Genetic Epidemiology of Plasmodium falciparum Asymptomatic Infections and Antimalarial Drug-resistance Markers in Kilifi, Kenya.

Wamae, Kevin Kariuki (2021). Genetic Epidemiology of Plasmodium falciparum Asymptomatic Infections and Antimalarial Drug-resistance Markers in Kilifi, Kenya. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0001263e

Abstract

Asymptomatic infections and anti-malarial drug resistance are impediments to malaria elimination. Markedly, asymptomatic infections often go undetected and untreated and this creates a parasite reservoir that fuels malaria transmission as well as being a risk factor for febrile malaria. On the other hand, drug-resistance renders antimalarial drugs ineffective and has been associated with increased morbidity and mortality in the past.

Using longitudinal malaria monitoring data and Plasmodium falciparum positive samples from Kilifi, Kenya, (i) an evaluation was conducted to assess the impact of age and malaria transmission intensity on the risk of developing febrile malaria in individuals harbouring asymptomatic infections, (ii) amplicon deep-sequencing was used to evaluate P. falciparum genetic diversity in asymptomatic and febrile infections and (iii) the diversity of twelve drug-resistance markers (crt, mdr1, dhps, nfs, k13, ap2mu, falcipain-2a, ubp-1, as well as four artemisinin resistance predisposing mutations; arps10 codon V127M, crt codon I356T, fd codon D193Y and mdr2 codon T484I) in Kilifi was evaluated using Sanger sequencing, including a neutral marker (serine-tRNA ligase) that is not under drug-pressure.

Analyses of the data revealed that in the moderate and high transmission intensity settings, asymptomatic infections were associated with a reduced risk of febrile malaria in older children (>3 years), while in the lower transmission setting, asymptomatic infections were associated with an increased risk of febrile malaria in children of all ages. Amplicon deep-sequencing revealed that P. falciparum genetic diversity in asymptomatic and febrile infections differ significantly, similar to previous reports. Also, a majority of the febrile cases (86%) were due to the introduction of P. falciparum clones that were not detected in the preceding asymptomatic episode. Regarding the analysis of antimalarial drug resistance markers, none of kelch 13 (k13) validated markers of artemisinin resistance were detected in the population, nonetheless, a single k13 allele, K189T, was maintained at a stable high frequency (>10%) over time. There was a distinct shift from chloroquine-resistant transporter (crt)-76, multi-drug resistant gene 1 (mdr1)-86 and mdr1-1246 chloroquine (CQ) resistance alleles to a 99% prevalence of CQ sensitive alleles in the population, following the withdrawal of CQ from routine use. In contrast, the dihydropteroate synthetase (dhps) double mutant (437G and 540E) associated with sulfadoxine-pyrimethamine (SP) resistance was maintained at a high frequency (>75%), after a change from SP to artemisinin combination therapies (ACTs). The novel cysteine desulfurase (nfs) K65 allele, implicated in resistance to lumefantrine in a West African study, showed a gradual significant decline in allele frequency pre- and post-ACT introduction (from 38% to 20%), suggesting evidence of directional selection in Kenya, potentially not due to lumefantrine. The frequency of AP-2 complex subunit mu (ap2-mu) S160N allele, a mutation that has been associated with directional selection after artemisinin combination therapy, was stable over time indicating that it is not under drug-pressure. On the other hand, the ubiquitin carboxyl-terminal hydrolase 1 (ubp-1) mutation at codon E1528D, also associated with directional selection after artemisinin combination therapy, was not detected. The S69Stop mutation in falcipain-2a that has been associated with artemisinin resistance, in vitro, was not detected and none of the artemisinin resistance predisposing mutations were identified.

Asymptomatic infections were found to be modified by transmission intensity and age, altering the risk of developing febrile episodes and this suggested that host immunity plays a prominent role in mediating this process. The differences in P. falciparum genetic diversity between asymptomatic and febrile malaria infections can be attributed to the broader spectrum of immunological memory in the form of antibodies that has been found to be higher in asymptomatic infections compared to febrile malaria infections. While evidence of apparent protection from developing febrile episodes was observed in children with asymptomatic infections, amplicon deep-sequencing revealed that this protection was offset when a new infection occurred. Lastly, due to lack of the validated molecular markers of artemisinin resistance, there appears to be no problem of resistance in the population, however, continued surveillance remains a requirement. To conclude, P. falciparum genetic epidemiology revealed the ability to characterise P. falciparum complexity of infection (COI), a proxy that can be used to characterise malaria transmission intensity. Additionally, frequent sampling of P. falciparum positive samples from a hospital setting coupled with the molecular genotyping of drug resistance markers revealed the utility of P. falciparum genetic epidemiology in surveillance of antimalarial drug resistance.

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