Using Next Generation Sequencing to study drug resistence malaria

• Autor(a) principal: DIAS, Brigite Maria Leitão
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/146195
• topic: Ciências biomédicas; Microbiologia médica; Biologia molecular; Malária; Domínio/Área Científica::Ciências Médicas

Malaria, a mosquito-borne disease caused by Plasmodium parasites, is a major public health threat. There are five Plasmodium species that cause malaria in humans, but P. falciparum causes the majority of mortality and morbidity. There were 241 million cases and 627,000 deaths reported in 2020 alone. Global malaria incidence rates have fallen in the last decade; however, progress has recently levelled and has reversed in some regions. Malaria control efforts focus on rapid diagnosis, effective treatments, and preventive medicine in susceptible groups, as well as vector control strategies. On October 2021, WHO recommended the use of the malaria vaccine RTS,S/AS01, to reduce severe malaria. The rapid emergence and spread of parasites resistant to antimalarial drugs is threatening the ability to treat malaria effectively. Artemisinin-based combination therapy (ACT) has replaced older antimalarial drugs and is the mainstay for malaria treatment in endemic regions. ACT has contributed significantly to the reduction in mortality. Temporal and geographical surveillance of antimalarial drug resistance, including geographic spreading of resistant parasites, requires high throughput SNP-profiling. Target amplicon together with next-generation sequencing (NGS) is particularly suited as a high throughput methodology for surveillance of antimalarial drug resistance SNPs. This methodology allows the selection of all relevant SNPs in target genes for parasite genetic barcoding and drug resistance profiling, as well as tracking of sequences pertaining to a specific infection. This project aims to use target amplicon sequencing combined with Illumina NGS technology to characterize parasites from patients infected with P. falciparum. The data generated were combined with those from global collections to characterize the genetic diversity of genes associated with antimalarial drug resistance in P. falciparum. Through the multiplex PCRs and NGS technology, eight samples from Mozambique were analysed for the pfmdr1 and pfk13 genes. The pfmdr1 Y184F and pfk13 571L mutations were identified in five and two samples, respectively. The predominant pfmdr1 haplotype was the NFD (62.5%). From the publicly available genomic global data set the SNPs frequencies in 10 genes were analysed. In Africa, the K76T pfcrt mutation was present in high frequency, as well as SNPs in the pfdhfr gene (>80%). The pfdhfr/pfdhps quintuple mutant was absent. The predominant haplotype in pfmdr1 was the NFD (>30%). No SNPs were detected in the pfk13 gene associated with antimalarial drug resistance. In the South East Asia region, the K76T pfcrt substitution was present in ~50% of samples, similar to SNP frequencies in the pfdhfr gene, The pfdhfr/pfdhps quintuple mutant was not observed. The pfmdr1 most frequent haplotype was the NYD. In the pk13 gene, the Y493H and R539T were present in low frequency (1%) and the C580Y mutation was more common (10%). The pfarps10 and pffd genes have SNPs present South East Asia with a frequency between 30- 40%. In the Western Pacific region, the SNP K76T is present in all samples and SNPs in the pfdhfr gene reached >80% frequencies, the quintuple mutant is also not present. The pfmdr1 gene had the NFD haplotype (42.7%). In the pfk13 gene, the SNP C580Y was common (~15%). The pffd and pfarps10 genes also have SNPs in 50% of the samples. In the Americas, the K76T mutation is also present in all samples, while the quintuple mutant was absent. The pfmdr1haplotype NFD has a frequency of 96% and the C580Y mutation was present at low frequency (<5%). Overall, this project has shown that the use of NGS methodologies can provide a high-throughput molecular surveillance method to monitor the emergence and spread of resistant alleles and inform malaria control programmes.