BackgroundTreatment failure of chloroquine for P. vivax infections has reached high levels in the eastern provinces of Indonesia, however, in vitro characterization of chloroquine resistance and its associated molecular profile have yet to be determined.MethodsUsing a modified schizont maturation assay we investigated the in vitro chloroquine susceptibility profile and molecular polymorphisms of P. vivax isolates collected from Papua, Indonesia, where high levels of clinical chloroquine treatment failure have been reported, and from Thailand, where chloroquine treatment is generally effective.ResultsThe geometric mean chloroquine IC50 for P. vivax isolates from Papua (n = 145) was 312 nM [95%CI: 237–411 nM] compared to 46.8 nM [95%CI: 34.7–63.1 nM] from Thailand (n = 81); p<0.001. Correlating with the known clinical efficacy of the area, a cut off for chloroquine resistance was defined as 220nM, a level exceeded in 13.6% (11/81) of Thai isolates and 65% (94/145) of Papuan isolates; p<0.001. Several sequence polymorphisms in pvcrt-o and pvmdr1, and difference in pvmdr1 copy number were identified. A Y976F mutation in pvmdr1 was present in 96% (123/128) of Papuan isolates and 25% (17/69) of Thai isolates; p<0.001. Overall, the geometric mean chloroquine IC50 in isolates with the Y976F mutation was 283 nM [95%CI: 211–379], compared to 44.5 nM [95%CI: 31.3–63.4] in isolates with the wild type; p< 0.001. Pvmdr1 amplification occurred in 23% (15/66) of Thai isolates compared to none (0/104) of Indonesian isolates (p<0.001), but was not associated with increased chloroquine resistance after controlling for geographical location.Conclusions In vitro susceptibility testing of P. vivax discriminates between populations with differing levels of clinical efficacy of chloroquine. The pvmdr1 polymorphism at Y976F may provide a useful tool to highlight areas of emerging chloroquine resistance, although further studies defining its clinical correlates are needed.

In Papua, Indonesia, the antimalarial susceptibility of Plasmodium vivax (n ‫؍‬ 216) and P. falciparum (n ‫؍‬ 277) was assessed using a modified schizont maturation assay for chloroquine, amodiaquine, artesunate, lumefantrine, mefloquine, and piperaquine. The most effective antimalarial against P. vivax and P. falciparum was artesunate, with geometric mean 50% inhibitory concentrations (IC 50 In vitro drug susceptibility assays assess antimicrobial activity in the absence of the confounding effects of the host. Although such assays have become useful for monitoring the antimalarial resistance of Plasmodium falciparum, the assay has been of limited use with P. vivax. This is in part a consequence of a perception of the importance of antimalarial drug resistance with P. vivax, compounded by difficulties in standardizing a field-based assay. Over the last decade, a number of clinical studies have demonstrated the emergence of highgrade chloroquine resistance in Papua, Indonesia, and Papua, New Guinea (1, 18, 21), and its spread to other regions of Asia (6) and South America (20). However, assessment of the clinical efficacy of antimalarial drugs against P. vivax infection is confounded by the occurrence of both reinfections and relapses, making the attributable fraction of recurrent infections due to intrinsic parasite resistance difficult to gauge (2, 3, 10). To confirm the emergence of the spread of antimalarial drug resistance of P. vivax and to investigate alternative antimalarial drugs, it is critical that a standardized in vitro assay be developed and validated. The aim of this study was to define the in vitro susceptibility profiles of a range of antimalarial drugs and to investigate the confounding factors that modulate the derived estimate of drug efficacy. MATERIALS AND METHODS Field location and sample collection. Between March 2004 and May 2007,Plasmodium isolates were collected from patients attending malaria clinics in Timika, located in the southern part of Papua province, Indonesia. Timika is a region of endemicity for multidrug-resistant strains of P. vivax and P. falciparum, with a risk of treatment failure of 65% within 28 days after chloroquine monotherapy for P. vivax malaria and 48% failure after multidrug therapy with chloroquine-sulfadoxine-pyrimethamine for P. falciparum malaria (16). In 2004, treatment guidelines were changed accordingly to recommend an artemisinin combination therapy for both P. falciparum and P. vivax infection, precluding further clinical studies of the use of chloroquine monotherapy in this region (15). Patients with symptomatic malaria who presented to an outpatient facility were recruited into the study if they were singly infected with P. falciparum or with P. vivax, with a parasitemia of between 2,000 l Ϫ1 and 80,000 l Ϫ1 . Patients treated with antimalarials in the previous 3 weeks were excluded from this study. Venous blood (5 ml) was collected by venipuncture and, after the host white blood cells were removed using a CF11 column, 2 ml of packed infected red bl...

Amplification of pvmdr1 and single-nucleotide polymorphisms are correlated with susceptibility of P. vivax to multiple antimalarial drugs. Chloroquine and mefloquine appear to exert competitive evolutionary pressure on pvmdr1, similar to that observed with pfmdr1 in Plasmodium falciparum.

Histone acetylation plays an important role in regulating gene transcription and silencing in Plasmodium falciparum. Histone deacetylase (HDAC) inhibitors, particularly those of the hydroxamate class, have been shown to have potent in vitro activity against drug-resistant and -sensitive laboratory strains of P. falciparum, raising their potential as a new class of antimalarial compounds. In the current study, stage-specific ex vivo susceptibility profiles of representative hydroxamate-based HDAC inhibitors suberoylanilide hydroxamic acid (SAHA), 2-ASA-9, and 2-ASA-14 (2-ASA-9 and 2-ASA-14 are 2-aminosuberic acid-based HDAC inhibitors) were assessed in multidrug-resistant clinical isolates of P. falciparum (n ‫؍‬ 24) and P. vivax (n ‫؍‬ 25) from Papua, Indonesia, using a modified schizont maturation assay. Submicromolar concentrations of SAHA, 2-ASA-9, and 2-ASA-14 inhibited the growth of both P. falciparum (median 50% inhibitory concentrations [IC 50 s] of 310, 533, and 266 nM) and P. vivax (median IC 50 s of 170, 503, and 278 nM). Inverse correlation patterns between HDAC inhibitors and chloroquine for P. falciparum and mefloquine for P. vivax indicate species-specific susceptibility profiles for HDAC inhibitors. These HDAC inhibitors were also found to be potent ex vivo against P. vivax schizont maturation, comparable to that in P. falciparum, suggesting that HDAC inhibitors may be promising candidates for antimalarial therapy in geographical locations where both species are endemic. Further studies optimizing the selectivity and in vivo efficacy of HDAC inhibitors in Plasmodium spp. and defining drug interaction with common antimalarial compounds are warranted to investigate the role of HDAC inhibitors in antimalarial therapy.

Pyronaridine, a Mannich base antimalarial, has demonstrated high in vivo and in vitro efficacy against chloroquine-resistant Plasmodium falciparum. Although this drug has the potential to become a prominent artemisinin combination therapy, little is known about its efficacy against drug-resistant Plasmodium vivax. The in vitro antimalarial susceptibility of pyronaridine was assessed in multidrug-resistant P. vivax (n ‫؍‬ 99) and P. falciparum (n ‫؍‬ 90) isolates from Papua, Indonesia, using a schizont maturation assay. The median 50% inhibitory concentration (IC 50 ) of pyronaridine was 1.92 nM (range, 0.24 to 13.8 nM) against P. falciparum and 2.58 nM (range, 0.13 to 43.6 nM) against P. vivax, with in vitro susceptibility correlating significantly with chloroquine, amodiaquine, and piperaquine (r s [Spearman's rank correlation coefficient] ‫؍‬ 0.45 to 0.62; P < 0.001). P. falciparum parasites initially at trophozoite stage had higher IC 50 s of pyronaridine than those exposed at the ring stage (8.9 nM [range, 0.6 to 8.9 nM] versus 1.6 nM [range, 0.6 to 8.9 nM], respectively; P ‫؍‬ 0.015), although this did not reach significance for P. vivax (4.7 nM [range, 1.4 to 18.7 nM] versus 2.5 nM [range, 1.4 to 15.6 nM], respectively; P ‫؍‬ 0.085). The excellent in vitro efficacy of pyronaridine against both chloroquine-resistant P. vivax and P. falciparum highlights the suitability of the drug as a novel partner for artemisinin-based combination therapy in regions where the two species are coendemic.

iThe 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.A pproximately 3.3 billion people (i.e., almost 50% of the world's population) are at risk of malaria with two Plasmodium species responsible for the majority of infections: P. falciparum and P. vivax (6,7,43). Traditionally, malaria control and research efforts have focused on P. falciparum, the dominant species in Africa. However, outside Africa, P. falciparum almost invariably coexists with P. vivax (7), with both species inflicting significant morbidity, particularly in infants and pregnant women (18,27).Chloroquine (CQ)-resistant P. falciparum is already well established, with emerging evidence that susceptibility to CQ in P. vivax is also declining across much of the world in which vivax is endemic. This combined threat is driving the investigation of alternative schizonticidal treatment regimens, such as artemisininbased combination therapy (ACT), for deployment against both P. falciparum and P. vivax (29). ACTs have become the mainstay of antimalarial chemotherapy, adopted in more than 100 countries worldwide (42). This huge demand for artemisinin and its derivatives relies on isolation from the plant source Artemisia annua and is vulnerable to harvest and production costs and intermittent supply (2, 11). Of particular concern are recent reports of prolonged ...

Ferroquine (FQ; SSR97193), a ferrocene-containing 4-aminoquinoline derivate, has potent in vitro efficacy against chloroquine (CQ)-resistant Plasmodium falciparum and CQ-sensitive P. vivax. In the current study, ex vivo FQ activity was tested in multidrug-resistant P. falciparum and P. vivax field isolates using a schizont maturation assay. Although FQ showed excellent activity against CQ-sensitive and -resistant P. falciparum and P. vivax (median 50% inhibitory concentrations [IC 50 s], 9.6 nM and 18.8 nM, respectively), there was significant cross-susceptibility with the quinoline-based drugs chloroquine, amodiaquine, and piperaquine (for P. falciparum, r ‫؍‬ 0.546 to 0.700, P < 0.001; for P. vivax, r ‫؍‬ 0.677 to 0.821, P < 0.001). The observed ex vivo cross-susceptibility is likely to reflect similar mechanisms of drug uptake/efflux and modes of drug action of this drug class. However, the potent activity of FQ against resistant isolates of both P. falciparum and P. vivax highlights a promising role for FQ as a lead antimalarial against CQ-resistant Plasmodium and a useful partner drug for artemisinin-based combination therapy.

Reports of potential drug-resistant strains ofPlasmodium malariaein western Indonesia raise concerns that chloroquine resistance may be emerging inP. malariaeandP. ovale. In order to assess this,in vivoandin vitroefficacy studies were conducted in patients with monoinfection in Papua, Indonesia. Consecutive patients with uncomplicated malaria due toP. ovaleorP. malariaewere enrolled in a prospective clinical trial, provided with supervised chloroquine treatment, and followed for 28 days. Blood from patients withP. malariaeorP. ovaleparasitemia greater than 1,000 per microliter underwentin vitroantimalarial drug susceptibility testing using a modified schizont maturation assay. Of the 57 evaluable patients in the clinical study (P. malariae,n= 46;P. ovale,n= 11), none had recurrence with the same species during follow-up. The mean parasite reduction ratio at 48 h was 86 (95% confidence interval [CI], 57 to 114) forP. malariaeand 150 (95% CI, 54 to 245) forP. ovale(P= 0.18). One patient infected withP. malariae, with 93% of parasites at the trophozoite stage, was still parasitemic on day 4.In vitrodrug susceptibility assays were carried out successfully for 40 isolates (34 infected withP. malariaeand 6 withP. ovale). TheP. malariaeinfections at trophozoite stages had significantly higher chloroquine 50% effective concentrations (EC50s) (median, 127.9 nM [range, 7.9 to 2,980]) than those initially exposed at the ring stage (median, 14.0 nM [range, 3.5 to 27.0];P= 0.01). The EC50for chloroquine inP. ovalewas also higher in an isolate initially at the trophozoite stage (23.2 nM) than in the three isolates predominantly at ring stage (7.8 nM). Chloroquine retains adequate efficacy againstP. ovaleandP. malariae, but its marked stage specificity of action may account for reports of delayed parasite clearance times.