Experience of healthcare personnel on Co-payment mechanism and the implications on its use in private drug outlets in Uganda.

BACKGROUND: Malaria treatment is faced with the challenge of access, affordability, availability, and quality of antimalarial medicines. Affordable medicines facility-malaria (AMFm) program and subsequently Co-payment mechanism were developed to help increase access to quality assured Artemisinin-based combination therapies (ACTs) in seven countries in sub-Saharan Africa. We explored through a qualitative study, experience of healthcare personnel on Co-payment mechanism and the implication on its use in private drug outlets in Uganda. METHOD: Private drug outlets that reported stocking antimalarial agents in moderate-to-high and low malaria transmission settings were purposively selected for inclusion in the study. In each drug outlet, data was collected from a pharmacist/dispenser through key informant interview. The interview was done using a key informant interview guide which covered the following areas, (i) sociodemographic characteristics, ii) awareness of healthcare personnel on the co-payment mechanism, (iii) awareness of healthcare personnel on quality assured artemisinin combination therapies (QAACT), (iv) antimalarial stocking in private drug outlets, (v) antimalarial dispensing prices, (vi) considerations made while stocking, and pricing antimalarial agents, vii) challenges in antimalarial dispensing, and (viii) access to antimalarial agents in private drug outlets. Data was managed using Atlas.ti and analyzed using framework methodology. RESULTS: Data was collected from 25 key informants (12 pharmacists and 13 dispensers). Five themes emerged following data analysis, (i) antimalarial stocking influenced by price and client demand, (ii) access and purchasing behavior of drug outlet clients, (iii) basis of dispensing antimalarial agents in private drug outlets, (iv) awareness of QAACT, and (v) awareness of Co-payment mechanism. None of the study participants was aware of the existence of Co-payment mechanism and QAACT in the private sector. Duocotecin brand of ACTs was the most mentioned and dispensed ACT among the study participants in private drug outlets. Nearly all the pharmacists/dispensers said that many clients who request to purchase ACTs don't come with a prescription and prefer buying cheaper antimalarial agents. Study participants reported stocking and selling both ACTs and non-ACT antimalarial agents in the drug outlets. Pharmacists/dispensers in the drug outlets reported that most clients could not afford buying a full dose of an ACT. None of the study participants considered using Co-payment mechanism while stocking ACTs in the drug outlets. CONCLUSION: There is lack of awareness and utilization of Co-payment mechanism in stocking, pricing, and dispensing of ACTs among pharmacists/dispensers in private drug outlets in Uganda. The antimalarial dispensing in drug outlets was mostly based on prescriptions, clients' preferences, and medicine affordability. The Ministry of Health needs to create demand for Co-payment mechanism through public awareness campaigns, training of healthcare personnel and behavior change communication in the private sector.

Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites.

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is associated with point mutations, mostly in kelch13 (k13) but also in other genes, like ubp1. K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. We tested 135 mutations in KICs but none conferred ART resistance. Double mutations of k13C580Y with k13R539T or k13C580Y with ubp1R3138H, did also not increase resistance. In contrast, k13C580Y parasites subjected to consecutive RSAs did, but the k13 sequence was not altered. Using isogenic parasites with different k13 mutations, we found correlations between K13 protein amount, resistance, and fitness cost. Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. While fitness cost of k13 mutations correlated with ART resistance, ubp1R3138H caused a disproportionately higher fitness cost. IMPORTANCE: Parasites with lowered sensitivity to artemisinin-based drugs are becoming widespread. However, even in these "resistant" parasites not all parasites survive treatment. We found that the proportion of surviving parasites correlates with the fitness cost of resistance-inducing mutations which might indicate that the growth disadvantages prevents resistance levels where all parasites survive treatment. We also found that combining two common resistance mutations did not increase resistance levels. However, selection through repeated ART-exposure did, even-though the known resistance genes, including k13, were not further altered, suggesting other causes of increased resistance. We also observed a disproportionally high fitness cost of a resistance mutation in resistance gene ubp1. Such high fitness costs may explain why mutations in ubp1 and other genes functioning in the same pathway as k13 are rare. This highlights that k13 mutations are unique in their ability to cause resistance at a comparably low fitness cost.

Access to quality-assured artemisinin-based combination therapy and associated factors among clients of selected private drug outlets in Uganda.

BACKGROUND: Malaria treatment in sub-Saharan Africa is faced with challenges including unreliable supply of efficacious agents, substandard medicines coupled with high price of artemisinin-based combinations. This affects access to effective treatment increasing risk of malaria parasite resistance development and adverse drug events. This study investigated access to quality-assured artemisinin-based combination therapy (QAACT) medicines among clients of selected private drug-outlets in Uganda. METHODS: This was a cross sectional study where exit interviews were conducted among clients of private drug outlets in low and high malaria transmission settings in Uganda. This study adapted the World Health Organization/Health Action International (WHO/HAI) standardized criteria. Data was collected using a validated questionnaire. Data entry screen with checks was created in Epi-data ver 4.2 software and data entered in duplicate. Data was transferred to STATA ver 14.0 and cleaned prior to analysis. The analysis was done at 95% level of significance. RESULTS: A total of 1114 exit interviews were conducted among systematically sampled drug outlet clients. Over half, 54.9% (611/1114) of the participants were males. Majority, 97.2% (1083/1114) purchased an artemisinin-based combination anti-malarial. Most, 55.5% (618/1114) of the participants had a laboratory diagnosis of malaria. Majority, 77.9% (868/1114) of the participants obtained anti-malarial agents without a prescription. Less than a third, 27.7% (309/1114) of the participants obtained a QAACT. Of the participants who obtained QAACT, more than half 56.9% (173/309) reported finding the medicine expensive. The predictors of accessing a QAACT anti-malarial among drug outlet clients include type of drug outlet visited (aPR = 0.74; 95%CI 0.6, 0.91), not obtaining full dose (3-day treatment) of ACT (aPR = 0.49; 95%CI 0.33, 0.73), not finding the ACT expensive (aPR = 1.24; 95%CI 1.03, 1.49), post-primary education (aPR = 1.29; 95%CI 1.07,1.56), business occupation (aPR = 1.24; 95%CI 1.02,1.50) and not having a prescription (aPR = 0.76; 95%CI 0.63, 0.92). CONCLUSION: Less than a third of the private drug outlet clients obtained a QAACT for management of malaria symptoms. Individuals who did not find artemisinin-based combinations to be expensive were more likely to obtain a QAACT anti-malarial. The Ministry of Health needs to conduct regular surveillance to monitor accessibility of QAACT anti-malarial agents under the current private sector copayment mechanism.

Copayment mechanism in selected districts of Uganda: Availability, market share and price of quality assured artemisinin-based combination therapies in private drug outlets.

BACKGROUND: Malaria remains one of the leading causes of morbidity, and mortality in Uganda. A large proportion of malaria symptomatic patients seek healthcare in private sector. However, availability and affordability are major barriers to access to effective treatment. The private sector copayment mechanism in Uganda aims to increase availability and affordability of antimalarial agents. Our study assessed availability, price, and market share of quality assured artemisinin-based combination therapies (QAACTs) in private drug outlets in selected districts during the implementation of copayment mechanism. METHODS: This was a cross-sectional survey of anti-malarial agents in private drug outlets in in selected moderate-to-high (Tororo, and Apac districts) and low (Kabale and Mbarara districts) malaria transmission settings. Following the World Health Organization/Health Action International (WHO/HAI) criteria, an audit of the antimalarial agents was done using a checklist to determine availability, price, and market share of QAACTs. Data were entered in Epi-data and analyzed in STATA ver 14.0 at 95% confidence level. RESULTS: A total of twenty-eight (28) private drug outlets (pharmacies and drug shops) were included in the survey. One in seven (20/144: 95%CI: 9.1, 20.6) of the antimalarial agents in private drug outlets were quality assured artemisinin-based combination therapies (QAACT). Artemether-lumefantrine (AL), 8.9% (11/124) and Artesunate-Amodiaquine (AQ), 7.3% (9/124) were the only QAACTs present in the drug outlets at the time of the survey. The majority, 86.1%% (124/144) of antimalarial agents present in stock in the drug outlets were artemisinin based. The most common, 38.9% (56/144) ACT in the drug outlets was Dihydroartemisinin-Piperaquine (DHP). Most, 69.4% (100/144) of the antimalarial agents were in high malaria transmission settings. The cost of ACT antimalarial agents is high in the country, USD 1.4 (Artemether-Lumefantrine, AL), USD 2.4 (Dihydroartemisinin-Piperaquine, DP), the first line and second-line agents respectively for treatment of uncomplicated malaria in Uganda. There was a statistically significant difference between the dispensing price of 'Green leaf' ACTs (QAACT) and the recommended price (p<0.001). Predictors of availability of QAACT in private drug outlets include pharmacy drug outlet (aPR:0.4; 95%CI: 0.2, 0.9) and dispensing price more than 3000UGX (USD 0.83) (aPR: 0.4, 95%CI: 0.1, 0.51). CONCLUSION: Quality assured artemisinin-based combination therapies (QAACTs) are not common in private drug outlets in selected districts in Uganda. All the drug outlets had at least one ACT antimalarial agent present on the day of the survey. The dispensing price of QAACTs was significantly higher than the recommended markup price. There is need for awareness creation, surveillance, and monitoring of the implementation of Copayment mechanism in the country.

Assessing availability, prices, and market share of quality-assured malaria ACT and RDT in the private retail sector in Nigeria and Uganda.

BACKGROUND: An estimated 50% of suspected malaria cases in sub-Saharan Africa first seek care in the private sector, especially in private medicine retail outlets. Quality of care in these outlets is generally unknown but considered poor with many patients not receiving a confirmatory diagnosis or the recommended first-line artemisinin-based combination therapy (ACT). In 2010, a subsidy pilot scheme, the Affordable Medicines Facility malaria, was introduced to crowd out the use of monotherapies in favour of WHO-pre-qualified artemisinin-based combinations (WHO-PQ-ACTs) in the private health sector. The scheme improved the availability, market share, and cost of WHO-PQ-ACTs in countries like Nigeria and Uganda, but in 2018, the subsidies were halted in Nigeria and significantly reduced in Uganda. This paper presents findings from six retail audit surveys conducted from 2014 to 2021 in Nigeria and Uganda to assess whether the impact of subsidies on the price, availability, and market share of artemisinin-based combinations has been sustained after the subsidies were reduced or discontinued. METHODS: Six independent retail audits were conducted in private medicine retail outlets, including pharmacies, drug shops, and clinics in Nigeria (2016, 2018, 2021), and Uganda (2014, 2019, 2020) to assess the availability, price, and market share of anti-malarials, including WHO-PQ-ACTs and non-WHO-PQ-ACTs, and malaria rapid diagnostic tests (RDTs). RESULTS: Between 2016 and 2021, there was a 57% decrease in WHO-PQ-ACT availability in Nigeria and a 9% decrease in Uganda. During the same period, non-WHO-PQ-ACT availability increased in Nigeria by 41% and by 34% in Uganda. The price of WHO-PQ-ACTs increased by 42% in Nigeria to $0.68 and increased in Uganda by 24% to $0.95. The price of non-WHO-PQ-ACTs decreased in Nigeria by 26% to $1.08 and decreased in Uganda by 64% to $1.23. There was a 76% decrease in the market share of WHO-PQ-ACTs in Nigeria and a 17% decrease in Uganda. Malaria RDT availability remained low throughout. CONCLUSION: With the reduction or termination of subsidies for WHO-PQ-ACTs in Uganda and Nigeria, retail prices have increased, and retail prices of non-WHO-PQ-ACTs decreased, likely contributing to a shift of higher availability and increased use of non-WHO-PQ-ACTs.

Chemical and spectroscopic characterization of (Artemisinin/Querctin/ Zinc) novel mixed ligand complex with assessment of its potent high antiviral activity against SARS-CoV-2 and antioxidant capacity against toxicity induced by acrylamide in male rats.

A novel Artemisinin/Quercetin/Zinc (Art/Q/Zn) mixed ligand complex was synthesized, tested for its antiviral activity against coronavirus (SARS-CoV-2), and investigated for its effect against toxicity and oxidative stress induced by acrylamide (Acy), which develops upon cooking starchy foods at high temperatures. The synthesized complex was chemically characterized by performing elemental analysis, conductance measurements, FT-IR, UV, magnetic measurements, and XRD. The morphological surface of the complex Art/Q/Zn was investigated using scanning and transmission electron microscopy (SEM and TEM) and energy dispersive X-ray analysis (XRD). The in vitro antiviral activity of the complex Art/Q/Zn against SARS-CoV-2 and its in vivo activity against Acy-induced toxicity in hepatic and pulmonary tissues were analyzed. An experimental model was used to evaluate the beneficial effects of the novel Art/Q/Zn novel complex on lung and liver toxicities of Acy. Forty male rats were randomly divided into four groups: control, Acy (500 mg/Kg), Art/Q/Zn (30 mg/kg), and a combination of Acy and Art/Q/Zn. The complex was orally administered for 30 days. Hepatic function and inflammation marker (CRP), tumor necrosis factor, interleukin-6 (IL-6), antioxidant enzyme (CAT, SOD, and GPx), marker of oxidative stress (MDA), and blood pressure levels were investigated. Histological and ultrastructure alterations and caspase-3 variations (immunological marker) were also investigated. FT-IR spectra revealed that Zn (II) is able to chelate through C=O and C-OH (Ring II) which are the carbonyl oxygen atoms of the quercetin ligand and carbonyl oxygen atom C=O of the Art ligand, forming Art/Q/Zn complex with the chemical formula [Zn(Q)(Art)(Cl)(H2O)2]⋅3H2O. The novel complex exhibited a potent anti-SARS-CoV-2 activity even at a low concentration (IC50 = 10.14 µg/ml) and was not cytotoxic to the cellular host (CC50 = 208.5 µg/ml). Art/Q/Zn may inhibit the viral replication and binding to the angiotensin-converting enzyme-2 (ACE2) receptor and the main protease inhibitor (MPro), thereby inhibiting the activity of SARS-CoV-2 and this proved by the molecular dynamics simulation. It alleviated Acy hepatic and pulmonary toxicity by improving all biochemical markers. Therefore, it can be concluded that the novel formula Art/Q/Zn complex is an effective antioxidant agent against the oxidative stress series, and it has high inhibitory effect against SARS-CoV-2.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India.

BACKGROUND: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. METHODS: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. RESULTS: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. CONCLUSION: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Bio Prospecting of Endophytes and PGPRs in Artemisinin Production for the Socio-economic Advancement.

The growing demand for Artemisia annua plants in healthcare, food, and pharmaceutical industries has led to increased cultivation efforts to extract a vital compound, Artemisinin. The efficacy of Artemisinin as a potent drug against malaria disease is well established but its limited natural abundance. However, the common practice of using chemical fertilizers for maximum yield has adverse effects on plant growth, development, and the quality of phytochemicals. To address these issues, the review discusses the alternative approach of harnessing beneficial rhizosphere microbiota, particularly plant growth-promoting rhizobacteria (PGPR). Microbes hold substantial biotechnological potential for augmenting medicinal plant production, offering an environmentally friendly and cost-effective means to enhance medicinal plant production. This review article aims to identify a suitable endophytic population capable of enabling Artemisia sp. to thrive amidst abiotic stress while simultaneously enhancing Artemisinin production, thereby broadening its availability to a larger population. Furthermore, by subjecting endophytes to diverse combinations of harsh conditions, this review sheds light on the modulation of essential artemisinin biosynthesis pathway genes, both up regulated and down regulated. The collective findings suggest that through the in vitro engineering of endophytic communities and their in vivo application to Artemisia plants cultivated in tribal population fields, artemisinin production can be significantly augmented. The overall aim of this review to explore the potential of harnessing microbial communities, their functions, and services to enhance the cultivation of medicinal plants. It outlines a promising path toward bolstering artemisinin production, which holds immense promise in the fight against malaria.

Assessment of genetic polymorphisms associated with malaria antifolate resistance among the population of Libreville, Gabon.

BACKGROUND: Gabon is a malaria-threatened country with a stable and hyperendemic transmission of Plasmodium falciparum monoinfection. Malaria drug resistance is widely spread in many endemic countries around the world, including Gabon. The molecular surveillance of drug resistance to antifolates and artemisinin-based combination therapy (ACT) is one of the strategies for combating malaria. As Plasmodium parasites continue to develop resistance to currently available anti-malarial drugs, this study evaluated the frequency of the polymorphisms and genetic diversity associated with this phenomenon among the parasites isolates in Gabon. METHODS: To assess the spread of resistant haplotypes among the malaria-infected population of Libreville, single nucleotide polymorphisms linked to sulfadoxine-pyrimethamine (SP) and artemisinin drugs resistance were screened for P. falciparum dihydrofolate reductase (Pfdhfr), P. falciparum dihydropteroate synthase (Pfdhps), and P. falciparum kelch 13-propeller domain (Pfk13) point mutations. RESULTS: The analysis of 70 malaria-positive patient samples screened for polymorphism showed 92.65% (n = 63) mutants vs. 7.35% (n = 5) wild parasite population in Pfdhfr, with high prevalence mutations at S108N(88.24%, n = 60), N51I(85.29%, n = 58), C59R(79.41%, n = 54); however, I164L(2.94%, n = 2) showed low frequency mutation. No wild haplotype existed for Pfdhps, and there were no mutations at the K540E, A581G, and A613T/S positions. However, the mutation rate at A437G(93.38%, n = 62) was the highest, followed by S436A/F(15.38%, n = 10). A higher frequency of quadruple IRNI-SGKAA (69.84%) than quintuple IRNI-(A/F)GKAA (7.94%) mutations was observed in the Pfdhfr-Pfdhps combination. Furthermore, none of the mutations associated with ACT resistance, especially those commonly found in Africa, were observed in Pfk13. CONCLUSIONS: High polymorphism frequencies of Pfdhfr and Pfdhps genes were observed, with alternative alanine/phenylalanine mutation at S436A/F (7.69%, n = 5) for the first time. Similar to that of other areas of the country, the patterns of multiple polymorphisms were consistent with selection owing to drug pressure. Although there was no evidence of a medication failure haplotype in the studied population, ACT drug efficacy should be regularly monitored in Libreville, Gabon.

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.

Health system readiness and the implementation of rectal artesunate for severe malaria in sub-Saharan Africa: an analysis of real-world costs and constraints.

BACKGROUND: Rectal artesunate, an efficacious pre-referral treatment for severe malaria in children, was deployed at scale in Uganda, Nigeria, and DR Congo. In addition to distributing rectal artesunate, implementation required additional investments in crucial but neglected components in the care for severe malaria. We examined the real-world costs and constraints to rectal artesunate implementation. METHODS: We collected primary data on baseline health system constraints and subsequent rectal artesunate implementation expenditures. We calculated the equivalent annual cost of rectal artesunate implementation per child younger than 5 years at risk of severe malaria, from a health system perspective, separating neglected routine health system components from incremental costs of rectal artesunate introduction. FINDINGS: The largest baseline constraints were irregular health worker supervisions, inadequate referral facility worker training, and inadequate malaria commodity supplies. Health worker training and behaviour change campaigns were the largest startup costs, while supervision and supply chain management accounted for most annual routine costs. The equivalent annual costs of preparing the health system for managing severe malaria with rectal artesunate were US$2·63, $2·20, and $4·19 per child at risk and $322, $219, and $464 per child treated in Uganda, Nigeria, and DR Congo, respectively. Strengthening the neglected, routine health system components accounted for the majority of these costs at 71·5%, 65·4%, and 76·4% of per-child costs, respectively. Incremental rectal artesunate costs accounted for the minority remainder. INTERPRETATION: Although rectal artesunate has been touted as a cost-effective pre-referral treatment for severe malaria in children, its real-world potential is limited by weak and under-financed health system components. Scaling up rectal artesunate or other interventions relying on community health-care providers only makes sense alongside additional, essential health system investments sustained over the long term. FUNDING: Unitaid. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Safflor yellow treating angina pectoris: A pharmacoeconomic evaluation and network meta-analysis.

BACKGROUND: Coronary heart disease (CHD) is a cardiovascular disease caused by myocardial ischemia. In China, safflor yellow and artemisinin-based combination therapies have been extensively used to treat angina pectoris. METHODS: Efficacies were provided by a network meta-analysis following the PRISMA 2020 checklist. Cost-effectiveness analysis was based on patient perspectives. Two-way and probabilistic sensitivity analyses were conducted to assess the robustness of the study results. RESULTS: Conventional treatment combined with safflower is a better choice against angina pectoris. Sensitivity analysis showed that the model was sensitive to the treatment efficacy rather than the drug cost. CONCLUSION: Conventional treatment combined with safflower injection is suggested to treat angina pectoris. Low molecular weight heparin or compound Danshen-dropping pills can be used to increase the recovery rate of angina pectoris, according to conventional treatment combined with safflower injection.

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.

Phytochemical screening, cytotoxicity assessment and evaluation of in vitro antiplasmodial and in vivo antimalarial activities of Mentha spicata L. methanolic leaf extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria causes extensive morbidity and mortality, and the decreasing efficacy of artemisinin and its partner drugs has posed a serious concern. Therefore, it is important to identify new antimalarials, and the natural compounds from plants provide a promising platform. Mentha spicata L. representing the Lamiaceae family has been used in traditional medicine for various diseases including malaria. AIM OF THE STUDY: This study was aimed at evaluating the antiplasmodial activity of M. spicata methanolic leaf extract using Plasmodium falciparum (Pf) cultures (Pf3D7 and artemisinin (ART)-resistant PfCam3.IR539T strains) and antimalarial activity using Plasmodium berghei (Pb)-infected mice. Dry leaf powder and methanolic leaf extract were examined for in vivo antimalarial activity and the efficacy of oral versus parenteral administration was compared. MATERIALS AND METHODS: Leaves of M. spicata were collected and extracted using 70% methanol in water (v/v). [3H]-hypoxanthine incorporation assays and Giemsa-stained smears were used to assess the in vitro antiplasmodial activity of M. spicata methanolic extract against Pf3D7 and ART-resistant PfCam3.IR539T strains. Cytotoxicity was evaluated in HeLa and HEK-293T cell lines using MTT assays. Hemolysis assays were performed using red blood cells (RBCs). In vivo antimalarial activities of M. spicata dry leaf powder and methanolic leaf extract were examined in P. berghei-infected mice by Rane's curative test and Peters' 4-day suppressive test. RESULTS: Phytochemical screening of M. spicata methanolic leaf extract indicated the presence of reducing sugars, phenolic compounds, flavonoids, glycosides, sterols, saponins, alkaloids, coumarins, tannins, carbohydrates, and proteins. In vitro studies carried out using Pf cultures showed that M. spicata methanolic leaf extract had significant antiplasmodial activity against Pf3D7 cultures with a 50% inhibitory concentration (IC50) of 57.99 ± 2.82 µg/ml. The extract was also effective against ART-resistant PfCam3.IR539T strain with an IC50 of 71.23 ± 3.85 µg/ml. The extract did not show significant in vitro cytotoxicity, hemolysis, and in vivo toxicity. In vivo studies performed using Pb-infected mice treated with M. spicata dry leaf powder and methanolic leaf extract showed ∼50% inhibition in parasite growth at 1500 mg/kg and 1000 mg/kg doses, respectively. There was also a significant delay in the mortality of treated mice. Parenteral administration was found to be appropriate for the in vivo treatment. CONCLUSIONS: Our in vitro and in vivo findings from Pf and Pb parasites suggested the therapeutic potential of M. spicata leaf extract as an antimalarial. M. spicata leaf extract could also inhibit the growth of ART-resistant Pf strain. Further studies on fractionation and active component analysis of M. spicata leaf extract would be required to identify the bioactive phytochemicals having pharmaceutical and therapeutic values. Such efforts would help us in developing new antimalarials to combat malaria.

Barcoding Genetically Distinct Plasmodium falciparum Strains for Comparative Assessment of Fitness and Antimalarial Drug Resistance.

The repeated emergence of antimalarial drug resistance in Plasmodium falciparum, including to the current frontline antimalarial artemisinin, is a perennial problem for malaria control. Next-generation sequencing has greatly accelerated the identification of polymorphisms in resistance-associated genes but has also highlighted the need for more sensitive and accurate laboratory tools to profile current and future antimalarials and to quantify the impact of drug resistance acquisition on parasite fitness. The interplay of fitness and drug response is of fundamental importance in understanding why particular genetic backgrounds are better at driving the evolution of drug resistance in natural populations, but the impact of parasite fitness landscapes on the epidemiology of drug resistance has typically been laborious to accurately quantify in the lab, with assays being limited in accuracy and throughput. Here we present a scalable method to profile fitness and drug response of genetically distinct P. falciparum strains with well-described sensitivities to several antimalarials. We leverage CRISPR/Cas9 genome-editing and barcode sequencing to track unique barcodes integrated into a nonessential gene (pfrh3). We validate this approach in multiplex competitive growth assays of three strains with distinct geographical origins. Furthermore, we demonstrate that this method can be a powerful approach for tracking artemisinin response as it can identify an artemisinin resistant strain within a mix of multiple parasite lines, suggesting an approach for scaling the laborious ring-stage survival assay across libraries of barcoded parasite lines. Overall, we present a novel high-throughput method for multiplexed competitive growth assays to evaluate parasite fitness and drug response. IMPORTANCE The complex interplay between antimalarial resistance and parasite fitness has important implications for understanding the development and spread of drug resistance alleles and the impact of genetic background on transmission. One limitation with current methodologies to measure parasite fitness is the ability to scale this beyond simple head-to-head competition experiments between a wildtype control line and test line, with a need for a scalable approach that allows tracking of parasite growth in complex mixtures. In our study, we have used CRISPR editing to insert unique DNA barcodes into a safe-harbor genomic locus to tag multiple parasite strains and use next-generation sequencing to read out strain dynamics. We observe inherent fitness differences between the strains, as well as sensitive modulation of responses to challenge with clinically relevant antimalarials, including artemisinin.

Assessment of the Anti-Malarial Properties of Dihydroartemisinin- Piperaquine Phosphate Solid Lipid-Based Tablets.

BACKGROUND: Artemisininbased combination therapies (ACTs) typified by dihydroartemisinin- piperaquine phosphate are first-line drugs used in the treatment of Plasmodium falciparum malaria. However, the emergence of drug resistance to ACTs shows the necessity to develop novel sustained release treatments in order to ensure maximum bioavailability. OBJECTIVES: To formulate dihydroartemisinin (DHA)-piperaquine phosphate (PQ) sustained release tablets based on solidified reverse micellar solutions (SRMS). METHODS: The SRMS was prepared by fusion using varying ratios of Phospholipon® 90H and Softisan® 154 and characterised. The tablets were prepared by using an in-house made and validated mould. The formulations were tested for uniformity of weight, hardness, friability, softening time, erosion time and in vitro-in vivo dissolution rate. Antimalarial properties were studied using modified Peter's 4-days suppressive test in mice. One-way analysis of variance (ANOVA) was used in the analysis of results. RESULTS: Smooth caplets, with average weight of 1300 ± 0.06 mg to 1312 ± 0.11 mg, drug content of 61 mg for DHA and t 450 mg for PQ. Tablet hardness ranged from 7.1 to 9.0 Kgf and softening time of 29.50 ± 1.90 min. Erosion time of 62.00 ± 2.58 to 152.00 ± 1.89 min were obtained for tablets formulated with Poloxamer 188 (Batches R2, S2 and T2) which significantly reduced the softening and erosion time (p < 0.05). In vitro release showed that the optimized formulations had a maximum release at 12 h. Formulations exhibited significantly higher parasitaemia clearance and in vivo absorption compared to marketed formulations at day 7 (p < 0.05). CONCLUSION: DHA-PQ tablets based on SRMS were much easier and relatively cheaper to produce than compressed tablets. They also showed exceptionally better treatment of malaria owing to their sustained release properties and improved bioavailability and are recommended to Pharmaceutical companies for further studies.

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.).

[Sustainable development roadmap of Chinese antimalarial products].

As the first country that has been certified malaria-free in the WHO Western Pacific Region for more than 3 decades, China should share its successful experiences of malaria control and provide globally recognized anti-malaria products by making full uses of its advantages in provision and production capacity of artemisinin raw materials, to fill the gaps of international demands for mosquito nets, rapid diagnostic reagents and antimalarial compounds. Increasing the investment of research and development of antimalarial products, building overseas bases for artemisinin raw material production, establishing international regulatory authority and promoting the local production of antimalarial products are needed to further promote the internationalization of Chinese antimalarial products, so as to achieve the equity and accessibility of Chinese antimalarial products in highburden regions for malaria.

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 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.