Artemisinin-based Combination Therapy Research Articles

From Plant to Yeast-Advances in Biosynthesis of Artemisinin.

Malaria is a life-threatening disease. Artemisinin-based combination therapy (ACT) is the preferred choice for malaria treatment recommended by the World Health Organization. At present, the main source of artemisinin is extracted from Artemisia annua; however, the artemisinin content in A. annua is only 0.1–1%, which cannot meet global demand. Meanwhile, the chemical synthesis of artemisinin has disadvantages such as complicated steps, high cost and low yield. Therefore, the application of the synthetic biology approach to produce artemisinin in vivo has magnificent prospects. In this review, the biosynthesis pathway of artemisinin was summarized. Then we discussed the advances in the heterologous biosynthesis of artemisinin using microorganisms (Escherichia coli and Saccharomyces cerevisiae) as chassis cells. With yeast as the cell factory, the production of artemisinin was transferred from plant to yeast. Through the optimization of the fermentation process, the yield of artemisinic acid reached 25 g/L, thereby producing the semi-synthesis of artemisinin. Moreover, we reviewed the genetic engineering in A. annua to improve the artemisinin content, which included overexpressing artemisinin biosynthesis pathway genes, blocking key genes in competitive pathways, and regulating the expression of transcription factors related to artemisinin biosynthesis. Finally, the research progress of artemisinin production in other plants (Nicotiana, Physcomitrella, etc.) was discussed. The current advances in artemisinin biosynthesis may help lay the foundation for the remarkable up-regulation of artemisinin production in A. annua through gene editing or molecular design breeding in the future.

In vitro susceptibility profile of Plasmodium falciparum clinical isolates from Ghana to antimalarial drugs and polymorphisms in resistance markers.

Drug resistance in Plasmodium falciparum compromises the effectiveness of antimalarial therapy. This study aimed to evaluate the extent of drug resistance in parasites obtained from international travelers returning from Ghana to guide the management of malaria cases. Eighty-two clinical parasite isolates were obtained from patients returning from Ghana in 2016–2018, of which 29 were adapted to continuous in vitro culture. Their geometric mean IC50 values to a panel of 11 antimalarial drugs, assessed using the standard SYBR Green-I drug sensitivity assay, were 2.1, 3.8, 1.0, 2.7, 17.2, 4.6, 8.3, 8.3, 19.6, 55.1, and 11,555 nM for artemether, artesunate, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, naphthoquine, pyronaridine, chloroquine, quinine, and pyrimethamine, respectively. Except for chloroquine and pyrimethamine, the IC50 values for other tested drugs were below the resistance threshold. The mean ring-stage survival assay value was 0.8%, with four isolates exceeding 1%. The mean piperaquine survival assay value was 2.1%, all below 10%. Mutations associated with chloroquine resistance (pfcrt K76T and pfmdr1 N86Y) were scarce, consistent with the discontinuation of chloroquine a decade ago. Instead, the pfmdr1 86N-184F-1246D haplotype was predominant, suggesting selection by the extensive use of artemether-lumefantrine. No mutations in the pfk13 propeller domain were detected. The pfdhfr/pfdhps quadruple mutant IRNGK associated with resistance to sulfadoxine-pyrimethamine reached an 82% prevalence. In addition, five isolates had pfgch1 gene amplification but, intriguingly, increased susceptibilities to pyrimethamine. This study showed that parasites originating from Ghana were susceptible to artemisinins and the partner drugs of artemisinin-based combination therapies. Genotyping drug resistance genes identified the signature of selection by artemether-lumefantrine. Parasites showed substantial levels of resistance to the antifolate drugs. Continuous resistance surveillance is necessary to guide timely changes in drug policy.

Overall and Gender-Specific Effects of Intermittent Preventive Treatment of Malaria with Artemisinin-Based Combination Therapies among Schoolchildren in Mali: A Three-Group Open Label Randomized Controlled Trial.

Intermittent preventive treatment of malaria among schoolchildren (IPTsc) reduces clinical malaria, asymptomatic parasitemia, and anemia. The effects of IPTsc by gender have not been studied longitudinally. We investigated overall IPTsc efficacy and conducted a secondary analysis to explore gender-specific differences. We enrolled schoolchildren aged 6-13 years in an open-label, rolling-cohort randomized controlled trial between September 2007 and February 2013 in Kolle, Mali. Annually, schoolchildren received two full-treatment courses of sulfadoxine-pyrimethamine (SP) plus artesunate, or amodiaquine (AQ) plus artesunate, or no malaria treatment as control. We used mixed-effects generalized linear models to estimate differences in treatment outcomes across groups with interaction terms to explore gender-specific differences associated with Plasmodium falciparum infection, hemoglobin, and grade point averages (GPA) based on standardized testing. Overall, 305 students contributed 4,564 observations. Compared with the control, SP plus artesunate and AQ plus artesunate reduced the odds of P. falciparum infection (odds ratio [OR]: 0.33, 95% CI: 0.26-0.43; OR: 0.46, 95% CI: 0.36-0.59). We found strong evidence of increased mean hemoglobin concentrations (g/dL) in the SP plus artesunate group versus control (difference +0.37, 95% CI: 0.13-0.58). Collectively, schoolchildren given AQ plus artesunate had higher mean GPA (difference +0.36, 95% CI: 0.02-0.69) relative to control. Schoolgirls, compared with schoolboys, given SP plus artesunate had greater improvement in GPA (+0.50, 95% CI: -0.02 to 1.02 versus -0.27, 95% CI: -0.71 to 0.16); interaction P = 0.048, respectively. The IPTsc decreases P. falciparum infections in schoolchildren. Treatment regimens that include longer-acting drugs may be more effective at decreasing malaria-related anemia and improving educational outcomes as observed among girls in this setting.

Effectiveness of rectal artesunate as pre-referral treatment for severe malaria in children under 5 years of age: a multi-country observational study

BackgroundTo prevent child deaths from severe malaria, early parenteral treatment is essential. Yet, in remote rural areas, accessing facilities offering parenteral antimalarials may be difficult. A randomised controlled trial found pre-referral treatment with rectal artesunate (RAS) to reduce deaths and disability in children who arrived at a referral facility with delay. This study examined the effectiveness of pre-referral RAS treatment implemented through routine procedures of established community-based health care systems.MethodsAn observational study accompanied the roll-out of RAS in the Democratic Republic of the Congo (DRC), Nigeria and Uganda. Children <5 years of age presenting to a community-based health provider with a positive malaria test and signs of severe malaria were enrolled and followed up during admission and after 28 days to assess their health status and treatment history. The primary outcome was death; covariates of interest included RAS use, referral completion, and post-referral treatment.ResultsPost-roll-out, RAS was administered to 88% of patients in DRC, 52% in Nigeria, and 70% in Uganda. The overall case fatality rate (CFR) was 6.7% (135/2011) in DRC, 11.7% (69/589) in Nigeria, and 0.5% (19/3686) in Uganda; 13.8% (865/6286) of patients were sick on day 28. The CFR was higher after RAS roll-out in Nigeria (16.1 vs. 4.2%) and stable in DRC (6.7 vs. 6.6%) and Uganda (0.7 vs. 0.3%). In DRC and Nigeria, children receiving RAS were more likely to die than those not receiving RAS (aOR=3.06, 95% CI 1.35–6.92 and aOR=2.16, 95% CI 1.11–4.21, respectively). Only in Uganda, RAS users were less likely to be dead or sick at follow-up (aOR=0.60, 95% CI 0.45–0.79). Post-referral parenteral antimalarials plus oral artemisinin-based combination therapy (ACT), a proxy for appropriate post-referral treatment, was protective. However, in referral health facilities, ACT was not consistently administered after parenteral treatment (DRC 68.4%, Nigeria 0%, Uganda 70.9%).ConclusionsImplemented at scale to the recommended target group, pre-referral RAS had no beneficial effect on child survival in three highly malaria-endemic settings. RAS is unlikely to reduce malaria deaths unless health system issues such as referral and quality of care at all levels are addressed.Trial registrationThe study is registered on ClinicalTrials.gov: NCT03568344.

East Africa International Center of Excellence for Malaria Research: Impact on Malaria Policy in Uganda.

Malaria is the leading cause of disease burden in sub-Saharan Africa. In 2010, the East Africa International Center of Excellence for Malaria Research, also known as the Program for Resistance, Immunology, Surveillance, and Modeling of Malaria (PRISM), was established to provide a comprehensive approach to malaria surveillance in Uganda. We instituted cohort studies and a robust malaria and entomological surveillance network at selected public health facilities that have provided a platform for monitoring trends in malaria morbidity and mortality, tracking the impact of malaria control interventions (indoor residual spraying of insecticide [IRS], use of long-lasting insecticidal nets [LLINs], and case management with artemisinin-based combination therapies [ACTs]), as well as monitoring of antimalarial drug and insecticide resistance. PRISM studies have informed Uganda's malaria treatment policies, guided selection of LLINs for national distribution campaigns, and revealed widespread pyrethroid resistance, which led to changes in insecticides delivered through IRS. Our continuous engagement and interaction with policy makers at the Ugandan Ministry of Health have enabled PRISM to share evidence, best practices, and lessons learned with key malaria stakeholders, participate in malaria control program reviews, and contribute to malaria policy and national guidelines. Here, we present an overview of interactions between PRISM team members and Ugandan policy makers to demonstrate how PRISM's research has influenced malaria policy and control in Uganda.

Multidisciplinary Investigations of Sustained Malaria Transmission in the Greater Mekong Subregion.

In the course of malaria elimination in the Greater Mekong Subregion (GMS), malaria epidemiology has experienced drastic spatiotemporal changes with residual transmission concentrated along international borders and the rising predominance of Plasmodium vivax. The emergence of Plasmodium falciparum parasites resistant to artemisinin and partner drugs renders artemisinin-based combination therapies less effective while the potential spread of multidrug-resistant parasites elicits concern. Vector behavioral changes and insecticide resistance have reduced the effectiveness of core vector control measures. In recognition of these problems, the Southeast Asian International Center of Excellence for Malaria Research (ICEMR) has been conducting multidisciplinary research to determine how human migration, antimalarial drug resistance, vector behavior, and insecticide resistance sustain malaria transmission at international borders. These efforts allow us to comprehensively understand the ecology of border malaria transmission and develop population genomics tools to identify and track parasite introduction. In addition to employing invivo, invitro, and molecular approaches to monitor the emergence and spread of drug-resistant parasites, we also use genomic and genetic methods to reveal novel mechanisms of antimalarial drug resistance of parasites. We also use omics and population genetics approaches to study insecticide resistance in malaria vectors and identify changes in mosquito community structure, vectorial potential, and seasonal dynamics. Collectively, the scientific findings from the ICEMR research activities offer a systematic view of the factors sustaining residual malaria transmission and identify potential solutions to these problems to accelerate malaria elimination in the GMS.

Impact and operational feasibility of adding malaria infection screening using an ultrasensitive RDT for placental and fetal outcomes in an area of high IPTP-SP coverage in Burkina Faso: the ASSER MALARIA pilot study protocol

BackgroundMalaria infection during pregnancy (MIP) is not only deleterious to the woman, but it also puts her fetus at increased risk of adverse outcomes, such as preterm delivery, low birth weight, and intrauterine growth retardation. Additionally, all-cause mortality during the first year of life in babies born to women with malaria during pregnancy is also increased. Many interventions such as IPTp-SP and long-lasting insecticidal nets have proven to be efficient at reducing malaria in pregnancy burden but adherence to recommended policies remains poor. In sub-Saharan Africa, malaria in pregnancy is often asymptomatic and many malaria infections may be missed due to the inadequate performance of the current rapid diagnostic test to detect low-level parasitemias. Therefore, additional strategies such as intermittent screening with ultrasensitive rapid diagnostic tests and treatment with an effective artemisinin-based combination therapy in addition to IPTp-SP could reduce placental malaria, peripheral malaria infection at delivery, and low birth weight.MethodsThis pilot 2-group randomized open trial with a nested qualitative social behavioral will be carried out in Nanoro district in which 340 pregnant women will be recruited. Pregnant women will be randomized into two groups and followed on a monthly basis until delivery. In the intervention group, monthly screening using ultrasensitive rapid diagnostic tests and treatment of those found to be infected with dihydroartemisinin-piperaquine will be performed. In addition, a reminder will be sent to increase the uptake of IPTp-SP doses per woman. During scheduled and unscheduled visits, malaria infection, hemoglobin level, and other clinical outcomes will be assessed and compared by the group. The primary feasibility outcome will evaluate the study site's capacity to enroll participants and the women’s perception and acceptability of the intervention. The primary clinical outcome will be the prevalence of placental malaria at delivery.DiscussionThe present protocol aims to evaluate the feasibility on a large-scale and also to demonstrate the impact and the operational feasibility of additional screening with ultrasensitive rapid diagnostic tests and treatment with DHA-PQ on placental malaria, low birth weight, and peripheral malaria infection at delivery in a high-burden setting in Burkina Faso.Trial registrationClinicalTrials.gov, ID: NCT04147546 (14 October 2019).

The Effect of Mass Testing, Treatment and Tracking on the Prevalence of Febrile Illness in Children under 15 in Ghana.

Background: Malaria remains a serious threat to children under 15 years of age in sub-Sahara Africa. Mass testing, treatment and tracking (MTTT) of malaria has been reported to reduce parasite load significantly. However, the impact of MTTT on the prevalence of febrile illnesses in children under 15 is not yet clear. This study explores the impact of MTTT complemented by prompt home-based management of malaria on febrile illnesses and their treatment in children under 15 years old. Methods: A cohort of 460 children under 15 years were recruited from the Pakro subdistrict in Ghana during a community-wide implementation of a quarterly MTTT intervention. The MTTT implementation involved testing all household members for malaria using RDTs, and positive cases were treated with Artemisinin-based combination therapy (ACT). Febrile illnesses among this cohort in the two weeks prior to the prevalence survey at baseline and endline were recorded to constitute date for analysis. Results: The prevalence of febrile illnesses, such chills, convulsion, fever, diarrhoea, headache, vomit, cough/rashes or stomachache, etc., were recorded). Asymptomatic parasitaemia prevalence at baseline was 53.3%, which dropped to 44.1% at evaluation. An overall decrease in the parasitaemia prevalence of 33.0% (OR = 0.67, CI = 0.50, 0.89) was observed at evaluation compared to baseline after adjusting for age, ITN use and temperature. A 67% decrease in severe anaemia cases (Hb < 7) was observed at evaluation. Conclusion: Our findings suggest that implementing MTTT complemented by home-based timely management of malaria does not only reduce febrile illnesses and for that matter malaria prevalence, but could also reduce severe anaemia in children under 15 years old.

Artemisinin resistance and malaria elimination: Where are we now?

The emergence of artemisinin resistance is a major obstacle to the global malaria eradication/elimination programs. Artemisinin is a very fast-acting antimalarial drug and is the most important drug in the treatment of severe and uncomplicated malaria. For the treatment of acute uncomplicated falciparum malaria, artemisinin derivatives are combined with long half-life partner drugs and widely used as artemisinin-based combination therapies (ACTs). Some ACTs have shown decreased efficacy in the Southeast Asian region. Fortunately, artemisinin has an excellent safety profile and resistant infections can still be treated successfully by modifying the ACT. This review describes the pharmacological properties of ACTs, mechanisms of artemisinin resistance and the potential changes needed in the treatment regimens to overcome resistance. The suggested ACT modifications are extension of the duration of the ACT course, alternating use of different ACT regimens, and addition of another antimalarial drug to the standard ACTs (Triple-ACT). Furthermore, a malaria vaccine (e.g., RTS,S vaccine) could be added to mass drug administration (MDA) campaigns to enhance the treatment efficacy and to prevent further artemisinin resistance development. This review concludes that artemisinin remains the most important antimalarial drug, despite the development of drug-resistant falciparum malaria.

Artemisinin-based combination therapy amongst medical students in the University of Benin, South-South Nigeria: A cross-sectional study

Since 2005, the recommended first-line therapy for uncomplicated malaria in Nigeria has been Artemisinin-based Combination Therapy (ACT). Previous research indicates that these therapies are widely accepted by health care providers and other end users. Nonetheless, few studies have examined the preferences of clinical students who will be future prescribers of these medications. This was a descriptive cross-sectional survey of medical students undergoing clinical placements at the University of Benin Teaching Hospital in Benin City, Edo State, South-South Nigeria, to assess ACT preferences, tolerability, and cost considerations. Consenting clinical medical students who were recruited sequentially were given a semi-structured questionnaire. The questionnaire collected information about the students’ demographics, previous episodes of malaria symptoms, diagnosis, and treatment. Their preferred antimalarial, tolerability to ACTs, and ACT cost. The information was presented descriptively. There were 475 registered clinical students, but only 416 agreed to participate in the survey, yielding an 87.6% response rate. The students’ mean (standard deviation) age was 24.3 (3.8) years, with a male preponderance of 250 (60.1%). The majority, 154 (37.0%), were in the 600 level, while the 500 and 400 levels were 130 (31.3%) and 132 (31.7%), respectively. The majority of students, 272 (65.5%), treated malaria presumptively, and the majority of students, 344 (82.7%), had treated one to five episodes of malaria in the previous 12 months. The ACTs were known to nearly all of the final year clinical students (97.4%). The majority, 289 (69.5%), had no adverse drug reactions with the ACTs. Among those who experienced adverse drug reactions, 23 (39.7%) discontinued their medication, while 2 (3.4%) required hospitalization. The mean cost of the ACT was ₦1263.4 (SD ± 529.6) (₦=Naira) (3.0 USD), with a range of ₦ 300- ₦3000 (0.72- 7.2 USD). This study demonstrates a high level of acceptance for ACTs, particularly artemether-lumefantrine. However, the adverse effects of ACTs, particularly other recommended ACTs, must be evaluated on a regular basis because they may have an impact on their continued use.

Safety of single-dose primaquine as a Plasmodium falciparum gametocytocide: a systematic review and meta-analysis of individual patient data

BackgroundIn 2012, the World Health Organization (WHO) recommended single low-dose (SLD, 0.25 mg/kg) primaquine to be added as a Plasmodium (P.) falciparum gametocytocide to artemisinin-based combination therapy (ACT) without glucose-6-phosphate dehydrogenase (G6PD) testing, to accelerate malaria elimination efforts and avoid the spread of artemisinin resistance. Uptake of this recommendation has been relatively slow primarily due to safety concerns.MethodsA systematic review and individual patient data (IPD) meta-analysis of single-dose (SD) primaquine studies for P. falciparum malaria were performed. Absolute and fractional changes in haemoglobin concentration within a week and adverse effects within 28 days of treatment initiation were characterised and compared between primaquine and no primaquine arms using random intercept models.ResultsData comprised 20 studies that enrolled 6406 participants, of whom 5129 (80.1%) had received a single target dose of primaquine ranging between 0.0625 and 0.75 mg/kg. There was no effect of primaquine in G6PD-normal participants on haemoglobin concentrations. However, among 194 G6PD-deficient African participants, a 0.25 mg/kg primaquine target dose resulted in an additional 0.53 g/dL (95% CI 0.17–0.89) reduction in haemoglobin concentration by day 7, with a 0.27 (95% CI 0.19–0.34) g/dL haemoglobin drop estimated for every 0.1 mg/kg increase in primaquine dose. Baseline haemoglobin, young age, and hyperparasitaemia were the main determinants of becoming anaemic (Hb < 10 g/dL), with the nadir observed on ACT day 2 or 3, regardless of G6PD status and exposure to primaquine. Time to recovery from anaemia took longer in young children and those with baseline anaemia or hyperparasitaemia. Serious adverse haematological events after primaquine were few (9/3, 113, 0.3%) and transitory. One blood transfusion was reported in the primaquine arms, and there were no primaquine-related deaths. In controlled studies, the proportions with either haematological or any serious adverse event were similar between primaquine and no primaquine arms.ConclusionsOur results support the WHO recommendation to use 0.25 mg/kg of primaquine as a P. falciparum gametocytocide, including in G6PD-deficient individuals. Although primaquine is associated with a transient reduction in haemoglobin levels in G6PD-deficient individuals, haemoglobin levels at clinical presentation are the major determinants of anaemia in these patients.Trial registrationPROSPERO, CRD42019128185

Incidence, pattern and determinants of self-medication for fevers in children: A survey of caregivers in an emergency unit in Benin City, Nigeria

Self-medication is common in medically underserved areas, with disastrous consequences. It is necessary to determine the frequency and risk factors in our area. The purpose of this study was to find out the frequency, pattern, and risk factors for self-medication for febrile childhood illnesses among caregivers seen in the emergency paediatric unit. A descriptive, cross-sectional design was used for this study. Participants included caregivers for children seen in the unit. A semi-structured questionnaire was used to collect data, which included sections on demography, determinants, pattern, and reasons for self-medication, as well as actual reports of self-medication in the previous three weeks. In order to identify risk factors for self-medication, inferential analyses were performed. P-values less than 0.05 were considered significant. The study included 153 caregivers, with an average (SD) age of 45.6 (13.8) years. Self-medication for fevers in children was 25.5%, with 20 (51.3%) of the children treated being under the age of five. Antipyretics (20.3%) and antimalarials (13.7%), particularly artemisinin-based combination therapy (ACT), were the most commonly used drugs. Place of residence (p= 0.011), living arrangement (p= 0.030), and awareness of an adverse drug event (p= 0.045) are factors that influence self-medication in the previous three weeks. Having health insurance, NHIS (OR = 2.734, 95% CI: 1.120-6.678; p=0.027), and using an insecticide-treated net (OR = 0.272, 95% CI: 0.117-0.634; p=0.003) are also independent predictors of self-medication. With multifactorial determinants in the setting, self-medication is common. There is a need for increased access to subsidized healthcare for children, as well as caregiver education on the potential harms of self-medication.

Naturally Acquired Kelch13 Mutations in Plasmodium falciparum Strains Modulate In Vitro Ring-Stage Artemisinin-Based Drug Tolerance and Parasite Survival in Response to Hyperoxia.

ABSTRACTThe ring-stage survival assay was utilized to assess the impact of physiological hyperoxic stress on dihydroartemisinin (DHA) tolerance for a panel of Plasmodium falciparum strains with and without Kelch13 mutations. Strains without naturally acquired Kelch13 mutations or the postulated genetic background associated with delayed parasite clearance time demonstrated reduced proliferation under hyperoxic conditions in the subsequent proliferation cycle. Dihydroartemisinin tolerance in three isolates with naturally acquired Kelch13 mutations but not two genetically manipulated laboratory strains was modulated by in vitro hyperoxic stress exposure of early-ring-stage parasites in the cycle before drug exposure. Reduced parasite tolerance to additional derivatives, including artemisinin, artesunate, and OZ277, was observed within the second proliferation cycle. OZ439 and epoxomicin completely prevented parasite survival under both hyperoxia and normoxic in vitro culture conditions, highlighting the unique relationship between DHA tolerance and Kelch13 mutation-associated genetic background.IMPORTANCE Artemisinin-based combination therapy (ACT) for treating malaria is under intense scrutiny following treatment failures in the Greater Mekong subregion of Asia. This is further compounded by the potential for extensive loss of life if treatment failures extend to the African continent. Although Plasmodium falciparum has become resistant to all antimalarial drugs, artemisinin “resistance” does not present in the same way as resistance to other antimalarial drugs. Instead, a partial resistance or tolerance is demonstrated, associated with the parasite’s genetic profile and linked to a molecular marker referred to as K13. It is suggested that parasites may have adapted to drug treatment, as well as the presence of underlying population health issues such as hemoglobinopathies, and/or environmental pressures, resulting in parasite tolerance to ACT. Understanding parasite evolution and control of artemisinin tolerance will provide innovative approaches to mitigate the development of artemisinin tolerance and thereby artemisinin-based drug treatment failure and loss of life globally to malaria infections.

Ethical considerations in deploying triple artemisinin-based combination therapies for malaria: An analysis of stakeholders' perspectives in Burkina Faso and Nigeria.

BackgroundArtemisinin-based combination therapies (ACTs) are the recommended treatment for uncomplicated Plasmodium falciparum malaria in all malaria endemic countries. Artemisinin resistance, partner drug resistance, and subsequent ACT failure are widespread in Southeast Asia. The more recent independent emergence of artemisinin resistance in Africa is alarming. In response, triple artemisinin-based combination therapies (TACTs) are being developed to mitigate the risks associated with increasing drug resistance. Since ACTs are still effective in Africa, where malaria is mainly a paediatric disease, the potential deployment of TACTs raises important ethical questions. This paper presents an analysis of stakeholders’ perspectives regarding key ethical considerations to be considered in the deployment of TACTs in Africa provided they are found to be safe, well-tolerated and effective for the treatment of uncomplicated malaria.MethodsWe conducted a qualitative study in Burkina Faso and Nigeria assessing stakeholders’ (policy makers, suppliers and end-users) perspectives on ethical issues regarding the potential future deployment of TACTs through 68 in-depth interviews and 11 focus group discussions.FindingsSome respondents suggested that there should be evidence of local artemisinin resistance before they consider deploying TACTs, while others suggested that TACTs should be deployed to protect the efficacy of current ACTs. Respondents suggested that additional side effects of TACTs compared to ACTs should be minimal and the cost of TACTs to end-users should not be higher than the cost of current ACTs. There was some disagreement among respondents regarding whether patients should have a choice of treatment options between ACTs and TACTs or only have TACTs available, while ACTs are still effective. The study also suggests that community, public and stakeholder engagement activities are essential to support the introduction and effective uptake of TACTs.ConclusionAddressing ethical issues regarding TACTs and engaging early with stakeholders will be important for their potential deployment in Africa.

Prevalence and Management of Falciparum Malaria among Febrile Patients in South-western Nigeria

The World Health Organization reported 241 million cases and 627,000 death from malaria in 2020 and Nigeria accounted for 27% of malaria death worldwide. Effective tools are continually developed through global efforts to have a malaria-free world. The study was aimed at assessing the prevalence and management of falciparum malaria among febrile patients in South-western Nigeria. This is a cross-sectional descriptive study conducted on febrile patients undergoing treatment at the outpatient units of selected hospitals in Ekiti and Ondo States, Nigeria between August 2019 and January, 2021. A total of 399 participants were enlisted in the study following informed consent. A structured questionnaire was administered to obtain socio-demographic information and the management of malaria among the participant and blood collected from them for malaria examination by microscopy and rapid test diagnosis. Two hundred and ninety-five (73.9%) of the subjects were females and 104(26.1%) males, with modal age of 21–28 years. There were 171(42.9%) and 228(57.1%) participants from Ekiti and Ondo States respectively. Subjects from Ondo State reported significantly higher occurrence of malaria than their counterparts from Ekiti State (p=0.005). Malaria occurrence was found to be significantly associated with age (p=0.047), occupation (p=0.001), individual’s perception of approach to malaria treatment (p=0.002) and use of local herbs (p=0.002). Artemisinin-based combination therapies (ACTs) were drugs of choice (76.3%) by the respondents; other drugs commonly used were Sulphadoxine-pyrimethamine (13.3%) and chloroquine/quinine (10.4%). The use of insecticide treated nets (26.6%), insecticide spray (25.3%), as well as, nets on windows and doors (15.8%) were reported as the major ways of controlling mosquito bites at home. The management of malaria among respondents is commendable; notwithstanding, healthcare professionals will need a combination of new approaches and tools in effective management of malaria.

Replenishing the malaria drug discovery pipeline: Screening and hit evaluation of the MMV Hit Generation Library 1 (HGL1) against asexual blood stage Plasmodium falciparum, using a nano luciferase reporter read-out.

A central challenge of antimalarial therapy is the emergence of resistance to the components of artemisinin-based combination therapies (ACTs) and the urgent need for new drugs acting through novel mechanism of action. Over the last decade, compounds identified in phenotypic high throughput screens (HTS) have provided the starting point for six candidate drugs currently in the Medicines for Malaria Venture (MMV) clinical development portfolio. However, the published screening data which provided much of the new chemical matter for malaria drug discovery projects have been extensively mined. Here we present a new screening and selection cascade for generation of hit compounds active against the blood stage of Plasmodium falciparum. In addition, we validate our approach by testing a library of 141,786 compounds not reported earlier as being tested against malaria. The Hit Generation Library 1 (HGL1) was designed to maximise the chemical diversity and novelty of compounds with physicochemical properties associated with potential for further development. A robust HTS cascade containing orthogonal efficacy and cytotoxicity assays, including a newly developed and validated nanoluciferase-based assay was used to profile the compounds. 75 compounds (Screening Active hit rate of 0.05%) were identified meeting our stringent selection criteria of potency in drug sensitive (NF54) and drug resistant (Dd2) parasite strains (IC50 ≤ 2 µM), rapid speed of action and cell viability in HepG2 cells (IC50 ≥ 10 µM). Following further profiling, 33 compounds were identified that meet the MMV Confirmed Active profile and are high quality starting points for new antimalarial drug discovery projects.

Preparation and application of a specific single-chain variable fragment against artemether

Artemether, an artemisinin derivative, is a component of the commonly used artemisinin-based combination therapy, artemether-lumefantrine. In this study, we cloned the VH and VL genes of a cell line (mAb 2G12E1) producing a monoclonal antibody specific to artemether, and used to construct a recombinant DNA of single-chain variable fragment (scFv). The scFv was constructed into prokaryotic expression vectors pET32a (+), pET22b (+), pGEX-2T, and pMAL-p5x, respectively. However, only the pMAL-p5x/scFv could be induced to express soluble scFv with comparable sensitivity and specificity to that of mAb 2G12E1. Based on the anti-artemether scFv, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The 50% of inhibition concentration (IC50) value and the working range based on IC20 to IC80 were 4.33 ng mL−1 and 1.05–22.65 ng mL−1, respectively. The artemether content in different drugs were determined by the developed icELISA, and the results were consistent to those determined by ultra performance liquid chromatography (UPLC). The anti-artemether scFv prepared in the current study could be a valuable genetically engineered antibody applied for artemether monitoring and specific binding mechanism studying.

Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA.

Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1. Using qPCR analysis, we measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages. We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His, while DAGK contained K270R and K292R mutations. PI4Kβ had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites. The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and their impact on drug responses require investigations using reverse genetics techniques in malaria parasites.

Lumefantrine pressure selects nonsynonymous mutation in cysteine desulfurase IscS gene in the rodent malaria parasite Plasmodium berghei ANKA

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ) are the essential long-acting partner drugs in the artemisinin-based combination therapies (ACTs) treatment regimens globally. Understanding the resistance mechanisms to partner drugs remains critical for tracking resistant parasites. Cysteine desulfurase IscS (nfs1), one of the proteins involved in the iron-sulfur (FeS) biogenesis pathway, has been implicated in mediating malaria parasite drug resistance. Methods: Using the rodent malaria parasites Plasmodium berghei ANKA in mice, we assessed whether the nfs1 gene is associated with LM, PQ, and AQ resistance. By means of PCR and sequencing analysis, we probed for single nucleotide polymorphisms (SNPs) within the nfs1 gene. Using qPCR, we then measured the expression of the nfs1 gene in resistant parasites relative to the drug-sensitive parent parasites. Results: Our analyses of nfs1 reveal a non-synonymous Gln142Arg mutation in the LM and PQ-resistant parasites. This mutation was not detected in the AQ-resistant parasites. The mRNA quantification of the nfs1 gene reveals significant downregulation in both LM and PQ-resistant parasites compared to the drug-sensitive wild-type (WT) parasites. Conversely, nfs1 expression was upregulated in the AQ-resistant schizont stage compared to the WT parasites. Conclusion: Our data suggest that LM and PQ selection pressure induces nonsynonymous mutation and nfs1 downregulation of its expression in Plasmodium berghei. Collectively, these findings provide a premise for investigating LM and PQ resistance mechanisms in both P. berghei and P. falciparum.

Contribution of child health interventions to under-five mortality decline in Ghana: A modeling study using lives saved and missed opportunity tools.

BackgroundIncreased coverage of interventions have been advocated to reduce under-five mortality. However, Ghana failed to achieve the Millennium Development Goal on child survival in 2015 despite improved coverage levels of some child health interventions. Therefore, there is the need to determine which interventions contributed the most to mortality reduction and those that can further rapidly reduce mortality to inform the prioritization of the scale-up of interventions.Materials and methodsDeterministic mathematical modeling was done using Lives Saved and Missed Opportunity Tools. Secondary data was used, and the period of the evaluation was between 2008 and 2014. Some of the interventions assessed were complementary feeding, skilled delivery, and rotavirus vaccine.ResultsA total of 48,084 lives were saved from changes in coverage of interventions and a reduction in the prevalence of stunting and wasting. Reduction in wasting prevalence saved 10,372(21.6%) lives, insecticide-treated net/indoor residual spraying 6,437(13.4%) lives saved, reduction in stunting 4,315(9%) lives saved and artemisinin-based combination therapy (ACTs) 4,325(9.0%) lives saved. If coverage levels of interventions in 2014 were scaled up to 90% in 2015, among neonates, full supportive care for prematurity (5,435 lives saved), full supportive care for neonatal sepsis/pneumonia (3,002 lives saved), and assisted vaginal delivery (2,163 lives saved), would have saved the most lives among neonates, while ACTs (4,925 lives saved), oral rehydration salts (ORS) (2,056 lives saved), and antibiotics for the treatment of pneumonia (1,805 lives saved) would have made the most impact on lives saved among children 1–59 months. Lastly, if all the interventions were at 100% coverage in 2014, the under-five mortality rate would have been 40.1 deaths per 1,000 live births in 2014.DiscussionThe state of the package of interventions will likely not lead to rapid mortality reduction. Coverage and quality of childbirth-related interventions should be increased. Additionally, avenues to further reduce stunting and wasting, including increased breastfeeding and complementary feeding, will be beneficial.