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.

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.

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.

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.

The Prevalence of α-Thalassemia and Its Relation to Plasmodium falciparum Infection in Patients Presenting to Clinics in Two Distinct Ecological Zones in Ghana.

Thalassemia and sickle cell disease constitute the most monogenic hemoglobin (Hb) disorders worldwide. Clinical symptoms of α(+)-thalassemia (α(+)-thal) are related to inadequate Hb production and accumulation of ß- and/or γ-globin subunits. The association of thalassemia with malaria remains contentious, though from its distribution it appears to have offered some protection against the disease. Data on the prevalence of thalassemia in Ghana and its link with malaria is scanty and restricted. It was an objective of this cross-sectional study to determine the prevalence of thalassemia in areas representing two of Ghana's distinct ecological zones. The relationship between thalassemia and Plasmodium falciparium (P. falciparum) infection was also ascertained. Overall, 277 patients presenting to health facilities in the study areas were recruited to participate. Tests were carried out to determine the presence of α(+)-thal, sickle cell and malaria parasites in the blood samples of participants. The outcome of this study showed an α(+)-thal frequency of 19.9% for heterozygotes (-α/αα) and 6.8% for homozygotes (-α/-α). Plasmodium falciparum was detected in 17.7% of the overall study population and 14.9% in those with α(+)-thal. No association was observed between those with α(+)-thal and the study sites (p > 0.05). A test of the Hardy-Weinberg law yielded no significant difference (p < 0.001). Findings from this study suggest a modest distribution of α(+)-thal in Ghana with no bias to the ecological zones. Although the prevalence and parasite density were relatively low in those with the disorder, no association was found between them.

Potent trypanocidal curcumin analogs bearing a monoenone linker motif act on trypanosoma brucei by forming an adduct with trypanothione.

We have previously reported that curcumin analogs with a C7 linker bearing a C4-C5 olefinic linker with a single keto group at C3 (enone linker) display midnanomolar activity against the bloodstream form of Trypanosoma brucei. However, no clear indication of their mechanism of action or superior antiparasitic activity relative to analogs with the original di-ketone curcumin linker was apparent. To further investigate their utility as antiparasitic agents, we compare the cellular effects of curcumin and the enone linker lead compound 1,7-bis(4-hydroxy-3-methoxyphenyl)hept-4-en-3-one (AS-HK014) here. An AS-HK014-resitant line, trypanosomes adapted to AS-HK014 (TA014), was developed by in vitro exposure to the drug. Metabolomic analysis revealed that exposure to AS-HK014, but not curcumin, rapidly depleted glutathione and trypanothione in the wild-type line, although almost all other metabolites were unchanged relative to control. In TA014 cells, thiol levels were similar to untreated wild-type cells and not significantly depleted by AS-HK014. Adducts of AS-HK014 with both glutathione and trypanothione were identified in AS-HK014-exposed wild-type cells and reproduced by chemical reaction. However, adduct accumulation in sensitive cells was much lower than in resistant cells. TA014 cells did not exhibit any changes in sequence or protein levels of glutathione synthetase and γ-glutamylcysteine synthetase relative to wild-type cells. We conclude that monoenone curcuminoids have a different mode of action than curcumin, rapidly and specifically depleting thiol levels in trypanosomes by forming an adduct. This adduct may ultimately be responsible for the highly potent trypanocidal and antiparasitic activity of the monoenone curcuminoids.

An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs.

BACKGROUND: The recently introduced SYBR Green1 (SG) assay for testing parasites susceptibility to anti-malarial drugs needs further improvement. This has been necessitated by various setbacks, the major one being the low fluorescence intensity associated with it use. This shortcoming diminishes the anticipated hope that this novel method was going to replace the more traditional ones, such as the isotopic and microscopy. In order to restore confidence in its use, series of experiments to determine conditions that give the best fluorescence intensity were conducted. METHODS: Conditions that yield the maximum fluorescent signal were ascertained by measuring the fluorescence after incubation of Plasmodium falciparum culture at different parasites concentration with lysis buffer containing SYBR Green (LBS) at different time period. In order to ascertain the effect of freeze-thaw on fluorescence intensity, P. falciparum culture was frozen for 1 h, thawed, incubated with LBS and the fluorescence measured. The optimized conditions determined in this study were then used to assess the susceptibility of clinical isolates of P. falciparum to artesunate, chloroquine and mefloquine. The concentration of anti-malarial drug inhibiting parasite growth by 50 % (IC50) for each drug was estimated using the online ICEstimator. The IC50 generated using the optimized SG method determined in this study was compared with that obtained using microscopic method and the previously reported standard SG method. RESULTS: Over all, the SG method was found to be easy to perform and sensitive. Freeze-thaw of parasite culture followed by incubation with lysis buffer containing the dye for 3 h was consistently observed to give the highest fluorescence signal. The IC50 values for chloroquine, mefloquine and artesunate determined were consistent and comparable with that determined with the previously reported standard SG method and the microscopic method. CONCLUSION: The authors conclude that freezing and thawing of parasite culture, followed by incubation with LBS in the dark for 3 h provided a significant improvement in fluorescence signal. The IC50 generated using the improved SG method is comparable with that from microscopy and the standard method.

Use of proscribed chloroquine is associated with an increased risk of pfcrt T76 mutation in some parts of Ghana.

BACKGROUND: After years of disuse of chloroquine (CQ) as first-line anti-malarial drug in Ghana, reports from molecular studies conducted in parts of the country indicate varying prevalence of T76 mutation in the pfcrt gene. This situation has several health implications, one being that mutations that confer resistance to CQ have been reported to show substantial cross-resistance to other anti-malarial drugs. It is important to identify some of the factors contributing to the continuous presence of CQ resistance markers in the country. This study determined the prevalence of T76 mutation in pfcrt gene of Plasmodium falciparum isolates collected from selected areas of the Central region of Ghana and correlated with the level of CQ use in these areas. METHODS: Plasmodium falciparum DNA was extracted from collected blood-blot filter paper samples in the study sites. The prevalence of T76 point mutation in pfcrt gene was assessed using nested PCR followed by RFLP. CQ from pharmacy and chemical shops was obtained using mystery buying method. The extent of CQ use by the participants was determined by measuring the level of the drug in their urine samples using the Saker-Solomon method. RESULTS: Of the 214 P. falciparum isolates analysed, 71.9% were found to have T76 mutation of pfcrt gene. The study revealed that 14.49% of community pharmacies and chemical shops had stocks of CQ for sale while 16.9% of the participants had CQ in their urine samples. There is five times more risks of becoming infected with CQ resistant strain for staying in an area where CQ is stocked for sale [RR = 0.20, p < 0.0001] and thirteen times more risks of having CQ-resistant mutant from those who still use CQ than non-users [OR = 0.08, p < 0.0001]. CONCLUSION: This study has shown that high variation in the prevalence of T76 mutations of P. falciparum is linked with the level of CQ stocking and usage within study area.

High-throughput analysis of antimalarial susceptibility data by the WorldWide Antimalarial Resistance Network (WWARN) in vitro analysis and reporting tool.

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Prevalence of congenital malaria in high-risk Ghanaian newborns: a cross-sectional study.

BACKGROUND: Congenital malaria is defined as malaria parasitaemia in the first week of life. The reported prevalence of congenital malaria in sub-Saharan Africa is variable (0 - 46%). Even though the clinical significance of congenital malaria parasitaemia is uncertain, anti-malarial drugs are empirically prescribed for sick newborns by frontline health care workers. Data on prevalence of congenital malaria in high-risk newborns will inform appropriate drug use and timely referral of sick newborns. METHODS: Blood samples of untreated newborns less than 1 week of age at the time of referral to Korle Bu Teaching hospital in Accra, Ghana during the peak malaria seasons (April to July) of 2008 and 2010 were examined for malaria parasites by, i) Giemsa-stained thick and thin blood smears for parasite count and species identification, ii) histidine-rich protein- and lactic dehydrogenase-based rapid diagnosis tests, or iii) polymerase chain reaction amplification of the merozoite surface protein 2 gene, for identification of sub-microscopic parasitaemia. Other investigations were also done as clinically indicated. RESULTS: In 2008, nine cases of Plasmodium falciparum parasitaemia were diagnosed by microscopy in 405 (2.2%) newborns. All the nine newborns had low parasite densities (≤ 50 per microlitre). In 2010, there was no case of parasitaemia by either microscopy or rapid diagnosis tests in 522 newborns; however, 56/467 (12%) cases of P. falciparum were detected by polymerase chain reaction. CONCLUSION: Congenital malaria is an uncommon cause of clinical illness in high-risk untreated newborns referred to a tertiary hospital in the first week of life. Empirical anti-malarial drug treatment for sick newborns without laboratory confirmation of parasitaemia is imprudent. Early referral of sick newborns to hospitals with resources and skills for appropriate care is recommended.

A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs.

BACKGROUND: Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy. METHODS: A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator. RESULTS: Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy, significant elevation of artesunate IC50 value was observed. The results also suggest the existence of possible cross-resistance among some of the test drugs. CONCLUSION: Ghanaian P. falciparum isolates, to some extent, have become susceptible to chloroquine in vitro, however the increasing trend in artesunate IC50 value observed should be of concern. Continuous monitoring of ACT in Ghana is recommended.

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy.

BACKGROUND: With the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations. METHODS: Archived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003-2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis. RESULTS: The percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003-04, 2005-06, 2007-08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×(2) = 96.31, p <0.001) and pfcrt K76 (×(2) = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (x(2) = 38.52, p <0.001) and pfcrt T76 (x(2) = 43.49, p <0.001) were observed from 2003-2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×(2) = 7.39,p=0.060; ×(2) = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×(2) = 20.75, p < 0.001). CONCLUSION: Increased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.

Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis.

OBJECTIVES: To evaluate existing protocols, based on Alamar blue (resazurin), for the routine determination of drug susceptibility in trichomonads, develop new ones and validate these by screening small antiprotozoal libraries. METHODS: The resazurin-based assay was evaluated by determining fluorescence development in Trichomonas media with various cell densities after various intervals and in the presence of metronidazole. Similar investigations were performed with the alternative fluorophores propidium iodide (PI) and resorufin. The optimized protocols were used to screen for new antitrichomonal compounds. RESULTS: Anaerobic cultures of Trichomonas vaginalis rapidly reduced blue resazurin to red, fluorescent resorufin. However, the ascorbic acid in the culture medium produced similar effects, even in the absence of cells, causing high background fluorescence and variability. Moreover, T. vaginalis rapidly metabolized resorufin to the non-fluorescent and colourless metabolite dihydroresorufin, making the fluorescent signal transient. In contrast, resorufin proved to be an excellent viability probe for Trichomonas due to its chemical stability in media and rapid metabolism by the parasite. We also show that staining with PI after cell permeabilization similarly constitutes a reliable measurement of trophozoite numbers. Using the PI and resorufin assays we determined reproducible EC(50) values and identified potent antitrichomonal compounds from a limited screen of phosphodiesterase inhibitors and phosphonium salts. CONCLUSIONS: The resorufin- and PI-based assays are suitable for routine and high-throughput drug screening, whereas resazurin-based assays are not. These assays constitute a major advance in the current protocols as demonstrated by a successful screen for new antitrichomonal lead compounds.

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.

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.

Symmetrical choline-derived dications display strong anti-kinetoplastid activity.

OBJECTIVES: to investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy. METHODS: from an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed. RESULTS: the activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC(50) values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24-48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation. CONCLUSIONS: the choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation.

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.

Uptake of purines in Plasmodium falciparum-infected human erythrocytes is mostly mediated by the human equilibrative nucleoside transporter and the human facilitative nucleobase transporter.

BACKGROUND: Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside transporter (hENT1), the human facilitative nucleobase transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three transporters to the uptake of adenosine and hypoxanthine. Using the appropriate transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated. METHODS: The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these transporters was also studied. RESULTS: Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 nucleobase transporter whereas adenosine entered predominantly through the hENT1 nucleoside transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC). It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1. CONCLUSION: Collectively, the data obtained in this study clearly show that the endogenous host erythrocyte transporters hENT1 and hFNT1, rather than the NPP, are the major route of entry of purine into parasitized RBC. Inhibitors of hENT1 and hFNT1, as well as the NPP, should be considered in the development of anti-malarials targeted to purine transport.

Detection of artemisinin-resistant Plasmodium falciparum in malarial infection: a brief review of methods.

The WHO has recommended artemisinin-based combination therapy (ACT) for the treatment of malaria in disease endemic countries. In an attempt to assess the sensitivity of Plasmodium falciparum to the drug, various methods have been used. It is the purpose of this short review to critically examine these methods and give recommendations.

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.