Anti-plasmodial Effect of C. limon and C. paradisi extracts on Plasmodium berghei-infected mice

Antiplasmodial effect of Citrus limon and Citrus paradisi extracts on Plasmodium berghei-infected mice was studied. Twenty five albino mice were randomized into five categories of G, L, GL, ACT (positive control) and NC which stand for grape, lemon, grape and lemon combined extracts, artemisinin combined therapy and negative control respectively. The NC group did not receive any intervention. Other treatments were administered orally for 12 days whereas administration of ACT lasted for 3 days. Blood was collected from the tail vein of the mice at a three day interval through venipuncture. Thick blood films were prepared and parasite densities were estimated using standard parasitological techniques. Results were analysed with ANOVA and Duncan multiple range tests. There was no significant difference (p>0.05) between parasite densities of the treatment groups and the negative control at baseline levels. However, as the treatment progressed from day 3 through day 9, there were significant reductions (p<0.05) in parasite densities among treatment groups when compared to the negative control. In this study, extracts of C. limon and C. paradisi in both single and combined strengths have been found to have antiplasmodial properties in mice. ACT possessed the highest antiplasmodial effect while C. limon as a single treatment ranked second in possession of antiplasmodial activity but exhibited increased RBC lysis. In combination, C. limoni and C. paradise extracts showed antiplasmodial activity that is slightly less than that exhibited by the lemon extract alone, but maintained normal RBC morphology whereas C. paradisi extract alone exhibited the lowest level of parasite clearance with atrophied red blood cells. Investigation of the effects of the extracts on liver, kidney and gastrointestinal tissues of mice is recommended before they could be prescribed as antimalaria for other animals and humans.

Artemether–lumefantrine and dihydroartemisinin–piperaquine treatment outcomes among children infected with uncomplicated Plasmodium falciparum malaria in Mwanza, Tanzania

Background Artemisinin based combination therapies (ACTs) have been a cornerstone in the treatment of malaria in the world. A rapid decline in dihydroartemisinin piperaquine (DHP) and artemether lumefantrine (ALU) efficacies has been reported in some parts of South East Asia, the historical epicenter for the antimalarial drug resistance. Prolonged drug use is associated with selection of resistant parasites due to exposure to inadequate drug levels hence effects on treatment outcomes in malaria. ALU and DHP are used as first line and alternative first line, respectively, in Tanzania. This study was carried in Igombe, Tanzania to assess the efficacies of ALU and DHP in routine treatment of uncomplicated malaria among children. Methods This was a prospective study involving children up to 10 years and followed up for 28 and 35 days as per the WHO protocol, 2015 for monitoring antimalarial drug efficacy. The primary end points were crude and adjusted Adequate Clinical and Parasitological Response (ACPR), parasite clearance rate and reported adverse events. Results A total of 205 children with uncomplicated malaria were enrolled. One hundred and sixteen participants were treated with ALU, while 89 participants were treated with DHP. Two participants in the ALU group were lost within the 24 h of follow-up. The PCR unadjusted ACPR was108 (94.7%) for ALU and 88 (98.9%) for DHP, while the PCR adjusted ACPR was 109(95.6%) and 88(98.9%) for ALU and DHP, respectively, at 28 day follow-up. No treatment failure was observed in both groups. Cumulative risk of recurrent parasitemia was similar in both groups (p = 0.32). Age and parasite density were strong predictors for persistent day 1 parasitemia (p = 0.034 and 0.026, respectively). Nausea and vomiting, abdominal pain and headache were the most clinical adverse events reported in both groups of patients. Conclusion The present study shows that ALU and DHP are still efficacious after more than a decade of use with PCR corrected efficacies greater than 95% implying a failure rate less than 5% which is below the WHO minimum threshold requirement for recommendation of a change in the treatment policy. Both drugs were well tolerated with no major adverse events reported.

Delayed Parasite Clearance Rates and Low Clinical and Parasitological Responses Following Treatment of Uncomplicated Falciparum Malaria With Artemether-lumefantrine in Ethiopian- Sudan Border, Western Ethiopia

BackgroundThe appearance of artemisinin-resistant parasites in Africa is catastrophic as many cases and deaths from malaria are usually reported in the continent. This study was the first to evaluate the status of artemether-lumefantrine (AL) efficacy, seventeen years after the introduction of this drug in the study area. This study aimed to assess PCR- corrected clinical and parasitological responses at 28 days following AL treatment.MethodsSixty uncomplicated falciparum malaria patients were enrolled, treated with standard doses of AL, and monitored for 28 days with clinical and parasitological assessments from September 15 to December 15, 2020. Molecular analysis was done on dried blood spots collected from each patient from day 0 and on follow-up days 1, 2, 3, 7, 14, 21, and 28. Descriptive statistics and binary logistic regressions were done using SPSS 20.0 statistical software. A p-value of less or equal to 0.05 was considered significant.ResultsSixty patients enrolled in the study, ten were lost to follow-up; and the results were analyzed for 50 patients. All of the patients were fever-free on day 3. But, two patients febrile on day 1 became febrile on day 7 of the follow-up period. Day 3 parasites positivity rate was zero and 60 % using microscopy and PCR, respectively. Parasite clearance on day 3 was more among patients ≥ 15 years as compared with < 15 years (AOR= 6.71, P=0.021). 14 of 50 patients tested were parasite positive on day 7 by PCR. In addition, 7 patients had persisted parasitemia by PCR from day 0 to day 28 of the follow-up. The PCR-corrected adequate clinical and parasitological response (ACPR) rate was 59.2 %. PCR adjusted AL treatment failures was 40.8% and it was noted as 0 early treatment failure, 10.2% late clinical failures, and 30.6% late parasitological failures. Recrudescence and new infection rate within 28 days was7/50(14%) and1/50 (2%) respectively.Conclusionartemisinin resistance is suspected in the study area as parasite positivity rate on day 3 was greater than 10%. A slow parasitological clearance was followed by low-density parasitemia persistence and recrudescences. AL treatment failure was greater than the 10% threshold for treatments in use as per WHO. These findings suggest probably a decreased sensitivity of the parasite for the artemisinin component and the partner drug and highlight the need for prompt reassessment of the efficacy of AL and other factors that may contribute to low parasite susceptibility to the drug.

E-NTPDases: Possible Roles on Host-Parasite Interactions and Therapeutic Opportunities

Belonging to the GDA1/CD39 protein superfamily, nucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP) and inorganic phosphate (Pi). Several NTPDase isoforms have been described in different cells, from pathogenic organisms to animals and plants. Biochemical characterization of nucleotidases/NTPDases has revealed the existence of isoforms with different specificities regarding divalent cations (such as calcium and magnesium) and substrates. In mammals, NTPDases have been implicated in the regulation of thrombosis and inflammation. In parasites, such as Trichomonas vaginalis, Trypanosoma spp., Leishmania spp., Schistosoma spp. and Toxoplasma gondii, NTPDases were found on the surface of the cell, and important processes like growth, infectivity, and virulence seem to depend on their activity. For instance, experimental evidence has indicated that parasite NTPDases can regulate the levels of ATP and Adenosine (Ado) of the host cell, leading to the modulation of the host immune response. In this work, we provide a comprehensive review showing the involvement of the nucleotidases/NTPDases in parasites infectivity and virulence, and how inhibition of NTPDases contributes to parasite clearance and the development of new antiparasitic drugs.

Plasmodium falciparum clearance time in Malawian children with cerebral malaria: a retrospective cohort study

Background Standard treatment for both uncomplicated and severe malaria is artemisinin derivatives. Delayed parasite clearance times preceded the appearance of artemisinin treatment failures in Southeast Asia. Most worldwide malaria cases are in sub-Saharan Africa (SSA), where clinically significant artemisinin resistance or treatment failure has not yet been detected. The recent emergence of a resistance-conferring genetic mutation in the Plasmodium falciparum parasite in Africa warrants continued monitoring throughout the continent. Methods An analysis was performed on data from a retrospective cohort study of Malawian children with cerebral malaria admitted between 2010 and 2019 to a public referral hospital, ascertaining parasite clearance times across years. Data were collected from patients treated for severe malaria with quinine or artesunate, an artemisinin derivative. Parasite density was determined at admission and every subsequent 6 h until parasitaemia was below 1000 parasites/µl.The mean parasite clearance time in all children admitted in any one year was compared to the parasite clearance time in 2014, the first year of artesunate use in Malawi. Results The median population parasite clearance time was slower from 2010 to 2013 (quinine-treated patients) compared to 2014, the first year of artesunate use in Malawi (30 h (95% CI: 30–30) vs 18 h (95% CI: 18–24)). After adjustment for admission parasite count, there was no statistically significant difference in the median population parasite clearance time when comparing 2014 with any subsequent year. Conclusion Malaria parasite clearance times in Malawian children with cerebral malaria remained constant between 2014 and 2019, arguing against evolving artemisinin resistance in parasites in this region.

Linking functional and molecular mechanisms of host resilience to malaria infection

It remains challenging to understand why some hosts suffer severe illnesses, while others are unscathed by the same infection. We fitted a mathematical model to longitudinal measurements of parasite and red blood cell density in murine hosts from diverse genetic backgrounds to identify aspects of within-host interactions that explain variation in host resilience and survival during acute malaria infection. Among eight mouse strains that collectively span 90% of the common genetic diversity of laboratory mice, we found that high host mortality was associated with either weak parasite clearance, or a strong, yet imprecise response that inadvertently removes uninfected cells in excess. Subsequent cross-sectional cytokine assays revealed that the two distinct functional mechanisms of poor survival were underpinned by low expression of either pro- or anti-inflammatory cytokines, respectively. By combining mathematical modelling and molecular immunology assays, our study uncovered proximate mechanisms of diverse infection outcomes across multiple host strains and biological scales.

Mutation in Plasmodium falciparum BTB/POZ domain of K13 protein confers artemisinin resistance

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 ( pfk13 ) gene. Here, we carried out in vitro selection over a one-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA 0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

Pyronaridine-Artesunate (Pyramax®) for the Treatment of Artemisinin- and Piperaquine-Resistant Plasmodium falciparum in the Central Highlands of Vietnam

The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine in Vietnam justifies the need to evaluate alternative artemisinin-based combination therapies. Between July 2018 and October 2019, a single-arm trial of pyronaridine-artesunate (Pyramax, PA) was conducted in Dak Nong province, Vietnam. PA (3-day course) was administered to adults and children infected with P. falciparum . PA was well tolerated by the participants. The proportion of patients with Day 42 PCR-corrected adequate clinical and parasitological response was 95.2% (95% confidence interval [CI], 82.3 to 98.8, n = 40/42) for treating falciparum malaria. The median parasite clearance half-life was 6.7 h (range, 2.6 to 11.9) and the median parasite clearance time was 72 h (range, 12 to 132) with 44.9% (22/49) of patients having positive blood films at 72 h. The two patients that recrudesced had comparable Day 7 blood pyronaridine concentrations (39.5 and 39.0 ng/ml) to the 40 patients who did not recrudesce (median 43.4 ng/ml, 95% CI, 35.1 to 54.9). Ring-stage and piperaquine survival assays revealed that of the 29 P. falciparum isolates collected from the patients before PA treatment, 22 (75.9%) had reduced susceptibility to artemisinins and 17 (58.6%) were resistant to piperaquine. Genotyping confirmed that 92.0% (46/50) of falciparum patients were infected with parasites bearing the Pfkelch13 C580Y mutation associated with artemisinin resistance. Of these, 56.0% (28/50) of the isolates also had multiple copies of the plasmepsin 2/3 genes responsible for piperaquine resistance. Overall, PA was effective in treating P. falciparum in the Central Highlands of Vietnam.

Biomarkers of cellular aging during a controlled human malaria infection

Cellular aging is difficult to study in individuals with natural infection, given the diversity of symptom duration and clinical presentation, and the high interference of aging-related processes with host and environmental factors. To address this challenge, we took advantage of the controlled human malaria infection (CHMI) model. This approach allowed us to characterize the relationship among cellular aging markers prior, during and post malaria pathophysiology in humans, controlling for infection dose, individual heterogeneity, previous exposure and co-infections. We demonstrate that already low levels of Plasmodium falciparum impact cellular aging by inducing high levels of inflammation and redox-imbalance; and that cellular senescence reversed after treatment and parasite clearance. This study provides insights into the complex relationship of telomere length, cellular senescence, telomerase expression and aging-related processes during a single malaria infection.

Dynamics of G6PD activity in patients receiving weekly primaquine for therapy of Plasmodium vivax malaria

Background Acute Plasmodium vivax malaria is associated with haemolysis, bone marrow suppression, reticulocytopenia, and post-treatment reticulocytosis leading to haemoglobin recovery. Little is known how malaria affects glucose-6-phosphate dehydrogenase (G6PD) activity and whether changes in activity when patients present may lead qualitative tests, like the fluorescent spot test (FST), to misdiagnose G6PD deficient (G6PDd) patients as G6PD normal (G6PDn). Giving primaquine or tafenoquine to such patients could result in severe haemolysis. Methods We investigated the G6PD genotype, G6PD enzyme activity over time and the baseline FST phenotype in Cambodians with acute P. vivax malaria treated with 3-day dihydroartemisinin piperaquine and weekly primaquine, 0·75 mg/kg x8 doses. Results Of 75 recruited patients (males 63), aged 5–63 years (median 24), 15 were G6PDd males (14 Viangchan, 1 Canton), 3 were G6PD Viangchan heterozygous females, and 57 were G6PDn; 6 patients had α/β-thalassaemia and 26 had HbE. Median (range) Day0 G6PD activities were 0·85 U/g Hb (0·10–1·36) and 11·4 U/g Hb (6·67–16·78) in G6PDd and G6PDn patients, respectively, rising significantly to 1·45 (0·36–5·54, p<0.01) and 12·0 (8·1–17·4, p = 0.04) U/g Hb on Day7, then falling to ~Day0 values by Day56. Day0 G6PD activity did not correlate (p = 0.28) with the Day0 reticulocyte counts but both correlated over time. The FST diagnosed correctly 17/18 G6PDd patients, misclassifying one heterozygous female as G6PDn. Conclusions In Cambodia, acute P. vivax malaria did not elevate G6PD activities in our small sample of G6PDd patients to levels that would result in a false normal qualitative test. Low G6PDd enzyme activity at disease presentation increases upon parasite clearance, parallel to reticulocytosis. More work is needed in G6PDd heterozygous females to ascertain the effect of P. vivax on their G6PD activities. Trial registration The trial was registered (ACTRN12613000003774) with the Australia New Zealand Clinical trials (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=363399&isReview=true).