Molecular Surveillance of Plasmodium falciparum Drug Resistance Markers in Clinical Samples from Botswana

Abstract

Drug-resistant Plasmodium falciparum is a major threat to global malaria control and elimination efforts. In Botswana, a southern African country approaching malaria elimination, P. falciparum molecular data are not available. Parasites were assessed through pollymerase chain reaction (PCR) for confirmation of positive rapid diagnostic tests, multiplicity of infection (MOI), and drug resistance markers among isolates from clinical uncomplicated malaria cases collected at health facilities. Of 211 dried blood spot samples selected for the study, 186 (88.2%) were PCR positive for P. falciparum. The mean MOI based on MSP1 genotyping was 2.3 and was not associated with age. A high prevalence of wildtype parasites for pfcrt and pfmdr1 was found, with a haplotype frequency (K76/N86) of 88.8% and 17.7% of the isolates having two copies of the pfmdr1 gene. For pfATPase6, all the parasites carried the wild-type S769 allele. Sequencing showed no evidence of non-synonymous mutations associated with reduced artemisinin derivative sensitivity in the P. falciparum k13 gene. In conclusion, we found that P. falciparum parasites in Botswana were mostly wild type for the drug resistance markers evaluated. Yet, there was a high rate of a molecular marker associated to reduced sensitivity to lumefantrine. Our results indicate the need for systematic drug efficacy surveillance to complement malaria elimination efforts.

Past and current biological factors affecting malaria in the low transmission setting of Botswana: A review

“…More importantly, P. falciparum may be generally tolerant to lumefantrine, which is one of the compounds utilized in first line treatment together with the highly efficacious artemether. The same study found two synonymous mutations -V555V and R513R -in the propeller domain of the P. falciparum Kelch13 gene (pfk13) (Tawe et al, 2018a(Tawe et al, , 2018b which is implicated in the resistance to artemisinin. It should be noted that neither of these genetic variants were associated with a delayed parasite clearance phenotype.…”

“…Admixture has occurred between the two ethnic groups to various extents (Schuster et al, 2010). As a consequence, there is a wider spectrum of genotypes and phenotypes when considering genetic characteristics such as Duffy antigen, sickle cell trait, glucose-6-phosphate dehydrogenase (G6PD), and detoxifying liver enzymes (Howes et al, 2011;Motshoge et al, 2018;Tawe et al, 2018aTawe et al, , 2018b. These human genetic factors play a pivotal role in susceptibility to malaria infection and associated morbidity (Haldane, 1949;Sirugo et al, 2014), as well as for metabolism of medications currently on the market (Tawe et al, 2018a(Tawe et al, , 2018b.…”

“…This is not surprising given the differences in life cycles of the two parasites, which directly impacts the severity of symptoms depending upon the species as well as the likelihood of patients presenting themselves for treatment (Antinori et al, 2012). In fact, subjects infected with P. falciparum are far more likely to develop symptomatic malaria in the low endemic context of Botswana, where malaria immunity is generally absent or very low (Tawe et al, 2018a). Biological determinants were long thought to limit P. vivax's pathogenicity.…”

“…A recent report on drug resistance markers in P. falciparum demonstrated that the parasite harbors certain variation in several of the genes analyzed. In the study, 95.8% of samples showed N86 polymorphism at Plasmodium falciparum multidrug resistance 1 gene (pfmdr1) (Tawe et al, 2018a(Tawe et al, , 2018b known to confer sensitivity to 4-aminoquinolines (Howes et al, 2015) (chloroquine and amodiaquine) but also associated to a reduced sensitivity to lumefantrine (Wongsrichanalai et al, 2002). This suggests that a similar situation to that seen in Malawi (Kublin et al, 2003) has unfolded in Botswana with regards to chloroquine.…”

Targeted deep amplicon sequencing of kelch 13 and cytochrome b in Plasmodium falciparum isolates from an endemic African country using the Malaria Resistance Surveillance (MaRS) protocol

“…Molecular surveillance of imported malaria cases is an important public health activity that can aid in the detection of drug-resistant P. falciparum parasites [21,22]. Recent advancements in next-generation sequencing are providing a new way to rapidly detect and characterize drug-resistant malaria parasites, including minor parasite populations in mixed infection P. falciparum cases [23,24].…”