A nanochitosan-D-galactose formulation increases the accumulation of primaquine in the liver.

Primaquine is the mainstream antimalarial drug to prevent Plasmodium vivax relapses. However, this drug can induce hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. Nanostructure formulations of primaquine loaded with D-galactose were used as a strategy to target the drug to the liver and decrease the hemolytic risks. Nanoemulsion (NE-Pq) and nanochitosan (NQ-Pq) formulations of primaquine diphosphate containing D-galactose were prepared and characterized by their physicochemistry properties. Pharmacokinetic and biodistribution studies were conducted using Swiss Webster mice. A single dose of 10 mg/kg of each nanoformulation or free primaquine solution was administered by gavage to the animals, which were killed at 0.5, 1, 2, 4, 8, and 24 hours. Blood samples and tissues were collected, processed, and analyzed by high-performance liquid chromatography. The nanoformulation showed sizes around 200 nm (NE-Pq) and 400 nm (NQ-Pq) and physicochemical stability for over 30 days. Free primaquine solution achieved higher primaquine Cmax in the liver than NE-Pq or NQ-Pq at 0.5 hours. However, the half-life and mean residence time (MRT) of primaquine in the liver were three times higher with the NQ-Pq formulation than with free primaquine, and the volume distribution was four times higher. Conversely, primaquine's half-life, MRT, and volume distribution in the plasma were lower for NQ-Pq than for free primaquine. NE-Pq, on the other hand, accumulated more in the lungs but not in the liver. Galactose-coated primaquine nanochitosan formulation showed increased drug targeting to the liver compared to free primaquine and may represent a promising strategy for a more efficient and safer radical cure for vivax malaria.

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.

Bioequivalence of a new coated 15 mg primaquine formulation for malaria elimination.

BACKGROUND: With only one 15 mg primaquine tablet registered by a stringent regulatory authority and marketed, more quality-assured primaquine is needed to meet the demands of malaria elimination. METHODS: A classic, two sequence, crossover study, with a 10-day wash out period, of 15 mg of IPCA-produced test primaquine tablets and 15 mg of Sanofi reference primaquine tablets was conducted. Healthy volunteers, aged 18-45 years, without glucose-6-phosphate dehydrogenase deficiency, a baseline haemoglobin ≥ 11 g/dL, creatinine clearance ≥ 70 mL/min/1.73 ms, and body mass index of 18.5-30 kg/m2 were randomized to either test or reference primaquine, administered on an empty stomach with 240 mL of water. Plasma primaquine and carboxyprimaquine concentrations were measured at baseline, then 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.333, 2.667, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 8.0, 10.0, 12.0, 16.0, 24.0, 36.0, 48.0 and 72.0 h by liquid chromatography coupled to tandem mass spectrometry. Primaquine pharmacokinetic profiles were evaluated by non-compartmental analysis and bioequivalence concluded if the 90% confidence intervals (CI) of geometric mean (GM) ratios of test vs. reference formulation for the peak concentrations (Cmax) and area under the drug concentration-time (AUC0-t) were within 80.00 to 125.00%. RESULTS: 47 of 50 volunteers, median age 33 years, completed both dosing rounds and were included in the bioequivalence analysis. For primaquine, GM Cmax values for test and reference formulations were 62.12 vs. 59.63 ng/mL, resulting in a GM ratio (90% CI) of 104.17% (96.92-111.96%); the corresponding GM AUC0-t values were 596.56 vs. 564.09 ngxh/mL, for a GM ratio of 105.76% (99.76-112.08%). Intra-subject coefficient of variation was 20.99% for Cmax and 16.83% for AUC0-t. Median clearances and volumes of distribution were similar between the test and reference products: 24.6 vs. 25.2 L/h, 189.4 vs. 191.0 L, whilst the median half-lives were the same, 5.2 h. CONCLUSION: IPCA primaquine was bioequivalent to the Sanofi primaquine. This opens the door to prequalification, registration in malaria endemic countries, and programmatic use for malaria elimination. Trial registration The trial registration reference is ISRCTN 54640699.

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 NCT02535767.

Efficacy and safety of chloroquine plus primaquine for the treatment of Plasmodium vivax malaria in Hamusit site, Northwestern Ethiopia.

BACKGROUND: Plasmodium vivax malaria is still an important public health problem in Ethiopia. Unlike Plasmodium falciparum, P. vivax has a dormant liver stage (hypnozoite) that can be a risk of recurrent vivax malaria unless treated by radical cure with primaquine. Drug resistance to chloroquine is threatening malaria control and elimination efforts. This study assessed the therapeutic efficacy and safety of chloroquine plus 14 days of primaquine on P. vivax infection based on parasitological, clinical, and haematological parameters. METHODS: A single-arm in vivo prospective therapeutic efficacy study was conducted to assess the clinical and parasitological response to the first-line treatment of P. vivax in Ethiopia, chloroquine plus 14 days low dose of (0.25 mg/kg/day) primaquine between December 2022 and March 2023 at Hamusit Health Centre using the standard World Health Organization (WHO) protocol. A total of 100 study participants with P. vivax mono-infection who were over 6 months old were enrolled and monitored for adequate clinical and parasitological responses for 42 days. The WHO double-entry Excel sheet and SPSS v.25 software were used for Kaplan-Meier survival analysis, and a paired t-test was used for analysis of haemoglobin improvements between follow up days. RESULTS: A total of 100 patients were enrolled among those, 96% cases were rural residents, 93% had previous malaria exposure, and predominant age group was 5-15 years (61%). 92.6% (95% CI 85.1-96.4%) of enrolled patients were adequate clinical and parasitological response, and 7.4% (95% CI 3.6-14.9%) recurrences were observed among treated patients. The fever and parasite clearance rate on day 3 were 98% and 94%, respectively. The baseline haemoglobin levels improved significantly compared to those days 14 and 42 (p < 0.001). No serious adverse event was observed during the study period. CONCLUSIONS: In this study, co-administration of chloroquine with primaquine was efficacious and well-tolerated with fast resolution of fever and high parasites clearance rate. However, the 7.4% failure is reported is alarming that warrant further monitoring of the therapeutic efficacy study of P. vivax.

Kinetics of glucose-6-phosphate dehydrogenase (G6PD) activity during Plasmodium vivax infection: implications for early radical malaria treatment.

BACKGROUND: Plasmodium vivax relapses due to dormant liver hypnozoites can be prevented with primaquine. However, the dose must be adjusted in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. In French Guiana, assessment of G6PD activity is typically delayed until day (D)14 to avoid the risk if misclassification. This study assessed the kinetics of G6PD activity throughout P. vivax infection to inform the timing of treatment. METHODS: For this retrospective monocentric study, data on G6PD activity between D1 and D28 after treatment initiation with chloroquine or artemisinin-based combination therapy were collected for patients followed at Cayenne Hospital, French Guiana, between January 2018 and December 2020. Patients were divided into three groups based on the number of available G6PD activity assessments: (i) at least two measurements during the P. vivax malaria infection; (ii) two measurements: one during the current infection and one previously; (iii) only one measurement during the malaria infection. RESULTS: In total, 210 patients were included (80, 20 and 110 in groups 1, 2 and 3, respectively). Data from group 1 showed that G6PD activity remained stable in each patient over time (D1, D3, D7, D14, D21, D28). None of the patients with normal G6PD activity during the initial phase (D1-D3) of the malaria episode (n = 44) was categorized as G6PD-deficient at D14. Patients with G6PD activity < 80% at D1 or D3 showed normal activity at D14. Sex and reticulocyte count were statistically associated with G6PD activity variation. In the whole sample (n = 210), no patient had severe G6PD deficiency (< 10%) and only three between 10 and 30%, giving a G6PD deficiency prevalence of 1.4%. Among the 100 patients from group 1 and 2, 30 patients (26.5%) were lost to follow-up before primaquine initiation. CONCLUSIONS: In patients treated for P. vivax infection, G6PD activity did not vary over time. Therefore, G6PD activity on D1 instead of D14 could be used for primaquine dose-adjustment. This could allow earlier radical treatment with primaquine, that could have a public health impact by decreasing early recurrences and patients lost to follow-up before primaquine initiation. This hypothesis needs to be confirmed in larger prospective studies.

The effect of single low-dose primaquine treatment for uncomplicated Plasmodium falciparum malaria on haemoglobin levels in Ethiopia: a longitudinal cohort study.

BACKGROUND: To interrupt residual malaria transmission and achieve successful elimination of Plasmodium falciparum in low-transmission settings, the World Health Organization (WHO) recommends the administration of a single dose of 0.25 mg/kg (or 15 mg/kg for adults) primaquine (PQ) combined with artemisinin-based combination therapy (ACT), without glucose-6-phosphate dehydrogenase (G6PD) testing. However, due to the risk of haemolysis in patients with G6PD deficiency (G6PDd), PQ use is uncommon. Thus, this study aimed to assess the safety of a single low dose of PQ administered to patients with G6PD deficiency. METHODS: An observational cohort study was conducted with patients treated for uncomplicated P. falciparum malaria with either single-dose PQ (0.25 mg/kg) (SLD PQ) + ACT or ACT alone. Microscopy-confirmed uncomplicated P. falciparum malaria patients visiting public health facilities in Arjo Didessa, Southwest Ethiopia, were enrolled in the study from September 2019 to November 2022. Patients with uncomplicated P. falciparum malaria were followed up for 28 days through clinical and laboratory diagnosis, such as measurements of G6PD levels and haemoglobin (Hb) concentrations. G6PD levels were measured by a quantiative CareSTART™ POCT S1 biosensor machine. Patient interviews were also conducted, and the type and frequency of clinical complaints were recorded. Hb data were taken on days (D) 7, 14, 21, and 28 following treatment with SLD-PQ + ACT or ACT alone. RESULTS: A total of 249 patients with uncomplicated P. falciparum malaria were enrolled in this study. Of these, 83 (33.3%) patients received ACT alone, and 166 (66.7%) received ACT combined with SLD-PQ treatment. The median age of the patients was 20 (IQR 28-15) years. G6PD deficiency was found in 17 (6.8%) patients, 14 males and 3 females. There were 6 (7.2%) and 11 (6.6%) phenotypic G6PD-deficient patients in the ACT alone and ACT + SLD-PQ arms, respectively. The mean Hb levels in patients treated with ACT + SLD-PQ were reduced by an average of 0.45 g/dl (95% CI = 0.39 to 0.52) in the posttreatment phase (D7) compared to a reduction of 0.30 g/dl (95% CI = 0.14 to - 0.47) in patients treated with ACT alone (P = 0.157). A greater mean Hb reduction was observed on day 7 in the G6PDd ACT + SLD-PQ group (- 0.60 g/dL) than in the G6PDd ACT alone group (- 0.48 g/dL); however, there was no statistically significant difference (P = 0.465). Overall, D14 losses were 0.10 g/dl (95% CI = - 0.00 to 0.20) and 0.05 g/dl (95% CI = - 0.123 to 0.22) in patients with and without SLD-PQ, respectively (P = 0.412). CONCLUSIONS: This study's findings indicate that using SLD-PQ in combination with ACT is safe for uncomplicated P. falciparum malaria regardless of the patient's G6PD status in Ethiopian settings. Caution should be taken in extrapolating this finding in other settings with diverse G6DP phenotypes.

Fish arginase constrains excessive production of nitric oxide and limits mitochondrial damage during Aeromonas hydrophila infection.

Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 µM of l-arginine and reached the highest yield at 300 and 3000 µM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 µM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.

Discovery of harmiprims, harmine-primaquine hybrids, as potent and selective anticancer and antimalarial compounds.

Although cancer and malaria are not etiologically nor pathophysiologically connected, due to their similarities successful repurposing of antimalarial drugs for cancer and vice-versa is known and used in clinical settings and drug research and discovery. With the growing resistance of cancer cells and Plasmodium to the known drugs, there is an urgent need to discover new chemotypes and enrich anticancer and antimalarial drug portfolios. In this paper, we present the design and synthesis of harmiprims, hybrids composed of harmine, an alkaloid of the ß-carboline type bearing anticancer and antiplasmodial activities, and primaquine, 8-aminoquinoline antimalarial drug with low antiproliferative activity, covalently bound via triazole or urea. Evaluation of their antiproliferative activities in vitro revealed that N-9 substituted triazole-type harmiprime was the most selective compound against MCF-7, whereas C1-substituted ureido-type hybrid was the most active compound against all cell lines tested. On the other hand, dimeric harmiprime was not toxic at all. Although spectrophotometric studies and thermal denaturation experiments indicated binding of harmiprims to the ds-DNA groove, cell localization showed that harmiprims do not enter cell nucleus nor mitochondria, thus no inhibition of DNA-related processes can be expected. Cell cycle analysis revealed that C1-substituted ureido-type hybrid induced a G1 arrest and reduced the number of cells in the S phase after 24 h, persisting at 48 h, albeit with a less significant increase in G1, possibly due to adaptive cellular responses. In contrast, N-9 substituted triazole-type harmiprime exhibited less pronounced effects on the cell cycle, particularly after 48 h, which is consistent with its moderate activity against the MCF-7 cell line. On the other hand, screening of their antiplasmodial activities against the erythrocytic, hepatic, and gametocytic stages of the Plasmodium life cycle showed that dimeric harmiprime exerts powerful triple-stage antiplasmodial activity, while computational analysis showed its binding within the ATP binding site of PfHsp90.

Effectiveness of unsupervised primaquine regimen for preventing Plasmodium vivax malaria relapses in northeast Myanmar, a single-arm non-randomized observational study.

BACKGROUND: Plasmodium vivax presents a significant challenge for malaria elimination in the Greater Mekong Subregion (GMS). We evaluated the effectiveness of primaquine (PQ) for reducing relapses of vivax malaria. METHODS: Patients with uncomplicated P. vivax malaria from eastern Myanmar received chloroquine (CQ, 25 mg base/kg given in 3 days) plus unsupervised PQ (0.25 mg/kg/day for 14 days) without screening for glucose-6-phosphate dehydrogenase deficiency and were followed for a year. RESULTS: Totally 556 patients were enrolled to receive the CQ/PQ treatment from February 2012 to August 2013. During the follow-up, 38 recurrences were detected, presenting a cumulative rate of recurrence of 9.1% (95% confidence interval, 4.1-14.1%). Genotyping at the pvmsp1 and pvmsp3α loci by Amplicon deep sequencing and model prediction indicated that 13 of the 27 recurrences with genotyping data were likely due to relapses. Notably, all confirmed relapses occurred within the first six months. CONCLUSIONS: The unsupervised standard dose of PQ was highly effective as a radical cure for P. vivax malaria in eastern Myanmar. The high presumed effectiveness might have benefited from the health messages delivered during the enrollment and follow-up activities. Six-month follow-ups in the GMS are sufficient for detecting most relapses.

Low prevalence of antimalarial-resistance mutations in India during 2014-15: Impact of combining first-line therapy with primaquine.

BACKGROUND: Antimalarial drug resistance surveillance and containment are crucial for countries aiming to eliminate malaria. Monitoring resistance evolution through studies before and after treatment policy changes is crucial. METHOD: A total of 939 P. falciparum-positive blood samples were collected between 2014 and 2015 across ten sites in India, categorized into four geographic clusters. PCR-amplified products were sequenced to identify point mutations at drug-resistance-conferring genes (Pfdhfr, Pfdhps, Pfmdr1, Pfk13). RESULT: Triple Pfdhfr mutants were found only in northeast India bordering Myanmar, while the wildtype was dominant in central India. Pfdhps wildtypes were prevalent in all areas, and no double mutants were found. Except in Northwest India, Pfmdr1 wildtype was dominant in all clusters. Nonsynonymous double mutations were only found in northwest India. Only synonymous mutations occurred in Pfk13. These were found in Central India at low frequency. The pattern of linkage disequilibrium and principal component analysis reflects low pressure for drug resistance and heterogeneity between the geographic clusters. CONCLUSION: Resistance levels were highest in Northeast India, close to the Myanmar border, where resistance is common. Primaquine has been widely used as a gametocidal and schizonticidal drug, has likely contributed to maintaining low drug resistance levels and preventing strong selection for resistance.

Factors affecting haemoglobin dynamics in African children with acute uncomplicated Plasmodium falciparum malaria treated with single low-dose primaquine or placebo.

BACKGROUND: Single low-dose primaquine (SLDPQ) effectively blocks the transmission of Plasmodium falciparum malaria, but anxiety remains regarding its haemolytic potential in patients with glucose-6-phopshate dehydrogenase (G6PD) deficiency. We, therefore, examined the independent effects of several factors on haemoglobin (Hb) dynamics in falciparum-infected children with a particular interest in SLDPQ and G6PD status. METHODS: This randomised, double-blind, placebo-controlled, safety trial was conducted in Congolese and Ugandan children aged 6 months-11 years with acute uncomplicated P. falciparum and day (D) 0 Hbs ≥ 6 g/dL who were treated with age-dosed SLDPQ/placebo and weight-dosed artemether lumefantrine (AL) or dihydroartemisinin piperaquine (DHAPP). Genotyping defined G6PD (G6PD c.202T allele), haemoglobin S (HbS), and α-thalassaemia status. Multivariable linear and logistic regression assessed factor independence for continuous Hb parameters and Hb recovery (D42 Hb > D0 Hb), respectively. RESULTS: One thousand one hundred thirty-seven children, whose median age was 5 years, were randomised to receive: AL + SLDPQ (n = 286), AL + placebo (286), DHAPP + SLDPQ (283), and DHAPP + placebo (282). By G6PD status, 284 were G6PD deficient (239 hemizygous males, 45 homozygous females), 119 were heterozygous females, 418 and 299 were normal males and females, respectively, and 17 were of unknown status. The mean D0 Hb was 10.6 (SD 1.6) g/dL and was lower in younger children with longer illnesses, lower mid-upper arm circumferences, splenomegaly, and α-thalassaemia trait, who were either G6PDd or heterozygous females. The initial fractional fall in Hb was greater in younger children with higher D0 Hbs and D0 parasitaemias and longer illnesses but less in sickle cell trait. Older G6PDd children with lower starting Hbs and greater factional falls were more likely to achieve Hb recovery, whilst lower D42 Hb concentrations were associated with younger G6PD normal children with lower fractional falls, sickle cell disease, α-thalassaemia silent carrier and trait, and late treatment failures. Ten blood transfusions were given in the first week (5 SLDPQ, 5 placebo). CONCLUSIONS: In these falciparum-infected African children, posttreatment Hb changes were unaffected by SLDPQ, and G6PDd patients had favourable posttreatment Hb changes and a higher probability of Hb recovery. These reassuring findings support SLDPQ deployment without G6PD screening in Africa. TRIAL REGISTRATION: The trial is registered at ISRCTN 11594437.

Treatment of uncomplicated Plasmodium vivax with chloroquine plus radical cure with primaquine without G6PDd testing is safe in Arba Minch, Ethiopia: assessment of clinical and parasitological response.

BACKGROUND: Ethiopia rolled out primaquine nationwide in 2018 for radical cure along with chloroquine for the treatment of uncomplicated Plasmodium vivax malaria in its bid for malaria elimination by 2030. The emergence of anti-malarial drug resistance would challenge the elimination goal. There is limited evidence on the emergence of chloroquine drug resistance. The clinical and parasitological outcomes of treatment of P. vivax with chloroquine plus radical cure using low dose 14 days primaquine were assessed in an endemic area of Ethiopia. METHODS: A semi-directly observed 42-days follow up in-vivo therapeutic efficacy study was conducted from October 2019 to February 2020. Plasmodium vivax mono-species infected patients (n = 102) treated with a 14 days low dose (0.25 mg/kg body weight per day) primaquine plus chloroquine (a total dose of 25 mg base/kg for 3 days) were followed for 42 days to examine clinical and parasitological outcomes. Samples collected at recruitment and days of recurrence were examined by 18 S based nested polymerase chain reaction (nPCR) and Pvmsp3α nPCR-restriction fragment length polymorphism. Asexual parasitaemia and the presence of gametocytes were assessed on the scheduled days using microscopy. Clinical symptoms, haemoglobin levels, and Hillmen urine test were also assessed. RESULTS: Of the 102 patients followed in this study, no early clinical and parasitological failure was observed. All patients had adequate clinical and parasitological responses within the 28 days of follow up. Late clinical (n = 3) and parasitological (n = 6) failures were observed only after day 28. The cumulative incidence of failure was 10.9% (95% confidence interval, 5.8-19.9%) on day 42. Among the paired recurrent samples, identical clones were detected only in two samples on day 0 and day of recurrences (day 30 and 42) using Pvmsp3α genotyping. No adverse effect was detected related to the low dose 14 days primaquine administrations. CONCLUSION: Co-administration of CQ with PQ in the study area is well tolerated and there was no recurrence of P. vivax before 28 days of follow up. Interpretation of CQ plus PQ efficacy should be done with caution especially when the recurrent parasitaemia occurs after day 28. Therapeutic efficacy studies with appropriate design might be informative to rule out chloroquine or primaquine drug resistance and/or metabolism in the study area.

Effect of adherence to primaquine on the risk of Plasmodium vivax recurrence: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Imperfect adherence is a major barrier to effective primaquine radical cure of Plasmodium vivax. This study investigated the effect of reduced adherence on the risk of P. vivax recurrence. METHODS: Efficacy studies of patients with uncomplicated P. vivax malaria, including a treatment arm with daily primaquine, published between January 1999 and March 2020 were identified. Individual patient data from eligible studies were pooled using standardized methodology. Adherence to primaquine was inferred from i) the percentage of supervised doses and ii) the total mg/kg dose received compared to the target total mg/kg dose per protocol. The effect of adherence to primaquine on the incidence of P. vivax recurrence between days 7 and 90 was investigated by Cox regression analysis. RESULTS: Of 82 eligible studies, 32 were available including 6917 patients from 18 countries. For adherence assessed by percentage of supervised primaquine, 2790 patients (40.3%) had poor adherence (≤ 50%) and 4127 (59.7%) had complete adherence. The risk of recurrence by day 90 was 14.0% [95% confidence interval: 12.1-16.1] in patients with poor adherence compared to 5.8% [5.0-6.7] following full adherence; p = 0.014. After controlling for age, sex, baseline parasitaemia, and total primaquine dose per protocol, the rate of the first recurrence was higher following poor adherence compared to patients with full adherence (adjusted hazard ratio (AHR) = 2.3 [1.8-2.9]). When adherence was quantified by total mg/kg dose received among 3706 patients, 347 (9.4%) had poor adherence, 88 (2.4%) had moderate adherence, and 3271 (88.2%) had complete adherence to treatment. The risks of recurrence by day 90 were 8.2% [4.3-15.2] in patients with poor adherence and 4.9% [4.1-5.8] in patients with full adherence; p < 0.001. CONCLUSION: Reduced adherence, including less supervision, increases the risk of vivax recurrence.

Interventions for promoting patients' adherence to 14-day primaquine treatment in a highly malaria-endemic township in Myanmar: a qualitative study among key stakeholders.

BACKGROUND: Plasmodium vivax malaria is considered a major threat to malaria eradication. The radical cure for P. vivax malaria normally requires a 14-day administration of primaquine (PQ) to clear hypnozoites. However, maintaining adherence to PQ treatment is a significant challenge, particularly in malaria-endemic rural areas. Hence, this study aimed to formulate interventions for promoting patients' commitment to PQ treatment in a highly malaria-endemic township in Myanmar. METHODS: A qualitative study was conducted in Waingmaw Township in northern Myanmar, where P. vivax malaria is highly endemic. Key stakeholders including public health officers and community members participated in focus group discussions (FGDs) and in-depth interviews (IDIs) in September 2022. Data were collected using validated guidelines, translated into English, and visualized through thematic analysis. RESULTS: Responsible individuals from different levels of the Myanmar National Malaria Control Programme participated in the IDIs. Most of them reported being aware of the markedly increasing trend of P. vivax and the possibility of relapse cases, especially among migrants who are lost to follow-up. Workload was a key concern surrounding intervention implementation. The respondents discussed possible interventions, such as implementing directly observed treatment (DOT) by family members, piloting a shorter PQ regimen, expanding the community's malaria volunteer network, and strengthening health education activities using local languages to promote reasonable drug adherence. FGDs among community members revealed that although people were knowledgeable about malaria symptoms, places to seek treatment, and the use of bed nets to prevent mosquito bites, most of them still preferred to be treated by quack doctors and rarely used insecticide-treated nets at worksites. Many often stopped taking the prescribed drugs once the symptoms disappeared. Nevertheless, some respondents requested more bed nets to be distributed and health promotion activities to be conducted. CONCLUSION: In rural areas where human resources are limited, interventions such as implementing family member DOT or shortening PQ regimens should be introduced to enhance the radical cure for the P. vivax infection. Disseminating information about the importance of taking the entire treatment course and emphasizing the burden of relapse is also essential.

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.

Community acceptability, participation, and adherence to mass drug administration with primaquine for Plasmodium vivax elimination in Southern Thailand: a mixed methods approach.

BACKGROUND: Mass drug administration (MDA) with primaquine (PQ) is being considered for accelerating Plasmodium vivax elimination in remaining active foci. This study aimed to determine the acceptability of MDA with PQ in malaria endemic villages in a malarious setting in the South of Thailand undergoing MDA with PQ. METHODS: A cross-sectional mixed-methods approach was conducted in seven malaria endemic villages where MDA with PQ was implemented. The data were collected from community villagers and health workers using structured questionnaires, in-depth interviews, and focus group discussions. Descriptive statistics and logistic regression models were used for quantitative data analysis. Thematic analysis was applied for qualitative data. RESULTS: Among a total of 469 participants from the MDA villages, 293 participants were eligible for MDA with PQ and 79.86% (234) completed 14-days of PQ. The logistic regressions indicated that males (adjusted odds ratio: 2.52 [95% confidence interval: 1.33-4.81]) and those who are farmers (2.57 [1.12-5.90]) were most likely to participate in the MDA. Among 293 participants in the post-MDA study, 74.06% had originally agreed to participate in the MDA with PQ while 25.94% had originally reported not wanting to participate in the MDA. Of those who originally reported being willing to participate in the MDA, 71.23% followed through with participation in the first or second round. Conversely, 93.24% of those who originally reported not being willing to participate in the MDA did in fact participate in the MDA. Factors contributing to higher odds of agreeing to participate and following through with participation included being male (1.98 [1.06-3.69]) and correctly responding that malaria is preventable (2.32 [1.01-5.35]) with some differences by village. Five key themes emerged from the qualitative analyses: concern about side effects from taking PQ; disbelief that malaria could be eliminated in this setting; low overall concern about malaria infections; misunderstandings about malaria; and a general need to tailor public health efforts for this unique context. CONCLUSION: While the reported likelihood of participating in MDA was high in this setting, actual follow-through was relatively moderate, partially because of eligibility (roughly 71% of those in the follow-up survey who originally agreed to participate actually followed through with participation). One of the largest concerns among study participants was PQ-related side effects-and these concerns likely heavily influenced participant adherence to the MDA. The results of this study can be used to tailor future MDAs, or other public health interventions, in this and potentially other similar settings.

Adherence to 14-day radical cure for Plasmodium vivax malaria in Papua, Indonesia: a mixed-methods study.

BACKGROUND: Reducing the risk of recurrent Plasmodium vivax malaria is critical for malaria control and elimination. Primaquine (PQ) is the only widely available drug against P. vivax dormant liver stages, but is recommended as a 14-day regimen, which can undermine adherence to a complete course of treatment. METHODS: This is a mixed-methods study to assess socio-cultural factors influencing adherence to a 14-day PQ regimen in a 3-arm, treatment effectiveness trial in Papua, Indonesia. The qualitative strand, consisting of interviews and participant observation was triangulated with a quantitative strand in which trial participants were surveyed using a questionnaire. RESULTS: Trial participants differentiated between two types of malaria: tersiana and tropika, equivalent to P. vivax and Plasmodium falciparum infection, respectively. The perceived severity of both types was similar with 44.0% (267/607) perceiving tersiana vs. 45.1% (274/607) perceiving tropika as more severe. There was no perceived differentiation whether malaria episodes were due to a new infection or relapse; and 71.3% (433/607) acknowledged the possibility of recurrence. Participants were familiar with malaria symptoms and delaying health facility visit by 1-2 days was perceived to increase the likelihood of a positive test. Prior to health facility visits, symptoms were treated with leftover drugs kept at home (40.4%; 245/607) or bought over the counter (17.0%; 103/607). Malaria was considered to be cured with 'blue drugs' (referring to dihydroartemisinin-piperaquine). Conversely, 'brown drugs,' referring to PQ, were not considered malaria medication and instead were perceived as supplements. Adherence to malaria treatment was 71.2% (131/184), in the supervised arm, 56.9% (91/160) in the unsupervised arm and 62.4% (164/263) in the control arm; p = 0.019. Adherence was 47.5% (47/99) among highland Papuans, 51.7% (76/147) among lowland Papuans, and 72.9% (263/361) among non-Papuans; p < 0.001. CONCLUSION: Adherence to malaria treatment was a socio-culturally embedded process during which patients (re-)evaluated the characteristics of the medicines in relation to the course of the illness, their past experiences with illness, and the perceived benefits of the treatment. Structural barriers that hinder the process of patient adherence are crucial to consider in the development and rollout of effective malaria treatment policies.

Association of G6PD status and haemolytic anaemia in patients receiving anti-malarial agents: a systematic review and meta-analysis.

BACKGROUND: Some anti-malarial drugs often cause haemolytic anaemia in glucose-6-phosphate-dehydrogenase deficiency (G6PDd) patients. This study aims to analyse the association of G6PDd and anaemia in malaria patients receiving anti-malarial drugs. METHODS: A literature search was performed in major database portals. All studies searched using keywords with Medical Subject Headings (MeSH) were included, without date or language restriction. Pooled mean difference of haemoglobin and risk ratio of anaemia were analysed using RevMan. RESULTS: Sixteen studies comprising 3474 malaria patients that included 398 (11.5%) with G6PDd were found. Mean difference of haemoglobin in G6PDd/G6PD normal (G6PDn) patients was - 0.16 g/dL (95% CI - 0.48, 0.15; I2 5%, p = 0.39), regardless of the type of malaria and dose of drugs. In particular with primaquine (PQ), mean difference of haemoglobin in G6PDd/G6PDn patients with dose < 0.5 mg/kg/day was - 0.04 (95% CI - 0.35, 0.27; I2 0%, p = 0.69). The risk ratio of developing anaemia in G6PDd patients was 1.02 (95% CI 0.75, 1.38; I2 0%, p = 0.79). CONCLUSION: Single or daily standard doses of PQ (0.25 mg/kg/day) and weekly PQ (0.75 mg/kg/week) did not increase the risk of anaemia in G6PDd patients.

Analysis of the relapse of imported Plasmodium vivax and Plasmodium ovale in five provinces of China.

BACKGROUND: The global battle against malaria is facing formidable challenges, particularly in controlling Plasmodium vivax and Plasmodium ovale, whose cases have not been reduced as effectively as Plasmodium falciparum because of their relapse. This study investigates the current situation and underlying factors contributing to relapse or recrudescence of imported cases of P. vivax and P. ovale, and seeks to provide a reference for reducing relapse or recrudescence in malaria-free areas and offers a scientific basis for designing strategies to prevent imported re-transmission. METHODS: This study analysed imported P. vivax and P. ovale in Anhui, Zhejiang, Henan, Hubei, and Guangxi provinces during 2014-2021 by retrospective analysis. A case-control study was conducted on patients who experienced relapse or recrudescence. RESULTS: From 2014 to 2021, 306 cases of P.vivax and 896 cases of P.ovale were included in the study, while 75 cases had relapse or recrudescence, including 49 cases of P. ovale (65.33%) and 26 cases of P. vivax (34.67%). Within less than 5 weeks after returning to the country, 122 cases of P. vivax (39.87%, 122/306) and 265 cases of P. ovale (29.58%, 265/896) occurred. Within less than 53 weeks, the ratio of P. vivax was 94.77% (290/306), and that of P. ovale was 89.96% (806/896). Among the cases experiencing relapse or recrudescence, only 1 case of P. vivax (1/26 3.85%) and 3 cases of P. ovale (3/49 6.12%) occurred within less than 5 weeks after the first onset, whereas 21 cases of P. vivax (21/26 80.77%) and 42 cases of P. ovale (42/49 85.71%) occurred within less than 53 weeks after the first onset. The difference in relapse or recrudescence due to different drugs and medication regimens and medical activities at various levels of medical institutions was statistically significant. CONCLUSION: In areas where malaria has been eliminated, routine health screening in a scientific time frame for people returning from at-risk areas can effectively improve the efficiency of preventing re-transmission, thereby reducing prevention costs and disease burden. Preventing patients from self-treating and strengthening medication regulations in health facilities are key measures to reduce relapse or recrudescence.