Investigação das características moleculares e filogenéticas dos isolamentos de Pythium insidiosum em casos de ceratite / Investigation of the molecular and phylogenetic characteristics of Pythium insidiosum isolates in cases of keratitis

Introdução: Pythium insidiosum é um microrganismo fenotipicamente semelhante aos fungos com hifas, mas este oomyceto pertencente ao filo Straminofila, filogeneticamente separado dos fungos verdadeiros. Usando metodologias moleculares, isolados de P. insidiosum foram colocados em três grupos filogenéticos de acordo com sua distribuição geográfica. Mais recentemente, quatro grupos filogenéticos foram relatados. Pythium insidiosum é capaz de causar infecções graves em humanos. Pode acometer os olhos na forma de ceratites e infecções dos tecidos adjacentes que ameaçam não apenas a visão, mas também a vida do paciente. Devido ao desenvolvimento de hifas, frequentemente a infecção por esse patógeno é confundida e diagnosticada erroneamente como infecção fúngica. Objetivo: Investigar as características moleculares e filogenéticas dos isolamentos de P. insidiosum recuperados de quatorze amostras coletadas na Índia em casos de ceratite. Materiais e Métodos: Foram avaliados quatorze isolamentos de P. insidiosum dos quais foram extraídos o DNA e, então, realizados PCR com primers universais. Prosseguiu-se com amplificação das sequências utilizando Internal Transcriber Spacers (ITS) e Citocromo C Oxidase subunidade II (COXII). As análises filogenéticas subsequentes, utilizando as sequências de ITS e COXII, foram analisadas pelo método estatístico Neighbor Joining (MEGAX). Resultados: As árvores filogenéticas usando as sequências de DNA amplificadas (ITS e COXII) mostraram resultados análogos. As sequências provenientes dos quatorze isolamentos analisados foram especificamente colocadas, com alto suporte de bootstrap, no grupo II (Ásia, Austrália e África) e no grupo IV (Tailândia, Israel), de acordo com a divisão atual de P. insidiosum. Do total dos quatorze isolamentos, dez foram colocados no grupo II e quatro no grupo IV. Interessantemente, nenhum dos isolamentos foi colocado nos grupos I e III (Américas). Foi possível comprovar que os dez isolados neste estudo foram identificados como P. insidiosum comprovando a hipótese inicial. No entanto, como foi postulado anteriormente, ao avaliar as quatro sequências localizadas no grupo IV a existência de uma espécie inteiramente nova é fortemente suportada. Para confirmar essa hipótese, são necessários novos estudos baseados em análises estatísticas. Conclusão: Pacientes com ceratite resistente ao tratamento convencional, de rápida evolução, com cultura de padrão de crescimento com presença de micélios submersos aderidos ao ágar devem alertar sobre a possibilidade de infecção por P. insidiosum. Para a confirmação de possíveis isolados de P. insidiosum, técnicas moleculares devem ser realizadas. Esses foram os passos seguidos no presente estudo tornando possível a identificação do agente etiológico das ceratites da Índia como P. insidiosum. Além disso, as técnicas filogenéticas usadas neste e em outros estudos, sugeriram a possibilidade de uma nova espécie dentro do complexo P. insidiosum, o que provavelmente é uma das contribuições mais importantes deste estudo.

Introduction: Pythium insidiosum is a microorganism phenotypically like fungi with hyphae, but this oomycete belongs to the phylum Straminopila phylogenetically away from true fungi. Using molecular methodologies, isolates of P. insidiosum were placed in three phylogenetic groups according to their geographic distribution. More recently, four phylogenetic groups were reported. Pythium insidiosum can cause serious infections in humans. It can affect the eyes in the form of keratitis and infections of adjacent tissues that threaten not only the eyesight, but also the patient's life. Due to the development of hyphae, infection with this pathogen is often mistaken and misdiagnosed as a fungal infection. Objective: To investigate the molecular and phylogenetic characteristics of the isolates of P. insidiosum recovered from fourteen samples collected in Indian cases of keratitis. Materials and Methods: Fourteen P. insidiosum isolates were evaluated, from which DNA was extracted and then PCR with universal primers was performed. PCR amplification was performed using Internal Transcriber Spacers (ITS) and Cytochrome C Oxidase S subunit II (COXII) primers and subsequently sequenced. Phylogenetic analyzes, using the aligned ITS and COXII sequences, were analyzed by the Neighbor Joining method (MEGAX). Results: Phylogenetic trees using the amplified DNA sequences (ITS and COXII) showed similar results. The DNA sequences from the fourteen analyzed isolates were specifically placed, with high bootstrap support, in group II (Asia, Australia and Africa) and in group IV (Thailand, Israel), according to the current phylogenetic cluster classification of P. insidiosum. Of the fourteen isolates, ten were placed in cluster II, and four of them in cluster IV. Interestingly, none of the isolations were placed in clusters I and III (The Americas). The ten isolates in this study were identified as P. insidiosum, which support the initial hypothesis. As was previously postulated, this study found that the four Indian DNA sequences located in cluster IV represent an entirely new specie. To confirm this finding, further studies based on statistical analysis are needed. Conclusion: Patients with keratitis of rapid clinical progression, resistant to conventional treatment, with the presence of submerged mycelia adhered to the agar should alert clinicians about the possibility of infection caused by P. insidiosum. To identify possible P. insidiosum isolates, molecular techniques must be performed. Molecular and phylogenetic analyses were used in the present study making it possible to identify the etiological agent of keratitis in India as P. insidiosum. In addition, phylogenetic analyses in this and other studies, suggested the possibility of a new species within P. insidiosum complex, which is probably one of the most important contributions of this study.

Genetic studies on the Cayo Santiago rhesus macaques: A review of 40 years of research.

Genetic studies not only contribute substantially to our current understanding of the natural variation in behavior and health in many species, they also provide the basis of numerous in vivo models of human traits. Despite the many challenges posed by the high level of biological and social complexity, a long lifespan and difficult access in the field, genetic studies of primates are particularly rewarding because of the close evolutionary relatedness of these species to humans. The free-ranging rhesus macaque (Macaca mulatta) population on Cayo Santiago (CS), Puerto Rico, provides a unique resource in this respect because several of the abovementioned caveats are of either minor importance there, or lacking altogether, thereby allowing long-term genetic research in a primate population under constant surveillance since 1956. This review summarizes more than 40 years of genetic research carried out on CS, from early blood group typing and the genetic characterization of skeletal material via population-wide paternity testing with DNA fingerprints and short tandem repeats (STRs) to the analysis of the highly polymorphic DQB1 locus within the major histocompatibility complex (MHC). The results of the paternity studies also facilitated subsequent studies of male dominance and other factors influencing male reproductive success, of male reproductive skew, paternal kin bias, and mechanisms of paternal kin recognition. More recently, the CS macaques have been the subjects of functional genetic and gene expression analyses and have played an important role in behavioral and quantitative genetic studies. In addition, the CS colony has been used as a natural model for human adult-onset macular degeneration, glaucoma, and circadian rhythm disorder. Our review finishes off with a discussion of potential future directions of research on CS, including the transition from STRs to single nucleotide polymorphism (SNP) typing and whole genome sequencing.