Putative molecular markers of Plasmodium falciparum resistance to antimalarial drugs in malaria parasites from Ghana.

Introduction: Antimalarial drugs including artemisinin-based combination therapy (ACT) regimens and sulphadoxine-pyrimethamine (SP) are used in Ghana for malaria therapeutics and prophylaxis respectively. The genetic basis of Plasmodium falciparum development of drug resistance involves single nucleotide polymorphisms in genes encoding proteins for multiple cellular and metabolic processes. The prevalence of single nucleotide polymorphisms in nine P. falciparum genes linked to ACT and SP resistance in the malaria parasite population was determined. Methods: Archived filter paper blood blot samples from patients aged 9 years and below with uncomplicated malaria reporting at 10 sentinel sites located in three ecological zones for the Malaria Therapeutic Efficacy Studies were used. The samples used were collected from 2007-2018 malaria transmission seasons and mutations in the genes were detected using PCR and Sanger sequencing. Results: In all 1,142 samples were used for the study. For falcipain-2 gene (pffp2), Sanger sequencing was successful for 872 samples and were further analysed. The prevalence of the mutants was 45% (392/872) with pffp2 markers V51I and S59F occurring in 15.0% (128/872) and 3.0% (26/872) of the samples respectively. Prevalence of other P. falciparum gene mutations: coronin (pfcoronin) was 44.8% (37/90); cysteine desulfurase (pfnfs) was 73.9% (68/92); apicoplast ribosomal protein S10 (pfarps10) was 36.8% (35/95); ferredoxin (pffd) was 8.8% (8/91); multidrug resistance protein-1 (pfmrp1) was 95.2.0% (80/84); multidrug resistance protein-2 (pfmrp2) was 91.4% (32/35); dihydrofolate reductase (pfdhfr) was 99.0% (84/85); dihydropteroate synthase (pfdhps) was 72% (68/95). Discussion: The observation of numerous mutations in these genes of interest in the Ghanaian isolates, some of which have been implicated in delayed parasite clearance is of great interest. The presence of these genotypes may account for the decline in the efficacies of ACT regimens being used to treat uncomplicated malaria in the country. The need for continuous monitoring of these genetic markers to give first-hand information on parasite susceptibility to antimalarial drugs to inform policy makers and stakeholders in malaria elimination in the country is further discussed.

Effectiveness of artemether-lumefantrine for treating uncomplicated malaria in low- and high-transmission areas of Ghana.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been effective in the supervised treatment of uncomplicated malaria in Ghana. Since ACT usage is primarily unsupervised, this study aimed to determine the effectiveness of artemether-lumefantrine (AL) for treating malaria patients in two transmission settings in Ghana. METHODS: Eighty-four individuals with uncomplicated Plasmodium falciparum malaria were recruited from Lekma Hospital (LH) in Accra (low-transmission area; N = 28), southern Ghana, and King's Medical Centre (KMC) in Kumbungu (high-transmission area; N = 56), northern Ghana. Participants were followed up for 28 days after unsupervised treatment with AL. The presence of asexual parasites was determined by microscopic examination of Giemsa-stained blood smears. Plasmodium species identification was confirmed using species-specific primers targeting the 18S rRNA gene. Parasite recrudescence or reinfection was determined by genotyping the Pfmsp 1 and Pfmsp 2 genes. RESULTS: After AL treatment, 3.6% (2/56) of the patients from KMC were parasitaemic on day 3 compared to none from the LH patients. One patient from KMC with delayed parasite clearance on day 3 remained parasite-positive by microscopy on day 7 but was parasite-free by day 14. While none of the patients from LH experienced parasite recurrence during the 28-day follow-up, three and two patients from KMC had recurrent parasitaemia on days 21 and 28, respectively. Percentage reduction in parasite densities from day 1, 2, and 3 for participants from the KMC was 63.2%, 89.5%, and 84.5%. Parasite densities for participants from the LH reduced from 98.2%, 99.8% on day 1, and 2 to 100% on day 3. The 28-day cumulative incidence rate of treatment failure for KMC was 12.8% (95% confidence interval: 1.9-23.7%), while the per-protocol effectiveness of AL in KMC was 89.47%. All recurrent cases were assigned to recrudescence after parasite genotyping by Pfmsp 1 and Pfmsp 2. CONCLUSION: While AL is efficacious in treating uncomplicated malaria in Ghana, when taken under unsupervised conditions, it showed an 89.4% PCR-corrected cure rate in northern Ghana, which is slightly below the WHO-defined threshold.

Assessment of artemisinin tolerance in Plasmodium falciparum clinical isolates in children with uncomplicated malaria in Ghana.

BACKGROUND: Artemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated malaria in Ghana. Artemisinin (ART) tolerance in Plasmodium falciparum has arisen in Southeast Asia and recently, in parts of East Africa. This is ascribed to the survival of ring-stage parasites post treatment. The present study sought to assess and characterize correlates of potential ART tolerance based on post-treatment parasite clearance, ex vivo and in vitro drug sensitivity, and molecular markers of drug resistance in P. falciparum isolates from children with uncomplicated malaria in Ghana. METHODS: Six months to fourteen years old children presenting with acute uncomplicated malaria (n = 115) were enrolled in two hospitals and a Health Centre in Ghana's Greater Accra region and treated with artemether-lumefantrine (AL) according to body weight. Pre- and post-treatment parasitaemia (day 0 and day 3) was confirmed by microscopy. The ex vivo ring-stage survival assay (RSA) was used to detect percent ring survival while the 72 h SYBR Green I assay was used to measure the 50% inhibition concentration (IC50s) of ART and its derivatives and partner drugs. Genetic markers of drug tolerance /resistance were evaluated using selective whole genome sequencing. RESULTS: Of the total of 115 participants, 85 were successfully followed up on day 3 post-treatment and 2/85 (2.4%) had parasitaemia. The IC50 values of ART, artesunate (AS), artemether (AM), dihydroartemisinin (DHA), amodiaquine (AQ), and lumefantrine (LUM) were not indicative of drug tolerance. However, 7/90 (7.8%) pre-treatment isolates had > 10% ring survival rates against DHA. Of the four isolates (2 RSA positive and 2 RSA negative) with high genomic coverage, P. falciparum (Pf) kelch 13 K188* and Pfcoronin V424I mutations were only present in the two RSA positive isolates with > 10% ring survival rates. CONCLUSIONS: The observed low proportion of participants with day-3 post-treatment parasitaemia is consistent with rapid ART clearance. However, the increased rates of survival observed in the ex vivo RSA against DHA, maybe a pointer of an early start of ART tolerance. Furthermore, the role of two novel mutations in PfK13 and Pfcoronin genes, harboured by the two RSA positive isolates that had high ring survival in the present study, remains to be elucidated.

Transcriptome-module phenotype association study implicates extracellular vesicles biogenesis in Plasmodium falciparum artemisinin resistance.

Plasmodium falciparum malaria is still an important disease in sub-Saharan Africa (sSA). Great strides have been made in its control spear-headed by artemisinin (ART)-based combination therapies (ACTs). However, concerns about the imminent spread of ART-resistant (ARTr) malaria parasites to sSA threaten gains already made. Attempts to mitigate this risk have highlighted the need to discover novel P. falciparum drug targets. Therefore, studies to deepen our understanding of the biology of P. falciparum are needed. The role of extracellular vesicles (EVs) in the biology of malaria parasites is not fully understood. Recently, the ART resistance-associated transcriptional profile has been reported to involve several biological processes connected to vesicular trafficking, proteotoxic stress, erythrocyte remodelling, and mitochondrial metabolism. We explored a role for EVs in developing the P. falciparum ARTr phenotype using bulk RNA sequencing of unsynchronized parasite cultures under untreated, 0.1% dimethyl sulfoxide and 700nM dihydroartemisinin treated conditions for six hours. As pathway and gene ontology analysis is limited in its curated knowledge repertoire on EVs biogenesis in P. falciparum, we used a modular (gene set) analysis approach to explore whether an EVs biogenesis module is associated with the ARTr phenotype in P. falciparum. We first generated well-defined EVs modules of interest and used statistical tools to determine differences in their expression among the parasite and treatment conditions. Then we used gene set enrichment analysis to determine the strength of the association between each EVs module of interest and the ARTr phenotype. This transcriptome-module phenotype association study (TMPAS) represents a well-powered approach to making meaningful discoveries out of bulk gene expression data. We identified four EVs module of interest and report that one module representing gene sets with correlated expression to PF3D7_1441800 - involved with EVs biogenesis in P. falciparum - is associated with the ARTr phenotype (R539T_DHA_treated versus R539T_untreated: normalized enrichment score (NES) = 1.1830174, FDR q-value < 0.25; C580R_DHA_treated versus C580R_untreated: NES = 1.2457103, FDR q-value < 0.25). PF3D7_1441800 has been reported to reduce EVs production when knocked out in P. falciparum. Altogether, our findings suggest a role for EVs in developing ART resistance and warrant further studies interrogating this association.

Plasmodium falciparum merozoite invasion ligands, linked antimalarial resistance loci and ex vivo responses to antimalarials in The Gambia.

BACKGROUND: Artemether/lumefantrine is the most commonly used artemisinin-based combination treatment (ACT) for malaria in sub-Saharan Africa. Drug resistance to ACT components is a major threat to malaria elimination efforts. Therefore, rigorous monitoring of drug efficacy is required for adequate management of malaria and to sustain the effectiveness of ACTs. OBJECTIVES: This study identified and described genomic loci that correlate with differences in ex vivo responses of natural Plasmodium falciparum isolates from The Gambia to antimalarial drugs. METHODS: Natural P. falciparum isolates from The Gambia were assayed for IC50 responses to four antimalarial drugs (artemether, dihydroartemisinin, amodiaquine and lumefantrine). Genome-wide SNPs from 56 of these P. falciparum isolates were applied to mixed-model regression and network analyses to determine linked loci correlating with drug responses. Genomic regions of shared haplotypes and positive selection within and between Gambian and Cambodian P. falciparum isolates were mapped by identity-by-descent (IBD) analysis of 209 genomes. RESULTS: SNPs in 71 genes, mostly involved in stress and drug resistance mechanisms correlated with drug responses. Additionally, erythrocyte invasion and permeability loci, including merozoite surface proteins (Pfdblmsp, Pfsurfin), and high-molecular-weight rhoptry protein 2 (Pfrhops2) were correlated with responses to multiple drugs. Haplotypes of pfdblmsp2 and known drug resistance loci (pfaat1, pfcrt and pfdhfr) from The Gambia showed high IBD with those from Cambodia, indicating co-ancestry, with significant linkage disequilibrium between their alleles. CONCLUSIONS: Multiple linked genic loci correlating with drug response phenotypes suggest a genomic backbone may be under selection by antimalarials. This calls for further analysis of molecular pathways to drug resistance in African P. falciparum.

Malaria artemisinin resistance: an extracellular vesicles export hypothesis.

Plasmodium falciparum causes malaria, and its resistance to artemisinin (ART) - a drug used for managing malaria - threatens to interfere with the effective control of malaria. ART resistance (ARTr) is driven by increased tolerance to oxidative stress and reduced haemoglobin trafficking to the food vacuole. We discuss how extracellular vesicles (EVs) may play a role in developing ARTr.

Assessing the Roles of Molecular Markers of Antimalarial Drug Resistance and the Host Pharmacogenetics in Drug-Resistant Malaria.

Malaria caused by the Plasmodium parasites is a major public health concern in malaria-endemic regions with P. falciparum causing the most severe form of the disease. The use of antimalarial drugs for the management of the disease proves to be one of the best methods to manage the disease. Unfortunately, P. falciparum has developed resistance to almost all the current in-use antimalarial drugs. Parasite development of resistance is primarily caused by both parasite and host genetic factors. The parasite genetic factors involve undergoing mutation in the drug target sites or increasing the drug target gene copy number to prevent the intended action of the antimalarial drugs. The host pharmacogenetic factors which determine how a particular antimalarial drug is metabolized could result in variations of drug plasma concentration and consequently contribute to variable treatment outcomes and the emergence or propagation of resistant parasites. Since both host and parasite genomes play a role in antimalarial drug action, a key question often asked is, "which of the two strongly drives or controls antimalarial drug resistance?" A major finding in our recent study published in the Malaria Journal indicates that the parasite's genetic factors rather than the host are likely to energize resistance to an antimalarial drug. However, others have reported contrary findings suggesting that the host genetic factors are the force behind resistance to antimalarial drugs. To bring clarity to these observations, there is the need for deciphering the major driving force behind antimalarial drug resistance through optimized strategies aimed at alleviating the phenomenon. In this direction, literature was systematically reviewed to establish the role and importance of each of the two factors aforementioned in the etiology of drug-resistant malaria. Using Internet search engines such as Pubmed and Google, we looked for terms likely to give the desired information which we herein present. We then went ahead to leverage the obtained information to discuss the globally avid aim of combating antimalarial drug resistance.

Novel pfk13 polymorphisms in Plasmodium falciparum population in Ghana.

The molecular determinants of Plasmodium falciparum artemisinin resistance are the single nucleotide polymorphisms in the parasite's kelch propeller domain, pfk13. Validated and candidate markers are under surveillance in malaria endemic countries using artemisinin-based combination therapy. However, pfk13 mutations which may confer parasite artemisinin resistance in Africa remains elusive. It has therefore become imperative to report all observed pfk13 gene polymorphisms in malaria therapeutic efficacy studies for functional characterization. We herein report all novel pfk13 mutations observed only in the Ghanaian parasite population. In all, 977 archived samples from children aged 12 years and below with uncomplicated malaria from 2007 to 2017 were used. PCR/Sanger sequencing analysis revealed 78% (763/977) of the samples analyzed were wild type (WT) for pfk13 gene. Of the 214 (22%) mutants, 78 were novel mutations observed only in Ghana. The novel SNPs include R404G, P413H, N458D/H/I, C473W/S, R529I, M579T/Y, C580R/V, D584L, N585H/I, Q661G/L. Some of the mutations were sites and ecological zones specific. There was low nucleotide diversity and purifying selection at the pfk13 locus in Ghanaian parasite population. With increasing drug pressure and its consequent parasite resistance, documenting these mutations as baseline data is crucial for future molecular surveillance of P. falciparum resistance to artemisinin in Ghana.

Therapeutic efficacy of dihydroartemisinin-piperaquine combination for the treatment of uncomplicated malaria in Ghana.

In 2020, Dihydroartemisinin-Piperaquine (DHAP) was adopted as a second-line antimalarial for treatment of uncomplicated malaria in Ghana following a review of the country's antimalarial medicines policy. Available data obtained in 2007 had shown PCR-uncorrected therapeutic efficacy of 93.3% using a 28-day follow-up schedule. In 2020, the standard 42-day follow-up schedule for DHAP was used to estimate efficacy levels among febrile children aged 6 months to 9 years in three malaria sentinel sites representing the three main ecological zones of the country- savannah, forest, and coastal. PCR genotyping distinguished between recrudescence and re-infection using merozoite surface protein 2 (MSP2)-specific primers for FC27 and 3D7 strains. Per protocol analyses showed day 28 efficacy of 100% in all three sentinel sites with day 42 PCR-corrected efficacy ranging between 90.3% (95% CI: 80.1 - 96.4%) in the savannah zone and 100% in the forest and coastal zones, yielding a national average of 97.0% (95% CI: 93.4 - 98.8). No day 3 parasitemia was observed in all three sites. Prevalence of measured fever (axillary temperature ≥ 37.5°C) declined from 50.0 - 98.8% on day 0 to 7.1-11.5% on day 1 whilst parasitemia declined from 100% on day 0 to 1.2 - 2.3% on day 1. Mean haemoglobin levels on days 28 and 42 were significantly higher than pre-treatment levels in all three sites. We conclude that DHAP is highly efficacious in the treatment of uncomplicated malaria in Ghana. This data will serve as baseline for subsequent DHAP efficacy studies in the country.

Genetic deletions and high diversity of Plasmodium falciparum histidine-rich proteins 2 and 3 genes in parasite populations in Ghana.

Rapid diagnostic tests (RDTs) are used to diagnose malaria in Ghana and other malaria endemic countries. Plasmodium falciparum histidine-rich protein 2 (PFHRP2) based RDTs are widely used, however the occurrence of deletions of the pfhrp2 gene in some parasites have resulted in false negative test results. Monoclonal antibodies of PFHRP2 cross reacts with PFHRP3 because they share structural similarities and this complements the detection of the parasites by RDT. These two genes were investigated in Ghanaian P. falciparum parasite population to detect deletions and the polymorphisms in exon 2 of the pfhrp2 and pfhrp3 genes. Parasite isolates (2,540) from children ≤ 12 years with uncomplicated malaria from 2015 to 2020 transmission seasons were used. Both genes were amplified using nested PCR and negative results indicated the presence of the deletion of genes. Amplified genes were sequenced for the detection of the amino acid repeats. Deletions were observed in 30.7% (780/2,540) and 17.2% (438/2,540) of the samples for pfhrp2 and pfhrp3 respectively with increasing trends over the three time periods (χ2 -10.305, p = 0.001). A total of 1,632 amplicons were sequenced for each gene, analysis was done on 1,124 and 1,307 good quality sequences for pfhrp2 and pfhrp3 respectively. Pfhrp2 repeat polymorphisms were dominantly of types 2 (AHHAHHAAD) and 7 (AHHAAD) with large numbers of variants. A novel variant of type 14 (AHHANHATD) was seen for pfhrp2. For the pfhrp3 repeat types, 16 (AHHAAN), 17 (AHHDG) and 18 (AHHDD) were the dominant types observed. Variants of type 16 (AHHAAH) and (AHHASH) were also dominant. Repeat types 1, 2, 3, 4, 5, 6, 7, 8, 11, 13, 15, 16, and 19 were observed be shared by both genes. The haplotype diversity of both genes ranged between 0.872 and 1 indicating high diversity of the polymorphisms in the isolates. The implication of the findings of the frequencies of the pfhrp2 and pfhrp3 deletions as well as the variants of the main epitopes of the monoclonal antibodies for the RDT (types 2 and 7) in our isolates is an indication of decreased sensitivity of the RDTs in diagnosing malaria infections in Ghana.

Trends and predictive factors for treatment failure following artemisinin-based combination therapy among children with uncomplicated malaria in Ghana: 2005-2018.

BACKGROUND: Since the introduction of artemisinin-based combination therapy (ACT) in Ghana in 2005 there has been a surveillance system by the National Malaria Control Programme (NMCP) and the University of Ghana Noguchi Memorial Institute for Medical Research (UG-NMIMR) to monitor the therapeutic efficacy of ACTs for the treatment of uncomplicated malaria in the country. We report trends and determinants of failure following treatment of Ghanaian children with artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) combinations. METHODS: Per protocol analyses as well as cumulative incidence of day 28 treatment failure from Kaplan Meier survival analyses were used to describe trends of failure over the surveillance period of 2005-2018. Univariable and multivariable cox regression analyses were used to assess the determinants of treatment failure over the period. RESULTS: Day 28 PCR-corrected failure, following treatment with ASAQ, significantly increased from 0.0% in 2005 to 2.0% (95% CI: 1.1-3.6) in 2015 (p = 0.013) but significantly decreased to 0.4% (95% CI: 0.1-1.6) in 2018 (p = 0.039). Failure, following treatment with AL, decreased from 4.5% (95% CI: 2.0-9.4) in 2010 to 2.7% (95% CI: 1.4-5.1) in 2018, though not statistically significant (p = 0.426). Risk of treatment failure, from multivariable cox regression analyses, was significantly lower among children receiving ASAQ compared with those receiving AL (HR = 0.24; 95% CI: 0.11-0.53; p < 0.001); lower among children with no parasitaemia on day 3 compared with those with parasitaemia on day 3 (HR = 0.02; 95% CI: 0.01-0.13; p < 0.001); and higher among children who received ASAQ and had axillary temperature ≥ 37.5 °C on day 1 compared with those with axillary temperature < 37.5 °C (HR = 3.96; 95% CI: 1.61-9.75; p = 0.003). CONCLUSIONS: Treatment failures for both ASAQ and AL have remained less than 5% (below WHO's threshold of 10%) in Ghana since 2005. Predictors of treatment failure that need to be considered in the management of uncomplicated malaria in the country should include type of ACT, day 3 parasitaemia, and day 1 axillary temperature of patients being treated.

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter.

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.

Malaria parasites both repress host CXCL10 and use it as a cue for growth acceleration.

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.

Implicating extracellular vesicles in Plasmodium falciparum artemisinin resistance development.

Plasmodium falciparum malaria remains a disease of significant public health impact today. With the risk of emerging artemisinin resistance stalling malaria control efforts, the need to deepen our understanding of the parasite's biology is dire. Extracellular vesicles (EVs) are vital to the biology of P. falciparum and play a role in the pathogenesis of malaria. Recent studies have also shown that EVs may play a role in the development of artemisinin resistance in P. falciparum. Here, we highlight evidence on EVs in P. falciparum biology and malaria pathogenesis and argue that there is sufficient ground to propose a role for EVs in the development of P. falciparum artemisinin resistance. We suggest that EVs are actively secreted functional organelles that contribute to cellular homeostasis in P. falciparum-infected red blood cells under artemisinin pressure. Further exploration of this hypothesized EVs-based molecular mechanism of artemisinin resistance will aid the discovery of novel antimalarial therapies.

Plasmodium falciparum Malaria Parasites in Ghana Show Signatures of Balancing Selection at Artemisinin Resistance Predisposing Background Genes.

Sub-Saharan Africa is courting the risk of artemisinin resistance (ARTr) emerging in Plasmodium falciparum malaria parasites. Current molecular surveillance efforts for ARTr have been built on the utility of P. falciparum kelch13 (pfk13) validated molecular markers. However, whether these molecular markers will serve the purpose of early detection of artemisinin-resistant parasites in Ghana is hinged on a pfk13 dependent evolution. Here, we tested the hypothesis that the background pfk13 genome may be present before the pfk13 ARTr-conferring variant(s) is selected and that signatures of balancing selection on these genomic loci may serve as an early warning signal of ARTr. We analyzed 12 198 single nucleotide polymorphisms (SNPs) in Ghanaian clinical isolates in the Pf3K MalariaGEN dataset that passed a stringent filtering regimen. We identified signatures of balancing selection in 2 genes (phosphatidylinositol 4-kinase and chloroquine resistance transporter) previously reported as background loci for ARTr. These genes showed statistically significant and high positive values for Tajima's D, Fu and Li's F, and Fu and Li's D. This indicates that the biodiversity required to establish a pfk13 background genome may have been primed in clinical isolates of P. falciparum from Ghana as of 2010. Despite the absence of ARTr in Ghana to date, our finding supports the current use of pfk13 for molecular surveillance of ARTr in Ghana and highlights the potential utility of monitoring malaria parasite populations for balancing selection in ARTr precursor background genes as early warning molecular signatures for the emergence of ARTr.

High Prevalence of Molecular Markers of Plasmodium falciparum Resistance to Sulphadoxine-Pyrimethamine in Parts of Ghana: A Threat to ITPTp-SP?

Malaria in pregnancy is a huge public health problem as it is the cause of maternal anaemia, still birth, premature delivery, low birth weight among others. To tackle this problem, WHO recommended the administration, during pregnancy, of intermittent preventive treatment with sulphadoxine-pyrimethamine (IPTp-SP). The introduction of this policy is likely to create SP drug pressure which may lead to the emergence of parasite strains resistant to the drug. This study investigated the prevalence of the molecular markers of SP resistance as pointers to potential failure of IPTp-SP among pregnant women attending antenatal clinic, women at the point of baby delivery and out patients department (OPD) attendees. The study was conducted in health facilities located in parts of Ghana. Prevalence of mutations in dhfr and dhps genes of Plasmodium falciparum was determined using the method described by Duraisingh et al. The outcome of the study indicated the presence of high prevalence of strains of P.falciparum with the resistant alleles of the dhfr or dhps genes in the three categories of participants. There was a high prevalence of triple mutations (IRN) in the dhfr gene of P.falciparum isolates: 71.4% in peripheral blood of antenatal attendees; 74.1% in placenta cord blood of delivering mothers and 71.1% in OPD attendees. Quintuple mutations were only found in 2 (0.5%) isolates from OPD attendees. This observation might have occurred due to the increased use of SP for IPTp among others. There is the need for an interventional measure in order to protect pregnant women and their unborn children.Lay summaryWhen pregnant women get infected with the malaria parasites they are exposed to all manner of dangers including pre-term delivery, still birth, maternal anaemia and low birth weight. Taking sulphadoxine-pyrimethamine (SP) at predetermined periods during pregnancy, referred to as 'intermittent preventive treatment with SP' (IPTp-SP)' helps to curtail these problems. However, the frequent taking of these drugs is likely to create SP drug pressure which may lead to the emergence of parasite strains that are not readily killed by the drugs. In order to ascertain this phenomenon and advice stakeholders, this study determined the prevalence of certain 'materials' certified as markers of parasite resistance to SP. Alarmingly, more than 5% of all the category of women recruited to participate in this study were found to harbour the parasites that causes malaria. The outcome, also suggest the existence of high levels of strains of the malaria parasite, carrying the materials that make them to become resistant to SP. Policy makers must pay attention to these observations and institute measures to avoid escalation of the situation.

Treatment of COVID-19 with Chloroquine: Implication for Malaria Chemotherapy Using ACTs in Disease Endemic Countries.

Based on reports of parasite resistance and on World Health Organization recommendation, chloroquine was replaced with the artemisinin-based combination therapies (ACTs) as the first choice of drugs for the treatment of uncomplicated malaria. Disuse of chloroquine led to restoration of drug-sensitive parasite to some extent in certain countries. Ever since chloroquine and hydroxychloroquine were touted as potential treatment for coronavirus disease 2019 (COVID-19), there has been a dramatic surge in demand for the drugs. Even in areas where chloroquine is proscribed, there has been an unexpected increase in demand and supply of the drug. This situation is quite worrying as the indiscriminate use of chloroquine may produce drug-resistant parasites which may impact negatively on the efficacy of amodiaquine due to cross-resistance. Amodiaquine is a partner drug in one of the ACTs and in some of the drugs used for intermittent preventive treatment. We herein discuss the consequences of the escalated use of chloroquine in the management of COVID-19 on chemotherapy or chemoprevention of malaria and offer an advice. We speculate that parasite strains resistant to chloroquine will escalate due to the increased and indiscriminate use of the drug and consequently lead to cross-resistance with amodiaquine which is present in some drug schemes aforementioned. Under the circumstance, the anticipated hope of reverting to the use of the 'resurrected chloroquine' to manage malaria in future is likely to diminish. The use of chloroquine and its derivatives for the management of COVID-19 should be controlled.

Plasmodium falciparum genetic factors rather than host factors are likely to drive resistance to ACT in Ghana.

BACKGROUND: Artemisinin-based combination therapy (ACT) partner drugs, currently used in Ghana are lumefantrine, amodiaquine and piperaquine. Plasmodium falciparum isolates with reduced susceptibility to these partner drugs may affect treatment outcome. Mutations in pfmdr1 gene is linked to reduced parasite susceptibility to amodiaquine and lumefantrine. In addition, the potency of the partner drugs in vivo depends on the metabolism by the cytochrome P450 (CYP) enzyme in the host. Mutations in the CYP2C8 and CYP3A4 genes are linked to reduced metabolism of amodiaquine and lumefantrine in vitro, respectively. This study investigated the host and parasite genetic factors affecting the susceptibility of the malaria parasite to ACT partner drugs. METHODS: Archived samples from 240 patients age ≤ 9 years participating in anti-malarial drug resistance survey in Ghana, and given artemether with lumefantrine (AL) or artesunate with amodiaquine (AA), were selected and analysed. Polymerase chain reaction (PCR) followed by Sanger sequencing was used to determine the polymorphisms in CYP2C8, CYP3A4 and pfmdr1 genes. RESULTS: For CYP3A4, all had wild type alleles, suggesting that the hosts are good metabolizers of lumefantrine. For CYP2C8 60% had wild type alleles, 35% heterozygous and 5% homozygous recessive alleles suggesting efficient metabolism of amodiaquine by the hosts. For pfmdr1 gene, at codon 86, 95% were wild type (N86) and 5% mutant (Y86). For codon 184, 36% were wild type (Y184) and 64% mutant (F184) while for codons 1034, 1042 and 1246, 100% (all) were wild type. The high prevalence of N86-F184-D1246 haplotype (NFD) suggest presence of parasites with reduced susceptibility to lumefantrine and not amodiaquine. Delayed clearance was observed in individuals with mutations in the pfmdr1 gene and not cytochrome 450 gene. Both synonymous and non-synonymous mutations were observed in the pfmdr1 at low prevalence. CONCLUSION: The outcome of this study indicates that the parasite's genetic factors rather than the host's are likely to drive resistance to ACT in Ghana.

Estimation of parasite age and synchrony status in Plasmodium falciparum infections.

Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management.

Plasmodium falciparum Kelch Propeller Polymorphisms in Clinical Isolates from Ghana from 2007 to 2016.

The continuous surveillance of polymorphisms in the kelch propeller domain of Plasmodium falciparum from Africa is important for the discovery of the actual markers of artemisinin resistance in the region. The information on the markers is crucial for control strategies involving chemotherapy and chemoprophylaxis for residents and nonimmune travelers to the country. Polymorphisms in the kelch propeller domain of Ghanaian malaria parasites from three different ecological zones at several time periods were assessed. A total of 854 archived samples (2007 to 2016) collected from uncomplicated malaria patients aged ≤9 years old from 10 sentinel sites were used. Eighty-four percent had wild-type sequences (PF3D7_1343700), while many of the mutants had mostly nonsynonymous mutations clustered around codons 404 to 650. Variants with different amino acid changes of the codons associated with artemisinin (ART) resistance validated markers were observed in Ghanaian isolates: frequencies for I543I, I543S, I543V, R561P, R561R, and C580V were 0.12% each and 0.6% for R539I. Mutations reported from African parasites, A578S (0.23%) and Q613L (0.23%), were also observed. Three persisting nonsynonymous (NS) mutations, N599Y (0.005%), K607E (0.004%), and V637G (0.004%), were observed in 3 of the 5 time periods nationally. The presence of variants of the validated markers of artemisinin resistance as well as persisting polymorphisms after 14 years of artemisinin-based combination therapy use argues for continuous surveillance of the markers. The molecular markers of artemisinin resistance and the observed variants will be monitored subsequently as part of ongoing surveillance of antimalarial drug efficacy/resistance studies in the country.