Deletions of Histidine-Rich Protein 2/3 Genes in Natural Plasmodium falciparum Populations from Cameroon and India: Role of Asymptomatic and Submicroscopic Infections.

The bulk of malaria rapid diagnostic tests (RDTs) target histidine-rich protein 2 of Plasmodium falciparum, the deadliest malaria species. The WHO considers pfhrp2/3 deletions as one of the main threats to successful malaria control and/or elimination; as such, parasites that lack part or all of the pfhrp2 gene are missed by pfHRP2-targeting RDTs. Such deletions have been reported in several African and Asian countries, but little is known in Cameroon and India. Blood samples were collected from individuals living in four areas of Cameroon (Douala, Maroua, Mayo-Oulo, Pette) and India (Mewat, Raipur, Ranchi, Rourkela). Deletions in pfhrp2/3 genes were confirmed if samples 1) had ≥100 parasites/µL by quantitative polymerase chain reaction (PCR), 2) PCR negative for pfhrp2/3, and 3) PCR positive for at least two single-copy genes. The overall proportion of pfhrp2 and pfhrp3 deletions in Cameroon was 13.5% and 3.1%. In India, the overall proportion was 8% for pfhrp2 and 4% for pfhrp3. The overall proportions of samples with both gene deletions (pfhrp2-/3-) were 3.1% in Cameroon and 1.3% in India. In Cameroon, pfhrp2-/3+ and pfhrp2-/3- deletions were common in Maroua (P = 0.02), in asymptomatic parasitemia (P = 0.006) and submicroscopic parasitemia (P <0.0001). In both countries, pfhrp2/3 deletions, including pfhrp2-/3- deletions, were mainly seen in monoclonal infections. This study outlines that double deletions that result in false negative RDTs are uncommon in our settings, and highlights the importance of active molecular surveillance for pfhrp2/3 deletions in Cameroon and India.

Genetic diversity and natural selection of apical membrane antigen-1 (ama-1) in Cameroonian Plasmodium falciparum isolates.

Antigenic variation associated with genetic diversity in global Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major impediment to designing an effective malaria vaccine. Here, we report the first study on genetic diversity and natural selection of the Pfama-1 gene in P. falciparum isolates from Cameroon. A total of 328 P. falciparum positive samples collected during 2016 and 2019 from five localities of Cameroon were analysed. The ectodomain coding fragment of Pfama-1 gene was amplified for polymorphism profiling and natural selection analysis. A total of 108 distinct haplotypes were found in 203 P. falciparum isolates with considerable nucleotide diversity (π = 0.016) and haplotype diversity (Hd = 0.976). Most amino acid substitutions detected were scattered in ectodomain-I and few specific mutations viz P145L, K148Q, K462I, L463F, N471K, S482L, E537G, K546R and I547F were seen only in Cameroonian isolates. A tendency of natural selection towards positive diversifying selection was observed (Taj-D = 2.058). Five positively selected codon sites (P145L, S283L, Q308E/K, P330S and I547F) were identified, which overlapped with predicted B-cell epitopes and red blood cell (RBC) binding sites, suggesting their potential implication in host immune pressure and parasite-RBC binding complex modulation. The Cameroonian P. falciparum populations indicated a moderate level of genetic differentiation when compared with global sequences, with few exceptions from Vietnam and Venezuela. Our findings provide baseline data on existing Pfama-1 gene polymorphisms in Cameroonian field isolates, which will be useful information for malaria vaccine design.

Rapid diagnostic tests for malaria diagnosis in Cameroon: impact of histidine rich protein 2/3 deletions and lactate dehydrogenase gene polymorphism.

Malaria rapid diagnostic tests (mRDT) play a vital role in malaria control in endemic areas. In this study, histidine-rich protein (hrp) and lactate dehydrogenase (ldh) genes were genotyped in Plasmodium falciparum (Pf) and Plasmodium ovale (Po) spp. isolates. Deletions in P. falciparum hrp2/3 (pfhrp2/3) proteins and single nucleotide polymorphisms (SNPs) were analyzed. Twenty-four samples were analyzed for pfhrp2/3 gene deletions and 25 for SNPs in ldh gene (18 Pf and 7 Po spp.). Deletions in pfhrp2/3 genes were observed in 1.9% malaria positive isolates. The pfldh gene sequences showed one SNP at codon 272 (D272N) in 22.2% of samples while in Po spp., sequences were 100% similar to P. ovale curtisi but when compared to P. ovale wallikeri reference sequence, SNPs at positions 143 (P143S), 168 (K168N), 204 (V204I) were found. Findings suggest low prevalence in pfhrp2/3 genes and highlight the circulation of P. ovale curtisi in the studies areas.

First Insight into Drug Resistance Genetic Markers, Glucose-6-Phosphate Dehydrogenase and Phylogenetic Patterns of Misdiagnosed Plasmodium vivax Malaria in Far North Region, Cameroon.

Plasmodium falciparum (Pf) is the predominant malaria species in Africa, but growing rates of non-falciparum species such as P. vivax (Pv) have been reported recently. This study aimed at characterizing drug resistance genes, glucose-6-phosphate dehydrogenase gene (G6PD), and phylogenetic patterns of a Pv + Pf co-infection misdiagnosed as a Pf mono-infection in the Far North region of Cameroon. Only one non-synonymous mutation in the pvdhps gene A383G was found. Pv drug resistance gene sequences were phylogenetically closer to the reference SAL-I strain and isolates from Southeast Asia and Western Pacific countries. Analyzing co-infecting Pf revealed no resistance mutations in Pfmdr1 and Pfk13 genes, but mutations in Pfcrt (C72V73I74E75T76) and Pfdhfr-Pfdhps genes (A16C50I51R59N108L164 - A436A437K540G581S613) were observed. No G6PD deficiency-related mutations were found. This is first study from Cameroon reporting presence of putative drug resistance mutations in Pv infections, especially in the pvdhps gene, and also outlined the absence of a G6PD-deficiency trait in patients.

Comparison of SYBR green I and lactate dehydrogenase antimalarial in vitro assay in Plasmodium falciparum field isolates.

Several assay methods are in use for monitoring the drug sensitivity of malaria parasites and screening new antimalarial drugs. Plasmodium lactate dehydrogenase (pLDH) and SYBR Green I in vitro assays were used to evaluate the drug efficacy of Chloroquine, Artemisinin and Azadirachta indica silver nano particles against Plasmodium falciparum 3D7 strain. The half-maximal inhibitory concentration (IC50) of each compound was estimated with non-linear regression model - dose-response analysis. The consistency between two methods was analysed with Cohen's kappa coefficient, interclass correlation and Bland-Altman plots. No statistical difference was found between IC50 values determined by both assays (p = 0.714). The proportion of resistant isolates to chloroquine according to SYBR green I (43.48%) and pLDH (34.78%) assays were similar (z = 0.302; p = 0.762) with significant concordant between methods (k = 0.819, p < 0.001). The results of pLDH Qualisa assay was comparable with classic SYBR green I assay and can be potentially useful in antimalarial drug efficacy surveillance.

Phytofabrication and characterization of Alchornea cordifolia silver nanoparticles and evaluation of antiplasmodial, hemocompatibility and larvicidal potential.

Purpose: The recent emergence of Plasmodium falciparum (Pf) parasites resistant to current artemisinin-based combination therapies in Africa justifies the need to develop new strategies for successful malaria control. We synthesized, characterized and evaluated medical applications of optimized silver nanoparticles using Alchornea cordifolia (AC-AgNPs), a plant largely used in African and Asian traditional medicine. Methods: Fresh leaves of A. cordifolia were used to prepare aqueous crude extract, which was mixed with silver nitrate for AC-AgNPs synthesis and optimization. The optimized AC-AgNPs were characterized using several techniques including ultraviolet-visible spectrophotometry (UV-Vis), scanning/transmission electron microscopy (SEM/TEM), powder X-ray diffraction (PXRD), selected area electron diffraction (SAED), energy dispersive X-ray spectroscopy (EDX), Fourier transformed infrared spectroscopy (FTIR), dynamic light scattering (DLS) and Zeta potential. Thereafter, AC-AgNPs were evaluated for their hemocompatibility and antiplasmodial activity against Pf malaria strains 3D7 and RKL9. Finally, lethal activity of AC-AgNPs was assessed against mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti which are vectors of neglected diseases such as dengue, filariasis and chikungunya. Results: The AC-AgNPs were mostly spheroidal, polycrystalline (84.13%), stable and polydispersed with size of 11.77 ± 5.57 nm. FTIR revealed the presence of several peaks corresponding to functional chemical groups characteristics of alkanoids, terpenoids, flavonoids, phenols, steroids, anthraquonones and saponins. The AC-AgNPs had a high antiplasmodial activity, with IC50 of 8.05 µg/mL and 10.31 µg/mL against 3D7 and RKL9 Plasmodium falciparum strains. Likewise, high larvicidal activity of AC-AgNPs was found after 24 h- and 48 h-exposure: LC50 = 18.41 µg/mL and 8.97 µg/mL (Culex quinquefasciatus), LC50 = 16.71 µg/mL and 7.52 µg/mL (Aedes aegypti) and LC50 = 10.67 µg/mL and 5.85 µg/mL (Anopheles stephensi). The AC-AgNPs were highly hemocompatible (HC50 > 500 µg/mL). Conclusion: In worrying context of resistance of parasite and mosquitoes, green nanotechnologies using plants could be a cutting-edge alternative for drug/insecticide discovery and development.

Plasmodium cynomolgi in humans: current knowledge and future directions of an emerging zoonotic malaria parasite.

Plasmodium cynomolgi (Pcy), a simian malaria parasite, is a recent perfect example of emerging zoonotic transfer in human. This review summarizes the current knowledge on the epidemiology of natural Pcy infections in humans, mosquitoes and monkeys, along with its biological, clinical and drug sensitivity patterns. Knowledge gaps and further studies on Pcy in humans are also discussed. This parasite currently seems to be geographically limited in South-East Asia (SEA) with a global prevalence in human ranging from 0 to 1.4%. The Pcy infections were reported in local SEA populations and European travelers, and range from asymptomatic carriage to mild/moderate attacks with no evidence of pathognomonic clinical and laboratory patterns but with Pcy strain-shaped clinical differences. Geographical distribution and competence of suitable mosquito vectors and non-primate hosts, globalization, climate change, and increased intrusion of humans into the habitat of monkeys are key determinants to emergence of Pcy parasites in humans, along with its expansion outside SEA. Sensitization/information campaigns coupled with training and assessment sessions of microscopists and clinicians on Pcy are greatly needed to improve data on the epidemiology and management of human Pcy infection. There is a need for development of sensitive and specific molecular tools for individual diagnosis and epidemiological studies. The development of safe and efficient anti-hypnozoite drugs is the main therapeutic challenge for controlling human relapsing malaria parasites. Experience gained from P. knowlesi malaria, development of integrated measures and strategies-ideally with components related to human, monkeys, mosquito vectors, and environment-could be very helpful to prevent emergence of Pcy malaria in humans through disruption of transmission chain from monkeys to humans and ultimately contain its expansion in SEA and potential outbreaks in a context of malaria elimination.

Intermittent preventive treatment with Sulfadoxine pyrimethamine for malaria: a global overview and challenges affecting optimal drug uptake in pregnant women.

Malaria in Pregnancy (MiP) leading to morbidity and mortality is a major public health problem that poses significant risk to pregnant women and their fetus. To cope with this alarming situation, administration of Sulfadoxine-pyrimethamine (SP) drugs to pregnant women as an intermittent preventive treatment (IPT) from 16 weeks of gestation is recommended by the World Health Organization (WHO) guidelines. We conducted a comprehensive search of published articles related to MiP in last 10 years with predefined keywords or their synonyms. The mapping of malaria in pregnant women showed a prevalence rate up to 35% in many countries. Although IPTp-SP has been implemented in endemic regions since several years but the IPTp-SP coverage percentage vary from country to country and continue to remain below the target of 80%. Major reasons for low IPTp-SP involve gestational age at first prenatal visit, level of education, place of residence, knowledge of IPTp-SP benefits, and use of antenatal services. Several challenges including the emergence of septuple and octuple SP-resistant parasites is reported from many countries which make the prophylactic use of IPTp-SP currently debatable. This narrative review addresses the barriers for optimal use of IPTp-SP and discusses alternative approaches to increase the use and effectiveness of SP intervention for preventing MiP. The COVID pandemic has drastically affected the public health disrupting the management of diseases worldwide. In view of this, a brief summary of COVID impact on MiP situation is also included.

Non-falciparum species and submicroscopic infections in three epidemiological malaria facets in Cameroon.

BACKGROUND: There are growing reports on the prevalence of non-falciparum species and submicroscopic infections in sub-Saharan African countries but little information is available from Cameroon. METHODS: A hospital-based cross-sectional study was carried out in four towns (Douala, Maroua, Mayo-Oulo, and Pette) from three malaria epidemiological strata (Forest, Sahelian, and Soudanian) of Cameroon. Malaria parasites were detected by Giemsa light microscopy and polymerase chain reaction (PCR) assay. Non-falciparum isolates were characterized and their 18S gene sequences were BLASTed for confirmatory diagnosis. RESULTS: PCR assay detected malaria parasites in 82.4% (98/119) patients, among them 12.2% (12/98) were asymptomatic cases. Three Plasmodium species viz. P. falciparum, P. ovale curtisi and P. vivax, and two co-infection types (P. falciparum + P. vivax and P. falciparum + P. ovale curtisi) were found. The remaining infections were mono-infections with either P. falciparum or P. ovale curtisi. All non-falciparum infections were symptomatic and microscopic. The overall proportion of submicroscopic infections was 11.8% (14/119). Most asymptomatic and submicroscopic infection cases were self-medicated with antimalarial drugs and/or medicinal plants. On analysis, P. ovale curtisi sequences were found to be phylogenetically closer to sequences from India while P. vivax isolates appeared closer to those from Nigeria, India, and Cameroon. No G6PD-d case was found among non-falciparum infections. CONCLUSIONS: This study confirms our previous work on circulation of P. vivax and P. ovale curtisi and the absence of P. knowlesi in Cameroon. More studies are needed to address non-falciparum malaria along with submicroscopic infections for effective malaria management and control in Cameroon.

In vitro antiplasmodial activity, hemocompatibility and temporal stability of Azadirachta indica silver nanoparticles.

Recently green nanotechnology has gained great interest as a promising tool for drug discovery. In the present study, we synthesized and characterized silver nanoparticles (AgNPs) using Azadirachta indica (AI) and evaluated their hemocompatibility and effect against Plasmodium falciparum strains. AI leaves and barks were used for aqueous extracts (AIL and AIB) and AgNPs synthesis. AgNPs were characterized using spectroscopic, diffraction, electron microscopic and electrostatic techniques. Anti-plasmodial and haemolytic activity were assessed following the SYBR Green I fluorescence assay and Miki et al. protocol, respectively. The normalized fluorescence counts were plotted against the log-transformed drug concentration and half-maximal inhibitory concentration (IC50) determined by analyzing the dose-response curves. AgNPs were stored for 120 days at room temperature-RT, +4 °C and -20 °C and subsequently their stability was evaluated by spectroscopy. Both NPs were predominantly spheroidal, crystalline in nature, stable, well dispersed with mean size of 13.01 nm for AIL-NPs and 19.30 nm for AIB-NPs and exhibited good antiplasmodial activity against 3D7 and RKL9 P. falciparum strains with IC50 of 9.27 µg/mL and 11.14 µg/mL for AIL-NPs, 8.10 µg/mL and 7.87 µg/mL for AIB-NPs, respectively. A. indica contain bioactive phyto-compounds indicating great potential for anti-malarial drug development through green nanotechnology. The AgNPs were structurally stable after 120 days but antiplasmodial activity was considerably affected. A significant haemolytic activity (>25%) was observed with AIL- and AIB-AgNPs at concentrations ≥125 µg/mL.

The spectrum of clinical biomarkers in severe malaria and new avenues for exploration.

Globally, malaria is a public health concern, with severe malaria (SM) contributing a major share of the disease burden in malaria endemic countries. In this context, identification and validation of SM biomarkers are essential in clinical practice. Some biomarkers (C-reactive protein, angiopoietin 2, angiopoietin-2/1 ratio, platelet count, histidine-rich protein 2) have yielded interesting results in the prognosis of Plasmodium falciparum severe malaria, but for severe P. vivax and P. knowlesi malaria, similar evidence is missing. The validation of these biomarkers is hindered by several factors such as low sample size, paucity of evidence-evaluating studies, suboptimal values of sensitivity/specificity, poor clinical practicality of measurement methods, mixed Plasmodium infections, and good clinical value of the biomarkers for concurrent infections (pneumonia and current COVID-19 pandemic). Most of these biomarkers are non-specific to pathogens as they are related to host response and hence should be regarded as prognostic/predictive biomarkers that complement but do not replace pathogen biomarkers for clinical evaluation of SM patients. This review highlights the importance of research on diagnostic/predictive/therapeutic biomarkers, neglected malaria species, and clinical practicality of measurement methods in future studies. Finally, the importance of omics technologies for faster identification/validation of SM biomarkers is also included.

Epidemiological, Physiological and Diagnostic Comparison of Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

Nowadays, Plasmodium ovale is divided into two non-recombinant sympatric species: Plasmodium ovale wallikeri and Plasmodium ovale curtisi. In this mini review, we summarize the available knowledge on the clinical/biological aspects of P. ovale spp. malaria and current techniques for the diagnosis/characterisation of P. ovale curtisi and P. ovale wallikeri. P. ovale wallikeri infections are characterized by a deeper thrombocytopenia and shorter latency compared to P. ovale curtisi infections, indicating that P. ovale wallikeri is more pathogenic than P. ovale curtisi. Rapid diagnosis for effective management is difficult for P. ovale spp., since specific rapid diagnostic tests are not available and microscopic diagnosis, which is recognized as the gold standard, requires expert microscopists to differentiate P. ovale spp. from other Plasmodium species. Neglect in addressing these issues in the prevalence of P. ovale spp. represents the existing gap in the fight against malaria.

Global trend of Plasmodium malariae and Plasmodium ovale spp. malaria infections in the last two decades (2000-2020): a systematic review and meta-analysis.

BACKGROUND: Recent studies indicate that the prevalence of non-falciparum malaria, including Plasmodium malariae and Plasmodium ovale spp., is increasing, with some complications in infected individuals. The aim of this review is to provide a better understanding of the malaria prevalence and disease burden due to P. malariae and P. ovale spp. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and the Joanna Briggs Institute prevalence study assessment tool were used to select and evaluate the studies, respectively. Six databases: PubMed, WHOLIS, Wiley Library, ScienceDirect, Web of Science and Google Scholar were used to screen articles published during the period January 2000-December 2020. The pooled prevalence estimates for P. malariae and P. ovale spp. were analysed using a random-effects model and the possible sources of heterogeneity were evaluated through subgroup analysis and meta-regression. RESULTS: Out of the 3297 studies screened, only 113 studies were included; among which 51.33% were from the African Region. The P. malariae and P. ovale spp. pooled prevalence were 2.01% (95% CI 1.31-2.85%) and 0.77% (95% CI 0.50-1.10%) respectively, with the highest prevalence in the African Region. P. malariae was equally distributed among adults (2.13%), children (2.90%) and pregnant women (2.77%) (p = 0.862), whereas P. ovale spp. was more prevalent in pregnant women (2.90%) than in children ≤ 15 years (0.97%) and in patients > 15 years old (0.39%) (p = 0.021). In this review, data analysis revealed that P. malariae and P. ovale spp. have decreased in the last 20 years, but not significantly, and these species were more commonly present with other Plasmodium species as co-infections. No difference in prevalence between symptomatic and asymptomatic patients was observed for either P. malariae or P. ovale spp. CONCLUSION: Our analysis suggests that knowledge of the worldwide burden of P. malariae and P. ovale spp. is very important for malaria elimination programmes and a particular focus towards improved tools for monitoring transmission for these non-falciparum species should be stressed upon to deal with increased infections in the future.