Applicability of the Platelia EIA® Aspergillus test for the diagnosis of aspergilosis in penguins / Aplicabilidade do Platelia EIA® Aspergillus como teste diagnostico da aspergilose em pinguins

Abstract Even today, an effective diagnostic test for aspergillosis in penguins is unknown, being the gold standard post-mortem examinations. The fungal antigen galactomannan (GM) has been used as a biomarker of disease in humans and is detected by the Platelia Aspergillus EIA (BioRad)®, a commercial kit based on the sandwich ELISA technique. It is standardized for use in neutropenic patients, however studies have demonstrated its usefulness also possible for birds. The aim of our study was to evaluate the effectiveness of Platelia Aspergillus EIA® test (BioRad-US) in the diagnosis of aspergillosis in Magellanic penguins, determining sensitivity, specificity, and positive and negative predictive values for different cut-off points. Were included in the study, blood serum samples (n = 29) Magellanic penguins in captivity that died by aspergillosis. Detection of GM was performed following manufacturer's instructions and the GM index was obtained by dividing the average value of OD of the duplicate of the clinical sample by duplicate OD of the average value of the cut-off sample provided by the kit. Through information database results were obtained for the presence of anti-Aspergillus fumigatus antibodies detected by agar gel immunodiffusion (AGID) for all serum samples. Results were analyzed using chi-square test and Kruskal-Wallis from SPSS 20.0, IBM®. ROC curve was obtained and from this, rates of sensitivity, specificity, positive and negative predictive values were also calculated based on four different cutoff points (0.5, 1.0, 1.5 and 2.0). The serum GM index did not differ between animals of the case and control group (pkw =0.097). In determining the ROC curve for serum GM detection the value of area under the curve was 0.635. From the values ​​determined by the coordinate of the curve, four different cut points (0.5, 1.0, 1.5 and 2.0) were analyzed, resulting in sensitivity rates ranging from 86.2 to 34.5% % and specificity between 87% and 26.1%. By comparing the serum GM index in group case as the presence or absence of antibodies detected by AGID was found p=0.503. The detection of GM the Platelia Aspergillus EIA® test seems is not be useful for the diagnosis of aspergillosis in naturally infected penguins.

Resumo Ainda hoje, um teste diagnóstico eficaz para aspergilose em pinguins não é conhecido, sendo o padrão-ouro os exames post-mortem. O antígeno fúngico galactomanana (GM) tem sido utilizado como biomarcador da doença em humanos, sendo detectado pelo Platelia Aspergillus EIA (BioRad)®, um kit comercial que se baseia na técnica ELISA sanduíche. É padronizado para utilização em pacientes neutropênicos, no entanto estudos tem demonstrado sua possível utilidade também para aves.O objetivo de nosso estudo foi avaliar a eficácia do teste Platelia Aspergillus EIA® (BioRad-US) no diagnóstico da aspergilose em pinguins-de-Magalhães, determinando sensibilidade, especificidade e valores preditivos positivos e negativos em diferentes pontos de corte. Foram incluídas no estudo, amostras de soro sanguíneo (n=29) de pinguins-de-Magalhães em cativeiro que vieram a óbito por aspergilose. A detecção de GM foi realizada seguindo instruções do fabricante e o índice de GM foi obtido dividindo o valor da média da DO da duplicata da amostra clínica pelo valor da média da DO da duplicata da amostra de cut-off fornecida pelo kit. Através de informações em banco de dados foram obtidos resultados sobre a presença de anticorpos anti-Aspergillus fumigatus, detectada por Imunodifusão em gel de ágar (IDGA) em todas as amostras séricas. Os resultados foram analisados utilizando-se teste de qui-quadrado e Kruskal-Wallis a partir do programa estatístico SPSS 20.0, IBM®. Curva ROC foi obtida e a partir desta, taxas de sensibilidade, especificidade, valores preditivo positivo e negativo foram igualmente calculados considerando quatro diferentes pontos de corte (0.5, 1.0, 1.5 e 2.0). O índice de GM sérica não diferiu entre os animais do grupo caso e controle (pKW = 0.097). Na determinação da curva ROC para detecção de GM sérica o valor da área sobre a curva foi de 0.635. A partir dos valores determinados pelas coordenadas da curva, quatro diferentes pontos de corte (0.5, 1.0, 1.5 e 2.0) foram analisados, resultando em taxas de sensibilidade variando de 86.2% a 34.5%, e de especificidade entre 87% e 26.1%. Ao comparar o índice de GM sérica nos animais do grupo caso quanto a presença ou não de anticorpos detectados pela IDGA foi encontrado p=0.503. A detecção de GM pelo teste Platelia Aspergillus EIA® não parece ser útil para o diagnóstico da aspergilose em pinguins naturalmente infectados.

Applicability of the Platelia EIA® Aspergillus test for the diagnosis of aspergilosis in penguins.

Even today, an effective diagnostic test for aspergillosis in penguins is unknown, being the gold standard post-mortem examinations. The fungal antigen galactomannan (GM) has been used as a biomarker of disease in humans and is detected by the Platelia Aspergillus EIA (BioRad)®, a commercial kit based on the sandwich ELISA technique. It is standardized for use in neutropenic patients, however studies have demonstrated its usefulness also possible for birds. The aim of our study was to evaluate the effectiveness of Platelia Aspergillus EIA® test (BioRad-US) in the diagnosis of aspergillosis in Magellanic penguins, determining sensitivity, specificity, and positive and negative predictive values for different cut-off points. Were included in the study, blood serum samples (n = 29) Magellanic penguins in captivity that died by aspergillosis. Detection of GM was performed following manufacturer's instructions and the GM index was obtained by dividing the average value of OD of the duplicate of the clinical sample by duplicate OD of the average value of the cut-off sample provided by the kit. Through information database results were obtained for the presence of anti-Aspergillus fumigatus antibodies detected by agar gel immunodiffusion (AGID) for all serum samples. Results were analyzed using chi-square test and Kruskal-Wallis from SPSS 20.0, IBM®. ROC curve was obtained and from this, rates of sensitivity, specificity, positive and negative predictive values were also calculated based on four different cutoff points (0.5, 1.0, 1.5 and 2.0). The serum GM index did not differ between animals of the case and control group (pkw =0.097). In determining the ROC curve for serum GM detection the value of area under the curve was 0.635. From the values ​​determined by the coordinate of the curve, four different cut points (0.5, 1.0, 1.5 and 2.0) were analyzed, resulting in sensitivity rates ranging from 86.2 to 34.5% % and specificity between 87% and 26.1%. By comparing the serum GM index in group case as the presence or absence of antibodies detected by AGID was found p=0.503. The detection of GM the Platelia Aspergillus EIA® test seems is not be useful for the diagnosis of aspergillosis in naturally infected penguins.

Study of bacteria isolated from the foot pad of Spheniscus magellanicus with and without bumblefoot / Estudo de bactérias isoladas de coxins plantares de Spheniscus magellanicus com e sem bumblefoot

The bumblefoot or pododermatitis is among the diseases with the highest morbidity in Magellanic penguins, sometimes evolving to septicemia and death. Therefore, this study aimed to relate the main species involved in the disorder, as well as the in vitro susceptibility profile of the microorganisms against routine antimicrobial usage in Veterinary Medicine. During two years in vivo material was harvested from 200 footpads (n=100 animals) for microbiological analysis and in vitro susceptibility tests against the Antibiotic enrofloxacin, streptomycin, penicillin and cephalosporin. Bacteria have been identified both as part of permanent and transient microbiota, also being associated to 100% of the pododermatitis cases. The most prevalent genus were Staphylococcus and Corynebacterium. The antibiograms of all the isolated bacteria resulted in greater susceptibility of the strains facing cephalosporin, followed by enrofloxacin, streptomycin and penicillin.

O bumblefoot ou pododermatite está entre as afecções de maior morbidade em pinguins-de-magalhães, podendo evoluir para septicemia e óbito. Portanto, o presente estudo objetivou relacionar as principais espécies bacterianas envolvidas na afecção, bem como o perfil de susceptibilidade in vitro destes microrganismos frente a antimicrobianos de uso rotineiro em medicina veterinária. Durante o período de dois anos, foi realizada colheita de material in vivo de 200 coxins plantares (n=100 animais) para análise microbiológica e testes de susceptibilidade in vitro frente aos antibióticos enrofloxacina, estreptomicina, penicilina e cefalosporina. Bactérias foram identificadas tanto como parte da microbiota permanente quanto da transitória, bem como estiveram associadas a 100% dos casos de pododermatite. Os gêneros mais prevalentes foram Staphylococcus e Corynebacterium. Os antibiogramas de todas as bactérias isoladas resultaram em maior sensibilidade das cepas frente à cefalosporina, seguida de enrofloxacina, estreptomicina e penicilina.