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Artemisinin and partner-drug resistance in Plasmodium falciparumare major threats to malaria control and elimination. Triple artemisinin-based combination therapies (TACTs), which combine existing co-formulated ACTs with a second partner drug that is slowly eliminated, might provide effective treatment and delay emergence of antimalarial drug resistance.

ABSTRACTIntroduction: RTS,S/AS01 is currently the most advanced malaria vaccine but provides incomplete, short-term protection. It was developed for use within the expanded program on immunizations (EPI) for African children. Another use could be adding mass RTS,S/AS01 vaccination to the integrated malaria elimination strategy in the Greater Mekong Subregion (GMS), where multidrug-resistant P.falciparumstrains have emerged and spread. Prior to evaluating RTS,S/AS01 in large-scale trials we assessed whether the vaccine, administered with and without antimalarial drugs, is safe and immunogenic in Asian populations.Methods: An open-label, randomized, controlled phase 2 trial was conducted in healthy, adult Thai volunteers. Seven vaccine regimens with and without antimalarial drugs (dihydroartemisinin-piperaquine plus a single low dose primaquine) were assessed. Antibody titres against the PfCSP full-length (NANP) 6, PfCSP anti-C–term, PfCSP full-length (N + C-Terminal) were measured by standard enzyme-linked immunosorbent assays. Liquid chromatography was used to measure piperaquine, primaquine and carboxy-primaquine concentrations.Results: 193 volunteers were enrolled and 186 study participants completed the 6 months follow-up period. One month after the last vaccination all study participants had seroconverted to the PfCSP (NANP)6, and the PfCSP Full Length (N + C-Terminal). More than 90% had seroconverted to the Pfanti-C-Term CSP. There was no indication that drug concentrations were influenced by vaccine regimens or the antibody levels by the drug regimens. Adverse events were similarly distributed between the seven treatment groups. No serious adverse events attributable to the study interventions were detected.Conclusion: This study found that RTS,S/AS01 with and without dihydroartemisinin-piperaquine plus a single low dose primaquine was safe and immunogenic in a healthy, adult Asian population.

Rapid and specific detection of single nucleotide polymorphisms (SNPs) related to drug resistance in infectious diseases is crucial for accurate prognostics, therapeutics and disease management at point-of-care. Here, we present a novel amplification method and provide universal guidelines for the detection of SNPs at isothermal conditions. This method, called USS-sbLAMP, consists of SNP-based loop-mediated isothermal amplification (sbLAMP) primers and unmodified self-stabilizing (USS) competitive primers that robustly delay or prevent unspecific amplification. Both sets of primers are incorporated into the same reaction mixture, but always targeting different alleles; one set specific to the wild type allele and the other to the mutant allele. The mechanism of action relies on thermodynamically favored hybridization of totally complementary primers, enabling allele-specific amplification. We successfully validate our method by detecting SNPs, C580Yand Y493H, in the Plasmodium falciparum kelch 13gene that are responsible for resistance to artemisinin-based combination therapies currently used globally in the treatment of malaria. USS-sbLAMP primers can efficiently discriminate between SNPs with high sensitivity (limit of detection of 5 × 101copies per reaction), efficiency, specificity and rapidness (<35 min) with the capability of quantitative measurements for point-of-care diagnosis, treatment guidance, and epidemiological reporting of drug-resistance.

ABSTRACTConventional 48-h in vitrosusceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivoparasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparumis prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P= 0.001). TMI IC50s correlated significantly with the in vivoresponses to artesunate (parasite clearance time [r= 0.44, P= 0.001] and parasite clearance half-life [r= 0.46, P= 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.

Intraerythrocytic maturation of the malaria parasite Plasmodium falciparumis associated with profound changes in the asymmetry of phospholipids in the lipid bilayer of the parasitized red blood cells pRBCs. These changes may contribute to adherence of pRBCs to endothelial cells. This study investigates the effect of febrile temperature and proinflammatory cytokines on phosphatidylserine PS expression on the exofacial surface of pRBCs during parasite maturation. The expression of PS on the pRBCs was determined by flow cytometry using fluoresceinlabeled annexin V, which specifically binds to PS and a vital nucleic acid fluorochrome for parasite staining. The results showed that PS expression on the surface of pRBCs increased in association with parasite maturation, especially at the late parasite stage. Furthermore, the growth of P. falciparumalso accelerated senescence of the uninfected RBCs in parasite cultures. Exposure to febrile temperature led to significant increases in the expression of PS on the surface of pRBCs, particularly at the late parasite stage associated with the virulence strain of the parasite. In contrast, proinflammatory cytokines had no detectable effect on PS expression on pRBCs. These data suggest that PS molecule expression is more dependent on fever, parasitemia, parasite strain, and virulence than on cytokine exposure. These findings contribute to our understanding of the factors that are involved in malaria pathogenesis. © 2010 International Society for Advancement of Cytometry

ABSTRACTMefloquine is widely used in combination with artemisinin derivatives for the treatment of falciparum malaria. Mefloquine resistance in Plasmodium falciparumhas been related to increased copy numbers of multidrug-resistant gene 1 (pfmdr1). We studied the ex vivo dynamics of pfmdr1gene amplification in culture-adapted P. falciparumin relation to mefloquine resistance and parasite fitness. A Thai P. falciparumisolate (isolate TM036) was assessed by the use of multiple genetic markers as a single genotype. Resistance was selected by exposure to stepwise increasing concentrations of mefloquine up to 30 ng/ml in continuous culture. The pfmdr1gene copy numbers increased as susceptibility to mefloquine declined (P= 0.03). No codon mutations at positions 86, 184, 1034, 1042, and 1246 in the pfmdr1gene were detected. Two subclones of selected parasites (average copy numbers, 2.3 and 3.1, respectively) showed a fitness disadvantage when they were grown together with the original parasites containing a single pfmdr1gene copy in the absence of mefloquine; the multiplication rates were 6.3% and 8.7% lower, respectively (P< 0.01). Modeling of the dynamics of the pfmdr1copy numbers over time in relation to the relative fitness of the parasites suggested that net pfmdr1gene amplification from one to two copies occurs once in every 108parasites and that amplification from two to three copies occurs once in every 103parasites. pfmdr1gene amplification in P. falciparumis a frequent event and confers mefloquine resistance. Parasites with multiple copies of the pfmdr1gene have decreased survival fitness in the absence of drug pressure.

ABSTRACTPlasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariaeand from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfrsequences in 35 isolates of P. malariaecollected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodiumspecies. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).

ABSTRACTThe infectivity of Plasmodium falciparumgametocytes after exposure in vitro to quinine, artesunate, and primaquine was assessed in Anopheles dirus, a major vector of malaria in Southeast Asia. Mature gametocytes (stage 5) of a Thai isolate of P. falciparumwere exposed to the drugs for 24 h in vitro before membrane feeding to A. dirus. After 10 days, the mosquito midguts were dissected and the oocysts were counted. In this system, artesunate showed the most potent transmission-blocking activity; the mean (standard deviation [SD]) 50% and 90% effective concentrations (EC50, and EC90, respectively, in nanograms per milliliter) were 0.1 (0.02) and 0.4 (0.15), respectively. Transmission-blocking activity of quinine and primaquine was observed at relatively high concentrations (SDs): EC50of quinine, 642 (111) ng/ml; EC50of primaquine, 181 (23) ng/ml; EC90of quinine, 816 (96) ng/ml; EC90of primaquine, 543 (43) ng/ml. Artesunate both prevents the maturation of immature P. falciparumgametocytes and reduces the transmission potential of mature gametocytes. Both of these effects may contribute to reducing malaria transmission.

Plasmodium vivax is the second most common species of malaria parasite and causes up to 80 million episodes of infection each year. New drug targets are urgently needed because of emerging resistance to current treatments. To study new potential targets, we have functionally characterized two natural variants of the hexose transporter of P. vivax (PvHT) after heterologous expression in Xenopus oocytes. We show that PvHT transports both glucose and fructose. Differences in the affinity for fructose between the two variants of PvHT establishes that sequence variation is associated with phenotypic plasticity. Mutation of a single glutamine residue, Gln167, predicted to lie in transmembrane helix 5, abolishes fructose transport by PvHT, although glucose uptake is preserved. In contrast, the exofacial site located between predicted helices 5 and 6 of PvHT is not an important determinant of substrate specificity, despite exhibiting sequence polymorphisms between hexose transporters of different Plasmodium spp. Indeed, replacement of twelve residues located within this region of PvHT by those found in the orthologous Plasmodium falciparum sequence (PfHT) is functionally silent with respect to affinity for hexoses. All PvHT variants are inhibited by compound 3361, a long-chain O-3 derivative of D-glucose effective against PfHT. Furthermore, compound 3361 kills short term cultures of P. vivax isolated from patients. These data provide unique insights into the function of hexose transporters of Plasmodium spp. as well as further evidence that they could be targeted by drugs.

ABSTRACTThe activities of primaquine in combination with quinine or artesunate against asexual- and sexual-stage parasites were assessed in 176 adult Thai patients with uncomplicated Plasmodium falciparummalaria. Patients were randomized to one of the six following 7-day oral treatment regimens: (i) quinine alone, (ii) quinine with tetracycline, (iii) quinine with primaquine at 15 mg/day, (iv) quinine with primaquine at 30 mg/day, (v) artesunate alone, or (vi) artesunate with primaquine. Clinical recovery occurred in all patients. There were no significant differences in fever clearance times, rates of P. falciparumreappearance, or recurrent vivax malaria between the six treatment groups. Patients treated with artesunate alone or in combination with primaquine had significantly shorter parasite clearance times (mean ± standard deviation = 65± 18 versus 79 ± 21 h) and lower gametocyte carriage rates (40 versus 62.7%) than those treated with quinine (P≤ 0.007). Primaquine did not affect the therapeutic response (P> 0.2). Gametocytemia was detected in 98 patients (56% [22% before treatment and 34% after treatment]). Artesunate reduced the appearance of gametocytemia (relative risk [95% confidence interval] = 0.34 [0.17 to 0.70]), whereas combinations containing primaquine resulted in shorter gametocyte clearance times (medians of 66 versus 271 h for quinine groups and 73 versus 137 h for artesunate groups; P≤ 0.038). These results suggest that artesunate predominantly inhibits gametocyte development whereas primaquine accelerates gametocyte clearance in P. falciparummalaria.

Nitric oxide (NO) is widely known to inhibit platelet and leukocyte adhesion to endothelium through its regulatory effect on adhesion molecule expression. The objective of the present study was to investigate if NO affects the cytoadherence of Plasmodium falciparum-infected erythrocytes (IRBCs) to human microvascular endothelium (HDMECs) under flow conditions in vitro. The effect of endogenous NO was studied using the NO synthase inhibitor l-NG-nitro-arginine-methyl-ester (l-NAME). Treatment of HDMECs with 3 mmol/L of l-NAMEfor 4 hours significantly enhanced IRBC adhesion and the effect could be reversed by an anti-P-selectin but not an anti-VCAM-1 antibody. The effect of exogenous NO on cytoadherence was studied by using the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PPN). PPN (300 μmol/L) treatment reduced the number of adherent IRBCs on resting HDMECs by down-regulating basal ICAM-1 expression, and on tumor necrosis factor-α-stimulated HDMECs by inhibition of VCAM-1 induction and down-regulation of ICAM-1 expression. The inhibitory effect of PPN on tumor necrosis factor-α-induced VCAM-1 expression at 24 hours was evident when the NO donor was added for as short as 2 hours. These findings suggest that NO may be protective against P. falciparuminfection by inhibiting cytoadherence, and underscore the therapeutic potential of NO in the treatment of severe falciparum malaria.

Hemoglobin E is very common in parts of Southeast Asia. The possible malaria protective effects of this and other inherited hemoglobin abnormalities prevalent in Thailand were assessed in a mixed erythrocyte invasion assay. In vitro, starting at 1% parasitemia,Plasmodium falciparum preferentially invaded normal (HbAA) compared to abnormal hemoglobin (HbH, AE, EE, HCS, β-thalassemia E) red cells (HRBCs). The median (range) ratio of parasitization of HRBCs (n = 109) compared to the controls of different major blood groups was 0.40 (0.08, 0.98), less than half that of the normal red cells (NRBCs) compared to their controls 0.88 (0.53, 1.4;P = .001). The median (range) parasitemia in the HRBCs was 2% (0.1%-9%) compared to 5.2% (1.2%-16.3%) in the NRBCs (P = .001). The proportion of the RBC population that is susceptible to malaria parasite invasion can be described by a selectivity index (SI; observed number of multiply invaded RBCs/number predicted). The heterozygote AE cells differed markedly from all the other cells tested with invasion restricted to approximately 25% of the RBCs; the median (range) SI was 3.8 (1-15) compared with 0.75 (0.1-0.9) for EE RBCs (P < .01). Despite their microcytosis, AE cells are functionally relatively normal in contrast to the RBCs from the other hemoglobinopathies studied. These findings suggest that HbAE erythrocytes have an unidentified membrane abnormality that renders the majority of the RBC population relatively resistant to invasion by P falciparum. This would not protect from uncomplicated malaria infections but would prevent the development of heavy parasite burdens and is consistent with the “Haldane” hypothesis of heterozygote protection against severe malaria for hemoglobin E.

A simple reproducible method for short-term ex-vivo Plasmodium vivaxculture is presented in which glucose, ascorbic acid, thiamine, hypoxanthine, and 50% human AB+serum are added to the standard P. falciparumin-vitro culture medium. Culture of freshly obtained blood samples from patients with acute vivax malaria with >0·5% parasitaemia resulted in >95% complete schizogony. Culture could be continued for 5–6 cycles without the addition of red cells. Criteria for staging the erythrocytic development of P. vivaxin the first schizogonic cycle based on synchronous ex-vivo culture are presented. The asexual cycle was divided into 7 morphological stages: tiny ring (0–6 h), small ring (6–12 h), large ring (12–18 h), early trophozoite (18–28 h), late trophozoite (28–36 h), early schizont (36–42 h) and mature schizont (42–48 h). This simple method of culturing P. vivax ex vivois suitable for antimalarial susceptibility and immunoparasitology studies.

Ring-infected erythrocyte surface antigen (RESA)-positive,Plasmodium falciparum–negative red blood cells (RBCs) are cells from which the malaria parasite has been removed by the host without the destruction of the erythrocyte (“pitting”). The survival of RESA-RBCs in vivo was assessed in 14 severe and 6 uncomplicated falciparum malaria patients. The mean RESA-RBC life of 183 hours (95% confidence interval [CI], 136-246) was longer than the median parasite clearance time of 66 hours (range, 30-108 hours) but shorter than the mean red cell life of 1027 hours (95% CI, 840-1213) (P= .0004), with a median ratio of 0.2:1.0 (range, 0.1-0.7). The estimated median percentage of parasites pitted/body transit was 0.003% (range, 0.001%-0.05%). The rate of rise of the RESA-RBC count during the first 24 hours after antimalarial treatment was significantly faster (P= .036) and the subsequent RESA-RBC survival significantly shorter (P= .017) after treatment with an artemisinin derivative than after treatment with quinine. Parasitization of red cells leads to changes in the erythrocyte that shorten their survival even if the parasite is removed subsequently.

Ring-infected erythrocyte surface antigen (RESA)-positive,Plasmodium falciparum–negative red blood cells (RBCs) are cells from which the malaria parasite has been removed by the host without the destruction of the erythrocyte (“pitting”). The survival of RESA-RBCs in vivo was assessed in 14 severe and 6 uncomplicated falciparum malaria patients. The mean RESA-RBC life of 183 hours (95% confidence interval [CI], 136-246) was longer than the median parasite clearance time of 66 hours (range, 30-108 hours) but shorter than the mean red cell life of 1027 hours (95% CI, 840-1213) (P = .0004), with a median ratio of 0.2:1.0 (range, 0.1-0.7). The estimated median percentage of parasites pitted/body transit was 0.003% (range, 0.001%-0.05%). The rate of rise of the RESA-RBC count during the first 24 hours after antimalarial treatment was significantly faster (P = .036) and the subsequent RESA-RBC survival significantly shorter (P = .017) after treatment with an artemisinin derivative than after treatment with quinine. Parasitization of red cells leads to changes in the erythrocyte that shorten their survival even if the parasite is removed subsequently.

To characterize red cell susceptibility to invasion in malaria, a selectivity index (SI) was calculated as the ratio of observed number of multiple-infected red cells to that expected from a random process (Poisson distribution). In patients with falciparum malaria (n= 100) SI decreased with increasing parasitaemia (P< 0·001), and correlated inversely with plasma lactate concentrations, chosen prospectively as a measure of disease severity (r= -0·36, P< 0·001). For parasitaemias <5%, the SI was lower in patients with severe malaria (geometric mean 1·35; 95% confidence interval 1· 01– 1·80) than in uncomplicated malaria (2·31; 1·89-2-81; P=0·003), despite similar parasite counts. The geometric mean (range) SI in vivax malaria (n= 20), 7·69 (1·67, 29·75), was significantly greater than that in falciparum malaria at comparable parasitaemias (=2%), 2·44 (0·45, 14·05), P< 0·001, suggesting that about 13% of circulating erythrocytes were susceptible to invasion by Plasmodium vivax. This translates into susceptibility for about 2 weeks after emergence from the bone marrow, if age is the sole determinant of this process. In falciparum malaria selectivity was inversely proportional to severity; lack of selectivity could reflect either a ‘favourable’ host red cell phenotype, or an indiscriminate parasite population. Both are dangerous for the host.

During acute falciparum malaria infection, red blood cells (RBC) containing abundant ring-infected erythrocyte surface antigen (Pf 155 or RESA), but no intracellular parasites, are present in the circulation. These RESA-positive parasite negative RBC are not seen in parasite cultures in vitro. This indicates that in acute falciparum malaria there is active removal of intraerythrocytic parasites by a host mechanism in vivo (probably the spleen) without destruction of the parasitized RBC. This may explain the observed disparity between the drop in hematocrit and decrease in parasite count in some hyperparasitemic patients. The fate of these “once-parasitized” RBC in vivo is not known.

Resistance to artemisinin-based combination therapy (ACT) in the Plasmodium falciparumparasite is threatening to reverse recent gains in reducing global deaths from malaria. While resistance manifests as delayed parasite clearance in patients, the phenotype can only spread geographically via the sexual stages and mosquito transmission. In addition to their asexual killing properties, artemisinin and its derivatives sterilize sexual male gametocytes. Whether resistant parasites overcome this sterilizing effect has not, however, been fully tested.

Artemisinin-based combination therapies (ACTs) have contributed substantially to the global decline in Plasmodium falciparummorbidity and mortality, but resistance to artemisinins and their partner drugs is increasing in Southeast Asia, threatening malaria control. New antimalarial compounds will not be generally available soon.

ABSTRACTArtemisinin (ART) resistance has spread through Southeast Asia, posing a serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparumkelch-13 (Pfk13) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients due to the reduced susceptibility of early ring-stage parasites to the active metabolite of ART dihydroartemisinin (DHA). Early rings can enter a state of quiescence upon DHA exposure and resume growth in its absence. These quiescent rings are referred to as dormant rings or DHA-pretreated rings (here called dormant rings). The imidazolopiperazines (IPZ) are a novel class of antimalarial drugs that have demonstrated efficacy in early clinical trials. Here, we characterized the stage of action of the IPZ GNF179 and evaluated its activity against rings and dormant rings in wild-type and ART-resistant parasites. Unlike DHA, GNF179 does not induce dormancy. We show that GNF179 is more rapidly cidal against schizonts than against ring and trophozoite stages. However, with 12 h of exposure, the compound effectively kills rings and dormant rings of both susceptible and ART-resistant parasites within 72 h. We further demonstrate that in combination with ART, GNF179 effectively prevents recrudescence of dormant rings, including those bearing pfk13propeller mutations.

ABSTRACTBackground: The use of obstetric ultrasound in the diagnosis of gestational trophoblastic disease (GTD) in high-income settings is well established, leading to prompt management and high survival rates. Evidence from low-income settings suggests ultrasound is essential in identifying complicated pregnancies, but with limited studies reviewing specific conditions including GTD.Objective: The aim of this study is to review the role of ultrasound in diagnosis and management of GTD in a marginalized population on the Thailand–Myanmar border. Antenatal ultrasound became available in this rural setting in 2001 and care for women with GTD has been provided by Thailand public hospitals for 20 years.Design:Retrospective record review.Results: The incidence of GTD was 103 of 57,004 pregnancies in Karen and Burmese women on the Thailand–Myanmar border from 1993–2013. This equates to a rate of 1.8 (95% CI 1.5–2.2) per 1000 or 1 in 553 pregnancies. Of the 102 women with known outcomes, one (1.0%) died of haemorrhage at home. The median number of days between first antenatal clinic attendance and referral to hospital was reduced from 20 (IQR 5–35; range 1–155) to 2 (IQR 2–6; range 1–179) days (p = 0.002) after the introduction of ultrasound. The proportion of severe outcomes (death and total abdominal hysterectomy) was 25% (3/12) before ultrasound compared to 8.9% (8/90) with ultrasound (p = 0.119). A recurrence rate of 2.5% (2/80) was observed in the assessable population. The presence of malaria parasites in maternal blood was not associated with GTD.Conclusions: The rate of GTD in pregnancy in this population is comparable to rates previously reported within South-East Asia. Referral time for uterine evacuation was significantly shorter for those women who had an ultrasound. Ultrasound is an effective method to improve diagnosis of GTD in low-income settings and an effort to increase availability in marginalized populations is required.