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      Teste de ELISA indireto para o diagnóstico sorológico de pitiose Translated title: Indirect ELISA for the serodiagnostic of pythiosis

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          Abstract

          A pitiose, doença granulomatosa de eqüinos causada pelo oomiceto Pythium insidiosum, tem como característica a evolução rápida seguida de morte dos animais. Estas mortes muitas vezes são causadas por diagnósticos errôneos ou demorados quando os doentes já não respondem ao tratamento. Este trabalho teve por objetivo a padronização do ensaio imunoenzimático indireto (ELISA) para diagnóstico sorológico de pitiose em eqüinos e coelhos, visando a diminuição de erros e de tempo necessário para o diagnóstico. Para o desenvolvimento e validação do teste foram utilizadas 72 amostras de soro de eqüinos saudáveis e 44 soros de eqüinos com pitiose confirmada. Os resultados da validação do ELISA para eqüinos foram: sensibilidade 97,72%, especificidade 90,27%, valor preditivo positivo 86%, valor preditivo negativo 98,4% e eficiência de 93,1%. Para coelhos, o teste foi padronizado com 48 amostras de soro de animais saudáveis e 24 amostras de coelhos imunizados com antígenos de P. insidiosum. Os resultados foram: sensibilidade 91,66%, especificidade 95,83%, valor preditivo positivo 91,66%, valor preditivo negativo 95,83% e eficiência de 94,44%. Os resultados deste trabalho demonstram que o ensaio imunoenzimático indireto é um método seguro e eficaz para o diagnóstico sorológico da pitiose.

          Translated abstract

          Pythiosis is a granulomatous disease caused by the oomycete Pythium insidiosum that affects humans and animals, especially horses. Deaths are very often the consequence of incorrect or late diagnosis when animals no longer respond to treatment. This study aimed standardization of the ELISA assay for the serodiagnostic of pythiosis in horses and rabbits, in order to minimize errors and delays in the diagnosis of the disease. Sera of 72 healthy and 44 of by pythiosis affected horses were used for development and evaluation of the test. The ELISA for equine diagnostic showed 97.72% sensitivity, 90.27% specificity, 86% positive predictive value, 98.4% negative predictive value, and 93.1% efficiency. The rabbit test was standardized with 48 sera of healthy rabbits and 24 sera of rabbits immunized with P. insidiosum antigens. The results were 91.66 % sensitivity, 95.83% specificity, 91.66% positive predictive value, 95.83% negative predictive value, and 94.44% efficiency. It can be concluded that ELISA is a reliable test for diagnostic and serological monitoring of pythiosis.

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          Human Pythiosis, Brazil

          Pythiosis, caused by Pythium insidiosum, occurs in humans and animals and is acquired from aquatic environments that harbor the emerging pathogen. Diagnosis is difficult because clinical and histopathologic features are not pathognomonic. We report the first human case of pythiosis from Brazil, diagnosed by using culture and rDNA sequencing.
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            Outbreaks of pythiosis in two flocks of sheep in northeastern Brazil.

            Two outbreaks of cutaneous pythiosis caused by Pythium insidiosum were diagnosed in two herds of crossbred hair wool sheep of different ages in the semiarid region of Northeastern Brazil. In one herd of 120 sheep, 40 were affected. The other outbreak affected six sheep out of 80. Local swellings with ulcerative lesions were observed in the limbs and abdominal and prescapular regions. Three sheep were necropsied. Two of them had lung metastasis characterized by multifocal nodules measuring 0.5-2 cm. In one animal, the prescapular lymph node was also affected. In another, the cutaneous lesion extended to the sesamoid bone. Microscopically, there were multifocal granulomas with intralesional P. insidiosum hyphae and Splendore-Hoeppli material surrounding the hyphae. The diagnosis was based on the histologic lesions, immunohistochemical identification, and culture of the etiologic agent. One sheep treated with potassium iodide recovered. Standing in swampy water for long periods in a warm aquatic environment seems to be the reason for the high prevalence of the disease.
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              Identification of emerging human-pathogenic Pythium insidiosum by serological and molecular assay-based methods.

              Pythium insidiosum is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of P. insidiosum. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the P. insidiosum isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described P. insidiosum DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for P. insidiosum was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.
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                Author and article information

                Journal
                pvb
                Pesquisa Veterinária Brasileira
                Pesq. Vet. Bras.
                Colégio Brasileiro de Patologia Animal - CBPA (Rio de Janeiro, RJ, Brazil )
                0100-736X
                1678-5150
                March 2006
                : 26
                : 1
                : 47-50
                Affiliations
                [03] orgnameUFSM orgdiv1Curso de Medicina Veterinária
                [02] orgnameUFSM orgdiv1Pesquisador do Laboratório de Pesquisas Micológicas
                [01] Santa Maria RS orgnameUniversidade Federal de Santa Maria orgdiv1Depto Microbiologia
                Article
                S0100-736X2006000100010 S0100-736X(06)02600110
                6d7a5915-0279-4080-836e-cf4a5ee6ca99

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

                History
                : 07 November 2005
                : 21 June 2005
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 26, Pages: 4
                Product

                SciELO Brazil

                Self URI: Texto completo somente em PDF (PT)

                ELISA,horses,rabbits,Pythium insidiosum,pitiose,eqüinos,pythiosis
                ELISA, horses, rabbits, Pythium insidiosum, pitiose, eqüinos, pythiosis

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