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BackgroundThe long-acting 8-aminoquinoline tafenoquine may be a good candidate for mass drug administration if it exhibits sufficient blood-stage antimalarial activity at doses low enough to be tolerated by glucose 6-phosphate dehydrogenase (G6PD)–deficient individuals.MethodsHealthy adults with normal levels of G6PD were inoculated with Plasmodium falciparum 3D7-infected erythrocytes on day 0. Different single oral doses of tafenoquine were administered on day 8. Parasitemia and concentrations of tafenoquine and the 5,6-orthoquinone metabolite in plasma/whole blood/urine were measured and standard safety assessments performed. Curative artemether-lumefantrine therapy was administered if parasite regrowth occurred, or on day 48 ± 2. Outcomes were parasite clearance kinetics, pharmacokinetic and pharmacokinetic/pharmacodynamic (PK/PD) parameters from modelling, and dose simulations in a theoretical endemic population.ResultsTwelve participants were inoculated and administered 200 mg (n = 3), 300 mg (n = 4), 400 mg (n = 2), or 600 mg (n = 3) tafenoquine. The parasite clearance half-life with 400 mg or 600 mg (5.4 hours and 4.2 hours, respectively) was faster than with 200 mg or 300 mg (11.8 hours and 9.6 hours, respectively). Parasite regrowth occurred after dosing with 200 mg (3/3 participants) and 300 mg (3/4 participants) but not after 400 mg or 600 mg. Simulations using the PK/PD model predicted that 460 mg and 540 mg would clear parasitaemia by a factor of 106 and 109, respectively, in a 60-kg adult.ConclusionsAlthough a single dose of tafenoquine exhibits potent P. falciparum blood-stage antimalarial activity, the estimated doses to effectively clear asexual parasitemia will require prior screening to exclude G6PD deficiency.Clinical Trials Registration. Australian and New Zealand Clinical Trials Registry (ACTRN12620000995976).

Background In 2018, the National Malaria Control Programme in Vietnam switched from prioritizing malaria control to elimination. However, with the ongoing elimination programme, there are still areas where residual malaria transmission persists, including the central highlands. This entomological survey was conducted to evaluate Anopheles diversity and host-seeking activity of Anopheles vectors in two communes with very low malaria transmission in Gia Lai Province. Methods Anopheles species were collected in Ia DReh commune and Ia KDam commune, Gia Lai Province in the central highlands of Vietnam. Collections were conducted using human-baited double net trap, light trap and manual aspiration collections around cattle sheds, in the dry and rainy season. Mosquito specimens were identified morphologically, and members of species complexes were distinguished molecularly. Mosquito night-feeding patterns were investigated during the dry and rainy seasons. Results Overall, 18,835 specimens including 19 taxa were collected in Ia KDam and Ia DReh communes. These included the primary malaria vectors, Anopheles dirus and Anopheles minimus, and other secondary vector species. Anopheles dirus was observed to be an anthropophilic species, whereas An. minimus and a number of secondary vectors were observed to be zoophilic. Anopheles vagus was the dominant species, followed by Anopheles sinensis and Anopheles peditaeniatus. The majority of specimens were collected in the rainy season due to the relatively large number of An. vagus, while An. peditaeniatus, An. dirus, Anopheles kochi, Anopheles monstrosus and Anopheles tessellatus were collected in greater numbers during the dry season. The peak of host-seeking activity for An. dirus, An. sinensis, and An. vagus was between 18.00 and 19.00 h. Conclusion This study provided information on the diversity, seasonal prevalence and behaviour of Anopheles at the study sites. Identifying the diverse mosquito fauna in the central highlands of Vietnam allows species-specific control measures to be implemented by the National Programme to reduce malaria in areas of very low malaria transmission. The peak Anopheles host-seeking activity observed in this study was between 18.00 and 23.00 h, which highlights the need to better characterize Anopheles behaviour in this region of Vietnam to inform on vector control strategies.

BackgroundThe long acting 8-aminoquinoline tafenoquine may be a good candidate for mass drug administration if it exhibits sufficient blood stage antimalarial activity at doses low enough to be tolerated by glucose 6-phosphate dehydrogenase (G6PD) deficient individuals.MethodsHealthy G6PD-normal adults were inoculated withPlasmodium falciparum3D7-infected erythrocytes on day 0. Different single oral doses of tafenoquine were administered on day 8. Parasitemia, and concentrations of tafenoquine and the 5,6-orthoquinone metabolite in plasma/whole blood/urine were measured and standard safety assessments performed. Curative artemether-lumefantrine therapy was administered if parasite regrowth occurred, or on day 48±2. Outcomes were parasite clearance kinetics, pharmacokinetic and pharmacokinetic/pharmacodynamic (PK/PD) parameters from modelling, and dose simulations in a theoretical endemic population.ResultsTwelve participants were inoculated and administered 200 mg (n=3), 300 mg (n=4), 400 mg (n=2), or 600 mg (n=3) tafenoquine. The parasite clearance half-life with 400 mg or 600 mg (5.4 h and 4.2 h respectively) was faster than with 200 mg or 300 mg (11.8 h and 9.6 h respectively). Parasite regrowth occurred after dosing with 200 mg (3/3 participants) and 300 mg (3/4 participants), but not after 400 mg or 600 mg. Simulations using the PK/PD model predicted that 460 mg and 540 mg would clear parasitaemia by a factor of 106and 109, respectively, in a 60 kg adult.ConclusionsAlthough a single dose or tafenoquine exhibits potentP. falciparumblood stage antimalarial activity, the estimated doses to effectively clear asexual parasitemia will require prior screening to exclude G6PD deficiency.Main pointA single oral dose of tafenoquine is effective against blood stagePlasmodium falciparuminfection. However, as the estimated dose to clear asexual parasitaemia is ≥460 mg (in adults), prior screening for glucose 6-phosphate dehydrogenase deficiency will be required.

Background Blocking the transmission of parasites from humans to mosquitoes is a key component of malaria control. Tafenoquine exhibits activity against all stages of the malaria parasite and may have utility as a transmission blocking agent. We aimed to characterize the transmission blocking activity of low-dose tafenoquine. Methods Healthy adults were inoculated with Plasmodium falciparum 3D7-infected erythrocytes on day 0. Piperaquine was administered on days 9 and 11 to clear asexual parasitemia while allowing gametocyte development. A single 50-mg oral dose of tafenoquine was administered on day 25. Transmission was determined by enriched membrane feeding assays predose and at 1, 4, and 7 days postdose. Artemether-lumefantrine was administered following the final assay. Outcomes were the reduction in mosquito infection and gametocytemia after tafenoquine and safety parameters. Results Six participants were enrolled, and all were infective to mosquitoes before tafenoquine, with a median 86% (range, 22–98) of mosquitoes positive for oocysts and 57% (range, 4–92) positive for sporozoites. By day 4 after tafenoquine, the oocyst and sporozoite positivity rate had reduced by a median 35% (interquartile range [IQR]: 16–46) and 52% (IQR: 40–62), respectively, and by day 7, 81% (IQR 36–92) and 77% (IQR 52–98), respectively. The decline in gametocyte density after tafenoquine was not significant. No significant participant safety concerns were identified. Conclusions Low-dose tafenoquine (50 mg) reduces P. falciparum transmission to mosquitoes, with a delay in effect.

The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine in Vietnam justifies the need to evaluate alternative artemisinin-based combination therapies. Between July 2018 and October 2019, a single-arm trial of pyronaridine-artesunate (Pyramax, PA) was conducted in Dak Nong province, Vietnam. PA (3-day course) was administered to adults and children infected with P. falciparum. PA was well tolerated by the participants. The proportion of patients with Day 42 PCR-corrected adequate clinical and parasitological response was 95.2% (95% confidence interval [CI], 82.3 to 98.8, n = 40/42) for treating falciparum malaria. The median parasite clearance half-life was 6.7 h (range, 2.6 to 11.9) and the median parasite clearance time was 72 h (range, 12 to 132) with 44.9% (22/49) of patients having positive blood films at 72 h. The two patients that recrudesced had comparable Day 7 blood pyronaridine concentrations (39.5 and 39.0 ng/ml) to the 40 patients who did not recrudesce (median 43.4 ng/ml, 95% CI, 35.1 to 54.9). Ring-stage and piperaquine survival assays revealed that of the 29 P. falciparum isolates collected from the patients before PA treatment, 22 (75.9%) had reduced susceptibility to artemisinins and 17 (58.6%) were resistant to piperaquine. Genotyping confirmed that 92.0% (46/50) of falciparum patients were infected with parasites bearing the Pfkelch13 C580Y mutation associated with artemisinin resistance. Of these, 56.0% (28/50) of the isolates also had multiple copies of the plasmepsin 2/3 genes responsible for piperaquine resistance. Overall, PA was effective in treating P. falciparum in the Central Highlands of Vietnam. (This study has been registered at AustralianClinicalTrials.gov.au under trial ID ACTRN12618001429246.).

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.

Background Malaria elimination by 2030 is an aim of many countries in the Greater Mekong Sub-region, including Vietnam. However, to achieve this goal and accelerate towards malaria elimination, countries need to determine the extent and prevalence of asymptomatic malaria as a potential reservoir for malaria transmission and the intensity of malaria transmission. The purpose of this study was to determine the prevalence of asymptomatic malaria and seropositivity rate in several districts of Gia Lai province in the Central Highlands of Vietnam. Methods A cross-sectional survey of asymptomatic malaria and serological testing was conducted in 3283 people living at 14 communes across seven districts in Gia Lai province in December 2016 to January 2017. Finger prick capillary blood samples were tested for malaria using rapid diagnostic testing and polymerase chain reaction (PCR), as well as detecting antibodies against 3 Plasmodium falciparum and 4 Plasmodium vivax antigens by indirect enzyme-linked immunosorbent assay (ELISA). Age-seroprevalence curves were fitted using reverse catalytic models with maximum likelihood. Results The study population was predominantly male (65.9%, 2165/3283), adults (88.7%, 2911/3283) and of a minority ethnicity (72.2%, 2371/3283), with most participants being farmers and outdoor government workers (90.2%, 2960/3283). Using a small volume of blood (≈ 10 µL) the PCR assay revealed that 1.74% (57/3283) of the participants had asymptomatic malaria (P. falciparum 1.07%, P. vivax 0.40%, Plasmodium malariae 0.15% and mixed infections 0.12%). In contrast, the annual malaria prevalence rates for clinical malaria in the communities where the participants lived were 0.12% (108/90,395) in 2016 and 0.22% (201/93,184) in 2017. Seropositivity for at least one P. falciparum or one P. vivax antigen was 38.5% (1257/3262) and 31.1% (1022/3282), respectively. Age-dependent trends in the proportion of seropositive individuals in five of the districts discriminated the three districts with sustained low malaria prevalence from the two districts with higher transmission. Conclusions Asymptomatic Plasmodium carriers were found to be substantially more prevalent than clinical cases in seven districts of Gia Lai province, and a third of the population had serological evidence of previous malaria exposure. The findings add knowledge on the extent of asymptomatic malaria and transmission for developing malaria elimination strategies for Vietnam.

Spectroscopy-based techniques are emerging diagnostic and surveillance tools for mosquito-borne diseases. This review has consolidated and summarised recent research in the application of Raman and infrared spectroscopy techniques including near- and mid-infrared spectroscopy for malaria and arboviruses, identified knowledge gaps, and recommended future research directions. Full-length peer-reviewed journal articles related to the application of Raman and infrared (near- and mid-infrared) spectroscopy for malaria and arboviruses were systematically searched in PUBMED, MEDILINE, and Web of Science databases using the PRISMA guidelines. In text review of identified studies included the methodology of spectroscopy technique used, data analysis applied, wavelengths used, and key findings for diagnosis of malaria and arboviruses and surveillance of mosquito vectors. A total of 58 studies met the inclusion criteria for our systematic literature search. Although there was an increased application of Raman and infrared spectroscopy-based techniques in the last 10 years, our review indicates that Raman spectroscopy (RS) technique has been applied exclusively for the diagnosis of malaria and arboviruses. The mid-infrared spectroscopy (MIRS) technique has been assessed for the diagnosis of malaria parasites in human blood and as a surveillance tool for malaria vectors, whereas the near-infrared spectroscopy (NIRS) technique has almost exclusively been applied as a surveillance tool for malaria and arbovirus vectors. Conclusions/Significance The potential of RS as a surveillance tool for malaria and arbovirus vectors and MIRS for the diagnosis and surveillance of arboviruses is yet to be assessed. NIRS capacity as a surveillance tool for malaria and arbovirus vectors should be validated under field conditions, and its potential as a diagnostic tool for malaria and arboviruses needs to be evaluated. It is recommended that all 3 techniques evaluated simultaneously using multiple machine learning techniques in multiple epidemiological settings to determine the most accurate technique for each application. Prior to their field application, a standardised protocol for spectra collection and data analysis should be developed. This will harmonise their application in multiple field settings allowing easy and faster integration into existing disease control platforms. Ultimately, development of rapid and cost-effective point-of-care diagnostic tools for malaria and arboviruses based on spectroscopy techniques may help combat current and future outbreaks of these infectious diseases., Author summary Malaria and many arboviruses such as Dengue virus, Zika virus, Chikungunya virus, and Ross River virus are persistent and detrimental to the global population. Rapid and accurate diagnosis of these infections in human populations and mosquito vectors is essential for understanding their epidemiology, for prompt treatment, and to improve and guide control and elimination strategies. Raman and infrared spectroscopy are rapid and cost-effective tools that have shown potential as diagnostic and surveillance tools for malaria and arboviruses. This systematic review presents up-to-date research conducted using RS, MIRS, and NIRS for the diagnosis of malaria parasite and arboviruses as well as for the surveillance of malaria and arbovirus vectors.

Novel 3,3'-disubstituted-5,5'-bi(1,2,4-triazine) compounds with potent in vitro activity against Plasmodium falciparum parasites were recently discovered. To improve the pharmacokinetic properties of the triazine derivatives, a new structure-activity relationship (SAR) investigation was initiated with a focus on enhancing the metabolic stability of lead compounds. These efforts led to the identification of second-generation highly potent antimalarial bis-triazines, exemplified by triazine 23, which exhibited significantly improved in vitro metabolic stability (8 and 42 μL/min/mg protein in human and mouse liver microsomes). The disubstituted triazine dimer 23 was also observed to suppress parasitemia in the Peters 4-day test with a mean ED50 value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of considerable interest.

Plasmodium falciparum resistance to dihydroartemisinin-piperaquine has spread through the Greater Mekong Subregion to southwestern Vietnam. In 2018 to 2019, we collected 127 P. falciparum isolates from Dak Nong (36), Dak Lak (55), Gia Lai (13), and Kon Tum (23) provinces in Vietnam’s Central Highlands and found parasites bearing the Pfkelch13 C580Y mutation and multiple plasmepsin 2/3 genes (mean prevalence, 17.9%; range, 4.3% to 27.8%), conferring resistance to dihydroartemisinin-piperaquine.

Parasites of the Plasmodium genus are unable to produce purine nucleotides de novo and depend completely on the salvage pathway. This fact makes plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT] a valuable target for development of antimalarial agents. A series of nucleotide analogues was designed, synthesized and evaluated as potential inhibitors of Plasmodium falciparum HGXPRT, P. vivax HGPRT and human HGPRT. These novel nucleoside phosphonates have a pyrrolidine, piperidine or piperazine ring incorporated into the linker connecting the purine base to a phosphonate group(s) and exhibited a broad range of Ki values between 0.15 and 72 μM. The corresponding phosphoramidate prodrugs, able to cross cell membranes, have been synthesized and evaluated in a P. falciparum infected human erythrocyte assay. Of the eight prodrugs evaluated seven exhibited in vitro antimalarial activity with IC50 values within the range of 2.5–12.1 μM. The bis-phosphoramidate prodrug 13a with a mean (SD) IC50 of 2.5 ± 0.7 μM against the chloroquine-resistant P. falciparum W2 strain exhibited low cytotoxicity in the human hepatocellular liver carcinoma (HepG2) and normal human dermal fibroblasts (NHDF) cell lines at a concentration of 100 μM suggesting good selectivity for further structure-activity relationship investigations.

High rates of artemisinin-based combination therapy (ACT) failures in the treatment of Plasmodium falciparum malaria in Southeast Asia have led to triple-drug strategies to extend the useful life of ACTs. In this study, we determined whether methylene blue [MB; 3,7-bis(dimethylamino)phenothiazin-5-ium chloride hydrate] alters the pharmacokinetics of artesunate-amodiaquine (ASAQ) and enhances the ex vivo antimalarial activity of ASAQ. In an open-label, randomized crossover design, a single oral dose of ASAQ (200 mg AS/540 mg AQ) alone or with MB (325 mg) was administered to 15 healthy Vietnamese volunteers. Serial blood samples were collected up to 28 days after dosing. Pharmacokinetic properties of the drugs were determined by noncompartmental analysis. After drug administration, plasma samples from seven participants were assessed for ex vivo antimalarial activity against the artemisinin-sensitive MRA1239 and the artemisinin-resistant MRA1240 P. falciparum lines, in vitro. MB significantly increased the mean area under the curve of the active metabolite of AS, dihydroartemisinin (1,246 ± 473 versus 917 ± 405 ng·h/ml, P = 0.009) but did not alter the pharmacokinetics of AQ, AS, or desethylamodiaquine. Comparing the antimalarial activities of the plasma samples from the participants collected up to 48 h after ASAQ plus MB (ASAQ+MB) and ASAQ dosing against the MRA1239 and MRA1240 lines, MB significantly enhanced the blood schizontocidal activity of ASAQ by 2.0-fold and 1.9-fold, respectively. The ring-stage survival assay also confirmed that MB enhanced the ex vivo antimalarial activity of ASAQ against MRA1240 by 2.9-fold to 3.8-fold, suggesting that the triple-drug combination has the potential to treat artemisinin-resistant malaria and for malaria elimination. (This study has been registered in the Australian New Zealand Clinical Trials Registry [https://anzctr.org.au/] under registration number ACTRN12612001298808.)

Nonimmune Aotus monkeys infected with Plasmodium falciparum and Plasmodium vivax were cured of their infections when treated with a single oral dose of 5 mg/kg and 10 mg/kg of the 2-aminomethylphenol, JPC-3210, respectively. Corresponding mean blood elimination half-lives of JPC-3210 were lengthy at 19.1 days and 20.5 days, respectively. This in vivo potency and lengthy half-life supports the further development of JPC-3210 as a promising, long-acting blood schizontocidal antimalarial for malaria treatment and prevention.

Background There is a high risk of Plasmodium vivax parasitaemia following treatment of falciparum malaria. Our study aimed to quantify this risk and the associated determinants using an individual patient data meta-analysis in order to identify populations in which a policy of universal radical cure, combining artemisinin-based combination therapy (ACT) with a hypnozoitocidal antimalarial drug, would be beneficial. Methods and findings A systematic review of Medline, Embase, Web of Science, and the Cochrane Database of Systematic Reviews identified efficacy studies of uncomplicated falciparum malaria treated with ACT that were undertaken in regions coendemic for P. vivax between 1 January 1960 and 5 January 2018. Data from eligible studies were pooled using standardised methodology. The risk of P. vivax parasitaemia at days 42 and 63 and associated risk factors were investigated by multivariable Cox regression analyses. Study quality was assessed using a tool developed by the Joanna Briggs Institute. The study was registered in the International Prospective Register of Systematic Reviews (PROSPERO: CRD42018097400). In total, 42 studies enrolling 15,341 patients were included in the analysis, including 30 randomised controlled trials and 12 cohort studies. Overall, 14,146 (92.2%) patients had P. falciparum monoinfection and 1,195 (7.8%) mixed infection with P. falciparum and P. vivax. The median age was 17.0 years (interquartile range [IQR] = 9.0–29.0 years; range = 0–80 years), with 1,584 (10.3%) patients younger than 5 years. 2,711 (17.7%) patients were treated with artemether-lumefantrine (AL, 13 studies), 651 (4.2%) with artesunate-amodiaquine (AA, 6 studies), 7,340 (47.8%) with artesunate-mefloquine (AM, 25 studies), and 4,639 (30.2%) with dihydroartemisinin-piperaquine (DP, 16 studies). 14,537 patients (94.8%) were enrolled from the Asia-Pacific region, 684 (4.5%) from the Americas, and 120 (0.8%) from Africa. At day 42, the cumulative risk of vivax parasitaemia following treatment of P. falciparum was 31.1% (95% CI 28.9–33.4) after AL, 14.1% (95% CI 10.8–18.3) after AA, 7.4% (95% CI 6.7–8.1) after AM, and 4.5% (95% CI 3.9–5.3) after DP. By day 63, the risks had risen to 39.9% (95% CI 36.6–43.3), 42.4% (95% CI 34.7–51.2), 22.8% (95% CI 21.2–24.4), and 12.8% (95% CI 11.4–14.5), respectively. In multivariable analyses, the highest rate of P. vivax parasitaemia over 42 days of follow-up was in patients residing in areas of short relapse periodicity (adjusted hazard ratio [AHR] = 6.2, 95% CI 2.0–19.5; p = 0.002); patients treated with AL (AHR = 6.2, 95% CI 4.6–8.5; p < 0.001), AA (AHR = 2.3, 95% CI 1.4–3.7; p = 0.001), or AM (AHR = 1.4, 95% CI 1.0–1.9; p = 0.028) compared with DP; and patients who did not clear their initial parasitaemia within 2 days (AHR = 1.8, 95% CI 1.4–2.3; p < 0.001). The analysis was limited by heterogeneity between study populations and lack of data from very low transmission settings. Study quality was high. Conclusions In this meta-analysis, we found a high risk of P. vivax parasitaemia after treatment of P. falciparum malaria that varied significantly between studies. These P. vivax infections are likely attributable to relapses that could be prevented with radical cure including a hypnozoitocidal agent; however, the benefits of such a novel strategy will vary considerably between geographical areas., Mohammad S. Hossain and colleagues explore P. vivax infection following P. falciparum treatment in this analysis of 42 published studies., Author summary Why was this study done? Plasmodium vivax is the most geographically widespread human malaria species; outside of sub-Saharan Africa, it almost invariably coexists with P. falciparum. A recent systematic review of 153 studies across 21 countries revealed that within 63 days of being treated for P. falciparum monoinfection, more than 15% of patients treated with an artemisinin-based combination therapy (ACT) had an episode of P. vivax malaria, far greater than expected for patients in similar locations who do not have acute malaria. We hypothesised that patients presenting with P. falciparum in coendemic locations are at high risk of carrying P. vivax dormant liver stages (hypnozoites) and would therefore potentially benefit from presumptive radical cure. What did the researchers do and find? We undertook an individual patient data meta-analysis to define the risk of vivax parasitaemia (blood stream infection) after treatment of P. falciparum malaria in different coendemic environments and to explore the factors underlying these risks. In total, 42 studies enrolling 15,341 patients were included in the analysis. By day 63, the risk of P. vivax ranged from 12.8% following dihydroartemisinin-piperaquine (DP) to 42.4% following artesunate-amodiaquine (AA). The highest rate of P. vivax malaria was in patients residing in areas with high risk of relapses and those who were slow to clear their initial parasitaemia. What do these findings mean? There is a high risk of vivax malaria after treatment of falciparum infection after all ACTs, although the risk varies substantially between locations. The correlation between the risk of P. vivax and the initial clearance of P. falciparum raises the possibility that the host response to acute malaria may be triggering the reactivation of P. vivax dormant liver stages. Universal radical cure with treatment to kill the liver stages may be warranted for reducing P. vivax, but the benefits will vary considerably between geographical locations, and further prospective clinical efficacy studies will be needed to determine the risks and benefits of such a strategy.

The increasing incidence of antimalarial drug resistance to the first-line artemisinin combination therapies underpins an urgent need for new antimalarial drugs, ideally with a novel mode of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, potent in vivo efficacy against murine malaria, and favorable preclinical pharmacokinetics including a lengthy plasma elimination half-life. To investigate the impact of JPC-3210 on biochemical pathways within P. falciparum-infected red blood cells, we have applied a "multi-omics" workflow based on high resolution orbitrap mass spectrometry combined with biochemical approaches. Metabolomics, peptidomics and hemoglobin fractionation analyses revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a specific depletion of short hemoglobin-derived peptides, peptidomics analysis revealed a depletion of longer hemoglobin-derived peptides, and the hemoglobin fractionation assay demonstrated decreases in hemoglobin, heme and hemozoin levels. To further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerization, about 10-fold less potent then the quinoline antimalarials, such as chloroquine and mefloquine. Further, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature compared with other known antimalarials. While JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. These studies revealed that the mode of action for JPC-3210 involves inhibition of the hemoglobin digestion pathway and elevation of regulators of protein translation. Importantly, JPC-3210 demonstrated rapid parasite killing kinetics compared with other quinolones, suggesting that JPC-3210 warrants further investigation as a potentially long acting partner drug for malaria treatment.

Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the foremost causative agents of malaria. Due to the development of resistance to current antimalarial medications, new drugs for this parasitic disease need to be discovered. The activity of hypoxanthine-guanine-[xanthine]-phosphoribosyltransferase, HG[X]PRT, is reported to be essential for the growth of both of these parasites, making it an excellent target for antimalarial drug discovery. Here, we have used rational structure-based methods to design an inhibitor, [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine, of PvHGPRT and PfHGXPRT that has Ki values of 8 and 7 nM, respectively, for these two enzymes. The crystal structure of PvHGPRT in complex with this compound has been determined to 2.85 A resolution. The corresponding complex with human HGPRT was also obtained to allow a direct comparison of the binding modes of this compound with the two enzymes. The tetra-(ethyl l-phenylalanine) tetraamide ...

Asymptomatic parasite carriers represent a “silent” infective reservoir for malaria transmission and contributes to malaria persistence. However, limited data are available on asymptomatic malaria in Vietnam. Between November 2018 and March 2019, we conducted a malaria epidemiological survey of asymptomatic people (children ≥ 10 years old and adults ≥18 years old, n = 2,809) residing in three communes in Tuy Duc district, Dak Nong province in the Central Highlands of Vietnam. Based on the national stratification of malaria risk, Dak Buk So, Dak Ngo and Quang Truc communes were classified by the National Malaria Control Programme as low, moderate and high malaria endemic areas, respectively. Using participants’ finger prick blood samples, malaria parasites were detected by one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The median age (Interquartile Range) for adults and children were 35 years (26–50) and 12 years (11–14), respectively. The prevalence of asymptomatic malaria was 1.7% (22/1,328), 3.5% (31/890) and 12.2% (72/591) for participants from Dak Buk So, Dak Ngo and Quang Truc, respectively. The prevalence of asymptomatic malaria was lower in children compared to adults: 2.6% (9/352) versus 4.7% (116/2,457) (Odds Ratio 0.53, 95% Confidence Interval 0.28 to1.02). Ownership of long-lasting insecticide-treated bed nets and hammocks was 97.1%, 99.0% and 94.7% for participants in Dak Buk So, Dak Ngo and Quang Truc, respectively, however, only 66.0%, 57.3% and 42.8% of the participants reported using bed nets every night. Of the several risk factors examined, going to the forest two weeks prior to enrolment into the study and sleeping in the forest had a significant association with participants being infected with asymptomatic malaria in Quang Truc, but not in the other two communes. Knowledge of the prevalence and distribution of asymptomatic malaria will help design and evaluate future intervention strategies for malaria elimination in Vietnam.

Two new series of symmetric acyclic nucleoside bisphosphonates (ANbPs) have been synthesised as potential inhibitors of the Plasmodium falciparum (Pf) and vivax (Pv) 6-oxopurine phosphoribosyltransferases. The structural variability between these symmetric ANbPs lies in the number of atoms in the two acyclic linkers connecting the N9 atom of the purine base to each of two phosphonate groups and the branching point of the acyclic moiety relative to the purine base, which occurs at either the alpha or beta positions. Within each series, six different 6-oxopurine bases have been attached. In general, the ANbPs with either guanine or hypoxanthine have lower Ki values than for those containing either the 8-bromo or 7-deaza 6-oxopurine bases. The lowest Ki values obtained for the two parasite enzymes were 0.1 μM (Pf) and 0.2 μM (Pv) for this series of compounds. Two phosphoramidate prodrugs of these inhibitors exhibited antimalarial activity against Pf in infected erythrocyte cell culture with IC50 values of 0.8 and 1.5 μM. These two compounds exhibited low cytotoxicity in human A549 cells having CC50 values of >300 μM resulting in an excellent selectivity index.

A series of 3,3′-disubstituted 5,5′-bi(1,2,4-triazine) derivatives was synthesized and screened against the erythrocytic stage of Plasmodium falciparum 3D7 line. The most potent dimer, 6k, with an IC50 (50% inhibitory concentration) of 0.008 μM, had high in vitro potency against P. falciparum lines resistant to chloroquine (W2, IC50 = 0.0047 ± 0.0011 μM) and artemisinin (MRA1240, IC50 = 0.0086 ± 0.0010 μM). Excellent ex vivo potency of 6k was shown against clinical field isolates of both P. falciparum (IC50 = 0.022–0.034 μM) and Plasmodium vivax (IC50 = 0.0093–0.031 μM) from the blood of outpatients with uncomplicated malaria. Despite 6k being cleared relatively rapidly in mice, it suppressed parasitemia in the Peters 4-day test, with a mean ED50 value (50% effective dose) of 1.47 mg kg–1 day–1 following oral administration. The disubstituted triazine dimer 6k represents a new class of orally available antimalarial compounds of considerable interest for further development.

Background Therapeutic efficacy studies in uncomplicated Plasmodium falciparum malaria are confounded by new infections, which constitute competing risk events since they can potentially preclude/pre-empt the detection of subsequent recrudescence of persistent, sub-microscopic primary infections. Methods Antimalarial studies typically report the risk of recrudescence derived using the Kaplan–Meier (K–M) method, which considers new infections acquired during the follow-up period as censored. Cumulative Incidence Function (CIF) provides an alternative approach for handling new infections, which accounts for them as a competing risk event. The complement of the estimate derived using the K–M method (1 minus K–M), and the CIF were used to derive the risk of recrudescence at the end of the follow-up period using data from studies collated in the WorldWide Antimalarial Resistance Network data repository. Absolute differences in the failure estimates derived using these two methods were quantified. In comparative studies, the equality of two K–M curves was assessed using the log-rank test, and the equality of CIFs using Gray’s k-sample test (both at 5% level of significance). Two different regression modelling strategies for recrudescence were considered: cause-specific Cox model and Fine and Gray’s sub-distributional hazard model. Results Data were available from 92 studies (233 treatment arms, 31,379 patients) conducted between 1996 and 2014. At the end of follow-up, the median absolute overestimation in the estimated risk of cumulative recrudescence by using 1 minus K–M approach was 0.04% (interquartile range (IQR): 0.00–0.27%, Range: 0.00–3.60%). The overestimation was correlated positively with the proportion of patients with recrudescence [Pearson’s correlation coefficient (ρ): 0.38, 95% Confidence Interval (CI) 0.30–0.46] or new infection [ρ: 0.43; 95% CI 0.35–0.54]. In three study arms, the point estimates of failure were greater than 10% (the WHO threshold for withdrawing antimalarials) when the K–M method was used, but remained below 10% when using the CIF approach, but the 95% confidence interval included this threshold. Conclusions The 1 minus K–M method resulted in a marginal overestimation of recrudescence that became increasingly pronounced as antimalarial efficacy declined, particularly when the observed proportion of new infection was high. The CIF approach provides an alternative approach for derivation of failure estimates in antimalarial trials, particularly in high transmission settings. Electronic supplementary material The online version of this article (10.1186/s12936-019-2837-4) contains supplementary material, which is available to authorized users.

ObjectivesDrug resistance exists to all current and investigational antimalarial drug classes. Consequently, we have set out to develop chemically and mechanistically discrete antimalarials. Here we report on the development of thiosemicarbazone (TSC) antimalarials, with TSC3 as the most advanced lead.MethodsThiosemicarbazones were generated through simple condensation reactions of thiosemicarbazides and ketones. TSC3 was selected and tested for in vitro antimalarial activities against MDR Plasmodium falciparum lines using the [3H]hypoxanthine growth assay, in vitro cytotoxicity against mammalian cell lines using the alamarBlue fluorescence cell viability assay, in vivo potency in the mouse–Plasmodium berghei model and blood exposure in mice measured by LC-MS for pharmacokinetic analysis.ResultsTSC3 showed potent in vitro activity against atovaquone-, dihydroartemisinin-, chloroquine- and mefloquine-resistant P. falciparum lines (EC50 500 in two of three mammalian cell lines. In P. berghei-infected mice, TSC3 showed potent activity in the Peters 4 day suppression test (ED50 1.2 mg/kg/day) and was as potent as artesunate and chloroquine in the curative modified Thompson test. A single oral dose of TSC3 at 16 mg/kg in healthy mice achieved a mean maximum blood concentration of 1883 ng/mL at 1 h after dosing and an elimination half-life of 48.7 h in groups of five mice.ConclusionsTSC3 shows promise as a persistent, potent and orally effective antimalarial. This, coupled with the extremely low cost of synthesis, suggests that the further development of antimalarial thiosemicarbazones is clearly warranted.

In vitro drug treatment with artemisinin derivatives, such as dihydroartemisinin (DHA), results in a temporary growth arrest (i.e., dormancy) at an early ring stage in Plasmodium falciparum . This response has been proposed to play a role in the recrudescence of P. falciparum infections following monotherapy with artesunate and may contribute to the development of artemisinin resistance in P. falciparum malaria. We demonstrate here that artemether does induce dormant rings, a finding which further supports the class effect of artemisinin derivatives in inducing the temporary growth arrest of P. falciparum parasites. In contrast and similarly to lumefantrine, the novel and fast-acting spiroindolone compound KAE609 does not induce growth arrest at the early ring stage of P. falciparum and prevents the recrudescence of DHA-arrested rings at a low concentration (50 nM). Our findings, together with previous clinical data showing that KAE609 is active against artemisinin-resistant K13 mutant parasites, suggest that KAE609 could be an effective partner drug with a broad range of antimalarials, including artemisinin derivatives, in the treatment of multidrug-resistant P. falciparum malaria.

Malaria is a disease caused by a protozoan of the Plasmodium genus and results in 0.5–0.7 million deaths per year. Increasing drug resistance of the parasite and insecticide resistance of mosquitoes necessitate alternative control measures. Numerous vaccine candidates have been identified but none have been able to induce robust, long-lived protection when evaluated in malaria endemic regions. Rodent studies have demonstrated that chemically attenuated blood stage parasites can persist at sub-patent levels and induce homologous and heterologous protection against malaria. Parasite-specific cellular responses were detected, with protection dependent on CD4+ T cells. To investigate this vaccine approach for Plasmodium falciparum, we characterised the persistence and immunogenicity of chemically attenuated P. falciparum FVO strain parasites (CAPs) in non-splenectomised Aotus nancymaae monkeys following administration of a single dose. Control monkeys received either normal red blood cells or wild-type parasites followed by drug treatment. Chemical attenuation was performed using tafuramycin A, which irreversibly binds to DNA. CAPs were detected in the peripheral blood for up to 2 days following inoculation as determined by thick blood smears, and for up to 8 days as determined by quantitative PCR. Parasite-specific IgG was not detected in monkeys that received CAPs; however, in vitro parasite-specific T cell proliferation was observed. Following challenge, the CAP monkeys developed an infection; however, one CAP monkey and the infection and drug-cure monkeys showed partial or complete resistance. These experiments lay the groundwork for further assessment of CAPs as a potential vaccine against malaria.

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a recognized target for antimalarial chemotherapeutics. It synthesises all of the 6-oxopurine nucleoside monophosphates, IMP, GMP and XMP needed by the malarial parasite, Plasmodium falciparum (Pf). PfHGXPRT is also indirectly responsible for the synthesis of the adenosine monophosphate, AMP. The acyclic nucleoside phosphonates (ANPs) are a class of PfHGXPRT inhibitors. Prodrugs of these compounds are able to arrest the growth of Pf in cell culture. In the search for new inhibitors of PfHGXPRT, a series of sulfur containing ANPs (thia-ANPs) has been designed and synthesized. These compounds are based on the structure of 2-(phosphonoethoxy)ethylguanine (PEEG) and PEEHx which consist of a purine base (i.e. guanine or hypoxanthine) linked to a phosphonate group by five atoms i.e. four carbons and one oxygen. Here, PEEG and PEEHx were modified by substituting a sulfide, sulfoxide or a sulfone bridge for the oxygen atom in the linker. The effect of these substitutions on the Ki values for human HGPRT and PfHGXPRT was investigated and showed that most of the thia-ANPs distinctively favour PfHGXPRT. For example, the thia-analogue of PEEHx has a Ki value of 0.2 μM for PfHGXPRT, a value 25-fold lower than for the human counterpart. Prodrugs of these compounds have IC50 values in the 4–6 μM range in antimalarial cell-based assays, making them attractive compounds for further development as antimalarial drug leads.

Background Reduced artemisinin susceptibility and artemisinin-based combination therapy (ACT)-resistance against Plasmodium falciparum and chloroquine (CQ)-resistant P. vivax malaria has been reported in Vietnam. Two therapeutic efficacy studies were conducted in Thuan Bac District (Ninh Thuan Province, Vietnam) in 2015 and 2016 to determine the extent of reduced artemisinin susceptibility and ACT resistant falciparum malaria, and CQ-resistant vivax malaria were present. Methods Twenty-seven patients with falciparum malaria were randomized to receive artesunate alone (AS ~ 4 mg/kg/day) for 4 days followed by dihydroartemisinin (DHA) (2.2 mg/kg)–piperaquine (PPQ) (18 mg/kg) daily for 3 days or artemether (AM) (1.7 mg/kg)–lumefantrine (LUM) (12 mg/kg) twice daily for 3 days. Sixteen subjects with vivax malaria received CQ (total 25 mg/kg over 3 days). The therapeutic efficacy study for treating falciparum malaria was complemented with molecular analysis for artemisinin and piperaquine resistance, and in vitro drug susceptibility testing. Patient’s drug exposure following both falciparum and vivax treatment studies was determined. Results Twenty-five of 27 patients treated with the artemisinin regimens completed the 42-day follow-up period. None had parasites present on day 3 after commencing treatment with no incidence of recrudescence (100% curative rate). One patient on AS + DHA–PPQ was lost to follow-up and one patient had Plasmodium falciparum and Plasmodium vivax infection on day 0 by PCR. Of the vivax patients, 15 of 16 completed CQ treatment and two had a recurrence of vivax malaria on day 28, a failure rate of 13.3% (2/15). No mutations in the Pfkelch-13 gene for artemisinin resistance or exo-E415G gene polymorphism and amplification in plasmepsins 2 and 3 for piperaquine resistance were observed. In vitro testing of patient’s falciparum parasites indicated susceptibility (low IC50 nM values) to dihydroartemisinin, lumefantrine, piperaquine and pyronaridine. Patient’s drug exposure to artesunate and lumefantrine was comparable to published data, however, blood CQ concentrations were lower. Conclusions Clinical findings, molecular analysis and in vitro testing revealed that the falciparum parasites at Phuoc Chien Commune were artemisinin susceptible. The clinical failure rate of the 15 vivax patients who completed CQ treatment was 13%. Further studies are required to determine whether CQ-resistant vivax malaria is present at the commune. Electronic supplementary material The online version of this article (10.1186/s12936-019-2640-2) contains supplementary material, which is available to authorized users.

The pharmacokinetics (PK) and ex vivo activity (pharmacodynamics [PD]) of two artemisinin combination therapies (ACTs) (artemisinin–piperaquine [ARN–PPQ] [Artequick(®)] and artesunate–amodiaquine [ARS–AQ] [Coarsucam(™)]) in healthy Vietnamese volunteers were compared following 3-day courses of the ACTs for the preselection of the drugs for falciparum malaria therapy. For PK analysis, serial plasma samples were collected from two separate groups of 22 volunteers after ACT administration. Of these volunteers, ex vivo activity was assessed in plasma samples from seven volunteers who received both ACTs. The area under the concentration–time curve (AUC(0–∞)) was 3.6-fold higher for dihydroartemisinin (active metabolite of ARS) than that for ARN, whereas the AUC(0–∞) of desethylamodiaquine (active metabolite of AQ) was 2.0-fold lower than that of PPQ. Based on the 50% inhibitory dilution values of the volunteers’ plasma samples collected from 0.25 to 3 hours after the last dose, the ex vivo activity of ARS–AQ was 2.9- to 16.2-fold more potent than that of ARN–PPQ against the drug-sensitive D6 Plasmodium falciparum line. In addition, at 1.5, 4.0, and 24 hours after the last dose, the ex vivo activity of ARS–AQ was 20.8-, 3.5-, and 8.5-fold more potent than that of ARN–PPQ against the ARN-sensitive MRA1239 line. By contrast, at 1.5 hours, the ex vivo activity of ARS–AQ was 5.4-fold more active than that of ARN–PPQ but had similar activities at 4 and 24 hours against the ARN-resistant MRA1240 line. The PK–PD data suggest that ARS–AQ possesses superior antimalarial activity than that of ARN–PPQ and would be the preferred ACT for further in vivo efficacy testing in multidrug-resistant falciparum malaria areas.

The new 2-aminomethylphenol, JPC-3210, has potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, and high in vivo efficacy against murine malaria. Here we report on the pharmacokinetics of JPC-3210 in mice and monkeys and the results of in vitro screening assays, including the inhibition of cytochrome P450 (CYP450) isozymes. In mice, JPC-3210 was rapidly absorbed and had an extensive tissue distribution, with a brain tissue-to-plasma concentration ratio of about 5.4. JPC-3210 had a lengthy plasma elimination half-life of about 4.5 days in mice and 11.8 days in monkeys. JPC-3210 exhibited linear single-oral-dose pharmacokinetics across the dose range of 5 to 40 mg/kg of body weight with high oral bioavailability (∼86%) in mice. Systemic blood exposure of JPC-3210 was 16.6% higher in P. berghei -infected mice than in healthy mice. In vitro studies with mice and human hepatocytes revealed little metabolism and the high metabolic stability of JPC-3210. The abundance of human metabolites from oxidation and glucuronidation was 2.0% and 2.5%, respectively. CYP450 studies in human liver microsomes showed JPC-3210 to be an inhibitor of CYP2D6 and, to a lesser extent, CYP3A4 isozymes, suggesting the possibility of a metabolic drug-drug interaction with drugs that are metabolized by these isozymes. In vitro studies showed that JPC-3210 is highly protein bound to human plasma (97%). These desirable pharmacological findings of a lengthy blood elimination half-life, high oral bioavailability, and low metabolism as well as high in vivo potency have led the Medicines for Malaria Venture to select JPC-3210 (MMV892646) for further advanced preclinical development.

Acyclic nucleoside phosphonates (ANPs) are a promising class of antimalarial therapeutic drug leads that exhibit a wide variety of Ki values for Plasmodium falciparum (Pf) and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases [HG(X)PRTs]. A novel series of ANPs, analogues of previously reported 2-(phosphonoethoxy)ethyl (PEE) and (R,S)-3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) derivatives, were designed and synthesized to evaluate their ability to act as inhibitors of these enzymes and to extend our ongoing antimalarial structure-activity relationship studies. In this series, (S)-3-hydroxy-2-(phosphonoethoxy)propyl (HPEP), (S)-2-(phosphonomethoxy)propanoic acid (CPME), or (S)-2-(phosphonoethoxy)propanoic acid (CPEE) are the acyclic moieties. Of this group, (S)-3-hydroxy-2-(phosphonoethoxy)propylguanine (HPEPG) exhibits the highest potency for PfHGXPRT, with a Ki value of 0.1 μM and a Ki value for human HGPRT of 0.6 μM. The crystal structures of HPEPG and HPEPHx (where Hx=hypoxanthine) in complex with human HGPRT were obtained, showing specific interactions with active site residues. Prodrugs for the HPEP and CPEE analogues were synthesized and tested for in vitro antimalarial activity. The lowest IC50 value (22 μM) in a chloroquine-resistant strain was observed for the bis-amidate prodrug of HPEPG.

A series of amide (8–32, 40–45) and urea (33, 34, 36–39) analogues based on the thiaplakortone A natural product scaffold were synthesised and screened for in vitro antimalarial activity against chloroquine-sensitive (3D7) and chloroquine- and mefloquine-resistant (Dd2) Plasmodium falciparum parasite lines. Several analogues displayed potent inhibition of P. falciparum growth (IC50500 nM) and good selectivity for P. falciparum versus human neonatal foreskin fibroblast cells (selectivity index100). Two of these compounds, 8 and 33, exhibited good aqueous solubility and metabolic stability, and when administered subcutaneously to mice (32 mg kg(-1)), plasma concentrations remained above 0.2 μM for at least 8 h. Both 8 and 33 were well tolerated in mice after subcutaneous administration of 32 mg kg(-1) twice daily for 4 days. Using this regimen blood stage P. berghei was suppressed by 52% for 8 and 26% for 33, relative to the vehicle control.

Hypoxanthine-guanine-[xanthine] phosphoribosyltransferase (HG[X]PRT) is considered an important target for antimalarial chemotherapy as it is the only pathway for the synthesis of the purine nucleoside monophosphates required for DNA/RNA production. Thus, inhibition of this enzyme should result in cessation of replication. The aza-acyclic nucleoside phosphonates (aza-ANPs) are good inhibitors of Plasmodium falciparum HGXPRT (PfHGXPRT), with Ki values as low as 0.08 and 0.01 μM for Plasmodium vivax HGPRT (PvHGPRT). Prodrugs of these aza-ANPs exhibit antimalarial activity against Pf lines with IC50 values (0.8-6.0 μM) and have low cytotoxicity against human cells. Crystal structures of six of these compounds in complex with human HGPRT have been determined. These suggest that the different affinities of these aza-ANPs could be due to the flexibility of the loops surrounding the active site as well as the flexibility of the inhibitors, allowing them to adapt to fit into three binding pockets of the enzyme(s).

Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the early ring-stage parasites can survive drug treatment by entering cell cycle arrest or dormancy. After drug removal, these parasites can resume growth. Dormancy has been hypothesized to be an adaptive physiological mechanism that has been linked to recrudescence of parasites after monotherapy with ART and, possibly contributes to ART resistance. Here, we evaluate the in vitro drug sensitivity profile of normally-developing P. falciparum ring stages and DHA-pretreated dormant rings (DP-rings) using a panel of antimalarial drugs, including the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. We report that while KDU691 shows no activity against rings, it is highly inhibitory against DP-rings; a drug effect opposite to that of ART. Moreover, we provide evidence that KDU691 also kills DP-rings of P. falciparum ART-resistant strains expressing mutant K13.

Acyclic nucleoside bisphosphonates (ANbPs) have previously been shown to be good inhibitors of human hypoxanthine–guanine phosphoribosyltransferase (HGPRT) and Plasmodium falciparum (Pf) hypoxanthine–guanine–xanthine phosphoribosyltransferase (PfHGXPRT). On the basis of this scaffold, a new series of ANbPs was synthesized. One of these new ANbPs, [3-(guanine-9-yl)-2-((2-phosphonoethoxy)methyl)propoxy]methylphosphonic acid, exhibited Ki values of 6 and 70 nM for human HGPRT and Pf HGXPRT, respectively. These low Ki values were achieved by inserting an extra carbon atom in the linker connecting the N9 atom of guanine to one of the phosphonate groups. The crystal structure of this ANbP in complex with human HGPRT was determined at 2.0 A resolution and shows that it fills three key pockets in the active site. The most potent phosphoramidate prodrugs of these compounds have IC50 values in the low micromolar range in Pf lines and low toxicity in human A549 cells, demonstrating that these ANbPs are excellent ant...

Wide-field fluorescence microscopy at high magnification was used to study the intracellular binding site of Rubb16 in Escherichia coli. Upon incubation of E. coli cells at the minimum inhibitory concentration, Rubb16 localised at ribosomes with no significant DNA binding observed. Furthermore, Rubb16 condensed the ribosomes when they existed as polysomes. It is postulated that the condensation of polysomes would halt protein production, and thereby inhibit bacterial growth. The results of this study indicate that the family of inert dinuclear ruthenium complexes Rubbn selectively target RNA over DNA in vivo. Selective RNA targeting could be advantageous for the development of therapeutic agents, and because of differences in ribosome structure between bacteria and eukaryotic cells, the Rubbn complexes could be selectively toxic to bacteria. In support of this hypothesis, the toxicity of Rubb16 was found to be significantly less to liver and kidney cell lines than against a range of bacteria.

Australian and US military medical services have collaborated since World War II to minimize vector-borne diseases such as malaria, dengue, and scrub typhus. In this review, collaboration over the last 30 years is discussed. The collaborative projects and exchange scientist programs have resulted in mutually beneficial outcomes in the fields of drug development and personal protection measures against vector-borne diseases.

OBJECTIVE: To determine the effects of late pregnancy and also oestrogen supplementation on the CYP2C19-mediated biotransformation of proguanil (PG) to its active antifol triazine metabolite cycloguanil (CG). METHODS: Case control study conducted on the NW border of Thailand; a single dose of PG (4 mg/kg) was administered to Karen women in late pregnancy and a single blood and urine sample taken 6 h later. Women were studied in late pregnancy (>36 weeks) and restudied 2 months after delivery. A separate cohort of Karen women newly attending a birth-control clinic were studied before and 3 weeks into their first course of oral contraceptives (OCP: levonorgestrel 0.15 mg and ethinyloestradiol 0.03 mg). Forty-five pregnant women and forty-two healthy OCP users were studied. RESULTS: The results were similar in both groups; pregnancy and OCP use were both associated with reduced formation of cycloguanil (CG). Impaired PG biotransformation was seen in women with the "extensive metaboliser" phenotype (urine PG/CG ratio

Summary To determine the level of antimalarial drug resistance in southern Papua, Indonesia, we assessed the therapeutic efficacy of chloroquine plus sulfadoxine–pyrimethamine (CQ+SP) for Plasmodium falciparum infections as well as CQ monotherapy for P. vivax infections. Patients with P. falciparum failing therapy were re-treated with unsupervised quinine ± doxycycline therapy and those with P. vivax with either unsupervised quinine ± doxycycline or amodiaquine. In total, 143 patients were enrolled in the study (103 treated with CQ+SP and 40 with CQ). Early treatment failures occurred in four patients (4%) with P. falciparum and six patients (15%) with P. vivax. The failure rate by Day 28 for P. vivax was 65% (95% CI 49–81). After PCR correction for re-infections, the Day 42 recrudescence rate for P. falciparum infections was 48% (95% CI 31–65). Re-treatment with unsupervised quinine ± doxycycline resulted in further recurrence of malaria in 48% (95% CI 31–65) of P. falciparum infections and 70% (95% CI 37–100) of P. vivax infections. Eleven patients with recurrent P. vivax were re-treated with amodiaquine; there were no early or late treatment failures. In southern Papua, a high prevalence of drug resistance of P. falciparum and P. vivax exists both to first- and second-line therapies. Preliminary data indicate that amodiaquine retains superior efficacy compared with CQ for CQ-resistant P. vivax.

A recent malaria epidemic in the Menoreh Hills of Central Java has increased concern about the re-emergence of endemic malaria on Java, which threatens the island's 120 million residents. A 28-day, in-vivo test of the efficacy of treatment of malaria with antimalarial drugs was conducted among 16 7 villagers in the Menoreh Hills. The treatments investigated, chloroquine (CQ) and sulfadoxine pyrimethamine (SP), constitute, respectively, the first- and second-line treatments for uncomplicated malaria in Indonesia. The prevalence of malaria among 1389 residents screened prior to enrollment was 33%. Treatment outcomes were assessed by microscopical diagnoses, PCR-based confirmation of the diagnoses, measurement of the whole-blood concentrations of CQ and desethylchloroquine (DCQ), and identification of the Plasmodium falciparum genotypes. The 28-day cumulative incidences of therapeutic failure for CQ and SP were, respectively, 47% (N = 36) and 22% (N = 50) in the treatment of P. falciparum, and 18% (N = 77) and 67% (N = 6) in the treatment of P. vivax. Chloroquine was thus an ineffective therapy for P. falciparum malaria, and the presence of CQ-resistant P. vivax and SP-resistant P. falciparum will further compromise efforts to control resurgent malaria on Java.

The continuing spread of multidrug resistance in Plasmodium falciparum malaria makes the search for alternative treatments ever more urgent. We have investigated the relative efficacy of halofantrine and mefloquine in two paired randomised trials on the Thai-Burmese border, a multidrug-resistant area. In the first trial, 198 patients with acute uncomplicated falciparum malaria were randomly assigned either the standard halofantrine regimen (24 mg/kg) or mefloquine (25 mg/kg). The cumulative failure rates by day 28 were 35% with halofantrine and 10% with mefloquine (p = 0.0002). In the second study of 437 patients, a higher dose of halofantrine (8 mg/kg every 8 h for 3 days = 72 mg/kg) was both more effective and better tolerated than mefloquine 25 mg/kg; the failure rates were 3% and 8% (p = 0.03), respectively, or 1% vs 6% after adjustment for possible reinfections (p = 0.009). The rate of failure was higher after retreatment than after primary treatment in all study groups. Halofantrine 72 mg/kg was especially effective in the retreatment of these recrudescent infections; the failure rate was 44% with mefloquine and 15% with high-dose halofantrine (relative risk 3.0 [95% CI 1.2-7.3], p = 0.008). Thus, high-dose halofantrine is better tolerated and more effective than mefloquine for the treatment of uncomplicated falciparum malaria in this area. However, evidence of possible cardiotoxicity will need to be investigated fully before a role can be established for halofantrine in the treatment of multidrug-resistant malaria.

High-grade chloroquine (CQ)-resistant Plasmodium vivax is prevalent in eastern Malaysia. Artesunate-mefloquine is an efficacious artemisinin combination therapy (ACT) for all malaria species. Wider CQ-efficacy surveillance is needed in vivax-endemic regions with earlier replacement with ACT when treatment failure is detected., Background. Chloroquine (CQ)-resistant Plasmodium vivax is increasingly reported throughout southeast Asia. The efficacy of CQ and alternative artemisinin combination therapies (ACTs) for vivax malaria in Malaysia is unknown. Methods. A randomized, controlled trial of CQ vs artesunate-mefloquine (AS-MQ) for uncomplicated vivax malaria was conducted in 3 district hospitals in Sabah, Malaysia. Primaquine was administered on day 28. The primary outcome was the cumulative risk of treatment failure by day 28 by Kaplan–Meier analysis. Results. From 2012 to 2014, 103 adults and children were enrolled. Treatment failure by day 28 was 61.1% (95% confidence interval [CI], 46.8–75.6) after CQ and 0% (95% CI, 0–.08) following AS-MQ (P < .001), of which 8.2% (95% CI, 2.5–9.6) were early treatment failures. All patients with treatment failure had therapeutic plasma CQ concentrations at day 7. Compared with CQ, AS-MQ was associated with faster parasite clearance (normalized clearance slope, 0.311 vs 0.127; P < .001) and fever clearance (mean, 19.0 vs 37.7 hours; P = .001) and with lower risk of anemia at day 28 (odds ratio = 3.7; 95% CI, 1.5–9.3; P = .005). Gametocytes were present at day 28 in 23.8% (10/42) of patients following CQ vs none with AS-MQ (P < .001). AS-MQ resulted in lower bed occupancy: 4037 vs 6510 days/1000 patients (incidence rate ratio 0.62; 95% CI, .60–.65; P < .001). One patient developed severe anemia not regarded as related to their AS-MQ treatment. Conclusions. High-grade CQ-resistant P. vivax is prevalent in eastern Malaysia. AS-MQ is an efficacious ACT for all malaria species. Wider CQ-efficacy surveillance is needed in vivax-endemic regions with earlier replacement with ACT when treatment failure is detected. Clinical Trials Registration. NCT01708876.

Safe and effective antimalarial drugs are required for the treatment of pregnant women. We report a 3-day regimen of artesunate (4 mg/kg/day)-azithromycin (25 mg/kg/day) (ASAZ) to be efficacious (polymerase chain reaction-corrected cure rate of 96.7%) and well tolerated in the treatment of Plasmodium falciparum malaria in children (N = 11) and adults (N = 19), in Vietnam in 2010. In comparison, the cure rate for artesunate (4 mg/kg on day 0, 2 mg/kg on days 1-6) was 90.0% in children (N = 7) and adults (N = 23). Because azithromycin is considered safe in pregnancy, our findings provide further evidence that ASAZ should be evaluated for the treatment of pregnant women with malaria.

Structure-activity relationship studies of trifluoromethyl-substituted pyridine and pyrimidine analogues of 2-aminomethylphenols (JPC-2997, JPC-3186, and JPC-3210) were conducted for preclinical development for malaria treatment and/or prevention. Of these compounds, JPC-3210 [4-( tert -butyl)-2-(( tert -butylamino)methyl)-6-(5-fluoro-6-(trifluoromethyl)pyridin-3-yl)phenol] was selected as the lead compound due to superior in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, lower in vitro cytotoxicity in mammalian cell lines, longer plasma elimination half-life, and greater in vivo efficacy against murine malaria.

A new bispyrroloiminoquinone alkaloid, tsitsikammamine C (1), displayed potent in vitro antimalarial activity with IC(50) values of 13 and 18 nM against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum, respectively. Tsitsikammamine C (1) displayed selectivity indices of200 against HEK293 cells and inhibited both ring and trophozoite stages of the malaria parasite life cycle. Previously reported compounds makaluvamines J (2), G (3), L (4), K (5) and damirones A (6) and B (7) were also isolated from the same marine sponge (Zyzzya sp.). Compounds 2-4 displayed potent growth inhibitory activity (IC(50)100 nM) against both P. falciparum lines and only moderate cytotoxicity against HEK293 cells (IC(50) = 1-4 μM). Makaluvamine G (3) was not toxic to mice and suppressed parasite growth in P. berghei infected mice following subcutaneous administration at 8 mg kg(-1) day(-1).