Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein.

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Plasmodium vivax Latent Liver Stage Infection and Relapse: Biological Insights and New Experimental Tools.

Plasmodium vivax is the most widespread human malaria parasite, in part because it can form latent liver stages known as hypnozoites after transmission by female anopheline mosquitoes to human hosts. These persistent stages can activate weeks, months, or even years after the primary clinical infection; replicate; and initiate relapses of blood stage infection, which causes disease and recurring transmission. Eliminating hypnozoites is a substantial obstacle for malaria treatment and eradication since the hypnozoite reservoir is undetectable and unaffected by most antimalarial drugs. Importantly, in some parts of the globe where P. vivax malaria is endemic, as many as 90% of P. vivax blood stage infections are thought to be relapses rather than primary infections, rendering the hypnozoite a major driver of P. vivax epidemiology. Here, we review the biology of the hypnozoite and recent discoveries concerning this enigmatic parasite stage. We discuss treatment and prevention challenges, novel animal models to study hypnozoites and relapse, and hypotheses related to hypnozoite formation and activation.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

The parasite intraerythrocytic cycle and human circadian cycle are coupled during malaria infection.

During infections with the malaria parasites Plasmodium vivax, patients exhibit rhythmic fevers every 48 h. These fever cycles correspond with the time the parasites take to traverse the intraerythrocytic cycle (IEC). In other Plasmodium species that infect either humans or mice, the IEC is likely guided by a parasite-intrinsic clock [Rijo-Ferreiraet al., Science 368, 746-753 (2020); Smith et al., Science 368, 754-759 (2020)], suggesting that intrinsic clock mechanisms may be a fundamental feature of malaria parasites. Moreover, because Plasmodium cycle times are multiples of 24 h, the IECs may be coordinated with the host circadian clock(s). Such coordination could explain the synchronization of the parasite population in the host and enable alignment of IEC and circadian cycle phases. We utilized an ex vivo culture of whole blood from patients infected with P. vivax to examine the dynamics of the host circadian transcriptome and the parasite IEC transcriptome. Transcriptome dynamics revealed that the phases of the host circadian cycle and the parasite IEC are correlated across multiple patients, showing that the cycles are phase coupled. In mouse model systems, host-parasite cycle coupling appears to provide a selective advantage for the parasite. Thus, understanding how host and parasite cycles are coupled in humans could enable antimalarial therapies that disrupt this coupling.

Vaccines and monoclonal antibodies: new tools for malaria control.

SUMMARYMalaria remains one of the biggest health problems in the world. While significant reductions in malaria morbidity and mortality had been achieved from 2000 to 2015, the favorable trend has stalled, rather significant increases in malaria cases are seen in multiple areas. In 2022, there were 249 million estimated cases, and 608,000 malaria-related deaths, mostly in infants and children aged under 5 years, globally. Therefore, in addition to the expansion of existing anti-malarial control measures, it is critical to develop new tools, such as vaccines and monoclonal antibodies (mAbs), to fight malaria. In the last 2 years, the first and second malaria vaccines, both targeting Plasmodium falciparum circumsporozoite proteins (PfCSP), have been recommended by the World Health Organization to prevent P. falciparum malaria in children living in moderate to high transmission areas. While the approval of the two malaria vaccines is a considerable milestone in vaccine development, they have much room for improvement in efficacy and durability. In addition to the two approved vaccines, recent clinical trials with mAbs against PfCSP, blood-stage vaccines against P. falciparum or P. vivax, and transmission-blocking vaccine or mAb against P. falciparum have shown promising results. This review summarizes the development of the anti-PfCSP vaccines and mAbs, and recent topics in the blood- and transmission-blocking-stage vaccine candidates and mAbs. We further discuss issues of the current vaccines and the directions for the development of next-generation vaccines.

Functional and immunological mapping of domains of the reticulocyte binding protein, Plasmodium vivax PvRBP2a.

We previously described a novel Plasmodium vivax invasion mechanism into human reticulocytes via the PvRBP2a-CD98 receptor-ligand pair. We assessed the PvRBP2a epitopes involved in CD98 binding and recognised by antibodies from infected patients using linear epitope mapping. We identified two epitope clusters mediating PvRBP2a-CD98 interaction. One cluster named cluster B (PvRBP2a431-448, TAALKEKGKLLANLYNKL) was the target of antibody responses in P. vivax-infected humans. Peptides from each cluster were able to prevent live parasite invasion of human reticulocytes. These results provide new insights for development of a malaria blood stage vaccine against P. vivax.

Changing Clinical Epidemiology of Plasmodium vivax Malaria as Transmission Decreases: Population-Based Prospective Panel Survey in the Brazilian Amazon.

BACKGROUND: Malarial infections are often missed by microscopy, and most parasite carriers are asymptomatic in low-endemicity settings. Whether parasite detectability and its ability to elicit symptoms change as transmission declines remains unclear. METHODS: We performed a prospective panel survey with repeated measurements on the same participants over 12 months to investigate whether Plasmodium vivax detectability by microscopy and risk of symptoms upon infection varied during a community-wide larviciding intervention in the Amazon basin of Brazil that markedly reduced vector density. We screened 1096 to 1400 residents in the intervention site for malaria by microscopy and quantitative TaqMan assays at baseline and twice during intervention. RESULTS: We found that more P vivax infections than expected from their parasite densities measured by TaqMan assays were missed by microscopy as transmission decreased. At lower transmission, study participants appeared to tolerate higher P vivax loads without developing symptoms. We hypothesize that changes in the ratio between circulating parasites and those that accumulate in the bone marrow and spleen, by avoiding peripheral blood microscopy detection, account for decreased parasite detectability and lower risk of symptoms under low transmission. CONCLUSIONS: P vivax infections are more likely to be subpatent and remain asymptomatic as malaria transmission decreases.

Naturally Acquired Transmission-Blocking Immunity Against Different Strains of Plasmodium vivax in a Malaria-Endemic Area in Thailand.

BACKGROUND: Human immunity triggered by natural malaria infections impedes parasite transmission from humans to mosquitoes, leading to interest in transmission-blocking vaccines. However, immunity characteristics, especially strain specificity, remain largely unexplored. We investigated naturally acquired transmission-blocking immunity (TBI) against Plasmodium vivax, a major malaria parasite. METHODS: Using the direct membrane-feeding assay, we assessed TBI in plasma samples and examined the role of antibodies by removing immunoglobulins through protein G/L adsorption before mosquito feeding. Strain specificity was evaluated by conducting a direct membrane-feeding assay with plasma exchange. RESULTS: Blood samples from 47 patients with P vivax were evaluated, with 37 plasma samples successfully infecting mosquitoes. Among these, 26 showed inhibition before immunoglobulin depletion. Despite substantial immunoglobulin removal, 4 samples still exhibited notable inhibition, while 22 had reduced blocking activity. Testing against heterologous strains revealed some plasma samples with broad TBI and others with strain-specific TBI. CONCLUSIONS: Our findings indicate that naturally acquired TBI is mainly mediated by antibodies, with possible contributions from other serum factors. The transmission-blocking activity of plasma samples varied by the tested parasite strain, suggesting single polymorphic or multiple targets for naturally acquired TBI. These observations improve understanding of immunity against P vivax and hold implications for transmission-blocking vaccine development.

Malaria Species Positivity Rates Among Symptomatic Individuals Across Regions of Differing Transmission Intensities in Mainland Tanzania.

BACKGROUND: Recent data indicate that non-Plasmodium falciparum species may be more prevalent than thought in sub-Saharan Africa. Although Plasmodium malariae, Plasmodium ovale spp., and Plasmodium vivax are less severe than P. falciparum, treatment and control are more challenging, and their geographic distributions are not well characterized. METHODS: We randomly selected 3284 of 12 845 samples collected from cross-sectional surveys in 100 health facilities across 10 regions of Mainland Tanzania and performed quantitative real-time PCR to determine presence and parasitemia of each malaria species. RESULTS: P. falciparum was most prevalent, but P. malariae and P. ovale were found in all but 1 region, with high levels (>5%) of P. ovale in 7 regions. The highest P. malariae positivity rate was 4.5% in Mara and 8 regions had positivity rates ≥1%. We only detected 3 P. vivax infections, all in Kilimanjaro. While most nonfalciparum malaria-positive samples were coinfected with P. falciparum, 23.6% (n = 13 of 55) of P. malariae and 14.7% (n = 24 of 163) of P. ovale spp. were monoinfections. CONCLUSIONS: P. falciparum remains by far the largest threat, but our data indicate that malaria elimination efforts in Tanzania will require increased surveillance and improved understanding of the biology of nonfalciparum species.

A Novel Ex Vivo Assay to Evaluate Functional Effectiveness of Plasmodium vivax Transmission-Blocking Vaccine Using Pvs25 Transgenic Plasmodium berghei.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs. METHODS: Transgenic murine malaria parasites (Plasmodium berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA). RESULTS: We describe the development of transmission-competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen. CONCLUSIONS: The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax-infected patients in endemic areas for evaluation.

Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45.

Gamete surface protein P48/45 has been shown to be important for male gamete fertility and a strong candidate for the development of a malaria transmission-blocking vaccine (TBV). However, TBV development for Plasmodium vivax homolog Pvs48/45 has been slow because of a number of challenges: availability of conformationally suitable recombinant protein; the lack of an in vivo challenge model; and the inability to produce P. vivax gametocytes in culture to test transmission-blocking activity of antibodies. To support ongoing efforts to develop Pvs48/45 as a potential vaccine candidate, we initiated efforts to develop much needed reagents to move the field forward. We generated monoclonal antibodies (mAbs) directed against Pvs48/45 and characterized putative functional domains in Pvs48/45 using recombinant fragments corresponding to domains D1-D3 and their biological functionality through ex vivo direct membrane feeding assays (DMFAs) using P. vivax parasites from patients in a field setting in Brazil. While some mAbs partially blocked oocyst development in the DMFA, one mAb caused a significant enhancement of the infectivity of gametocytes in the mosquitoes. Individual mAbs exhibiting blocking and enhancing activities recognized non-overlapping epitopes in Pvs48/45. Further characterization of precise epitopes recognized by transmission-reducing and -enhancing antibodies will be crucial to design an effective immunogen with optimum transmission-reducing potential.

Autochthonous Plasmodium vivax Infections, Florida, USA, 2023.

During May-July 2023, a cluster of 7 patients at local hospitals in Florida, USA, received a diagnosis of Plasmodium vivax malaria. Whole-genome sequencing of the organism from 4 patients and phylogenetic analysis with worldwide representative P. vivax genomes indicated probable single parasite introduction from Central/South America.

Plasmodium vivax Infections among Immigrants from China Traveling to the United States.

Beginning in 2023, we observed increased Plasmodium vivax malaria cases at an institution in Los Angeles, California, USA. Most cases were among migrants from China who traveled to the United States through South and Central America. US clinicians should be aware of possible P. vivax malaria among immigrants from China.

Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

Therapeutic efficacy of pyronaridine-artesunate (Pyramax) in treating Plasmodium vivax malaria in the central highlands of Vietnam.

The emergence and spread of chloroquine-resistant Plasmodium vivax have necessitated the assessment of alternative blood schizonticidal drugs. In Vietnam, chloroquine-resistant P. vivax malaria has been reported. In an open-label, single-arm trial, the safety, tolerability, and efficacy of pyronaridine-artesunate (Pyramax, PA) was evaluated in Dak Nong province, Vietnam. A 3-day course of PA was administered to adults and children (≥20 kg) infected with P. vivax. Patients also received primaquine (0.25 mg/kg daily for 14 days). PA was well tolerated with transient asymptomatic increases in liver transaminases. The per-protocol proportion of patients with day 42 PCR-unadjusted adequate clinical and parasitological response was 96.0% (95% CI, 84.9%-99.0%, n = 48/50). The median parasite clearance time was 12 h (range, 12-36 h), with a median fever clearance time of 24 h (range, 12-60 h). Single nucleotide polymorphisms (SNPs) as potential genetic markers of reduced drug susceptibility were analyzed in three putative drug resistance markers, Pvcrt-o, Pvmdr1, and PvK12. Insertion at position K10 of the Pvcrt-o gene was found in 74.6% (44/59) of isolates. Pvmdr1 SNPs at Y976F and F1076L were present in 61% (36/59) and 78% (46/59), respectively. Amplification of Pvmdr1 gene (two copies) was found in 5.1% (3/59) of parasite samples. Only 5.1% (3/59) of isolates had mutation 552I of the PvK12 gene. Overall, PA rapidly cleared P. vivax blood asexual stages and was highly efficacious in treating vivax malaria, with no evidence of artemisinin resistance found. PA provides an alternative to chloroquine treatment for vivax malaria in Vietnam. CLINICAL TRIALS: This study is registered with the Australian New Zealand Clinical Trials Registry as ACTRN12618001429246.

Delayed gametocyte clearance in Plasmodium vivax malaria is associated with polymorphisms in the cytochrome P450 reductase (CPR).

Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.

The human malaria-Aotus monkey model: a historical perspective in antimalarial chemotherapy research at the Gorgas Memorial Laboratory-Panama.

The human malaria-Aotus monkey model has served the malaria research community since its inception in 1966 at the Gorgas Memorial Laboratory (GML) in Panama. Spanning over five decades, this model has been instrumental in evaluating the in vivo efficacy and pharmacokinetics of a wide array of candidate antimalarial drugs, whether used singly or in combination. The animal model could be infected with drug-resistant and susceptible Plasmodium falciparum and Plasmodium vivax strains that follow a characteristic and reproducible course of infection, remarkably like human untreated and treated infections. Over the years, the model has enabled the evaluation of several synthetic and semisynthetic endoperoxides, for instance, artelinic acid, artesunate, artemether, arteether, and artemisone. These compounds have been evaluated alone and in combination with long-acting partner drugs, commonly referred to as artemisinin-based combination therapies, which are recommended as first-line treatment against uncomplicated malaria. Further, the model has also supported the evaluation of the primaquine analog tafenoquine against blood stages of P. vivax, contributing to its progression to clinical trials and eventual approval. Besides, the P. falciparum/Aotus model at GML has also played a pivotal role in exploring the biology, immunology, and pathogenesis of malaria and in the characterization of drug-resistant P. falciparum and P. vivax strains. This minireview offers a historical overview of the most significant contributions made by the Panamanian owl monkey (Aotus lemurinus lemurinus) to malaria chemotherapy research.

Plasmodium vivax infection alters the peripheral immunoregulatory network of CD4 T follicular cells and B cells.

Regulatory and effector cell responses to Plasmodium vivax, the most common human malaria parasite outside Africa, remain understudied in naturally infected populations. Here, we describe peripheral CD4+ T- and B-cell populations during and shortly after an uncomplicated P. vivax infection in 38 continuously exposed adult Amazonians. Consistent with previous observations, we found an increased frequency in CD4+ CD45RA- CD25+ FoxP3+ T regulatory cells that express the inhibitory molecule CTLA-4 during the acute infection, with a sustained expansion of CD21- CD27- atypical memory cells within the CD19+ B-cell compartment. Both Th1- and Th2-type subsets of CXCR5+ ICOShi PD-1+ circulating T follicular helper (cTfh) cells, which are thought to contribute to antibody production, were induced during P. vivax infection, with a positive correlation between overall cTfh cell frequency and IgG antibody titers to the P. vivax blood-stage antigen MSP119 . We identified significant changes in cell populations that had not been described in human malaria, such as an increased frequency of CTLA-4+ T follicular regulatory cells that antagonize Tfh cells, and a decreased frequency of circulating CD24hi CD27+ B regulatory cells in response to acute infection. In conclusion, we disclose a complex immunoregulatory network that is critical to understand how naturally acquired immunity develops in P. vivax malaria.

Evidence of Plasmodium vivax circulation in western and eastern regions of Senegal: implications for malaria control.

BACKGROUND: Malaria elimination in Senegal requires accurate diagnosis of all Plasmodium species. Plasmodium falciparum is the most prevalent species in Senegal, although Plasmodium malariae, Plasmodium ovale, and recently Plasmodium vivax have also been reported. Nonetheless, most malaria control tools, such as Histidine Rich Protein 2 rapid diagnosis test (PfHRP2-RDT,) can only diagnose P. falciparum. Thus, PfHRP2-RDT misses non-falciparum species and P. falciparum infections that fall below the limit of detection. These limitations can be addressed using highly sensitive Next Generation Sequencing (NGS). This study assesses the burden of the four different Plasmodium species in western and eastern regions of Senegal using targeted PCR amplicon sequencing. METHODS: Three thousand samples from symptomatic and asymptomatic individuals in 2021 from three sites in Senegal (Sessene, Diourbel region; Parcelles Assainies, Kaolack region; Gabou, Tambacounda region) were collected. All samples were tested using PfHRP2-RDT and photoinduced electron transfer polymerase chain reaction (PET-PCR), which detects all Plasmodium species. Targeted sequencing of the nuclear 18S rRNA and the mitochondrial cytochrome B genes was performed on PET-PCR positive samples. RESULTS: Malaria prevalence by PfHRP2-RDT showed 9.4% (94/1000) and 0.2% (2/1000) in Diourbel (DBL) and Kaolack (KL), respectively. In Tambacounda (TAM) patients who had malaria symptoms and had a negative PfHRP2-RDT were enrolled. The PET-PCR had a positivity rate of 23.5% (295/1255) overall. The PET-PCR positivity rate was 37.6%, 12.3%, and 22.8% in Diourbel, Kaolack, and Tambacounda, respectively. Successful sequencing of 121/295 positive samples detected P. falciparum (93%), P. vivax (2.6%), P. malariae (4.4%), and P. ovale wallikeri (0.9%). Plasmodium vivax was co-identified with P. falciparum in thirteen samples. Sequencing also detected two PfHRP2-RDT-negative mono-infections of P. vivax in Tambacounda and Kaolack. CONCLUSION: The findings demonstrate the circulation of P. vivax in western and eastern Senegal, highlighting the need for improved malaria control strategies and accurate diagnostic tools to better understand the prevalence of non-falciparum species countrywide.

Efficacy and safety of chloroquine plus primaquine for the treatment of Plasmodium vivax malaria in Hamusit site, Northwestern Ethiopia.

BACKGROUND: Plasmodium vivax malaria is still an important public health problem in Ethiopia. Unlike Plasmodium falciparum, P. vivax has a dormant liver stage (hypnozoite) that can be a risk of recurrent vivax malaria unless treated by radical cure with primaquine. Drug resistance to chloroquine is threatening malaria control and elimination efforts. This study assessed the therapeutic efficacy and safety of chloroquine plus 14 days of primaquine on P. vivax infection based on parasitological, clinical, and haematological parameters. METHODS: A single-arm in vivo prospective therapeutic efficacy study was conducted to assess the clinical and parasitological response to the first-line treatment of P. vivax in Ethiopia, chloroquine plus 14 days low dose of (0.25 mg/kg/day) primaquine between December 2022 and March 2023 at Hamusit Health Centre using the standard World Health Organization (WHO) protocol. A total of 100 study participants with P. vivax mono-infection who were over 6 months old were enrolled and monitored for adequate clinical and parasitological responses for 42 days. The WHO double-entry Excel sheet and SPSS v.25 software were used for Kaplan-Meier survival analysis, and a paired t-test was used for analysis of haemoglobin improvements between follow up days. RESULTS: A total of 100 patients were enrolled among those, 96% cases were rural residents, 93% had previous malaria exposure, and predominant age group was 5-15 years (61%). 92.6% (95% CI 85.1-96.4%) of enrolled patients were adequate clinical and parasitological response, and 7.4% (95% CI 3.6-14.9%) recurrences were observed among treated patients. The fever and parasite clearance rate on day 3 were 98% and 94%, respectively. The baseline haemoglobin levels improved significantly compared to those days 14 and 42 (p < 0.001). No serious adverse event was observed during the study period. CONCLUSIONS: In this study, co-administration of chloroquine with primaquine was efficacious and well-tolerated with fast resolution of fever and high parasites clearance rate. However, the 7.4% failure is reported is alarming that warrant further monitoring of the therapeutic efficacy study of P. vivax.