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. Prior 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 surrogate endpoint for 8-aminoquinoline antihypnozoite activity to prevent P. vivax recurrence.  Methods 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 (log2 transformed), adjusted for the partner schizontocidal drug, the primaquine regimen duration as a proxy for the total primaquine dose (mg/kg), the daily primaquine dose (mg/kg), and other factors. The systematic review protocol was registered with PROSPERO (CRD42023345956).  Findings We identified 219 P. vivax efficacy studies, of which eight 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 dataset, there were 1747 G6PD-normal patients enrolled from 24 study sites across 8 different countries (Indonesia, Brazil, Vietnam, Thailand, Peru, Colombia, Ethiopia, 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 vivax recurrence (adjusted hazard ratio = 0.70; 95% 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%.  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 surrogate endpoint for primaquine antihypnozoite activity in G6PD normal patients with P. vivax malaria. ### Competing Interest Statement I have read the journal's policy and the authors of this manuscript have the following competing interests: JAG and GCKWK are former employees of GSK and hold shares in GSK and AstraZeneca. GCKWK reports travel support from AstraZeneca. JKB and KT receive institutional research funding from Medicines for Malaria Venture. JKB reports GSK, Wellcome Trust, and Sanaria participation on the US National Institutes of Health data safety monitoring board and membership of the editorial board of Travel Medicine and Infectious Disease and the guidelines development group for malaria control and elimination, Global Malaria Programme, WHO. RJC, JKB, and RNP report contributions to Up-to-Date. All other authors declare no competing interests. ### Clinical Protocols ### Funding Statement Yes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All studies included in our meta-analysis provided pseudonymised individual data and had obtained ethical approvals from the corresponding site of origin. Therefore, additional ethical approval was not required for the current analysis, as per the Oxford Tropical Research Ethics Committee. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Pseudonymised participant data used in this study can be accessed via the WorldWide Antimalarial Resistance Network (wwarn.org). Requests for access will be reviewed by a data access committee to ensure that use of data protects the interests of the participants and researchers according to the terms of ethics approval and principles of equitable data sharing. Requests can be submitted by email to malariaDAC@iddo.org via the data access form available at https://www.wwarn.org/working-together/sharing-accessing-data/accessing-data. WWARN is registered with the Registry of Research Data Repositories (https://www.re3data.org/).