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Michael Fokuo Ofori,1,* Emma E Kploanyi,1 Benedicta A Mensah,2 Emmanuel K Dickson,1 Eric Kyei-Baafour,1 Sampson Gyabaa,3 Mary Tetteh,4 Kwadwo A Koram,2 Benjamin K Abuaku,2,* Anita Ghansah1,5,* 1Immunology Department, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana; 2Epidemiology Department, Noguchi Memorial Institute for Medical Research,University of Ghana, Legon, Accra, Ghana; 3Ewim Polyclinic, Ghana Health Service, Cape Coast, Ghana; 4Begoro District Hospital, Ghana Health Service, Begoro, Ghana; 5Parasitology Department, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana*These authors contributed equally to this workCorrespondence: Michael Fokuo OforiImmunology Department, Noguchi Memorial Institute for Medical Research, University of Ghana, Post Office Box LG581, Legon, GhanaTel +233 244 715975Fax +233 302 502182Email mofori@noguchi.ug.edu.ghPurpose: Malaria continues to be a major health issue globally with almost 85% of the global burden and deaths borne by sub-Saharan Africa and India. Although the current artemisinin derived combination therapies in Ghana are still efficacious against the Plasmodium falciparum (Pf) parasite, compounding evidence of artemisinin and amodiaquine resistance establish the need for a full, up-to-date understanding and monitoring of antimalarial resistance to provide evidence for planning control strategies.Materials and Methods: The study was cross-sectional and was conducted during the peak malaria transmission seasons of 2015, 2016, and 2017 in two ecological zones of Ghana. Study participants included children aged 6 months to 14 years. Using ex vivo 4,6-diamidino-2-phenylindole (DAPI) drug sensitivity assay, 330 Pf isolates were used to investigate susceptibility to five antimalarial drugs: chloroquine (CQ), amodiaquine (AMD) dihydroartemisinin (DHA), artesunate (ART) and mefloquine (MFQ).Results: The pooled geometric mean IC50S (GMIC50) of the five drugs against the parasites from Cape Coast and Begoro were 15.5, 42.4, 18.9, 4.6 and 27.3nM for CQ, AMD, DHA, ART, and MFQ, respectively. The GMIC50 values for CQ (p< 0.001), ART (p< 0.011) and DHA (p< 0.018) were significantly higher for Cape Coast isolates as compared to Begoro isolates. However, GMIC50 estimates for MFQ (p< 0.022) were significantly higher for Begoro isolates. Positive correlations were found between each pair of drugs with the weakest found between MFQ and DHA (r = 0.34;p< 0.001), and the strongest between ART and DHA (r =0.66; p< 0.001).Conclusion: The parasites showed reduced sensitivities to three (AMD, DHA and MFQ) out of the five drugs assessed. The study also demonstrated the continual return of chloroquine-sensitive parasites after 13 years of its withdrawal as the first-line drug for the treatment of uncomplicated malaria in Ghana. The ex vivo DAPI assay is a reliable method for assessing antimalarial drug sensitivities of Pf field isolates under field settings.Keywords: malaria, Plasmodium falciparum, anti-malarial drugs, field isolates, DAPI, uncomplicated malaria

Background & objectives: The in vitro assays for susceptibility of Plasmodium falciparum to antimalarial drugs are important tools for monitoring drug resistance. During the present study, efforts were made to establish long-term continuous in vitro culture of Indian field isolates of P. falciparum and to determine their sensitivity to standard antimalarial drugs and antibiotics. Methods: Four (MZR-I, -II, -III and -IV) P. falciparum isolates were obtained from four patients who showed artemisinin-based combination therapy (ACT) from Mizoram, a north-eastern State of India, and characterized for their in vitro susceptibility to chloroquine diphosphate (CQ), quinine hydrochloride dehydrate, mefloquine, piperaquine, artemether, arteether, dihydro-artemisinin (DHA), lumefantrine and atovaquone and antibiotics, azithromycin and doxycycline. These patients showed ACT treatment failure. Two-fold serial dilutions of each drug were tested and the effect was evaluated using the malaria SYBR Green I fluorescence assay. K1 (chloroquine-resistant) and 3D7 (chloroquine-sensitive) reference strains were used as controls. Results: Growth profile of all field isolates was identical to that of reference parasites. The IC50 values of all the drugs were also similar against field isolates and reference parasite strains, except K1, exhibited high IC50 value (275±12.5 nM) of CQ for which it was resistant. All field isolates exhibited higher IC50 values of CQ, quinine hydrochloride dihydrate and DHA compared to reference strains. The resistance index of field isolates with respect to 3D7 ranged between 260.55 and 403.78 to CQ, 39.83 and 46.42 to quinine, and 2.98 and 4.16 to DHA, and with respect to K1 strain ranged between 6.51 and 10.08, 39.26 and 45.75, and 2.65 and 3.71. MZR-I isolate exhibited highest resistance index. Interpretation & conclusions: As the increase in IC50 and IC90 values of DHA against field isolates of P falciparum was not significant, the tolerance to DHA-piperaquine (PPQ) combination might be because of PPQ only. Further study is required on more number of such isolates to generate data for a meaningful conclusion. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin. Methods: Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots. Results: The manual and automated quantification showed strong, positive correlation (r² = 0.959, p <0.001) and with similar detection limit and precision. The automated assay showed the expected dose-dependent reduction in binding to CHO cells when blocking with soluble chondroitin sulfate A or anti-CD36 antibody. Quantification of binding to endothelial cells showed clear distinction between selected vs. non-selected parasite lines. Importantly, the assay was sufficiently sensitive to detect adhesion of field isolates to endothelial cells. Conclusions: The assay is simple and in a reproducible manner quantifies erythrocyte adhesion to several types of immobilized cells. [ABSTRACT FROM AUTHOR]

Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992). Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b). We have studied the diversity of this SERP region in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa. The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced. Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of anti-blood stage malaria vaccine.

Abstract: Plasmodium merozoites attach to and invade red blood cells (RBCs) during the erythrocytic cycle. The invasion process requires recognition of RBC surface receptors by proteins of the Plasmodium Duffy binding like erythrocyte binding like (DBL-EBP) family. Clones and isolates of Plasmodium falciparum have varying abilities to utilize different RBC receptors, and multiple distinct pathways so far identified depend on glycophorins A, B, C, and as yet unidentified receptors. At present, five members of the DBL-EBP family have been identified in the P. falciparum genome, based on gene structure and amino acid sequence homology. The cardinal features of this family consist of conserved 5′ and 3′ cysteine-rich regions (regions II and VI, respectively) whose cysteine residues are highly conserved along with the majority of aromatic amino acids. In contrast to the single DBL-EBP family member in Plasmodium vivax, in P. falciparum all DBL-EBP family members have a duplication of the conserved 5′ cysteine-rich region denoted as the F1 and F2 domains. These cysteine-rich regions are considered crucial in recognition of erythrocyte receptors and it has been shown that several bind to glycophorins on the erythrocyte surface. Several studies, on both field isolates and laboratory strains have uncovered a relatively high degree of sequence polymorphism in the DBP-EBL genes. This study is now extended to include field isolates collected from sites within Kenya. DNA isolated from blood samples of infected patients was utilized to amplify the region I sequence of ebl-1 gene in order to investigate polymorphism in the region immediately adjacent to the 5′ cysteine-rich domains, and to determine the prevalence of an insertion mutant that effectively knocks out the gene. [Copyright &y& Elsevier]

Plasmodium falciparum infection can lead to a life threatening disease and the pathogenetic mechanisms of severe manifestations are not fully understood. Here, we investigated the capacity of P. falciparum -parasitized red blood cells (PRBC) from 45 children with clinical malaria to induce endothelial cell (EC) apoptosis. In all subjects, PRBC that cytoadhered to ECs could be found albeit to a variable degree. By contrast, PRBC that induce EC apoptosis were found only in nine (20%) subjects. Interestingly, children with neurological manifestations were significantly more likely to harbour apoptogenic strains. There was no quantitative relationship between the capacity of these isolates to cytoadhere and apoptosis induction. We hypothesize that P. falciparum -encoded molecules could be responsible for apoptosis induction and therefore suggest new insights in the pathogenesis of P. falciparum malaria. Further investigations are currently in progress to determine whether these results can be confirmed and to identify putative parasite apoptogenic factors. [ABSTRACT FROM AUTHOR]

Relapse variants in chronic Plasmodium falciparum infections are antigenically distinct from the parental parasites. The variable antigen PfEMP1 expressed at the surface of the infected erythrocyte (IE) is encoded by the var gene family with &ap;60 copies per haploid genome. Placental isolates commonly express DBLγ containing subtypes of var genes with homology to either 3D7var5.2 (varCOMMON) or FCR3varCSA. Here we report that varCOMMON related genes are constitutively transcribed in &ap;60% of malaria infected children in Gabon. varCOMMON is conserved in field isolates over at least 2.1 kb. In 3D7 parasites varCOMMON is present on chromosome 5 (var5.2) and constitutively transcribed in the opposite direction to most other var genes. It lacks a regulatory intron, an acidic terminal segment and ends in telomeric repeat sequences. varCOMMON encodes a large, hypothetical PfEMP1 of a structure similar to previous placenta-binding PfEMP1s but it is not present at the IE-surface. IE of a 3D7 clone (3D7S8) transcribe varCOMMON but express a PfEMP1 distinct from varCOMMON at the surface and adhere to placental tissues through varCOMMON independent novel mechanisms. Our report suggests that expression of varCOMMON type genes is not restricted to placental malaria. [Copyright &y& Elsevier]