Avaliação do teste Platelia EIA® Aspergillus para diagnóstico da aspergilose em pinguins e interferência da contaminação ambiental

Abstract

Aspergillosis is a non-contagious infectious disease caused by fungi of the genus Aspergillus, especially those belonging to the Fumigati section and has been describe in several species, among them penguins. The high mortality rates attributed to this disease are due in particular to the difficulty of performing an early diagnosis. Direct diagnostic techniques for the detection of galactomannan (GM) fungal antigen in clinical samples have been increase used for the diagnosis of invasive aspergillosis (AI) in humans. GM is a polysaccharide present in the cell wall of fungi of the genus Aspergillus. Although its applicability from the demonstration of circulating GM kinetics is already establish for the early diagnosis of AI in humans, a single serum test by Platelia® Aspergillus EIA should be interpreted with caution, since the technique has some limitations. In animals, its applicability is still not well understand. The objective of this study was to evaluate the effectiveness of the Platelia EIA® Aspergillus test for the diagnosis of aspergillosis in penguins and the influence of environmental contamination on the results. Serological samples of 29 Magellanic penguins that died of aspergillosis (case group) and 23 healthy (control group) were included in the diagnostic study, which were tested from the micro plate sandwich immunoenzymatic assay kit (Platelia Aspergillus EIA ®, Bio-Rad) according to the manufacturer's instructions. For the environmental interference experiment, 12 isolates of A. fumigatus from the mycology laboratory of the FURG / FAMED School of Medicine including standard strains, clinical isolates of penguins, humans and environmental isolates were use. The filtration, sedimentation, Pour-plate and spectrophotometry steps was use to standardize the inoculum according to CLSI, 2008. GM detection was perform on three successive dilutions of the inoculums following the manufacturer's instructions. At the end, the GM index was calculated by dividing the mean OD value of the sample duplicate (clinical or strain tested) by the mean OD value of the duplicate cut-off sample provided by the kit. The serum GM index did not differ between the animals in the control and the case group (pKW = 0.097). From the values determined by the coordinates of the ROC curve, four different cutoff 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%. In the evaluation of environmental interference, the lowest concentration of fungal propagates of Aspergillus fumigatus capable of generating a positive result was 480 conidia, with a median concentration of 4.8x103. It is conclude that the serum detection of GM by the Platelia Aspergillus EIA® test does not seem to be useful for the diagnosis of aspergillosis in naturally infected penguins, resulting in many falsepositives. And, in relation to the amount of A. fumigatus conidia required to determine false-positive results in the GM detection test for AI diagnosis, it was shown that the degree of environmental contamination capable of causing interference in the Platelia Aspergillus EIA® test Be robust, of about 500 conidia.