The spread of drug-resistant Plasmodium falciparum, the most virulent malaria parasite, represents a serious concern for the treatment and control of falciparum malaria. It is generally believed that the emergence of drug-resistant P. falciparum is rare and geographically restricted 1 2 3 4 . Clinical resistance to chloroquine (CQ) was first identified simultaneously in two different geographic regions in the late 1950s; Southeast Asia (Thailand-Cambodia border) 5 and South America 6 . CQ resistance then expanded to neighbouring countries in the 1960s, and nearly all Southeast Asian countries by the mid-1970s 4 . In Melanesia, resistance to CQ was reported in the early 1960s in Indonesian West Papua, shortly after mass administration of CQ in medicated table salt 7 . Subsequently, it spread to Papua New Guinea 8 and the Solomon Islands 4 in 1976 and 1980, respectively. In Africa, CQ resistance was first reported in the late 1970s in Tanzania 9 10 , and it was found to be have been introduced from Southeast Asia 11 .

Since the discovery of P. falciparum chloroquine transporter (PfCRT) as a primary target of CQ resistance 12 , reports on the geographic origins and spread of CQ-resistant P. falciparum have accumulated 1 . PfCRT is localized to the parasite food vacuole and is known to have > 10 polymorphic amino acid sites 12 . Among these, an amino acid change from Lys (K) to Thr (T) at position 76 (K76T) plays a decisive role in conferring resistance to CQ 12 ; the mutation greatly reduces the accumulation of CQ in the parasite food vacuole by accelerating efflux of CQ 13 . Microsatellite (MS) analysis flanking the PfCRT gene, pfcrt, has revealed that the geographic origin of CQ resistance is quite limited, with only four CQ resistant lineages initially identified: one in Indochina/Africa, one in Melanesia, and two in South America (Brazil/Peru and Ecuador/Colombia) 1 . Subsequently, one distinct CQ-resistant lineage was discovered in isolates originating in the Philippines 14 .

These CQ resistant lineages harbour one of four mutant pfcrt genotypes at positions 72-76 (CV

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This possibility can be inferred from the previous finding of a laboratory-maintained CQ-sensitive P. falciparum clone (106/1) originating in Sudan that apparently underwent a back mutation from T to K at position 76 12 . It is thus likely that novel pfcrt genotypes may be identified through surveys of large numbers of P. falciparum isolates from diverse geographic areas. In support of this, recent large-scale surveys of genotypes of the dihydrofolate reductase gene dhfr, a target of pyrimethamine, and of the dihydropteroate gene dhps, a target of sulphadoxine, have identified several novel genotypes from wide geographic areas 24 25 , and have provided insights into the evolutionary history of drug resistance in P. falciparum.

In this study, genotyping of pfcrt and haplotyping of MS markers flanking pfcrt were performed in P. falciparum isolates collected from Asia (Bangladesh, Cambodia, Lao P.D.R., Philippines and Thailand), Melanesia (Papua New Guinea and Vanuatu), and Africa (Ghana). Results showed that there were only three CQ-resistant lineages, all of which were identified previously 1 14 . No evidence of back mutation was observed at position 76. Importantly, however, among pfcrt genotypes having the K76T mutation, amino acid changes other than K76T appear to have been recently generated on at least several occasions, producing novel pfcrt mutant genotypes.

The present study using 256 P. falciparum isolates collected from Asia, Melanesia and Africa revealed only three major lineages of CQ resistance originating in Indochina, Melanesia and the Philippines. No novel pfcrt genotype was identified in the present samples collected from large-scale survey. The number of CQ-mutant lineages observed in this study was identical to that in two previous studies that analysed 48 laboratory-adapted CQ-resistant parasite lines 1 and field isolates in the Philippines 14 . This observation supports the hypothesis that the emergence of CQ resistance is a rare event, but the reasons for this rare emergence of CQ resistance remain to be clarified.

This study also revealed that a single CQ resistance lineage contained two mutant pfcrt genotypes; the CV

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The role of pfcrt mutations other than K76T remains to be fully elucidated. They may confer some benefit to CQ-resistant parasites, e.g., a small effect on CQ tolerance or compensation for impaired protein activity after the acquisition of the critical mutation K76T. For the conventional anti-malarial drug pyrimethamine, it is known that levels of resistance increase as mutations progressively accumulate in dhfr; relative IC₅₀ values of pyrimethamine for mutant dhfr genotypes, as compared to the wild dhfr genotype, are 35-fold higher in a single mutant and 1,111-fold higher in a quadruple mutant (highest resistance dhfr genotype) 38 . Similarly, association between the accumulation of mutations in dhps and progressive increases of sulphadoxine resistance have also been reported 39 . Further studies are necessary to clarify the potential association between the accumulation of additional pfcrt mutations and augmentation of CQ resistance.

The present analysis did not identify a back mutation at position 76 in pfcrt, as observed in previous studies 26 27 28 . However, there remains the possibility that the back mutation has occurred in geographic areas not studied here. Most of the isolates sampled in the present study came from Asia and Melanesia, where CQ is still being used for treatment of Plasmodium vivax. Thus, the continuous CQ pressure present in Asia and Melanesia may be suppressing the expression of this back mutation. Further molecular epidemiological studies in different endemic areas having different histories of CQ usage may be necessary in order to better understand the possible back mutation at the critical position 76, which would indicate the recovery of CQ susceptibility.

The conclusions of the present study are limited to samples collected from the late 1990s to the mid 2000s in geographical areas where samples were collected. Although the intercontinental migration of CQ resistance from Asia to Africa was already accomplished, the selection and spread of CQ resistance was not homogenously advanced in Asia. In fact, at the time of our sampling, while mutant pfcrt alleles were almost fixed in Thailand and Cambodia, the selection seemed to be ongoing in Lao PDR, where nearly half of P. falciparum still harboured the wild pfcrt genotype.

The present molecular analysis using samples across Asia, Africa and Melanesia provided two important insights into CQ resistance in terms of malaria control. First, the emergence of novel CQ-mutant lineages among P. falciparum isolates is rare. Second, in isolates harbouring the K76T mutation, additional mutations in pfcrt other than K76T have readily been generated (in the last 30 years). These new mutations may confer stronger resistance to CQ than the K76T mutant. Further molecular monitoring of pfcrt genotypes should provide valuable information with regard to the current situation of CQ resistance and to the potential emergence of CQ-sensitive P. falciparum isolates in areas where CQ use has been withdrawn.

The authors declare that they have no competing interests.

NT performed experiments, data analysis and paper writing. TT1, MD, LD, BK, JS, MN, MS, JK, FH, TT2 and AK coordination of sampling. KT critically reviewed the manuscript. KT, HE and TM participated in acquisition of funding. TM made substantial contributions to study design, coordination of sampling, data analysis, paper writing and reviewing. All authors have read and approved the final manuscript.