Effect of Fluorescent Dyes on In Vitro-Differentiated,Late-Stage Plasmodium falciparum Gametocytes

Plasmodium falciparum gametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates of P. falciparum with a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC₅₀) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC₅₀s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC₅₀s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly high in vitro activity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds.     

Comparative Ex Vivo Activity of Novel Endoperoxides in Multidrug-Resistant Plasmodium falciparum and P. vivax

The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC(50)s) in both species (median IC(50)s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicity.     

Ex Vivo Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Western,Northern, and Eastern Cambodia, 2011-2012: Association with Molecular Markers

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC₅₀s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC₅₀s (GMIC₅₀s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC₅₀s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC₅₀ and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.     

Decreasing pfmdr1 Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is Regaining In Vitro Susceptibility to Mefloquine

Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies—a molecular marker for MQ resistance—in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ.     

Antimalarial efficacy and drug interactions of the novel semi-synthetic endoperoxide artemisone in vitro and in vivo

OBJECTIVES: The in vitro and in vivo efficacy and drug-drug interactions of the novel semi-synthetic endoperoxide artemisone with standard antimalarials were investigated in order to provide the basis for the selection of the best partner drug. METHODS: Antimalarial activity and drug interactions were evaluated in vitro against Plasmodium falciparum by the incorporation of [(3)H]hypoxanthine. In vivo efficacy and drug interactions were assessed using the standard 4-day Peters' test. RESULTS: Artemisone was 10 times more potent than artesunate in vitro against a panel of 12 P. falciparum strains, independent of their susceptibility profile to antimalarial drugs, and consistently 4 to 10 times more potent than artesunate in rodent models against drug-susceptible and primaquine- or sulfadoxine/pyrimethamine-resistant Plasmodium berghei lines and chloroquine- or artemisinin-resistant lines of Plasmodium yoelii. Slight antagonistic trends were found between artemisone and chloroquine, amodiaquine, tafenoquine, atovaquone or pyrimethamine and additive to slight synergistic trends with artemisone and mefloquine, lumefantrine or quinine. Various degrees of synergy were observed in vivo between artemisone and mefloquine, chloroquine or clindamycin. CONCLUSIONS: These results confirm the increased efficacy of artemisone over artesunate against multidrug-resistant P. falciparum and provide the basis for the selection of potential partner drugs for future deployment in areas of multidrug-resistant malaria. Artemisone represents an important addition to the repertoire of artemisinin combination therapies currently in use, as it has enhanced antimalarial activity, improved bioavailability and stability over current endoperoxides.     

Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment

Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia (P <10⁻³). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC₅₀) of PP were independent of treatment outcomes, in contrast to median mefloquine IC₅₀, which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P = 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P = 0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.)     

Assessment of the Induction of Dormant Ring Stages in Plasmodium falciparum Parasites by Artemisone and Artemisone Entrapped in Pheroid Vesicles In Vitro

The in vitro antimalarial activities of artemisone and artemisone entrapped in Pheroid vesicles were compared, as was their ability to induce dormancy in Plasmodium falciparum. There was no increase in the activity of artemisone entrapped in Pheroid vesicles against multidrug-resistant P. falciparum lines. Artemisone induced the formation of dormant ring stages similar to dihydroartemisinin. Thus, the Pheroid delivery system neither improved the activity of artemisone nor prevented the induction of dormant rings.     

Treatment of Murine Cerebral Malaria by Artemisone in Combination with Conventional Antimalarial Drugs: Antiplasmodial Effects and Immune Responses

The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.     

In Vitro Susceptibility of Plasmodium vivax to Antimalarials in Colombia

The in vitro susceptibilities of 30 isolates of Plasmodium vivax to a number of antimalarials (chloroquine [CQ], mefloquine, amodiaquine, quinine, and artesunate [AS]) were evaluated. The isolates came from the region of Urabá in Colombia, in which malaria is endemic, and were evaluated by the schizont maturation test. The 50% inhibitory concentration (IC₅₀) was 0.6 nM (95% confidence interval [CI], 0.3 to 1.0 nM) for artesunate, 8.5 nM (95% CI, 5.6 to 13.0 nM) for amodiaquine, 23.3 nM (95% CI, 12.4 to 44.1 nM) for chloroquine, 55.6 nM (95% CI, 36.8 to 84.1 nM) for mefloquine, and 115.3 nM (95% CI, 57.7 to 230.5 nM) for quinine. The isolates were classified according to whether the initial parasites were mature or immature trophozoites (Tfz). It was found that the IC₅₀s for chloroquine and artesunate were significantly different in the two aforementioned groups (P < 0.001). The IC₅₀s of CQ and AS were higher in the isolates from mature Tfz (CQ, 39.3 nM versus 17 nM; AS, 1.4 nM versus 0.3 nM), and 10% of the isolates showed lower susceptibilities to one of the antimalarial drugs, 13.3% to two antimalarial drugs, and 3.3% to more than three antimalarial drugs. It should be highlighted that despite the extensive use of chloroquine in Colombia, P. vivax continues to be susceptible to antimalarials. This is the first report, to our knowledge, showing in vitro susceptibilities of P. vivax isolates to antimalarials in Colombia.     

First assessment in humans of the safety, tolerability, pharmacokinetics, and ex vivo pharmacodynamic antimalarial activity of the new artemisinin derivative artemisone

In preclinical studies, artemisone (BAY 44-9585), a new artemisinin derivative, was shown to possess enhanced efficacy over artesunate, and it does not possess the neurotoxicity characteristic of the current artemisinins. In a phase I program with double-blind, randomized, placebo-controlled, single and multiple ascending oral-dose studies, we evaluated the safety, tolerability, pharmacokinetics, and ex vivo pharmacodynamic antimalarial activity of artemisone. Single doses (10, 20, 30, 40, and 80 mg) and multiple doses (40 and 80 mg daily for 3 days) of artemisone were administered orally to healthy subjects. Plasma concentrations of artemisone and its metabolites were measured by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Artemisone was well tolerated, with no serious adverse events and no clinically relevant changes in laboratory and vital parameters. The pharmacokinetics of artemisone over the 10- to 80-mg range demonstrated dose linearity. After the single 80-mg dose, artemisone had a geometric mean maximum concentration of 140.2 ng/ml (range, 86.6 to 391.0), a short elimination half-life (t_1/2) of 2.79 h (range, 1.56 to 4.88), a high oral clearance of 284.1 liters/h (range, 106.7 to 546.7), and a large volume of distribution of 14.50 liters/kg (range, 3.21 to 51.58). Due to artemisone's short t_1/2, its pharmacokinetics were comparable after single and multiple dosing. Plasma samples taken after multiple dosing showed marked ex vivo pharmacodynamic antimalarial activities against two multidrug-resistant Plasmodium falciparum lines. Artemisone equivalent concentrations measured by bioassay revealed higher activity than artemisone measured by LC/MS-MS, confirming the presence of active metabolites. Comparable to those of other artemisinin's, artemisone's t_1/2 is well suited for artemisinin-based combination therapy for the treatment of P. falciparum malaria.     

Laboratory Detection of Artemisinin-Resistant Plasmodium falciparum

Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite 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. falciparum is 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 vivo responses 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.     

The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand

On the western border of Thailand, Plasmodium falciparum has become resistant to almost all antimalarial agents. The molecular basis of resistance in these parasite populations has not been well characterized. This study assessed genetic polymorphisms in the pfmdr1 gene in 54 parasites collected from the western border of Thailand to determine the relationship of pfmdr1 copy number and codon mutations with parasite sensitivities to mefloquine, chloroquine, halofantrine, quinine, and artesunate assessed in vitro. A point mutation at codon 86 (resulting in a change of Asn to Tyr) was associated with a significantly lower 50% inhibitory concentration (IC(50)) of mefloquine (median, 9 ng/ml versus 52.4 ng/ml; P = 0.003). Overall 35% of the isolates (19 of 54) had an increase in pfmdr1 copy number, and all 19 carried the wild-type allele at codon 86. Increased pfmdr1 copy number was associated with higher IC(50)s of mefloquine (P = 0.04) and artesunate (P = 0.005), independent of polymorphism at codon 86. The relationship between pfmdr1 and resistance to structurally distinct antimalarial agents confirms the presence of a true multidrug-resistant phenotype.     

Repeat Polymorphisms in the Low-Complexity Regions of Plasmodium falciparum ABC Transporters and Associations with In Vitro Antimalarial Responses

The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC₅₀) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required.     

pfmdr1 Amplification and Fixation of pfcrt Chloroquine Resistance Alleles in Plasmodium falciparum in Venezuela

Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: S_tctVMNT (91%) and S_agtVMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites.Amplification of the Plasmodium falciparum multidrug resistance gene (pfmdr1) has been implicated in mefloquine (MQ) resistance in Thailand and Cambodia (1, 17, 27, 28, 34, 41), but not elsewhere. It is not known if amplification has occurred in Venezuela, where MQ monotherapy was used between 2001 and 2004 and the combination of artesunate (AS) and MQ thereafter. pfmdr1 duplication is also implicated in resistance to lumefantrine, halofantrine, quinine, and AS (39) and may decrease resistance to chloroquine (CQ) (43). Also, single-nucleotide mutations in pfmdr1, such as N86Y, Y184F, S1034C, N1042D, and D1246Y (the mutated amino acid is shown in boldface type), are postulated to modulate drug response. While these mutations may or may not contribute to CQ resistance (40), mutations at codons 1034, 1042, and 1246 make parasites more sensitive to MQ (40). Studies suggest at least two lineages of mutant pfmdr1 genotypes have evolved in South America (4, 21).In South America, CQ and sulfadoxine-pyrimethamine (SP) were used to treat P. falciparum prior to the use of artemisinin-based combination therapy (ACT). Resistance to CQ and SP evolved independently in South America (18, 23). Point mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene are correlated with CQ resistance (10). The pfcrt point mutation K76T is critical, but C72S, M74I, N75E, and N75K are also associated with resistance (48). There are at least four different origins of CQ resistance pfcrt alleles: one in Papua New Guinea (SVMNT), where the genotype represents amino acids at codons 72 to 76, one in Southeast Asia (CVIET) that spread to Africa, and two in South America (SVMNT/CVMNT in Brazil/Peru and CVMET/CVMNT in Ecuador/Colombia) (49).Molecular surveillance showed that, after drug removal, CQ resistance genotypes, in Malawi and China (16, 46), and SP resistance genotypes, in the Peruvian Amazon (52), declined. Therefore, the reduction in the frequency of resistant parasites likely occurred because resistant parasite populations are at a fitness disadvantage in the absence of drug pressure. In Bolivar State, Venezuela, mutant pfcrt alleles remained after the removal of CQ in 1986 (6) and mutant dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes remained fixed after SP removal (19). Whether the recent use of MQ and AS-MQ led to the evolution of pfmdr1 genotypes associated with AS and MQ resistance is unknown.This study in the state of Bolivar, Venezuela assessed the following: (i) whether pfmdr1 duplication has occurred, (ii) the frequency of pfmdr1 and pfcrt mutations, (iii) whether MQ and CQ drug pressure has affected variation surrounding these genes, and (iv) linkage disequilibrium between dhfr, dhps, pfcrt, and pfmdr1 alleles.(Part of this research [some data pertaining to pfmdr1 and pfcrt genotypes, microsatellites, and copy number] was presented at the 57th Annual Meeting of the American Society of Tropical Medicine and Hygiene, New Orleans, LA, 2008, and the 58th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Washington, DC, 2009.)     

Antimalarial Activity of Tigecycline,a Novel Glycylcycline Antibiotic

Tigecycline is a novel glycylcycline antibiotic with a broad antibacterial spectrum. Tigecycline was tested with 66 clinical isolates of Plasmodium falciparum from Bangladesh using the histidine-rich protein 2 in vitro drug susceptibility assay. The 50% and 90% inhibitory concentrations of tigecycline were 699 (95% confidence interval, 496 to 986) and 5,905 nM (4,344 to 8,028). Tigecycline shows no activity correlation with traditional antimalarials and has substantial antimalarial activity on its own.The spreading resistance of Plasmodium falciparum to existing drugs (15) and first reports of artemisinin resistance (7) call for the search for novel antimalarial drugs. Antibiotics with antimalarial activity, such as azithromycin, doxycyline, and clindamycin, in combination with traditional antimalarial drugs are an interesting option for treating multidrug-resistant falciparum malaria (11, 13, 14). Particularly, tetracyclines, azithromycin, or clindamycin in combination with quinine or artesunate is considered to be a potential second-line therapy for the treatment of uncomplicated falciparum malaria (16).Tigecycline is the first member of a new class of antimicrobials, the glycylcyclines, and is currently registered only for intravenous therapy. It is a semisynthetic derivate of minocycline with a unique and novel mechanism of action in bacteria. These tetracycline analogues are specifically designed to overcome two common mechanisms of tetracycline resistance, namely, resistance mediated by acquired efflux pumps and/or ribosomal protection (4). Clinical studies have shown that intravenously administrated tigecycline has an expanded spectrum of in vitro and in vivo activity against gram-positive, gram-negative, atypical, anaerobic, and other difficult-to-treat pathogens. With a twice-daily dosing regimen, tigecycline is relatively easy to administer and generally well tolerated (6).The project was carried out at the MARIB (Malaria Research Initiative Bandarban) field site in Bandarban in southeastern Bangladesh. Written informed consent was obtained from all study participants or their legal representatives, and the study protocol was approved by the Ethical Review Committees of the Medical University of Vienna and the International Centre for Diarrhoeal Disease Research, Bangladesh. Blood samples were taken from male and nonpregnant female patients of ages 8 to 65 years who presented with microscopically confirmed P. falciparum monoinfections with a parasite density of 100 to 100,000 asexual parasites per μl. Parasite samples with more than 1% parasitemia were diluted with uninfected red blood cells. Pregnant or breastfeeding women and patients with malaria drug therapy in the preceding 30 days were excluded from the study. All samples were tested in the histidine-rich protein 2 (HRP2) in vitro drug susceptibility assay. The culture and enzyme-linked immunosorbent assay (ELISA) were carried out as described previously (8, 9). Fresh P. falciparum isolates were cultured in the presence of threefold serial dilutions of the antimalarial drugs tigecycline (34.3 to 25,000 nM), doxycycline (68.6 to 50,000 nM), azithromycin (68.6 to 50,000 nM), dihydroartemisinin (0.014 to 10.0 nM), chloroquine (3.4 to 2,500 nM), quinine (3.4 to 2,500 nM), and mefloquine (0.3 to 250 nM). Culture was carried out at a 1.5% hematocrit value in complete RPMI 1640 medium with 0.5% Albumax (Albumax I; Gibco Bangkok, Thailand) and 25 mg/liter of gentamicin without freezing, washing, dilution, the addition of serum, or preculture. After 72 h of culture, the plates were frozen and stored at −20°C. The plates were then thawed, and parasite growth inhibition was quantified by using a highly sensitive histidine-rich protein 2 ELISA. The optical density was measured at 450 nm using a standard ELISA plate reader (Tecan Sunrise; Tecan Austria). The optical density readings were used for estimating inhibitory concentrations by nonlinear regression analysis (10).From a total of 94 patients, 66 samples (70.2%; 95% confidence interval [CI], 59.8 to 79.0) with a geometric mean parasite density of 10,684 per μl (95% CI, 8,311 to 13,735) were successfully tested. The geometric mean 50% inhibitory concentration (IC₅₀) for tigecycline was 699.11 nM (n = 66; 95% CI, 495.74 to 985.91), and the corresponding value for doxycycline was 4,276.01 nM (n = 33; 95% CI, 3,084.28 to 5,928.21). IC₅₀s, 90% inhibitory concentrations (IC₉₀s), and 99% inhibitory concentrations (IC₉₉s) with 95% confidence intervals for all drugs tested are shown in Table ​Table1.1. No correlation was found between parasite density and IC₅₀s of tigecycline (r = −0.001; P = −0.994) and doxycycline (r = 0.23; P = 0.188), suggesting little influence of the inoculum size on the outcome of in vitro assays (2). Individual inhibitory concentrations were calculated for all drugs tested in parallel and correlated by nonparametric correlation analysis to determine potential cross-sensitivity and/or cross-resistance patterns between the drugs at IC₉₀ levels. Tigecycline showed a significant activity correlation with doxycycline (r = 0.51; P = 0.003; n = 32) but no evidence of a correlation with any of the other tested antimalarials (for dihydroartemisinin, r = 0.038; P = 0.775; and n = 58; for mefloquine, r = −0.159; P = 0.234; and n = 58; for quinine, r = 0.210; P = 0.114; and n = 58; for chloroquine, r = 0.198; P = 0.135; and n = 58; and for azithromycin, r = 0.252; P = 0.057; and n = 58).

TABLE 1.

Geometric mean inhibitory concentrations for various drugs against fresh P. falciparum isolates from Bangladesh

Absence of Association between Piperaquine In Vitro Responses and Polymorphisms in the pfcrt,pfmdr1, pfmrp,and pfnhe Genes in Plasmodium falciparum

We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC₅₀s) for PPQ ranged from 29 to 98 nM (geometric mean = 57.8 nM, 95% confidence interval [CI] = 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean = 1.8 nM, 95% CI = 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r² = 0.17; P = 0.0495) and between the responses to PPQ and DOX (r² = 0.41; P = 0.001). We did not find a significant association between the PPQ IC₅₀ (0.0525 < P < 0.9247) or the DHA IC₅₀ (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes. There was an absence of cross-resistance with quinolines, and the IC₅₀s for PPQ and DHA were found to be unrelated to mutations in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 transport protein genes, which are involved in quinoline antimalarial drug resistance. These results confirm the interest in and the efficacy of the combination of PPQ and DHA for areas in which parasites are resistant to chloroquine or other quinolines.Over the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine (CQ) and other antimalarial drugs (32). This development has prompted the search for an effective alternative antimalarial drug with minimal side effects. One strategy for reducing the prevalence of malaria is the combinatorial use of drugs, which is thought to protect against the development of resistance to each drug and to reduce the overall rate of transmission of malaria (52). Since 2001, more than 60 countries have officially adopted artemisinin-based combination therapies (ACTs) for the treatment of falciparum malaria (40), and the official first-line antimalarial treatment in Africa is now ACT (18). The artemisinin derivatives cause rapid and effective reductions in the parasite biomass and the level of gametocyte carriage, while the partner drug, which has a longer duration of action, achieves effective clinical and parasitological cure. Several different ACTs have been evaluated, including artesunate-sulfadoxine-pyrimethamine (PY) (50), artesunate-amodiaquine (7), artemether-lumefantrine (LMF) (53), artesunate-mefloquine (MQ) (2), artesunate-chlorproguanil-dapsone (42), artesunate-atovaquone (ATV)-proguanil, artesunate-pyronaridine (44), and dihydroartemisinin (DHA)-piperaquine (PPQ) (1, 22, 47).However, individual P. falciparum isolates resistant to artemisinin in vitro and the first clinical failures have been described in Cambodia (10, 16, 39, 45). In addition, prior therapy with an amodiaquine-containing ACT has been found to select for a reduced response to monodesethylamodiaquine (MDAQ), suggesting that amodiaquine-containing regimens may rapidly lose efficacy in Africa (38). This emergence of parasites resistant to ACTs underlines the fact that novel compounds and combinations must be discovered and developed.DHA-PPQ is an inexpensive, safe, and highly effective treatment for uncomplicated falciparum and vivax malaria (36, 43). DHA-PPQ has been shown to offer a longer posttreatment prophylactic effect following therapy than artemether-lumefantrine (27, 53, 54) or artesunate-amodiaquine (23). The significantly lower risk of recurrent parasitemia after treatment with DHA-PPQ is likely explained by differences in the pharmacokinetics of the nonartemisinin partner drugs. PPQ, a bisquinoline, is estimated to have an elimination half-life of 17 to 33 days (28, 47, 48), while the elimination half-life of lumefantrine is 4 to 10 days (19) and that of amodiaquine is 1 to 6 h (the half-life of its active metabolite, monodesethylamodiaquine, is 1 to 10 days). Bisquinolines are compounds with two quinoline nuclei bound by a covalent aliphatic or aromatic link.The first aim of the present work was to assess the in vitro cross-resistance of PPQ with other quinoline drugs, whether they are artemisinin partners or not. The following drugs were tested: CQ, quinine (QN), MQ, MDAQ, LMF, DHA, ATV, PY, and doxycycline (DOX). The second aim was to identify genetic polymorphisms in the Plasmodium falciparum crt (pfcrt) pfcrt, pfmrp, pfmdr1, and pfnhe-1 genes, which are known to be associated with reduced quinoline susceptibility, that could be associated with decreased susceptibility to PPQ with the goal of identifying molecular markers of PPQ resistance for use in resistance surveillance.     

In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity

Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203 Plasmodium falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms in P. falciparum crt (Pfcrt), Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with the Pfcrt wild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P < 0.0001), and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild-type N allele in Pfmdr-1 codon 86 (93.5%), with only 7 (3.50%) samples with the N86Y mutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Y mutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean of 0.907 ± 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya.     

Evaluation of Antimalarial Resistance Marker Polymorphism in Returned Migrant Workers in China

Imported malaria has been a great challenge for public health in China due to decreased locally transmitted cases and frequent exchange worldwide. Plasmodium falciparum has been mainly responsible for the increasing impact. Currently, artesunate plus amodiaquine, one of the artemisinin combination therapies recommended by the World Health Organization, has been mainly used against uncomplicated P. falciparum malaria in China. However, drug resistance marker polymorphism in returning migrant workers has not been demonstrated. Here, we have evaluated the prevalence of pfmdr1 and pfcrt polymorphisms, as well as the K13 propeller gene, a molecular marker of artemisinin resistance, in migrant workers returned from Ghana to Shanglin County, Guangxi Province, China, in 2013. A total of 118 blood samples were randomly selected and used for the assay. Mutations of the pfmdr1 gene that covered codons 86, 184, 1034, and 1246 were found in 11 isolates. Mutations at codon N86Y (9.7%) were more frequent than at others, and Y₈₆Y₁₈₄S₁₀₃₄D₁₂₄₆ was the most prevalent (63.6%) of the four haplotypes. Mutations of the pfcrt gene that covered codons 74, 75, and 76 were observed in 17 isolates, and M₇₄N₇₅T₇₆ was common (70.6%) in three haplotypes. Eight different genotypes of the K13 propeller were first observed in 10 samples in China, 2 synonymous mutations (V487V and A627A) and 6 nonsynonymous mutations. C580Y was the most prevalent (2.7%) in all the samples. The data presented might be helpful for enrichment of molecular surveillance of antimalarial resistance and will be useful for developing and updating antimalarial guidance in China.