Disclosure of cinnamic acid/4,9-diaminoacridine conjugates as multi-stage antiplasmodial hits.

4,9-diaminoacridines with reported antiplasmodial activity were coupled to different trans-cinnamic acids, delivering a new series of conjugates inspired by the covalent bitherapy concept. The new compounds were more potent than primaquine against hepatic stages of Plasmodium berghei, although this was accompanied by cytotoxic effects on Huh-7 hepatocytes. Relevantly, the conjugates displayed nanomolar activities against blood stage P. falciparum parasites, with no evidence of hemolytic effects below 100 µM. Moreover, the new compounds were at least 25-fold more potent than primaquine against P. falciparum gametocytes. Thus, the new antiplasmodial hits disclosed herein emerge as valuable templates for the development of multi-stage antiplasmodial drug candidates.

Superior protection in a relapsing Plasmodium cynomolgi rhesus macaque model by a chemoprophylaxis with sporozoite immunization regimen with atovaquone-proguanil followed by primaquine.

BACKGROUND: To gain a deeper understanding of protective immunity against relapsing malaria, this study examined sporozoite-specific T cell responses induced by a chemoprophylaxis with sporozoite (CPS) immunization in a relapsing Plasmodium cynomolgi rhesus macaque model. METHODS: The animals received three CPS immunizations with P. cynomolgi sporozoites, administered by mosquito bite, while under two anti-malarial drug regimens. Group 1 (n = 6) received artesunate/chloroquine (AS/CQ) followed by a radical cure with CQ plus primaquine (PQ). Group 2 (n = 6) received atovaquone-proguanil (AP) followed by PQ. After the final immunization, the animals were challenged with intravenous injection of 104 P. cynomolgi sporozoites, the dose that induced reliable infection and relapse rate. These animals, along with control animals (n = 6), were monitored for primary infection and subsequent relapses. Immunogenicity blood draws were done after each of the three CPS session, before and after the challenge, with liver, spleen and bone marrow sampling and analysis done after the challenge. RESULTS: Group 2 animals demonstrated superior protection, with two achieving protection and two experiencing partial protection, while only one animal in group 1 had partial protection. These animals displayed high sporozoite-specific IFN-γ T cell responses in the liver, spleen, and bone marrow after the challenge with one protected animal having the highest frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. Partially protected animals also demonstrated a relatively high frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. It is important to highlight that the second animal in group 2, which experienced protection, exhibited deficient sporozoite-specific T cell responses in the liver while displaying average to high T cell responses in the spleen and bone marrow. CONCLUSIONS: This research supports the notion that local liver T cell immunity plays a crucial role in defending against liver-stage infection. Nevertheless, there is an instance where protection occurs independently of T cell responses in the liver, suggesting the involvement of the liver's innate immunity. The relapsing P. cynomolgi rhesus macaque model holds promise for informing the development of vaccines against relapsing 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.

Primaquine in glucose-6-phosphate dehydrogenase deficiency: an adaptive pharmacometric assessment of ascending dose regimens in healthy volunteers.

Background: Primaquine is an 8-aminoquinoline antimalarial. It is the only widely available treatment to prevent relapses of Plasmodium vivax malaria. The 8-aminoquinolines cause dose-dependent haemolysis in glucose-6-phosphate dehydrogenase deficiency (G6PDd). G6PDd is common in malaria endemic areas but testing is often not available. As a consequence primaquine is underused. Methods: We conducted an adaptive pharmacometric study to characterise the relationship between primaquine dose and haemolysis in G6PDd. The aim was to explore shorter and safer primaquine radical cure regimens compared to the currently recommended 8-weekly regimen (0.75 mg/kg once weekly), potentially obviating the need for G6PD testing. Hemizygous G6PDd healthy adult Thai and Burmese male volunteers were admitted to the Hospital for Tropical Diseases in Bangkok. In Part 1, volunteers were given ascending dose primaquine regimens whereby daily doses were increased from 7.5 mg up to 45 mg over 15-20 days. In Part 2 conducted at least 6 months later, a single primaquine 45 mg dose was given. Results: 24 volunteers were enrolled in Part 1, and 16 in Part 2 (13 participated in both studies). In three volunteers, the ascending dose regimen was stopped because of haemolysis (n=1) and asymptomatic increases in transaminases (n=2; one was hepatitis E positive). Otherwise the ascending regimens were well tolerated with no drug-related serious adverse events. In Part 1, the median haemoglobin concentration decline was 3.7 g/dL (range: 2.1-5.9; relative decline of 26% [range: 15-40%]). Primaquine doses up to 0.87 mg/kg/day were tolerated subsequently without clinically significant further falls in haemoglobin. In Part 2, the median haemoglobin concentration decline was 1.7 g/dL (range 0.9-4.1; relative fall of 12% [range: 7-30% decrease]). The ascending dose primaquine regimens gave seven times more drug but resulted in only double the haemoglobin decline. Conclusions: In patients with Southeast Asian G6PDd variants, full radical cure treatment can be given in under 3 weeks compared with the current 8-week regimen. Funding: Medical Research Council of the United Kingdom (MR/R015252/1) and Wellcome (093956/Z/10/C, 223253/Z/21/Z). Clinical trial number: Thai Clinical Trial Registry: TCTR20170830002 and TCTR20220317004.

Prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Gia Lai Province, Vietnam.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is one of the most common X-linked hereditary disorders worldwide. G6PD deficiency provides resistance against severe malaria, but paradoxically, G6PD deficiency is also a stumbling block in fighting against malaria. Primaquine (PQ), a drug for the radical cure of Plasmodium vivax, can cause lethal acute hemolytic anemia in malaria patients with inherited G6PD deficiency. In this study, we analyzed the phenotypic and genotypic G6PD deficiency status in 1721 individuals (963 males and 758 females) residing in three malaria-endemic areas within the Gia Lai province, Vietnam. The G6PD activity in individuals ranged from 3.04 to 47.82 U/g Hb, with the adjusted male median (AMM) of 7.89 U/g Hb. Based on the G6PD activity assay results, no phenotypic G6PD deficiency was detected. However, the multiplex polymerase chain reaction to detect G6PD variations in the gene level revealed that 26 individuals (7 males, 19 females) had Viangchan mutations (871 G > A). Sequencing analyses suggested that all the males were hemizygous Viangchan, whereas one was homozygous, and 18 were heterozygous Viangchan in females. These results suggested a relatively low prevalence of G6PD deficiency mutation rate (1.51%) in the minor ethnic populations residing in the Gia Lai province, Vietnam. However, considering these areas are high-risk malaria endemic, concern for proper and safe use of PQ as a radical cure of malaria is needed by combining a G6PD deficiency test before PQ prescription.

A Case Report of Primaquine Associated Methemoglobinemia in a Man With Pneumocystis Jirovecii Pneumonia Following Cardiac and Renal Transplantation.

We describe the case of a 51-year-old Caucasian man with a background of a cardiac and renal transplant who developed Enterocytozoon bieneusi colitis and pneumocystis jirovecii (PJP) pneumonia following treatment for suspected rejection. The patient developed methemoglobinemia which was attributed to primaquine. He was treated with intravenous methylene blue leading to clinical and biochemical resolution. We describe in detail the pathophysiological mechanism for methemoglobinemia and its treatment, in particular with methylene blue.

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.

Operational feasibility of Plasmodium vivax radical cure with tafenoquine or primaquine following point-of-care, quantitative glucose-6-phosphate dehydrogenase testing in the Brazilian Amazon: a real-life retrospective analysis.

BACKGROUND: To achieve malaria elimination, Brazil must implement Plasmodium vivax radical cure. We aimed to investigate the operational feasibility of point-of-care, quantitative, glucose-6-phosphate dehydrogenase (G6PD) testing followed by chloroquine plus tafenoquine or primaquine. METHODS: This non-interventional, observational study was done at 43 health facilities in Manaus (Amazonas State) and Porto Velho (Rondônia State), Brazil, implementing a new P vivax treatment algorithm incorporating point-of-care quantitative G6PD testing to identify G6PD status and single-dose tafenoquine (G6PD normal, aged ≥16 years, and not pregnant or breastfeeding) or primaquine (intermediate or normal G6PD, aged ≥6 months, not pregnant, or breastfeeding >1 month). Following training of health-care providers, we collated routine patient records from the malaria epidemiological surveillance system (SIVEP-Malaria) retrospectively for all consenting patients aged at least 6 months with parasitologically confirmed P vivax malaria mono-infection or P vivax plus P falciparum mixed infection, presenting between Sept 9, 2021, and Aug 31, 2022. The primary endpoint was the proportion of patients aged at least 16 years with P vivax mono-infection treated or not treated appropriately with tafenoquine in accordance with their G6PD status. The trial is registered with ClinicalTrials.gov, NCT05096702, and is completed. FINDINGS: Of 6075 patients enrolled, 6026 (99·2%) had P vivax mono-infection, 2685 (44·6%) of whom were administered tafenoquine. G6PD status was identified in 2685 (100%) of 2685 patients treated with tafenoquine. The proportion of patients aged at least 16 years with P vivax mono-infection who were treated or not treated appropriately with tafenoquine in accordance with their G6PD status was 99·7% (95% CI 99·4-99·8; 4664/4680). INTERPRETATION: Quantitative G6PD testing before tafenoquine administration was operationally feasible, with high adherence to the treatment algorithm, supporting deployment throughout the Brazilian health system. FUNDING: Brazilian Ministry of Health, Municipal and State Health Secretariats; Fiocruz; Medicines for Malaria Venture; Bill & Melinda Gates Foundation; Newcrest Mining; and the UK Government. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Comment on 'The clinical pharmacology of tafenoquine in the radical cure of Plasmodium vivax malaria: An individual patient data meta-analysis'.

A single 300 mg dose of tafenoquine, in combination with chloroquine, is currently approved in several countries for the radical cure (prevention of relapse) of Plasmodium vivax malaria in patients aged ≥16 years. Recently, however, Watson et al. suggested that the approved dose of tafenoquine is insufficient for radical cure, and that a higher 450 mg dose could reduce P. vivax recurrences substantially (Watson et al., 2022). In this response, we challenge Watson et al.'s assertion based on empirical evidence from dose-ranging and pivotal studies (published) as well as real-world evidence from post-approval studies (ongoing, therefore currently unpublished). We assert that, collectively, these data confirm that the benefit-risk profile of a single 300 mg dose of tafenoquine, co-administered with chloroquine, for the radical cure of P. vivax malaria in patients who are not G6PD-deficient, continues to be favourable where chloroquine is indicated for P. vivax malaria. If real-world evidence of sub-optimal efficacy in certain regions is observed or dose-optimisation with other blood-stage therapies is required, then well-designed clinical studies assessing safety and efficacy will be required before higher doses are approved for clinical use.

Response to comment on 'The clinical pharmacology of tafenoquine in the radical cure of Plasmodium vivax malaria: An individual patient data meta-analysis'.

In our recent paper on the clinical pharmacology of tafenoquine (Watson et al., 2022), we used all available individual patient pharmacometric data from the tafenoquine pre-registration clinical efficacy trials to characterise the determinants of anti-relapse efficacy in tropical vivax malaria. We concluded that the currently recommended dose of tafenoquine (300 mg in adults, average dose of 5 mg/kg) is insufficient for cure in all adults, and a 50% increase to 450 mg (7.5 mg/kg) would halve the risk of vivax recurrence by four months. We recommended that clinical trials of higher doses should be carried out to assess their safety and tolerability. Sharma and colleagues at the pharmaceutical company GSK defend the currently recommended adult dose of 300 mg as the optimum balance between radical curative efficacy and haemolytic toxicity (Sharma et al., 2024). We contend that the relative haemolytic risks of the 300 mg and 450 mg doses have not been sufficiently well characterised to justify this opinion. In contrast, we provided evidence that the currently recommended 300 mg dose results in sub-maximal efficacy, and that prospective clinical trials of higher doses are warranted to assess their risks and benefits.

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.

Artemisinin-naphthoquine plus lower-dose primaquine to treat and prevent recurrence of Plasmodium vivax malaria: an open-label randomized and non-inferiority trial.

BACKGROUND: Plasmodium vivax malaria, with the widest geographic distribution, can cause severe disease and death. Primaquine is the main licensed antimalarial drug that can kill hypnozoites. The dose-dependent acute haemolysis in individuals with glucose-6-phospate dehydrogenase (G6PD) deficiency is the main safety concern when using primaquine. The recommended treatment regimen for P. vivax malaria is chloroquine plus primaquine for 14 days (CQPQ14) in Myanmar. The study aimed to evaluate the therapeutic efficacy, safety and adherence for the regimen of artemisinin-naphthoquine plus primaquine for 3 days (ANPQ3) in patients with P. vivax infections compared to those with CQPQ14. METHODS: The patients in the ANPQ3 group were given fixed-dose artemisinin-naphthoquine (a total 24.5 mg/kg bodyweight) plus a lower total primaquine dose (0.9 mg/kg bodyweight) for 3 days. The patients in the CQPQ14 group were given a total chloroquine dose of 30 mg/kg body weight for 3 days plus a total primaquine dose of 4.2 mg/kg bodyweight for 14 days. All patients were followed up for 365 days. RESULTS: A total of 288 patients completed follow-up, 172 in the ANPQ3 group and 116 in the CQPQ14 group. The first recurrence patients were detected by day 58 in both groups. By day 182, 16 recurrences had been recorded: 12 (7.0%) patients in the ANPQ3 group and 4 (3.4%) in the CQPQ14 group. The difference in recurrence-free patients was 3.5 (-8.6 to 1.5) percentage points between ANPQ3 and CQPQ14 group (P = 0.2946). By day 365, the percentage of recurrence-free patients was not significant between the two groups (P = 0.2257). Mean fever and parasite clearance time of ANPQ3 group were shorter than those in CQPQ14 group (P ≤ 0.001). No severe adverse effect was observed in ANPQ3 group, but five (3.9%) patients had acute haemolysis in CQPQ14 group (P = 0.013). Medication percentage of ANPQ3 group was significantly higher than that of CQPQ14 group (P < 0.0001). CONCLUSIONS: Both ANPQ3 and CQPQ14 promised clinical cure efficacy, and the radical cure efficacy was similar between the ANPQ3 and CQPQ14 group. ANPQ3 clears fever and parasites faster than CQPQ14. ANPQ3 is safer and shows better patient adherence to the regimen for treatment of P. vivax malaria along the China-Myanmar border. TRIAL REGISTRATION: ChiCTR-INR-17012523. Registered 31 August 2017, https://www.chictr.org.cn/showproj.html?proj=21352.

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.

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.

Mass Relapse Prevention to Reduce Transmission of Plasmodium vivax- A Systematic Review.

Several temperate countries have used mass chemoprevention interventions with medicines of the 8-aminoquinoline class that prevent relapses from Plasmodium vivax before peak transmission to reduce transmission of malaria. The WHO commissioned a systematic review of the literature and evidence synthesis to inform development of recommendations regarding this intervention referred to as "mass relapse prevention" (MRP). Electronic databases were searched, 866 articles screened, and 25 assessed for eligibility after a full-text review. Two nonrandomized studies were included, one from the Democratic People's Republic of Korea (391,357 participants) and the second from the Azerbaijan Soviet Socialist Republic (∼30,000 participants). The two studies administered a single round of primaquine over 14 days (0.25 mg/kg per day). From 1 to 3 months after the treatment round, the incidence of P. vivax infections was significantly lower in areas that received MRP than those that did not (pooled rate ratio [RR] 0.08, 95% CI 0.07-0.08). At 4 to 12 months after the treatment round, the prevalence of P. vivax infection was significantly lower in MRP villages than non-MRP villages (odds ratio 0.12, 95% CI 0.03-0.52). No severe adverse events were found. The certainty of evidence for all outcomes was very low and no conclusions as to the effectiveness or safety of MRP could be drawn. However, it is not likely that this intervention will be needed in the future as most temperate countries where P. vivax is transmitted are nearing or have already eliminated malaria.

Prevalence of G6PD deficiency and submicroscopic malaria parasites carriage in malaria hotspot area in Northwest, Tanzania.

BACKGROUND: The use of primaquine for mass drug administration (MDA) is being considered as a key strategy for malaria elimination. In addition to being the only drug active against the dormant and relapsing forms of Plasmodium vivax, primaquine is the sole potent drug against mature/infectious Plasmodium falciparum gametocytes. It may prevent onward transmission and help contain the spread of artemisinin resistance. However, higher dose of primaquine is associated with the risk of acute haemolytic anaemia in individuals with a deficiency in glucose-6-phosphate dehydrogenase. In many P. falciparum endemic areas there is paucity of information about the distribution of individuals at risk of primaquine-induced haemolysis at higher dose 45 mg of primaquine. METHODS: A retrospective cross-sectional study was carried out using archived samples to establish the prevalence of G6PD deficiency in a malaria hotspot area in Misungwi district, located in Mwanza region, Tanzania. Blood samples collected from individuals recruited between August and November 2010 were genotyped for G6PD deficiency and submicroscopic parasites carriage using polymerase chain reaction. RESULTS: A total of 263 individuals aged between 0 and 87 were recruited. The overall prevalence of the X-linked G6PD A- mutation was 83.7% (220/263) wild type, 8% (21/263) heterozygous and 8.4% (22/263) homozygous or hemizygous. Although, assessment of the enzymatic activity to assign the phenotypes according to severity and clinical manifestation as per WHO was not carried out, the overall genotype and allele frequency for the G6PD deficiency was 16.4% and 13. 2%, respectively. There was no statistically significant difference in among the different G6PD genotypes (p > 0.05). Out of 248 samples analysed for submicroscopic parasites carriage, 58.1% (144/248) were P. falciparum positive by PCR. G6PD heterozygous deficiency were associated with carriage of submicroscopic P. falciparum (p = 0.029). CONCLUSIONS: This study showed that 16.4% of the population in this part of North-western Tanzania carry the G6PD A- mutation, within the range of 15-32% seen in other parts of Africa. G6PD gene mutation is widespread and heterogeneous across the study area where primaquine would be valuable for malaria control and elimination. The maps and population estimates presented here reflect potential risk of higher dose of primaquine being associated with the risk of acute haemolytic anaemia (AHA) in individuals with a deficiency in glucose-6-phosphate dehydrogenase and call further research on mapping of G6PD deficiency in Tanzania. Therefore, screening and education programmes for G6PD deficiency is warranted in a programme of malaria elimination using a higher primaquine dose.

Prevalence and molecular heterogeneity of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Senoi Malaysian Orang Asli population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder characterized by reduced G6PD enzyme levels in the blood. This condition is common in populations exposed to malaria; an acute febrile disease caused by Plasmodium parasites. G6PD-deficient individuals may suffer from acute hemolysis following the prescription of Primaquine, an antimalarial treatment. The population at risk for such a condition includes the Senoi group of Orang Asli, a remote indigenous community in Malaysia. This study aimed to elucidate the G6PD molecular heterogeneity in this subethnic group which is important for malaria elimination. A total of 662 blood samples (369 males and 293 females) from the Senoi subethnic group were screened for G6PD deficiency using a quantitative G6PD assay, OSMMR2000-D kit with Hb normalization. After excluding the family members, the overall prevalence of G6PD deficiency in the studied population was 15.2% (95% CI: 11-19%; 56 of 369), with males (30 of 172; 17.4%) outnumbering females (26 of 197; 13.2%). The adjusted male median (AMM), defined as 100% G6PD activity, was 11.8 IU/gHb. A total of 36 participants (9.6%; 26 male and 10 female) were deficient (<30% of AMM) and 20 participants (5.4%; 4 male and 16 female) were G6PD-intermediate (30-70% of AMM). A total of 87 samples were genotyped, of which 18 showed no mutation. Seven mutations were found among 69 genotyped samples; IVS11 T93C (47.1%; n = 41), rs1050757 (3'UTR +357A>G)(39.1%; n = 34), G6PD Viangchan (c.871G>A)(25.3%; n = 22), G6PD Union (c.1360C>T)(21.8%; n = 19), c.1311C>T(20.7%; n = 18), G6PD Kaiping (c.1388G>A)(8.0%; n = 7), and G6PD Coimbra (c.592C>T)(2.3%; n = 2). Our analysis revealed 27 hemizygote males, 18 heterozygote females, 7 homozygote females, and 2 compound heterozygote females. This study confirms the high prevalence of G6PD deficiency among the Senoi Malaysian Orang Asli, with a significant degree of molecular heterogeneity. More emphasis should be placed on screening for G6PD status and proper and safe use of Primaquine in the elimination of malaria among this indigenous population.

Can incorporating genotyping data into efficacy estimators improve efficiency of early phase malaria vaccine trials?

BACKGROUND: Early phase malaria vaccine field trials typically measure malaria infection by PCR or thick blood smear microscopy performed on serially sampled blood. Vaccine efficacy (VE) is the proportion reduction in an endpoint due to vaccination and is often calculated as VEHR = 1-hazard ratio or VERR = 1-risk ratio. Genotyping information can distinguish different clones and distinguish multiple infections over time, potentially increasing statistical power. This paper investigates two alternative VE endpoints incorporating genotyping information: VEmolFOI, the vaccine-induced proportion reduction in incidence of new clones acquired over time, and VEC, the vaccine-induced proportion reduction in mean number of infecting clones per exposure. METHODS: Power of VEmolFOI and VEC was compared to that of VEHR and VERR by simulations and analytic derivations, and the four VE methods were applied to three data sets: a Phase 3 trial of RTS,S malaria vaccine in 6912 African infants, a Phase 2 trial of PfSPZ Vaccine in 80 Burkina Faso adults, and a trial comparing Plasmodium vivax incidence in 466 Papua New Guinean children after receiving chloroquine + artemether lumefantrine with or without primaquine (as these VE methods can also quantify effects of other prevention measures). By destroying hibernating liver-stage P. vivax, primaquine reduces subsequent reactivations after treatment completion. RESULTS: In the trial of RTS,S vaccine, a significantly reduced number of clones at first infection was observed, but this was not the case in trials of PfSPZ Vaccine or primaquine, although the PfSPZ trial lacked power to show a reduction. Resampling smaller data sets from the large RTS,S trial to simulate phase 2 trials showed modest power gains from VEC compared to VEHR for data like those from RTS,S, but VEC is less powerful than VEHR for trials in which the number of clones at first infection is not reduced. VEmolFOI was most powerful in model-based simulations, but only the primaquine trial collected enough serial samples to precisely estimate VEmolFOI. The primaquine VEmolFOI estimate decreased after most control arm liver-stage infections reactivated (which mathematically resembles a waning vaccine), preventing VEmolFOI from improving power. CONCLUSIONS: The power gain from the genotyping methods depends on the context. Because input parameters for early phase power calculations are often uncertain, these estimators are not recommended as primary endpoints for small trials unless supported by targeted data analysis. TRIAL REGISTRATIONS: NCT00866619, NCT02663700, NCT02143934.