From multisystem inflammatory syndrome in children to secondary hemophagocytic lymphohistiocytosis a series of misfortunate events: case report and review of literature.

Multisystem inflammatory syndrome of childhood (MIS-C) is a recently described entity in pediatrics post-COVID-19 pandemic. Hemophagocytic lymphohistiocytosis (HLH) is a clinical syndrome caused by an unregulated proliferation of macrophages as well as T lymphocytes. Both entities can be considered overlapping, although distinct criteria for each can be found in the literature. Herein, we report a patient with MIS-C post-COVID-19 infection, complicated with HLH secondary to Plasmodium falciparum malaria from a blood transfusion.

How to manage adult patients with malaria in the non-endemic setting.

BACKGROUND: The diagnosis and management of malaria in non-endemic countries presents a continuing challenge. Plasmodium falciparum, which is capable to rapidly induce severe and life-threatening multiorgan disease, is the species most frequently diagnosed in Europe and North America. OBJECTIVES: To summarise the more relevant diagnostic findings and clinical features of malaria observed in non-endemic settings and to provide an up-date of the key management decision points using three illustrative clinical scenarios of uncomplicated and severe malaria. SOURCES: The discussion is based on relevant literature search spanning the last twenty years. Recommendations are based on available clinical guidelines including those of the World Health Organization (WHO), on observational studies conducted in non-endemic settings and, when available, with extrapolation from randomised studies from malaria endemic settings. CONTENT: The following topics are covered: diagnosis, including the use of molecular biology; clinical characteristics; management with specific focus on complicated (severe) and uncomplicated malaria and on areas of resistance to available antimalarial drugs. IMPLICATIONS: Malaria imported to non-endemic settings, especially Plasmodium falciparum malaria, is sometimes initially overlooked and the delayed diagnosis is responsible every year of preventable deaths. This review aims to raise awareness of malaria outside endemic countries and to provide clinicians with a practical guide for efficient diagnosis and targeted therapy to the different species involved.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and 3D Focused Ion Beam-Scanning Electron Microscopy, we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or break-down revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

Genetic analysis of the circumsporozoite gene in Plasmodium falciparum isolates from Cameroon: Implications for efficacy and deployment of RTS,S/AS01 vaccine.

Current understanding of genetic polymorphisms and natural selection in Plasmodium falciparum circumsporozoite (PfCSP), the leading malaria vaccine, is crucial for the development of next-generation vaccines, and such data is lacking in Africa. Blood samples were collected among Plasmodium-infected individuals living in four Cameroonian areas (Douala, Maroua, Mayo-Oulo, Pette). DNA samples were amplified using nested PCR protocols, sequenced, and BLASTed. Single nucleotide polymorphisms (SNPs) were analysed in each PfCSP region, and their impact on PfCSP function/structure was predicted in silico. The N-terminal region showed a limited polymorphism with four haplotypes, and three novel SNPs (N68Y, R87W, K93E) were found. Thirty-five haplotypes were identified in the central region, with several variants (e.g., NVNP and KANP). The C-terminal region was also highly diverse, with 25 haplotypes and eight novel SNPs (N290D, N308I, S312G, K317A, V344I, D356E, E357L, D359Y). Most polymorphic codon sites were mainly observed in the Th2R subregion in isolates from Douala and Pette. The codon site 321 was under episodic positive selection. One novel (E357L) and three known (K322I, G349D, D359Y) SNPs show an impact on function/structure. This study showed extensive genetic diversity with geographical patterns and evidence of the selection of Cameroonian PfCSP central and C-terminal regions.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain.

BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

DNA N-glycosylases Ogg1 and EndoIII as components of base excision repair in Plasmodium falciparum organelles.

The integrity of genomes of the two crucial organelles of the malaria parasite - an apicoplast and mitochondrion in each cell - must be maintained by DNA repair mediated by proteins targeted to these compartments. We explored the localisation and function of Plasmodium falciparum base excision repair (BER) DNA N-glycosylase homologs PfEndoIII and PfOgg1. These N-glycosylases would putatively recognise DNA lesions prior to the action of apurinic/apyrimidinic (AP)-endonucleases. Both Ape1 and Apn1 endonucleases have earlier been shown to function solely in the parasite mitochondrion. Immunofluorescence localisation showed that PfEndoIII was exclusively mitochondrial. PfOgg1 was not seen clearly in mitochondria when expressed as a PfOgg1leader-GFP fusion, although chromatin immunoprecipitation assays showed that it could interact with both mitochondrial and apicoplast DNA. Recombinant PfEndoIII functioned as a DNA N-glycosylase as well as an AP-lyase on thymine glycol (Tg) lesions. We further studied the importance of Ogg1 in the malaria life cycle using reverse genetic approaches in Plasmodium berghei. Targeted disruption of PbOgg1 resulted in loss of 8-oxo-G specific DNA glycosylase/lyase activity. PbOgg1 knockout did not affect blood, mosquito or liver stage development but caused reduced blood stage infection after inoculation of sporozoites in mice. A significant reduction in erythrocyte infectivity by PbOgg1 knockout hepatic merozoites was also observed, thus showing that PbOgg1 ensures smooth transition from liver to blood stage infection. Our results strengthen the view that the Plasmodium mitochondrial genome is an important site for DNA repair by the BER pathway.

Infectivity of Plasmodium parasites to Aedes aegypti and Anopheles stephensi mosquitoes maintained on blood-free meals of SkitoSnack.

BACKGROUND: Aedes and Anopheles mosquitoes are responsible for tremendous global health burdens from their transmission of pathogens causing malaria, lymphatic filariasis, dengue, and yellow fever. Innovative vector control strategies will help to reduce the prevalence of these diseases. Mass rearing of mosquitoes for research and support of these strategies presently depends on meals of vertebrate blood, which is subject to acquisition, handling, and storage issues. Various blood-free replacements have been formulated for these mosquitoes, but none of these replacements are in wide use, and little is known about their potential impact on competence of the mosquitoes for Plasmodium infection. METHODS: Colonies of Aedes aegypti and Anopheles stephensi were continuously maintained on a blood-free replacement (SkitoSnack; SS) or bovine blood (BB) and monitored for engorgement and hatch rates. Infections of Ae. aegypti and An. stephensi were assessed with Plasmodium gallinaceum and P. falciparum, respectively. RESULTS: Replicate colonies of mosquitoes were maintained on BB or SS for 10 generations of Ae. aegypti and more than 63 generations of An. stephensi. The odds of engorgement by SS- relative to BB-maintained mosquitoes were higher for both Ae. aegypti (OR = 2.6, 95% CI 1.3-5.2) and An. stephensi (OR 2.7, 95% CI 1.4-5.5), while lower odds of hatching were found for eggs from the SS-maintained mosquitoes of both species (Ae. aegypti OR = 0.40, 95% CI 0.26-0.62; An. stephensi OR = 0.59, 95% CI 0.36-0.96). Oocyst counts were similar for P. gallinaceum infections of Ae. aegypti mosquitoes maintained on SS or BB (mean ratio = [mean on SS]/[mean on BB] = 1.11, 95% CI 0.85-1.49). Similar oocyst counts were also observed from the P. falciparum infections of SS- or BB-maintained An. stephensi (mean ratio = 0.76, 95% CI 0.44-1.37). The average counts of sporozoites/mosquito showed no evidence of reductions in the SS-maintained relative to BB-maintained mosquitoes of both species. CONCLUSIONS: Aedes aegypti and An. stephensi can be reliably maintained on SS over multiple generations and are as competent for Plasmodium infection as mosquitoes maintained on BB. Use of SS alleviates the need to acquire and preserve blood for mosquito husbandry and may support new initiatives in fundamental and applied research, including novel manipulations of midgut microbiota and factors important to the mosquito life cycle and pathogen susceptibility.

Impact on pregnancy outcomes of intermittent preventive treatment with sulfadoxine-pyrimethamine in urban and peri-urban Papua New Guinea: a retrospective cohort study.

BACKGROUND: Intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) reduces malaria-attributable adverse pregnancy outcomes and may also prevent low birth weight (< 2,500 g) through mechanisms independent of malaria. Malaria transmission in Papua New Guinea (PNG) is highly heterogeneous. The impact of IPTp-SP on adverse birth outcomes in settings with little or no malaria transmission, such as PNG's capital city Port Moresby, is unknown. METHODS: A retrospective cohort study was conducted amongst HIV-negative women with a singleton pregnancy who delivered at Port Moresby General Hospital between 18 July and 21 August 2022. The impact of IPTp-SP doses on adverse birth outcomes and anaemia was assessed using logistic and linear regression models, as appropriate. RESULTS: Of 1,140 eligible women amongst 1,228 consecutive births, 1,110 had a live birth with a documented birth weight. A total of 156 women (13.7%) did not receive any IPTp-SP, 347 women (30.4%) received one, 333 (29.2%) received two, and 304 (26.7%) received the recommended ≥ 3 doses of IPTp-SP. A total of 65 of 1,110 liveborn babies (5.9%) had low birth weight and there were 34 perinatal deaths (3.0%). Anaemia (haemoglobin < 100 g/L) was observed in 30.6% (243/793) of women, and 14 (1.2%) had clinical malaria in pregnancy. Compared to women receiving 0-1 dose of IPTp-SP, women receiving ≥ 2 doses had lower odds of LBW (adjusted odds ratio [aOR] 0.50; 95% confidence interval [CI] 0.26, 0.96), preterm birth (aOR 0.58; 95% CI 0.32, 1.04), perinatal death (aOR 0.49; 95% CI 0.18, 1.38), LBW/perinatal death (aOR 0.55; 95% CI 0.27, 1.12), and anaemia (OR 0.50; 95% CI 0.36, 0.69). Women who received 2 doses versus 0-1 had 45% lower odds of LBW (aOR 0.55, 95% CI 0.27, 1.10), and a 16% further (total 61%) reduction with ≥ 3 doses (aOR 0.39, 95% CI 0.14, 1.05). Birth weights for women who received 2 or ≥ 3 doses versus 0-1 were 81 g (95% CI -3, 166) higher, and 151 g (58, 246) higher, respectively. CONCLUSIONS: Provision of IPTp-SP in a low malaria-transmission setting in PNG appears to translate into substantial health benefits, in a dose-response manner, supporting the strengthening IPTp-SP uptake across all transmission settings in PNG.

Detection of Duffy blood group genotypes and submicroscopic Plasmodium infections using molecular diagnostic assays in febrile malaria patients.

BACKGROUND: Malaria remains a severe parasitic disease, posing a significant threat to public health and hindering economic development in sub-Saharan Africa. Ethiopia, a malaria endemic country, is facing a resurgence of the disease with a steadily rising incidence. Conventional diagnostic methods, such as microscopy, have become less effective due to low parasite density, particularly among Duffy-negative human populations in Africa. To develop comprehensive control strategies, it is crucial to generate data on the distribution and clinical occurrence of Plasmodium vivax and Plasmodium falciparum infections in regions where the disease is prevalent. This study assessed Plasmodium infections and Duffy antigen genotypes in febrile patients in Ethiopia. METHODS: Three hundred febrile patients visiting four health facilities in Jimma town of southwestern Ethiopia were randomly selected during the malaria transmission season (Apr-Oct). Sociodemographic information was collected, and microscopic examination was performed for all study participants. Plasmodium species and parasitaemia as well as the Duffy genotype were assessed by quantitative polymerase chain reaction (qPCR) for all samples. Data were analysed using Fisher's exact test and kappa statistics. RESULTS: The Plasmodium infection rate by qPCR was 16% (48/300) among febrile patients, of which 19 (39.6%) were P. vivax, 25 (52.1%) were P. falciparum, and 4 (8.3%) were mixed (P. vivax and P. falciparum) infections. Among the 48 qPCR-positive samples, 39 (13%) were negative by microscopy. The results of bivariate logistic regression analysis showed that agriculture-related occupation, relapse and recurrence were significantly associated with Plasmodium infection (P < 0.001). Of the 300 febrile patients, 85 (28.3%) were Duffy negative, of whom two had P. vivax, six had P. falciparum, and one had mixed infections. Except for one patient with P. falciparum infection, Plasmodium infections in Duffy-negative individuals were all submicroscopic with low parasitaemia. CONCLUSIONS: The present study revealed a high prevalence of submicroscopic malaria infections. Plasmodium vivax infections in Duffy-negative individuals were not detected due to low parasitaemia. In this study, an improved molecular diagnostic tool was used to detect and characterize Plasmodium infections, with the goal of quantifying P. vivax infection in Duffy-negative individuals. Advanced molecular diagnostic techniques, such as multiplex real-time PCR, loop-mediated isothermal amplification (LAMP), and CRISPR-based diagnostic methods. These techniques offer increased sensitivity, specificity, and the ability to detect low-parasite-density infections compared to the employed methodologies.

Plasma metabolites in childhood Burkitt lymphoma cases and cancer-free controls in Uganda.

INTRODUCTION: Burkitt lymphoma (BL) is an aggressive non-Hodgkin lymphoma associated with Plasmodium falciparum and Epstein-Barr virus, both of which affect metabolic pathways. The metabolomic patterns of BL is unknown. MATERIALS AND METHODS: We measured 627 metabolites in pre-chemotherapy treatment plasma samples from 25 male children (6-11 years) with BL and 25 cancer-free area- and age-frequency-matched male controls from the Epidemiology of Burkitt Lymphoma in East African Children and Minors study in Uganda using liquid chromatography-tandem mass spectrometry. Unconditional, age-adjusted logistic regression analysis was used to estimate odds ratios (ORs) and their 95% confidence intervals (CIs) for the BL association with 1-standard deviation increase in the log-metabolite concentration, adjusting for multiple comparisons using false discovery rate (FDR) thresholds and Bonferroni correction. RESULTS: Compared to controls, levels for 42 metabolite concentrations differed in BL cases (FDR < 0.001), including triacylglyceride (18:0_38:6), alpha-aminobutyric acid (AABA), ceramide (d18:1/20:0), phosphatidylcholine ae C40:6 and phosphatidylcholine C38:6 as the top signals associated with BL (ORs = 6.9 to 14.7, P < 2.4✕10- 4). Two metabolites (triacylglyceride (18:0_38:6) and AABA) selected using stepwise logistic regression discriminated BL cases from controls with an area under the curve of 0.97 (95% CI: 0.94, 1.00). CONCLUSION: Our findings warrant further examination of plasma metabolites as potential biomarkers for BL risk/diagnosis.

Synthesis, design, and optimization of a potent and selective series of pyridylpiperazines as promising antimalarial agents.

An optimization of the pyridylpiperazine series against Plasmodium falciparum has been performed, exploring a structure-activity relationship carried out on the toluyl fragment of hit 1, a compound with low micromolar activity against Plasmodium falciparum discovered by high-throughput screening. After confirming the crucial role played by this aryl fragment in the antiplasmodial activity, the replacement of the ortho-methyl substituent of 1 by halogenated ones led to an improvement for four analogs, either in terms of potency, expected pharmacokinetics profile, or both. Further introduction of endocyclic nitrogens in this fragment identified two more optimized compounds, 20 and 23, which are expected to be much more metabolically stable than 1. Additional assessment of the cytotoxicity, Ligand Lipophilic Efficiency, potency against the chloroquine-resistant Dd2 strain and in silico ADMET predictions revealed a satisfactory profile for most compounds, ultimately identifying the four optimized compounds 7, 9, 20 and 23 as promising compounds for further lead optimization of this series against Plasmodium falciparum.

High prevalence and risk of malaria among asymptomatic individuals from villages with high prevalence of artemisinin partial resistance in Kyerwa district of Kagera region, north-western Tanzania.

BACKGROUND: Although Tanzania adopted and has been implementing effective interventions to control and eventually eliminate malaria, the disease is still a leading public health problem, and the country experiences heterogeneous transmission. Recent studies reported the emergence of parasites with artemisinin partial resistance (ART-R) in Kagera region with high prevalence (> 10.0%) in two districts of Karagwe and Kyerwa. This study assessed the prevalence and predictors/risk of malaria infections among asymptomatic individuals living in a hyperendemic area where ART-R has emerged in Kyerwa District of Kagera region, north-western Tanzania. METHODS: This was a community-based cross-sectional survey which was conducted in July and August 2023 and involved individuals aged ≥ 6 months from five villages in Kyerwa district. Demographic, anthropometric, clinical, parasitological, type of house inhabited and socio-economic status (SES) data were collected using electronic capture tools run on Open Data Kit (ODK) software. Predictors/risks of malaria infections were determined by univariate and multivariate logistic regression, and the results were presented as crude (cORs) and adjusted odds ratios (aORs), with 95% confidence intervals (CIs). RESULTS: Overall, 4454 individuals were tested using rapid diagnostic tests (RDTs), and 1979 (44.4%) had positive results. The prevalence of malaria infections ranged from 14.4% to 68.5% and varied significantly among the villages (p < 0.001). The prevalence and odds of infections were significantly higher in males (aOR = 1.28, 95% CI 1.08 -1.51, p = 0.003), school children (aged 5-≤10 years (aOR = 3.88, 95% CI 3.07-4.91, p < 0.001) and 10-≤15 years (aOR = 4.06, 95% CI 3.22-5.13, p < 0.001)) and among individuals who were not using bed nets (aOR = 1.22, 95% CI 1.03-1.46, p = 0.024). The odds of malaria infections were also higher in individuals with lower SES (aOR = 1.42, 95% CI 1.17-1.72, p < 0.001), and living in houses without windows (aOR = 2.08, 95% CI 1.46-2.96, p < 0.001), partially open (aOR = 1.33, 95% CI 1.11-1.58, p = 0.002) or fully open windows (aOR = 1.30, 95%CI 1.05-1.61, p = 0.015). CONCLUSION: The five villages had a high prevalence of malaria infections and heterogeneity at micro-geographic levels. Groups with higher odds of malaria infections included school children, males, and individuals with low SES, living in poorly constructed houses or non-bed net users. These are important baseline data from an area with high prevalence of parasites with ART-R and will be useful in planning interventions for these groups, and in future studies to monitor the trends and potential spread of such parasites, and in designing a response to ART-R.

Transcriptional responses of brain endothelium to Plasmodium falciparum patient-derived isolates in vitro.

A hallmark of cerebral malaria (CM) is sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature. Binding of IE to endothelium reduces microvascular flow and, combined with an inflammatory response, perturbs endothelial barrier function, resulting in breakdown of the blood-brain barrier (BBB). Cytoadherence leads to activation of the endothelium and alters a range of cell processes affecting signaling pathways, receptor expression, coagulation, and disruption of BBB integrity. Here, we investigated whether CM-derived parasites elicit differential effects on human brain microvascular endothelial cells (HBMECs), as compared to uncomplicated malaria (UM)-derived parasites. Patient-derived IE from UM and CM clinical cases, as well as non-binding skeleton-binding protein 1 knockout parasites, were overlaid onto tumour necrosis factor (TNF)-activated HBMECs. Gene expression analysis of endothelial responses was performed using probe-based assays of a panel of genes involved in inflammation, apoptosis, endothelial barrier function, and prostacyclin synthesis pathway. We observed a significant effect on endothelial transcriptional responses in the presence of IE, yet there was no significant correlation between HBMEC responses and type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and level of IE binding to HBMECs, as we detected the same change in endothelial responses when employing both binding and non-binding parasites. Our results suggest that interaction of IE with endothelial cells in this co-culture model induces some endothelial responses that are independent of clinical origin and independent of the expression of the major variant antigen Plasmodium falciparum erythrocyte membrane protein 1 on the IE surface. IMPORTANCE: Cerebral malaria (CM) is the most prevalent and deadly complication of severe Plasmodium falciparum infection. A hallmark of this disease is sequestration of P. falciparum-infected erythrocytes (IE) in brain microvasculature that ultimately results in breakdown of the blood-brain barrier. Here, we compared the effect of P. falciparum parasites derived from uncomplicated malaria (UM) and CM cases on the relative gene expression of human brain microvascular endothelial cells (HBMECs) for a panel of genes. We observed a significant effect on the endothelial transcriptional response in the presence of IE, yet there is no significant correlation between HBMEC responses and the type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and the level of IE binding to HBMECs. Our results suggest that interaction of IE with endothelial cells induces endothelial responses that are independent of clinical origin and not entirely driven by surface Plasmodium falciparum erythrocyte membrane protein 1 expression.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission. Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018. Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors. Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel.

Lack of selection of antimalarial drug resistance markers after intermittent preventive treatment of schoolchildren (IPTsc) against malaria in northeastern Tanzania.

OBJECTIVE: Intermittent Preventive Treatment of schoolchildren (IPTsc) is recommended by WHO as a strategy to protect against malaria; to explore whether IPTsc with dihydroartemisinin-piperaquine (DP) or artesunate-amodiaquine (ASAQ) cause a selection of molecular markers in Plasmodium falciparum genes associated with resistance in children in seven schools in Tanga region, Tanzania. METHODS: SNPs in P. falciparum genes Pfmdr1, Pfexo, Pfkelch13, and Pfcrt and copy number variations in Pfplasmepsin-2 and Pfmdr1 were assessed in samples collected at 12 months (visit 4, n=74) and 20 months (visit 6, n=364) after initiation of IPTsc and compared with the baseline prevalence (n=379). RESULTS: The prevalence of Pfmdr1 N86 and Pfexo 415G was >99% and 0%, respectively without any temporal differences observed. The prevalence of Pfmdr1 184F changed significantly from baseline (52.2%) to visit 6 (64.6%) (χ2=6.11, P=0.013), but no differences were observed between the treatment arms (χ2=0.05, P=0.98). Finally, only minor differences in the amplification of Pfmdr1 were observed; from 10.2% at baseline to 16.7% at visit 6 (χ2=0.98, P=0.32). CONCLUSIONS: The IPTsc strategy does not seem to pose a risk for the selection of markers associated with DP or ASAQ resistance. Continuously and timely surveillance of markers of antimalarial drug resistance is recommended.

A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

Phenotypic changes of γδ T cells in Plasmodium falciparum placental malaria and pregnancy outcomes in women at delivery in Cameroon.

Introduction: Depending on the microenvironment, γδ T cells may assume characteristics similar to those of Th1, Th2, Th17, regulatory T cells or antigen presenting cells. Despite the wide documentation of the effect of Th1/Th2 balance on pregnancy associated malaria and outcomes, there are no reports on the relationship between γδ T cell phenotype change and Placental Malaria (PM) with pregnancy outcomes. This study sought to investigate the involvement of γδ T cells and its subsets in placental Plasmodium falciparum malaria. Methods: In a case-control study conducted in Yaoundé, Cameroon from March 2022 to May 2023, peripheral, placental and cord blood samples were collected from 50 women at delivery (29 PM negative: PM- and 21 PM positive: PM+; as diagnosed by light microscopy). Hemoglobin levels were measured using hemoglobinometer. PBMCs, IVBMCs and CBMCs were isolated using histopaque-1077 and used to characterize total γδ T cell populations and subsets (Vδ1+, Vδ2+, Vδ1-Vδ2-) by flow cytometry. Results: Placental Plasmodium falciparum infection was associated with significant increase in the frequency of total γδ T cells in IVBMC and of the Vδ1+ subset in PBMC and IVBMC, but decreased frequency of the Vδ2+ subset in PBMC and IVBMC. The expression of the activation marker: HLA-DR, and the exhaustion markers (PD1 and TIM3) within total γδ T cells and subsets were significantly up-regulated in PM+ compared to PM- group. The frequency of total γδ T cells in IVBMC, TIM-3 expression within total γδ T cells and subsets in IVBMC, as well as HLA-DR expression within total γδ T cells and Vδ2+ subset in IVBMC were negatively associated with maternal hemoglobin levels. Furthermore, the frequency of total γδ T cells in PBMC and PD1 expression within the Vδ2+ subset in CBMC were negatively associated with birth weight contrary to the frequency of Vδ1-Vδ2- subset in PBMC and HLA-DR expression within the Vδ2+ subset in IVBMC which positively associated with maternal hemoglobin level and birth weight, respectively. Conclusion: The data indicate up-regulation of activated and exhausted γδ T cells in Plasmodium falciparum placental malaria, with effects on pregnancy outcomes including maternal hemoglobin level and birth weight.

The human malaria-Aotus monkey model: a historical perspective in antimalarial chemotherapy research at the Gorgas Memorial Laboratory-Panama.

The human malaria-Aotus monkey model has served the malaria research community since its inception in 1966 at the Gorgas Memorial Laboratory (GML) in Panama. Spanning over five decades, this model has been instrumental in evaluating the in vivo efficacy and pharmacokinetics of a wide array of candidate antimalarial drugs, whether used singly or in combination. The animal model could be infected with drug-resistant and susceptible Plasmodium falciparum and Plasmodium vivax strains that follow a characteristic and reproducible course of infection, remarkably like human untreated and treated infections. Over the years, the model has enabled the evaluation of several synthetic and semisynthetic endoperoxides, for instance, artelinic acid, artesunate, artemether, arteether, and artemisone. These compounds have been evaluated alone and in combination with long-acting partner drugs, commonly referred to as artemisinin-based combination therapies, which are recommended as first-line treatment against uncomplicated malaria. Further, the model has also supported the evaluation of the primaquine analog tafenoquine against blood stages of P. vivax, contributing to its progression to clinical trials and eventual approval. Besides, the P. falciparum/Aotus model at GML has also played a pivotal role in exploring the biology, immunology, and pathogenesis of malaria and in the characterization of drug-resistant P. falciparum and P. vivax strains. This minireview offers a historical overview of the most significant contributions made by the Panamanian owl monkey (Aotus lemurinus lemurinus) to malaria chemotherapy research.