ORIGINAL ARTICLE Year : 2023  |  Volume : 16  |  Issue : 2  |  Page : 72-79

Mutations in Plasmodium knowlesi Kelch protein 13 and the dihydropteroate synthase gene in clinical samples

Ahmed Saif
Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Najran University, P.O. Box 1988, Najran 61441, Saudi Arabia

Date of Submission06-Sep-2022Date of Decision26-Dec-2022Date of Acceptance21-Jan-2023Date of Web Publication27-Feb-2023

Correspondence Address:
Ahmed Saif
Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Najran University, P.O. Box 1988, Najran 61441
Saudi Arabia

Source of Support: This study was supported by the institutional funding committee of Najran University, Najran, Saudi Arabia (Project code: NU/IFC/ ENT/01/007), Conflict of Interest: None

DOI: 10.4103/1995-7645.370146

Objective: To determine the genetic diversity, natural selection and mutations in Plasmodium (P.) knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia.
Methods: P. knowlesi full-length gene sequences Kelch 13 gene (PkK13) from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases. Genetic diversity, natural selection, and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2.
Results: Seventy-two single nucleotide polymorphic sites (SNPs) across the full-length PkK13 gene (63 synonymous substitutions and 9 non-synonymous substitutions) with nucleotide diversity of π~0.005 was observed. Analysis of the full-length Pkdhps gene revealed 73 SNPs and π~0.006 (44 synonymous substitutions and 29 non-synonymous substitutions). A high number of haplotypes (PkK13; H=37 and Pkdhps; H=29) with haplotype diversity of Hd ~0.99 were found in both genes, indicating population expansion. Nine mutant alleles were identified in PkK13 amino acid alignment of which, 7 (Asp3Glu, Lys50Gln, Lys53Glu, Ser123Thr, Ser127Pro, Ser149Thr and Ala169Thr) were within the Plasmodium specific domain, 2 (VaI372Ile and Lys424Asn) were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain. The 29 non-synonymous mutations in the Pkdhps gene were novel and only presented in exon 1 and 2.
Conclusions: Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P. knowlesi.

Keywords: Plasmodium knowlesi; Kelch 13; dhps; Dihydropteroate synthase; Drug resistance; Mutation

How to cite this article:
Saif A. Mutations in Plasmodium knowlesi Kelch protein 13 and the dihydropteroate synthase gene in clinical samples. Asian Pac J Trop Med 2023;16:72-9
How to cite this URL:
Saif A. Mutations in Plasmodium knowlesi Kelch protein 13 and the dihydropteroate synthase gene in clinical samples. Asian Pac J Trop Med [serial online] 2023 [cited 2023 Feb 28];16:72-9. Available from: https://www.apjtm.org/text.asp?2023/16/2/72/370146

  1. Introduction 

Emerging infectious diseases originating in wildlife pose a serious threat to the world health, security, and economic growth, so preventing their spread is a top concern for public health agencies. Humans and non-human primates share a high degree of genetic and physiological similarities, which makes them vulnerable to the majority of infections that can cross primate species boundaries. One such example is Plasmodium (P.)knowlesi (Pk) which crossed the species boundaries and started infecting humans in Southeast Asian countries and is now the most common malaria parasite in the region[1]. It poses a challenge to malaria elimination efforts in Southeast Asia. Infection with P. knowlesi can quickly lead to high parasitaemia, and the risk of severe disease in adults is as high as that of falciparum malaria[2]. Thus, early diagnosis and treatment are critical in terms of reducing morbidity and mortality.

Proguanil was used in Malaysia in the late 1940s, however, resistance developed quickly. Pyrimethamine resistance was noted in the middle of the 1970s[3], although sulfadoxine-pyrimethamine (SP) was used as a first-line drug for more than 30 years until artemisinin-combination therapy (ACT) was launched in 2009. As per the Ministry of Health, Malaysia, the first-line treatment for P. knowlesi and P. malariae suspected cases is ACT (artemether-lumefantrine or artesunate-mefloquine), for the states of Sabah and Sarawak in Peninsular Malaysia. However, in remote regions of Sabah and Sarawak in Malaysian Borneo, single dose pre-referral SP for falciparum malaria is still being used[4]. Since P. knowlesi and P. falciparum are morphologically indistinguishable microscopically during the early ring stages and because the two parasites can coinfect, it is quite possible that P. knowlesi parasite populations in Malaysia were exposed to SP. The enzyme dihydropteroate synthase is the target of sulfadoxine, and sequencing of the dihydropteroate synthase gene has shown amino acid mutations in codons 436, 437, 540, 581 and 613 confer drug resistance in P. falciparum[5],[6]. A triple dihydropholate reductase mutation (N51I/C59R/S108N) combined with a double dhps mutation (A437G/K540E) is a useful predictor of clinical SP therapy failure in P. falciparum[7].

Over the last two decades, ACTs have made a considerable contribution to lowering malaria’s global impact[8],[9]. The first synthetic antimalarial, chloroquine, was used to treat malaria on a global scale. P. falciparum, the parasite that causes the most lethal type of human malaria, has developed resistance to chloroquine and, other antimalarial drugs such as sulfadoxine and pyrimethamine, mefloquine, and quinine became resistant in various malaria endemic countries[10]. Parasite resistance to artemisinin, the most effective and novel drug used against malaria infection worldwide, is now being reported in many research studies; as a result, first-line treatment strategies, including ACT, are becoming less effective in areas where drug resistance is widespread[11],[12]. Artemisinin resistance has been found in Cambodia, Lao People’s Democratic Republic, Myanmar, Thailand, and Vietnam[15]. The use of artemisinin in monotherapy and in combination (ACT), the most effective and newest drug in use, is being reported in many research studies. It should be noted that the WHO advises against the use of artemisinins in monotherapy, as it is one of the causes of increasing resistance to this drug, and may jeopardise the use of ACTs[13],[14]. Artemisinin resistance has been found in Cambodia, Lao People’s Democratic Republic, Myanmar, Thailand, and Vietnam[15]. Recent research has revealed that mutations in the P. falciparum Kelch 13 propeller region as a molecular marker of ACT resistance, which is associated with delayed parasite clearance in vitro and in vivo[13]. The kelch gene is composed of three domains: a Plasmodium-specific domain, a BTB/POZ (Broad-complex, tramtrack and bric-à-brac) domain, and the kelch propeller domain[16]. Researches have reported several SNPs across the full-length P. falciparum K13 gene, however, only 20 of the 124 non-synonymous Kelch 13 propeller mutations have been linked to resistance to artemisinin so far[17],[18],[19],[20]. Thus, mutations within the propeller region are established as a strong and reliable marker for P. falciparum artemisinin resistance in field isolates. In the past 9 years (2011-2019), 8473 indigenous P. knowlesi malaria cases have been reported from Sarawak, Malaysian Borneo which accounted for more than 60%-70% malaria cases in Malaysia[21],[22],[23]. Previous drug resistance studies on P. knowlesi were limited to clinical efficacy studies and antimalarial drugs such as artemisinins (Artesunate-mefloquine) and chloroquine were found to be effective for uncomplicated knowlesi cases[24],[25]. A study on Pkdhfr gene polymorphism from Sabah identified 14 non-synonymous mutations across the full-length gene along with a moderate level of genetic diversity and no selection pressure[4]. Given the importance of studying the Kelch 13 and dhps gene polymorphisms in other Plasmodium species, this study was conducted to report on the pattern of polymorphisms across the full-length gene and determine genetic diversity and natural selection in P. knowlesi clinical samples. The study provides new knowledge and information crucial for malaria control strategies in Southeast Asian countries.

  2. Subjects and methods 

2.1. Sequence data

Forty full-length PkK13 gene (2 139 bp) sequences and 30 Pkdhps (2 696 bp) sequences were retrieved from public genomic databases (https://www.ebi.ac.uk/ena/browser/home), with majority of them originating from clinical isolates from Malaysian Borneo (n=36) and long-time isolated parasite lines from Peninsular Malaysia (n=4) including the reference sequence PkK13; PKNH_1257700 and Pkdhps; PKNH_1429900) for H-strain[26]. Only high quality DNA sequences were retrieved for analysis. Accession numbers of sequences used for this study are listed in Supplementary Table 1 [Additional file 1].

2.2. Data analysis

The sequence data were aligned by using the CLUSTAL-W program in MegAlign Lasergene v 7.0 (DNASTAR) and converted to FASTA format for further analysis. Sequence diversity (π) was determined using the DnaSP v5.0 software[27], as a measure of genetic diversity. DnaSP software was also used to determine the number of polymorphic sites, synonymous substitutions (silent mutations), non-synonymous substitutions (replacement changes), singleton sites, haplotypes (H), and the haplotype diversity in P. knowlesi Kech13 and dhps gene using DNAsp software. The same software was used to generate a graphical representation of nucleotide diversity, with a window length of 100 bp and a step size of 50 bp.

Natural selection was determined by calculating the rates of synonymous substitutions per synonymous site (dS) and nonsynonymous substitutions per non-synonymous site (dN) as computed by using Nei and Gojobori’s method and robustness were estimated by the bootstrap method with 1 000 pseudo replicates as implemented in the MEGA 5.0 software[28]. The difference between dN and dS was determined by applying codon-based Z-test (P<0.05) in MEGA software v.5 with 1 000 bootstrap replications[28]. The Tajima’s D, Fu & Li’s D* and F* neutrality tests were performed as implemented in DnaSP v5.10 software. Tajima’s D is expected to be zero under neutrality. When Tajima’s D values are positive and statistically significant, which indicates positive/balancing selection, whereas negative values indicate negative selection or population expansion. For domain-wise analysis of P. knowlesi Kelch 13 mutations within the clinical isolates, deduced amino acid from all 40 sequences were aligned along with the P. falciparum Kelch 13 sequence (PF3D7_1343700), and amino acid changes observed in each domain are shown as a schematic diagram. A similar analysis was conducted for 30 amino acid sequences of Pkdhps.

2.3. Phylogenetic analyses

Phylogenetic analyses of the P. knowlesi Kelch 13 were performed using deduced amino acid sequences using maximum likelihood approach based on the Poisson correction model as in MEGA 5.0. [28] along with the closest ortholog sequences of P. coatneyi, P. cynomolgi, P. vivax and P. falciparum.

  3. Results 

3.1. Sequence alignment, polymorphism and genetic diversity analysis of PkK13 and Pkdhps

Seventy-two SNPs (63 synonymous and 9 nonsynonymous mutations) were identified in DNA sequence alignment of 40 full-length P. knowlesi Kelch with 13 sequences [Table 1]. Thirty-three singleton sites (site having only one nucleotide in among the sequences) were found in 38 parsimony informative sites (sites that have a minimum of two nucleotides that are present at least twice), and among them, 32 had two variants and 1 had three variants (C573A/T). The overall nucleotide diversity was found to be π=0.005 02. The graphical representation of nucleotide diversity indicated that SNP variations were throughout the gene; however, the central BTB/POZ domain had higher number of SNPs [Figure 1]A. The number of haplotypes was found to be high (H=37), leading to higher haplotype diversity (Hd=0.995) [Table 1]. Alignment and comparison of 40 full-length deduced amino acid sequences of P. knowlesi Kelch 13 protein revealed that there were 9 mutations (due to 9 non-synonymous substitutions). Domain-wise analysis showed that out of the 9 mutations, 7 were at the Plasmodium specific region (Asp3Glu, Lys50Gln, Lys53Glu, Ser123Thr, Ser127Pro, Ser149Thr and Ala169Thr) and 2 mutations (Val372Ile and Lys424Asn) at the BTB/POZ domain [Figure 1]B. The frequency of each allele was observed within the 40 clinical samples [Table 2].

Table 1: Estimates of nucleotide diversity, haplotype diversity and neutrality indices of PkK13 and Pkdhps gene.

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Table 2: Domain wise alleles observed within clinical isolates of Plasmodium knowlesi Kelch 13 protein.

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Figure 1: Graphical representation of nucleotide diversity across the Plasmodium knowlesi kelch gene. (A) The three domains: Plasmodium specific domain, BTB/POZ domain and Kelch propeller domain. (B) Schematic representation of Plasmodium knowlesi K13 gene mutations from 40 samples.

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DNA sequence alignment of 30 full-length P. knowlesi dhps sequences revealed 73 SNPs (44 synonymous and 29 nonsynonymous mutations) [Table 1], which was higher compared to the Kelch 13 gene, 35 parsimony informative sites and 37 were singleton sites. Among the 37 singleton sites, 35 were two variants and 2 were three variants (A1886G/C). The overall nucleotide diversity was found to be π=0.006 73. The graphical representation of nucleotide diversity indicated that SNP variations were found across the full-length dhps gene [Figure 2]A. The graph indicated that nucleotide diversity was higher towards the 3’ end of the gene owing to the presence of a repeat region comprising the peptide sequence GSLNWLM. The non-synonymous substitutions (mutations) in each exon were shown in [Figure 2]B. No non-synonymous substitutions were noted in exon 3. The DNA polymorphism and amino acid polymorphism across the full-length gene were shown in Supplementary Figures 1 [Additional file 2] and 2 [Additional file 3], respectively. The number of haplotypes was found to be high (H=29), leading to higher haplotype diversity (Hd=0.998) which was similar to the PkK13 gene [Table 1].

Figure 2: Graphical representation of nucleotide diversity across the Plasmodium knowlesi dhps gene. (A) The diversity within the three exons: exon 1, exon 2 and exon 3. (B) Schematic representation of Plasmodium knowlesi dhps gene mutations from 30 samples. Amino acid with tri-variants or triple mutants are shown in blue.

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3.2. Natural selection in PkK13 and Pkdhps

Natural selection analysis indicated strong purifying selection for both of the genes. dS-dN, Tajima’s D as well as Li and Fu’s D* and F* values were negative, indicative of negative/purifying selection [Table 1]. Li and Fu’s D* and F* and the high number of haplotypes due to the presence of high singleton sites were indicative of population expansion.

3.3. Phylogenetic analyses

Phylogenetic analysis of 40 deduced amino acid sequences of P. knowlesi K13 clinical isolates with other ortholog species using maximum likelihood indicated that they formed a single clade with closest ortholog in P. coatneyi, followed by P. cynomolgi, P. vivax and P. falciparum [Figure 3].

Figure 3: Phylogenetic relationship of Plasmodium knowlesi Kelch amino acid sequences from clinical isolates of Malaysia and its ortholog in other Plasmodium species using maximum likelihood method.

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  4. Discussion 

P. knowlesi infections cases are increasing in Southeast Asian countries and multiple drug combinations have been reported to be effective such as artemether-lumefantrine, artesunate-mefloquine and chloroquine plus primaquine[25],[29],[30]. In the present study Kelch 13 and dhps gene diversity and mutations associated with artemisinin and sulphadoxine resistance were studied from clinical samples of Malaysia. Though no mutations (non-synonymous substitutions) were observed with the Kelch propeller domain, indicating the ACT-therapy is still an effective treatment for P. knowlesi infections. All 29 mutations (non-synonymous substitutions) identified within the Pkdhps gene were novel and may indicate drug failure due to previous uncontrolled use of SP in remote areas of Malaysia. However, clinical efficacy study along with monitoring of mutations in dhfr and dhps genes would be necessary to confirm the association of these mutations with sulphadoxine and pyremethamine failure.

High levels of SNPs across the Kelch 13 and dhps gene was indicative of recent population expansion (negative values for Tajima’s D, Li and Fu’s D* and F* values). Similar observation on recent population expansion was also seen in P. knowlesi dhfr gene which is a molecular marker of anti-folate drug resistance to pyremethamine[4]. The genetic diversity observed across the full-length PkK13 and Pkdhps was similar to the Pkdhfr, indicating that both anti-folate as well as artemisinin derivatives could be effective antimalarials against knowlesi infections[4]. The absence of strong positive selection within the PkK13 gene reflected the same effectiveness. It is worth noting that despite strong negative selection within the PkK13 sequences, we did not find geographical clustering across sequences from Malaysian Borneo and Peninsular Malaysia in maximum likelihood based phylogeny. Geographical clustering of gene sequences was observed in multiple genes of P. knowlesi[3l],[32],[33],[34],[35],[36],[37],[38]. Since this is the first study to report on the polymorphism across the full-length Kelch 13 and dhps gene, the mutations observed would serve as a starting point for future surveillance studies with a higher number of clinical samples in all P. knowlesi affected countries of Southeast Asia. And since the data is from clinical samples from an endemic region (Sarawak, Malaysian Borneo), these polymorphisms would also serve as a baseline data for future interventions. Though no mutations were noted within the Kelch propeller domain, indicating ACT is still an effective drug for P. knowlesi infections, and periodic monitoring of mutations from clinical samples will be necessary to ascertain artemisinin and SP resistance in P. knowlesi.

In conclusion, moderate levels of diversity were observed for both PkK13 and Pkdhps genes, and strong purifying/negative natural selection was observed for both the genes, indicative of population expansion. Future studies should focus on time to time monitoring of point mutations on these drug resistance molecular markers with samples originating throughout P. knowlesi endemic regions.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgement

The author would like to extend his appreciation to the Ministry Agency for Research and Innovation, Ministry of Education, Saudi Arabia and Najran University, Najran, Saudi Arabia.

Funding

This study was supported by the institutional funding committee of Najran University, Najran, Saudi Arabia (Project code: NU/IFC/ ENT/01/007).

Author’s contributions

AS contributed to the study conception, design, implemented the study, analyzed the data, interpreted the data and wrote the manuscript.

 

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  [Figure 1], [Figure 2], [Figure 3]
 
 
  [Table 1], [Table 2]