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.

Higher-Dose Primaquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: In most of the Americas, the recommended treatment to prevent relapse of Plasmodium vivax malaria is primaquine at a total dose of 3.5 mg per kilogram of body weight, despite evidence of only moderate efficacy. METHODS: In this trial conducted in Brazil, we evaluated three primaquine regimens to prevent relapse of P. vivax malaria in children at least 5 years of age and in adults with microscopy-confirmed P. vivax monoinfection. All the patients received directly observed chloroquine for 3 days (total dose, 25 mg per kilogram). Group 1 received a total primaquine dose of 3.5 mg per kilogram (0.5 mg per kilogram per day) over 7 days with unobserved administration; group 2 received the same regimen as group 1 but with observed administration; and group 3 received a total primaquine dose of 7.0 mg per kilogram over 14 days (also 0.5 mg per kilogram per day) with observed administration. We monitored the patients for 168 days. RESULTS: We enrolled 63 patients in group 1, 96 in group 2, and 95 in group 3. The median age of the patients was 22.4 years (range, 5.4 to 79.8). By day 28, three P. vivax recurrences were observed: 2 in group 1 and 1 in group 2. By day 168, a total of 70 recurrences had occurred: 24 in group 1, 34 in group 2, and 12 in group 3. No serious adverse events were noted. On day 168, the percentage of patients without recurrence was 58% (95% confidence interval [CI], 44 to 70) in group 1, 59% (95% CI, 47 to 69) in group 2, and 86% (95% CI, 76 to 92) in group 3. Survival analysis showed a difference in the day 168 recurrence-free percentage of 27 percentage points (97.5% CI, 10 to 44; P<0.001) between group 1 and group 3 and a difference of 27 percentage points (97.5% CI, 12 to 42; P<0.001) between group 2 and group 3. CONCLUSIONS: The administration of primaquine at a total dose of 7.0 mg per kilogram had higher efficacy in preventing relapse of P. vivax malaria than a total dose of 3.5 mg per kilogram through day 168. (Supported by the U.S. Agency for International Development; ClinicalTrials.gov number, NCT03610399.).

Tafenoquine following G6PD screening versus primaquine for the treatment of vivax malaria in Brazil: A cost-effectiveness analysis using a transmission model.

BACKGROUND: Malaria transmission modelling has demonstrated the potential impact of semiquantitative glucose-6-phosphate dehydrogenase (G6PD) testing and treatment with single-dose tafenoquine for Plasmodium vivax radical cure but has not investigated the associated costs. This study evaluated the cost-effectiveness of P. vivax treatment with tafenoquine after G6PD testing using a transmission model. METHODS AND FINDINGS: We explored the cost-effectiveness of using tafenoquine after G6PD screening as compared to usual practice (7-day low-dose primaquine (0.5 mg/kg/day) without G6PD screening) in Brazil using a 10-year time horizon with 5% discounting considering 4 scenarios: (1) tafenoquine for adults only assuming 66.7% primaquine treatment adherence; (2) tafenoquine for adults and children aged >2 years assuming 66.7% primaquine adherence; (3) tafenoquine for adults only assuming 90% primaquine adherence; and (4) tafenoquine for adults only assuming 30% primaquine adherence. The incremental cost-effectiveness ratios (ICERs) were estimated by dividing the incremental costs by the disability-adjusted life years (DALYs) averted. These were compared to a willingness to pay (WTP) threshold of US$7,800 for Brazil, and one-way and probabilistic sensitivity analyses were performed. All 4 scenarios were cost-effective in the base case analysis using this WTP threshold with ICERs ranging from US$154 to US$1,836. One-way sensitivity analyses showed that the results were most sensitive to severity and mortality due to vivax malaria, the lifetime and number of semiquantitative G6PD analysers needed, cost per malaria episode and per G6PD test strips, and life expectancy. All scenarios had a 100% likelihood of being cost-effective at the WTP threshold. The main limitations of this study are due to parameter uncertainty around our cost estimates for low transmission settings, the costs of G6PD screening, and the severity of vivax malaria. CONCLUSIONS: In our modelling study that incorporated impact on transmission, tafenoquine prescribed after a semiquantitative G6PD testing was highly likely to be cost-effective in Brazil. These results demonstrate the potential health and economic importance of ensuring safe and effective radical cure.

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.

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.

Comparison of lumefantrine, mefloquine, and piperaquine concentrations between capillary plasma and venous plasma samples in pregnant women with uncomplicated falciparum and vivax malaria.

Capillary samples offer practical benefits compared with venous samples for the measurement of drug concentrations, but the relationship between the two measures varies between different drugs. We measured the concentrations of lumefantrine, mefloquine, piperaquine in 270 pairs of venous plasma and concurrent capillary plasma samples collected from 270 pregnant women with uncomplicated falciparum or vivax malaria. The median and range of venous plasma concentrations included in this study were 447.5 ng/mL (8.81-3,370) for lumefantrine (day 7, n = 76, median total dose received 96.0 mg/kg), 17.9 ng/mL (1.72-181) for desbutyl-lumefantrine, 1,885 ng/mL (762-4,830) for mefloquine (days 3-21, n = 90, median total dose 24.9 mg/kg), 641 ng/mL (79.9-1,950) for carboxy-mefloquine, and 51.8 ng/mL (3.57-851) for piperaquine (days 3-21, n = 89, median total dose 52.2 mg/kg). Although venous and capillary plasma concentrations showed a high correlation (Pearson's correlation coefficient: 0.90-0.99) for all antimalarials and their primary metabolites, they were not directly interchangeable. Using the concurrent capillary plasma concentrations and other variables, the proportions of venous plasma samples predicted within a ±10% precision range was 34% (26/76) for lumefantrine, 36% (32/89) for desbutyl-lumefantrine, 74% (67/90) for mefloquine, 82% (74/90) for carboxy-mefloquine, and 24% (21/89) for piperaquine. Venous plasma concentrations of mefloquine, but not lumefantrine and piperaquine, could be predicted by capillary plasma samples with an acceptable level of agreement. Capillary plasma samples can be utilized for pharmacokinetic and clinical studies, but caution surrounding cut-off values is required at the individual level.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT01054248.

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.

Malaria.

Malaria is resurging in many African and South American countries, exacerbated by COVID-19-related health service disruption. In 2021, there were an estimated 247 million malaria cases and 619 000 deaths in 84 endemic countries. Plasmodium falciparum strains partly resistant to artemisinins are entrenched in the Greater Mekong region and have emerged in Africa, while Anopheles mosquito vectors continue to evolve physiological and behavioural resistance to insecticides. Elimination of Plasmodium vivax malaria is hindered by impractical and potentially toxic antirelapse regimens. Parasitological diagnosis and treatment with oral or parenteral artemisinin-based therapy is the mainstay of patient management. Timely blood transfusion, renal replacement therapy, and restrictive fluid therapy can improve survival in severe malaria. Rigorous use of intermittent preventive treatment in pregnancy and infancy and seasonal chemoprevention, potentially combined with pre-erythrocytic vaccines endorsed by WHO in 2021 and 2023, can substantially reduce malaria morbidity. Improved surveillance, better access to effective treatment, more labour-efficient vector control, continued drug development, targeted mass drug administration, and sustained political commitment are required to achieve targets for malaria reduction by the end of this decade.

Primaquine radical cure in patients with Plasmodium falciparum malaria in areas co-endemic for P falciparum and Plasmodium vivax (PRIMA): a multicentre, open-label, superiority randomised controlled trial.

BACKGROUND: In areas co-endemic for Plasmodium vivax and Plasmodium falciparum there is an increased risk of P vivax parasitaemia following P falciparum malaria. Radical cure is currently only recommended for patients presenting with P vivax malaria. Expanding the indication for radical cure to patients presenting with P falciparum malaria could reduce their risk of subsequent P vivax parasitaemia. METHODS: We did a multicentre, open-label, superiority randomised controlled trial in five health clinics in Bangladesh, Indonesia, and Ethiopia. In Bangladesh and Indonesia, patients were excluded if they were younger than 1 year, whereas in Ethiopia patients were excluded if they were younger than 18 years. Patients with uncomplicated P falciparum monoinfection who had fever or a history of fever in the 48 h preceding clinic visit were eligible for enrolment and were required to have a glucose-6-dehydrogenase (G6PD) activity of 70% or greater. Patients received blood schizontocidal treatment (artemether-lumefantrine in Ethiopia and Bangladesh and dihydroartemisinin-piperaquine in Indonesia) and were randomly assigned (1:1) to receive either high-dose short-course oral primaquine (intervention arm; total dose 7 mg/kg over 7 days) or standard care (standard care arm; single dose oral primaquine of 0·25 mg/kg). Random assignment was done by an independent statistician in blocks of eight by use of sealed envelopes. All randomly assigned and eligible patients were included in the primary and safety analyses. The per-protocol analysis excluded those who did not complete treatment or had substantial protocol violations. The primary endpoint was the incidence risk of P vivax parasitaemia on day 63. This trial is registered at ClinicalTrials.gov, NCT03916003. FINDINGS: Between Aug 18, 2019, and March 14, 2022, a total of 500 patients were enrolled and randomly assigned, and 495 eligible patients were included in the intention-to-treat analysis (246 intervention and 249 control). The incidence risk of P vivax parasitaemia at day 63 was 11·0% (95% CI 7·5-15·9) in the standard care arm compared with 2·5% (1·0-5·9) in the intervention arm (hazard ratio 0·20, 95% CI 0·08-0·51; p=0·0009). The effect size differed with blood schizontocidal treatment and site. Routine symptom reporting on day 2 and day 7 were similar between groups. In the first 42 days, there were a total of four primaquine-related adverse events reported in the standard care arm and 26 in the intervention arm; 132 (92%) of all 143 adverse events were mild. There were two serious adverse events in the intervention arm, which were considered unrelated to the study drug. None of the patients developed severe anaemia (defined as haemoglobin <5 g/dL). INTERPRETATION: In patients with a G6PD activity of 70% or greater, high-dose short-course primaquine was safe and relatively well tolerated and reduced the risk of subsequent P vivax parasitaemia within 63 days by five fold. Universal radical cure therefore potentially offers substantial clinical, public health, and operational benefits, but these benefits will vary with endemic setting. FUNDING: Australian Academy of Science Regional Collaborations Program, Bill & Melinda Gates Foundation, and National Health and Medical Research Council.

Chloroquine has shown high therapeutic efficacy against uncomplicated Plasmodium vivax malaria in southern Ethiopia: seven decades after its introduction.

BACKGROUND: Plasmodium vivax malaria is a leading cause of morbidity in Ethiopia. The first-line treatment for P. vivax is chloroquine (CQ) and primaquine (PQ), but there have been local reports of CQ resistance. A clinical study was conducted to determine the efficacy of CQ for the treatment of P. vivax malaria in southern Ethiopia. METHODS: In 2021, patients with P. vivax mono-infection and uncomplicated malaria were enrolled and treated with 25 mg/kg CQ for 3 consecutive days. Patients were followed for 28 days according to WHO guidelines. The data were analysed using per-protocol (PP) and Kaplan‒Meier (K‒M) analyses to estimate the risk of recurrent P. vivax parasitaemia on day 28. RESULTS: A total of 88 patients were enrolled, 78 (88.6%) of whom completed the 28 days of follow-up. Overall, 76 (97.4%) patients had adequate clinical and parasitological responses, and two patients had late parasitological failures. The initial therapeutic response was rapid, with 100% clearance of asexual parasitaemia within 48 h. CONCLUSION: Despite previous reports of declining chloroquine efficacy against P. vivax, CQ retains high therapeutic efficacy in southern Ethiopia, supporting the current national treatment guidelines. Ongoing clinical monitoring of CQ efficacy supported by advanced molecular methods is warranted to inform national surveillance and ensure optimal treatment guidelines.

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.

Radical cure for Plasmodium vivax malaria after G6PD qualitative testing in four provinces in Cambodia, results from Phase I implementation.

BACKGROUND: Cambodia aims to eliminate all forms of malaria by 2025. In 2020, 90% of all malaria cases were Plasmodium vivax. Thus, preventing P. vivax and relapse malaria is a top priority for elimination. 14-day primaquine, a World Health Organization-recommended radical cure treatment regimen, specifically targets dormant hypnozoites in the liver to prevent relapse. Cambodia introduced P. vivax radical cure with primaquine after glucose-6-phosphate dehydrogenase (G6PD) qualitative testing in 2019. This paper presents Cambodia's radical cure Phase I implementation results and assesses the safety, effectiveness, and feasibility of the programme prior to nationwide scale up. METHODS: Phase I implementation was carried out in 88 select health facilities (HFs) across four provinces. Males over 20kgs with confirmed P. vivax or mixed (P. vivax and Plasmodium falciparum) infections were enrolled. A descriptive analysis evaluated the following: successful referral to health facilities, G6PD testing results, and self-reported 14-day treatment adherence. P. vivax incidence was compared before and after radical cure rollout and a controlled interrupted time series analysis compared the estimated relapse rate between implementation and non-implementation provinces before and after radical cure. RESULTS: In the 4 provinces from November 2019 to December 2020, 3,239 P. vivax/mixed infections were reported, 1,282 patients underwent G6PD deficiency testing, and 959 patients received radical cure, achieving 29.6% radical cure coverage among all P. vivax/mixed cases and 98.8% coverage among G6PD normal patients. Among those who initiated radical cure, 747 patients (78%) completed treatment. Six patients reported side effects. In implementation provinces, an average 31.8 relapse cases per month were estimated signaling a 90% (286 cases) reduction in relapse compared to what would be expected if radical cure was not implemented. CONCLUSIONS: Plasmodium vivax radical cure is a crucial tool for malaria elimination in Cambodia. The high coverage of radical cure initiation and adherence among G6PD normal patients demonstrated the high feasibility of providing radical cure at point of care in Cambodia. Incomplete referral from community to HFs and limited capacity of HF staff to conduct G6PD testing in high burden areas led to lower coverage of G6PD testing. Phase I implementation informed approaches to improve referral completion and patient adherence during the nationwide expansion of radical cure in 2021.

Kinetics of glucose-6-phosphate dehydrogenase (G6PD) activity during Plasmodium vivax infection: implications for early radical malaria treatment.

BACKGROUND: Plasmodium vivax relapses due to dormant liver hypnozoites can be prevented with primaquine. However, the dose must be adjusted in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. In French Guiana, assessment of G6PD activity is typically delayed until day (D)14 to avoid the risk if misclassification. This study assessed the kinetics of G6PD activity throughout P. vivax infection to inform the timing of treatment. METHODS: For this retrospective monocentric study, data on G6PD activity between D1 and D28 after treatment initiation with chloroquine or artemisinin-based combination therapy were collected for patients followed at Cayenne Hospital, French Guiana, between January 2018 and December 2020. Patients were divided into three groups based on the number of available G6PD activity assessments: (i) at least two measurements during the P. vivax malaria infection; (ii) two measurements: one during the current infection and one previously; (iii) only one measurement during the malaria infection. RESULTS: In total, 210 patients were included (80, 20 and 110 in groups 1, 2 and 3, respectively). Data from group 1 showed that G6PD activity remained stable in each patient over time (D1, D3, D7, D14, D21, D28). None of the patients with normal G6PD activity during the initial phase (D1-D3) of the malaria episode (n = 44) was categorized as G6PD-deficient at D14. Patients with G6PD activity < 80% at D1 or D3 showed normal activity at D14. Sex and reticulocyte count were statistically associated with G6PD activity variation. In the whole sample (n = 210), no patient had severe G6PD deficiency (< 10%) and only three between 10 and 30%, giving a G6PD deficiency prevalence of 1.4%. Among the 100 patients from group 1 and 2, 30 patients (26.5%) were lost to follow-up before primaquine initiation. CONCLUSIONS: In patients treated for P. vivax infection, G6PD activity did not vary over time. Therefore, G6PD activity on D1 instead of D14 could be used for primaquine dose-adjustment. This could allow earlier radical treatment with primaquine, that could have a public health impact by decreasing early recurrences and patients lost to follow-up before primaquine initiation. This hypothesis needs to be confirmed in larger prospective studies.

Optimal balance of benefit versus risk for tafenoquine in the treatment of Plasmodium vivax malaria.

A single 300 mg dose of tafenoquine (an 8-aminoquinoline), in combination with a standard 3-day course of chloroquine, is approved in several countries for the radical cure (prevention of relapse) of Plasmodium vivax malaria in patients aged ≥ 16 years. Despite this, questions have arisen on the optimal dose of tafenoquine. Before the availability of tafenoquine, a 3-day course of chloroquine in combination with the 8-aminoquinoline primaquine was the only effective radical cure for vivax malaria. The World Health Organization (WHO)-recommended standard regimen is 14 days of primaquine 0.25 mg/kg/day or 7 days of primaquine 0.5 mg/kg/day in most regions, or 14 days of primaquine 0.5 mg/kg/day in East Asia and Oceania, however the long treatment courses of 7 or 14 days may result in poor adherence and, therefore, low treatment efficacy. A single dose of tafenoquine 300 mg in combination with a 3-day course of chloroquine is an important advancement for the radical cure of vivax malaria in patients without glucose-6-phosphate dehydrogenase (G6PD) deficiency, as the use of a single-dose treatment will improve adherence. Selection of a single 300 mg dose of tafenoquine for the radical cure of P. vivax malaria was based on collective efficacy and safety data from 33 studies involving more than 4000 trial participants who received tafenoquine, including over 800 subjects who received the 300 mg single dose. The safety profile of single-dose tafenoquine 300 mg is similar to that of standard-dosage primaquine 0.25 mg/kg/day for 14 days. Both primaquine and tafenoquine can cause acute haemolytic anaemia in individuals with G6PD deficiency; severe haemolysis can lead to anaemia, kidney damage, and, in some cases, death. Therefore, relapse prevention using an 8-aminoquinoline must be balanced with the need to avoid clinical haemolysis associated with G6PD deficiency. To minimize this risk, the WHO recommends G6PD testing for all individuals before the administration of curative doses of 8-aminoquinolines. In this article, the authors review key efficacy and safety data from the pivotal trials of tafenoquine and argue that the currently approved dose represents a favourable benefit-risk profile.

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.

Impact of Chemoprophylaxis on Plasmodium vivax and Plasmodium ovale Infection Among Civilian Travelers: A Nested Case-Control Study With a Counterfactual Approach on 862 Patients.

BACKGROUND: The impact of chemoprophylaxis targeting Plasmodium falciparum on Plasmodium vivax and Plasmodium ovale, which may remain quiescent as hypnozoites in the liver, is debated. METHODS: We conducted a nested case-control analysis of the outcomes of P. vivax and P. ovale infections in imported malaria cases in France among civilian travelers from 1 January 2006, to 31 December 2017. Using adjusted logistic regression, we assessed the effect of chemoprophylaxis on the incubation period, time from symptoms to diagnosis, management, blood results, symptoms, and hospitalization duration. We analyzed the effect of blood-stage drugs (doxycycline, mefloquine, chloroquine, chloroquine-proguanil) or atovaquone-proguanil on the incubation period. We used a counterfactual approach to ascertain the causal effect of chemoprophylaxis on postinfection characteristics. RESULTS: Among 247 P. vivax- and 615 P. ovale-infected travelers, 30% and 47%, respectively, used chemoprophylaxis, and 7 (3%) and 8 (1%) were severe cases. Chemoprophylaxis users had a greater risk of presenting symptoms >2 months after returning for both species (P. vivax odds ratio [OR], 2.91 [95% confidence interval {CI}, 1.22-6.95], P = .02; P. ovale OR, 2.28 [95% CI, 1.47-3.53], P < .001). Using drugs only acting on the blood stage was associated with delayed symptom onset after 60 days, while using atovaquone-proguanil was not. CONCLUSIONS: Civilian travelers infected with P. vivax or P. ovale reporting chemoprophylaxis use, especially of blood-stage agents, had a greater risk of delayed onset of illness. The impact of chemoprophylaxis on the outcomes of infection with relapse-causing species calls for new chemoprophylaxis acting against erythrocytic and liver stages.

Novel Transmission-Blocking Antimalarials Identified by High-Throughput Screening of Plasmodium berghei Ookluc.

Safe and effective malaria transmission-blocking chemotherapeutics would allow a community-level approach to malaria control and eradication efforts by targeting the mosquito sexual stage of the parasite life cycle. However, only a single drug, primaquine, is currently approved for use in reducing transmission, and drug toxicity limits its widespread implementation. To address this limitation in antimalarial chemotherapeutics, we used a recently developed transgenic Plasmodium berghei line, Ookluc, to perform a series of high-throughput in vitro screens for compounds that inhibit parasite fertilization, the initial step of parasite development within the mosquito. Screens of antimalarial compounds, approved drug collections, and drug-like molecule libraries identified 185 compounds that inhibit parasite maturation to the zygote form. Seven compounds were further characterized to block gametocyte activation or to be cytotoxic to formed zygotes. These were further validated in mosquito membrane-feeding assays using Plasmodium falciparum and P. vivax. This work demonstrates that high-throughput screens using the Ookluc line can identify compounds that are active against the two most relevant human Plasmodium species and provides a list of compounds that can be explored for the development of new antimalarials to block transmission.

Cytochrome P450 2D6 profiles and anti-relapse efficacy of tafenoquine against Plasmodium vivax in Australian Defence Force personnel.

Plasmodium vivax infections and relapses remain a major health problem for malaria-endemic countries, deployed military personnel, and travelers. Presumptive anti-relapse therapy and radical cure using the 8-aminoquinoline drugs primaquine and tafenoquine are necessary to prevent relapses. Although it has been demonstrated that the efficacy of primaquine is associated with Cytochrome P450 2D6 (CYP2D6) activity, there is insufficient data on the role of CYP2D6 in the anti-relapse efficacy of tafenoquine. We investigated the relationship between CYP2D6 activity status and tafenoquine efficacy in preventing P. vivax relapses retrospectively using plasma samples collected from Australian Defence Force personnel deployed to Papua New Guinea and Timor-Leste who participated in clinical trials of tafenoquine during 1999-2001. The CYP2D6 gene was amplified from plasma samples and fully sequenced from 92 participant samples, comprised of relapse (n = 31) and non-relapse (n = 61) samples, revealing 14 different alleles. CYP2D6 phenotypes deduced from combinations of CYP2D6 alleles predicted that among 92 participants 67, 15, and 10 were normal, intermediate, and poor metabolizers, respectively. The deduced CYP2D6 phenotype did not correlate with the corresponding participant's plasma tafenoquine concentrations that were determined in the early 2000s by high-performance liquid chromatography or liquid chromatography-mass spectrometry. Furthermore, the deduced CYP2D6 phenotype did not associate with P. vivax relapse outcomes. Our results indicate that CYP2D6 does not affect plasma tafenoquine concentrations and the efficacy of tafenoquine in preventing P. vivax relapses in the assessed Australian Defence Force personnel.

Antimalarial drug efficacy and resistance in malaria-endemic countries in HANMAT-PIAM_net countries of the Eastern Mediterranean Region 2016-2020: Clinical and genetic studies.

INTRODUCTION: The World Health Organization recommends regular monitoring of the efficacy of nationally recommended antimalarial drugs. We present the results of studies on the efficacy of recommended antimalarials and molecular markers of artemisinin and partner resistance in Afghanistan, Pakistan, Somalia, Sudan and Yemen. METHODS: Single-arm prospective studies were conducted to evaluate the efficacy of artesunate-sulfadoxine-pyrimethamine (ASSP) in Afghanistan and Pakistan, artemether-lumefantrine (AL) in all countries, or dihydroartemisinin-piperaquine (DP) in Sudan for the treatment of Plasmodium falciparum. The efficacy of chloroquine (CQ) and AL for the treatment of Plasmodium vivax was evaluated in Afghanistan and Somalia, respectively. Patients were treated and monitored for 28 (CQ, ASSP and AL) or 42 (DP) days. Polymerase chain reaction (PCR)-corrected cure rate and parasite positivity rate at Day 3 were estimated. Mutations in the P. falciparum kelch 13 (Pfk13) gene and amplifications of plasmepsin (Pfpm2) and multidrug resistance-1 (Pfmdr-1) genes were also studied. RESULTS: A total of 1680 (249 for ASSP, 1079 for AL and 352 for DP) falciparum cases were successfully assessed. A PCR-adjusted ASSP cure rate of 100% was observed in Afghanistan and Pakistan. For AL, the cure rate was 100% in all but four sites in Sudan, where cure rates ranged from 92.1% to 98.8%. All but one patient were parasite-free at Day 3. For P. vivax, cure rates were 98.2% for CQ and 100% for AL. None of the samples from Afghanistan, Pakistan and Yemen had a Pfk13 mutation known to be associated with artemisinin resistance. In Sudan, the validated Pfk13 R622I mutation accounted for 53.8% (14/26) of the detected non-synonymous Pfk13 mutations, most of which were repeatedly detected in Gadaref. A prevalence of 2.7% and 9.3% of Pfmdr1 amplification was observed in Pakistan and Yemen, respectively. CONCLUSION: High efficacy of ASSP, AL and DP in the treatment of uncomplicated falciparum infection and of CQ and AL in the treatment of P. vivax was observed in the respective countries. The repeated detection of a relatively high rate of Pfk13 R622I mutation in Sudan underscores the need for close monitoring of the efficacy of recommended ACTs, parasite clearance rates and Pfk13 mutations in Sudan and beyond. Registration numbers of the trials: ACTRN12622000944730 and ACTRN12622000873729 for Afghanistan, ACTRN12620000426987 and ACTRN12617001025325 for Pakistan, ACTRN12618001224213 for Somalia, ACTRN12617000276358, ACTRN12622000930785 and ACTRN12618001800213 for Sudan and ACTRN12617000283370 for Yemen.

Analysis of the relapse of imported Plasmodium vivax and Plasmodium ovale in five provinces of China.

BACKGROUND: The global battle against malaria is facing formidable challenges, particularly in controlling Plasmodium vivax and Plasmodium ovale, whose cases have not been reduced as effectively as Plasmodium falciparum because of their relapse. This study investigates the current situation and underlying factors contributing to relapse or recrudescence of imported cases of P. vivax and P. ovale, and seeks to provide a reference for reducing relapse or recrudescence in malaria-free areas and offers a scientific basis for designing strategies to prevent imported re-transmission. METHODS: This study analysed imported P. vivax and P. ovale in Anhui, Zhejiang, Henan, Hubei, and Guangxi provinces during 2014-2021 by retrospective analysis. A case-control study was conducted on patients who experienced relapse or recrudescence. RESULTS: From 2014 to 2021, 306 cases of P.vivax and 896 cases of P.ovale were included in the study, while 75 cases had relapse or recrudescence, including 49 cases of P. ovale (65.33%) and 26 cases of P. vivax (34.67%). Within less than 5 weeks after returning to the country, 122 cases of P. vivax (39.87%, 122/306) and 265 cases of P. ovale (29.58%, 265/896) occurred. Within less than 53 weeks, the ratio of P. vivax was 94.77% (290/306), and that of P. ovale was 89.96% (806/896). Among the cases experiencing relapse or recrudescence, only 1 case of P. vivax (1/26 3.85%) and 3 cases of P. ovale (3/49 6.12%) occurred within less than 5 weeks after the first onset, whereas 21 cases of P. vivax (21/26 80.77%) and 42 cases of P. ovale (42/49 85.71%) occurred within less than 53 weeks after the first onset. The difference in relapse or recrudescence due to different drugs and medication regimens and medical activities at various levels of medical institutions was statistically significant. CONCLUSION: In areas where malaria has been eliminated, routine health screening in a scientific time frame for people returning from at-risk areas can effectively improve the efficiency of preventing re-transmission, thereby reducing prevention costs and disease burden. Preventing patients from self-treating and strengthening medication regulations in health facilities are key measures to reduce relapse or recrudescence.