Prevalence of Plasmodium falciparum delayed clearance associated polymorphisms in adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1) genes in Ghanaian isolates

Abstract

Background: Plasmodium falciparum delayed clearance with the use of artemisinin-based combination therapy (ACTs) has been reported in some African countries. Single nucleotide polymorphisms (SNPs) in two genes, P. falciparum adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1), have been linked to delayed clearance with ACT use in Kenya and recurrent imported malaria in Britain. With over 12 years of ACT use in Ghana, this study investigated the prevalence of SNPs in the pfap2mu and pfubp1 in Ghanaian clinical P. falciparum isolates to provide baseline data for antimalarial drug resistance surveillance in the country.

Methods: Filter paper blood blots collected in 2015-2016 from children aged below 9 years presenting with uncomplicated malaria at hospitals in three sentinel sites Begoro, Cape Coast and Navrongo were used. Parasite DNA was extracted from 120 samples followed by nested polymerase chain reaction (nPCR). Sanger sequencing was performed to detect and identify SNPs in pfap2mu and pfubp1 genes.

Results: In all, 11.1% (9/81) of the isolates carried the wildtype genotypes for both genes. A total of 164 pfap2mu mutations were detected in 67 isolates whilst 271 pfubp1 mutations were observed in 72 isolates. The majority of the mutations were non-synonymous (NS): 78% (128/164) for pfap2mu and 92.3% (250/271) for pfubp1. Five unique samples had a total of 215 pfap2mu SNPs, ranging between 15 and 63 SNPs per sample. Genotypes reportedly associated with ART resistance detected in this study included pfap2mu S160N (7.4%, 6/81) and pfubp1 E1528D (7.4%, 6/81) as well as D1525E (4.9%, 4/81). There was no significant difference in the prevalence of the SNPs between the three ecologically distinct study sites (pfap2mu: χ² = 6.905, df = 2, P = 0.546; pfubp1: χ² = 4.883, df = 2, P = 0.769).

Conclusions: The detection of pfap2mu and pfubp1 genotypes associated with ACT delayed parasite clearance is evidence of gradual nascent emergence of resistance in Ghana. The results will serve as baseline data for surveillance and the selection of the genotypes with drug pressure over time. The pfap2mu S160N, pfubp1 E1528D and D1525E must be monitored in Ghanaian isolates in ACT susceptibility studies, especially when cure rates of ACTs, particularly AL, is less than 100%.

Keywords: ACT; Antimalarial drug resistance; Artemisinin; Ghana; Mutations; Plasmodium falciparum; pfap2mu; pfubp1.

Fig. 1

The map of Ghana showing the three study sites, Navrongo, Begoro and Cape Coast in three different ecological areas, guinea savannah, forest and coastal savanna

Fig. 2

Distribution of pfap2mu and pfubp1 NS and SYN mutations in isolates from the three sites. a pfap2mu. b pfubp1

Fig. 3

Proportion of isolates from the three sites with varying number of pfap2mu and pfubp1 mutations. a pfap2mu. b pfubp1

Fig. 4

Proportion of isolates from the three sites with shared pfap2mu and pfubp1 mutations. a pfap2mu. b pfubp1

Fig. 5

A sequence alignment of pfap2mu gene showing amino acid changes due to single nucleotide polymorphisms. The alignment was done using pfap2mu reference sequence of the 3D7 strain (PF3D7_1218300). Mutations present at codons 185–282, nucleotide positions 553–849 of pfap2mu for 21 samples. Samples C314 and G026 had a frameshift, samples C312. C314, C315, C408, G009, G011, G012, G033, N030 and N102 had an asparagine (N) insertion at codon 233, as well as a lysine (K) insertion at the same position for sample N055

Fig. 6

A sequence alignment for 5 samples with multiple mutations. The alignment was done using pfap2mu reference sequence of the 3D7 strain (PF3D7_1218300). About 215 SNPs were observed in these isolates ranging from 15 to 63 SNPs per isolate. These mutations were found from codons 227–324, nucleotide positions 679–970 and are likely due to multiplicity of infection

Fig. 7

A sequence alignment of pfubp1 gene showing amino acid changes due to single nucleotide polymorphisms. The alignment was done using pfubp1 reference sequence of the 3D7 strain (PF3D7_0104300). Mutations from codons 1483–1549 at nucleotide positions 4449–4647 is shown. The gaps are a result of the insertions of amino acids which resulted in a frameshift. The gap between 1519E and 1520K are therefore a result of a 6 amino acid insertion in C356, G001 and G025. The known mutations D1525E and E1528D are shown in the isolates N061 and N096 respectively. A frameshift mutation is observed in N084, after codon 1536 as a result of a deletion