptococcus, além de outros como Geotrichum, Pichia e Trichosporon. O objetivo do trabalho é revisar os aspectos gerais sobre a mastite micótica causada por leveduras e fungos leveduriformes, tais como etiologia, causas predisponentes, patogenicidade, diagnóstico, tratamento e profilaxia por meio de uma abordagem cronológica dos primeiros relatos, mas com enfoque principal nos resultados descritos recentemente na literatura veterinária nacional e internacional. O controle da mastite micótica é fundamentado em métodos preventivos, como adequado manejo da ordenha, correta higienização dos equipamentos e das instalações, com a finalidade de reduzir o número de animais acometidos, além de garantir a qualidade e a inocuidade dos produtos lácteos.

The veterinary literature registers sporadic cases of infections caused by environmental microorganisms in which the main agents involved are yeasts, yeasts-like and filamentous fungi. In relation to dairy animals, the yeasts are more frequently incriminated as the aetiological agents of mycotic mastitis. Most cases occur under the form of localized outbreaks and / or after treatment with antimicrobial agents. The main genera involved in mastitis are Candida and Cryptococcus, besides others such as Geotrichum, Pichia and Trichosporon. The purpose of this review is to point out the main aspects of mastitis caused by yeasts and yeast-like fungi, such as etiology, predisposing factors, pathogenicity, diagnosis, treatment and prophylaxis, through a chronological approach of the first reports, with main focus on results described recently in national and international veterinary literature. The mycotic mastitis control should be mainly focused on preventive methods, especially based on adequate management of essential factors like proper milking procedures and a high level of hygiene of the environment and equipments, with the aim of reducing the number of affected animals, assuring the quality and innocuity of dairy food safety.

Perfil de las onicomicosis basado en 2106 exámenes micológicos / Onychomycoses profile based on 2106 mycological studies

Introducción. Las onicomicosis representan el 50% de las patologías ungueales, su incidencia se calcula en el 4% de la población mundial y es aún más elevada en las personas mayores de 50 años. Son causadas por tres grupos de hongos: dermatofitos, levaduras y hongos miceliales no dermatofitos (HFND). Objetivos. El objetivo es dar a conocer los resultados de exámenes micológicos de uñas en una muestra significativa de una parte de la población de la ciudad de Buenos Aires, donde se consignan además las características clínicas de las lesiones observadas, los agentes causales, los factores predisponentes y los principales diagnósticos diferenciales. Resultados. Se estudiaron 1753 lesiones en los pies y 353 onicodistrofias de manos. En las manos la alteración ungueal más frecuente fue la onicolisis (75%); en los pies, las más comunes fueron onicolisis (42%) y compromiso distal subungueal (37,7%). El 50,3% de los exámenes micológicos fue negativo. La correlación entre los exámenes microscópicos directos y los cultivos fue de 79,3%; el 31% de los exámenes directos evidenciaron dermatofitos o hifas hialinas que tuvieron cultivos negativos. Los aislamientos más frecuentes fueron: Trichophyton rubrum (419), Candida spp (140), HFND (94: Acremonium spp y Fusarium spp fueron responsables del 70%) y Trichophyton mentagrophytes (50). Conclusiones. La mitad de los exámenes micológicos de uñas fueron negativos. La mayor parte de las onicomicosis de los pies fue ocasionada por T. rubrum, en tanto que en las manos predominó Candida sp. El papel desempeñado por Candida y los HFND en la producción de onicodistrofias no es claro. No hubo ninguna lesión patognomónica de onicomicosis

Introduction. Approximately 50% of nail diseases are fungal infections, its incidence is 4% of human population and it is higher in persons older than 50 years old. Onychomycoses are caused by three groups of fungi: Dermatophytes, yeasts and nondermatophytes mycelial fungi (NDMF). Objectives. To report the results of mycological examinations of the nails of a representative sample of Buenos Aires city population; etiologic agents, clinical characteristics, risk factors and differential diagnosis were considered. Results. 1753 toenails studies and 353 fingernails examinations were performed. Onycholysis was the most frequent fingernail alteration, 75% of the cases, while onycholysis 42% and distal or lateral subungueal dystrophies were the most common lesions in the toenails. Negative mycological studies were detected in 50,3% of the studies and in 79,3% of the exams, the microscopicobservation agreed with the cultures results; 31% of the cases which presented dermatophytes or hyaline hyphae in the direct microscopic study, did not show any fungal growth in cultures. The fungal species more frequently identified were Trichophytonrubrum 419 cases, Candida spp 140, NDMF 94 and Trichophyton mentagrophytes in 50 patients. Acremonium spp and Fusariumspp were the etiologic agents of 70% of onychomycoses due to NDMF.Conclusions. 50% of mycologic studies were negative. The majority of toenails onychomycoses was due to T. rubrum and the most frequent etiologic agent of fingernails onychomycosis was Candida sp. The role of Candida sp and NDMF in the production of onychodistrophies is not clear. There was not any typical nail alteration of onychomycosis.

Bacteriostasis Test in vitro of Deep Yeast-like Fungi by Radix Scutellariae,Radix Sophorae Flavescentis and Flos Lonicerae in Gansu / 中华医院感染学杂志

OBJECTIVE To find the active principle in Radix Scutellariae,Radix Sophorae Flavescentis and Flos Lonicerae in Gansu inhibiting yeast-like fungi of deep infection.METHODS Radix Scutellariae,Radix Sophorae Flavescentis and Flos Lonicerae were separately cleaned,toasted and grinded to fine power.Five kinds of(8 strains) yeast-like fungi that were collected and spread into 90mm Sabouraud plate.On aseptic condition,bored holes on Sabouraud plate with the drilling instrument of 6mm diameter,and filled every holes with 25.73mg fine powder of Radix Scutellariae,Radix Sophorae Flavescentis and Flos Lonicerae,and added 3 to 5 drops of distilled water in the medicinal powder.Under 35℃,the bacteria were cultured for 24 to 48 hours,and the size of bacteriostatic ring was observed.RESULTS The diameters of the bacteriostatic ring of Radix Scutellariae powder inhibiting five kinds of yeast-like fungi were separately 16.0mm,13.5mm,13.0mm,13.0mm and 10.5mm,respectively,and the diameter of Radix Sophorae Flavescentis and Flos Lonicerae powder was 0.CONCLUSIONS There is the active principle inhibiting yeast-like fungi in Radix Scutellariae,but not that in Radix Sophorae Flavescentis and Flos Lonicerae.

Phylogenetic position of the yeast-like symbiotes of Tagosodes orizicolus (Homoptera: Delphacidae) based on 18S ribosomal DNA partial sequences

Tagosodes orizicolus Muir (Homoptera: Delphacidae), the endemic delphacid species of tropical America carries yeast-like symbiotes (YLS) in the abdominal fat bodies and the ovarial tissues, like other rice planthoppers of Asia. These YLS are obligate symbiotes, which are transmitted transovarially, and maintain a mutualistic relationship with the insect host. This characteristic has made in vitro culture and classification of YLS rather difficult using conventional methods. Nevertheless, microorganisms of similar characteristics have been successfully classified by using molecular taxonomy. In the present work, the YLS of Tagosodes orizicolus (YLSTo) were purified on Percoll gradients, and specific segments of 18S rDNA were amplified by PCR, cloned and sequenced. Sequences were aligned by means of the CLUSTAL V (DNASTAR) program; phylogenetic trees were constructed with the Phylogeny Inference Package (PHYLIP), showing that YLSTo belong to the fungi class Pyrenomycetes, phylum Ascomycota. Similarities between 98% and 100% were observed among YLS of the rice delphacids Tagosodes orizicolus, Nilaparvata lugens, Laodelphax striatellus and Sogatella fur cifera, and between 89.8% and 90.8% when comparing the above to YLS of the aphid Hamiltonaphis styraci. These comparisons revealed that delphacid YLS are a highly conserved monophyletic group within the Pyrenomycetes and are closely related to Hypomyces chrysospermus.

Disc Diffusion on Fluconazole Susceptibility Test of Yeast-like Fungi: Evaluation and Clinical Practice / 中华医院感染学杂志

OBJECTIVE To evaluate the disc diffusion method on the clinical antifungal susceptibility test for yeast-like fungi.METHODS The fluconazole susceptibility test was detected by disc diffusion method and NCCLS broth microdilution method with 113 yeast-like fungus strains isolated from clinical specimens.Three standard strains were used as the quality control.RESULTS The coincidence rate of the two methods was 89.4%.The sensitive strains detected by one method didn′t show resistance in another detection method,and the resistant strains detected by one method yet didn′t show sensitivity in another one.CONCLUSIONS Disc diffusion method can be used in clinical detection instead of NCCLS broth microdilution method.