Estimated distribution of malaria cases among children in sub-Saharan Africa by specified age categories using data from the Global Burden of Diseases 2019.

BACKGROUND: Children in sub-Saharan Africa (SSA) remain the most vulnerable to malaria and malaria mortality. This study estimated the disease burden and distribution of Plasmodium falciparum malaria among children with age categories (0 to < 2 years, 2 to < 6 years, 6 to < 12 years, ≥ 12 years) in SSA. METHODS: Data on the number of cases and incidence rates of P. falciparum malaria by age group from the Institute of Health Metrics and Evaluation (GBD 2019) for 11 countries in SSA was employed in this study. The best-fitting distribution of P. falciparum malaria cases by prespecified age categories was derived using a combination of a Log-normal and Weibull distribution. RESULTS: Plasmodium falciparum malaria was 15.4% for ages 0 to < 2 years, 30.5% for 2 to < 6 years, 17.6% for 6 to < 12 years, and 36.5% for ≥ 12 years based on data from countries in SSA. The results have important implications for the current drive by the FDA and EMA to ensure the representativeness of real-world populations in clinical trials evaluating the safety and efficacy of medication exposure. CONCLUSIONS: The theoretical distributions of P. falciparum malaria will help guide researchers in ensuring that children are appropriately represented in clinical trials and other interventions aiming to address the current burden of malaria in SSA.

In silico prediction and in vitro assessment of novel heterocyclics with antimalarial activity.

The development of new antimalarials is paramount to keep the goals on reduction of malaria cases in endemic regions. The search for quality hits has been challenging as many inhibitory molecules may not progress to the next development stage. The aim of this work was to screen an in-house library of heterocyclic compounds (HCUV) for antimalarial activity combining computational predictions and phenotypic techniques to find quality hits. The physicochemical determinants, pharmacokinetic properties (ADME), and drug-likeness of HCUV were evaluated in silico, and compounds were selected for structure-based virtual screening and in vitro analysis. Seven Plasmodium target proteins were selected from the DrugBank Database, and ligands and receptors were processed using UCSF Chimera and Open Babel before being subjected to docking using Autodock Vina and Autodock 4. Growth inhibition of P. falciparum (3D7) cultures was tested by SYBR Green assays, and toxicity was assessed using hemolytic activity tests and the Galleria mellonella in vivo model. From a total of 792 compounds, 341 with good ADME properties, drug-likeness, and no interference structures were subjected to in vitro analysis. Eight compounds showed IC50 ranging from 0.175 to 0.990 µM, and active compounds included pyridyl-diaminopyrimido-diazepines, pyridyl-N-acetyl- and pyridyl-N-phenyl-pyrazoline derivatives. The most potent compound (UV802, IC50 0.178 µM) showed no toxicophoric and was predicted to interact with P. falciparum 1-cysperoxidredoxin (PfPrx1). For the remaining 7 hits (IC50 < 1 µM), 3 showed in silico binding to PfPrx1, one was predicted to bind the haloacid dehalogenase-like hydrolase and plasmepsin II, and one interacted with the plasmodial heat shock protein 90.

Assessment of artemisinin tolerance in Plasmodium falciparum clinical isolates in children with uncomplicated malaria in Ghana.

BACKGROUND: Artemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated malaria in Ghana. Artemisinin (ART) tolerance in Plasmodium falciparum has arisen in Southeast Asia and recently, in parts of East Africa. This is ascribed to the survival of ring-stage parasites post treatment. The present study sought to assess and characterize correlates of potential ART tolerance based on post-treatment parasite clearance, ex vivo and in vitro drug sensitivity, and molecular markers of drug resistance in P. falciparum isolates from children with uncomplicated malaria in Ghana. METHODS: Six months to fourteen years old children presenting with acute uncomplicated malaria (n = 115) were enrolled in two hospitals and a Health Centre in Ghana's Greater Accra region and treated with artemether-lumefantrine (AL) according to body weight. Pre- and post-treatment parasitaemia (day 0 and day 3) was confirmed by microscopy. The ex vivo ring-stage survival assay (RSA) was used to detect percent ring survival while the 72 h SYBR Green I assay was used to measure the 50% inhibition concentration (IC50s) of ART and its derivatives and partner drugs. Genetic markers of drug tolerance /resistance were evaluated using selective whole genome sequencing. RESULTS: Of the total of 115 participants, 85 were successfully followed up on day 3 post-treatment and 2/85 (2.4%) had parasitaemia. The IC50 values of ART, artesunate (AS), artemether (AM), dihydroartemisinin (DHA), amodiaquine (AQ), and lumefantrine (LUM) were not indicative of drug tolerance. However, 7/90 (7.8%) pre-treatment isolates had > 10% ring survival rates against DHA. Of the four isolates (2 RSA positive and 2 RSA negative) with high genomic coverage, P. falciparum (Pf) kelch 13 K188* and Pfcoronin V424I mutations were only present in the two RSA positive isolates with > 10% ring survival rates. CONCLUSIONS: The observed low proportion of participants with day-3 post-treatment parasitaemia is consistent with rapid ART clearance. However, the increased rates of survival observed in the ex vivo RSA against DHA, maybe a pointer of an early start of ART tolerance. Furthermore, the role of two novel mutations in PfK13 and Pfcoronin genes, harboured by the two RSA positive isolates that had high ring survival in the present study, remains to be elucidated.

Antimalarial efficacy and toxicological assessment of medicinal plant ingredients of Prabchompoothaweep remedy as a candidate for antimalarial drug development.

BACKGROUND: Drug resistance exists in almost all antimalarial drugs currently in use, leading to an urgent need to identify new antimalarial drugs. Medicinal plant use is an alternative approach to antimalarial chemotherapy. This study aimed to explore potent medicinal plants from Prabchompoothaweep remedy for antimalarial drug development. METHODS: Forty-eight crude extracts from Prabchompoothaweep remedy and its 23 plants ingredients were investigated in vitro for antimalarial properties using Plasmodium lactate dehydrogenase (pLDH) enzyme against Plasmodium falciparum K1 strain and toxicity effects were evaluated in Vero cells. The plant with promising antimalarial activity was further investigated using gas chromatography-mass spectrometry (GC-MS) to identify phytochemicals. Antimalarial activity in mice was evaluated using a four-day suppressive test against Plasmodium berghei ANKA at dose of 200, 400, and 600 mg/kg body weight, and acute toxicity was analyzed. RESULTS: Of the 48 crude extracts, 13 (27.08%) showed high antimalarial activity against the K1 strain of P. falciparum (IC50 <  10 µg/ml) and 9 extracts (18.75%) were moderately active (IC50 = 11-50 µg/ml). Additionally, the ethanolic extract of Prabchompoothaweep remedy showed moderate antimalarial activity against the K1 strain of P. falciparum (IC50 = 14.13 µg/ml). Based on in vitro antimalarial and toxicity results, antimalarial activity of the aqueous fruit extract of Terminalia arjuna (IC50 = 4.05 µg/ml and CC50 = 219.6 µg/ml) was further studied in mice. GC-MS analysis of T. arjuna extract identified 22 compounds. The most abundant compounds were pyrogallol, gallic acid, shikimic acid, oleamide, 5-hydroxymethylfurfural, 1,1-diethoxy-ethane, quinic acid, and furfural. Analysis of the four-day suppressive test indicated that T. arjuna extract at dose of 200, 400, and 600 mg/kg body weight significantly suppressed the Plasmodium parasites by 28.33, 45.77, and 67.95%, respectively. In the acute toxicity study, T. arjuna extract was non-toxic at 2000 mg/kg body weight. CONCLUSIONS: The aqueous fruit extract of T. arjuna exerts antimalarial activity against Plasmodium parasites found in humans (P. falciparum K1) and mice (P. berghei ANKA). Acute toxicity studies showed that T. arjuna extract did not show any lethality or adverse effects up to a dose of 2000 mg/kg.

New In Vitro Interaction-Parasite Reduction Ratio Assay for Early Derisk in Clinical Development of Antimalarial Combinations.

The development and spread of drug-resistant phenotypes substantially threaten malaria control efforts. Combination therapies have the potential to minimize the risk of resistance development but require intensive preclinical studies to determine optimal combination and dosing regimens. To support the selection of new combinations, we developed a novel in vitro-in silico combination approach to help identify the pharmacodynamic interactions of the two antimalarial drugs in a combination which can be plugged into a pharmacokinetic/pharmacodynamic model built with human monotherapy parasitological data to predict the parasitological endpoints of the combination. This makes it possible to optimally select drug combinations and doses for the clinical development of antimalarials. With this assay, we successfully predicted the endpoints of two phase 2 clinical trials in patients with the artefenomel-piperaquine and artefenomel-ferroquine drug combinations. In addition, the predictive performance of our novel in vitro model was equivalent to that of the humanized mouse model outcome. Last, our more informative in vitro combination assay provided additional insights into the pharmacodynamic drug interactions compared to the in vivo systems, e.g., a concentration-dependent change in the maximum killing effect (Emax) and the concentration producing 50% of the killing maximum effect (EC50) of piperaquine or artefenomel or a directional reduction of the EC50 of ferroquine by artefenomel and a directional reduction of Emax of ferroquine by artefenomel. Overall, this novel in vitro-in silico-based technology will significantly improve and streamline the economic development of new drug combinations for malaria and potentially also in other therapeutic areas.

In vitro and in silico assessment of new beta amino ketones with antiplasmodial activity.

BACKGROUND: Based on the current need for new drugs against malaria, our study evaluated eight beta amino ketones in silico and in vitro for potential antimalarial activity. METHODS: Using the Brazilian Malaria Molecular Targets (BraMMT) and OCTOPUS® software programs, the pattern of interactions of beta-amino ketones was described against different proteins of P. falciparum and screened to evaluate their physicochemical properties. The in vitro antiplasmodial activities of the compounds were evaluated using a SYBR Green-based assay. In parallel, in vitro cytotoxic data were obtained using the MTT assay. RESULTS: Among the eight compounds, compound 1 was the most active and selective against P. falciparum (IC50 = 0.98 µM; SI > 60). Six targets were identified in BraMMT that interact with compounds exhibiting a stronger binding energy than the crystallographic ligand: P. falciparum triophosphate phosphoglycolate complex (1LYX), P. falciparum reductase (2OK8), PfPK7 (2PML), P. falciparum glutaredoxin (4N0Z), PfATP6, and PfHT. CONCLUSIONS: The physicochemical properties of compound 1 were compatible with the set of criteria established by the Lipinski rule and demonstrated its potential as a drug prototype for antiplasmodial activity.

Derivados de artemisinina para el tratamiento de malaria (Plasmodium falciparum) en gestantes en el 1er, 2ndo y 3er trimestre de gestación / Artemisinin derivatives for the treatment of malaria (Plasmodium falciparum) in pregnant women in the 1st, 2nd and 3rd years trimester of pregnancy

INTRODUCCIÓN: Este documento técnico se realiza a solicitud de la Estrategia sanitaria nacional de prevención y control de enfermedades metaxénicas y otras transmitidas por vectores. a. Cuadro clínico: La malaria es una enfermedad infecciosa causada por protozoos del género Plasmodium spp. Se estima que, globalmente, la infección por Plasmodium falciparum representa más del 90% de la mortalidad por malaria. Algunos grupos corren un riesgo considerablemente mayor que la población general de contraer la malaria y presentar un cuadro clínico grave, incluidas las mujeres gestantes. La malaria en este grupo tiene efectos severos tanto en la salud maternal como en la salud del naciente, y existen probabilidades más altas de desarrollar malaria severa especialmente en el segundo y tercer trimestre de gestación. b. Tecnología sanitária: El mejor tratamiento existente, en especial para la malaria causada por P. falciparum, es el tratamiento combinado con artemisinina (TCA), que tiene por principal objetivo lograr una rápida y completa eliminación de los parásitos para evitar que un caso sin complicaciones derive en una forma grave o mortal de la enfermedad. Actualmente, la OMS recomienda el uso de TCAs para el tratamiento de la malaria por P. falciparum sin complicaciones en niños y adultos (incluidas gestantes en el segundo y tercer trimestre), y el uso de quinina-clindamicina en gestantes en el primer trimestre de gestación. Los TCAs solo se recomiendan en gestantes en el primer trimestre si la quinina-clindamicina no está disponible o hay una falla del tratamiento. Esto es debido a que el primer trimestre es el momento de mayor preocupación por posible teratogenicidad. Aun así, la evidencia con respecto a la seguridad, eficacia y farmacocinética de la mayoría de los medicamentos antipalúdicos en gestantes es limitado. OBJETIVO El objetivo del presente documento es evaluar la eficacia y seguridad, así como documentos relacionados a la decisión del uso de los derivados de la artemisinina para el tratamiento de la malaria por P. falciparum en gestantes en el primer, segundo y tercer trimestre de gestación. METODOLOGÍA Se realizó una búsqueda en las principales bases de datos bibliográficas: MEDLINE, LILACS, COCHRANE, así como en buscadores genéricos de Internet incluyendo Google Scholar y TRIPDATABASE. Adicionalmente, se hizo una búsqueda dentro de la información generada por las principales instituciones internacionales de infectología, y agencias de tecnologías sanitarias que realizan revisiones sistemáticas (RS), evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC). RESULTADOS: Se seleccionaron cuatro metaanálisis, una revisión sistemática (RS), tres ECAs y cuatro guías de práctica clínica (GPC). Saito et al. publicaron dos metaanálisis con datos de pacientes individuales en 2020. El primer metaanálisis comparaba la eficacia y tolerabilidad de los derivados de la artemisinina y quinina como tratamientos para la malaria causada por P. falciparum no complicada en gestantes. Con datos derivados de 19 estudios realizados en África sub-Sahariana y Asia, se compararon un total de diez medicamentos antipalúdicos. El análisis encontró que la eficacia de los TCAs y quinina-clindamicina (QC) fue mayor del 90% en el día 28, 42 y 63 del tratamiento, mientras que la monoterapia con quinina tuvo la eficacia más baja en el día 28. El riesgo de falla de tratamiento comparado con el tratamiento estándar de artemisina-lumefantrina (AL) fue ligeramente menor para el tratamiento con artesunato-amodiaquina (ASAQ) y artesunato-mefloquina (ASMQ), y mayor para la monoterapia con quinina. En cuanto a la tolerabilidad, el tratamiento con AL tuvo el menor riesgo de eventos adversos, parecido al de la monoterapia con artesunato, y seguido de dihidroartemisinina-piperaquina (DP). Los tratamientos con quinina, ASAQ y ASMQ estuvieron asociados a un mayor riesgo de eventos adversos. El segundo metaanálisis publicado por Saito et al. comparaba los resultados del embarazo y riesgo de malaria placentaria después del tratamiento con derivados de la artemisinina y quinina para la malaria causada por P. falciparum no complicada en gestantes. Se utilizaron datos derivados de 16 estudios realizados en África subSahariana y Asia. No se identificaron diferencias estadísticamente significativas en la proporción de anomalías congénitas (p=0,68), aborto espontáneo (p=0,60), nacimiento de un niño muerto, parto prematuro moderado a tardío, ni en el riesgo de tener un bebé pequeño para la edad gestacional o de la presencia de pigmento de malaria en la placenta, entre los tratamientos antipalúdicos más comunes (AL, ASAQ, ASMQ y DP) en gestantes en el segundo y tercer trimestre. Los resultados para otros tratamientos requieren una interpretación cuidadosa debido al pequeño número de mujeres incluidas en los análisis, pero en general se asociaron con riesgos similares al tratamiento con AL. En 2017, Dellicour et al. publicaron un metaanálisis de estudios observacionales que investigaban el riesgo de eventos adversos en los resultados del embarazo relacionados al uso de derivados de la artemisinina y quinina en gestantes en el primer trimestre. Se incluyeron cinco estudios realizados en África sub-Sahariana y en la frontera entre Tailandia y Myanmar. No se encontraron diferencias significativas en el riesgo de aborto espontáneo asociado al uso de TCAs comparado con quinina en el primer trimestre de gestación, en el riesgo de nacimiento de un niño muerto, ni en la combinación de ambos eventos adversos. La prevalencia de anomalías congénitas mayores también fue similar entre ambos tratamientos. Sin embargo, el riesgo de aborto espontáneo fue mayor en gestantes tratadas con quinina comparado con gestantes no tratadas con ningún antipalúdico, mientras que esta relación no se observó en gestantes tratadas con TCAs. Burger et al. realizaron una RS en 2016 para investigar los efectos del embarazo sobre la farmacocinética de TCAs para el tratamiento de malaria en gestantes. El estudio concluyó que los regímenes actuales de artesunato, lumefantrina, sulfadoxina, atovacuona y proguanil resultan en una infradosificación del fármaco en gestantes, definida como una concentración plasmática del fármaco en el día 7 del tratamiento menor de 280 ng/ml, lo que es un umbral asociado con un mayor riesgo de recrudecimiento de la malaria. A base de la evidencia de los efectos del embarazo sobre la farmacocinética y eficacia de los TCAs para el tratamiento de la malaria en gestantes, Kloprogge et al. realizaron un metaanálisis en 2018 para investigar mejoras en el actual régimen estándar de AL, el TCA más común. Los autores encontraron que la concentración venosa de lumefantrina en el día 7 de tratamiento con un régimen estándar de AL era 20,2% menor en gestantes en el segundo y tercer trimestre de gestación comparado con adultos no gestantes (p<0,001). Los autores desarrollaron un modelo farmacocinético-farmacodinámico para comparar tres regímenes de dosificación alternativos: una dosis aumentada, un tratamiento prolongado y un tratamiento intensificado. El modelo concluyó que la dosis aumentada en gestantes no resultó en concentraciones de lumefantrina equivalentes a las de la población adulta no gestante, el tratamiento intensificado resultó en concentraciones similares, y el tratamiento prolongado mostró la probabilidad más alta de alcanzar concentraciones equivalentes. CONCLUSIONES: La evidencia con respecto a la seguridad, eficacia y farmacocinética de la mayoría de los medicamentos antipalúdicos en gestantes, incluidos los TCAs, es limitada. Dos metaanálisis publicados en 2020 encontraron una alta eficacia y tolerabilidad de los TCAs más comunes en gestantes en el segundo y tercer trimestre con malaria por P. falciparum no complicada, y no se identificaron diferencias significativas en los resultados del embarazo y riesgo de malaria placentaria entre el tratamiento con TCAs y los derivados de la quinina. En cuanto al tratamiento de gestantes en el primer trimestre de gestación, la evidencia es aún más escasa, aunque un metaanálisis de estudios observacionales tampoco encontró diferencias significativas en los riesgos de eventos adversos entre el tratamiento con TCAs y los derivados de la quinina en esta población. El embarazo causa una variedad de cambios fisiológicos que pueden afectar a la farmacocinética de los medicamentos tomados en este periodo, y que pueden reducir su eficacia. Una RS concluyó que los regímenes actuales de los TCAs más comunes también resultan en una infradosificación del fármaco en gestantes. Un tratamiento prolongado de los TCAs podría resultar en concentraciones más altas en gestantes. Debido a la escasez de la evidencia en gestantes en el primer trimestre, las actuales GPCs solo recomiendan el uso de TCAs en gestantes en el segundo y tercer trimestre de gestación. La quinina-clindamicina esta recomendada para gestantes en el primer trimestre de gestación. Tampoco se recomienda la modificación de las dosis estándar en este momento.

Evaluación de tecnología sanitaria de mefloquina vs doxiciclina para profilaxis de malaria falciparum en personal militar / Health technology evaluation of mefloquine vs doxycycline for falciparum malaria prophylaxis in military personnel

INTRODUCCIÓN: a. Cuadro clínico: La malaria es una enfermedad infecciosa causada por protozoos del género Plasmodium spp. Se estima que, globalmente, la infección por P. falciparum representa más del 90% de la mortalidad por malaria. Algunos grupos corren un riesgo considerablemente mayor que la población general de contraer la malaria, incluidas las poblaciones itinerantes como la población militar. b. Tecnología sanitária: En general, no existe consenso con respecto a profilaxis en población militar. Muchos estudios reportan que la preferencia de una tecnología a otra depende del lugar geográfico donde se establezcan los militares y el riesgo de malaria de la zona. La mefloquina es un antipalúdico ampliamente utilizado sin un modo de acción conocido. Mientras que la doxiciclina es un antibiotico lipofílico que actúa intracelularmente, uniéndose al ARNm ribosómico e inhibiendo la síntesis de proteínas. Este medicamento es ampliamente utilizado para la profilaxis de malaria también. OBJETIVO: El objetivo del presente documento es evaluar la eficacia y seguridad, así como documentos relacionados a la decisión del uso de mefloquina vs doxiciclina para profilaxis de malaria falciparum en personal militar. METODOLOGÍA: Se realizó una búsqueda en las principales bases de datos bibliográficas: MEDLINE, LILACS, COCHRANE, así como en buscadores genéricos de Internet incluyendo Google Scholar y TRIPDATABASE. Adicionalmente, se hizo una búsqueda dentro de la información generada por las principales instituciones internacionales de infectología, y agencias de tecnologías sanitarias que realizan revisiones sistemáticas (RS), evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC). RESULTADOS: Se seleccionaron dos estudios comparativos de mefloquina vs doxiciclina en personal militar. Además, se identificaron dos guías de práctica clínica. No se identificaron evaluaciones de tecnología sanitarias ni evaluaciones económicas de la región. Un ECA publicado en el año 1997 compara mefloquina y doxiciclina para la profilaxis de malaria en el Noreste de Irian Jaya, Indonesia. Se incluyeron 204 soldados. Después del tratamiento curativo radical, los participantes fueron asignados al azar para recibir 100 mg de doxiciclina por día y un placebo de mefloquina; 250 mg de mefloquina por semana (precedidos de una dosis de carga de 250 mg/día durante 3 días) y placebo de doxiciclina; o placebos para ambos fármacos. La profilaxis duró aproximadamente 13 semanas. En el grupo de placebo, 53/69 soldados desarrollaron malaria (9,1 años-persona), lo que resultó en una tasa de ataque de 5,8 casos por año-persona (IC95%; 4,3 a 7,7 casos por año-persona). No se presentó malaria en los 68 soldados (16,9 años-persona) en el grupo de mefloquina; por lo que la eficacia protectora de la mefloquina fue del 100 % (IC95%; 96 % a 100 %). En el grupo de doxiciclina, malaria por P. falciparum ocurrió en 1 de 67 soldados (16,0 años-persona), lo que resultó en una eficacia protectora del 99 % (IC95%, 94 % a 100 %). Ambos fármacos fueron muy bien tolerados CONCLUSIONES: La evidencia comparativa de mefloquina y doxiciclina para profilaxis de malaria por P. falciparum es escasa y se basa en un ECA. Este estudio muestra que estas dos alternativas son efectivas como profilaxis de malaria y que los eventos adversos son similares. Un estudio observacional con una muestra grande encontró que los dos comparadores fueron eficaces, pero sin embargo, los pacientes que tomaron doxiciclina tuvieron mayor frecuencia de eventos adversos no severos. Las principales guías de práctica clínica en el mundo y Perú de malaria no mencionan recomendaciones específicas para profilaxis en poblaciones militares específicamente. Sin embargo, documentos militares de UK mencionan a mefloquina como una opción si los militares tienen que ir a un lugar endémico de malaria con alta resistencia a cloroquina. Otro documento de Estados Unidos menciona a la doxiciclina como segunda línea y mefloquina como tercera línea para profilaxis de malaria en esta población, sin embargo, esta recomendación no está basada en literatura.

Parasite Viability as a Measure of In Vivo Drug Activity in Preclinical and Early Clinical Antimalarial Drug Assessment.

The rate at which parasitemia declines in a host after treatment with an antimalarial drug is a major metric for assessment of antimalarial drug activity in preclinical models and in early clinical trials. However, this metric does not distinguish between viable and nonviable parasites. Thus, enumeration of parasites may result in underestimation of drug activity for some compounds, potentially confounding its use as a metric for assessing antimalarial activity in vivo. Here, we report a study of the effect of artesunate on Plasmodium falciparum viability in humans and in mice. We first measured the drug effect in mice by estimating the decrease in parasite viability after treatment using two independent approaches to estimate viability. We demonstrate that, as previously reported in humans, parasite viability declines much faster after artesunate treatment than does the decline in parasitemia (termed parasite clearance). We also observed that artesunate kills parasites faster at higher concentrations, which is not discernible from the traditional parasite clearance curve and that each subsequent dose of artesunate maintains its killing effect. Furthermore, based on measures of parasite viability, we could accurately predict the in vivo recrudescence of infection. Finally, using pharmacometrics modeling, we show that the apparent differences in the antimalarial activity of artesunate in mice and humans are partly explained by differences in host removal of dead parasites in the two hosts. However, these differences, along with different pharmacokinetic profiles, do not fully account for the differences in activity. (This study has been registered with the Australian New Zealand Clinical Trials Registry under identifier ACTRN12617001394336.).

The assessment of antimalarial drug efficacy in vivo.

Currently recommended methods of assessing the efficacy of uncomplicated falciparum malaria treatment work less well in high-transmission than in low-transmission settings. There is also uncertainty how to assess intermittent preventive therapies and seasonal malaria chemoprevention (SMC), and Plasmodium vivax radical cure. A pharmacometric antimalarial resistance monitoring (PARM) approach is proposed specifically for evaluating slowly eliminated antimalarial drugs in areas of high transmission. In PARM antimalarial drug concentrations at recurrent parasitaemia are measured to identify outliers (i.e., recurrent parasitaemias in the presence of normally suppressive drug concentrations) and to evaluate changes over time. PARM requires characterization of pharmacometric profiles but should be simpler and more sensitive than current molecular genotyping-based methodologies. PARM does not require parasite genotyping and can be applied to the assessment of both prevention and treatment.

Nutrient Limitation Magnifies Fitness Costs of Antimalarial Drug Resistance Mutations.

Drug resistance mutations tend to disrupt key physiological processes and frequently carry fitness costs, which are a central determinant of the rate of spread of these mutations in natural populations. Head-to-head competition assays provide a standard approach to measuring fitness for malaria parasites. These assays typically use a standardized culture medium containing RPMI 1640, which has a 1.4- to 5.5-fold higher concentration of amino acids than human blood. In this rich medium, we predict that fitness costs will be underestimated because resource competition is weak. We tested this prediction using an artemisinin-sensitive parasite edited to contain kelch-C580Y or R561H mutations conferring resistance to artemisinin or synonymous control mutations. We examined the impact of these single amino acid mutations on fitness, using replicated head-to-head competition experiments conducted in media containing (i) normal RPMI, (ii) modified RPMI with reduced amino acid concentration, (iii) RPMI containing only isoleucine, or (iv) 3-fold diluted RPMI. We found a significant 1.3- to 1.4-fold increase in fitness costs measured in modified and isoleucine-only media relative to normal media, while fitness costs were 2.5-fold higher in diluted media. We conclude that fitness costs are strongly affected by media composition and will be significantly underestimated in normal RPMI. Several components differed between media, including pABA and sodium bicarbonate concentrations, so we cannot directly determine which is responsible. Elevated fitness costs in nature will limit spread of artemisinin (ART) resistance but will also promote evolution of compensatory mutations that restore fitness and can be exploited to maximize selection in laboratory experiments.

Assessment of quantitative and semi-quantitative biological test methods of artesunate in vitro.

Artesunate is the current most potent antimalarial drug widely used for the treatment of malaria. Considering the emergence of artemisinin resistance, several situations may require a simple method for artesunate quantification. We thus developed a quantitative and a semi-quantitative biological method for the determination of artesunate in liquid samples. The tests are based on the measurement of samples' antimalarial activity on Plasmodium falciparum 3D7 using a modified SYBR Green I drug susceptibility test. For the quantitative test, we established a standard curve that resulted from a dose-response curve and evaluated its performances using controls samples. Whereas the linear regression analysis between artesunate concentration and antimalarial activity showed promising results (linearity range 1.5-24.6 ng/mL, r2 = 0.9373), we found that artesunate content of the controls was significantly overestimated (p = 0.0313). For the semi-quantitative test, we compared the antimalarial activities of samples collected during permeation studies of artesunate to that of a reference (artesunate IC50) by statistical analysis. We demonstrated that antimalarial activities of samples from permeation tests using a powder formulation of artesunate were greater than those of samples from tests using a solution formulation. Bioassays can be simple techniques to assess artesunate in liquid samples, particularly in resource-limited settings. Comparison with reference methods is still recommended when accurate drug quantification is required.

Title: Évaluation de méthodes de tests biologiques quantitatives et semi-quantitatives de l'artésunate in vitro. Abstract: L'artésunate est le médicament antipaludique le plus puissant actuellement, largement utilisé pour le traitement du paludisme. Compte tenu de l'émergence de la résistance à l'artémisinine, plusieurs situations peuvent nécessiter une méthode simple de quantification de l'artésunate. Nous avons ainsi développé un test biologique quantitatif et un test semi-quantitatif pour le dosage de l'artésunate dans des échantillons liquides. Les méthodes sont basées sur la mesure de l'activité antipaludique des échantillons sur Plasmodium falciparum 3D7 à l'aide d'un test de sensibilité aux médicaments SYBR Green I modifié. Pour le test quantitatif, nous avons établi une courbe standard issue d'une courbe dose-réponse et évalué ses performances à l'aide d'échantillons témoins. Alors que l'analyse de régression linéaire entre la concentration d'artésunate et l'activité antipaludique a montré des résultats prometteurs (gamme de linéarité de 1,5 à 24,6 ng/mL, r2 = 0,9373), nous avons constaté que la teneur en artésunate des témoins était significativement surestimée (p = 0,0313). Pour le test semi-quantitatif, nous avons comparé les activités antipaludiques d'échantillons collectés lors des études de perméation de l'artésunate à celle d'une référence (artésunate IC50) par analyse statistique. Nous avons démontré que les activités antipaludiques des échantillons provenant de tests de perméation utilisant une formulation en poudre d'artésunate étaient supérieures à celles des échantillons provenant de tests utilisant une formulation en solution. Les dosages biologiques peuvent être des techniques simples pour évaluer l'artésunate dans des échantillons liquides, en particulier dans les milieux à ressources limitées. La comparaison avec des méthodes de référence est toujours recommandée lorsqu'une quantification précise du médicament est requise.

Assessment In Vitro of the Antimalarial and Transmission-Blocking Activities of Cipargamin and Ganaplacide in Artemisinin-Resistant Plasmodium falciparum.

Artemisinin resistance in Plasmodium falciparum has emerged and spread widely in the Greater Mekong Subregion, threatening current first-line artemisinin combination treatments. New antimalarial drugs are needed urgently. Cipargamin (KAE609) and ganaplacide (KAF156) are promising novel antimalarial compounds in advanced stages of development. Both compounds have potent asexual blood stage activities, inhibit P. falciparum gametocytogenesis, and reduce oocyst development in anopheline mosquitoes. In this study, we compared the asexual and sexual stage activities of cipargamin, ganaplacide, and artesunate in artemisinin-resistant P. falciparum isolates (n = 6; K13 mutations C580Y, G449A, and R539T) from Thailand and Cambodia. Asexual blood stage antimalarial activity was evaluated in a SYBR-green I-based 72-h in vitro assay, and the effects on male and female mature stage V gametocytes were assessed in the P. falciparum dual gamete formation assay. Ganaplacide had higher activities than cipargamin and artesunate, with mean (standard deviation [SD]) 50% inhibitory concentrations (IC50s) against asexual stages of 5.6 (1.2) nM and 6.9 (3.8) nM for male gametocytes and 47.5 (54.7) nM for female gametocytes. Cipargamin had a similar potency against male and female gametocytes, with mean (SD) IC50s of 115.6 (66.9) nM for male gametocytes, 104.9 (84.3) nM for female gametocytes, and 2.4 (0.7) nM for asexual stages. Both cipargamin and ganaplacide showed significant transmission-blocking activities against artemisinin-resistant P. falciparum in vitro.

Model-based assessment of the safety of community interventions with primaquine in sub-Saharan Africa.

BACKGROUND: Single low-dose primaquine (SLD-PQ) is recommended in combination with artemisinin-based combination therapy to reduce Plasmodium falciparum transmission in areas threatened by artemisinin resistance or aiming for malaria elimination. SLD-PQ may be beneficial in mass drug administration (MDA) campaigns to prevent malaria transmission but uptake is limited by concerns of hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. The aim of this study was to improve the evidence on the safety of MDA with SLD-PQ in a sub-Saharan African setting. METHODS: A nonlinear mixed-effects model describing the pharmacokinetics and treatment-induced hemolysis of primaquine was developed using data from an adult (n = 16, G6PD deficient) and pediatric study (n = 38, G6PD normal). The relationship between primaquine pharmacokinetics and hemolysis was modeled using an established erythrocyte lifespan model. The safety of MDA with SLD-PQ was explored through Monte Carlo simulations for SLD-PQ at 0.25 or 0.4 mg/kg using baseline data from a Tanzanian setting with detailed information on hemoglobin concentrations and G6PD status. RESULTS: The predicted reduction in hemoglobin levels following SLD-PQ was small and returned to pre-treatment levels after 25 days. G6PD deficiency (African A- variant) was associated with a 2.5-fold (95% CI 1.2-8.2) larger reduction in hemoglobin levels. In the Tanzanian setting where 43% of the population had at least mild anemia (hemoglobin < 11-13 g/dl depending on age and sex) and 2.73% had severe anemia (hemoglobin < 7-8 g/dl depending on age and sex), an additional 3.7% and 6.0% of the population were predicted to develop at least mild anemia and 0.25% and 0.41% to develop severe anemia after 0.25 and 0.4 mg/kg SLD-PQ, respectively. Children < 5 years of age and women ≥ 15 years of age were found to have a higher chance to have low pre-treatment hemoglobin. CONCLUSIONS: This study supports the feasibility of MDA with SLD-PQ in a sub-Saharan African setting by predicting small and transient reductions in hemoglobin levels. In a setting where a substantial proportion of the population had low hemoglobin concentrations, our simulations suggest treatment with SLD-PQ would result in small increases in the prevalence of anemia which would most likely be transient.

Protocol for a quasi-experimental study to assess the feasibility, acceptability and costs of multiple first-lines artemisinin-based combination therapies for uncomplicated malaria in the Kaya health district, Burkina Faso.

INTRODUCTION: As demonstrated in mathematical models, the simultaneous deployment of multiple first-line therapies (MFT) for uncomplicated malaria, using artemisinin-based combination therapies (ACTs), may extend the useful therapeutic life of the current ACTs. This is possible by reducing drug pressure and slowing the spread of resistance without putting patients' life at risk. We hypothesised that a simultaneous deployment of three different ACTs is feasible, acceptable and can achieve high coverage rate if potential barriers are properly identified and addressed. METHODS AND ANALYSIS: We plan to conduct a quasi-experimental study in the Kaya health district in Burkina Faso. We will investigate a simultaneous deployment of three ACTs, artemether-lumefantrine, pyronaridine-artesunate, dihydroartesinin-piperaquine, targeting three segments of the population: pregnant women, children under five and individuals aged five years and above. The study will include four overlapping phases: the formative phase, the MFT deployment phase, the monitoring and evaluation phase and the post-evaluation phase. The formative phase will help generate baseline information and develop MFT deployment tools. It will be followed by the MFT deployment phase in the study area. The monitoring and evaluation phase will be conducted as the deployment of MFT progresses. Cross-sectional surveys including desk reviews as well as qualitative and quantitative research methods will be used to assess the study outcomes. Quantitatives study outcomes will be measured using univariate, bivariate and multivariate analysis, including logistic regression and interrupted time series analysis approach. Content analysis will be performed on the qualitative data. ETHICS AND DISSEMINATION: The Health Research Ethics Committee in Burkina Faso approved the study (Clearance no. 2018-8-113). Study findings will be disseminated through feedback meetings with local communities, national workshops, oral presentations at congresses, seminars and publications in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: NCT04265573.

The impact of delayed treatment of uncomplicated P. falciparum malaria on progression to severe malaria: A systematic review and a pooled multicentre individual-patient meta-analysis.

BACKGROUND: Delay in receiving treatment for uncomplicated malaria (UM) is often reported to increase the risk of developing severe malaria (SM), but access to treatment remains low in most high-burden areas. Understanding the contribution of treatment delay on progression to severe disease is critical to determine how quickly patients need to receive treatment and to quantify the impact of widely implemented treatment interventions, such as 'test-and-treat' policies administered by community health workers (CHWs). We conducted a pooled individual-participant meta-analysis to estimate the association between treatment delay and presenting with SM. METHODS AND FINDINGS: A search using Ovid MEDLINE and Embase was initially conducted to identify studies on severe Plasmodium falciparum malaria that included information on treatment delay, such as fever duration (inception to 22nd September 2017). Studies identified included 5 case-control and 8 other observational clinical studies of SM and UM cases. Risk of bias was assessed using the Newcastle-Ottawa scale, and all studies were ranked as 'Good', scoring ≥7/10. Individual-patient data (IPD) were pooled from 13 studies of 3,989 (94.1% aged <15 years) SM patients and 5,780 (79.6% aged <15 years) UM cases in Benin, Malaysia, Mozambique, Tanzania, The Gambia, Uganda, Yemen, and Zambia. Definitions of SM were standardised across studies to compare treatment delay in patients with UM and different SM phenotypes using age-adjusted mixed-effects regression. The odds of any SM phenotype were significantly higher in children with longer delays between initial symptoms and arrival at the health facility (odds ratio [OR] = 1.33, 95% CI: 1.07-1.64 for a delay of >24 hours versus ≤24 hours; p = 0.009). Reported illness duration was a strong predictor of presenting with severe malarial anaemia (SMA) in children, with an OR of 2.79 (95% CI:1.92-4.06; p < 0.001) for a delay of 2-3 days and 5.46 (95% CI: 3.49-8.53; p < 0.001) for a delay of >7 days, compared with receiving treatment within 24 hours from symptom onset. We estimate that 42.8% of childhood SMA cases and 48.5% of adult SMA cases in the study areas would have been averted if all individuals were able to access treatment within the first day of symptom onset, if the association is fully causal. In studies specifically recording onset of nonsevere symptoms, long treatment delay was moderately associated with other SM phenotypes (OR [95% CI] >3 to ≤4 days versus ≤24 hours: cerebral malaria [CM] = 2.42 [1.24-4.72], p = 0.01; respiratory distress syndrome [RDS] = 4.09 [1.70-9.82], p = 0.002). In addition to unmeasured confounding, which is commonly present in observational studies, a key limitation is that many severe cases and deaths occur outside healthcare facilities in endemic countries, where the effect of delayed or no treatment is difficult to quantify. CONCLUSIONS: Our results quantify the relationship between rapid access to treatment and reduced risk of severe disease, which was particularly strong for SMA. There was some evidence to suggest that progression to other severe phenotypes may also be prevented by prompt treatment, though the association was not as strong, which may be explained by potential selection bias, sample size issues, or a difference in underlying pathology. These findings may help assess the impact of interventions that improve access to treatment.

Assessment of the Acceptability of Testing and Treatment during a Mass Drug Administration Trial for Malaria in Zambia Using Mixed Methods.

From 2014 to 2016, a community-randomized controlled trial in Southern Province, Zambia, compared mass drug administration (MDA) and focal MDA (fMDA) with the standard of care. Acceptability of the intervention was assessed quantitatively using closed-ended and Likert scale-based questions posed during three household surveys conducted from April to May in 2014, 2015, and 2016 in 40 health catchments that implemented MDA and fMDA and 20 catchments that served as trial controls. In 2014 and 2015, 47 households per catchment were selected, targeting 1,880 households in MDA and fMDA trial arms; in 2016, 55 households per catchment were selected for a target of 2,200 households in MDA and fMDA trial arms. Concurrently, 27 focus group discussions and 23 in-depth interviews with 248 participants were conducted on reasons for testing and treatment refusal, reasons for nonadherence, and community perception of the MDA campaign. Results demonstrated that the MDA campaign was highly accepted with more than 99% of respondents stating that they would take treatment if positive for malaria. High acceptability at baseline could be associated with test-and-treat campaigns recently conducted in the study area. There was a large increase in the acceptability of prophylactic treatment if negative for malaria from the baseline to follow-up survey for adults and children, from 62% to 96% for each. This likely resulted from an intensive community-wide sensitization program that occurred before the first treatment round at each household during community health worker visits.

Cost-Effectiveness of Focal Mass Drug Administration and Mass Drug Administration with Dihydroartemisinin-Piperaquine for Malaria Prevention in Southern Province, Zambia: Results of a Community-Randomized Controlled Trial.

Community-wide administration of antimalarial drugs in therapeutic doses is a potential tool to prevent malaria infection and reduce the malaria parasite reservoir. To measure the effectiveness and cost of using the antimalarial drug combination dihydroartemisinin-piperaquine (DHAp) through different community-wide distribution strategies, Zambia's National Malaria Control Centre conducted a three-armed community-randomized controlled trial. The trial arms were as follows: 1) standard of care (SoC) malaria interventions, 2) SoC plus focal mass drug administration (fMDA), and 3) SoC plus MDA. Mass drug administration consisted of offering all eligible individuals DHAP, irrespective of a rapid diagnostic test (RDT) result. Focal mass drug administration consisted of offering DHAP to all eligible individuals who resided in a household where anyone tested positive by RDT. Results indicate that the costs of fMDA and MDA per person targeted and reached are similar (US$9.01 versus US$8.49 per person, respectively, P = 0.87), but that MDA was superior in all cost-effectiveness measures, including cost per infection averted, cost per case averted, cost per death averted, and cost per disability-adjusted life year averted. Subsequent costing of the MDA intervention in a non-trial, operational setting yielded significantly lower costs per person reached (US$2.90). Mass drug administration with DHAp also met the WHO thresholds for "cost-effective interventions" in the Zambian setting in 90% of simulations conducted using a probabilistic sensitivity analysis based on trial costs, whereas fMDA met these criteria in approximately 50% of simulations. A sensitivity analysis using costs from operational deployment and trial effectiveness yielded improved cost-effectiveness estimates. Mass drug administration may be a cost-effective intervention in the Zambian context and can help reduce the parasite reservoir substantially. Mass drug administration was more cost-effective in relatively higher transmission settings. In all scenarios examined, the cost-effectiveness of MDA was superior to that of fMDA.

Screening Marine Natural Products for New Drug Leads against Trypanosomatids and Malaria.

Neglected Tropical Diseases (NTD) represent a serious threat to humans, especially for those living in poor or developing countries. Almost one-sixth of the world population is at risk of suffering from these diseases and many thousands die because of NTDs, to which we should add the sanitary, labor and social issues that hinder the economic development of these countries. Protozoan-borne diseases are responsible for more than one million deaths every year. Visceral leishmaniasis, Chagas disease or sleeping sickness are among the most lethal NTDs. Despite not being considered an NTD by the World Health Organization (WHO), malaria must be added to this sinister group. Malaria, caused by the apicomplexan parasite Plasmodium falciparum, is responsible for thousands of deaths each year. The treatment of this disease has been losing effectiveness year after year. Many of the medicines currently in use are obsolete due to their gradual loss of efficacy, their intrinsic toxicity and the emergence of drug resistance or a lack of adherence to treatment. Therefore, there is an urgent and global need for new drugs. Despite this, the scant interest shown by most of the stakeholders involved in the pharmaceutical industry makes our present therapeutic arsenal scarce, and until recently, the search for new drugs has not been seriously addressed. The sources of new drugs for these and other pathologies include natural products, synthetic molecules or repurposing drugs. The most frequent sources of natural products are microorganisms, e.g., bacteria, fungi, yeasts, algae and plants, which are able to synthesize many drugs that are currently in use (e.g. antimicrobials, antitumor, immunosuppressants, etc.). The marine environment is another well-established source of bioactive natural products, with recent applications against parasites, bacteria and other pathogens which affect humans and animals. Drug discovery techniques have rapidly advanced since the beginning of the millennium. The combination of novel techniques that include the genetic modification of pathogens, bioimaging and robotics has given rise to the standardization of High-Performance Screening platforms in the discovery of drugs. These advancements have accelerated the discovery of new chemical entities with antiparasitic effects. This review presents critical updates regarding the use of High-Throughput Screening (HTS) in the discovery of drugs for NTDs transmitted by protozoa, including malaria, and its application in the discovery of new drugs of marine origin.

Assessment of the risk posed to Singapore by the emergence of artemisinin-resistant malaria in the Greater Mekong Subregion.

OBJECTIVE: To assess the public health risk to Singapore posed by the emergence of artemisinin-resistant (ART-R) malaria in the Greater Mekong Subregion (GMS). METHODS: We assessed the likelihood of importation of drug-resistant malaria into Singapore and the impact on public health of its subsequent secondary spread in Singapore. Literature on the epidemiology and contextual factors associated with ART-R malaria was reviewed. The epidemiology of malaria cases in Singapore was analysed. The vulnerability and receptivity of Singapore were examined, including the connectivity with countries reporting ART-R malaria, as well as the preparedness of Singaporean health authorities. Sources of information include international journals, World Health Organization guidelines, data from the Singapore Ministry of Health and National Public Health Laboratory of the National Centre for Infectious Diseases, and the International Air Transport Association. RESULTS: The importation of ART-R malaria into Singapore is possible given the close proximity and significant travel volume between Singapore and the GMS countries reporting artemisinin resistance. Singapore's vulnerability is further enhanced by the presence of foreign workers from neighbouring endemic countries. Nonetheless, the overall likelihood of such an event is low based on the rarity and decreasing trend of imported malaria incidence.​: With the presence of Anopheles vectors in Singapore, imported cases of drug-resistant malaria could cause secondary transmission. Nevertheless, the risk of sustained spread is likely to be mitigated by the comprehensive surveillance and control system in place for both infected vectors and human cases. DISCUSSION: This risk assessment highlights the need for a continued high degree of vigilance of ART-R malaria locally and globally to minimize the risk and public health impact of drug-resistant malaria in Singapore.