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.

Clinical antiviral efficacy of favipiravir in early COVID-19 (PLATCOV): an open-label, randomised, controlled, adaptive platform trial.

In early symptomatic COVID-19 treatment, high dose oral favipiravir did not accelerate viral clearance. BACKGROUND: Favipiravir, an anti-influenza drug, has in vitro antiviral activity against SARS-CoV-2. Clinical trial evidence to date is inconclusive. Favipiravir has been recommended for the treatment of COVID-19 in some countries. METHODS: In a multicentre open-label, randomised, controlled, adaptive platform trial, low-risk adult patients with early symptomatic COVID-19 were randomised to one of ten treatment arms including high dose oral favipiravir (3.6g on day 0 followed by 1.6g daily to complete 7 days treatment) or no study drug. The primary outcome was the rate of viral clearance (derived under a linear mixed-effects model from the daily log10 viral densities in standardised duplicate oropharyngeal swab eluates taken daily over 8 days [18 swabs per patient]), assessed in a modified intention-to-treat population (mITT). The safety population included all patients who received at least one dose of the allocated intervention. This ongoing adaptive platform trial was registered at ClinicalTrials.gov (NCT05041907) on 13/09/2021. RESULTS: In the final analysis, the mITT population contained data from 114 patients randomised to favipiravir and 126 patients randomised concurrently to no study drug. Under the linear mixed-effects model fitted to all oropharyngeal viral density estimates in the first 8 days from randomisation (4,318 swabs), there was no difference in the rate of viral clearance between patients given favipiravir and patients receiving no study drug; a -1% (95% credible interval: -14 to 14%) difference. High dose favipiravir was well-tolerated. INTERPRETATION: Favipiravir does not accelerate viral clearance in early symptomatic COVID-19. The viral clearance rate estimated from quantitative measurements of oropharyngeal eluate viral densities assesses the antiviral efficacy of drugs in vivo with comparatively few studied patients.

Clinical Antiviral Efficacy of Remdesivir in Coronavirus Disease 2019: An Open-Label, Randomized Controlled Adaptive Platform Trial (PLATCOV).

BACKGROUND: Uncertainty over the therapeutic benefit of parenteral remdesivir in coronavirus disease 2019 (COVID-19) has resulted in varying treatment guidelines. METHODS: In a multicenter open-label, controlled, adaptive, pharmacometric platform trial, low-risk adult patients with early symptomatic COVID-19 were randomized to 1 of 8 treatment arms including intravenous remdesivir (200 mg followed by 100 mg daily for 5 days) or no study drug. The primary outcome was the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance (estimated under a linear model fit to the daily log10 viral densities, days 0-7) in standardized duplicate oropharyngeal swab eluates, in a modified intention-to-treat population. This ongoing adaptive trial is registered at ClinicalTrials.gov (NCT05041907). RESULTS: The 2 study arms enrolled 131 patients (remdesivir n = 67, no study drug n = 64) and estimated viral clearance rates from a median of 18 swab samples per patient (a total of 2356 quantitative polymerase chain reactions). Under the linear model, compared with the contemporaneous control arm (no study drug), remdesivir accelerated mean estimated viral clearance by 42% (95% credible interval, 18%-73%). CONCLUSIONS: Parenteral remdesivir accelerates viral clearance in early symptomatic COVID-19. Pharmacometric assessment of therapeutics using the method described can determine in vivo clinical antiviral efficacy rapidly and efficiently.

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.

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.

Cardiovascular concentration-effect relationships of amodiaquine and its metabolite desethylamodiaquine: Clinical and preclinical studies.

AIMS: Amodiaquine is a 4-aminoquinoline used extensively for the treatment and prevention of malaria. Orally administered amodiaquine is largely converted to the active metabolite desethylamodiaquine. Amodiaquine can cause bradycardia, hypotension, and electrocardiograph QT interval prolongation, but the relationship of these changes to drug concentrations is not well characterized. METHODS: We conducted a secondary analysis of a pharmacokinetic study of the cardiac safety of amodiaquine (10 mg base/kg/day over 3 days) in 54 Kenyan adults (≥18 years) with uncomplicated malaria. Nonlinear mixed effects modelling was used to assess amodiaquine and desethylamodiaquine concentration-effect relationships for vital sign (pulse rate, blood pressure) and electrocardiograph interval (QT, QRS, PR) outcomes. We also measured the spontaneous beating heart rate after cumulative dosing of amodiaquine and desethylamodiaquine in isolated mouse atrial preparations. RESULTS: Amodiaquine and desethylamodiaquine caused concentration-dependent mean decreases in pulse rate (1.9 beats/min per 100 nmol/L; 95% confidence interval: 1.5-2.4), supine systolic blood pressure (1.7 mmHg per 100 nmol/L; 1.2-2.1), erect systolic blood pressure (1.5 mmHg per 100 nmol/L; 1.0-2.0) and erect diastolic blood pressure (1.4 mmHg per 100 nmol/L; 1.0-1.7). The mean QT interval prolongation was 1.4 ms per 100 nmol/L irrespective of correction factor after adjustment for residual heart rate dependency. There was no significant effect of drug concentration on postural change in blood pressure or PR and QRS intervals. In mouse atria, the spontaneous beating rate was significantly reduced by amodiaquine (n = 6) and desethylamodiaquine (n = 8) at 3 µmol/L (amodiaquine: 10 ± 2%; desethylamodiaquine: 12 ± 3%) and 10 µmol/L (amodiaquine: 50 ± 7%; desethylamodiaquine: 46 ± 6%) concentrations with no significant difference in potency between the 2 compounds. CONCLUSION: Amodiaquine and desethylamodiaquine have concentration-dependent effects on heart rate, blood pressure, and ventricular repolarization.

The heterogeneity of symptom reporting across study sites: a secondary analysis of a randomised placebo-controlled multicentre antimalarial trial.

INTRODUCTION: Symptoms reported following the administration of investigational drugs play an important role in decisions for registration and treatment guidelines. However, symptoms are subjective, and interview methods to quantify them are difficult to standardise. We explored differences in symptom reporting across study sites of a multicentre antimalarial trial, with the aim of informing trial design and the interpretation of safety and tolerability data. METHODS: Data were derived from the IMPROV trial, a randomised, placebo-controlled double blinded trial of high dose primaquine to prevent Plasmodium vivax recurrence conducted in eight study sites in Afghanistan, Ethiopia, Indonesia and Vietnam. At each follow up visit a 13-point symptom questionnaire was completed. The number and percentage of patients with clinically relevant symptoms following the administration of primaquine or placebo, were reported by study site including vomiting, diarrhoea, anorexia, nausea, abdominal pain and dizziness. Multivariable logistic regression was used to estimate the confounder-adjusted site-specific proportion of each symptom. RESULTS: A total of 2,336 patients were included. The greatest variation between sites in the proportion of patients reporting symptoms was for anorexia between day 0 and day 13: 97.3% (361/371) of patients in Arba Minch, Ethiopia, reported the symptom compared with 4.7% (5/106) of patients in Krong Pa, Vietnam. Differences attenuated slightly after adjusting for treatment arm, age, sex, day 0 parasite density and fever; with the adjusted proportion for anorexia ranging from 4.8% to 97.0%. Differences between sites were greater for symptoms graded as mild or moderate compared to those rated as severe. Differences in symptom reporting were greater between study sites than between treatment arms within the same study site. CONCLUSION: Despite standardised training, there was large variation in symptom reporting across trial sites. The reporting of severe symptoms was less skewed compared to mild and moderate symptoms, which are likely to be more subjective. Trialists should clearly distinguish between safety and tolerability outcomes. Differences between trial arms were much less variable across sites, suggesting that the relative difference in reported symptoms between intervention and control group is more relevant than absolute numbers and should be reported when possible. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01814683; March 20th, 2013.

Prevalence and associated risk factors of soil-transmitted helminth infections in Kandahar, Afghanistan.

BACKGROUND: Soil-transmitted helminth (STH) infections are still a major health problem, especially in resource-limited countries. The community-based prevalence of STH is unknown in Afghanistan. Main objectives of this study were to estimate the prevalence and associated factors of STH among children in Daman district of Kandahar province in Afghanistan. METHODS: This was a community-based cross-sectional study, with data collected during five months (June-October, 2020) from children living in five villages of Daman district in Kandahar, Afghanistan. All the stool samples were examined by saline wet mount method. Data were analyzed by using descriptive statistics, Chi square test, and multivariate logistic regression. RESULTS: A total of 1426 children were studied, with majority (61.8%) of males and the mean age of 6.3 years. The overall prevalence of any intestinal parasitic infection was 39.8%. The overall prevalence of STH infection was 22.7%, with Ascaris lumbricoides (18.7%) as the most prevalent STH species, followed by hookworm (7.5%) and Trichuris trichiura (1.4%). Single, double, and triple STH infections were present in 14.9%, 7.2%, and 0.6% of the children, respectively. Multivariate logistic regression revealed that not washing hands after defecating/before eating (AOR 7.0, 95% CI 3.4-14.0), living in mud house (AOR 3.5, 95% CI 1.6-7.4), walking barefoot (AOR 2.2, 95% CI 1.6-3.1), living in overcrowded house (AOR 1.6, 95% CI 1.1-2.3), and practicing open defecation (AOR 1.4, 95% CI 1.1-2.0) as the risk factors associated with the predisposition of rural children for getting STH in Daman district of Afghanistan. CONCLUSIONS: Prevalence of STH is high among children of Daman district in Afghanistan. Most of the risk factors are related to poverty, decreased sanitation, and improper hygiene. Improvement of socioeconomic status, sanitation, and health education to promote public awareness about health and hygiene together with periodic mass deworming programs are better strategies for the control of STH infections in Afghanistan. Also, government and international donor agencies in Afghanistan should help in improving socio-economic status of the rural areas through provision of basic facilities such as piped water, electricity, good housing, and proper toilets.

Factors affecting the electrocardiographic QT interval in malaria: A systematic review and meta-analysis of individual patient data.

BACKGROUND: Electrocardiographic QT interval prolongation is the most widely used risk marker for ventricular arrhythmia potential and thus an important component of drug cardiotoxicity assessments. Several antimalarial medicines are associated with QT interval prolongation. However, interpretation of electrocardiographic changes is confounded by the coincidence of peak antimalarial drug concentrations with recovery from malaria. We therefore reviewed all available data to characterise the effects of malaria disease and demographic factors on the QT interval in order to improve assessment of electrocardiographic changes in the treatment and prevention of malaria. METHODS AND FINDINGS: We conducted a systematic review and meta-analysis of individual patient data. We searched clinical bibliographic databases (last on August 21, 2017) for studies of the quinoline and structurally related antimalarials for malaria-related indications in human participants in which electrocardiograms were systematically recorded. Unpublished studies were identified by the World Health Organization (WHO) Evidence Review Group (ERG) on the Cardiotoxicity of Antimalarials. Risk of bias was assessed using the Pharmacoepidemiological Research on Outcomes of Therapeutics by a European Consortium (PROTECT) checklist for adverse drug events. Bayesian hierarchical multivariable regression with generalised additive models was used to investigate the effects of malaria and demographic factors on the pretreatment QT interval. The meta-analysis included 10,452 individuals (9,778 malaria patients, including 343 with severe disease, and 674 healthy participants) from 43 studies. 7,170 (68.6%) had fever (body temperature ≥ 37.5°C), and none developed ventricular arrhythmia after antimalarial treatment. Compared to healthy participants, patients with uncomplicated falciparum malaria had shorter QT intervals (-61.77 milliseconds; 95% credible interval [CI]: -80.71 to -42.83) and increased sensitivity of the QT interval to heart rate changes. These effects were greater in severe malaria (-110.89 milliseconds; 95% CI: -140.38 to -81.25). Body temperature was associated independently with clinically significant QT shortening of 2.80 milliseconds (95% CI: -3.17 to -2.42) per 1°C increase. Study limitations include that it was not possible to assess the effect of other factors that may affect the QT interval but are not consistently collected in malaria clinical trials. CONCLUSIONS: Adjustment for malaria and fever-recovery-related QT lengthening is necessary to avoid misattributing malaria-disease-related QT changes to antimalarial drug effects. This would improve risk assessments of antimalarial-related cardiotoxicity in clinical research and practice. Similar adjustments may be indicated for other febrile illnesses for which QT-interval-prolonging medications are important therapeutic options.

Significant Efficacy of a Single Low Dose of Primaquine Compared to Stand-Alone Artemisinin Combination Therapy in Reducing Gametocyte Carriage in Cambodian Patients with Uncomplicated Multidrug-Resistant Plasmodium falciparum Malaria.

Since 2012, a single low dose of primaquine (SLDPQ; 0.25 mg/kg of body weight) with artemisinin-based combination therapies has been recommended as the first-line treatment of acute uncomplicated Plasmodium falciparum malaria to interrupt its transmission, especially in low-transmission settings of multidrug resistance, including artemisinin resistance. Policy makers in Cambodia have been reluctant to implement this recommendation due to primaquine safety concerns and a lack of data on its efficacy. In this randomized controlled trial, 109 Cambodians with acute uncomplicated P. falciparum malaria received dihydroartemisinin-piperaquine (DP) alone or combined with SLDPQ on the first treatment day. The transmission-blocking efficacy of SLDPQ was evaluated on days 0, 1, 2, 3, 7, 14, 21, and 28, and recrudescence by reverse transcriptase PCR (RT-PCR) (gametocyte prevalence) and membrane feeding assays with Anopheles minimus mosquitoes (gametocyte infectivity). Without the influence of recrudescent infections, DP-SLDPQ reduced gametocyte carriage 3-fold compared to that achieved with DP. Of 48 patients tested on day 0, only 3 patients were infectious to mosquitoes (∼6%). Posttreatment, three patients were infectious on day 14 (3.5%, 1/29) and on the 1st and 7th days of recrudescence (8.3%, 1/12 for each); this overall low infectivity precluded our ability to assess its transmission-blocking efficacy. Our study confirms the effective gametocyte clearance of SLDPQ when combined with DP in multidrug-resistant P. falciparum infections and the negative impact of recrudescent infections due to poor DP efficacy. Artesunate-mefloquine (ASMQ) has replaced DP, and ASMQ-SLDPQ has been deployed to treat all patients with symptomatic P. falciparum infections to further support the elimination of multidrug-resistant P. falciparum in Cambodia. (This study has been registered at ClinicalTrials.gov under identifier NCT02434952.).

Short-course primaquine for the radical cure of Plasmodium vivax malaria: a multicentre, randomised, placebo-controlled non-inferiority trial.

BACKGROUND: Primaquine is the only widely used drug that prevents Plasmodium vivax malaria relapses, but adherence to the standard 14-day regimen is poor. We aimed to assess the efficacy of a shorter course (7 days) of primaquine for radical cure of vivax malaria. METHODS: We did a randomised, double-blind, placebo-controlled, non-inferiority trial in eight health-care clinics (two each in Afghanistan, Ethiopia, Indonesia, and Vietnam). Patients (aged ≥6 months) with normal glucose-6-phosphate dehydrogenase (G6PD) and presenting with uncomplicated vivax malaria were enrolled. Patients were given standard blood schizontocidal treatment and randomly assigned (2:2:1) to receive 7 days of supervised primaquine (1·0 mg/kg per day), 14 days of supervised primaquine (0·5 mg/kg per day), or placebo. The primary endpoint was the incidence rate of symptomatic P vivax parasitaemia during the 12-month follow-up period, assessed in the intention-to-treat population. A margin of 0·07 recurrences per person-year was used to establish non-inferiority of the 7-day regimen compared with the 14-day regimen. This trial is registered at ClinicalTrials.gov (NCT01814683). FINDINGS: Between July 20, 2014, and Nov 25, 2017, 2336 patients were enrolled. The incidence rate of symptomatic recurrent P vivax malaria was 0·18 (95% CI 0·15 to 0·21) recurrences per person-year for 935 patients in the 7-day primaquine group and 0·16 (0·13 to 0·18) for 937 patients in the 14-day primaquine group, a difference of 0·02 (-0·02 to 0·05, p=0·3405). The incidence rate for 464 patients in the placebo group was 0·96 (95% CI 0·83 to 1·08) recurrences per person-year. Potentially drug-related serious adverse events within 42 days of starting treatment were reported in nine (1·0%) of 935 patients in the 7-day group, one (0·1%) of 937 in the 14-day group and none of 464 in the control arm. Four of the serious adverse events were significant haemolysis (three in the 7-day group and one in the 14-day group). INTERPRETATION: In patients with normal G6PD, 7-day primaquine was well tolerated and non-inferior to 14-day primaquine. The short-course regimen might improve adherence and therefore the effectiveness of primaquine for radical cure of P vivax malaria. FUNDING: UK Department for International Development, UK Medical Research Council, UK National Institute for Health Research, and the Wellcome Trust through the Joint Global Health Trials Scheme (MR/K007424/1) and the Bill & Melinda Gates Foundation (OPP1054404).

Hemolytic Dynamics of Weekly Primaquine Antirelapse Therapy Among Cambodians With Acute Plasmodium vivax Malaria With or Without Glucose-6-Phosphate Dehydrogenase Deficiency.

BACKGROUND: Hemoglobin (Hb) data are limited in Southeast Asian glucose-6-phosphate dehydrogenase (G6PD) deficient (G6PD-) patients treated weekly with the World Health Organization-recommended primaquine regimen (ie, 0.75 mg/kg/week for 8 weeks [PQ 0.75]). METHODS: We treated Cambodians who had acute Plasmodium vivax infection with PQ0.75 and a 3-day course of dihydroartemisinin/piperaquine and determined the Hb level, reticulocyte count, G6PD genotype, and Hb type. RESULTS: Seventy-five patients (male sex, 63) aged 5-63 years (median, 24 years) were enrolled. Eighteen were G6PD deficient (including 17 with G6PD Viangchan) and 57 were not G6PD deficient; 26 had HbE (of whom 25 were heterozygous), and 6 had α-/ß-thalassemia. Mean Hb concentrations at baseline (ie, day 0) were similar between G6PD deficient and G6PD normal patients (12.9 g/dL [range, 9‒16.3 g/dL] and 13.26 g/dL [range, 9.6‒16 g/dL], respectively; P = .46). G6PD deficiency (P = <.001), higher Hb concentration at baseline (P = <.001), higher parasitemia level at baseline (P = .02), and thalassemia (P = .027) influenced the initial decrease in Hb level, calculated as the nadir level minus the baseline level (range, -5.8-0 g/dL; mean, -1.88 g/dL). By day 14, the mean difference from the day 7 level (calculated as the day 14 level minus the day 7 level) was 0.03 g/dL (range, -0.25‒0.32 g/dL). Reticulocyte counts decreased from days 1 to 3, peaking on day 7 (in the G6PD normal group) and day 14 (in the G6PD deficient group); reticulocytemia at baseline (P = .001), G6PD deficiency (P = <.001), and female sex (P = .034) correlated with higher counts. One symptomatic, G6PD-deficient, anemic male patient was transfused on day 4. CONCLUSIONS: The first PQ0.75 exposure was associated with the greatest decrease in Hb level and 1 blood transfusion, followed by clinically insignificant decreases in Hb levels. PQ0.75 requires monitoring during the week after treatment. Safer antirelapse regimens are needed in Southeast Asia. CLINICAL TRIALS REGISTRATION: ACTRN12613000003774.

The haematological consequences of Plasmodium vivax malaria after chloroquine treatment with and without primaquine: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Malaria causes a reduction in haemoglobin that is compounded by primaquine, particularly in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The aim of this study was to determine the relative contributions to red cell loss of malaria and primaquine in patients with uncomplicated Plasmodium vivax. METHODS: A systematic review identified P. vivax efficacy studies of chloroquine with or without primaquine published between January 2000 and March 2017. Individual patient data were pooled using standardised methodology, and the haematological response versus time was quantified using a multivariable linear mixed effects model with non-linear terms for time. Mean differences in haemoglobin between treatment groups at day of nadir and day 42 were estimated from this model. RESULTS: In total, 3421 patients from 29 studies were included: 1692 (49.5%) with normal G6PD status, 1701 (49.7%) with unknown status and 28 (0.8%) deficient or borderline individuals. Of 1975 patients treated with chloroquine alone, the mean haemoglobin fell from 12.22 g/dL [95% CI 11.93, 12.50] on day 0 to a nadir of 11.64 g/dL [11.36, 11.93] on day 2, before rising to 12.88 g/dL [12.60, 13.17] on day 42. In comparison to chloroquine alone, the mean haemoglobin in 1446 patients treated with chloroquine plus primaquine was - 0.13 g/dL [- 0.27, 0.01] lower at day of nadir (p = 0.072), but 0.49 g/dL [0.28, 0.69] higher by day 42 (p < 0.001). On day 42, patients with recurrent parasitaemia had a mean haemoglobin concentration - 0.72 g/dL [- 0.90, - 0.54] lower than patients without recurrence (p < 0.001). Seven days after starting primaquine, G6PD normal patients had a 0.3% (1/389) risk of clinically significant haemolysis (fall in haemoglobin > 25% to < 7 g/dL) and a 1% (4/389) risk of a fall in haemoglobin > 5 g/dL. CONCLUSIONS: Primaquine has the potential to reduce malaria-related anaemia at day 42 and beyond by preventing recurrent parasitaemia. Its widespread implementation will require accurate diagnosis of G6PD deficiency to reduce the risk of drug-induced haemolysis in vulnerable individuals. TRIAL REGISTRATION: This trial was registered with PROSPERO: CRD42016053312. The date of the first registration was 23 December 2016.

The tolerability of single low dose primaquine in glucose-6-phosphate deficient and normal falciparum-infected Cambodians.

BACKGROUND: The WHO recommends single low-dose primaquine (SLDPQ, 0.25 mg/kg body weight) in falciparum-infected patients to block malaria transmission and contribute to eliminating multidrug resistant Plasmodium falciparum from the Greater Mekong Sub region (GMS). However, the anxiety regarding PQ-induced acute haemolytic anaemia in glucose-6-phosphate dehydrogenase deficiency (G6PDd) has hindered its use. Therefore, we assessed the tolerability of SLDPQ in Cambodia to inform national policy. METHODS: This open randomised trial of dihydroartemisinin-piperaquine (DHAPP) + SLDPQ vs. DHAPP alone recruited Cambodians aged ≥1 year with acute uncomplicated P. falciparum. Randomisation was 4:1 DHAPP+SLDPQ: DHAPP for G6PDd patients and 1:1 for G6PDn patients, according to the results of the qualitative fluorescent spot test. Definitive G6PD status was determined by genotyping. Day (D) 7 haemoglobin (Hb) concentration was the primary outcome measure. RESULTS: One hundred nine patients (88 males, 21 females), aged 4-76 years (median 23) were enrolled; 12 were G6PDd Viangchan (9 hemizygous males, 3 heterozygous females). Mean nadir Hb occurred on D7 [11.6 (range 6.4 ─ 15.6) g/dL] and was significantly lower (p = 0.040) in G6PDd (n = 9) vs. G6PDn (n = 46) DHAPP+SLDPQ recipients: 10.9 vs. 12.05 g/dL, Δ = -1.15 (95% CI: -2.24 ─ -0.05) g/dL. Three G6PDn patients had D7 Hb concentrations < 8 g/dL; D7-D0 Hbs were 6.4 ─ 6.9, 7.4 ─ 7.4, and 7.5 ─ 8.2 g/dL. For all patients, mean (range) D7-D0 Hb decline was -1.45 (-4.8 ─ 2.4) g/dL, associated significantly with higher D0 Hb, higher D0 parasitaemia, and receiving DHAPP; G6PDd was not a factor. No patient required a blood transfusion. CONCLUSIONS: DHAPP+SLDPQ was associated with modest Hb declines in G6PD Viangchan, a moderately severe variant. Our data augment growing evidence that SLDPQ in SE Asia is well tolerated and appears safe in G6PDd patients. Cambodia is now deploying SLDPQ and this should encourage other GMS countries to follow suit. TRIAL REGISTRATION: The clinicaltrials.gov reference number is NCT02434952 .

Contribution to Malaria Transmission of Symptomatic and Asymptomatic Parasite Carriers in Cambodia.

Background: Eliminating falciparum malaria in Cambodia is a top priority, requiring the implementation of novel tools and strategies to interrupt its transmission. To date, few data are available regarding the contributions to malaria transmission of symptomatic and asymptomatic carriers. Methods: Direct-membrane and skin feeding assays (DMFAs, SFAs) were performed, using Anopheles minimus and Anopheles dirus, to determine infectivity of symptomatic falciparum-infected patients and malaria asymptomatic carriers; a subset of the latter were followed up for 2 months to assess their transmission potential. Results: By microscopy and real-time polymerase chain reaction, Plasmodium falciparum gametocyte prevalence rates were, respectively, 19.3% (n = 21/109) and 44% (n = 47/109) on day (D) 0 and 17.9% (n = 5/28) and 89.3% (n = 25/28) in recrudescent patients (Drec) (RT-PCR Drec vs D0 P = .002). Falciparum malaria patient infectivity was low on D0 (6.2%; n = 3/48) and in Drec (8.3%; n = 1/12). Direct-membrane feeding assays and SFAs gave similar results. None of the falciparum (n = 0/19) and 3 of 28 Plasmodium vivax asymptomatic carriers were infectious to mosquitoes, including those that were followed up for 2 months. Overall, P. falciparum gametocytemias were low except in a few symptomatic carriers. Conclusions: Only symptomatic falciparum malaria patients were infectious to mosquito vectors at baseline and recrudescence, highlighting the need to detect promptly and treat effectively P. falciparum patients.

Quantifying primaquine effectiveness and improving adherence: a round table discussion of the APMEN Vivax Working Group.

The goal to eliminate malaria from the Asia-Pacific by 2030 will require the safe and widespread delivery of effective radical cure of malaria. In October 2017, the Asia Pacific Malaria Elimination Network Vivax Working Group met to discuss the impediments to primaquine (PQ) radical cure, how these can be overcome and the methodological difficulties in assessing clinical effectiveness of radical cure. The salient discussions of this meeting which involved 110 representatives from 18 partner countries and 21 institutional partner organizations are reported. Context specific strategies to improve adherence are needed to increase understanding and awareness of PQ within affected communities; these must include education and health promotion programs. Lessons learned from other disease programs highlight that a package of approaches has the greatest potential to change patient and prescriber habits, however optimizing the components of this approach and quantifying their effectiveness is challenging. In a trial setting, the reactivity of participants results in patients altering their behaviour and creates inherent bias. Although bias can be reduced by integrating data collection into the routine health care and surveillance systems, this comes at a cost of decreasing the detection of clinical outcomes. Measuring adherence and the factors that relate to it, also requires an in-depth understanding of the context and the underlying sociocultural logic that supports it. Reaching the elimination goal will require innovative approaches to improve radical cure for vivax malaria, as well as the methods to evaluate its effectiveness.

Population structures of Leishmania infantum and Leishmania tropica the causative agents of kala-azar in Southwest Iran.

Visceral leishmaniasis (VL) is endemic in Iran and is caused predominantly by Leishmania infantum, but L. tropica is emerging as an important cause. We studied the intra-species population structure of Leishmania spp. causing VL in southwest Iran by sequence analysis of the internal transcribed spacer (ITS) 1 of DNA samples from 29 bone marrow aspiration smears. L. infantum (n = 25) and L. tropica (n = 4) were identified, consisting of 10 and three ITS1 sequence types (STs), respectively. Compared to GenBank ITS1 STs, our L. infantum parasites displayed high heterogeneity but less heterogeneity compared than northwest Iranian isolates. VL affects mostly nomadic populations in southwest Iran, and their mobility may explain partly the L. infantum heterogeneity. The VL causing L. tropica was also genetically heterogeneous but genetically indistinguishable from L. tropica strains causing anthroponotic cutaneous leishmaniasis from southwest Iran.

Quantitative imaging of deuterated metabolic tracers in biological tissues with nanoscale secondary ion mass spectrometry.

In the field of secondary ion mass spectrometry at nanometer scale (NanoSIMS), configuration of parallel detectors to routinely measure isotope ratios in sub-100 nm domains brings classical stable isotope tracer studies from the whole tissue level down to the suborganelle level. Over the past decade, the marriage of stable isotope tracers with NanoSIMS has been applied to a range of fundamental biological questions that were largely inaccessible by other means. Although multiplexed measurement of different stable isotope tracers is feasible, in practice there remains a gap in the current analytical capacity to efficiently measure stable isotopes commonly utilized in tracer studies. One such example is the measurement of deuterated tracers. The most obvious approach to measuring deuterium/hydrogen isotope ratios is at mass 2/1. However, the radius of the magnetic sector limits concomitant measurement of other masses critical to multiplexed exploration of biological samples. Here we determine the experimental parameters to measure deuterated tracers in biological samples using the C2H- polyatomic ion species (C2D-/C2H-) while operating the NanoSIMS at a reduced Mass Resolving Power of 14,000. Through control of the sputtering parameters, we demonstrate that there is an analytical window during which the C2D-/C2H- isotope ratio can be measured with sufficient precision for biological studies where the degree of D-labeling is typically well above natural abundance. We provide validation of this method by comparing the C2D measurement of D-water labeling in the murine small intestine relative to measurements of native D/H conducted in the same analytical fields. Additional proof-of-concept demonstrations include measurement of D-water, D-glucose, and D-thymidine in biological specimens. Therefore, this study provides a practical template for deuterium-based tracer studies in biological systems.