ABSTRACT
Cryptosporidium is a leading cause of waterborne outbreaks globally, and Cryptosporidium hominis and C. parvum are the principal cause of human cryptosporidiosis on the planet. Thanks to the advances in Next-Generation Sequencing (NGS) sequencing and bioinformatic software development, more than 100 genomes have been generated in the last decade using a metagenomic-like strategy. This procedure involves the parasite oocyst enrichment from stool samples of infected individuals, NGS sequencing, metagenomic assembly, parasite genome computational filtering, and comparative genomic analysis. Following this approach, genomes of infected individuals of all continents have been generated, although with striking different quality results. In this study, we performed a thorough comparison, in terms of assembly quality and purity, of 100+ de novo assembled genomes of C. hominis. Remarkably, after quality genome filtering, a comprehensive phylogenomic analysis allowed us to discover that C. hominis encompasses two lineages with continental segregation. These lineages were named based on the observed continental distribution bias as C. hominis Euro-American (EA) and the C. hominis Afro-Asian (AA) lineages.
ABSTRACT
Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.
Isolados de Cryptosporidium identificados em quatorze amostras de fezes, coletadas de cinco pacientes com infecção por HIV e de nove crianças imunocompetentes, residentes no estado de São Paulo, Brasil, foram submetidos a análise molecular por Nested-PCR, seguido da caracterização genética por polimorfismo do tamanho do fragmento de restrição (RFLP). A análise foi baseada na digestão, com a enzima de restrição RsaI, de um fragmento de DNA amplificado do gene que codifica a proteína de parede do oocisto de Cryptosporidium (COWP). Baseado nesta análise, quando comparadas aos padrões utilizados, quatro amostras foram identificadas como Cryptosporidium parvum, oito como Cryptosporidium hominis e duas apresentaram um perfil correspondente ao de Cryptosporidium meleagridis. O uso de métodos moleculares pode ser útil para identificar a fonte das infecções e os fatores de risco relacionados à infecção por Cryptosporidium em nossas comunidades.