Population pharmacokinetics of primaquine and its metabolites in African males

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 NCT0253576

A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes.

BACKGROUND: The transmission of malaria to mosquitoes depends on the presence of gametocytes that circulate in the peripheral blood of infected human hosts. Sensitive estimates of the densities of female gametocytes (FG) and male gametocytes (MG) may allow the prediction of infectivity to mosquitoes and thus a molecular estimate of the human infectious reservoir for transmission. METHODS: A novel multiplex qRT-PCR assay with intron-spanning primers was developed for the parallel quantification of FG and MG. CCp4 (PF3D7_0903800) transcripts specific for FG and PfMGET (PF3D7_1469900) transcripts specific for MG were quantified in total nucleic acids. The assay was validated on sex-sorted gametocytes from culture material and on samples from clinical trials with gametocytocidal drugs. Synthetic RNA standards were generated for the two targets genes and calibrated against known gametocyte quantities. RESULTS: The limit of detection was determined at 0.1 male and 0.1 female gametocyte/µL, which was equal to the limit of quantification (LOQ) for MG, while the LOQ for FG was 1 FG/µL. Results from previously reported clinical trials that used separate gametocyte qRT-PCR assays for FG (targeting Pfs25) and MG (targeting PfMGET) were reproduced with the multiplex assay. High levels of agreement between separate assays and the multiplex approach were observed (R2 = 0.9473, 95% CI 0.9314-0.9632, for FG measured by transcript levels of Pfs25 in qRT-PCR or CCp4 in multiplex; R2 = 0.8869, 95% CI 0.8541-0.9197, for MG measured by PfMGET in either single or multiplex qRT-PCR). FG and MG transcripts were detected in pure ring stage parasites at 10,000- and 100,000-fold reduced frequency for CCp4 and PfMGET, respectively. The CCp4 and PfMGET transcripts were equally stable under suboptimal storage conditions. CONCLUSIONS: Gametocyte densities and their sex ratios can be determined in the presented one-step multiplex assay with higher throughput than single assays. The interpretation of low gametocyte densities at asexual parasite densities above 1000 parasites/µL requires caution to avoid false positive gametocyte signals from spurious transcript levels in ring stage parasites

Mechanistic modeling of primaquine pharmacokinetics, gametocytocidal activity, and mosquito infectivity

Clinical studies have shown that adding a single 0.25 mg base/kg dose of primaquine to standard antimalarial regimens rapidly sterilizes Plasmodium falciparum gametocytes. However, the mechanism of action and overall impact on malaria transmission is still unknown. Using data from 81 adult Malians with P. falciparum falciparum gametocytemia who received the standard dihydroartemisinin-piperaquine treatment course and were randomized to receive either a single dose of primaquine between 0.0625 and 0.5 mg base/kg or placebo, we characterized the pharmacokinetic-pharmacodynamic relationships for transmission blocking activity. Both gametocyte clearance and mosquito infectivity were assessed. A mechanistically linked pharmacokinetic-pharmacodynamic model adequately described primaquine and carboxy-primaquine pharmacokinetics, gametocyte dynamics, and mosquito infectivity at different clinical doses of primaquine. Primaquine showed a dose-dependent gametocytocidal effect that precedes clearance. A single low dose of primaquine (0.25 mg/kg) rapidly prevented P. falciparum transmissibility.</p

Persistence of mRNA indicative of Plasmodium falciparum ring-stage parasites 42 days after artemisinin and non-artemisinin combination therapy in naturally infected Malians

Contains fulltext : 230372.pdf (publisher's version ) (Open Access

Transmission-blocking effects of primaquine and methylene blue suggest P. falciparum gametocyte sterilisation rather than effects on sex ratio

Gametocyte density and sex-ratio can predict the proportion of mosquitoes that become infected after feeding on blood of patients receiving non-gametocytocidal drugs. Because primaquine and methylene blue sterilize gametocytes before affecting their density and sex-ratio, mosquito feeding experiments are required to demonstrate their early transmission-blocking effects

A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes

Contains fulltext : 199051.pdf (publisher's version ) (Open Access

Genome-wide genetic variation and molecular surveillance of drug resistance in Plasmodium falciparum isolates from asymptomatic individuals in Ouélessébougou, Mali.

Sequence analysis of Plasmodium falciparum parasites is informative in ensuring sustained success of malaria control programmes. Whole-genome sequencing technologies provide insights into the epidemiology and genome-wide variation of P. falciparum populations and can characterise geographical as well as temporal changes. This is particularly important to monitor the emergence and spread of drug resistant P. falciparum parasites which is threatening malaria control programmes world-wide. Here, we provide a detailed characterisation of genome-wide genetic variation and drug resistance profiles in asymptomatic individuals in South-Western Mali, where malaria transmission is intense and seasonal, and case numbers have recently increased. Samples collected from Ouélessébougou, Mali (2019-2020; n = 87) were sequenced and placed in the context of older Malian (2007-2017; n = 876) and African-wide (n = 711) P. falciparum isolates. Our analysis revealed high multiclonality and low relatedness between isolates, in addition to increased frequencies of molecular markers for sulfadoxine-pyrimethamine and lumefantrine resistance, compared to older Malian isolates. Furthermore, 21 genes under selective pressure were identified, including a transmission-blocking vaccine candidate (pfCelTOS) and an erythrocyte invasion locus (pfdblmsp2). Overall, our work provides the most recent assessment of P. falciparum genetic diversity in Mali, a country with the second highest burden of malaria in West Africa, thereby informing malaria control activities

Mechanistic Modeling of Primaquine Pharmacokinetics, Gametocytocidal Activity, and Mosquito Infectivity.

Clinical studies have shown that adding a single 0.25 mg base/kg dose of primaquine to standard antimalarial regimens rapidly sterilizes Plasmodium falciparum gametocytes. However, the mechanism of action and overall impact on malaria transmission is still unknown. Using data from 81 adult Malians with P. falciparum gametocytemia who received the standard dihydroartemisinin-piperaquine treatment course and were randomized to receive either a single dose of primaquine between 0.0625 and 0.5 mg base/kg or placebo, we characterized the pharmacokinetic-pharmacodynamic relationships for transmission blocking activity. Both gametocyte clearance and mosquito infectivity were assessed. A mechanistically linked pharmacokinetic-pharmacodynamic model adequately described primaquine and carboxy-primaquine pharmacokinetics, gametocyte dynamics, and mosquito infectivity at different clinical doses of primaquine. Primaquine showed a dose-dependent gametocytocidal effect that precedes clearance. A single low dose of primaquine (0.25 mg/kg) rapidly prevented P. falciparum transmissibility

Measuring the impact of seasonal malaria chemoprevention as part of routine malaria control in Kita, Mali

Abstract Background Seasonal malaria chemoprevention (SMC) is a new strategy recommended by WHO in areas of highly seasonal transmission in March 2012. Although randomized controlled trials (RCTs) have shown SMC to be highly effective, evidence and experience from routine implementation of SMC are limited. Methods A non-randomized pragmatic trial with pre-post design was used, with one intervention district (Kita), where four rounds of SMC with sulfadoxine + amodiaquine (SP + AQ) took place in August–November 2014, and one comparison district (Bafoulabe). The primary aims were to evaluate SMC coverage and reductions in prevalence of malaria and anaemia when SMC is delivered through routine programmes using existing community health workers. Children aged 3–59 months from 15 selected localities per district, sampled with probability proportional to size, were surveyed and blood samples collected for malaria blood smears, haemoglobin (Hb) measurement, and molecular markers of drug resistance in two cross-sectional surveys, one before SMC (July 2014) and one after SMC (December 2014). Difference-in-differences regression models were used to assess and compare changes in malaria and anaemia in the intervention and comparison districts. Adherence and tolerability of SMC were assessed by cross-sectional surveys 4–7 days after each SMC round. Coverage of SMC was assessed in the post-SMC survey. Results During round 1, 84% of targeted children received at least the first SMC dose, but coverage declined to 67% by round 4. Across the four treatment rounds, 54% of children received four complete SMC courses. Prevalence of parasitaemia was similar in intervention and comparison districts prior to SMC (23.4 vs 29.5%, p = 0.34) as was the prevalence of malaria illness (2.4 vs 1.9%, p = 0.75). After SMC, parasitaemia prevalence fell to 18% in the intervention district and increased to 46% in the comparison district [difference-in-differences (DD) OR = 0.35; 95% CI 0.20–0.60]. Prevalence of malaria illness fell to a greater degree in the intervention district versus the comparison district (DD OR = 0.20; 95% CI 0.04–0.94) and the same for moderate anaemia (Hb < 8 g/dL) (DD OR = 0.26, 95% CI 0.11–0.65). The frequency of the quintuple mutation (dhfr N51I, C59R and S108N + dhps A437G and K540E) remained low (5%) before and after intervention in both districts. Conclusions Routine implementation of SMC in Mali substantially reduced malaria and anaemia, with reductions of similar magnitude to those seen in previous RCTs. Improving coverage could further strengthen SMC impact. Trial registration clinical trial registration number NCT0289429

IFN-λ4 is associated with increased risk and earlier occurrence of several common infections in African children

Genetic polymorphisms within the IFNL3/IFNL4 genomic region, which encodes type III interferons, have been strongly associated with clearance of hepatitis C virus. We hypothesized that type III interferons might be important for the immune response to other pathogens as well. In a cohort of 914 Malian children, we genotyped functional variants IFNL4-rs368234815, IFNL4-rs117648444, and IFNL3-rs4803217 and analyzed episodes of malaria, gastrointestinal, and respiratory infections recorded at 30,626 clinic visits from birth up to 5 years of age. Compared to children with the rs368234815-TT/TT genotype (IFN-λ4-Null), rs368234815-dG allele was most strongly associated with an earlier time-to-first episode of gastrointestinal infections (p = 0.003). The risk of experiencing an infection episode during the follow-up was also significantly increased with rs368234815-dG allele, with OR = 1.53, 95%CI (1.13-2.07), p = 0.005 for gastrointestinal infections and OR = 1.30, 95%CI (1.02-1.65), p = 0.033 for malaria. All the associations for the moderately linked rs4803217 (r2 = 0.78 in this set) were weaker and lost significance after adjusting for rs368234815. We also analyzed all outcomes in relation to IFN-λ4-P70S groups. Our results implicate IFN-λ4 and not IFN-λ3 as the primary functional cause of genetic associations with increased overall risk and younger age at first clinical episodes but not with recurrence or intensity of several common pediatric infections