The Primaquine Methaemoglobin Study Group was formed in September 2022. Research groups with relevant data sets were contacted. Data gathering, curation, and analysis has now finished. The final results have been published in PLoS Medicine. The pooled individual patient data meta-analysis strongly supports the use of methaemoglobin as a surrogate outcome for clinical trials of 8-aminoquinoline drugs in vivax malaria. These results will help accelerate determining the optimal dose of primaquine and tafenoquine.

Plasmodium vivax is the most geographically widespread cause of human malaria. Its characteristic ability to form dormant liver-stage hypnozoites leads to cumulative morbidity and mortality over time, as well as confounding malaria control activities, since acute episodes may recur (relapse) weeks to months after an initial infection. Currently, the only widely available drug to prevent relapses is the 8-aminoquinoline primaquine. Primaquine is a prodrug whose active metabolites remain largely unknown and uncharacterised. Preliminary data suggest that methaemoglobin production may be a correlate of the production and exposure to the active metabolites driving drug efficacy (1). This review will synthesise available longitudinal methaemoglobin data in patients with symptomatic vivax malaria treated with primaquine to investigate the utility of using day-7 methemoglobinemia as a pharmacodynamic proxy measurement that predicts treatment failure following radical cure.

The primary aim of this study is to investigate the utility of day-7 methemoglobinemia as a pharmacodynamic proxy measure of primaquine antihypnozoite activity in patients with symptomatic Plasmodium vivax malaria.

Embase, Medline, Web of Science, and the Cochrane Library will be systematically searched based on an existing and living systematic review (2); to identify prospective, clinical efficacy studies of acute, uncomplicated vivax malaria published between 1 January 2000 and 29 September 2022 (inclusive) in any language with a minimum active follow-up of 42 days that record methaemoglobin data following daily primaquine administration. Relevant data from unpublished studies will be obtained where possible. Studies that fulfil the study criteria will be targeted through direct email to the corresponding author and/or principal investigator.

Essential criteria of eligible studies 

• Prospective clinical efficacy studies of uncomplicated vivax malaria; including randomised and non-randomised therapeutic trials, and prospective cohort studies with active follow-up
• A minimum follow-up of 42 days
• Treatment with a daily primaquine regimen given within the first three days of a schizontocidal treatment, i.e., chloroquine or one of six common artemisinin-based combination therapies (artemether-lumefantrine, artesunate-mefloquine, artesunate-amodiaquine, dihydroartemisinin-piperaquine, artesunate-sulfadoxine-pyrimethamine, artesunate-pyronaridine)

Essential data for inclusion in the analysis 

• Information on schizontocidal treatment and timing, dose, and duration of primaquine
• Baseline data on patient, weight, age, and sex
• Methaemoglobin concentrations measured at baseline and at least once in the first week of follow-up between days 5 and 9

PROSPERO ID: CRD42023345956

Desirable data for inclusion in the analysis

• Individual tablet or mg dosing
• Baseline parasitaemia
• Documentation of the supervision of drug administration
• Haemoglobin or haematocrit measured on day 0 and at least once during follow-up
• Qualitative or quantitative assessment of G6PD status
• CYP2D6 status
• Food administration
• History of fever within the last 24 hours at baseline and during follow-up
• Data on vomiting within 1 hour post-administration
• Primaquine/metabolite drug concentrations
• Partner drug levels in terminal elimination phase (e.g., day-7 levels)

Once data are uploaded into the WWARN repository, they will be curated and standardised using the WWARN Data Management and Statistical Analysis Plans (3) for clinical data and pooled into a single database of quality-assured individual patient data.

Stratified by primaquine dosing regimen, the conditional predictive value of the 7-day methaemoglobin concentration for the time to first Plasmodium vivax recurrence will be estimated by multivariable Cox regression analysis. The covariates in the model include: age, sex, daily primaquine dose, type of schizontocidal drug, baseline parasite density; with shared frailty for study site. Adjusted hazard ratios in each stratum will be presented along with a meta-analytic estimate. The analysis will be restricted to G6PD normal patients (≥ 30% activity or a negative qualitative test).

Additional analyses will explore other secondary endpoints using the main analysis framework above:

• Any Plasmodium vivax recurrence by 4 months (binary endpoint), restricting to studies with at least 4 months follow-up using multivariable logistic regression.
• Absolute change (continuous endpoint) in haemoglobin from baseline to the minimum haemoglobin concentration on day 2 or 3, representing the safety outcome using multivariable linear regression. The model will be adjusted for baseline Hb.

Additional analyses will investigate alternative summary statistics (AUC[0, ∞), Cmax) based on population nonlinear mixed-effects modelling that may be predictive of the efficacy and safety endpoints, using the main analysis-framework.

Additional analyses will also explore the determinants and other correlates of methaemoglobin production, by specifying linear mixed-effects models with day-7 methaemoglobin concentrations as the endpoint. We will investigate how G6PD status, primaquine and carboxyprimaquine trough levels, haemoglobin concentrations, and CYP2D6 activity scores relate to methaemoglobin production.

A detailed statistical analysis plan has been developed.

The Primaquine Methaemoglobin Study Group comprises participating investigators who contribute relevant data sets to the pooled analysis. Data sets will remain the property of the investigator and will not be shared without their consent. Ihsan Fadilah, James Watson, Rob Commons, Kevin Baird, Ric Price, and Nicholas White will oversee the statistical analyses. Participating investigators will be recognised in the publication as contributors under the banner of the Primaquine Methaemoglobin Study Group. A Writing Committee will coordinate activities including data analysis and drafting of publications and reports for complete group review. The Writing Committee will comprise Ihsan Fadilah, James Watson, Rob Commons, Kevin Baird, Ric Price, Nicholas White and other interested investigators. They are responsible for undertaking the data analysis and preparation of the manuscript. Authors will be recognised according to the ICMJE guidelines and the WWARN publication policy.

For further information, please contact Ihsan Fadilah on email: ifadilah@oucru.org

1. Chu CS, Watson JA, Phyo AP, Win HH, Yotyingaphiram W, Thinraow S, et al. Determinants of Primaquine and Carboxyprimaquine Exposures in Children and Adults with Plasmodium vivax Malaria. Antimicrobial agents and chemotherapy. 2021;65(11):e0130221. 

2. Commons RJ, Thriemer K, Humphreys G, Suay I, Sibley CH, Guerin PJ, et al. The Vivax Surveyor: Online mapping database for Plasmodium vivax clinical trials. International Journal for Parasitology: Drugs and Drug Resistance. 2017 Aug;7(2):181–90. 

3. WorldWide Antimalarial Resistance Network (WWARN). Clinical Module: Data Management and Statistical Analysis Plan – Version 1.2. [Internet]. 2012. Available from: https://www.wwarn.org/sites/default/files/ClinicalDMSAP.pdf