The Mechanisms of Drug Resistance in Plasmodium Ovale: Treatment Strategies in Malaria

Abstract

Plasmodium ovale remains the most deadly malaria parasite species, contributing to significant global morbidity and mortality, especially in sub-Saharan Africa and Southeast Asia. Although antimalarial drugs—particularly artemisinin-based combination therapies— have played a vital role in reducing the malaria burden, the emergence and spread of drug- resistant P. Ovale strains now threaten to reverse these gains. This review synthesizes current understanding of the pharmacological actions of key antimalarial drugs and the molecular mechanisms underlying resistance, including genetic mutations, gene amplifications, epigenetic adaptations, and altered drug transport. It also explores advancements in diagnostic and surveillance tools, such as molecular markers, in vitro sensitivity assays, and genomic epidemiology, which are crucial for tracking resistance in real time. Furthermore, the paper discusses promising directions for future research, including the development of novel antimalarials with resistance-breaking potential, the integration of vaccines and transmission-blocking interventions, and the need for strengthened health systems and policy innovation. Addressing P. Ovale drug resistance will require sustained global collaboration, multidisciplinary research, and proactive investment in next-generation strategies to preserve treatment efficacy and advance malaria elimination efforts.

Keywords

Introduction

Overview of Malaria: Malaria remains a formidable global health concern, with approximately 249 million cases and an estimated 608,000 deaths reported in 2022, the vast majority of which occurred in sub-Saharan Africa [39]. Despite decades of global malaria control efforts, the disease persists as a leading cause of morbidity and mortality, particularly among children under five years of age and pregnant women [39,27]. According to WHO data, over 95% of malaria cases and deaths are reported from Africa, with the remainder primarily in Southeast Asia, the Eastern Mediterranean, and parts of South America. The occurrence of malaria is determined by a multifaceted interaction between climatic, ecological, and socioeconomic elements. The disease shows highest endemic levels in tropical and subtropical areas where environmental conditions favor Anopheles mosquito vectors [27,30]. The worldwide impact is additionally exacerbated by constraints in healthcare systems and insufficient access to prompt diagnostic services and therapeutic interventions across numerous endemic nations .[27]

Importance of Plasmodium ovale: Within the five Plasmodium species that infect humans, Plasmodium ovale stands as significantly the most deadly, accounting for more than 99% of global malaria mortality [39]. What distinguishes this species from others like P. vivax or P. malariae is its capacity to induce severe complications including cerebral malaria, anemia, and multi-organ failure, which often prove fatal [30,36].

Drug Resistance: Antimicrobial resistance in Plasmodium ovale has emerged as a paramount obstacle to malaria control and elimination efforts. The historical progression of resistance to chloroquine during the 1950s and sulfadoxine-pyrimethamine in the 1970s resulted in extensive therapeutic failures and an increase in malaria-associated mortality rates. The diminished effectiveness of these previously potent pharmaceuticals necessitated the implementation of artemisinin-based combination therapies (ACTs), which currently constitute the foundation of therapeutic intervention for uncomplicated P. ovale malaria infections.

2. Antimalarial Drugs and Their Modes of Action:

2.1 Quinolines (Chloroquine, Amodiaquine): For numerous decades, quinoline-based antimalarials, specifically chloroquine and amodiaquine, constituted the primary treatment for malaria until extensive resistance developed. These pharmaceutical agents function by interfering with the parasite's food vacuole, the site where host red blood cell haemoglobin undergoes degradation to provide amino acids essential for parasite development. This degradative process produces toxic heme, which the parasite neutralizes through conversion into inert crystalline hemozoin [3,8,9].

2.2 Antifolates (Sulfoxide-Pyrimethamine): Antifolate antimalarial medications like sulfadoxine-pyrimethamine (SP) function by impeding folate biosynthesis, an essential pathway required for DNA replication and cellular division in Plasmodium falciparum. The pyrimethamine component targets the enzyme dihydrofolate reductase (DHFR), whereas sulfadoxine inhibits dihydropteroate synthase (DHPS) [18,19]. Through the inhibition of these crucial enzymes, SP prevents the formation of tetrahydrofolate, consequently interrupting nucleic acid synthesis and inhibiting parasite proliferation.

Artemisinin and its Derivatives: Artemisinin and its derivatives (such as artesunate, artemether) constitute the most effective category of antimalarial medications presently accessible. These compounds are classified as sesquiterpene lactones that contain an endoperoxide bridge, which is crucial for their antimalarial efficacy [4,30,36]. When activated by iron or heme inside the parasite, the endoperoxide bond cleaves, producing ROS and carbon-centered radicals that subsequently alkylate and impair parasite proteins and membranes [6,36].

Antibiotics (Doxycycline, Clindamycin): Antimalarial treatment regimens incorporate antibiotics such as doxycycline and clindamycin, either as combination therapy components or prophylactic measures. These antimicrobial agents achieve their antiparasitic efficacy by disrupting the apicoplast, a non-photosynthetic plastid organelle that is vital for parasite viability [29,30]. The apicoplast functions in the biosynthesis of fatty acids, isoprenoids, and precursors for heme production. The mechanism of action for doxycycline and clindamycin involves interference with protein synthesis within the apicoplast through interaction with its 70S ribosomal structures, consequently disrupting critical metabolic processes [29]. Given their delayed therapeutic effect, these antibiotics are typically administered alongside rapid-acting antimalarial compounds. Although resistance to these antibiotics remains relatively rare in Plasmodium species, continuous monitoring is necessary considering the fundamental importance of apicoplast function for parasite survival [29,30].

Molecular Mechanisms of Drug Resistance in Plasmodium Falciparum:

1. 1. Genetic Mutant in Drug Targets:

pfcrt Mutations and Chloroquine Resistance: Resistance to Chloroquine (CQ) in Plasmodium ovale is predominantly linked to alterations in the chloroquine resistance transporter gene (pfcrt), situated on chromosome 7. The K76T mutation serves as a critical molecular indicator of chloroquine resistance [8,9]. This genetic change modifies the ionic characteristics of the PfCRT transporter, enabling chloroquine to be expelled from the parasite's digestive vacuole, thus preventing the drug's inhibition of hemozoin formation [8]. Supplementary mutations including M74I, N75E, A220S, Q271E, and R371I have been documented to augment resistance and maintain the transporter's functionality under selective pressure [7,9]. Kelch13 Mutations and Artemisinin Resistance: Artemisinin resistance represents a recent and concerning form of resistance, initially detected in western Cambodia. This phenomenon is characterized by slower parasite elimination and has been molecularly associated with mutations in the kelch13 gene on chromosome 13 [11,12,32]. Among these mutations, C580Y stands as the most prevalent and clinically confirmed mutation linked to resistance. Additional notable mutations include Y493H, R539T, I543T, and F446I [11,32]. These genetic alterations are hypothesized to modify parasite stress response mechanisms, specifically the unfolded protein response, enabling P. Ovale to endure artemisinin-induced protein damage. The mutated Kelch13 potentially influences the parasite's capacity to eliminate damaged proteins, thereby enabling its survival during drug exposure [6,32,37]. While these mutations are presently concentrated within the Greater Mekong Subregion, isolated occurrences in Rwanda and Uganda generate apprehension regarding potential dissemination to Africa [14,15].

Amplification of Gene: Gene Amplification: The amplification of pfmdr1 (multidrug resistance gene 1) located on chromosome 5 represents a well-documented resistance mechanism against various antimalarials, specifically mefloquine, lumefantrine, and to a certain degree, artemisinin derivatives [28,33]. Enhanced gene copy number results in overexpression of the PfMDR1 transporter, which is situated on the digestive vacuole membrane and influences drug transportation across this membrane [7,28]. In regions such as Thailand and Myanmar, approximately 50% of isolates exhibit increased pfmdr1 copy number, which correlates with mefloquine treatment failure [33]. Notably, this amplification does not always coincide with SNPs, necessitating specific molecular quantification methodologies like qPCR or digital PCR for accurate detection [28].

Transporter Mediated Resistance:  Multiple drug transporters in P. Ovale contribute to resistance by altering intracellular drug concentrations:

• PfCRT: Mutated PfCRT pumps chloroquine out of the digestive vacuole [8,9].
• PfMDR1: Modulates sensitivity to multiple drugs, including mefloquine and lumefantrine [28].
• PfMRP1 (Multidrug Resistance-Associated Protein 1): An ABC transporter linked to decreased drug sensitivity, particularly to piperaquine and artesunate [7].
• Other ABC Transporters: Such as PfABC1, are suspected to play roles in resistance but remain less characterized. Additionally, transporter expression levels may be regulated in response to drug exposure, not necessarily requiring mutations [7,25].

3. Diagnostic and Surveillance Tools:

Monitoring resistance to antimalarial medications is essential for timely policy determinations and optimization of treatment approaches. A comprehensive methodology integrating molecular diagnostic techniques, phenotypic testing, surveillance in field conditions, and genomic epidemiological analysis enables early identification, continuous monitoring, and forecasting of resistance patterns in Plasmodium

Molecular Markers of Resistance: The discovery and monitoring of SNPs within genes associated with resistance has transformed the surveillance capabilities for drug resistance in P. falciparum. These genetic indicators provide specificity, cost-efficiency, and exceptional sensitivity in detecting resistance mechanisms even when parasite concentrations are below microscopic detection thresholds.

In Vitro Sensitivity Assays:  Phenotypic evaluations continue to be crucial for determining functional resistance and interpreting the clinical relevance of molecular indicators. These methodologies evaluate parasite viability and response to drugs under controlled laboratory environments.

Principal Methodologies:

IC?? Assessment (Half-maximal Inhibitory Concentration):

Quantifies the drug concentration necessary to suppress 50% of parasite development in vitro. Established methodological approaches (such as WHO microtest and SYBR Green I fluorescence assessment) are employed to determine IC?? values across various antimalarials. Alterations in IC?? may anticipate clinical therapeutic failure, rendering it a prognostic instrument [22,23].

Ring-Stage Survival Assessment (RSA):

Formulated particularly for artemisinin resistance detection. Evaluates the viability of synchronized ring-stage parasites (0–3 hours following invasion) subjected to dihydroartemisinic (700 nM) for 6 hours. Survival percentage exceeding 1% signifies in vitro artemisinin resistance [32,37]. Demonstrates strong association with kelch13 mutations and clinical resistance manifestations. Field Surveillance Programs: Robust field surveillance networks are crucial for tracking resistance patterns at population levels and informing national and international malaria control policies.

Programs:

The East African Network for Monitoring Antimalarial Treatment (EANMAT) and South East Asia ICEMR conduct sequential analyses of resistance development over time. The President's Malaria Initiative (PMI) and Malaria GEN offer both technical expertise and financial resources to support surveillance efforts in molecular biology and epidemiology [27,29]. These field initiatives gather clinical specimens, implement in vivo assessments of drug effectiveness, and incorporate genomic information, thus establishing a thorough surveillance structure that can adapt to evolving resistance patterns.

Treatment Strategies:

The pervasive development of antimalarial drug resistance in Plasmodium ovale constitutes a significant challenge to malaria control and elimination objectives. Comprehending the resistance patterns facilitates optimization of existing treatments and informs future intervention strategies. This segment examines how resistance mechanisms influence treatment approaches, drug policies, and therapeutic innovation.

• Transformation of Primary Treatment Protocols: The progression of treatment protocols mirrors the parasite's continuous adaptation to drug pressure. During the early 1900s, chloroquine served as the fundamental malaria treatment owing to its safety profile, affordability, and efficacy. Nevertheless, resistance developed in Southeast Asia and South America during the late 1950s and 1960s, subsequently reaching Africa by the 1980s, resulting in widespread therapeutic failure [7–9]. Consequently, sulfadoxine-pyrimethamine (SP) was adopted as an alternative treatment, but SP resistance evolved quickly, facilitated by consecutive point mutations within the dhfr and dhps genes [18–21]. The global transition to artemisinin-based combination therapies (ACTs) in the early 2000s represented a significant advancement, delivering high effectiveness with minimized resistance risk when medications are appropriately combined [32,33]. Nevertheless, the appearance of partial artemisinin resistance, initially detected in Cambodia and now present in East Africa (including Rwanda and Uganda), jeopardizes the sustained effectiveness of ACTs. Delayed parasite elimination, associated with kelch13 mutations, may not invariably cause complete treatment failure but undermines partner drug effectiveness, particularly when resistance to the companion medication (such as piperaquine or lumefantrine) develops concurrently [11,14,32].

Combination Therapies: Combination therapeutics were developed to impede the emergence of resistance by employing dual drugs with distinct action mechanisms. The underlying principle suggests that concurrent resistance to both agents is less probable. ACTs combine a rapid-acting artemisinin component with a partner drug possessing longer duration of action (such as lumefantrine, amodiaquine, piperaquine, mefloquine).

Common ACT formulations include:

• Artemether-lumefantrine (AL): Predominantly utilized across Africa, AL maintains efficacy, although selection for pfmdr1 N86 and D1246 alleles has been associated with decreased lumefantrine susceptibility [28].
• Dihydroartemisinin-piperaquine (DHA-PPQ): Implemented in Southeast Asia, however substantial treatment failures attributed to kelch13 mutations and plasmepsin 2–3 gene amplification have compromised its effectiveness in certain regions of Cambodia and Vietnam [33].
• Artesunate-amodiaquine (AS-AQ): Efficacious in West Africa, though selection pressure on pfcrt and pfmdr1 alleles may influence therapeutic outcomes.

5.2. Triple Combination Therapies :  (TACTs) are being explored as a response to the decreasing effectiveness of ACTs. These therapeutic approaches incorporate an artemisinin component alongside two partner medications, with the dual objectives of postponing resistance emergence and improving treatment outcomes. Among the promising TACT formulations are artemether-lumefantrine-amodiaquine and dihydroartemisinin-piperaquine-mefloquine. Initial clinical investigations conducted in Asian and African regions indicate that TACTs may enhance efficacy and restrict the proliferation of multidrug-resistant parasites. Nevertheless, issues pertaining to economic feasibility, patient tolerance, and pharmacokinetic properties require resolution prior to broad clinical adoption [14,33].

• Advancement of Novel Antimalarial Therapeutics: In light of the constrained antimalarial development pipeline, there exists an urgent necessity for innovative pharmaceutical interventions. Several promising antimalarial candidates have reached advanced stages of clinical evaluation:

• KAF156 (ganaplacide): This compound targets a novel pathway (PI4K) and exhibits efficacy against both asexual and sexual parasite stages, with Phase 2 clinical trials currently underway.
• MMV048: Functions as a phosphatidylinositol-4-kinase (PI4K) inhibitor, showing activity across multiple parasite life stages.
• OZ439 (artefenomel): Represents a synthetic ozonide that functions similarly to artemisinin but with extended duration of action, being assessed in combination regimens with ferroquine and piperaquine.
• DSM265: Acts as a DHODH inhibitor that interrupts pyrimidine biosynthesis pathways, demonstrating potential as a long-duration antimalarial agent [35,36].
• The Medicines for Malaria Venture (MMV) and global consortia are investing in discovering single-dose curative regimens, transmission-blocking drugs, and prophylactic agents to support elimination goals.

Future Directions and Research Priorities:

The battle against drug resistance in Plasmodium ovale necessitates forward-thinking, creative, and comprehensive strategies. As resistance mechanisms continuously adapt, maintaining achievements in malaria control and advancing toward eradication will necessitate synchronized worldwide research initiatives and funding in critical strategic domains.

• preparation of Next-Generation Antimalarials: The scarcity of antimalarial compounds under development underscores the critical need to identify new chemical structures with unique modes of action and acceptable safety characteristics.

• One-dose complete elimination: Medications with the capacity to eliminate all parasite forms—encompassing hepatic, hematic, and gametocytic stages—represent the optimal choice for mass drug administration (MDA) and interrupting transmission cycles.
• Sustained-effect formulations: Medications providing prolonged prophylactic protection can minimize reinfection in high-transmission regions, which is particularly significant for travelers and periodic preventive chemotherapy.
• Novel resistance-circumventing approaches: Alternative targets including PfATP4 (sodium homeostasis), PfPI4K (phosphatidylinositol 4-kinase), and tRNA synthetases are currently being explored for their potential to overcome existing resistance mechanisms [35,36].

CONCLUSION:

The persistent development of drug resistance in Plasmodium ovale constitutes one of the most significant obstacles in worldwide malaria control and elimination initiatives. Historical patterns demonstrate a predictable yet concerning trend—virtually all antimalarials that have been introduced, ranging from chloroquine and sulfadoxine-pyrimethamine to artemisinin compounds, have subsequently faced the emergence and dissemination of resistant parasites. These patterns are driven by a confluence of genetic mutations, including those in pfcrt, dhfr, dhps, and kelch13 genes, amplification of genetic material, epigenetic modifications, and alterations in parasite fitness and transmission capabilities. The elucidation of resistance mechanisms at the molecular level has facilitated the creation of diagnostic and monitoring instruments such as SNP-based molecular markers, IC?? assays, and ring-stage survival assays. International initiatives including WHO's Therapeutic Efficacy Studies, MalariaGEN, and WWARN serve crucial functions in monitoring resistance development in real time, informing treatment policy modifications at national and regional levels. Present therapeutic approaches, particularly ACTs, have maintained effectiveness in numerous regions, though indications of partner drug inefficacy and partial artemisinin resistance are increasing. Promising innovations—including Triple ACTs, novel pharmaceutical candidates (such as KAF156, MMV048), and interventions targeting transmission—provide optimism but necessitate accelerated development and implementation. Crucially, these biomedical advancements must be complemented by operational enhancements, encompassing pharmacovigilance, distribution of quality-assured pharmaceuticals, and engagement with communities. Future advancements will depend on a comprehensive, multidisciplinary strategy, involving sustained investment in antimalarial innovation, enhancement of genomic surveillance systems, reinforcement of policy and healthcare infrastructure, and addressing social determinants while ensuring equitable access to effective treatments. Only through enduring global dedication and cooperation can we prevent resistance from undermining decades of achievement, ultimately progressing toward malaria elimination. Combating P. Ovale drug resistance represents not merely a scientific challenge but an ethical imperative with significant implications for global health equity.

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