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BACKGROUND: Primaquine (PQ) is the prototype 8-aminoquinoline drug, a class which targets gametocytes and hypnozoites. The World Health Organization (WHO) recommends adding a single low dose of primaquine to the standard artemisinin-based combination therapy (ACT) in order to block malaria transmission in regions with low malaria transmission. However, the haemolytic toxicity is a major adverse outcome of primaquine in glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects. This study aimed to characterize the pharmacokinetic properties of primaquine and its major metabolites in G6PD-deficient subjects. METHODS: A single low-dose of primaquine (0.4-0.5 mg/kg) was administered in twenty-eight African males. Venous and capillary plasma were sampled up to 24 h after the drug administration. Haemoglobin levels were observed up to 28 days after drug administration. Only PQ, carboxy-primaquine (CPQ), and primaquine carbamoyl-glucuronide (PQCG) were present in plasma samples and measured using liquid chromatography mass spectrometry. Drug and metabolites pharmacokinetic properties were investigated using nonlinear mixed-effects modelling. RESULTS: Population pharmacokinetic properties of PQ, CPQ, and PQCG can be described by one-compartment disposition kinetics with a transit-absorption model. Body weight was implemented as an allometric function on the clearance and volume parameters for all compounds. None of the covariates significantly affected the pharmacokinetic parameters. No significant correlations were detected between the exposures of the measured compounds and the change in haemoglobin or methaemoglobin levels. There was no significant haemoglobin drop in the G6PD-deficient patients after administration of a single low dose of PQ. CONCLUSIONS: A single low-dose of PQ was haematologically safe in this population of G6PD-normal and G6PD-deficient African males without malaria. Trial registration NCT02535767.

Abstract Background Strengthening malaria control activities in Tanzania has dramatically declined human malaria infections. However, there is an increasing epidemiological shift in the burden on school-age children. The underlying causes for such an epidemiological shift remain unknown in this context. This study explored activities and behaviours that could increase the vulnerability of school-age children to transmission risk to provide insight into protection gap with existing interventions and opportunities for supplementary interventions. Methods This cross-sectional study conducted twenty-four focus group discussions (FGDs) in three districts of Rufiji, Kibiti and Kilwa in south-eastern Tanzania. Sixteen FGDs worked with school-age children (13 to 18 years) separating girls and boys and eight FGDs with their parents in mixed-gender groups. A total of 205 community members participated in FGDs across the study area. Of them, 72 participants were parents, while 133 were school-age children (65 boys and 68 girls). Results Routine domestic activities such as fetching water, washing kitchen utensils, cooking, and recreational activities such as playing and watching television and studying were the reported activities that kept school-age children outdoors early evening to night hours (between 18:00 and 23:00). Likewise, the social and cultural events including initiation ceremonies and livelihood activities also kept this age group outdoors from late evening to early night and sometimes past midnight hours. Parents migrating to farms from December to June, leaving behind school-age children unsupervised affecting their net use behaviour plus spending more time outdoors at night, and the behaviour of children sprawling legs and hands while sleeping inside treated bed nets were identified as potential risks to infectious mosquito bites. Conclusion The risky activities, behaviours, and social events mostly occurring outdoors might increase school-age children’s vulnerability to malaria infections. The findings provide preliminary insight on potential risk factors for persisting transmission. Further studies to quantify the risk behaviour and activities are recommended to establish the magnitude and anticipated impact on supplementary control strategies to control infection in school-age children.

Abstract Background Spatial repellents that create airborne concentrations of an active ingredient (AI) within a space offer a scalable solution to further reduce transmission of malaria, by disrupting mosquito behaviours in ways that ultimately lead to reduced human-vector contact. Passive emanator spatial repellents can protect multiple people within the treated space and can last for multiple weeks without the need for daily user touchpoints, making them less intrusive interventions. They may be particularly advantageous in certain use cases where implementation of core tools may be constrained, such as in humanitarian emergencies and among mobile at-risk populations. The purpose of this study was to assess the efficacy of Mosquito Shield™ deployed in experimental huts against wild, free-flying, pyrethroid-resistant Anopheles arabiensis mosquitoes in Tanzania over 1 month. Methods The efficacy of Mosquito Shield™ transfluthrin spatial repellent in reducing mosquito lands and blood-feeding was evaluated using 24 huts: sixteen huts were allocated to Human Landing Catch (HLC) collections and eight huts to estimating blood-feeding. In both experiments, half of the huts received no intervention (control) while the remaining received the intervention randomly allocated to huts and remained fixed for the study duration. Outcomes measured were mosquito landings, blood-fed, resting and dead mosquitoes. Data were analysed by multilevel mixed effects regression with appropriate dispersion and link function accounting for volunteer, hut and day. Results Landing inhibition was estimated to be 70% (57–78%) [IRR 0.30 (95% CI 0.22–0.43); p

Abstract Background Primaquine (PQ) has been used for the radical cure of relapsing Plasmodium vivax malaria for more than 60 years. PQ is also recommended for prophylaxis and prevention of transmission of Plasmodium falciparum. However, clinical utility of PQ has been limited due to toxicity in individuals with genetic deficiencies in glucose 6-phosphate dehydrogenase (G6PD). PQ is currently approved for clinical use as a racemic mixture. Recent studies in animals as well as humans have established differential pharmacological and toxicological properties of the two enantiomers of PQ. This has been attributed to differential metabolism and pharmacokinetics of individual PQ enantiomers. The aim of the current study is to evaluate the comparative pharmacokinetics (PK), tissue distribution and metabolic profiles of the individual enantiomers in mice. Methods Two groups of 21 male Albino ND4 Swiss mice were dosed orally with 45 mg/kg of S-(+)-PQ and R-(−)PQ respectively. Each of the enantiomers was comprised of a 50:50 mixture of 12C- and 13C- stable isotope labelled species (at 6 carbons on the benzene ring of the quinoline core). Three mice were euthanized from each group at different time points (at 0, 0.5, 1, 2, 4, 8, 24 h) and blood was collected by terminal cardiac bleed. Liver, spleen, lungs, kidneys and brain were removed, extracted and analysed using UPLC/MS. The metabolites were profiled by tandem mass (MS/MS) fragmentation profile and fragments with 12C–13C twin peaks. Non-compartmental analysis was performed using the Phoenix WinNonLin PK software module. Results The plasma AUC0-last (µg h/mL) (1.6 vs. 0.6), T1/2 (h) (1.9 vs. 0.45), and Tmax (h) (1 vs. 0.5) were greater for SPQ as compared to RPQ. Generally, the concentration of SPQ was higher in all tissues. At Tmax, (0.5–1 h in all tissues), the level of SPQ was 3 times that of RPQ in the liver. Measured Cmax of SPQ and RPQ in the liver were about 100 and 40 times the Cmax values in plasma, respectively. Similar observations were recorded in other tissues where the concentration of SPQ was higher compared to RPQ (2× in the spleen, 6× in the kidneys, and 49× in the lungs) than in the plasma. CPQ, the major metabolite, was preferentially generated from RPQ, with higher levels in all tissues (> 10× in the liver, and 3.5× in the plasma) than from SPQ. The PQ-o-quinone was preferentially formed from the SPQ (> 4× compared to RPQ), with higher concentrations in the liver. Conclusion These studies show that in mice, PQ enantiomers are differentially biodistributed and metabolized, which may contribute to differential pharmacologic and toxicity profiles of PQ enantiomers. The findings on higher levels of PQ-o-quinone in liver and RBCs compared to plasma and preferential generation of this metabolite from SPQ are consistent with the higher anti-malarial efficacy of SPQ observed in the mouse causal prophylaxis test, and higher haemolytic toxicity in the humanized mouse model of G6PD deficiency. Potential relevance of these findings to clinical use of racemic PQ and other 8-aminoquinolines vis-à-vis need for further clinical evaluation of individual enantiomers are discussed.

Abstract Background This study aimed to estimate the socio-economic costs of uncomplicated malaria and to explore health care-seeking behaviours that are likely to influence these costs in the Democratic Republic of Congo (DRC), a country ranked worldwide as the second most affected by malaria. Methods In 2017, a cross-sectional survey included patients with uncomplicated malaria in 64 healthcare facilities from 10 sentinel sites of the National Malaria Control Programme (NMCP) in the DRC. A standard questionnaire was used to assess health care-seeking behaviours of patients. Health-related quality of life (HRQL) and disutility weights (DW) of illness were evaluated by using the EuroQol Group’s descriptive system (EQ-5D-3L) and its visual analogue scale (EQ VAS). Malaria costs were estimated from a patient’s perspective. Probabilistic sensitivity analyses (PSA) evaluated the uncertainty around the cost estimates. Generalized regression models were fitted to assess the effect of potential predictive factors on the time lost and the DW during illness. Results In total, 1080 patients (age: 13.1 ± 14 years; M/F ratio: 1.1) were included. The average total costs amounted to US$ 36.3 [95% CI 35.5–37.2] per malaria episode, including US$ 16.7 [95% CI 16.3–17.1] as direct costs and US$ 19.6 [95% CI 18.9–20.3] indirect costs. During care seeking, economically active patients and their relatives lost respectively 3.3 ± 1.8 and 3.4 ± 2.1 working days. This time loss occurred mostly at the pre-hospital stage and was the parameter associated the most with the uncertainty around malaria cost estimates. Patients self-rated an average 0.36 ± 0.2 DW and an average 0.62 ± 0.3 EQ-5D index score per episode. A lack of health insurance coverage (896 out of 1080; 82.9%) incurred substantially higher costs, lower quality of life, and heavier DW while leading to longer time lost during illness. Residing in rural areas incurred a disproportionally higher socioeconomic burden of uncomplicated malaria with longer time lost due to illness and limited access to health insurance mechanisms. Conclusion Uncomplicated malaria is associated with high economic costs of care in the DRC. Efforts to reduce the cost-of-illness should target time lost at the pre-hospital stage and social disparities in the population, while reinforcing measures for malaria control in the country.

Abstract Background The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. Methods From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. Results Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed. Conclusion No molecular markers currently known to be associated with resistance to the first-line ACT in use were detected in isolates of P. falciparum from treatment failure patients. Regular monitoring through in vivo drug efficacy and molecular studies must continue to ensure the effectiveness of malaria treatment in Democratic Republic of Congo.

Abstract Background The loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as a first-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72–76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of Plasmodium falciparum, 16 years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype. Methods In 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was first detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72–76 was amplified by conventional PCR before sequencing. Results A total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI 25.4%–31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. Conclusion Overall, the frequency of the P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy.

Abstract Background The activity and haemolytic toxicity associated with primaquine has been linked to its reactive metabolites. The reactive metabolites are thought to be primarily formed through the action of cytochrome P450-mediated pathways. Human erythrocytes generally are not considered a significant contributor to drug biotransformation. As erythrocytes are the target of primaquine toxicity, the ability of erythrocytes to mediate the formation of reactive oxidative primaquine metabolites in the absence of hepatic enzymes, was evaluated. Methods Primaquine and its enantiomers were incubated separately with human red blood cells and haemoglobin. Post-incubation analysis was performed with UPLC–MS/MS to identify products of biotransformation. Results The major metabolite detected was identified as primaquine-5,6-orthoquinone, reflecting the pathway yielding putative active and haematotoxic metabolites of primaquine, which was formed by oxidative demethylation of 5-hydroxyprimaquine. Incubation of primaquine with haemoglobin in a cell-free system yielded similar results. It appears that the observed biotransformation is due to non-enzymatic processes, perhaps due to reactive oxygen species (ROS) present in erythrocytes or in the haemoglobin incubates. Conclusion This study presents new evidence that primaquine-5,6-orthoquinone, the metabolite of primaquine reflecting the oxidative biotransformation pathway, is generated in erythrocytes, probably by non-enzymatic means, and may not require transport from the liver or other tissues.

Abstract Background Primaquine (PQ), an 8-aminoquinoline, is the only drug approved by the United States Food and Drug Administration for radical cure and prevention of relapse in Plasmodium vivax infections. Knowledge of the metabolism of PQ is critical for understanding the therapeutic efficacy and hemolytic toxicity of this drug. Recent in vitro studies with primary human hepatocytes have been useful for developing the ultra high-performance liquid chromatography coupled with high-resolution mass spectrometric (UHPLC-QToF-MS) methods for simultaneous determination of PQ and its metabolites generated through phase I and phase II pathways for drug metabolism. Methods These methods were further optimized and applied for phenotyping PQ metabolites from plasma and urine from healthy human volunteers treated with single 45 mg dose of PQ. Identity of the metabolites was predicted by MetaboLynx using LC–MS/MS fragmentation patterns. Selected metabolites were confirmed with appropriate standards. Results Besides PQ and carboxy PQ (cPQ), the major plasma metabolite, thirty-four additional metabolites were identified in human plasma and urine. Based on these metabolites, PQ is viewed as metabolized in humans via three pathways. Pathway 1 involves direct glucuronide/glucose/carbamate/acetate conjugation of PQ. Pathway 2 involves hydroxylation (likely cytochrome P450-mediated) at different positions on the quinoline ring, with mono-, di-, or even tri-hydroxylations possible, and subsequent glucuronide conjugation of the hydroxylated metabolites. Pathway 3 involves the monoamine oxidase catalyzed oxidative deamination of PQ resulting in formation of PQ-aldehyde, PQ alcohol and cPQ, which are further metabolized through additional phase I hydroxylations and/or phase II glucuronide conjugations. Conclusion This approach and these findings augment our understanding and provide comprehensive view of pathways for PQ metabolism in humans. These will advance the clinical studies of PQ metabolism in different populations for different therapeutic regimens and an understanding of the role these play in PQ efficacy and safety outcomes, and their possible relation to metabolizing enzyme polymorphisms.

Abstract Background Malaria is rampant in Africa and causes untold mortality and morbidity. Vector-borne diseases are climate sensitive and this has raised considerable concern over the implications of climate change on future disease risk. The problem of malaria vectors (Anopheles mosquitoes) shifting from their traditional locations to invade new zones is an important concern. The vision of this study was to exploit the sets of information previously generated by entomologists, e.g. on geographical range of vectors and malaria distribution, to build models that will enable prediction and mapping the potential redistribution of Anopheles mosquitoes in Africa. Methods The development of the modelling tool was carried out through calibration of CLIMEX parameters. The model helped estimate the potential geographical distribution and seasonal abundance of the species in relation to climatic factors. These included temperature, rainfall and relative humidity, which characterized the living environment for Anopheles mosquitoes. The same parameters were used in determining the ecoclimatic index (EI). The EI values were exported to a GIS package for special analysis and proper mapping of the potential future distribution of Anopheles gambiae and Anophles arabiensis within the African continent under three climate change scenarios. Results These results have shown that shifts in these species boundaries southward and eastward of Africa may occur rather than jumps into quite different climatic environments. In the absence of adequate control, these predictions are crucial in understanding the possible future geographical range of the vectors and the disease, which could facilitate planning for various adaptation options. Conclusion Thus, the outputs from this study will be helpful at various levels of decision making, for example, in setting up of an early warning and sustainable strategies for climate change and climate change adaptation for malaria vectors control programmes in Africa.