Development of a bioinformatics pipeline for the identification of emerging infectious diseases in patients submitted to chronic transfusion

O risco do surgimento de novas doenças infecciosas tem sido um fator de preocupação há várias décadas. Atualmente, diversas condições como urbanização, desflorestamento, aumento da mobilidade aérea e alta densidade de criação de animais para abate são alguns dos fatores que corroboram para surgimento de doenças infecciosas emergentes em um espaço de tempo cada vez menor. Esse risco potencial é ainda mais acentuado nas regiões equatoriais e tropicais do globo, onde os vírus de origem zoonótica possuem maiores chances de contato com a população humana, desafiando medidas de biossegurança de diversas áreas da saúde, inclusive de bancos de sangue, devido à possibilidade de transmissão transfusional desses agentes. Portanto, esse estudo visa a análise do viroma de pacientes com hemoglobinopatias submetidos a um regime transfusional crônico e hemofilia utilizando uma pipeline de bioinformática para a análise computacional de dados de Sequenciamento de Nova Geração (SNG). Esses pacientes possuem alto risco de aquisição de doenças transmissíveis via transfusão devido ao alto volume de sangue que recebem durante o tratamento clínico. A pipeline utilizada demonstrou êxito na análise bioinformática contendo os seguintes programas para cada um dos respectivos processos: Controle de qualidade (FastQC), Filtragem (Trimmomatic, Prinseq, Deconseq), Classificação e montagem (Kraken 2, SPAdes, BLASTn, BLASTx). Os agentes mais importantes identificados foram classificados utilizando a análise Filogenética. Nesse projeto foram avaliadas 75 amostras, provenientes de pacientes com hemoglobinopatias (30 de pacientes com beta-talassemia e 45 com anemia falciforme) submetidos a regime de transfusão crônica e 5 amostras de pacientes com hemofilia tratados com fatores plasmáticos. Como controle testamos 76 amostras de doadores de sangue. A análise metagenômica identificou vírus pertencentes a duas famílias virais principais: Anneloviridae e Flaviviridae. Os anelovírus foram os vírus mais abundantes nos pacientes com beta-talassemia. Em segundo lugar nós conseguimos identificar o flavivírus pegivirus humano 1 (HPgV-1, GBV-C ou vírus da hepatite G). No grupo de pacientes com beta-talassemia conseguimos identificar viremia por Hepatite B e Hepatite C. No grupo de pacientes com anemia falciforme, o vírus HPgV-1 foi o mais representativo. Também em pacientes com anemia falciforme conseguimos identificar contaminantes do sequenciamento como plasmídeos, correspondendo majoritariamente a vetores de clonagem utilizados na prática laboratorial. A abundância viral foi semelhante entre doadores de sangue e pacientes que recebem hemocomponentes o que sugere que o surgimento de vírus que podem ameaçar a segurança transfusional é um evento de baixa probabilidade, porém a análise metagenômica pode ser utilizada para identificar futuros agentes que podem ter impacto na hemoterapia.The emergence of new infectious diseases has been a factor of concern for several decades. Currently, many conditions, such as the high density of animals for meat consumption, facilitated aerial mobility and the rate of deforestation, corroborate to a reality where new diseases may emerge in increasingly short periods of time, especially in countries located in the tropical regions, where viruses of zoonotic origin have even more favorable conditions to emerge due to the very close contact between the population and the environment. In this scenario, the threat of the emergence of these viruses defies biosafety measures in health systems, including blood banks, due to the possibility of transfusion transmission of these agents. This study aims to develop and improve a pipeline for computational analysis of Next Generation Sequencing (SNG) data from patients with hemoglobinopathies undergoing chronic transfusion regimen. The pipeline introduced demonstrated success in fulfilling three main steps for bioinformatics analysis with the subsequent programs used: Quality control (FastQC), Filtration (Trimmomatic, Prinseq, Deconseq), Classification and assembly (Kraken 2, CLARK, SPAdes, BLASTn, BLASTx ) and Phylogenetic Analysis (MAFFT, IQ-Tree, Tree-Puzzle, Bioedit, FigTree). In this project, 75 samples were evaluated, from patients with hemoglobinopathies (30 from patients with beta-thalassemia and 45 with sickle cell anemia) who underwent a chronic transfusion regimen, 76 samples from blood donors as a control group, and 5 samples from patients with hemophilia treated with plasma factors. The implemented pipeline was able to identify two predominant viral families: Anneloviridae and Flaviviridae. Firstly, anelloviruses were the most representative viruses in patients with beta-thalassemia. Second, we were able to identify the human pegivirus flavivirus 1 or HPgV-1 (formerly referred to as GBV-C or hepatitis G virus). In the group of patients with beta-thalassemia, we were able to identify Hepatitis B and Hepatitis C viruses. In the group of patients with sickle cell anemia, the HPgV-1 virus was the most representative. It was possible to identify agents of contaminating origin as plasmids, mostly corresponding to cloning vectors used in the laboratory. Phylogenetic analysis confirmed the most prevalent genotypes of each of the identified viral groups. There were no significant differences between the viral families identified in the groups of poly-transfused patients and blood donors

Deep sequencing applied to the analysis of viromes in patients with beta-thalassemia

International audienceTo date, blood banks apply routine diagnosis to a specific spectrum of transfusion-transmitted viruses. Even though this measure is considered highly efficient to control their transmission, the threat imposed by emerging viruses is increasing globally, which can impact transfusion safety, especially in the light of the accelerated viral discovery by novel sequencing technologies. One of the most important groups of patients, who may indicate the presence of emerging viruses in the field of blood transfusion, is the group of individuals who receive multiple transfusions due to hereditary hemoglobinopathies. It is possible that they harbor unknown or unsuspected parenterally-transmitted viruses. In order to elucidate this, nucleic acids from 30 patients with beta-thalassemia were analyzed by Illumina next-generation sequencing and bioinformatics analysis. Three major viral families: Anelloviridae, Flaviviridae and Hepadnaviridae were identified. Among them, anelloviruses were the most representative, being detected with high number of reads in all tested samples. Human Pegivirus 1 (HPgV-1, or GBV-C), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) were also identified. HBV and HCV detection was expected due to the high seroprevalence in patients with beta thalassemia. Our results do not confirm the presence of emerging or unsuspected viruses threatening the transfusion safety at present, but can be used to actively search for viruses that threaten blood transfusion safety. We believe that the application of viral metagenomics in multiple-transfused patients is highly useful to monitor possible viral transfusion threats and for the annotation of their virome composition