Confirmed Plasmodium vivax Resistance to Chloroquine in Central Vietnam

Plasmodium vivax resistance to chloroquine (CQ) is currently reported in almost all countries where P. vivax is endemic. In Vietnam, despite a first report on P. vivax resistance to chloroquine published in the early 2000s, P. vivax was still considered sensitive to CQ. Between May 2009 and December 2011, a 2-year cohort study was conducted in central Vietnam to assess the recommended radical cure regimen based on a 10-day course of primaquine (0.5 mg/kg/day) together with 3 days of CQ (25 mg/kg). Here we report the results of the first 28-day follow-up estimating the cumulative risk of P. vivax recurrences together with the corresponding CQ blood concentrations, among other endpoints. Out of 260 recruited P. vivax patients, 240 completed treatment and were followed up to day 28 according to the WHO guidelines. Eight patients (3.45%) had a recurrent P. vivax infection, at day 14 (n = 2), day 21 (n = 1), and day 28 (n = 5). Chloroquine blood concentrations, available for 3/8 recurrent infections (days 14, 21, and 28), were above the MIC (>100 ng/ml whole blood) in all of these cases. Fever and parasitemia (both sexual and asexual stages) were cleared by day 3. Anemia was common at day 0 (35.8%), especially in children under 10 years (50%), and hemoglobin (Hb) recovery at day 28 was substantial among anemic patients (median change from day 0 to 28, +1.7 g/dl; interquartile range [IQR], +0.7 to +3.2). This report, based on CQ blood levels measured at the time of recurrences, confirms for the first time P. vivax CQ resistance in central Vietnam and calls for further studies using standardized protocols for accurately monitoring the extent and evolution of P. vivax resistance to chloroquine in Vietnam. These results, together with the mounting evidence of artemisinin resistance in central Vietnam, further highlight the increasing threat of antimalarial drug resistance to malaria elimination in Vietnam.     

Chloroquine Remains Effective for Treating Plasmodium vivax Malaria in Pursat Province,Western Cambodia

Chloroquine (CQ) is used to treat Plasmodium vivax malaria in areas where CQ resistance has not been reported. The use of artemisinin (ART)-based combination therapies (ACTs) to treat CQ-sensitive P. vivax infections is effective and convenient but may promote the emergence and worsening of ART resistance in sympatric Plasmodium falciparum populations. Here, we show that CQ effectively treats P. vivax malaria in Pursat Province, western Cambodia, where ART-resistant P. falciparum is highly prevalent and spreading. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00663546","term_id":"NCT00663546"}}NCT00663546.)     

3-Halo Chloroquine Derivatives Overcome Plasmodium falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in P. falciparum

Polymorphism in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was shown to cause chloroquine resistance. In this report, we examined the antimalarial potential of novel 3-halo chloroquine derivatives (3-chloro, 3-bromo, and 3-iodo) against chloroquine-susceptible and -resistant P. falciparum. All three derivatives inhibited the proliferation of P. falciparum; with 3-iodo chloroquine being most effective. Moreover, 3-iodo chloroquine was highly effective at potentiating and reversing chloroquine toxicity of drug-susceptible and -resistant P. falciparum.     

Analysis of Single-Nucleotide Polymorphisms in the crt-o and mdr1 Genes of Plasmodium vivax among Chloroquine-Resistant Isolates from the Brazilian Amazon Region

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.Plasmodium vivax is the most widely distributed human malaria parasite, causing approximately 80 to 300 million clinical cases of malaria each year (17). Numerous factors indicate that this burden will increase due to the emergence and spread of chloroquine-resistant parasites (3, 17).More than 50% of the malaria cases in Latin America occur in Brazil, and P. vivax predominates as the causative agent (16, 21). Notably, failures of chloroquine treatment of P. vivax malaria in the Brazilian Amazon city of Manaus have been reported recently (1). The local confirmation of the presence of active P. vivax parasites resisting chloroquine at the proposed minimal effective concentration in plasma for sensitive strains is a public health concern deserving attention.Point mutations in two digestive-vacuole membrane proteins of P. falciparum, the P. falciparum chloroquine resistance transporter (PfCRT) and multidrug resistance 1 protein (PfMDR1), have been associated with chloroquine resistance (CQR), albeit to different extents (2, 10). Orthologues of these proteins in P. vivax (P. vivax CRT-O [PvCRT-O] and PvMDR1) have been identified previously (6, 15, 18), and recently, pvmdr1 mutant alleles were suggested to be associated with both in vitro and in vivo CQR in Southeast Asia (6, 20).Here, we report a single-nucleotide polymorphism (SNP) analysis of pvmdr1 and pvcrt-o genes in P. vivax isolates from chloroquine-treated patients with and without recrudescent disease in the Brazilian Amazon region. In addition to complete coding sequences, we analyzed sequences from 5′ flanking regions and introns.Field isolates were collected during a 28-day in vivo chloroquine efficacy study conducted in the city of Manaus, Brazil (8). Plasmatic chloroquine levels in all volunteers were measured by high-performance liquid chromatography on day 3 to confirm adequate dosing and good absorption of the oral chloroquine intake (three doses of 10, 7.5, and 7.5 mg/kg of body weight in 150-mg tablet form at 24-h intervals). Clinical treatment failure was defined as the occurrence of a positive blood smear result (confirmed by PCR diagnostic analysis) on day 14, 21, or 28 and the presence of parasites in peripheral blood (collected on the same day as the positive blood smear) containing >10 ng/ml of chloroquine as determined by high-performance liquid chromatography (7). Measurements of chloroquine and its active metabolite desethylchloroquine in whole blood were not obtained, as plasma samples were collected and processed at and transported from remote field sites. The presence of drug-resistant isolates in plasma samples with a mean chloroquine concentration ± standard deviation of 356.6 ± 296.1 ng/ml, however, undoubtedly confirms CQR (4). Using these stringent criteria, we selected seven samples (four obtained prior to treatment [on day 0] from patients with nonrecrudescent disease and three obtained after treatment [on days 21 and 28] from other patients with recrudescent disease). Different sets of primers, PCR conditions, and algorithms were used to amplify coding and noncoding regions and to generate and analyze sequences (see the supplemental material).Analyses of the complete coding sequences of the pvmdr1 gene demonstrated that these sequences contained 24 SNPs and a single conserved microsatellite sequence. Notably, 17 (73%) of the 24 SNPs detected were nonsynonymous; 11 were contained in predicted extracellular loops in the parasite digestive-vacuole cytosol, and 5 were present in transmembrane domains (TMDs) (Fig. ​(Fig.1;1; also see Table S2 in the supplemental material). Despite the high frequency of SNPs, however, none were found in ABC conserved motifs (see Table S3 in the supplemental material).Open in a separate windowFIG. 1.Predicted structures of and representative polymorphisms in PvMDR1 and PfMDR1. PvMDR1, like PfMDR1, has two hydrophobic homologous domains, each with six transmembrane α-helices, and a cytosolic domain harboring nucleotide-binding domain 1 (NBD₁) and NBD₂, each containing an ATP-binding site with characteristic Walker motifs A and B and the S signature (ATP-binding cassette) of these transporters. In this figure, boxes with dotted lines in the diagram of PvMDR1 delimit predicted NBD locations. In the PfMDR1 illustration, closed dots represent point mutations associated with CQR in P. falciparum. In the PvMDR1 illustration, open dots represent SNPs described previously (5, 6, 11, 18, 20), open triangles represent SNPs identified in this study, and patterned dots represent SNPs described in other studies (5, 6, 11, 18, 20).To relate these polymorphisms to protein function, we mapped selective constraints throughout coding sequences by calculating a K_d/K_s (ω) ratio for each amino acid substitution (see Fig. S1 in the supplemental material). These results revealed ω values of ≥1, indicating higher accelerated rates of nonsynonymous substitutions than expected to result from chance at the sites of most SNPs (P < 0.00001). Indeed, an ω value of 1.80811 (P < 0.00001), evidencing positive selection, was calculated for nucleotide positions 2722 to 2727, which correspond to amino acids 907 and 908 in PvMDR1. Presently, however, it is difficult to ensure that this positive selection is advantageous for the CQR phenotype and not for another metabolic aspect(s) related to the function of the PvCRT-O and PvMDR1 transporters. Like other authors (5, 6, 11, 18), we did not find SNPs at homologous positions of PfMDR1. We did, however, find a polymorphism in PvMDR1 at amino acid position 89, which is located very near and in the same intravacuolar loop, between TMDs I and II, as an SNP at the corresponding position in PfMDR1 (amino acid position 86), which has partial correlation with the CQR phenotype (12, 20). In addition, SNP Y976F, proposed previously as an early marker of CQR (6, 20), was found in only one isolate from a patient with nonrecrudescent disease. It is thus clear that the value of these polymorphisms as markers of CQR in P. vivax needs to be further explored.Complete sequencing of the 14 exons of pvcrt-o revealed the presence of one synonymous transition and five nonsynonymous substitutions (Fig. ​(Fig.2;2; see also Table S4 in the supplemental material). Interestingly, the lysine (K) insertion at amino acid 10 of PvCRT-O, highly prevalent in Thai isolates (20), was also found in a sample from a chloroquine-treated patient from Brazil with recrudescent disease. Moreover, the rate of nonsynonymous substitutions in TMD VII was significantly higher than expected to result from chance (P < 0.05) (see Fig. S2 in the supplemental material). Despite these differences, however, we were unable to find an association between chloroquine treatment failure and the SNPs detected. Similar results following SNP analyses of pvcrt-o in monkey-adapted strains and human isolates have been reported previously (5, 15, 20).Open in a separate windowFIG. 2.Predicted structures of and representative polymorphisms in PvCRT-O and PfCRT. Like PfCRT, PvCRT-O has 10 predicted transmembrane helices, with C- and N-terminal domains located in the parasite cytoplasm. In the diagram of PvCRT-O, open dots represent SNPs detected in chloroquine-resistant and chloroquine-sensitive samples by Nomura et al. (15), and open triangles represent (named) SNPs described in this work. Closed dots in the PfCRT illustration represent point mutations that have been strongly associated with CQR in P. falciparum.The lack of mutations in coding sequences of pvmdr1 and pvcrt-o associated with treatment failure prompted us to look for these associations in introns and 5′ flanking sequences, as they influence gene expression in malaria (7, 13). Interestingly, 6 (46%) of 13 introns contained SNPs, nine of which were transitions and one of which was a transversion (see Table S5 in the supplemental material). The remaining introns contained conserved microsatellites (see Table S6 in the supplemental material). Yet differences in length at the microsatellite in intron 12 (see Fig. S3 in the supplemental material) among samples sharing the same evolutionary spatial and temporal distributions suggest that this microsatellite marker may be a good candidate for the study of this locus. Moreover, analyses of 5′ flanking sequences from pvmdr1 and pvcrt-o revealed a single SNP (A→G) at nucleotide position 566 upstream from the ATG start codon in pvcrt-o. This high degree of conservation is consistent with the results of another study which analyzed untranslated regions of pfmdr1 in six reference strains with different chloroquine phenotypes (14). Together, these results suggest the existence of functional constraints at these genome loci that may play an important role in gene regulation.In conclusion, we have analyzed the complete coding and noncoding sequences of the pvmdr1 and pvcrt-o genes from Brazilian P. vivax isolates that fulfilled rigorous criteria for chloroquine sensitivity and resistance. Although we did not genotype these isolates to determine if CQR isolates represent clonal as opposed to complex populations, it is clear that CQR isolates are already circulating in the Brazilian Amazon basin. Therefore, these sequences can be used as a baseline for future prospective studies of drug resistance in this region. Our analysis revealed, however, that there was no correlation between CQR and pvmdr1 and pvcrt-o mutations in these specific isolates, even if some mutations had been not randomly selected. The striking differences in the topologies of SNPs in the MDR1 and CRT transporter genes between P. vivax and P. falciparum thus indicate that mechanisms other than mutations may be implicated in the appearance of CQR in these two human malaria parasites (likely candidates are gene amplification and changes in expression levels [9, 11, 19]) or that other genes are associated with this virulent phenotype in P. vivax.     

Therapeutic Responses of Plasmodium vivax Malaria to Chloroquine and Primaquine Treatment in Northeastern Myanmar

Chloroquine-primaquine (CQ-PQ) continues to be the frontline therapy for radical cure of Plasmodium vivax malaria. Emergence of CQ-resistant (CQR) P. vivax parasites requires a shift to artemisinin combination therapies (ACTs), which imposes a significant financial, logistical, and safety burden. Monitoring the therapeutic efficacy of CQ is thus important. Here, we evaluated the therapeutic efficacy of CQ-PQ for P. vivax malaria in northeast Myanmar. We recruited 587 patients with P. vivax monoinfection attending local malaria clinics during 2012 to 2013. These patients received three daily doses of CQ at a total dose of 24 mg of base/kg of body weight and an 8-day PQ treatment (0.375 mg/kg/day) commencing at the same time as the first CQ dose. Of the 401 patients who finished the 28-day follow-up, the cumulative incidence of recurrent parasitemia was 5.20% (95% confidence interval [CI], 3.04% to 7.36%). Among 361 (61%) patients finishing a 42-day follow-up, the cumulative incidence of recurrent blood-stage infection reached 7.98% (95% CI, 5.20% to 10.76%). The cumulative risk of gametocyte carriage at days 28 and 42 was 2.21% (95% CI, 0.78% to 3.64%) and 3.93% (95% CI, 1.94% to 5.92%), respectively. Interestingly, for all 15 patients with recurrent gametocytemia, this was associated with concurrent asexual stages. Genotyping of recurrent parasites at the merozoite surface protein 3α gene locus from 12 patients with recurrent parasitemia within 28 days revealed that 10 of these were the same genotype as at day 0, suggesting recrudescence or relapse. Similar studies in 70 patients in the same area in 2007 showed no recurrent parasitemias within 28 days. The sensitivity to chloroquine of P. vivax in northeastern Myanmar may be deteriorating.     

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.)     

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.     

Chloroquine resistance in the malarial parasite,Plasmodium falciparum

Malarial parasites remain a health problem of staggering proportions. Worldwide, they infect about 500 million, incapacitate tens of millions, and kill approximately 2.5 million (mostly children) annually. Four species infect humans, but most deaths are caused by one particular species, Plasmodium falciparum. The rising number of malarial deaths is due in part to increased drug resistance in P. falciparum. There are many varieties of antimalarial drug resistance, and there may very well be several molecular level contributions to each variety. This is because there are a number of different drugs with different mechanisms of action in use, and more than one molecular event may sometimes be relevant for resistance to any one class of drugs. Thus, "multidrug" resistance in a clinical setting likely entails complex combinations of overlapping resistance pathways, each specific for one class of drug, that then add together to confer the particular multidrug resistance phenotype. Nonetheless, rapid progress has been made in recent years in elucidating mechanisms of resistance to specific classes of antimalarial drugs. As one example, resistance to the antimalarial drug chloroquine, which has been the mainstay therapy for decades, is becoming well understood. This article focuses on recent advances in determining the molecular mechanism of chloroquine resistance, with particular attention to the biochemistry and biophysics of the P. falciparum digestive vacuole, wherein changes in pH have recently been found to be associated with chloroquine resistance.     

Profound thrombocytopenia in Plasmodium vivax malaria

In India, malaria is endemic and commonly caused by Plasmodium vivax and P. falciparum. Thrombocytopenia is a common finding in falciparum infection but is rare in P. vivax infection. We report profound thrombocytopenia in a 43-year-old female patient due to P. vivax infection. The platelet count was as low as 5 x 10(9)/liter, such severe thrombocytopenia has never been reported in vivax malaria.     

Evidence of Selective Sweeps in Genes Conferring Resistance to Chloroquine and Pyrimethamine in Plasmodium falciparum Isolates in India

Treatment of Plasmodium falciparum is complicated by the emergence and spread of parasite resistance to many of the first-line drugs used to treat malaria. Antimalarial drug resistance has been associated with specific point mutations in several genes, suggesting that these single nucleotide polymorphisms can be useful in tracking the emergence of drug resistance. In India, P. falciparum infection can manifest itself as asymptomatic, mild, or severe malaria, with or without cerebral involvement. We tested whether chloroquine- and antifolate drug-resistant genotypes would be more commonly associated with cases of cerebral malaria than with cases of mild malaria in the province of Jabalpur, India, by genotyping the dhps, dhfr, pfmdr-1, and pfcrt genes using pyrosequencing, direct sequencing, and real-time PCR. Further, we used microsatellites surrounding the genes to determine the origins and spread of the drug-resistant genotypes in this area. Resistance to chloroquine was essentially fixed, with 95% of the isolates harboring the pfcrt K76T mutation. Resistant genotypes of dhfr, dhps, and pfmdr-1 were found in 94%, 17%, and 77% of the isolates, respectively. Drug-resistant genotypes were equally likely to be associated with cerebral malaria as with mild malaria. We found evidence of a selective sweep in pfcrt and, to a lesser degree, in dhfr, indicating high levels of resistance to chloroquine and evolving resistance to pyrimethamine. Microsatellites surrounding pfcrt indicate that the resistant genotypes (SVMNT) were most similar to those found in Papua New Guinea.Malaria is arguably the most important vector-borne disease in the world, with annual morbidity and mortality estimates surpassing 300 and 1 million, respectively (20, 61). Over 90% of the total malaria incidence is reported from sub-Saharan and tropical Africa; however, each year Southeast Asia, including the Indian subcontinent, reports approximately 2.5 million malaria cases, 75% of which are from India (20). Further, Plasmodium falciparum incidence in India has increased dramatically over the past few years, including the spread of drug-resistant strains (1, 2, 23, 46, 47).Efforts to control malaria have been hindered by the rapid rise and spread of drug-resistant P. falciparum strains. Chloroquine (CQ)-resistant strains of P. falciparum first appeared in the late 1950s, almost simultaneously in Southeast Asia and South America (51, 58, 64), and subsequently spread through most regions where P. falciparum is endemic. Sulfadoxine-pyrimethamine (SP) was next used as the drug of choice against CQ-resistant malaria; however, resistance quickly emerged on the Thai-Cambodian border around 1980 and is now found throughout most of Southeast Asia, the Amazonian basin of South America, and Africa (1, 2, 7, 17, 40, 53). In India, CQ and SP resistance was first documented in 1973 (42) and 1979 (10), respectively, in the northeast region of the country. Now, studies using molecular markers suggest that CQ and SP resistance is widespread across India (1, 23, 55). However, in India CQ still remains the first line of treatment for Plasmodium vivax malaria and for P. falciparum in low-risk and CQ-sensitive areas. In light of reports of CQ treatment failures, artesunate plus SP (artesunate combination therapy [ACT]) has been introduced in states with high burdens of P. falciparum malaria (6, 45) and is being implemented for other districts with high prevalences of P. falciparum.Resistance to chloroquine has been associated with point mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene (16), while resistance to SP has been linked to the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes (32, 52). Point mutations in P. falciparum multidrug resistance gene 1 (pfmdr-1) have been reported to modulate resistance to different antimalarial drugs, and variations in copy number appear to be associated with mefloquine resistance (12, 36).In order to combat drug resistance in Plasmodium falciparum, it is important to understand the genetic basis and the evolutionary forces affecting loci governing resistance. The discovery of new drug targets and the development of effective drugs and vaccines require careful study of the population genetics of P. falciparum. Investigations regarding point mutations in genes conferring drug resistance and the microsatellite loci that surround these genes can provide information on selection pressures, rates of recombination, and the potential origin of the resistant alleles or mutations.P. falciparum infection can manifest as asymptomatic, mild (uncomplicated) malaria or severe malaria, with or without cerebral involvement; little is known about the factors involved in these clinical manifestations. Cerebral malaria (CM) is one of the most common complications of P. falciparum infection in India, besides severe malaria anemia and multiorgan failure (26, 27, 60, 61). It is not known whether drug-resistant parasites also contribute to the increased risk for CM, especially because patients may receive inadequate treatment with drugs of reduced efficacy. In this context, we were interested in determining whether parasites with resistant genotypes were more often associated with patients diagnosed with CM than with patients with mild malaria (MM). We hypothesized that individuals harboring resistant parasites may be more likely to progress to severe disease due to treatment failure than those with wild-type parasites. Additionally, we wanted to determine whether drug-resistant genotypes in India have evolved locally or have been influenced by gene flow from other regions. To this end, we genotyped four genes associated with drug resistance (pfcrt, dhfr, dhps, and pfmdr-1) and assessed the genetic diversity of microsatellites surrounding pfcrt, dhfr, and dhps from P. falciparum-positive blood samples taken from patients enrolled in a hospital-based study to assess neurological disorders associated with cerebral malaria in central India.     

多重巢式PCR检测恶性疟和间日疟原虫

目的建立鉴别诊断恶性疟原虫(P.f)和间日疟原虫(P.v)的多重巢式PCR法。方法针对P.f、P.v 18S rRNA基因设计外引物和内引物,优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重巢式PCR,并检测54例疑似疟疾临床标本,以镜检法为金标准评价敏感性和特异性等指标。结果该方法可扩增出162 bp(P.f)和112 bp(P.v)基因片段,并能检出混合感染。该方法检测P.f,敏感性为87.50%、特异性为63.33%;检测P.v,敏感性为69.23%、特异性为68.29%。结论所建立的多重巢式PCR方法能可靠诊断疟疾并鉴别虫种,敏感性高,在混合感染的诊断方面具有优越性。     

Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and Plasmodium vivax

Monoclonal antibodies were raised against sporozoites of two species of malaria parasites, Plasmodium falciparum and Plasmodium vivax. The antibodies reacted with polypeptides (circumsporozoite proteins) that are uniformly distributed over the entire surface of sporozoites, as shown by indirect immunofluorescence and by the circumsporozoite precipitin reaction. The epitopes recognized by the monoclonal antibodies were expressed on sporozoites from different geographical isolates of the homologous species but were not detected on sporozoites of heterologous species nor on blood forms of the parasite. The monoclonal antibody to P. falciparum specifically immunoprecipitated two polypeptides of apparent 67,000 mol wt (Pf67) and 58,000 mol wt (Pf58) from extracts of [35S]methionine-labeled P. falciparum sporozoites. Similarly, the anti-P. vivax monoclonal immunoprecipitated two proteins of 51,000 mol wt (Pv51) and 45,000 mol wt (Pv45) from extracts of metabolically labeled P. vivax sporozoites. The extracts were also reacted with the serum of human volunteers successfully vaccinated with sporozoites of either P. vivax or P. falciparum. The patterns of immunoprecipitation were almost identical to those obtained with the corresponding monoclonal antibodies. The circumsporozoite proteins of P. falciparum and P. vivax play a role in immune protection. Incubation of the appropriate monoclonal antibody with viable sporozoites of the homologous species significantly reduced parasite infectivity, as determined by sporozoite neutralization assays carried out in splenectomized chimpanzees.     

Simultaneous Administration of 2-Aminoethyl Diphenylborinate and Chloroquine Reverses Chloroquine Resistance in Malaria Parasites

A nearly complete reversal of chloroquine (CQ) resistance in the CQ-resistant Plasmodium falciparum K-1 strain, with a significant decrease in the mean ± standard deviation (SD) 50% inhibitory concentration (IC₅₀) from 1,050 ± 95 nM to 14 ± 2 nM, was achieved in vitro by the simultaneous administration of 2-aminoethyl diphenylborinate (2-APB). The CQ resistance-reversing activity of 2-APB, which showed the same efficacy as verapamil, was also observed in an in vivo mouse infection model with the CQ-resistant Plasmodium chabaudi AS(30CQ) strain.     

In Vitro Activity of Pyronaridine against Multidrug-Resistant Plasmodium falciparum and Plasmodium vivax

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 (IC₅₀) 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 IC₅₀s 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.Almost 40% of the world''s population is at risk for infection by Plasmodium vivax, with an estimated 132 to 391 million clinical infections each year (19). Although chloroquine (CQ) remains the treatment of choice in most of the P. vivax-endemic world, this status is now being undermined by the emergence and spread of chloroquine-resistant (CQR) P. vivax. First reported in the 1980s on the island of New Guinea (2, 23), CQR P. vivax has since spread to other parts of Asia and recently to South America (1). In Papua, Indonesia, CQ resistance in P. vivax has reached levels precluding the use of CQ in most of the province (22, 30). There is an urgency to assess the efficacies of alternative antimalarial agents against drug-resistant P. vivax and to develop new strategies to combat the parasite.Pyronaridine (Pyr), a Mannich base synthesized in China in the 1970s (3, 16), is being developed as a novel antimalarial for multidrug-resistant malaria. It demonstrates potent in vitro activity against erythrocytic stages of Plasmodium falciparum (8, 24, 26, 36), retaining efficacy against CQR isolates (12, 17, 18). Clinical trials have shown excellent efficacy of monotherapy against multidrug-resistant falciparum malaria (14, 24, 25), with the early therapeutic response faster when combined with artesunate (20). Phase III studies with a coformulation of Pyramax (Shin Poong Pharmaceuticals) containing artesunate plus pyronaridine have recently been completed (34).Less is known of the antimalarial properties of pyronaridine against P. vivax, although early clinical studies in China demonstrated a rapid therapeutic response (3). To investigate the activity of pyronaridine against CQR P. vivax, we applied a modified schizont maturation assay on fresh field isolates from Papua, Indonesia, where CQR P. vivax is highly prevalent.     

Synergism between pyronaridine and retinol in Plasmodium vivax in vitro

Estimates of the annual number of infections with Plasmodium vivax reach 391 million. So far the blood-schizontocidal therapy with chloroquine remained effective in most parts of the world, but reports about emerging resistance are increasing. The study had the objective of determining the pharmacodynamic interaction between pyronaridine and retinol in Plasmodium vivax, since pyronaridine is a potential alternative for chloroquine and an earlier study had shown strong synergism between pyronaridine and retinol in Plasmodium falciparum. The study was conducted at the Malaria Clinic of Mae Sot, Tak Province, Thailand, near the border to Myanmar. The in vitro observations followed the method of Tasanor. Successful tests were performed with 44 isolates. The mean IC(50), IC(90) and IC(99) values for pyronaridine were 9.8, 2069.6 and 162446.5 nM. The mean IC(50), IC(90) and IC(99) values for the combinations with retinol (corresponding to the 50th, 65th and 80th percentile of the physiological retinol levels in healthy adults) were 1.7, 542.8 and 59379.5 nM for pyronaridine + retinol "low", for the combination with retinol "medium" they were 0.5, 313.7 and 58891.4 nM and for the combination with retinol "high" they were 0.2, 96.7 and 16754.3 nM. These results suggest strong synergism between the two substances.     

Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion

Plasmodium vivax and the related monkey malaria, P. knowlesi, require interaction with the Duffy blood group antigen, a receptor for a family of chemokines that includes interleukin 8, to invade human erythrocytes. One P. vivax and three P. knowlesi proteins that serve as erythrocyte binding ligands in such interactions share sequence homology. Expression of different regions of the P. vivax protein in COS7 cells identified a cysteine-rich domain that bound Duffy blood group-positive but not Duffy blood group-negative human erythrocytes. The homologous domain of the P. knowlesi proteins also bound erythrocytes, but had different specificities. The P. vivax and P. knowlesi binding domains lie in one of two regions of homology with the P. falciparum sialic acid binding protein, another erythrocyte binding ligand, indicating conservation of the domain for erythrocyte binding in evolutionarily distant malaria species. The binding domains of these malaria ligands represent potential vaccine candidates and targets for receptor-blockade therapy.     

Platelet alloantigen frequencies in Amazon Indians and Brazilian blood donors

The frequencies of human platelet-specific alloantigens (HPAs) vary between different ethnic groups, and genotyping using DNA techniques has been preferred over immunophenotyping methods for population studies. Using a polymerase chain reaction with allele-specific primers (PCR-ASP) method, we determined the allelic polymorphisms of five HPA systems among 174 unrelated individuals of two different Brazilian ethnic groups including Amazon Indians (n = 95) and blood donors (n = 79). Comparison of the calculated gene frequencies of the two alleles of HPA-1, -2, -3, -4 and -5 systems for Amazon Indians and Brazilian blood donors showed that gene frequencies obtained for the two alleles of HPA-1 (P<0.001), HPA-2 (P = 0.001) and HPA-5 (P<0.001) were significantly different between the two groups of individuals. All natives tested carried the HPA-2a and the HPA-5a alleles, but the HPA-1b and HPA-4b alleles are absent from the Indian population. It was also observed that all blood donors carried the HPA-1a, HPA-4a and HPA-5a alleles. In conclusion, the present data indicate differences in the frequency of the HPA systems between Amazon Indians and Brazilian subjects who present a high rate of racial admixture. While the frequencies of the HPA-1 and HPA-5 genes seen in Amazon Indians are similar to those reported for Oriental populations, the frequencies of the HPAs alleles in Brazilian blood donors are comparable to those reported for populations in North America and Europe.     

Antimalarial effects of rifampin in Plasmodium vivax malaria.

The antimalarial effects of rifampin in 60 adults with Plasmodium vivax malaria were assessed. There were three treatment groups: rifampin (20 and 15 mg/kg of body weight per day for 1 and 4 days, respectively; n = 5); rifampin followed by primaquine (15 mg of base per day for 14 days; n = 25), and chloroquine (25 mg of base per kg over 3 days) followed by primaquine (n = 30). All patients were hospitalized till clearance of fever and parasites, and 45 patients stayed in the hospital for 1 month. Despite initial clearance of fever in all patients and a > or = 6-fold reduction in parasitemia per 48-h life cycle, rifampin alone was not effective: all five patients had subsequent R2-like parasitological responses. All patients treated with rifampin-primaquine cleared both fever and parasitemia, but the therapeutic responses were slower than those following treatment with chloroquine-primaquine. Final fever clearance times were significantly longer (mean [standard deviation] = 43 [35] versus 27 [19] h; P = 0.046), and the parasite clearance times (to 50 and 90% of admission parasite counts and to a level undetectable in a peripheral blood smear) were also significantly greater (P = 0.053 to < 0.001). However, reappearance of infection occurred in only one patient treated with rifampin-primaquine. The results of this study suggest that rifampin at the usual therapeutic doses has partial activity against blood stages of P. vivax in humans but that used alone it is insufficient for cure. Rifampin might therefore be of value in combination antimalarial therapy.