Malaria Challenge Research Articles

Virus-like particle-based vaccines targeting the Anopheles mosquito salivary protein, TRIO.

Malaria is a highly lethal infectious disease caused by Plasmodium parasites. These parasites are transmitted to vertebrate hosts when mosquitoes of the Anopheles genus probe for a blood meal. Sporozoites, the infectious stage of Plasmodium , transit to the liver within hours of injection into the dermis. Vaccine efforts are hindered by the complexity of the parasite's lifecycle and the speed at which the infection is established in the liver. In an effort to enhance immunity against Plasmodium , we produced a virus-like particle (VLP)-based vaccine displaying an epitope of TRIO, an Anopheles salivary protein which has been shown to enhance mobility and dispersal of sporozoites in the dermis. Previous work demonstrated that passive immunization with TRIO offered protection from liver infection and acted synergistically with a Plasmodium targeted vaccine. Immunization of mice with TRIO VLPs resulted in high-titer and long-lasting antibody responses that did not significantly drop for over 18 months post-immunization. TRIO VLPs were similarly immunogenic when combined with an anti-malaria vaccine targeting the L9 epitope of the Plasmodium falciparum circumsporozoite protein.However, when used in a malaria challenge mouse model, TRIO VLPs only provided modest protection from infection and did not boost the protection provided by L9 VLPs.

Spatial Pattern and Distribution of Malaria Cases in Owo Local Government Area, Nigeria

This study investigates the spatial pattern and distribution of malaria cases in Owo Local Government Area, Nigeria. A multi-stage sampling technique was used, such that 50% of the 7 urban political wards were selected. The selected 4 urban political wards comprise of 12,466 household heads. Also, 10% of the 127 rural settlements were randomly selected making 14 rural settlements with 9,095 household heads. In all, 1.5% of the total household heads, comprising 187 in the urban areas and 136 in the rural settlements were interviewed using systematic random sampling techniques. A secondary data source obtained from relevant healthcare facilities was used for spatial analysis. Specifically, 59 selected residential settlements were used as the input feature class, while the records on malaria cases in each residential settlement during the reviewed year serve as the input field for the analyses. Spatial statistical functions provided by ArcGIS 10.8 and Geospatial tool are utilized for data analysis. Findings revealed preponderance of malaria within the urban core area of Owo. This can be attributed to inadequate wastewater management, poor waste disposal methods, and inadequate environmental education and awareness. On the other hand, malaria cases in rural areas were relatively low due to the use of alternative disease treatment methods. To ameliorate these challenges, there is a need for improved access to a safe and high-quality environment, implement suitable sanitation facilities and solid waste disposal methods, promote community-based educational initiatives and awareness campaigns, and encouraging hygienic behaviors. Through these, the health challenges of malaria can be mitigated.

Efficacy of ivermectin and its metabolites against Plasmodium falciparum liver stages in primary human hepatocytes.

Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3″-O-demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC50) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro.

Malaria prevalence and associated population and ecological risk factors among women and children under 5 years in Rwanda

BackgroundMalaria continues to pose a substantial public health concern in Rwanda, despite substantial progress in recent years. Little is known about effect of ecological factors and their interaction in malaria transmission. Understanding the prevalence and identifying risk factors, both population-based and ecological such as zooprophylaxis and irrigation are crucial for targeted intervention strategies. MethodsThis study analyzed the 6th iteration of the Demographic and Health Survey conducted in Rwanda between 2019 and 2020. The study employed a nationally representative sample, utilizing rapid diagnostic tests and blood smear microscopy to determine malaria prevalence among women and under 5 years old children. Logistic regression analysis was used in R version 4.3.1 to evaluate population and ecological risk factors that are associated with malaria prevalence. Additionally, interactive effects of ecological factors on malaria were evaluated. ResultsThe analysis revealed a notable malaria prevalence in Rwanda, emphasizing the continued significance of malaria control efforts. Approximately 1.79 % of the population tested positive for malaria. Proximity to irrigation sites and lowland were identified as significant risk factors to malaria with adjusted odds ratio (AOR) 1.47(1.00–2.15) and AOR 5.44(4.01–8.61) respectively however cattle ownership exhibited a protective effect AOR 0.41(0.23–0.72). Interactive effects of livestock and irrigation on malaria prevalence were revealed. Additionally, population-based risk factors, including age, household wealth, utilization of Insecticide Treated Nets, were associated with varying malaria risks. ConclusionThis study underscores the persistent challenge of malaria in Rwanda and the importance of tailored intervention strategies. To effectively combat malaria, efforts must consider the interplay of ecological factors, such as high cattle density, and demographic factors, targeting high-risk populations, especially those living in proximity to lowlands and irrigation areas. These findings provide critical insights for advancing malaria elimination efforts in Rwanda and serve as a basis for comprehensive public health planning and action.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

Full-length MSP1 is a major target of protective immunity after controlled human malaria infection.

The merozoite surface protein 1 (MSP1) is the most abundant protein on the surface of the invasive merozoite stages of Plasmodium falciparum and has long been considered a key target of protective immunity. We used samples from a single controlled human malaria challenge study to test whether the full-length version of MSP1 (MSP1FL) induced antibodies that mediated Fc-IgG functional activity in five independent assays. We found that anti-MSP1FL antibodies induced complement fixation via C1q, monocyte-mediated phagocytosis, neutrophil respiratory burst, and natural killer cell degranulation as well as IFNγ production. Activity in each of these assays was strongly associated with protection. The breadth of MSP1-specific Fc-mediated effector functions was more strongly associated with protection than the individual measures and closely mirrored what we have previously reported using the same assays against merozoites. Our findings suggest that MSP1FL is an important target of functional antibodies that contribute to a protective immune response against malaria.

Enhancing impact of <i>Prosopis africana</i> fraction on biochemical indices and oxidative balance in <i>Plasmodium berghei</i>-infected mice

This study addresses the ongoing global health challenge of malaria by exploring the therapeutic potential of the Prosopis africana fraction in mice infected with Plasmodium berghei. Using 42 male mice divided into six groups, including a control group, infected groups without treatment, infected treated with 20 mg/kg bw chloroquine, and infected groups treated with different doses of Prosopis africana fraction at 6.25 mg/kg,12.5mg/kg and 25 mg/kg bw respectively. The mice were induced by NK-65 chloroquine sensitive Plasmodium berghei and administered oral treatment for four days. After the treatment period, biochemical analysis of blood samples and tissues was conducted, focusing on liver and kidney function markers, as well as oxidative stress indicators. Mice infected with Plasmodium berghei exhibited significant alterations in biochemical indices and oxidative balance compared to the control group. However, treatment with Prosopis africana fraction mitigated these changes, restoring biochemical parameters and reestablishing oxidative balance. Notably, the dose at 12.5 mg/kg bw of Prosopis africana fraction demonstrated superior efficacy in reversing the alterations induced by malaria infection. These findings suggest that Prosopis africana fraction holds therapeutic potential in mitigating the biochemical disruptions and oxidative stress associated with malaria infection.

Diagnostic challenges of recurrent malaria in non-endemic areas†.

In Spain, a patient’s unexpected relapse with Plasmodium falciparum raises questions about genetic influences on treatment efficacy. Meanwhile, in a separate case, a recurrence of Plasmodium malariae prompts speculation on latent reservoirs and treatment strategies. Here, we delve into these complex cases that underscore the evolving landscape of malaria.

Recombinant Full-length Plasmodium falciparum Circumsporozoite Protein-Based Vaccine Adjuvanted With Glucopyranosyl Lipid A-Liposome Quillaja saponaria 21: Results of Phase 1 Testing With Malaria Challenge.

Malaria is preventable yet causes >600 000 deaths annually. RTS,S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. We conducted an open-label, dose escalation phase 1 study of a full-length recombinant circumsporozoite protein vaccine (rCSP) administered with adjuvant glucopyranosyl lipid A-liposome Quillaja saponaria 21 formulation (GLA-LSQ) on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naive adults. The primary end points were safety and reactogenicity. The secondary end points were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection. Participants were enrolled into 4 groups receiving rCSP/GLA-LSQ: 10 µg × 3 (n = 20), 30 µg × 3 (n = 10), 60 µg × 3 (n = 10), or 60 µg × 2 (n = 9); 10 participants received 30 µg rCSP alone × 3, and there were 6 infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent controlled human malaria infection 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher immunoglobulin G titers but did not achieve previously established RTS,S benchmarks. rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess whether adjuvant or schedule adjustments improve efficacy. NCT03589794.

Syndemic Challenges of Malaria and COVID-19 and Reported Coinfection of Both: Experience from a Tertiary Care Center

Syndemic Challenges of Malaria and COVID-19 and Reported Coinfection of Both: Experience from a Tertiary Care Center

Impact of Malaria on Hematocrit Levels in HIV-Infected Individuals: A Comprehensive Review

His comprehensive review explores the intricate relationship between malaria infection and hematocrit levels in individuals co-infected with HIV. Malaria and HIV, both prevalent in resource-limited regions, exhibit a synergistic impact on the immune system, leading to exacerbated anemia and subsequent reductions in hematocrit levels. The interplay between these infections involves complex immune responses, compromising the ability to combat anemia effectively. Accurate diagnosis presents challenges due to overlapping symptoms, necessitating integrated diagnostic approaches. Treatment strategies, including antiretroviral therapy and antimalarial drugs, must be carefully chosen to avoid adverse interactions. Preventive measures, such as malaria prevention methods and nutritional interventions, play a crucial role in mitigating the risk of co-infection and anemia. The review emphasizes the need for a multidimensional approach in both diagnosis and treatment to enhance the overall well-being of individuals facing the complex challenge of malaria and HIV co-infection.

Epidemiology of malaria in saravan city and its suburbs from 2018 to 2023, Southeast Iran.

Malaria, transmitted by Plasmodium parasites and anopheline mosquitoes, continues to be a leading cause of global disease and death. This retrospective investigation from 2018 to 2023 examines the epidemiological attributes of malaria in Saravan, southeastern Iran. It seeks to evaluate the prevalence, transmission causes, local population impact, and health system effects. Blood samples from suspected malaria cases in Saravan health centers were collected for this analysis. Each positive case was detailed with demographic data in a questionnaire. The SPSS 26 statistical program scrutinized data with t-tests comparing the variables. The study indicated fluctuating malaria cases peaking in 2023, with an annual parasite incidence. (API) of 17.27. Plasmodium vivax was the predominant species (P < 0.001), with the majority of cases in individuals over 15, notably males. A significant number of cases were reported in September (20.7%). The findings emphasize the persistent malaria challenges in Saravan, accentuating the urgent need to strengthen prevention and control strategies. Reducing disease burden demands focused approaches, including improving prevention and treatment programs, enhancing surveillance systems, developing health infrastructures, and implementing localized therapies, especially considering recent climatic and rainfall patterns.

Perspectives of African stakeholders on gene drives for malaria control and elimination: a multi-country survey

BackgroundGene drive modified mosquitoes (GDMMs) have the potential to address Africa’s persistent malaria problem, but are still in early stages of development and testing. Continuous engagement of African stakeholders is crucial for successful evaluation and implementation of these technologies. The aim of this multi-country study was, therefore, to explore the insights and recommendations of key stakeholders across Africa on the potential of GDMMs for malaria control and elimination in the continent.MethodsA concurrent mixed-methods study design was used, involving a structured survey administered to 180 stakeholders in 25 countries in sub-Saharan Africa, followed by 18 in-depth discussions with selected groups and individuals. Stakeholders were drawn from academia, research and regulatory institutions, government ministries of health and environment, media and advocacy groups. Thematic content analysis was used to identify key topics from the in-depth discussions, and descriptive analysis was done to summarize information from the survey data.ResultsDespite high levels of awareness of GDMMs among the stakeholders (76.7%), there was a relatively low-level of understanding of their key attributes and potential for malaria control (28.3%). When more information about GDMMs was provided to the stakeholders, they readily discussed their insights and concerns, and offered several recommendations to ensure successful research and implementation of the technology. These included: (i) increasing relevant technical expertise within Africa, (ii) generating local evidence on safety, applicability, and effectiveness of GDMMs, and (iii) developing country-specific regulations for safe and effective governance of GDMMs. A majority of the respondents (92.9%) stated that they would support field trials or implementation of GDMMs in their respective countries. This study also identified significant misconceptions regarding the phase of GDMM testing in Africa, as several participants incorrectly asserted that GDMMs were already present in Africa, either within laboratories or released into the field.ConclusionIncorporating views and recommendations of African stakeholders in the ongoing research and development of GDMMs is crucial for instilling stakeholder confidence on their potential application. These findings will enable improved planning for GDMMs in Africa as well as improved target product profiles for the technologies to maximize their potential for solving Africa’s enduring malaria challenge.

China’s hidden role in malaria control and elimination in Africa

BackgroundInsufficient funding is hindering the achievement of malaria elimination targets in Africa, despite the pressing need for increased investment in malaria control. While Western donors attribute their inaction to financial...

Anopheles mosquito diversity, entomological indicators of malaria transmission and challenges of morphological identification in southwestern Ethiopia

BackgroundA number of Anopheles species play either a primary or secondary role in malaria transmission. This necessitates understanding the species composition, bionomics, and behaviors of malaria mosquitoes in a particular geographic area, which is relevant to design and implement tailored intervention tools. This study aimed to assess the species composition, sporozoite infection rate, and blood meal origins of malaria mosquitoes in two malaria-endemic villages of Boreda district in Gamo Zone, southwest Ethiopia. MethodsThirty houses, 20 for Center for Disease Control and Prevention (CDC) light traps and 10 for Pyrethrum Spray Catches (PSC) were randomly selected for bimonthly mosquito collection from October 2019 to February 2020. An enzyme-linked immunosorbent assay (ELISA) was carried out to detect the blood meal origins and circumsporozoite proteins (CSPs). The entomological inoculation rate (EIR) was calculated by multiplying the sporozoite and human biting rates from PSCs. Anopheles gambiae complex and An. funestus group samples were further identified to species by the polymerase chain reaction (PCR). Anopheles species with some morphological similarity with An. gambiae complex or An. funestus group were tested using the primers of the two species complexes.ResultsA total of 14 Anopheles species were documented, of which An. demeilloni was found to be the dominant species. An. arabiensis was found to be positive for P. falciparum CSP with the overall CSP rate of 0.53% (1/190: 95% CI 0.01–2.9). The overall estimated P. falciparum EIR of An. arabiensis from PSC was 1.5 infectious bites/person/5 months. Of the 145 freshly fed Anopheles mosquitoes tested for blood meal sources, 57.9% (84/145) had bovine blood meal, 15.2% (22/145) had human blood meal origin alone, and 16.5% (24/145) had a mixed blood meal origin of human and bovine. Anopheles demeilloni were more likely to feed on blood meals of bovine origin (102/126 = 80.9%), while An. arabiensis were more likely to have blood meals of human origin. Eleven samples (2.6%; 11/420) were morphologically categorized as An. demeilloni, but it has been identified as An. leesoni (the only An. funestus group identified in the area) by PCR, though it requires additional verification by sequencing, because different species genes may have amplified for these species specific primers. Similarly, a small number of An. arabiensis were morphologically identified as An. salbaii, An. maculipalpis and An. fuscivenosus.Conclusions and recommendationsIn spite of the wide variety of Anopheles mosquito species, An. arabiensis dominates indoor malaria transmission, necessitating additional interventions targeting this species. In addition, increasing entomological knowledge may make morphological identification less difficult.

Distinct immune responses associated with vaccination status and protection outcomes after malaria challenge.

Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malarial infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with type I and II interferon and NK cell responses were increased in prior to CHMI while T and B cell signatures were decreased as early as one day following CHMI in protected vaccinees. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of vδ2+ γδ T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that vaccine-induced immune response is heterogenous between protected and non-protected vaccinees and that inducted-malaria protection by PfRAS is associated with early and rapid changes in interferon, NK cell and adaptive immune responses. Trial Registration: ClinicalTrials.gov NCT01994525.

Plasmablast Ig repertoire dynamics through repeat Plasmodium falciparum challenges

Abstract The development of natural immunity against malaria requires multiple exposures and even after protection develops against clinical disease individuals continue to be susceptible to infections. To study the dynamics of the immune response to repetitive P. falciparum (PF) infected mosquito exposures, we used controlled human malaria challenges with the same strain of PF. The homologous strain was used to eliminate differences in immune responses due to strain polymorphisms and focus on the maturation of the human immune response to repeat exposures. Consistent with the gradual immune response observed in the field, after 4 sequential exposures over the course of 2 years, symptoms decreased and the time to parasite detection on a blood smear increased significantly, but all the volunteers continued to be susceptible to infections. Anti-PF antibody levels increased over time and were able to inhibit liver cell invasion in an in vitro assay. The Ig repertoire of plasmablasts collected 7 days after peak parasitemia demonstrated large clonal expansions in one or two sequential infections that decreased after subsequent exposures. This pattern suggests a marked difference in the lineage of plasmablast and memory cells populations. Naïve B cells stimulated by PF to proliferate and differentiate into plasmablasts may fail to differentiate into memory cells that can produce both plasmablasts and memory cells following subsequent exposures. This differentiation pattern would lead to new sets of expanded clones at each PF exposure, instead of further maturation of the same antibodies generated during the first response. Such a response could enhance the diversity of the Ig pool, but would delay the generation of a strong, durable memory response U0110852

Current Status and Challenges of Malaria in Zambia

Malaria is a mosquito-borne infection caused by the Plasmodium parasite, which infects a person carrying the parasite through mosquito bites or blood transfusions. It is the world's leading parasitic disease. There are four species of Plasmodium in the human body, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. More than 100 countries and regions are reported to be affected by malaria to varying degrees. Malaria has a high incidence in Zambia, which seriously endangers people's life and health, and increases the social and economic burden of the affected communities to a certain extent. In addition, the number of malaria deaths remains high, the number of malaria cases has even risen, and the eradication of malaria has stagnated. Therefore, timely and accurate diagnosis is of great significance to effectively prevent malaria, control its spread, and give patients early treatment. This article describes the current progress and challenges of malaria elimination in Zambia, and Outlines current malaria elimination strategies and key interventions.

Imported malaria from land bordering countries in China: A challenge in preventing the reestablishment of malaria transmission

ObjectiveNo indigenous malaria cases have been reported since 2017 in China, but a large number of imported cases are still reported every year, including those from the land bordering countries. To characterize their epidemiological profiles will provide evidence for the development of appropriate strategies to effectively address the challenges of border malaria in the post-elimination phase. MethodsIndividual-level data of imported malaria cases from the land bordering countries were collected from 2017 to 2021 in China via the web-based surveillance systems, and analyzed by SPSS, ArcGIS and WPS software, to explore their epidemiological profiles. ResultsA total of 1170 malaria cases imported into China from six of the fourteen land bordering countries were reported between 2017 and 2021 with a decline trend. Overall, cases were widely distributed in 31–97 counties from 11 to 21 provinces but mainly in Yunnan. Moreover, these imported cases were mainly infected with P. vivax (94.8%), and a total of 68 recurrent cases were reported in 6–14 counties from 4 to 8 provinces. In addition, nearly 57.1% of the total reported cases could seek healthcare within 2 days of getting sick, and 71.3% of the reported cases could be confirmed as malaria on the day they sought medical care. ConclusionsChina still needs to attach great importance to the risk and challenge of the imported malaria from bordering countries particularly from Myanmar in preventing reestablishment of malaria transmission in the post-elimination phase. It is necessary not only to strengthen collaboration and cooperation with the bordering countries, but also coordinate multiple departments at home to improve malaria surveillance and response system and prevent the reestablishment of malaria transmission in China.

In vitro activity of rhinacanthin analogues against drug resistant Plasmodium falciparum isolates from Northeast Thailand

BackgroundNew anti-malarial drugs are needed urgently to address the increasing challenges of drug-resistant falciparum malaria. Two rhinacanthin analogues containing a naphthoquinone moiety resembling atovaquone showed promising in-vitro activity against a P. falciparum laboratory reference strain (K1). The anti-malarial activity of these 2 compounds was further evaluated for P. falciparum field isolates from an area of multi-drug resistance in Northeast Thailand.MethodsUsing a pLDH enzyme-linked immunosorbent assay, four P. falciparum isolates from Northeast Thailand in 2018 were tested for in vitro sensitivity to the two synthetic rhinacanthin analogues 1 and 2 as well as established anti-malarials. Mutations in the P. falciparum cytochrome b gene, a marker for atovaquone (ATQ) resistance, were genotyped in all four field isolates as well as 100 other clinical isolates from the same area using PCR-artificial Restriction Fragment Length Polymorphisms. Pfkelch13 mutations, a marker for artemisinin (ART) resistance, were also examined in all isolates.ResultsThe 50% inhibitory concentrations (IC50) of P. falciparum field isolates for rhinacanthin analogue 1 was 321.9–791.1 nM (median = 403.1 nM). Parasites were more sensitive to analogue 2: IC50 48.6–63.3 nM (median = 52.2 nM). Similar results were obtained against P. falciparum reference laboratory strains 3D7 and W2. The ART-resistant IPC-5202 laboratory strain was more sensitive to these compounds with a median IC50 45.9 and 3.3 nM for rhinacanthin analogues 1 and 2, respectively. The ATQ-resistant C2B laboratory strain showed high-grade resistance towards both compounds (IC50 > 15,000 nM), and there was a strong positive correlation between the IC50 values for these compounds and ATQ (r = 0.83–0.97, P < 0.001). There were no P. falciparum cytochrome b mutations observed in the field isolates, indicating that P. falciparum isolates from this area remained ATQ-sensitive. Pfkelch13 mutations and the ring-stage survival assay confirmed that most isolates were resistant to ART.ConclusionsTwo rhinacanthin analogues showed parasiticidal activity against multi-drug resistant P. falciparum isolates, although less potent than ATQ. Rhinacanthin analogue 2 was more potent than analogue 1, and can be a lead compound for further optimization as an anti-malarial in areas with multidrug resistance.