Investigação de casos de doenças febris agudas e doenças neurológicas graves com etiologia desconhecida por sequenciamento metagenômico por nanoporos

Abstract

Acute Febrile Illness (AFI) and Severe Neurological Diseases (SND) pose significant diagnostic challenges due to the diversity of infectious agents that can cause them and the limitations of conventional diagnostic tests, which are often negative. In view of this, nanopore metagenomic sequencing has emerged as a promising tool for identifying the etiological agents of unknown infectious diseases, offering a variety of strategies for analyzing and characterizing these agents. The aim of this study was to identify the etiological agents associated with cases of hospitalization for AFI and SND in the Eduardo de Menezes Hospital, Belo Horizonte, Minas Gerais. Biological samples from 16 hospitalized patients were analyzed. These samples were subjected to two different protocols: shotgun sequencing and RNA enrichment using the SMART-9N protocol, followed by nanopore sequencing. Subsequent steps included quality control, removal of host genes, de novo assembly of the genome to form contigs, two taxonomic classifications to minimize false positives and phylogenetic analysis. In cases of AFI, we identified a spectrum of disease-associated microbes including Escherichia coli, Streptococcus sp., Human Immunodeficiency Virus 1 (Subtype B), and Human Pegivirus (Genotype 2B). In DNG cases, the pathogens identified included Escherichia coli, Clostridium sp. and dengue virus type 2 (Genotype-II lineage). This study demonstrates the effectiveness and speed of metagenomic analysis, highlighting its ability to identify pathogens associated with various diseases, the feasibility of recovering almost complete genomes of RNA viruses and the importance of this technique for more accurate diagnoses and personalized treatments.