Comparison of primers for RAPD-PCR from environmental isolates of Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii complex

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e.g., CAV2 for C. albidus) may not to be applied to some species.

Cryptococcus spp. E ECTOPARASITOS ASSOCIADOS A POMBOS (Columba livia) EM HOSPITAIS, ESCOLAS, PRAÇAS, IGREJAS E PRÉDIOS ANTIGOS NA CIDADE DE SALVADOR, BAHIA

Objetivou-se neste estudo avaliar o risco da ocorrência de Cryptococcus spp. e ectoparasitos associados à presença de pombos (Columba livia) em hospitais, escolas, praças, igrejas e prédios antigos na cidade de Salvador, nordeste do Brasil. No período de 2015 a 2016, foram coletadas 851 amostras, sendo: de swab da cloaca de pombo (n= 229), material aderido às patas de pombo (n=26), fezes de pombos do ambiente (n= 315), poeira do interior de recintos (n=218) e detrito de árvores (n=63). Nos hospitais todos os tipos de amostras foram coletados o que variou para os demais locais de estudo. Os pombos foram capturados com rede, anilhados e coletadas amostras de cloaca e de resíduos de patas. Em três exemplares foram acoplados mini-GPSs. Foi realizada identificação fenotípica e bioquímica de Cryptococcus spp. O Risco Relativo [RR] de contaminação de Cryptococcus spp. foi calculado entre os locais com a presença de pombos e suas fezes em relação à poeira de recintos internos em hospitais e escolas, bem como, para locais com a presença de pombos e suas fezes em igrejas e prédios antigos em relação às praças. A positividade geral nas amostras foi de 15,0% (128/851), sendo 72,3% (99/137) de C. albidus, 17,5% (24/137) de C. laurentii, 10,2% (14/137) de C. neoformans, nove amostras apresentaram contaminação mista. Segundo os resultados de isolamento, as áreas externas dos hospitais apresentaram 3,6 vezes risco mais elevado de contaminação por Cryptococcus spp. em relação aos seus recintos internos (RR 3·6, 95% IC 1·5 - 7·4; p = 0·001). As igrejas e prédios antigos apresentaram duas vezes mais risco de contaminação em relação às praças (RR 20, 95% CI 10 39; p=0036). C. albidus e C. laurentii foram isolados em amostras de cloaca (56/229), patas de pombos (8/26) e detritos de árvores (2/63). C. neoformans (n=14) foi isolado apenas em amostras de fezes. Foram registradas infestações nosocomiais por Ornithonyssus bursa, Pseudolynchia canariensis e Peckia intermutans em um dos hospitais estudados, todas associadas à presença de Columba livia. Medidas estruturais são necessárias para evitar a permanência de pombos e a formação de seus ninhos, além de ações de educação no sentido de não alimentar essas aves e aprimorar o manejo do lixo em espaços públicos e nos hospitais, evitando a exposição das pessoas às contaminações e infestações associadas a Columba livia.

The objective of this study was to evaluate the risk of Cryptococcus spp. And ectoparasites associated with the presence of Columba livia pigeons in hospitals, schools, squares, churches and old buildings in the city of Salvador, northeastern Brazil. In the period from 2015 to 2016, 851 samples of pigeon cloaca swab (n = 229), material adhered to the pigeon paws (n=26), pigeon feces from the environment (n = 315), dust from the interior of rooms (n = 218) and detritus of trees (n=63). In the hospitals, all types of samples were collected, which varied for the other study sites. The pigeons were captured with net, ringed and collected samples of cloaca and paws. In three birds were coupled mini-GPS. Phenotypic and biochemical identification of Cryptococcus spp. The Relative Risk [RR] of contamination of Cryptococcus spp. Was calculated between sites with the presence of pigeons and their feces in relation to dust from interior of rooms in hospitals and schools, as well as for sites with pigeons and their feces in churches and old buildings in relation to squares. The overall positivity in the samples was 15.0% (128/851), with 72.3% (99/137) of C. albidus, 17.5% (24/137) of C. laurentii, 10.2% (14/137) of C. neoformans, nine samples showed mixed contamination. According to the isolation results, the external areas of the hospitals presented a 3.6 times higher risk of Cryptococcus spp. contamination in relation to its interior of rooms (RR 3 · 6, 95% CI 1 · 5 - 7 · 4, p = 0 · 001). Churches and old buildings had twice the risk of contamination compared to squares (RR 2 0, 95% CI 1 0 - 3 9, p = 0 036). C. albidus and C. laurentii were isolated in cloacal samples (56/229), pigeon paws (8/26) and tree debris (2/63). C. neoformans (n = 14) was isolated only in feces samples. Nosocomial infestations by Ornithonyssus bursa, Pseudolynchia canariensis and Peckia intermutans were recorded in one of the hospitals studied, all associated with the presence of Columba livia. Structural measures are necessary to prevent pigeons from permanence and nesting, as well as educational actions in order not to feed these birds and improve waste management in public spaces and hospitals, avoiding the exposure of people to the contamination and infestations associated with Columba livia.

Cryptococcus gattii and Cryptococcus albidus in Captive Domestic Pigeon (Columba livia)

Cryptococcosis in animals is an important fungal disease caused by the encapsulated yeast Cryptococcus neoformans. This report describes the occurrence of Cryptococcus gattii and Cryptococcus albidus in domestic pigeon (Columba livia), living together with other birds in a breeding center. The animal presented a pinkish, vascularized mass with gelatinous aspect localized subcutaneously under the right lower eyelid, with approximately 2cm in diameter. At microbiological exam it was isolated Cryptococcus gatti from the eyelid mass, lungs and liver, C. albidus from the trachea and both Cryptococcus species from muscle and kidney.(AU)

Infecção fúngica mista por Sporothrix schenckii e Cryptococcus albidus em um canino / Mixed fungal infection by Sporothrix schenckii and Cryptococcus albidus in a dog

Background: The dimorphic fungus Sporothrix schenckii and the yeasts of the Cryptococcus complex are important fungal pathogens of humans and animals that cause sporotrichosis and cryptococcosis, respectively. Both fungi are saprophytes of soils rich in organic matter and infect the host through traumatic injuries and/or inhalation. Sporotrichosis has been recognized as an important disease in the small animal clinic and in public health. Cases of this mycosis in cats and dogs have been reported in various Brazilian States. Our research group diagnosed eleven cases of sporotrichosis in dogs in Rio Grande do Sul in a five-year period. On the other hand, clinical cases of cryptococcosis in dogs and cats have not often been described in Brazil. In addition, infections caused by non- neoformans species have rarely been reported throughout the world. The aim of this study is to describe the first case of concomitant infection by Sporothrix schenckii and Cryptococcus albidus in a dog. Case: In 2008, a veterinary clinic of Rio Grande municipality received a 5-year-old female Dalmatian which had presented an increase of the nasal plan, sneezing, dyspnea and a serous nasal discharge for 30 days. Upon clinical examination, abnormalities in retal temperature, cardiopulmonary auscultation and frequency of the animal were not observed. The presence of an eritematous non-ulcerated mass partially occluding the airways was verified in the nasal cavity. Differential diagnosis included sporotrichosis, cryptococcosis and transmissible venereal tumor (TVT). Diagnosis confirmation was performed by laboratory tests which included radiographic, cytological and mycological analyses. The nasal radiograph revealed a mass of 20 mm in diameter with adjacent bone compromising. Ovoid and round cells suggestive of yeasts were found in cytology. Colonies with macroscopic and microscopic morphology compatible with Sporothrix schenckii and Cryptococcus albidus were obtained in mycological analysis. Confirmation of the species C. albidus was obtained by the API32 commercial system. Antifungal therapy with itraconazole at a 10 mg kg -1 oral dosage for 90 days was successfully performed, resulting in the regression of the lesions. Discussion: Sporotrichosis has been frequently diagnosed in cats and dogs in the south and southeast of Brazil. However, mixed infection cases with Sporothrix schenckii are rare. In Brazil, one case was described in a cat with sporotrichosis, demodicosis and pediculosis which was co-infected with the feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Mixed fungal infection by Cryptococcus neoformans and was described only in a dog in Canada that presented lesions similar to those reported in this study. Cryptococcosis cases in animals and humans caused by non-neoformans species with C. albidus and C. magnus have been described worldwide. Systemic, renal and cutaneous infections by C. albidus have been reported in dogs, cats and horses. The present study reports the first case of a concomitant infection of Sporothrix schenckii and Cryptococcus albidus in a dog, and warns small animal doctors of the importance of laboratory exams so that a final infectious disease diagnosis, especially of fungal infections, can be reached.(AU)

Infecção fúngica mista por Sporothrix schenckii e Cryptococcus albidus em um canino / Mixed fungal infection by Sporothrix schenckii and Cryptococcus albidus in a dog

Background: The dimorphic fungus Sporothrix schenckii and the yeasts of the Cryptococcus complex are important fungal pathogens of humans and animals that cause sporotrichosis and cryptococcosis, respectively. Both fungi are saprophytes of soils rich in organic matter and infect the host through traumatic injuries and/or inhalation. Sporotrichosis has been recognized as an important disease in the small animal clinic and in public health. Cases of this mycosis in cats and dogs have been reported in various Brazilian States. Our research group diagnosed eleven cases of sporotrichosis in dogs in Rio Grande do Sul in a five-year period. On the other hand, clinical cases of cryptococcosis in dogs and cats have not often been described in Brazil. In addition, infections caused by non- neoformans species have rarely been reported throughout the world. The aim of this study is to describe the first case of concomitant infection by Sporothrix schenckii and Cryptococcus albidus in a dog. Case: In 2008, a veterinary clinic of Rio Grande municipality received a 5-year-old female Dalmatian which had presented an increase of the nasal plan, sneezing, dyspnea and a serous nasal discharge for 30 days. Upon clinical examination, abnormalities in retal temperature, cardiopulmonary auscultation and frequency of the animal were not observed. The presence of an eritematous non-ulcerated mass partially occluding the airways was verified in the nasal cavity. Differential diagnosis included sporotrichosis, cryptococcosis and transmissible venereal tumor (TVT). Diagnosis confirmation was performed by laboratory tests which included radiographic, cytological and mycological analyses. The nasal radiograph revealed a mass of 20 mm in diameter with adjacent bone compromising. Ovoid and round cells suggestive of yeasts were found in cytology. Colonies with macroscopic and microscopic morphology compatible with Sporothrix schenckii and Cryptococcus albidus were obtained in mycological analysis. Confirmation of the species C. albidus was obtained by the API32 commercial system. Antifungal therapy with itraconazole at a 10 mg kg -1 oral dosage for 90 days was successfully performed, resulting in the regression of the lesions. Discussion: Sporotrichosis has been frequently diagnosed in cats and dogs in the south and southeast of Brazil. However, mixed infection cases with Sporothrix schenckii are rare. In Brazil, one case was described in a cat with sporotrichosis, demodicosis and pediculosis which was co-infected with the feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Mixed fungal infection by Cryptococcus neoformans and was described only in a dog in Canada that presented lesions similar to those reported in this study. Cryptococcosis cases in animals and humans caused by non-neoformans species with C. albidus and C. magnus have been described worldwide. Systemic, renal and cutaneous infections by C. albidus have been reported in dogs, cats and horses. The present study reports the first case of a concomitant infection of Sporothrix schenckii and Cryptococcus albidus in a dog, and warns small animal doctors of the importance of laboratory exams so that a final infectious disease diagnosis, especially of fungal infections, can be reached.

Cryptococcus gattii and Cryptococcus albidus in captive domestic pigeon (Columba livia)

Cryptococcosis in animals is an important fungal disease caused by the encapsulated yeast Cryptococcus neoformans. This report describes the occurrence of Cryptococcus gattii and Cryptococcus albidus in domestic pigeon (Columba livia), living together with other birds in a breeding center. The animal presented a pinkish, vascularized mass with gelatinous aspect localized subcutaneously under the right lower eyelid, with approximately 2cm in diameter. At microbiological exam it was isolated Cryptococcus gatti from the eyelid mass, lungs and liver, C. albidus from the trachea and both Cryptococcus species from muscle and kidney.