Antimalarial resistance genes polymorphism and imunoendocrine biomarker profile in patients with uncomplicated plasmodium falciparum malaria in Lagos, Nigeria
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Date
2020-01
Authors
Idowu, A.O.
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School of Postgraduate Studies of the University of Lagos, Akoka
Abstract
The threat of a possible emergence of resistance to currently recommended artemisinin based combination therapies (ACTs) against malaria, has made proactive surveillance for resistance markers imperative for early detection of resistance before they become widespread. The changing pattern of malaria transmission due to reduction in cases has also made reassessment of cytokine and endocrine response in malaria infection important. This study assessed antimalarial resistance genes polymorphism and immunoendocrine biomarker profile in patients with uncomplicated Plasmodium falciparum malaria in Lagos, Nigeria. Blood samples collected between July, 2015 and August, 2016 in 3 health facilities in Lagos from a cross section of 2,534 consenting patients with symptoms consistent with malaria were screened for P. falciparum by microscopy and RDT. Thirty-seven samples positive for P. falciparum from patients with self- reported antimalarial medication use were selected for molecular studies. Genomic DNA of malaria positive samples were extracted from dried blood spots (DBS) using QIAamp DNA mini-kit (Qiagen, Valencia, CA). Sanger and next generation sequencing (NGS) methods were used to screen for mutations in three genes (Pfcrt, Pfmdr1 and Pfk13) that have been previously associated with antimalarial resistance. Amplification and sequencing of the Pfk13, Pfcrt, and
Pfmdr1 genes was successfully performed in 26 samples. Plasma concentration for cytokines (IL-12p70, IFN-γ, TNF-α, IL-10, TGF-β), and corticosteroids (cortisol and dexamethasone) were evaluated using enzyme-linked immunosorbent assay (ELISA) in twenty-six patients’ samples categorized as treated, untreated and control. Of the common fragments sequenced by both methods, more mutations and haplotypes were detected by NGS than by Sanger. The NGS
method detected three Pfk13, seven Pfcrt, and six Pfmdr1 mutations in our clinical isolates which include rare Pfmdr1 mutations, N504K, N649D, F938Y and S967N, which, to our knowledge, were previously unreported. Nineteen of the 26 (73.1%) isolates had one or more Pfk13 mutations, 9 of the 26 (34.6%) isolates had one or more Pfcrt mutations, and 22 of the 26 (84.6%) isolates had one or more Pfmdr1 mutations. There was moderate prevalence of the K76T mutation (34.6%) and CVIET haplotype (30.8%) associated with chloroquine and amodiaquine resistance and high prevalence of the Pfmdr1 N86 wild type allele (92.3%) and NF (84.6%) haplotype associated with lumefantrine resistance. None of the mutations in the Pfk13 gene associated or confirmed with resistance to artemisinin in South-East Asia was seen. There
was a correlation pattern showing that IL-10, IFN-γ and TGF-β levels were markedly lower while the level of cortisol increased but that of dexamethasone-induced protein decreased in treated compared with untreated patients. This result suggests that the interplay between cytokine and endocrine response could have a modulating effect on malaria unresponsive to treatment. This study describes polymorphism in three antimalarial resistance associated genes and how
sensitive detection methods such as the NGS can strengthen surveillance for resistant alleles and enhance malaria control efforts.
Description
A thesis submitted to the School of Postgraduate Studies, University of Lagos, Nigeria in fulfillment of the requirements for the award of Doctor of Philosophy Degree in Pharmaceutics and pharmaceutical Microbiology
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Citation
Idowu, A.O. (2020). Antimalarial resistance genes polymorphism and imunoendocrine biomarker profile in patients with uncomplicated plasmodium falciparum malaria in Lagos, Nigeria. A thesis submitted to the School of Postgraduate Studies, University of Lagos, Nigeria in fulfillment of the requirements for the award of Doctor of Philosophy Degree in Pharmaceutics and pharmaceutical Microbiology, 208p.