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Introduction: Malaria remains a widespread health problem with a huge burden. Severe or complicated malaria is highly lethal and encompasses a variety of pathological processes, including immune activation, inflammation, and dysmetabolism. Previously, we showed that adrenal hormones, in particular glucocorticoids (GCs), play critical roles to maintain disease tolerance during Plasmodium infection in mice. Here, GC responses were studied in Cameroon in children with uncomplicated malaria (UM), severe malaria (SM) and asymptomatic controls (AC)., Methods: To determine the sensitivity of leukocytes to GC signaling on a transcriptional level, we measured the ex vivo induction of glucocorticoid induced leucine zipper (GILZ) and FK506-binding protein 5 (FKBP5) by GCs in human and murine leukocytes. Targeted tracer metabolomics on peripheral blood mononuclear cells (PBMCs) was performed to detect metabolic changes induced by GCs., Results: Total cortisol levels increased in patients with clinical malaria compared to AC and were higher in the SM versus UM group, while cortisol binding globulin levels were unchanged and adrenocorticotropic hormone (ACTH) levels were heterogeneous. Induction of both GILZ and FKBP5 by GCs was significantly reduced in patients with clinical malaria compared to AC and in malaria-infected mice compared to uninfected controls. Increased activity in the pentose phosphate pathway was found in the patients, but this was not affected by ex vivo stimulation with physiological levels of hydrocortisone. Interestingly, hydrocortisone induced increased levels of cAMP in AC, but not in clinical malaria patients., Discussion: Altogether, this study shows that patients with SM have increased cortisol levels, but also a decreased sensitivity to GCs, which may clearly contribute to the severity of disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vandermosten, Prenen, Fogang, Dagneau de Richecour, Knoops, Donkeu, Nguefack, Taguebue, Ndombo, Ghesquière, Ayong and Van den Steen.)

Tithonia diversifolia is widely used in African traditional medicine for the treatment of a large number of ailments and disorders, including malaria. In the present study, we evaluated the repellent activity of essential oils (EO) of this plant against Anopheles coluzzii, a major vector of malaria in Africa. Fresh leaves of T. diversifolia were used to extract EO, which were used to perform repellency assays in the laboratory and in the field using commercially available N,N-Diethyl-meta-toluamide (DEET) and Cymbopogon (C.) citratus EO as positive controls and vaseline as negative control. The repellency rates and durations of protection of the human volunteers involved were used as measures of repellent activity. Chemical composition of the T. diversifolia EO was established further by gas chromatography coupled with mass spectrometry. The moisture content and oil yield were 81% and 0.02% respectively. A total of 29 compounds in the T. diversifolia EO was identified, with D-limonene (20.1%), α-Copaene (10.3%) and o-Cymene (10.0%) as the most represented. In field studies, the mean time of protection against mosquito bites was significantly lower in T. diversifolia EO-treated volunteers compared to treatments with C. citratus EO (71 min versus 125 min, p = 0.04), but significantly higher when compared with the non-treated volunteers (71 min vs 0.5 min, p = 0.03). The same pattern was found in laboratory repellency assays against A. coluzzii. In contrast, repulsion rates were statistically similar between T. diversifolia EO and positive controls. In conclusion, the study suggests promising repellent potential of leaves of T. diversifolia EO against A. coluzzii., (© 2023. The Author(s).)

In pregnancy-associated malaria, chemokines such as CXCL-4, CXCL-13, CXCL-16, and CCL-24 play critical roles in leucocyte trafficking to tissue sites in the infected placenta where inflammatory reactions are active. However, how plasma levels of these chemokines associate with Plasmodium falciparum placental malaria and pregnancy outcomes remains not well understood. The present study analyzed the plasma levels of CXCL-4, CXCL-13, CXCL-16, and CCL-24 chemokines in matched peripheral, placental and cord blood in relation with placental malaria (PM), and with submicroscopic parasitaemia. This was a retrospective case-control study (1:3 ratio) involving samples from 134 women (34 PM+ and 100 PM-) enrolled at delivery at the Marie Reine Health Center in Yaoundé, Cameroon between June 2013 and October 2018. Samples were collected just after delivery and used to diagnose microscopic and submicroscopic Plasmodium falciparum infections. Submicroscopic infections were detected by reverse transcription LAMP whereas chemokine levels were determined by Magnetic Luminex Screening Assay. Overall, PM was associated with increased plasma levels of CXCL-13 and CXCL-16 and low levels of CXCL-4 and CCL-24 in both peripheral and placental blood (0.0002 ≤ p ≤ 0.042). Similarly, CCL-24 levels in peripheral and placental blood samples were significantly lower in submicroscopically infected women compared to healthy controls (p = 0.04 and 0.02, respectively). Maternal hemoglobin levels increased with peripheral plasma levels of CXCL-4 (p = 0.005), CXCL-16 (p = 0.03), and CCL-24 (p = 0.002) while birth weight was lower for babies born from women with high levels of peripheral CXCL-13 (p = 0.0006) and low levels of cord CXCL-4 and CCL-24 (p = 0.02 and 0.08, respectively). Together the data suggest that low levels of CXCL-4 and CCL-24 coupled with high plasma levels of CXCL-13 and for a lesser extend CXCL-16 represent signatures of PM in the study population. These findings are relevant for understanding the immunopathogenesis of PM and developing new therapeutic or preventive strategies against severe PM outcomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Megnekou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Malaria control relies on passive case detection, and this strategy fails detecting asymptomatic infections. In addition, infections in endemic areas harbor multiple parasite genotypes that could affect case management and malaria epidemiology. Here, we performed AmpSeq genotyping to capture polymorphisms associated with antimalarial resistance and the genetic diversity within natural Plasmodium falciparum infections. Known genetic polymorphisms associated with altered drug susceptibility were screened for the five most common marker genes, pfdhfr , pfdhps , pfmdr1 , pfcrt , and pfK13 , and genetic diversity was established from two known AmpSeq markers, cpmp and csp . Relative abundance of the different genotypes within mixed infections was calculated from the number of reads per genotype. Genotyping was performed on 117 samples, 63 from asymptomatic and 54 from symptomatic individuals. We identified up to 15 genotypes within an infection, and the median multiplicity of infection was higher in asymptomatic infections (median MOI = 5 in asymptomatics versus median MOI = 2 in symptomatics, P <  0.001). No genetic differentiation on parasites from asymptomatic and symptomatic individuals was found. No mutation associated with ART resistance was identified. Prevalence of the P. falciparum chloroquine resistance wild-type genotype (CVMNK) reached 80%, confirming a return to chloroquine (CQ) sensitive parasites in Cameroon. In addition, the CQ-associated resistant genotype (CVIET) was present at very low density in polyclonal infections. Persistence of low-density chloroquine resistant parasites indicates competition-survival trade-offs may contribute to maintaining genetic diversity in natura . Thus, monitoring the expansion of these low-density genotypes in different immune backgrounds will be critical to evaluate drug policy changes.

Background: Dihydroartemisinin/piperaquine is increasingly used for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. The efficacy of this combination in Cameroon is poorly documented, while resistance to dihydroartemisinin/piperaquine readily spreads in Southeast Asia., Objectives: This study evaluated the clinical efficacy of dihydroartemisinin/piperaquine in Cameroon, as well as the molecular profile and phenotypic susceptibility of collected isolates to dihydroartemisinin and piperaquine., Patients and Methods: Dihydroartemisinin/piperaquine efficacy in 42 days was followed-up for 138 patients presenting non-complicated falciparum malaria. Piperaquine concentration was determined at day 7 for 124 patients. kelch13 gene polymorphisms (n = 150) and plasmepsin2 gene amplification (n = 148) were determined as molecular markers of resistance to dihydroartemisinin and piperaquine, respectively. Parasite susceptibility to dihydroartemisinin and piperaquine was determined using validated in vitro survival assays., Results: The efficacy of dihydroartemisinin/piperaquine treatment was 100% after PCR correction. The reinfections were not associated with a variation of piperaquine concentration at day 7. Ninety-six percent (144/150) of the samples presented a WT allele of the kelch13 gene. Two percent (3/150) presented the non-synonymous mutation A578S, which is not associated with resistance to dihydroartemisinin. No duplication of the plasmepsin2 gene was observed (0/148). All the samples tested in vitro by survival assays (n = 87) were susceptible to dihydroartemisinin and piperaquine., Conclusions: Dihydroartemisinin/piperaquine has demonstrated excellent therapeutic efficacy with no evidence of emerging artemisinin or piperaquine resistance in Yaoundé, Cameroon. This observation suggests that dihydroartemisinin/piperaquine could be a sustainable therapeutic solution for P. falciparum malaria if implemented in areas previously free of artemisinin- and piperaquine-resistant parasites, unlike Southeast Asia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Malaria remains a serious public health problem in Cameroon. Implementation of control interventions requires prior knowledge of the local epidemiological situation. Here we report the results of epidemiological and entomological surveys carried out in Tibati, Adamawa Region, Cameroon, an area where malaria transmission is seasonal, 6 years after the introduction of long-lasting insecticidal bed nets., Methods: Cross-sectional studies were carried out in July 2015 and 2017 in Tibati. Thick blood smears and dried blood spots were collected from asymptomatic and symptomatic individuals in the community and at health centers, respectively, and used for the molecular diagnosis of Plasmodium species. Adult mosquitoes were collected by indoor residual spraying and identified morphologically and molecularly. The infection status of Plasmodium spp. was determined by quantitative PCR, and positivity of PCR-positive samples was confirmed by Sanger sequencing., Results: Overall malaria prevalence in our study population was 55.0% (752/1367) and Plasmodium falciparum was the most prevalent parasite species (94.3%), followed by P. malariae (17.7%) and P. ovale (0.8%); 92 (12.7%) infections were mixed infections. Infection parameters varied according to clinical status (symptomatic/asymptomatic) and age of the sampled population and the collection sites. Infection prevalence was higher in asymptomatic carriers (60.8%), but asexual and sexual parasite densities were lower. Prevalence and intensity of infection decreased with age in both the symptomatic and asymptomatic groups. Heterogeneity in infections was observed at the neighborhood level, revealing hotspots of transmission. Among the 592 Anopheles mosquitoes collected, 212 (35.8%) were An. gambiae, 172 (29.1%) were An. coluzzii and 208 (35.1%) were An. funestus (s.s.). A total of 26 (4.39%) mosquito specimens were infected by Plasmodium sp. and the three Anopheles mosquitoes transmitted Plasmodium at equal efficiency. Surprisingly, we found an An. coluzzii specimen infected by Plasmodium vivax, which confirms circulation of this species in Cameroon. The positivity of all 26 PCR-positive Plasmodium-infected mosquitoes was successively confirmed by sequencing analysis., Conclusion: Our study presents the baseline malaria parasite burden in Tibati, Adamawa Region, Cameroon. Our results highlight the high malaria endemicity in the area, and hotspots of disease transmission are identified. Parasitological indices suggest low bednet usage and that implementation of control interventions in the area is needed to reduce malaria burden. We also report for the first time a mosquito vector with naturally acquired P. vivax infection in Cameroon.

Diagnosing malaria is a key component of effective case management and monitoring of antimalarial programs worldwide. This chapter features the different diagnostic approaches currently in use or under testing for use in case management and/or epidemiological studies of malaria. Emphasis is laid on the basic principles of each diagnostic approach as well as their operational limits under different malaria endemicity settings. The discussed methods are defined as "conventional" or "unconventional" depending on their widespread use in malaria case management. The chapter therefore provides a practical guide to students, health practitioners, and field researchers involved in the fight against malaria through community-based interventions.

Malaria infection is one of the major causes of deaths in the African continent. The high burden of malaria in Africa is due to P. falciparum, which adapts and cospecializes with Anopheles gambiae, the most effective and widespread malaria vector. Since 2000, the incidence of malaria has been reduced by 17% and malaria mortality rates by 26%. However, the rate of decline has stalled and even reversed in some regions since 2014. In 2017 as described by the latest World malaria report, 219 million malaria cases were reported, up from 2017 million cases reported in 2016 in 91 countries, and the global tally of malaria deaths reached 435,000 deaths, compared with 451,000 estimated deaths in 2016. Despite these achievements, the African region continues to account for about 92% of malaria cases and deaths worldwide. Therefore, it is important to master the current situation of malaria in Africa to see how to better plan its elimination. In this chapter, we present the current situation and prospective means to improve it, including a salutogenesis approach.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

We screened a collection of synthetic compounds consisting of natural-product-like substructural motifs to identify a spirocyclic chromane as a novel antiplasmodial pharmacophore using an unbiased cell-based assay. The most active spirocyclic compound UCF 201 exhibits a 50% effective concentration (EC50) of 350 nM against the chloroquine-resistant Dd2 strain and a selectivity over 50 using human liver HepG2 cells. Our analyses of physicochemical properties of UCF 201 showed that it is in compliance with Lipinski's parameters and has an acceptable physicochemical profile. We have performed a limited structure-activity-relationship study with commercially available chromanes preserving the spirocyclic motif. Our evaluation of stage specificities of UCF 201 indicated that the compound is early-acting in blocking parasite development at ring, trophozoite and schizont stages of development as well as merozoite invasion. SPC is an attractive lead candidate scaffold because of its ability to act on all stages of parasite's aexual life cycle unlike current antimalarials.

Background: The emerging resistance of Plasmodium species to currently available anti-malarials remains a public health concern, hence the need for new effective, safe and affordable drugs. Natural products remain a reliable source of drugs. Nefang is a polyherbal anti-malarial of the Cameroonian folklore medicine with demonstrated in vitro antiplasmodial and antioxidant activities. It is composed of Mangifera indica (bark and leaf), Psidium guajava, Carica papaya, Cymbopogon citratus, Citrus sinensis, Ocimum gratissimum (leaves). This study aimed at investigating the suppressive, prophylactic and curative activities of Nefang in Plasmodium infected rodent models., Methods: Systemic acute oral toxicity of Nefang aqueous and ethanol extracts was assessed in mice up to a dose of 5,000 mgkg(-1) body weight. BALB/c mice and Wistar rats were inoculated with Plasmodium chabaudi chabaudi and Plasmodium berghei, respectively, and treated with Nefang, the Mangifera indica bark/Psidium guajava combination and a Psidium guajava leaf aqueous extracts (75, 150, 300 and 600 mgkg(-1) bwt). Their schizonticidal activity was then evaluated using the Peter's 4-day suppressive test). The prophylactic and curative (Rane's Test) activity of Nefang was also evaluated by determining the parasitaemia, survival time, body weight and temperature in pre-treated rodents., Results: Acute oral toxicity of the extract did not cause any observed adverse effects. Percent suppressions of parasitaemia at 600 mgkg(-1) bwt were as follows (P. berghei/P. chabaudi): Nefang - 82.9/86.3, Mangifera indica bark/Psidium guajava leaf combination extract - 79.5/81.2 and Psidium guajava leaf - 58.9/67.4. Nefang exhibited a prophylactic activity of 79.5% and its chemotherapeutic effects ranged from 61.2 - 86.1% with maximum effect observed at the highest experimental dose., Conclusion: These results indicate that Nefang has excellent in vivo anti-malarial activities against P. berghei and P. chabaudi, upholding earlier in vitro antiplasmodial activities against multi-drug resistant P. falciparum parasites as well as its traditional use. Hence, Nefang represents a promising source of new anti-malarial agents.

Disruption of blood-brain barrier (BBB) function is a key feature of cerebral malaria. Increased barrier permeability occurs due to disassembly of tight and adherens junctions between endothelial cells, yet the mechanisms governing junction disassembly and vascular permeability during cerebral malaria remain poorly characterized. We found that EphA2 is a principal receptor tyrosine kinase mediating BBB breakdown during Plasmodium infection. Upregulated on brain microvascular endothelial cells in response to inflammatory cytokines, EphA2 is required for the loss of junction proteins on mouse and human brain microvascular endothelial cells. Furthermore, EphA2 is necessary for CD8+ T cell brain infiltration and subsequent BBB breakdown in a mouse model of cerebral malaria. Blocking EphA2 protects against BBB breakdown highlighting EphA2 as a potential therapeutic target for cerebral malaria.

Abstract Background The need to start treatment early for pregnant women who present with clinical features of malaria usually conflicts with the need to confirm diagnosis by microscopy (MP) before treatment, due to delays in obtaining results. Parasite sequestration in the placenta is also a problem. Rapid diagnostic tests (RDT), which detect soluble antigens, are a valuable alternative. The objective of this study was to evaluate pretreatment parasite prevalence by microscopy and by RDT and to assess the accuracy of RDT with MP as reference. Methods A prospective cross-sectional study was carried out at the obstetrical unit of the Central Hospital in Yaoundé, during the period January-August 2015. Consenting patients with symptoms of suspected malaria in pregnancy were recruited and a blood sample taken for MP and RDT before treatment was started. The estimates of diagnostic performance (with 95% confidence interval) were calculated in OpenEpi online software using the Wilson’s score. The agreement, as reflected by the Cohen’s kappa, was calculated and interpreted using known intervals. Results The results showed that, out of the 104 patients recruited, 69.2% (95%CI: 59.1–77.5) were MP positive while 77.94% (95%CI: 63.1–80.9) were RDT positive. The sensitivity of the malaria RDT was 91.67% (95%CI: 83.69–96.77) while the specificity was 53.13% (95%CI: 31.39–65.57). The diagnostic accuracy of the RDT with MP as reference was 79.81% (95%CI: 70.0–86.1). All cases were due to Plasmodium falciparum. A Cohen’s kappa of 0.45 (95%CI: 0.26–0.64) was obtained, consistent with a moderate agreement between the tests. Conclusions The diagnostic accuracy of the CareStart™ malaria Pf/PAN compared to microscopy was high, but not as desirable, with a false negative RDT at very high parasitaemia. In tertiary facilities, RDTs appear to provide a better diagnostic solution compared to microscopy. However, future studies with larger sample sizes should make this observation more generalizable; as missing a case could have serious consequences on pregnancy outcome.

The spread of Plasmodium falciparum resistant parasites remains one of the major challenges for malaria control and elimination in Sub Saharan Africa. Monitoring of molecular markers conferring resistance to different antimalarials is important to track the spread of resistant parasites and to optimize the therapeutic lifespan of current drugs. This study aimed to evaluate the prevalence of known mutations in the drug resistance genes Pfcrt, Pfmdr1, Pfdhfr and Pfdhps in two different epidemiological settings in Cameroon. Dried blood spots collected in 2018 and 2019 from asymptomatic individuals were used for DNA extraction and then the Plasmodium infection status was determined byPCR. Detection of SNPs was performed by nested PCR followed by allele-specific restriction analysis (ASRA). The prevalence of each genotype was compared between sites using the Chi square and Fisher's exact tests. A high prevalence of the Pfcrt K76 wild type allele was found in both sites (88.5 and 62.29% respectively; P< 0,0001). The prevalence of Pfmdr1 mutations 86Y and 1246Y was respectively 55.83 and 1.45% in Mfou and 45.87 and 5.97% in Tibati, with significant difference between the studied areas (P<0.0001). Overall, the Pfdhfr triple-mutant genotype (51I/59R/108N) was highly prevalent (> 96%), however no SNP was detected at codon 164. In Pfdhps, the prevalence of the 437G mutation reached (90%) and was at higher frequency in Mfou (P< 0.0001). Overall, the Pfdhps mutations 540E and 581G were less common (0.33 and 3.26%, respectively). The quadruple resistant genotype (Pfdhfr 51I/59R/108N+Pfdhp437G) was found almost 90% of the samples. The wild-type genotype (Pfdhfr N51/C59/S108/164I+Pfdhps A437/K540/A581) was never identified and the sextuple mutant (Pfdhfr 51I/59R/108N+Pfdhp437G/540E/581G), kwon as super resistant appeared in two samples from Tibati. These findings demonstrate declining trends in the prevalence of mutations conferring resistance to 4-aminoquinolines, especially to chloroquine. However, a high level of mutations in P. falciparum genes related to SP resistance was detected and this raises concerns about the future efficacy of IPTp-SP and SMC in Cameroon. [ABSTRACT FROM AUTHOR]

Light microscopy and rapid diagnostic tests are the two commonly used methods for malaria diagnosis that rely on the direct use of unprocessed blood samples. However, both methods do not have the level of sensitivity required for malaria diagnosis in cases of low density parasitaemia. We report here the diagnostic performance of a whole blood-based reverse transcription loop-mediated isothermal amplification method for Plasmodium falciparum malaria diagnosis in apparently healthy blood donors and febrile neonates in Cameroon. The presence of malaria parasites in whole blood samples was determined by light microscopy, antigen-based rapid diagnostic test (RDT), and by RT-LAMP using a "lyse and amplify" experimental protocol. Of the 256 blood donors tested, 36 (14.1%) were positive for malaria parasites by light microscopy, 38 (14.8%) were positive by RDT whereas 78 (30.5%) were positive by RT-LAMP. Only light microscopy and RT-LAMP detected infection among the febrile neonates (279 neonates, median age: 2 days, range: 1–9 days), with positivity rates of 8.6% and 12.2%, respectively. The overall concordance between the three methods were 75.9% for RT-LAMP and light microscopy, 75.1% for RT-LAMP and RDT, and 83.9% for light microscopy and RDT. Blood parasite densities were significantly lower in the neonates (mean: 97.6, range: 61–192 parasites/μL) compared to the blood donors (mean: 447.8, range: 63–11 000 parasites/μL). Together, the study demonstrates the usefulness of whole blood RT-LAMP for use in rapid pre-screening of blood donors and suspected neonates to avert severe consequences of P. falciparum infections. [ABSTRACT FROM AUTHOR]

The specific immune response to the Anopheles salivary peptide could be a pertinent and complementary tool to assess the risk of malaria transmission and the effectiveness of vector control strategies. This study aimed to obtain first reliable data on the current state of the Anopheles gSG6-P1 biomarker for assess the level of exposure to Anopheles bites in high malaria endemic areas in Cameroon. Blood smears were collected from people living in the neighborhoods of Youpwe (suburban area, continental) and Manoka (rural area, Island), both areas in the coastal region of Cameroon. Malaria infection was determined using thick blood smear microscopy, whereas the level of specific IgG response to gSG-P1 peptide was assessed by enzyme-linked immunosorbent assay from the dried blood spots. Of 266 (153 from Youpwe, 113 from Manoka) malaria endemic residents (mean age: 22.8±19.8 years, age range: 6 months–94 years, male/female sex ratio: 1/1.2, with Manoka mean age: 23.71±20.53, male/female sex ratio:1/1.13 and Youpwe mean age: 22.12±19.22, male/female sex ratio 1/0.67) randomly included in the study, Plasmodium infection prevalence was significantly higher in Manoka than in Youpwe (64.6% vs 12,4%, p = 0.0001). The anti-gSG6-P1 IgG response showed a high inter-individual heterogeneity and was significantly higher among individuals from Manoka than those from Youpwe (p = 0.023). Malaria infected individuals presented a higher anti-gSG6-P1 IgG antibody response than non-infected (p = 0.0004). No significant difference in the level of specific IgG response to gSG-P1 was observed according to long lasting insecticidal nets use. Taken together, the data revealed that human IgG antibody response to Anopheles gSG-P1 salivary peptide could be also used to assess human exposure to malaria vectors in Central African region. This finding strengthens the relevance of this candidate biomarker to be used for measuring human exposure to malaria vectors worldwide. [ABSTRACT FROM AUTHOR]

The emergence of artemisinin-resistant parasites since the late 2000s at the border of Cambodia and Thailand poses serious threats to malaria control globally, particularly in Africa which bears the highest malaria transmission burden. This study aimed to obtain reliable data on the current state of the kelch13 molecular marker for artemisinin resistance in Plasmodium falciparum in Cameroon. DNA was extracted from the dried blood spots collected from epidemiologically distinct endemic areas in the Center, Littoral and North regions of Cameroon. Nested PCR products from the Kelch13-propeller gene were sequenced and analyzed on an ABI 3730XL automatic sequencer. Of 219 dried blood spots, 175 were sequenced successfully. We identified six K13 mutations in 2.9% (5/175) of samples, including 2 non-synonymous, the V589I allele had been reported in Africa already and one new allele E612K had not been reported yet. These two non-synonymous mutations were uniquely found in parasites from the Littoral region. One sample showed two synonymous mutations within the kelch13 gene. We also observed two infected samples with mixed K13 mutant and K13 wild-type infection. Taken together, our data suggested the circulation of the non-synonymous K13 mutations in Cameroon. Albeit no mutations known to be associated with parasite clearance delays in the study population, there is need for continuous surveillance for earlier detection of resistance as long as ACTs are used and scaled up in the community. [ABSTRACT FROM AUTHOR]

Presence of mature gametocyte forms of malaria parasites in peripheral blood is a key requirement for malaria transmission. Yet, studies conducted in most malaria transmission zones report the absence of gametocyte in the majority of patients. We therefore sought to determine the risk factors of both all-stage and mature gametocyte carriage in an area with high stable transmission of Plasmodium falciparum in Cameroon. Gametocyte positivity was determined using three complementary methods: thick blood smear microscopy, RT-PCR and RT-LAMP, whereas exposure to the infection was assessed by enzyme-linked immunosorbent assay. Of 361 malaria endemic residents randomly included in the study (mean age: 28±23 years, age range: 2–100 years, male/female sex ratio: 1.1), 87.8% were diagnosed with P. falciparum infection, of whom 45.7% presented with fever (axillary body temperature ≥37.5°C). Mature gametocyte positivity was 1.9% by thick blood smear microscopy and 8.9% by RT-PCR targeting the mature gametocyte transcript, Pfs25. The gametocyte positivity rate was 24.1% and 36.3% by RT-PCR or RT-LAMP, respectively, when targeting the sexual stage marker, Pfs16. Multivariate analyses revealed anemia as a common independent risk factor for both mature and all-stage gametocyte carriage, whereas fever and low anti-gametocyte antibody levels were independently associated with all-stage gametocyte carriage only. Taken together, the data suggest important differences in risk factors of gametocyte carriage depending on stage analyzed, with anemia, fever and low antiplasmodial plasma antibody levels representing the major contributing risk factors. [ABSTRACT FROM AUTHOR]

Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

With more than 40% of the world’s population at risk, 200–300 million infections each year, and an estimated 1.2 million deaths annually, malaria remains one of the most important public health problems of mankind today. With the propensity of malaria parasites to rapidly develop resistance to newly developed therapies, and the recent failures of artemisinin-based drugs in Southeast Asia, there is an urgent need for new antimalarial compounds with novel mechanisms of action to be developed against multidrug resistant malaria. We present here a novel image analysis algorithm for the quantitative detection and classification of Plasmodium lifecycle stages in culture as well as discriminating between viable and dead parasites in drug-treated samples. This new algorithm reliably estimates the number of red blood cells (isolated or clustered) per fluorescence image field, and accurately identifies parasitized erythrocytes on the basis of high intensity DAPI-stained parasite nuclei spots and Mitotracker-stained mitochondrial in viable parasites. We validated the performance of the algorithm by manual counting of the infected and non-infected red blood cells in multiple image fields, and the quantitative analyses of the different parasite stages (early rings, rings, trophozoites, schizonts) at various time-point post-merozoite invasion, in tightly synchronized cultures. Additionally, the developed algorithm provided parasitological effective concentration 50 (EC50) values for both chloroquine and artemisinin, that were similar to known growth inhibitory EC50 values for these compounds as determined using conventional SYBR Green I and lactate dehydrogenase-based assays. [ABSTRACT FROM AUTHOR]

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high- throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence- specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria. [ABSTRACT FROM AUTHOR]