Padronização e aplicação de uma reação em cadeia da polimerase espécie-específica para diferenciação entre as espécies Ascaris lumbricoides e Ascaris suum

Abstract

Ascaris lumbricoides and A. suum are described as the species of Ascaris infecting humans and pigs, respectively. It is estimated that about 447 million people are infected with A. lumbricoides, being predominant in children living in developing countries, characterizing a serious economic and public health problem in the affected countries. However, there is an increasing number of cases of human ascariasis, even in countries whose rates for this parasitosis were low or even zero. In these locations, pigs have been blamed as the main source of infection for humans. Despite the adult forms of both species presenting subtle morphological differences, obtaining is not trivial. In addition, parasitological tests, commonly used in clinical practice, are unable to differentiate Ascaris species from the eggs found in the feces. However, molecular techniques have been proposed to carry out such differentiation. Therefore, the present study aimed to standardize and apply a species-specific polymerase chain reaction (PCR) assay, based on the allele-specific PCR (AS-PCR) methodology, with the Transcribed Internal Space as a molecular target 1 (ITS-1) of ribosomal DNA for scanning and identification of A. lumbricoides and A. suum species from the egg. The positive controls were synthesized by means of conventional PCR and cloning for standardization of the technique and use in reactions. Positive stool samples were obtained from 68 patients from seven Brazilian states to identify the species. Stool samples from six pigs from two farms in Minas Gerais were also used as a way of validating the technique. After the recovery and incubation processes of Ascaris eggs, single egg DNA was used in species-specific PCR. After the reactions, all samples obtained from humans were genotyped as A. lumbricoides and all samples obtained from pigs were genotyped as A. suum. A percentage (2.9%) of these samples were sequenced for validation of the technique. The results found were consistent with the current literature, which demonstrates that in endemic regions the transmission cycles are separated, although the presence of a limited gene flow between species is assumed. Thus, the execution of this work enabled the standardization of a useful methodology for the differential diagnosis of species, contributing to the characterization of the real epidemiological profile of human and swine ascariasis in Brazil. The data presented here can assist in the implementation of future control strategies.