Caracterização de múltiplos polimorfismos de Pneumocystis Jirovecii por multiplex-PCR/Single base extension

Characterization of multiple polymorphisms in Pneumocystis jirovecii by Multiplex-PCR/Single Base Extension Camila Borges Fernandes Pneumocystis jirovecii is known for causing specific infections in the respiratory tract of humans, mostly in immunocompromised patients, leading to a severe, and often fatal, pneumonia known as Pneumocystis pneumonia. The characterization of the genetic diversity of P. jirovecii has shown that some specific single nucleotide polymorphisms have been recognized as the molecular markers of choice to study the geographical distribution, modes of transmission, drug susceptibility/resistance, virulence factors and population genetics of specific genetic subtypes. The gold of the present work was to characterize P. jirovecii polymorphisms, using the methodology Multiplex PCR/Single Base Extension, having as main purpose, the detection of possible associations between single nucleotide polymorphisms and multilocus genotypes with demographic and clinical data of infection by P. jirovecii. Sixty six pulmonary specimens previously identified as positive for P. jirovecii, collected between 2001 and 2012, from immunocompromised portuguese patients were randomly selected and included in this multilocus study. Multiplex-PCR was used for simultaneous amplification of tree genomic regions: mitochondrial large-subunit rRNA, superoxide dismutase and dihydropteroate synthase. Five single nucleotide polymorphisms correlated previously with parameters of disease were genotyped by Single Base Exrension: mt85, SOD110, SOD215, DHPS165 and DHPS171. A total of 330 single nucleotide polymorphisms and 29 potential P. jirovecii multilocus genotypes were identified and characterized in the samples studied. The polymorphisms distribution patterns were analyzed and the temporal and/or geographic variation of their allelic forms have been considered. There was a large genotypic diversity among isolates of P. jirovecii that may have influenced the epidemiology of infection. Statistical associations between mt85/multilocus genotypes and some demographic and clinical informations were observed. The most relevant correlation was observed between mt85C with low to moderate parasite load, whilst mt85T was associated with high parasite load. MLG5, MLG9 and MLG13 were associated with low, moderate and high parasite load, respectively. Such associations demonstrate that optimal molecular markers of infection by P. jirovecii exist and specific polymorphisms/genotypes may determine specific epidemiological profiles of Pneumocystis pneumonia. The cross-genetic analysis demonstrated statistically significant associations among single nucleotide polymorphisms. The polymorphisms SOD110T and SOD215C, SOD110C and SOD215T, DHPS165A and DHPS171C, DHPS165G and DHPS171T, were statistically associated. The most prevalent multilocus genotypes identified were considered in the recombination test d1. Two multilocus genotypes (MLG7 and MLG9) were observed in higher frequency, and the genetic analysis indicated at these multilocus genotypes are over-represented in the population of P. jirovecii studied. These evidences indicate that linkage disequilibrium and clonal propagation of genetic genotypes is common in this population, considering that the species P. jirovecii could be represented by an epidemic population structure. The present work confirmed the importance of the study of polymorphisms in P. jirovecii, suggesting that the multilocus characterization can provide useful information to understanding the patterns, causes and control of infection, enhancing the research of this important pathogen.