EVALUATION OF VIRULENCE OF ISOLATES OF Acanthamoeba GENOTYPES T3, T4 AND T5 FROM CLINICAL AND ENVIRONMENTAL SAMPLES
Name: DÉBORA DA VITORIA DE MELO FERNANDES
Publication date: 23/03/2018
Examining board:
Name |
Role |
|---|---|
| BLIMA FUX | Examinador Interno |
| CINTHIA FURST LEROY GOMES BUELONI | Coorientador |
| GUSTAVO ROCHA LEITE | Examinador Externo |
Summary: The genus Acanthamoeba comprises protozoa that are widely distributed in the most diverse environments and on all continents and that are capable of causing infections in humans, such as keratitis and granulomatous encephalitis. The pathogenesis of Acanthamoeba is a multifactorial process, with factors that involve both the amoeba and the host, however, this pathogenicity mechanism has not yet been fully elucidated. The objective of this study was to identify the virulence of six isolates of Acanthamoeba of clinical and environmental origins, with three different genotypes, T3, T4 and T5 – representing the 20 types currently discovered, and with two doses of amoeba on three different types of mammalian cell lines, MDCK, VERO and CHO, for cytotoxic and cytopathic effect tests. Clinical samples from corneal scraping cultures of patients diagnosed with amoebic keratitis and environmental samples from tap water, floodwater and dust were collected and axenized between 2014 and 2017. Considering that prolonged cultivation of Acanthamoeba isolates can cause a decrease or possible loss of virulence, the amoebas were passaged in an MDCK cell line to reactivate the virulence of the isolates in prolonged culture. The cytotoxic effect demonstrated that there are differences in results depending on the cell line used, but not on the isolates. Passage in an MDCK cell line was able to cause an increase in the virulence of the isolates Mnus4 (T3-environmental), Krt15.DFNL (T3-clinical), Krt12.ROS (T4-clinical) and Krt16.PEN (T5-clinical) in cytotoxicity tests. Our results demonstrated that the cell line most susceptible to the conditioned medium (cytotoxic effect) was MDCK, followed by VERO and CHO. However, for the cytopathogenicity test, CHO was the most susceptible to exposure to Acanthamoeba trophozoites, followed by VERO and MDCK. In the cytopathic effect, the results varied according to the isolate used, the dose of trophozoites used and the passage in the cell line. The increase in the number of amoeba trophozoites in incubation with the cell lines was able to cause an increase in the virulence of the isolates of the three genotypes in the cytopathic effect test, mainly on the VERO and CHO lines. In the cytopathic effect test, the most virulent genotype was T5, followed by T4 and T3, with the most virulent isolate being A3P4 (T5) of environmental origin. Thus, it is concluded that the results obtained with the cytopathic and cytotoxic effect tests with the different mammalian cell lines have variations related to the type of mammalian cell line used, as well as the inherent characteristics of each isolate. Thus, the results obtained here may be useful for planning future research related to studies of the pathogenicity of Acanthamoeba.
