Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges / Desenvolvimento e aplicação da reação em cadeia da polimerase para detecção de Conidiobolus lamprauges

Conidiobolomycosis is a granulomatous disease caused by the fungus Conidiobolus spp. in humans and animals. Traditional technique for diagnosis of the disease is isolation of the agent associated with the presence of typical clinical signs and pathological conditions. The aim of this study was to describe the development of a specific polymerase chain reaction (PCR) test for Conidiobolus lamprauges to detect the fungus in clinical samples. Samples from suspected animals were collected and submitted to isolation, histopathological analysis and amplification by PCR. DNA from tissues was subjected to PCR with fungi universal primers 18S rDNA gene, and specific primers were designed based on the same gene in C. lamprauges that generated products of about 540 bp and 222 bp respectively. The culture was positive in 26.6% of clinical samples. The PCR technique for C. lamprauges showed amplification of DNA from fresh tissues (80%) and paraffin sections (44.4%). In conclusion, the PCR technique described here demonstrated a high sensitivity and specificity for detection of fungal DNA in tissue samples, providing a tool for the rapid diagnosis of C. lamprauges.

A conidiobolomicose é uma doença granulomatosa causada pelo fungo Conidiobolus spp., observada em humanos e animais. As técnicas tradicionais de diagnóstico da doença são o isolamento do agente associado à presença de sinais clínicos típicos e condições patológicas. O objetivo deste trabalho é descrever o desenvolvimento de um teste da reação em cadeia da polimerase (PCR) específico para Conidiobolus lamprauges em amostras clínicas. As amostras de animais suspeitos foram coletadas e submetidas ao isolamento, análise histopatológica e amplificação pela PCR. O DNA de tecidos foi submetido a PCR com os iniciadores universais de fungos baseados no gene 18S rDNA e iniciadores específicos foram concebidos com base no mesmo gene em C. lamprauges que gerou produtos de aproximadamente 540 pb e 222 pb, respectivamente. A cultura foi positiva em 26,6% das amostras clínicas. A técnica de PCR para C. lamprauges mostrou a amplificação de DNA a partir de tecidos frescos (80%) e secções de parafina (44,4%). Em conclusão, a técnica de PCR aqui descrita demonstrou elevada sensibilidade e especificidade na detecção de DNA de fungos em amostras de tecido, proporcionando uma ferramenta rápida para o diagnóstico de C. lamprauges.

Hemograma, proteinograma, ionograma e dosagens bioquímicas e enzimáticas de ovinos acometidos por conidiobolomicose no nordeste do Brasil: [revisão] / Hematology and blood biochemistry in sheep with conidiobolomycosis in northeastern Brazil: [revision]

Foram realizados eritrogramas, leucogramas, dosagens séricas de proteínas totais, albumina, globulinas, cálcio, fósforo, magnésio, cloretos, uréia, creatinina, bilirrubina total, direta e indireta e atividade sérica de aspartato aminotransferase (AST), fosfatase alcalina (FA) e gama-glutamiltransferase (GGT) de 56 ovinos afetados por conidiobolomicose e de 371 ovinos sadios provenientes dos mesmos rebanhos dos casos da doença. Os resultados revelaram que os ovinos com conidiobolomicose apresentam anemia arregenerativa normocítica normocrômica, leucocitose com neutrofilia e discreto desvio à esquerda regenerativo, com elevação da relação neutrófilo:linfócito, monocitose moderada, trombocitose, hipoproteinemia (hipoglobulinemia), hipomagnesemia, hipocalcemia e elevação dos teores de fósforo. As atividades séricas de AST e GGT estavam aumentadas e as de FA diminuídas. Não houve alteração nos níveis de uréia e creatinina, mas ocorreu hipoglicemia e hiperbilirrubinemia. Estes resultados podem ser utilizados para estudos experimentais da doença, em ensaios com tratamentos e para identificar casos precoces da enfermidade que não apresentem sinais clínicos. Além disso, os valores encontrados em 371 ovinos deslanados hígidos podem ser utilizados como valores referenciais para este tipo de ovinos na região semi-árida do Brasil.

Hematologic values, serum levels of albumin, globulins, total proteins, calcium, phosphorus, magnesium, chloride, urea nitrogen, creatinine, total, direct and indirect bilirrubin, and serum activities of aspartate aminotransferase (AST), alkalin phosphatase (AF) and gama-glutamyltransferase (GGT) were determined in 56 hair sheep with conidiobolomycosis and 371 healthy hair sheep from the same flocks. Sheep with conidiobolomycosis had nonregenerative, normocytic and normochromic anemia, leucocytosis with neutrophilia and increased nutrophil:lymphocyte rate, moderate monocytosis, moderate thrombocytosis, hypoproteinemia (hypoglobulinemia), hypomagnesemia, hypocalcemia and increased values of phosphorus. Serum activities of AST and GGT were increased and serum activity of FA decreased. Serum values of urea and creatinine were within normal values. Hypoglycemia and hyperbilirrubinemia were also observed. These results can be used for experimental studies with the disease, for treatments trials, and to detect early cases of the disease without clinical signs. Otherwise the hematologic and blood biochemistry values of healthy sheep can be used as reference values for hair sheep in the semi-arid region of Brazil.

Epidemiologia e sinais clínicos da conidiobolomicose em ovinos no Estado do Piauí / Epidemiology and symptoms of conidiobolomycosis in sheep in the State of Piauí, Brazil

Foi realizado um estudo de ocorrência da conidiobolomicose ovina em 25 rebanhos no Estado do Piauí, de janeiro de 2002 a dezembro de 2004. A enfermidade acometeu apenas ovinos e ocorreu principalmente em abril-julho. A incidência média anual foi de 2,80 por cento e a incidência semestral foi significativamente mais alta (P <0,05) no primeiro semestre (2,10 por cento), durante a época chuvosa, do que no segundo semestre (0,69 por cento), durante a seca. A incidência entre rebanhos variou de 0,1-14,3 por cento e a letalidade foi de 100 por cento. Ao exame clínico, os animais apresentavam apatia, emagrecimento progressivo, secreção nasal serosa, mucosa e/ou hemorrágica, dificuldade respiratória, respiração ruidosa, febre e na maioria dos casos assimetria crânio-facial e exoftalmia. Alguns ovinos permaneciam com a cabeça baixa ou a pressionavam contra objetos. O curso clínico foi de 1-5 semanas. As lesões macroscópicas, microscópicas e ultrastructurais e a identificação do agente são descritas separadamente. Este é o primeiro registro de conidiobolomicose em ovinos no Brasil, enfermidade endêmica no Estado do Piauí, associada à alta pluviosidade (1000-1600mm anuais) e alta temperatura (19-36ºC).

Conidiobolomycosis is reported in 25 farms, from January 2002 to December 2004, in the state of Piauí. The disease affects only sheep, mainly in April-June. The mean morbidity rate was 2.80 percent, but was higher in the first semester (2.1 percent), during the raining period, than in the second one (0.69 percent), during the dry period. Morbidity rate among flocks varied from 0.1-14.3 percent. Case fatality rate was 100 percent, and the clinical manifestation period varied from 1-5 weeks. Clinical signs were serous, mucous and/or bloody nasal secretion, respiratory distress, snoring respiration, cranium-facial asymmetry, exophthalmia, fever and progressive emaciation. Marked depression, sometimes with the head down or head pressing was observed in some cases. Gross, microscopic and ultrastructural lesions and identification of the agent are reported elsewhere. This is the first report of conidiobolomycosis in Brazil, which is endemic and has a high frequency in sheep in the State of Piauí, associated with high rainfalls (1000-1600mm annually) and high temperature (19-36ºC).

Rhinoentomophthoromycosis.

A sixty year old patient presented with a slowly progressive swelling of the nose, of one year duration, suggesting a clinical diagnosis of subcutaneous zygomycosis. On investigation, the tissue fungal culture grew Conidiobolus coronatus, confirming the diagnosis as rhinoentomophthoromycosis. He was treated with a combination of oral fluconazole and oral potassium iodide for a total period of 5 months. His symptoms subsided completely. Serial CT scanning of paranasal sinuses showed the gradual resolution of the swelling, in response to the treatment. Early detection of the disease and combination therapy gave rapid and good results. This is the first case of its kind to be reported from Kerala, the southern state of India.

Entomoftoromicose (zigomicose) causada por Conidiobolus coronatus em Mato Grosso (Brasil): relato de caso / Entomophthoramycosis (zygomycosis) caused by Conidiobolus coronatus in Mato Grosso (Brazil): case report

Zigomicoses são micoses subcutâneas causadas por fungos do solo, que geralmente manifestam-se como uma infiltração granulomatosa crônica da submucosa nasal, estendendo-se para o tecido subcutâneo e pele da face. Descreve-se aqui o primeiro caso de zigomicose nasofacial causada pelo Conidiobolus coronatus, ocorrendo em Mato Grosso, Brasil.

Entomophthoromycosis in South Bengal (Eastern India): a 9 years study.

Subcutaneous mycoses caused by the family Entomophthoraceae is very rare type of disease and is being reported sporadically from various Tropical countries including India. Here we report 8 cases of rhinoentomophthoromycosis caused by Conidiobolous coronatus and 7 cases of chronic subcutaneous phycomycosis caused by Basidiobolus ranarum. Cases were detected during a span of 9 years between 1991 to 1999, from 9 districts in and around Kolkata (Eastern India). Former type of lesions were detected among 20 to 65 age group of healthy individuals, predominantly males (7:1). In the latter type, male-female ratio was 2:5, and except for one all cases belonged to below 20 years age group of healthy individuals. Several cases were detected only after examination of repeat biopsy samples. With high degree of clinical suspicion, right approach is needed for laboratory confirmation of diagnosis.

Zygomycosis due to Conidiobolus coronatus in west Bengal.

Zygomycosis of upper respiratory tract, caused by Conidiobolus coronatus, in eight Bengalee males and one female is described. The clinical syndrome of the cases included initial coryza, epistaxis, intranasal tumor, nasal obstruction, broadening of bridge of nose with swelling of paranasal sinuses, dorsum of nose, alae nasi, cheek and glabella in all or majority of the cases. In addition upper lip or both lips and pharynx were involved in some cases. Swellings were fixed to the skin in seven cases and to the deeper tissues in five cases. The fungus was demonstrated by direct microscopy in the nasal mucosa, and was isolated in culture. The isolates produced conidia, replicative conidia and villose conidia but not multiplicative conidia. The disease was cured with KI and/or ketoconazole/fluconazole. The cases described here constitute the first report on zygomycosis due to C. coronatus from North-eastern India.