Methaemoglobin as a surrogate marker of primaquine antihypnozoite activity in Plasmodium vivax malaria: A systematic review and individual patient data meta-analysis.

BACKGROUND: The 8-aminoquinolines, primaquine and tafenoquine, are the only available drugs for the radical cure of Plasmodium vivax hypnozoites. Previous evidence suggests that there is dose-dependent 8-aminoquinoline induced methaemoglobinaemia and that higher methaemoglobin concentrations are associated with a lower risk of P. vivax recurrence. We undertook a systematic review and individual patient data meta-analysis to examine the utility of methaemoglobin as a population-level surrogate endpoint for 8-aminoquinoline antihypnozoite activity to prevent P. vivax recurrence. METHODS AND FINDINGS: We conducted a systematic search of Medline, Embase, Web of Science, and the Cochrane Library, from 1 January 2000 to 29 September 2022, inclusive, of prospective clinical efficacy studies of acute, uncomplicated P. vivax malaria mono-infections treated with radical curative doses of primaquine. The day 7 methaemoglobin concentration was the primary surrogate outcome of interest. The primary clinical outcome was the time to first P. vivax recurrence between day 7 and day 120 after enrolment. We used multivariable Cox proportional-hazards regression with site random-effects to characterise the time to first recurrence as a function of the day 7 methaemoglobin percentage (log base 2 transformed), adjusted for the partner schizonticidal drug, the primaquine regimen duration as a proxy for the total primaquine dose (mg base/kg), the daily primaquine dose (mg/kg), and other factors. The systematic review protocol was registered with PROSPERO (CRD42023345956). We identified 219 P. vivax efficacy studies, of which 8 provided relevant individual-level data from patients treated with primaquine; all were randomised, parallel arm clinical trials assessed as having low or moderate risk of bias. In the primary analysis data set, there were 1,747 patients with normal glucose-6-phosphate dehydrogenase (G6PD) activity enrolled from 24 study sites across 8 different countries (Indonesia, Brazil, Vietnam, Thailand, Peru, Colombia, Ethiopia, and India). We observed an increasing dose-response relationship between the daily weight-adjusted primaquine dose and day 7 methaemoglobin level. For a given primaquine dose regimen, an observed doubling in day 7 methaemoglobin percentage was associated with an estimated 30% reduction in the risk of P. vivax recurrence (adjusted hazard ratio = 0.70; 95% confidence interval [CI] [0.57, 0.86]; p = 0.0005). These pooled estimates were largely consistent across the study sites. Using day 7 methaemoglobin as a surrogate endpoint for recurrence would reduce required sample sizes by approximately 40%. Study limitations include the inability to distinguish between recrudescence, reinfection, and relapse in P. vivax recurrences. CONCLUSIONS: For a given primaquine regimen, higher methaemoglobin on day 7 was associated with a reduced risk of P. vivax recurrence. Under our proposed causal model, this justifies the use of methaemoglobin as a population-level surrogate endpoint for primaquine antihypnozoite activity in patients with P. vivax malaria who have normal G6PD activity.

Primaquine for uncomplicated Plasmodium vivax malaria in children younger than 15 years: a systematic review and individual patient data meta-analysis.

BACKGROUND: Primaquine, the only widely available treatment to prevent relapsing Plasmodium vivax malaria, is produced as 15 mg tablets, and new paediatric formulations are being developed. To inform the optimal primaquine dosing regimen for children, we aimed to determine the efficacy and safety of different primaquine dose strategies in children younger than 15 years. METHODS: We undertook a systematic review (Jan 1, 2000-July 26, 2024) for P vivax efficacy studies with at least one treatment group that was administered primaquine over multiple days, that enrolled children younger than 15 years, that followed up patients for at least 28 days, and that had data available for inclusion by June 30, 2022. Patients were excluded if they were aged 15 years or older, presented with severe malaria, received adjunctive antimalarials within 14 days of diagnosis, commenced primaquine more than 7 days after starting schizontocidal treatment, had a protocol violation in the original study, or were missing data on age, sex, or primaquine dose. Available individual patient data were collated and standardised. To evaluate efficacy, the risk of recurrent P vivax parasitaemia between days 7 and 180 was assessed by time-to-event analysis for different total mg/kg primaquine doses (low total dose of ∼3·5 mg/kg and high total dose of ∼7 mg/kg). To evaluate tolerability and safety, the following were assessed by daily mg/kg primaquine dose (low daily dose of ∼0·25 mg/kg, intermediate daily dose of ∼0·5 mg/kg, and high daily dose of ∼1 mg/kg): gastrointestinal symptoms (vomiting, anorexia, or diarrhoea) on days 5-7, haemoglobin decrease of at least 25% to less than 7g/dL (severe haemolysis), absolute change in haemoglobin from day 0 to days 2-3 or days 5-7, and any serious adverse events within 28 days. This study is registered with PROSPERO, CRD42021278085. FINDINGS: In total, 3514 children from 27 studies and 15 countries were included. The cumulative incidence of recurrence by day 180 was 51·4% (95% CI 47·0-55·9) following treatment without primaquine, 16·0% (12·4-20·3) following a low total dose of primaquine, and 10·2% (8·4-12·3) following a high total dose of primaquine. The hazard of recurrent P vivax parasitaemia in children younger than 15 years was reduced following primaquine at low total doses (adjusted hazard ratio [HR] 0·17, 95% CI 0·11-0·25) and high total doses (0·09, 0·07-0·12), compared with no primaquine. In 525 children younger than 5 years, the relative rates of recurrence were also reduced, with an adjusted HR of 0·33 (95% CI 0·18-0·59) for a low total dose and 0·13 (0·08-0·21) for a high total dose of primaquine compared with no primaquine. The rate of recurrence following a high total dose was reduced compared with a low dose in children younger than 15 years (adjusted HR 0·54, 95% CI 0·35-0·85) and children younger than 5 years (0·41, 0·21-0·78). Compared with no primaquine, children treated with any dose of primaquine had a greater risk of gastrointestinal symptoms on days 5-7 after adjustment for confounders, with adjusted risks of 3·9% (95% CI 0-8·6) in children not treated with primaquine, 9·2% (0-18·7) with a low daily dose of primaquine, 6·8% (1·7-12·0) with an intermediate daily dose of primaquine, and 9·6% (4·8-14·3) with a high daily dose of primaquine. In children with 30% or higher glucose-6-phosphate dehydrogenase (G6PD) activity, there were few episodes of severe haemolysis following no primaquine (0·4%, 95% CI 0·1-1·5), a low daily dose (0·0%, 0·0-1·6), an intermediate daily dose (0·5%, 0·1-1·4), or a high daily dose (0·7%, 0·2-1·9). Of 15 possibly drug-related serious adverse events in children, two occurred following a low, four following an intermediate, and nine following a high daily dose of primaquine. INTERPRETATION: A high total dose of primaquine was highly efficacious in reducing recurrent P vivax parasitaemia in children compared with a low dose, particularly in children younger than 5 years. In children treated with high and intermediate daily primaquine doses compared with low daily doses, there was no increase in gastrointestinal symptoms or haemolysis (in children with 30% or higher G6PD activity), but there were more serious adverse events. FUNDING: Medicines for Malaria Venture, Bill & Melinda Gates Foundation, and Australian National Health and Medical Research Council.

Lineage-informative microhaplotypes for recurrence classification and spatio-temporal surveillance of Plasmodium vivax malaria parasites.

Challenges in classifying recurrent Plasmodium vivax infections constrain surveillance of antimalarial efficacy and transmission. Recurrent infections may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or reinfection. Molecular inference of familial relatedness (identity-by-descent or IBD) can help resolve the probable origin of recurrences. As whole genome sequencing of P. vivax remains challenging, targeted genotyping methods are needed for scalability. We describe a P. vivax marker discovery framework to identify and select panels of microhaplotypes (multi-allelic markers within small, amplifiable segments of the genome) that can accurately capture IBD. We evaluate panels of 50-250 microhaplotypes discovered in a global set of 615 P. vivax genomes. A candidate global 100-microhaplotype panel exhibits high marker diversity in the Asia-Pacific, Latin America and horn of Africa (median HE = 0.70-0.81) and identifies 89% of the polyclonal infections detected with genome-wide datasets. Data simulations reveal lower error in estimating pairwise IBD using microhaplotypes relative to traditional biallelic SNP barcodes. The candidate global panel also exhibits high accuracy in predicting geographic origin and captures local infection outbreak and bottlenecking events. Our framework is open-source enabling customised microhaplotype discovery and selection, with potential for porting to other species or data resources.

Implementation of Glucose-6-Phosphate Dehydrogenase (G6PD) testing for Plasmodium vivax case management, a mixed method study from Cambodia.

Plasmodium vivax remains a challenge for malaria elimination since it forms dormant liver stages (hypnozoites) that can reactivate after initial infection. 8-aminoquinolone drugs kill hypnozoites but can cause severe hemolysis in individuals with Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency. The STANDARD G6PD test (Biosensor) is a novel point-of-care diagnostic capable of identifying G6PD deficiency prior to treatment. In 2021, Cambodia implemented the Biosensor to facilitate radical cure treatment for vivax malaria. To assess the Biosensor's implementation after its national rollout, a mixed-methods study was conducted in eight districts across three provinces in Cambodia. Interviews, focus group discussions, and observations explored stakeholders' experiences with G6PD testing and factors influencing its implementation. Quantitative data illustrative of test implementation were gathered from routine surveillance forms and key proportions derived. Qualitative data were analyzed thematically. The main challenge to implementing G6PD testing was that only 49.2% (437/888) of eligible patients reached health centers for G6PD testing following malaria diagnosis by community health workers. Factors influencing this included road conditions and long distances to the health center, compounded by the cost of seeking further care and patients' perceptions of vivax malaria and its treatment. 93.9% (790/841) of eligible vivax malaria patients who successfully completed referral (429/434) and directly presented to the health center (360/407) were G6PD tested. Key enabling factors included the test's acceptability among health workers and their understanding of the rationale for testing. Only 36.5% (443/1213) of eligible vivax episodes appropriately received primaquine. 70.5% (165/234) of female patients and all children under 20 kilograms never received primaquine. Our findings suggest that access to radical cure requires robust infrastructure and income security, which would likely improve referral rates to health centers enabling access. Bringing treatment closer to patients, through community health workers and nuanced community engagement, would improve access to curative treatment of vivax malaria.

The PreQuine Platform: A novel diagnostic tool for measuring glucose-6-phosphate dehydrogenase (G6PD) activity and hemoglobin concentration.

Quantitative diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency is essential for the safe administration of 8-aminoquinoline based radical cure for the treatment of Plasmodium vivax infections. Here, we present the PreQuine Platform (IVDS, USA), a quantitative biosensor that uses a dual-analyte assay for the simultaneous measurement of Hemoglobin (Hgb) levels and G6PD enzyme activity within the same sample. The platform relies on a downloadable mobile application. The device requires 10µl of whole blood and works with a reflectance-based meter. Comparing the G6PD measurement normalized by Hgb of 12 samples from the PreQuine Platform with reference measurements methods (spectrophotometry, Pointe Scientific, USA and hemoglobin meter, HemoCue, Sweden) showed a positive and significant agreement with a slope of 1.0091 and an intercept of -0.0379 under laboratory conditions. Next steps will be to conduct field trials in Bangladesh, Cambodia, and the USA to assess diagnostic performance, user friendliness and acceptance.

On-target, dual aminopeptidase inhibition provides cross-species antimalarial activity.

To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum, is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817-resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817, and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial.

Field evaluation of a novel semi-quantitative point-of-care diagnostic for G6PD deficiency in Indonesia.

BACKGROUND: The WHO recommends routine testing of G6PD activity to guide radical cure in patients with Plasmodium vivax malaria. Females may have intermediate G6PD enzyme activity and to date, only complex diagnostics are able to reliably identify them. The semi-quantitative G6PD diagnostic "One Step G6PD Test" (Humasis, RoK; "RDT") is a lateral flow assay that can distinguish deficient, intermediate, and normal G6PD status and offers a simpler diagnostic alternative. METHODS: G6PD status of participants enrolled in Malinau and Nunukan Regencies and the capital Jakarta was assessed with the RDT, and G6PD activity was measured in duplicate by reference spectrophotometry. The adjusted male median (AMM) of the spectrophotometry measurements was defined as 100% activity; 70% and 30% of the AMM were defined as thresholds for intermediate and deficient G6PD status, respectively. Results were compared to those derived from spectrophotometry at the clinically relevant G6PD activity thresholds of 30% and 70%. RESULTS: Of the 161 participants enrolled, 10 (6.2%) were G6PD deficient and 12 (7.5%) had intermediate G6PD activity by spectrophotometry. At the 30% threshold, the sensitivity of the RDT was 10.0% (95%CI: 0.3-44.5%) with a specificity of 99.3% (95%CI: 96.4-100.0%); the positive predictive value was 50.0% (95%CI: 1.3-98.7%) and the negative predictive value 94.3% (95%CI: 89.5-97.4%). The corresponding figures at the 70% threshold were 22.7% (95%CI: 7.8-45.4%), 100.0% (95%CI: 97.4-100.0%), 100.0% (95%CI: 47.8-100.0%) and 89.1% (95%CI: 83.1-93.5%), respectively. CONCLUSION: While there is a dire need for an easy-to-use, economical, semi-quantitative diagnostic for the point of care, the observed performance of the "One Step G6PD Test" in its current form was insufficient to guide antimalarial treatment.

Evidence uptake is only part of the process: Stakeholders' insights on WHO treatment guideline recommendation processes for radical cure of P. vivax malaria.

Health policy processes should be evidence-informed, transparent and timely, but these processes are often unclear to stakeholders outside the immediate policymaking environment. We spoke to 36 international malaria stakeholders to gain insights on the processes involved in the World Health Organization's Global Malaria Programme's recommendations for their treatment guidelines of P. vivax malaria. Four key themes which drew on the 3i policy framework and Shiffman's four factors that influence global and national policymaking were identified to understand these processes. Triggers for policy change and change prioritisation, evidence types that inform policy, effects of funding on decision-making processes, and transparency and communication of these processes to external stakeholders. Results indicate that more clarity is needed on what triggers global malaria policy change processes, a clearer justification of evidence types used to inform policymaking, better understanding of the impact of the WHO's funding model on policymaking and further transparency and improved communication of these processes to external stakeholders is also needed. We suggest that global malaria policymaking could be improved by using the following strategies: ensuring that identified triggers actually initiate the policy change process, expediting decision-making timelines by developing a priority framework for assessing new evidence, adopting suitable frameworks to assess contextual evidence, and increasing the transparency of the role of non-state funders in policy decision-making processes and when publishing new recommendations.

A randomised controlled trial to compare the efficacy, safety, and tolerability of low dose, short course primaquine in adults with uncomplicated P. vivax malaria in two hospitals in India.

BACKGROUND: Plasmodium vivax remains a major challenge for malaria control and elimination due to its ability to cause relapsing illness. To prevent relapses the Indian National Center for Vector Borne Diseases Control (NCVBDC) recommends treatment with primaquine at a dose of 0.25 mg/kg/day provided over 14 days. Shorter treatment courses may improve adherence and treatment effectiveness. METHODS: This is a hospital-based, randomised, controlled, open-label trial in two centres in India. Patients above the age of 16 years, with uncomplicated vivax malaria, G6PD activity of ≥ 30% of the adjusted male median (AMM) and haemoglobin levels ≥ 8 g/dL will be recruited into the study and randomised in a 1:1 ratio to receive standard schizonticidal treatment plus 7-day primaquine at 0.50 mg/kg/day or standard care with schizonticidal treatment plus 14-day primaquine at 0.25 mg/kg/day. Patients will be followed up for 6 months. The primary endpoint is the incidence risk of any P. vivax parasitaemia at 6 months. Safety outcomes include the incidence risk of severe anaemia (haemoglobin < 8 g/dL), the risk of blood transfusion, a > 25% fall in haemoglobin and an acute drop in haemoglobin of > 5 g/dL during primaquine treatment. DISCUSSION: This study will evaluate the efficacy and safety of a 7-day primaquine regimen compared to the standard 14-day regimen in India. Results from this trial are likely to directly inform national treatment guidelines. TRIAL REGISTRATION: Trial is registered on CTRI portal, Registration No: CTRI/2022/12/048283.

Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testing.

In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care diagnostic STANDARD G6PD (SDBiosensor, RoK), and evaluated recommended cut-offs. We tested capillary blood from fingerpricks (Standard Method) and a microtainer (BD, USA; Method 1), venous blood from a vacutainer (BD, USA; Method 2), varied sample application methods (Methods 3), and used micropipettes rather than the test's single-use pipette (Method 4). Repeatability was assessed by comparing median differences between paired measurements. All methods were tested 20 times under laboratory conditions on three volunteers. The Standard Method and the method with best repeatability were tested in Indonesia and Nepal. In Indonesia 60 participants were tested in duplicate by both methods, in Nepal 120 participants were tested in duplicate by either method. The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity. In Indonesia, the difference between paired readings of the Standard and modified methods was compared to assess the impact of delayed testing. In the pilot study repeatability didn't differ significantly (p = 0.381); Method 3 showed lowest variability. One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity). Repeatability didn't differ significantly in Indonesia (Standard: 0.2U/gHb [IQR: 0.1-0.4]; Method 3: 0.3U/gHb [IQR: 0.1-0.5]; p = 0.425) or Nepal (Standard: 0.4U/gHb [IQR: 0.2-0.6]; Method 3: 0.3U/gHb [IQR: 0.1-0.6]; p = 0.330). Median G6PD measurements by Method 3 were 0.4U/gHb (IQR: -0.2 to 0.7, p = 0.005) higher after a 5-hour delay compared to the Standard Method. The definition of 100% activity by the Standard Method matched the manufacturer-recommended cut-off for 70% activity. We couldn't improve repeatability. Delays of up to 5 hours didn't result in a clinically relevant difference in measured G6PD activity. The manufacturer's recommended cut-off for intermediate deficiency is conservative.

Effect of primaquine dose on the risk of recurrence in patients with uncomplicated Plasmodium vivax: a systematic review and individual patient data meta-analysis.

BACKGROUND: Primaquine is used to eliminate Plasmodium vivax hypnozoites, but its optimal dosing regimen remains unclear. We undertook a systematic review and individual patient data meta-analysis to investigate the efficacy and tolerability of different primaquine dosing regimens to prevent P vivax recurrence. METHODS: For this systematic review and individual patient data meta-analysis, we searched MEDLINE, Web of Science, Embase, and Cochrane Central for prospective clinical studies of uncomplicated P vivax from endemic countries published between Jan 1, 2000, and June 8, 2023. We included studies if they had active follow-up of at least 28 days, and if they included a treatment group with daily primaquine given over multiple days, where primaquine was commenced within 7 days of schizontocidal treatment and was given alone or coadministered with chloroquine or one of four artemisinin-based combination therapies (ie, artemether-lumefantrine, artesunate-mefloquine, artesunate-amodiaquine, or dihydroartemisinin-piperaquine). We excluded studies if they were on prevention, prophylaxis, or patients with severe malaria, or if data were extracted retrospectively from medical records outside of a planned trial. For the meta-analysis, we contacted the investigators of eligible trials to request individual patient data and we then pooled data that were made available by Aug 23, 2021. We assessed the effects of total dose and duration of primaquine regimens on the rate of first P vivax recurrence between day 7 and day 180 by Cox's proportional hazards regression (efficacy analysis). The effect of primaquine daily dose on gastrointestinal symptoms on days 5-7 was assessed by modified Poisson regression (tolerability analysis). The study was registered with PROSPERO, CRD42019154470. FINDINGS: Of 226 identified studies, 23 studies with patient-level data from 6879 patients from 16 countries were included in the efficacy analysis. At day 180, the risk of recurrence was 51·0% (95% CI 48·2-53·9) in 1470 patients treated without primaquine, 19·3% (16·9-21·9) in 2569 patients treated with a low total dose of primaquine (approximately 3·5 mg/kg), and 8·1% (7·0-9·4) in 2811 patients treated with a high total dose of primaquine (approximately 7 mg/kg), regardless of primaquine treatment duration. Compared with treatment without primaquine, the rate of P vivax recurrence was lower after treatment with low-dose primaquine (adjusted hazard ratio 0·21, 95% CI 0·17-0·27; p<0·0001) and high-dose primaquine (0·10, 0·08-0·12; p<0·0001). High-dose primaquine had greater efficacy than low-dose primaquine in regions with high and low relapse periodicity (ie, the time from initial infection to vivax relapse). 16 studies with patient-level data from 5609 patients from ten countries were included in the tolerability analysis. Gastrointestinal symptoms on days 5-7 were reported by 4·0% (95% CI 0·0-8·7) of 893 patients treated without primaquine, 6·2% (0·5-12·0) of 737 patients treated with a low daily dose of primaquine (approximately 0·25 mg/kg per day), 5·9% (1·8-10·1) of 1123 patients treated with an intermediate daily dose (approximately 0·5 mg/kg per day) and 10·9% (5·7-16·1) of 1178 patients treated with a high daily dose (approximately 1 mg/kg per day). 20 of 23 studies included in the efficacy analysis and 15 of 16 in the tolerability analysis had a low or unclear risk of bias. INTERPRETATION: Increasing the total dose of primaquine from 3·5 mg/kg to 7 mg/kg can reduce P vivax recurrences by more than 50% in most endemic regions, with a small associated increase in gastrointestinal symptoms. FUNDING: Australian National Health and Medical Research Council, Bill & Melinda Gates Foundation, and Medicines for Malaria Venture.

Primaquine dose and the risk of haemolysis in patients with uncomplicated Plasmodium vivax malaria: a systematic review and individual patient data meta-analysis.

BACKGROUND: Primaquine radical cure is used to treat dormant liver-stage parasites and prevent relapsing Plasmodium vivax malaria but is limited by concerns of haemolysis. We undertook a systematic review and individual patient data meta-analysis to investigate the haematological safety of different primaquine regimens for P vivax radical cure. METHODS: For this systematic review and individual patient data meta-analysis, we searched MEDLINE, Web of Science, Embase, and Cochrane Central for prospective clinical studies of uncomplicated P vivax from endemic countries published between Jan 1, 2000, and June 8, 2023. We included studies if they had active follow-up of at least 28 days, if they included a treatment group with daily primaquine given over multiple days where primaquine was commenced within 3 days of schizontocidal treatment and was given alone or coadministered with chloroquine or one of four artemisinin-based combination therapies (ie, artemether-lumefantrine, artesunate-mefloquine, artesunate-amodiaquine, or dihydroartemisinin-piperaquine), and if they recorded haemoglobin or haematocrit concentrations on day 0. We excluded studies if they were on prevention, prophylaxis, or patients with severe malaria, or if data were extracted retrospectively from medical records outside of a planned trial. For the meta-analysis, we contacted the investigators of eligible trials to request individual patient data and we then pooled data that were made available by Aug 23, 2021. The main outcome was haemoglobin reduction of more than 25% to a concentration of less than 7 g/dL by day 14. Haemoglobin concentration changes between day 0 and days 2-3 and between day 0 and days 5-7 were assessed by mixed-effects linear regression for patients with glucose-6-phosphate dehydrogenase (G6PD) activity of (1) 30% or higher and (2) between 30% and less than 70%. The study was registered with PROSPERO, CRD42019154470 and CRD42022303680. FINDINGS: Of 226 identified studies, 18 studies with patient-level data from 5462 patients from 15 countries were included in the analysis. A haemoglobin reduction of more than 25% to a concentration of less than 7 g/dL occurred in one (0·1%) of 1208 patients treated without primaquine, none of 893 patients treated with a low daily dose of primaquine (<0·375 mg/kg per day), five (0·3%) of 1464 patients treated with an intermediate daily dose (0·375 mg/kg per day to <0·75 mg/kg per day), and six (0·5%) of 1269 patients treated with a high daily dose (≥0·75 mg/kg per day). The covariate-adjusted mean estimated haemoglobin changes at days 2-3 were -0·6 g/dL (95% CI -0·7 to -0·5), -0·7 g/dL (-0·8 to -0·5), -0·6 g/dL (-0·7 to -0·4), and -0·5 g/dL (-0·7 to -0·4), respectively. In 51 patients with G6PD activity between 30% and less than 70%, the adjusted mean haemoglobin concentration on days 2-3 decreased as G6PD activity decreased; two patients in this group who were treated with a high daily dose of primaquine had a reduction of more than 25% to a concentration of less than 7 g/dL. 17 of 18 included studies had a low or unclear risk of bias. INTERPRETATION: Treatment of patients with G6PD activity of 30% or higher with 0·25-0·5 mg/kg per day primaquine regimens and patients with G6PD activity of 70% or higher with 0·25-1 mg/kg per day regimens were associated with similar risks of haemolysis to those in patients treated without primaquine, supporting the safe use of primaquine radical cure at these doses. FUNDING: Australian National Health and Medical Research Council, Bill & Melinda Gates Foundation, and Medicines for Malaria Venture.

Retention of uninfected red blood cells causing congestive splenomegaly is the major mechanism of anemia in malaria.

Splenomegaly frequently occurs in patients with Plasmodium falciparum (Pf) or P. vivax (Pv) malarial anemia, but mechanisms underlying this co-occurrence are unclear. In malaria-endemic Papua, Indonesia, we prospectively analyzed red blood cell (RBC) concentrations in the spleen and spleen-mimetic retention in 37 subjects splenectomized for trauma or hyperreactive splenomegaly, most of whom were infected with Plasmodium. Splenomegaly (median 357 g [range: 80-1918 g]) was correlated positively with the proportion of red-pulp on histological sections (median 88.1% [range: 74%-99.4%]; r = .59, p = .0003) and correlated negatively with the proportion of white-pulp (median 8.3% [range: 0.4%-22.9%]; r = -.50, p = .002). The number of RBC per microscopic field (>95% uninfected) was correlated positively with spleen weight in both Pf-infected (r = .73; p = .017) and Pv-infected spleens (r = .94; p = .006). The median estimated proportion of total-body RBCs retained in Pf-infected spleens was 8.2% (range: 1.0%-33.6%), significantly higher than in Pv-infected (2.6% [range: 0.6%-23.8%]; p = .015) and PCR-negative subjects (2.5% [range: 1.0%-3.3%]; p = .006). Retained RBCs accounted for over half of circulating RBC loss seen in Pf infections. The proportion of total-body RBC retained in Pf- and Pv-infected spleens correlated negatively with hemoglobin concentrations (r = -.56, p = .0003), hematocrit (r = -.58, p = .0002), and circulating RBC counts (r = -.56, p = .0003). Splenic CD71-positive reticulocyte concentrations correlated with spleen weight in Pf (r = 1.0; p = .003). Retention rates of peripheral and splenic RBCs were correlated negatively with circulating RBC counts (r = -.69, p = .07 and r = -.83, p = .008, respectively). In conclusion, retention of mostly uninfected RBC in the spleen, leading to marked congestion of the red-pulp, was associated with splenomegaly and is the major mechanism of anemia in subjects infected with Plasmodium, particularly Pf.

Imported malaria into Australia: surveillance insights and opportunities.

BACKGROUND: Malaria continues to pose a significant burden in endemic countries, many of which lack access to molecular surveillance. Insights from malaria cases in travellers returning to non-endemic areas can provide valuable data to inform endemic country programmes. To evaluate the potential for novel global insights into malaria, we examined epidemiological and molecular data from imported malaria cases to Australia. METHODS: We analysed malaria cases reported in Australia from 2012 to 2022 using National Notifiable Disease Surveillance System data. Molecular data on imported malaria cases were obtained from literature searches. RESULTS: Between 2012 and 2022, 3204 malaria cases were reported in Australia. Most cases (69%) were male and 44% occurred in young adults aged 20-39 years. Incidence rates initially declined between 2012 and 2015, then increased until 2019. During 2012-2019, the incidence in travellers ranged from 1.34 to 7.71 per 100 000 trips. Cases were primarily acquired in Sub-Saharan Africa (n = 1433; 45%), Oceania (n = 569; 18%) and Southern and Central Asia (n = 367; 12%). The most common countries of acquisition were Papua New Guinea (n = 474) and India (n = 277). Plasmodium falciparum accounted for 58% (1871/3204) of cases and was predominantly acquired in Sub-Saharan Africa, and Plasmodium vivax accounted for 32% (1016/3204), predominantly from Oceania and Asia. Molecular studies of imported malaria cases to Australia identified genetic mutations and deletions associated with drug resistance and false-negative rapid diagnostic test results, and led to the establishment of reference genomes for P. vivax and Plasmodium malariae. CONCLUSIONS: Our analysis highlights the continuing burden of imported malaria into Australia. Molecular studies have offered valuable insights into drug resistance and diagnostic limitations, and established reference genomes. Integrating molecular data into national surveillance systems could provide important infectious disease intelligence to optimize treatment guidelines for returning travellers and support endemic country surveillance programmes.

Safety and efficacy of primaquine in patients with Plasmodium vivax malaria from South Asia: a systematic review and individual patient data meta-analysis.

BACKGROUND: The optimal dosing of primaquine to prevent relapsing Plasmodium vivax malaria in South Asia remains unclear. We investigated the efficacy and safety of different primaquine regimens to prevent P. vivax relapse. METHODS: A systematic review identified P. vivax efficacy studies from South Asia published between 1 January 2000 and 23 August 2021. In a one-stage meta-analysis of available individual patient data, the cumulative risks of P. vivax recurrence at day 42 and 180 were assessed by primaquine total mg/kg dose and duration. The risk of recurrence by day 180 was also determined in a two-stage meta-analysis. Patients with a >25% drop in haemoglobin to <70 g/L, or an absolute drop of >50 g/L between days 1 and 14 were categorised by daily mg/kg primaquine dose. RESULTS: In 791 patients from 7 studies in the one-stage meta-analysis, the day 180 cumulative risk of recurrence was 61.1% (95% CI 42.2% to 80.4%; 201 patients; 25 recurrences) after treatment without primaquine, 28.8% (95% CI 8.2% to 74.1%; 398 patients; 4 recurrences) following low total (2 to <5 mg/kg) and 0% (96 patients; 0 recurrences) following high total dose primaquine (≥5 mg/kg). In the subsequent two-stage meta-analysis of nine studies (3529 patients), the pooled proportions of P. vivax recurrences by day 180 were 12.1% (95% CI 7.7% to 17.2%), 2.3% (95% CI 0.3% to 5.4%) and 0.7% (95% CI 0% to 6.1%), respectively. No patients had a >25% drop in haemoglobin to <70 g/L. CONCLUSIONS: Primaquine treatment led to a marked decrease in P. vivax recurrences following low (~3.5 mg/kg) and high (~7 mg/kg) total doses, with no reported severe haemolytic events. PROSPERO REGISTRATION NUMBER: CRD42022313730.

Genomics of Plasmodium vivax in Colombia reveals evidence of local bottle-necking and inter-country connectivity in the Americas.

Colombia aims to eliminate malaria by 2030 but remains one of the highest burden countries in the Americas. Plasmodium vivax contributes half of all malaria cases, with its control challenged by relapsing parasitaemia, drug resistance and cross-border spread. Using 64 Colombian P. vivax genomes collected between 2013 and 2017, we explored diversity and selection in two major foci of transmission: Chocó and Córdoba. Open-access data from other countries were used for comparative assessment of drug resistance candidates and to assess cross-border spread. Across Colombia, polyclonal infections were infrequent (12%), and infection connectivity was relatively high (median IBD = 5%), consistent with low endemicity. Chocó exhibited a higher frequency of polyclonal infections (23%) than Córdoba (7%), although the difference was not significant (P = 0.300). Most Colombian infections carried double pvdhfr (95%) and single pvdhps (71%) mutants, but other drug resistance mutations were less prevalent (< 10%). There was no evidence of selection at the pvaat1 gene, whose P. falciparum orthologue has recently been implicated in chloroquine resistance. Global population comparisons identified other putative adaptations. Within the Americas, low-level connectivity was observed between Colombia and Peru, highlighting potential for cross-border spread. Our findings demonstrate the potential of molecular data to inform on infection spread and adaptation.

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.

Genomic analysis of Plasmodium vivax describes patterns of connectivity and putative drivers of adaptation in Ethiopia.

Ethiopia has the greatest burden of Plasmodium vivax in Africa, but little is known about the epidemiological landscape of parasites across the country. We analysed the genomic diversity of 137 P. vivax isolates collected nine Ethiopian districts from 2012 to 2016. Signatures of selection were detected by cross-country comparisons with isolates from Thailand (n = 104) and Indonesia (n = 111), representing regions with low and high chloroquine resistance respectively. 26% (35/137) of Ethiopian infections were polyclonal, and 48.5% (17/35) of these comprised highly related clones (within-host identity-by-descent > 25%), indicating frequent co-transmission and superinfection. Parasite gene flow between districts could not be explained entirely by geographic distance, with economic and cultural factors hypothesised to have an impact on connectivity. Amplification of the duffy binding protein gene (pvdbp1) was prevalent across all districts (16-75%). Cross-population haplotype homozygosity revealed positive selection in a region proximal to the putative chloroquine resistance transporter gene (pvcrt-o). An S25P variant in amino acid transporter 1 (pvaat1), whose homologue has recently been implicated in P. falciparum chloroquine resistance evolution, was prevalent in Ethiopia (96%) but not Thailand or Indonesia (35-53%). The genomic architecture in Ethiopia highlights circulating variants of potential public health concern in an endemic setting with evidence of stable transmission.

The effect and control of malaria in pregnancy and lactating women in the Asia-Pacific region.

Half of all pregnancies at risk of malaria worldwide occur in the Asia-Pacific region, where Plasmodium falciparum and Plasmodium vivax co-exist. Despite substantial reductions in transmission, malaria remains an important cause of adverse health outcomes for mothers and offspring, including pre-eclampsia. Malaria transmission is heterogeneous, and infections are commonly subpatent and asymptomatic. High-grade antimalarial resistance poses a formidable challenge to malaria control in pregnancy in the region. Intermittent preventive treatment in pregnancy reduces infection risk in meso-endemic New Guinea, whereas screen-and-treat strategies will require more sensitive point-of-care tests to control malaria in pregnancy. In the first trimester, artemether-lumefantrine is approved, and safety data are accumulating for other artemisinin-based combinations. Safety of novel antimalarials to treat artemisinin-resistant P falciparum during pregnancy, and of 8-aminoquinolines during lactation, needs to be established. A more systematic approach to the prevention of malaria in pregnancy in the Asia-Pacific is required.