Abstract

Chloroquine (CQ): a drug of choice for malaria treatment - Malaria is an infectious disease that has been present in the tropics for much of history. It varies widely in epidemiology and clinical manifestation and is responsible for an estimated 216 million clinical episodes and approximately 655,000 deaths per year, of which approximately 90% occur in Africa (WHO 2011). The variability in the spectrum of malarial diseases is the result of several factors, including the distribution of the two primary species of malaria parasites ( Plasmodium falciparum and Plasmodium vivax ), their levels of susceptibility to antimalarial drugs, the distribution and efficiency of mosquito vectors, climate and other environmental conditions and the behaviour and level of acquired immunity of the exposed human populations (Bloland 2001). Due to the lack of an effective vaccine, malaria is currently incurable and thus its case management depends solely on anti-malarials (WHO 1973, 1984). In the Western world, the first anti-malarial used to treat human malaria was quinine, which is extracted from the bark of the cinchona tree and was described as early as 1632 (Baird et al. 1996). In Chinese medicine, the use of Artemisia annua (qinghao) plants for the treatment of intermittent fever/malaria was described as early as 283-343 AD. Around 340 AD, in Hong Ge’s Handbook of Prescriptions for Emergency Treatment , a cold extraction method of qinghao was described for the treatment of intermittent fevers (Klayman 1985). Although primaquine and quinacrine were produced after World War I (1914-1918) and remained effective for malaria treatment for a period of time, the intense demand for other anti-malarials led to the discovery of CQ by Bayer in Germany (Thompson & Werbel 1972).

CQ, a 4-aminoquinoline derivative of quinine, was first synthesised in 1934 ( Thompson & Werbel 1972Thompson PE, Werbel LM 1972. Antimalarial agents: chemistry and pharmacology , Academic Press Inc, New York, 395 pp. ) and has since become the most widely used antimalarial drug. Historically, CQ was used to combat malaria in 1946 after the Second World War. Since then, it has been considered to be the drug of choice for the treatment of non-severe, un-complicated malaria and for chemoprophylaxis. Apart from certain toxic side effects, such as retinal and psychiatric symptoms, cardiac disorders, respiratory depression, neurological problems and severe gastro-intestinal irritation ( Telgt et al. 2005Telgt DS, van der Ven AJ, Schimmer B, Droogleever-Fortuyn HA, Sauerwein RW 2005. Serious psychiatric symptoms after chloroquine treatment following experimental malaria infection. Ann Pharmacother 39 : 551-554. ), certain unique properties, including high efficacy, wide distribution, ready availability, quick metabolism, inexpensiveness and high therapeutic index ( Payne 1987Payne D 1987. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol Today 3 : 241-246. ), made CQ the drug of choice for treating malaria ( Coatney 1963Coatney GR 1963. Pitfalls in a discovery: the chronicle of chloroquine. Am J Trop Med Hyg 12: 121-128. ). Over the years, CQ has proven to be one of the most successful and important drugs ever deployed against malaria, especially in the highly endemic areas of Africa, where the malaria parasite P. falciparum infects nearly every child ( Wellems & Plowe 2001Wellems TE, Plowe CV 2001. Chloroquine resistant malaria. J Infect Dis 184 : 770-776. ). This efficiency of CQ also prompted the World Health Organization (WHO) to spearhead projects and establish large mass drug administration programs using CQ ( Litsios 1996Litsios S 1996. The tomorrow of malaria , Pacific Press, Wellington, 181 pp. , WHO 2002WHO - World Health Organization 2002. Southern Africa Malaria Control Programme. WHO/CDS/RBM/2002.42. ). The introduction of CQ near the end of Second World War brought dramatic new power to malaria control programs ( Wellems et al. 2009Wellems TE, Hayton K, Fairhurst RM 2009. The impact of malaria parasitism: from corpuscles to communities. J Clin Invest 119 : 2496-2505. ) and these efforts further reduced the incidence of malaria in most of the endemic regions in the world. However, the malaria eradication campaign started by the WHO in the 1950s excluded Africa, the continent with the highest burden of malaria and focused on the rest of the world. As such, by the late 1950s and early 1960s, malaria was eradicated in most of the Western world and was reduced to its historically lowest level in Asia and the Americas, but remained at approximately the same level in Africa ( Talisuna et al. 2004Talisuna AO, Bloland P, D’Alessandro U 2004. History, dynamics and public health importance of malaria parasite resistance. Clin Microbiol Rev 17 : 235-254. ).

Mechanism of action of CQ - CQ acts on the endolysosomal system of malaria parasites, causing morphologic changes and haemoglobin accumulation in endocytic vesicles ( Fitch 2004Fitch CD 2004. Ferriprotoporphyrin IX, phospholipids and the antimalarial actions of quinoline drugs. Life Sci 74 : 1957-1972. , Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ). Being alkaline in nature, CQ accumulates in high concentrations within the digestive vacuole (DV) of the parasite and raises its pH. Because the DV is acidic in pH, the deprotonation of CQ renders the DV alkaline ( Orjih et al. 1994Orjih AU, Ryerse JS, Fitch CD 1994. Hemoglobin catabolism and the killing of intraerythrocytic Plasmodium falciparum by chloroquine. Experientia 50 : 34-39. , Ecker et al. 2012Orjih AU, Ryerse JS, Fitch CD 1994. Hemoglobin catabolism and the killing of intraerythrocytic Plasmodium falciparum by chloroquine. Experientia 50 : 34-39. ). CQ then induces the rapid clumping of the malarial pigment and eventually inhibits the parasitic enzyme haeme polymerase, which normally converts the toxic ferric haeme (ferriprotoporphyrin IX) into the non-toxic haemozoin (5-haematin). This inhibition results in the accumulation of toxic ferric haeme, leading to lysis and, ultimately, parasite death ( Roepe 2009Roepe PD 2009. Molecular and physiologic basis of quinoline drug resistance in Plasmodium falciparum malaria. Future Microbiol 4 : 441-455. , Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ). Studies have suggested that the mechanism of action of CQ relies heavily on the accumulation of high concentrations of the drug ( Fitch 2004Fitch CD 2004. Ferriprotoporphyrin IX, phospholipids and the antimalarial actions of quinoline drugs. Life Sci 74 : 1957-1972. , Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ).

CQ resistance (CQR) is a major hurdle to malaria control - The tremendous success of CQ and its heavy use for almost 12 years ( Wongsrichanalai et al. 2002Wongsrichanalai C, Pickard AL, Wernsdorfer WH 2002. Epidemiology of drug resistant malaria. Lancet Infect Dis 2 : 209-218. ) led to the development of resistance in P. falciparum during the late 1950s ( Maberti 1960Maberti S 1960. Development of resistance to pyrimethamine. Presentation of 15 cases studied in Trujillo, Venezuela. Arch Venez Med Trop Parasitol Med 3 : 239-259. , Moore & Lanier 1961Moore DV, Lanier JE 1961. Observations on the two Plasmodium falciparum infections with an abnormal response to chloroquine. Am J Trop Med Hyg 10 : 5-9. , Young & Moore 1961Young MD, Moore DV 1961. Chloroquine resistance in Plasmodium falciparum . Am J Trop Med Hyg 10 : 317-320. , Reyes 1981Reyes S 1981. Malarial infections with Plasmodium falciparum resistant to chloroquine treatment. The situation in Brazil (1960-1981). Rev Bras Malariol Doencas Trop 33 : 109-130. , Peters 1987Peters W 1987. Chemotherapy and drug resistance in malaria , 2nd ed., Academic Press, London, 542 pp. ). The contribution of the extensive use and misuse of CQ to the selection of resistant parasites became particularly evident during the Global Malaria Eradication Campaign, which was launched by the WHO in 1955. CQR was implicated in the spread of malaria to new areas and the re-emergence of malaria in areas where the disease was previously eradicated due to population movement ( Bloland 2001Bloland PB 2001. Drug resistance in malaria , WHO/CDS/CRS/DRS, Geneva, 32 pp. , Tatem & Smith 2010Tatem AJ, Smith DL 2010. International population movements and regional Plasmodium falciparum malaria elimination strategies. Proc Natl Acad Sci USA 107 : 12222-12227. ). CQR was reported for the first time at the Thailand-Cambodia border in 1957 and the Venezuela-Colombia border in 1959 and eventually spread to other countries throughout the world ( Wernsdorfer & Payne 1991Wernsdorfer WH 1994. Epidemiology of drug resistance in malaria. Acta Trop 56 : 143-156. , Mehlotra et al. 2001Mehlotra RK, Fujioka H, Roepe PD 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with Pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci USA 98 : 12689-12694. , Ridley 2002Ridley RG 2002. Medical need, scientific opportunity and the drive for anti-malarial drugs. Nature 415 : 686-693. ). Moreover, several recent molecular epidemiological studies have identified at least six independent origins of CQR from different regions of the world ( Mehlotra et al. 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. , Wellems et al. 2009Wellems TE, Hayton K, Fairhurst RM 2009. The impact of malaria parasitism: from corpuscles to communities. J Clin Invest 119 : 2496-2505. ). Despite the suggested multiple independent origins of CQR, CQR parasites share some common phenotypes, such as increased 50% inhibitory concentration (IC ₅₀ ), chemosensitisation, reduced CQ accumulation, low pH in the DV and similar genetic mutations ( ^{Jiang et al. 2006}Jiang H, Joy DA, Furuya T, Su X-z 2006. Current understanding of the molecular basis of chloroquine-resistance in Plasmodium falciparum . J Postgrad Med 52 : 271-276. ). Drug pressure in the field is also considered to be an essential prerequisite for the development of resistance ( Wellems 2002Wellems TE 2002. Plasmodium chloroquine resistance and the search for a replacement antimalarial drug . Science 298 : 124-126. , Plowe 2009Plowe CV 2009. The evolution of drug-resistant malaria. Trans R Soc Trop Med Hyg 1035 : 11-14. ). However, the rate at which drug resistance spreads and how the resistant mutants survive in nature are still a matter of investigation ( Talisuna et al. 2004Talisuna AO, Bloland P, D’Alessandro U 2004. History, dynamics and public health importance of malaria parasite resistance. Clin Microbiol Rev 17 : 235-254. , Anderson & Roper 2005Anderson TJ, Nair S, Qin H, Singlam S, Brockman A, Paiphun L, Nosten F 2005. Are transporter genes other than the chloroquine resistance locus ( pfcrt ) and multidrug resistance gene ( pfmdr ) associated with antimalarial drug resistance? Antimicrob Agents Chemother 49 : 2180-2188. , Hyde 2005Hyde JE 2005. Drug-resistant malaria. Trends Parasitol 21 : 494-498. ). Several models, including the degree of drug use, drug elimination half-life, host heterogeneity ( Hastings et al. 2002Hastings IM, Watkins WM, White NJ 2002. The evolution of drug-resistant malaria: the role of drug elimination half-life. Philos Trans R Soc Lond B Biol Sci 357 : 505-519. ), parasite biomass ( Hastings & D’Alessandro 2000Hastings IM, D’Alessandro U 2000. Modeling a predictable disaster: the rise and spread of drug-resistant malaria. Parasitol Today 16 : 340-347. ), parasite fitness ( Walliker et al. 2005Walliker D, Hunt P, Babiker H 2005. Fitness of drug-resistant malaria parasites . Acta Trop 94 : 251-259. ), malaria transmission intensity ( Hastings & Watkins 2005Hastings IM, Watkins WM 2005. Intensity of malaria transmission and the evolution of drug resistance. Acta Trop 94 : 218-229. ), host immunity and intrahost dynamics ( Hastings 1997Hastings IM 1997. A model for the origins and spread of drug resistant malaria. Parasitology 115 : 133-141. ), were developed to better understand this drug resistance ( Talisuna et al. 2003Talisuna AO, Langi P, Mutabingwa TK, Van Marck E, Speybroeck N, Egwang TG, Watkins WW, Hastings IM, D’Alessandro U 2003. Intensity of transmission and spread of gene mutations linked to chloroquine and sulphadoxine-pyrimethamine resistance in falciparum malaria. Int J Parasitol 33 : 1051-1058. , 2004Talisuna AO, Bloland P, D’Alessandro U 2004. History, dynamics and public health importance of malaria parasite resistance. Clin Microbiol Rev 17 : 235-254. , 2007Talisuna AO, Okello PE, Erhart A, Coosemans M, D’Alessandro U 2007. Intensity of malaria transmission and the spread of Plasmodium falciparum -resistant malaria: a review of epidemiologic field evidence. Am J Trop Med Hyg 77 : 170-180. ). Because CQR parasites have been experimentally shown to have greater fitness potential in CQ environments than CQS parasites ( Walliker et al. 2005Walliker D, Hunt P, Babiker H 2005. Fitness of drug-resistant malaria parasites . Acta Trop 94 : 251-259. ), the resistant parasites were able to spread and establish themselves throughout the P. falciparum malaria-endemic zones. Following the emergence and spread of CQR, the drug policies of many countries were revised and several new drugs were introduced in the field either as single agents or in combination therapies. Gradually, P. falciparum developed resistance to nearly all anti-malarials in use ( Table I ), although the geographical distribution of resistance to any single-agent antimalarial drug varies greatly ( Bloland 2001Bloland PB 2001. Drug resistance in malaria , WHO/CDS/CRS/DRS, Geneva, 32 pp. , Mita & Tanabe 2012Mita T, Tanabe K 2012. Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin resistance. Jpn J Infect Dis 65 : 465-475. ). Like CQ, the extensive deployment of other antimalarial drugs also placed tremendous selection pressure on P. falciparum to evolve mechanisms of resistance ( Anderson 2009Anderson TJ 2009. Mapping the spread of malaria drug resistance. PLoS Med 6 : e1000054. ). Additionally, cross-resistance and the genetic plasticity of the parasite contributed to CQR ( White 2004White NJ 2004. Antimalarial drug resistance. J Clin Invest 113 : 1084-1092. ). Despite the prevalence of CQR in P. falciparum , CQ still remains the drug of choice for the treatment of non-severe P. falciparum and non- P. falciparum infections in many malaria-endemic countries and its several unique properties make it advantageous over all other anti-malarial drugs. Despite the introduction of several therapies to treat complicated and non-complicated malaria, CQ still has a prominent place in malaria treatment. Thus, the development of CQR poses a great hurdle to malaria control measures and has contributed to rollbacks in malaria programmes ( Talisuna et al. 2004Talisuna AO, Bloland P, D’Alessandro U 2004. History, dynamics and public health importance of malaria parasite resistance. Clin Microbiol Rev 17 : 235-254. ).

Genetics of CQR in P. falciparum - Identification of the Pfcrt gene - Soon after CQR P. falciparum isolates were found to be widespread in malaria-endemic zones, the mutagenic basis of CQR was made evident by several clinical and epidemiological studies ( Wellems et al. 1991Wellems T, Walker-Jonah A, Panton LJ 1991. Genetic mapping of the chloroquine resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci USA 88 : 3382-3386. , Fidock et al. 2000Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su X-z, Wootton JC, Roepe PD, Wellems TE 2000. Mutations in the P. falcipa rum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 : 861-871. ). Phenotypic studies involving genetic crosses between CQR and CQ-sensitive (CQS) strains further supported the hypothesis of the genetic basis of CQR ( Wellems et al. 1991Wellems T, Walker-Jonah A, Panton LJ 1991. Genetic mapping of the chloroquine resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci USA 88 : 3382-3386. ) and genetic loci on chromosome 13 ( Pfnhe 1 gene) ( Fig. 1 ) and chromosome 5 ( Pfmdr 1 gene) ( Fig. 1 ) were proposed to be associated with higher IC ₅₀ values in the progeny of genetic crosses ( Wellems et al. 1991Wellems T, Walker-Jonah A, Panton LJ 1991. Genetic mapping of the chloroquine resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci USA 88 : 3382-3386. , Ferdig et al. 2004Ferdig MT, Cooper RA, Mu J, Deng B, Joy DA, Su XZ, Wellems TE 2004. Dissecting the loci of low-level quinine resistance in malaria parasites. Mol Microbiol 52 : 985-997. ). However, a direct association between Pfnhe 1 gene mutations and CQR could not be established. Instead, Pfnhe1 mutations were found to correlate well with quinine in several studies ( Cooper et al. 2002Cooper RA, Ferdig MT, Su XZ, Ursos LM, Mu J, Nomura T, Fujioka H, Fidock DA, Roepe PD, Wellems TE 2002. Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum . Mol Pharmacol 61 : 35-42. , Hayton & Su 2004Hayton K, Su X-z 2004. Genetic and biochemical aspects of drug resistance in malaria parasites. Curr Drug Targets Infect Disord 4 : 1-10. , 2008Hayton K, Su X-z 2008. Drug resistance and genetic mapping in Plasmodium falciparum . Curr Genet 54 : 223-239. ). Mutations in Pfmdr1 , which encodes a homolog of the human multidrug resistance p -glycoprotein ( PfPgh 1), were also found to be associated with CQR ( Djimde et al. 2001Djimde AA, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourté Y, Coulibaly D, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV 2001. A molecular marker for chloroquine resistant falciparum malaria. N Engl J Med 344 : 257-263. , Mu et al. 2003Mu J, Ferdig MT, Feng X, Joy DA, Duan J, Furuya T, Subramanian G, Aravind L, Cooper RA, Wootton JC, Xiong M, Su X-z 2003. Multiple transporters associated with malaria parasite responses to chloroquine and quinine. Mol Microbiol 49 : 977-989. , Duraisingh & Cowman 2005Duraisingh MT, Cowman AF 2005. Contribution of the pfmdr 1 gene to antimalarial drug-resistance. Acta Trop 94 : 181-190. , Sidhu et al. 2005Sidhu AB, Valderramos SG, Fidock DA 2005. Pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol 57 : 913-926. , Valderramos & Fidock 2006Valderramos SG, Fidock DA 2006. Transporters involved in resistance to antimalarial drugs. Trends Pharmacol Sci 27 : 594-601. ), but the contribution of Pfmdr1 in modulating CQR remains debatable ( Hayton & Su 2004Hayton K, Su X-z 2004. Genetic and biochemical aspects of drug resistance in malaria parasites. Curr Drug Targets Infect Disord 4 : 1-10. , 2008Hayton K, Su X-z 2008. Drug resistance and genetic mapping in Plasmodium falciparum . Curr Genet 54 : 223-239. ). It was later established that CQR is inherited as a single locus in a genetic cross between the CQR Dd2 (Indochina) and CQS HB3 (Honduras) clones and this locus was identified to be the single determinant of CQ sensitivity ( Wellems et al. 1991Wellems T, Walker-Jonah A, Panton LJ 1991. Genetic mapping of the chloroquine resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci USA 88 : 3382-3386. , Su et al. 1997Su X-z, Kirkman LA, Fujioka H, Wellems TE 1997. Complex polymorphisms in an approximately 330 kDa protein are linked to chloroquine-resistant P. falciparum in Southeast Asia and Africa. Cell 91 : 593-603. , Fidock et al . 2000Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su X-z, Wootton JC, Roepe PD, Wellems TE 2000. Mutations in the P. falcipa rum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 : 861-871. ). The CQR phenotype was further mapped to a 48-Kb chromosomal locus harbouring the highly interrupted gene Pfcrt ( P. falciparum CQR transporter) ( Figs 1 , 2 ). This gene is present on chromosome 7, spans 3.1 kb and has 13 exons ranging in size from 45-269 bp. It produces a 1,275-bp cDNA that encodes the 424-amino acid 48.6-kDa Pf CRT protein, which has 10 transmembrane domains (TMDs) ( Wellems et al. 1991Wellems T, Walker-Jonah A, Panton LJ 1991. Genetic mapping of the chloroquine resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci USA 88 : 3382-3386. , Su et al. 1997Su X-z, Kirkman LA, Fujioka H, Wellems TE 1997. Complex polymorphisms in an approximately 330 kDa protein are linked to chloroquine-resistant P. falciparum in Southeast Asia and Africa. Cell 91 : 593-603. , Fidock et al . 2000Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su X-z, Wootton JC, Roepe PD, Wellems TE 2000. Mutations in the P. falcipa rum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 : 861-871. , Bray et al. 2005Bray PG, Martin RE, Tilley L, Ward SA, Kirk K, Fidock DA 2005. Defining the role of Pf CRT in Plasmodium falciparum chloroquine resistance . Mol Microbiol 56 : 323-333. ). Further evidence establishing Pfcrt as a CQR determinant came from studies of culture-adapted field isolates, which showed that CQR P. falciparum isolates had extensive linkage disequilibrium (LD) surrounding a 36-Kb segment of Pfcrt ( Wootton et al. 2002Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su X-z 2002. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum . Nature 418 : 320-323. ).

Putative functional role of PfCRT in P. falciparum - The endogenous function of Pf CRT remains unknown, but its transmembrane structure and cellular location suggest that it is involved in the transport of critical metabolites, such as drugs and maintains the pH balance in the DV of P. falciparum ( Dzekunov et al. 2000Dzekunov SM, Ursos LM, Roepe PD 2000. Digestive vacuolar pH of intact intraerythrocytic P. falciparum either sensitive or resistant to chloroquine. Mol Biochem Parasitol 110 : 107-124. , Bennett et al. 2004Bennett TN, Kosar AD, Ursos LM, Dzekunov S, Sidhu ABS, Fidock DA, Roepe PD 2004. Drug resistance-associated PfCRT mutations confer decreased Plasmodium falciparum digestive vacuolar pH. Mol Biochem Parasitol 133 : 99-114. , Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ). Other potential roles for Pf CRT include the expulsion of amino acids resulting from haemoglobin digestion from the DV and indirect involvement in maintaining H ⁺ balance in the DV ( Jiang et al. 2008Jiang H, Patel JJ, Yi M, Mu J, Ding J, Stephens R, Cooper RA, Ferdig MT, Su X-z 2008. Genome-wide compensatory changes accompany drug-selected mutations in the Plasmodium falciparum crt gene. PLoS ONE 3 : e2484. ). The roles of other transporters, such as Pf VP2, the Ca ⁺² /H ⁺ antiporter VCX1, PFE0785c and ATPase/synthase (PF11_0412 and PFC0840w), which might also play crucial roles in CQR, have also been well documented ( Jiang et al. 2008Jiang H, Patel JJ, Yi M, Mu J, Ding J, Stephens R, Cooper RA, Ferdig MT, Su X-z 2008. Genome-wide compensatory changes accompany drug-selected mutations in the Plasmodium falciparum crt gene. PLoS ONE 3 : e2484. ). Moreover, phylogenetic analyses predict Pf CRT to be a member of the drug/metabolite transporter superfamily of electrochemical potential driven transporters, thus supporting its hypothesised roles in P. falciparum ( Martin & Kirk 2004Martin RE, Kirk K 2004. The malaria parasite’s chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol Biol Evol 21 : 1938-1949. ).

PfCRT, glutathione (GSH) and the human immune response - Human immune responses play an important role in shaping the ability of the host to resolve drug-resistant infections harbouring mutant Pfcrt ( Djimde et al. 2003Djimde AA, Doumbo OK, Traore O, Guindo AB, Kayentao K, Diourte Y, Niare-Doumbo S, Coulibaly D, Kone AK, Cissoko Y, Tekete M, Fofana B, Dicko A, Diallo DA, Wellems TE, Kwiatkowski D, Plowe CV 2003. Clearance of drug-resistant parasites as a model for protective immunity in Plasmodium falciparum malaria. Am J Trop Med Hyg 69 : 558-563. ), as failures in treatment are generally associated with specific polymorphisms in the parasite genome or gene copy number ( Picot et al. 2009Picot S, Olliaro P, de Monbrison F, Bienvenu AL, Price RN, Ringwald P 2009. A systematic review and meta-analysis of evidence for correlation between molecular markers of parasite resistance and treatment outcome in falciparum malaria. Malar J 8 : 89. ). A low level of CQR P. falciparum and acquired protective immunity can explain why CQ treatment is able to successfully cure some infections harbouring mutant Pfcrt parasites in semi-immune individuals ( Wellems & Plowe 2001Wellems TE, Plowe CV 2001. Chloroquine resistant malaria. J Infect Dis 184 : 770-776. , Djimde et al. 2003Djimde AA, Doumbo OK, Traore O, Guindo AB, Kayentao K, Diourte Y, Niare-Doumbo S, Coulibaly D, Kone AK, Cissoko Y, Tekete M, Fofana B, Dicko A, Diallo DA, Wellems TE, Kwiatkowski D, Plowe CV 2003. Clearance of drug-resistant parasites as a model for protective immunity in Plasmodium falciparum malaria. Am J Trop Med Hyg 69 : 558-563. ) and why, at other times, the immune response allows a relatively ineffective drug to clear an infection without any therapy ( Schofield & Mueller 2006Schofield L, Mueller I 2006. Clinical immunity to malaria. Curr Mol Med 6 : 205-221. , Greenhouse et al. 2009Greenhouse B, Slater M, Njama-Meya D, Nzarubara B, Maiteki-Sebuguzi C, Clark TD, Staedke SG, Kamya MR, Hubbard A, Rosenthal PJ, Dorsey G 2009. Decreasing efficacy of antimalarial combination therapy in Uganda is explained by decreasing host immunity rather than increasing drug resistance. J Infect Dis 199 : 758-765. ). Altered intracellular levels of GSH have been shown to cause a corresponding shift in CQ susceptibility in P. falciparum ( Ginsburg et al. 1998Ginsburg H, Famin O, Zhang J, Krugliak M 1998. Inhibition of glutathione dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action. Biochem Pharmacol 56 : 1305-1313. ). Additional indirect evidence has suggested a potential link between CQR and GSH ( Ginsburg & Golenser 2003Ginsburg H, Golenser J 2003. Glutathione is involved in the antimalarial action of chloroquine and its modulation affects drug sensitivity of human and murine species of Plasmodium . Redox Rep 8 : 276-279. ), which originated from the observation that the Pfmrp gene, which is localised to the parasite surface, is disrupted in CQR ( Raj et al. 2009Raj DK, Mu J, Jiang H, Kabat J, Singh S, Sullivan M, Fay MP, McCutchan TF, Su X-z 2009. Disruption of a Plasmodium falciparum multidrug resistance-associated protein ( PfMRP ) alters its fitness and transport of antimalarial drugs and glutathione. J Biol Chem 284 : 7687-7696. ). Moreover, a recent report found that Pf CRT homologs in Arabidopsis thaliana mediate GSH transport and stress tolerance when assayed in Xenopus oocysts ( Maughan et al. 2010Maughan SC, Pasternak M, Cairns N, Kiddle G, Brach T, Jarvis R, Haas F, Nieuwland J, Lim B, Müller C, Salcedo-Sora E, Kruse C, Orsel M, Hell R, Miller AJ, Bray P, Foyer CH, Murray JA, Meyer AJ, Cobbett CS 2010. Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, Pf CRT, are required for glutathione homeostasis and stress responses. Proc Natl Acad Sci USA 107 : 2331-2336. ).

The K76T mutation in PfCRT: a key factor? - Sequence comparisons of Pf CRT in CQR and CQS P. falciparum have identified several mutations, among which the mutation of residue 76 (wild type K to the mutant T) could be directly associated with CQR. This finding was confirmed by allelic exchange studies ( Fidock et al. 2000Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su X-z, Wootton JC, Roepe PD, Wellems TE 2000. Mutations in the P. falcipa rum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 : 861-871. , Sidhu et al . 2002Sidhu AB, Verdier-Pinard D, Fidock DA 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 298 : 210-213. , Lakshmanan et al. 2005Lakshmanan V, Bray PG, Verdier-Pinard D, Johnson DJ, Horrocks P, Muhle RA, Alakpa GE, Hughes RH, Ward SA, Krogstad DJ, Sidhu ABS, Fidock DA 2005. A critical role for PfCRT K76T in Plasmodium falciparum verapamil reversible chloroquine resistance. EMBO J 24 : 2294-2305. ). Other additional single nucleotide polymorphisms (SNPs) present in exons 2, 3, 4, 6, 9, 10 and 11 of the Pfcrt gene have also been proposed to have some association with CQR. Similarly, at the protein level, approximately 32 mutations in the 10 α-helical TMD of Pf CRT have been reported to be associated with CQR. Studies have suggested that these mutations might epistatically interact with the K76T mutation and might also evolve to maintain homeostasis ( Fidock et al. 2000Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su X-z, Wootton JC, Roepe PD, Wellems TE 2000. Mutations in the P. falcipa rum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 : 861-871. , Wootton et al. 2002Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su X-z 2002. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum . Nature 418 : 320-323. ). However, these mutations have been casually associated with resistance in vitro and in vivo and even with altered drug accumulation ( Sanchez et al. 2003Sanchez CP, Stein W, Lanzer M 2003. Trans stimulation provides evidence for a drug efflux carrier as the mechanism of chloroquine resistance in Plasmodium falciparum . Biochemistry 42 : 9383-9394. , 2004Sanchez CP, McLean JE, Stein W, Lanzer M 2004. Evidence for a substrate specific and inhibitable drug efflux system in chloroquine resistant Plasmodium falciparum strains. Biochemistry 43 : 16365-16373. , 2005Sanchez CP, McLean JE, Rohrbach P, Fidock DA, Stein WD, Lanzer M 2005. Evidence for a pfcrt -associated chloroquine efflux system in the human malarial parasite Plasmodium falciparum . Biochemistry 44 : 9862-9870. ). Regardless of the exact knowledge of these mutations, in general, it was found that parasites that are resistant to CQ and that bear mutations in Pf CRT accumulate less CQ due to either active energy-dependent CQ efflux ( Krogstad et al. 1987Krogstad DJ, Gluzman IY, Kyle DE, Oduola AM, Martin SK, Milhous WK, Schlesinger PH 1987. Efflux of chloroquine from Plasmodium falciparum : mechanism of chloroquine resistance. Science 238 : 1283-1285. , Sanchez et al. 2003Sanchez CP, Stein W, Lanzer M 2003. Trans stimulation provides evidence for a drug efflux carrier as the mechanism of chloroquine resistance in Plasmodium falciparum . Biochemistry 42 : 9383-9394. ) or the passive efflux of diprotic CQ ( Sanchez et al. 2010Peters W 1987. Chemotherapy and drug resistance in malaria , 2nd ed., Academic Press, London, 542 pp. ).

Compensatory mutations within Pfcrt and Pfmdr1 - Recent studies have suggested that Pf CRT mutations may affect parasite survival by switching back to their CQS form in the absence of drug pressure, perhaps owing to the low fitness properties of the resistant Pf CRT in competing against the sensitive Pf CRT, as observed in Malawi, Kenya and Hainan ( Kublin et al. 2003Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimdé AA, Kouriba B, Taylor TE, Plowe CV 2003. Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis 187 : 1870-1875. , Mita et al. 2003Mita T, Kaneko A, Lum JK, Bwijo B, Takechi M, Zungu IL, Tsukahara T, Tanabe K, Kobayakawa T, Bjorkman A 2003. Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi. Am J Trop Med Hyg 68 : 413-415. , 2004Mita T, Kaneko A, Lum JK, Zungu IL, Tsukahara T, Eto H, Kobayakawa T, Bjorkman A, Tanabe K 2004. Expansion of wild type allele rather than back mutation in pfcrt explains the recent recovery of chloroquine sensitivity of Plasmodium falciparum in Malawi. Mol Biochem Parasitol 135 : 159-163. , Wang et al. 2005Wang X, Mu J, Li G, Chen P, Guo X, Fu L, Chen L, Su X, Wellems TE 2005. Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People’s Republic of China. Am J Trop Med Hyg 72 : 410-414. , Laufer et al. 2006Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjalamala FK, Takala SL, Taylor TE, Plowe CV 2006. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med 355 : 1959-1966. , Mwai et al. 2009Mwai L, Ochong E, Abdirahman A, Kiara SM, Ward S, Kokwaro G, Sasi P, Marsh K, Borrmann S, Mackinnon M, Nzila A 2009. Chloroquine resistance before and after its withdrawal in Kenya. Malar J 8 : 106. ). On the contrary, some parasite lines ( e.g ., FCB and Dd2) grow well in vitro, even in the absence of drug pressure, suggesting the presence of potential compensatory mutations within Pf CRT. These compensatory changes within Pf CRT may not fully restore the biological functions of the protein and further changes in other parts of the genome may be required ( Jiang et al. 2008Jiang H, Patel JJ, Yi M, Mu J, Ding J, Stephens R, Cooper RA, Ferdig MT, Su X-z 2008. Genome-wide compensatory changes accompany drug-selected mutations in the Plasmodium falciparum crt gene. PLoS ONE 3 : e2484. ). This compensatory role is believed to be played by the Pfmdr1 gene. This hypothesis is supported by the fact that strong LD was found between variants of both the Pfcrt and Pfmdr1 genes ( Duraisingh et al. 2000Duraisingh MT, von Seidlein LV, Jepson A, Jones P, Sambou I, Pinder M, Warhurst DC 2000. Linkage disequilibrium between two chromosomally distinct loci associated with increased resistance to chloroquine in Plasmodium falciparum . Parasitology 121 : 1-7. , Adagut & Warhurst 2001Adagut IS, Warhurst DC 2001. Plasmodium falciparum : linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria. Parasitology 123 : 219-224. , Duraisingh & Refour 2005Duraisingh MT, Cowman AF 2005. Contribution of the pfmdr 1 gene to antimalarial drug-resistance. Acta Trop 94 : 181-190. , Mu et al. 2005Mu J, Awadalla P, Duan J 2005. Recombination hotspots and population structure in Plasmodium falciparum . PLoS Biol 3 : e335. , Sutar et al. 2011Sutar SKD, Gupta B, Ranjit M, Kar SK, Das A 2011. Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India. Mem Inst Oswaldo Cruz 106 : 78-84. ). Furthermore, Pfmdr1 was observed to be non-randomly associated with the mutant Pfcrt gene and directly related to CQR to improve parasite fitness ( Ekland & Fidock 2007Ekland EH, Fidock DA 2007. Advances in understanding the genetic basis of antimalarial drug resistance. Curr Opin Microbiol 10 : 363-370. ). Both genes also combine in a region-specific manner to create higher levels of drug resistance ( Sa et al. 2009Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE 2009. Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci USA 106 : 18883-18889. ). However, the precise role of the Pfmdr1 gene in the efflux mechanism of CQ in P. falciparum is still unclear ( Krogstad 1990Krogstad DJ 1990. Chloroquine resistance not linked to mdr -like genes in a Plasmodium falciparum cross. Nature 345 : 253-255. , Krogstad et al. 1992Krogstad DJ, Gluzman IY, Herwaldt BL, Schlesinger PH, Wellems TE 1992. Energy dependence of chloroquine accumulation and chloroquine efflux in Plasmodium falciparum. Biochem Pharmacol 43 : 57-62. ). It has been proposed that copy number variations influence Pfmdr1 expression in response to CQ and mefloquine selection or mutations in Pfcrt , suggesting a direct association between these two genes ( Cowman et al. 1994Cowman AF, Galatis D, Thompson JK 1994. Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr 1 gene and cross-resistance to halofantrine and quinine . Proc Natl Acad Sci USA 91 : 1143-1147. , Price et al. 2004Price RN, Uhlemann AC, Brockman A, McGready R, Ashley E, Phaipun L, Patel R, Laing K, Looareesuwan S, White NJ, Nosten F, Krishna S 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364 : 438-447. , Anderson et al. 2005Anderson TJ, Nair S, Qin H, Singlam S, Brockman A, Paiphun L, Nosten F 2005. Are transporter genes other than the chloroquine resistance locus ( pfcrt ) and multidrug resistance gene ( pfmdr ) associated with antimalarial drug resistance? Antimicrob Agents Chemother 49 : 2180-2188. , Duraisingh & Cowman 2005Duraisingh MT, Cowman AF 2005. Contribution of the pfmdr 1 gene to antimalarial drug-resistance. Acta Trop 94 : 181-190. , Hayton & Su 2008Hayton K, Su X-z 2008. Drug resistance and genetic mapping in Plasmodium falciparum . Curr Genet 54 : 223-239. ). Moreover, a low copy number Pfmdr1 in P. falciparum also increases its susceptibility to other drugs ( Sidhu et al. 2006Sidhu AB, Valderramos SG, Fidock DA 2005. Pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol 57 : 913-926. ). Thus, it seems that Pfcrt has a causal effect on CQR, while Pfmdr1 acts as a secondary modulator ( Babiker et al. 2001Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D 2001. High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr 1 . J Infect Dis 183 : 1535-1538. , Ngo et al. 2003Ngo T, Duraisingh M, Reed M, Hipgrave D, Biggs B, Cowman AF 2003. Analysis of pfcrt , pfmdr 1, dhfr and dhps mutations and drug sensitivities in Plasmodium falciparum isolates from patients in Vietnam before and after treatment with artemisinin. Am J Trop Med Hyg 68 : 350-356. , Holmgren et al. 2006Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Björkman A 2006. Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr 1 86Y . Infect Genet Evol 6 : 309-314. , Jiang et al. 2006Jiang H, Joy DA, Furuya T, Su X-z 2006. Current understanding of the molecular basis of chloroquine-resistance in Plasmodium falciparum . J Postgrad Med 52 : 271-276. ). Surprisingly, apart from Pfmdr1 , no other gene has been found to be associated with CQR, although quantitative CQ responses differ in CQR and CQS strains, even when the Pfcrt and Pfmdr1 genes remain unchanged. This finding indicates that the level of the CQ response may be influenced by additional genes ( Foote et al. 1990Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cowman AF 1990. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum . Nature 345 : 255-258. , Reed et al. 2000Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403 : 906-909. , Mu et al. 2003Mu J, Ferdig MT, Feng X, Joy DA, Duan J, Furuya T, Subramanian G, Aravind L, Cooper RA, Wootton JC, Xiong M, Su X-z 2003. Multiple transporters associated with malaria parasite responses to chloroquine and quinine. Mol Microbiol 49 : 977-989. ). Moreover, studies on culture-adapted isolates that harbour the mutant Pfcrt gene reported low CQ IC ₅₀ values that failed to meet the standard criteria for CQR, providing indubitable evidence that mutant Pfcrt is insufficient to confer CQR to all genetic backgrounds, even though the strains showed high CQ tolerance and recrudescence under CQ pressure ( Valderramos et al. 2010Valderramos SG, Valderramos JC, Musset L, Purcell LA, Mercereau-Puijalon O, Legrand E, Fidock DA 2010. Identification of a mutant Pfcrt -mediated chloroquine tolerance phenotype in Plasmodium falciparum . PLoS Pathog 6 : e1000887. ).

Pfcrt mutations and haplotypes: global distribution of different haplotypes - Amino acid polymorphisms have been found in exon 2 of the Pfcrt gene at residues 72, 74, 75 and 76 in P. falciparum isolates, suggesting that they may be involved in the genetic characterisation of CQR and CQS ( Fig. 2 ). Accordingly, whereas the C ₇₂ V ₇₃ M ₇₄ N ₇₅ K ₇₆ haplotype is considered to be CQS, parasites with polymorphisms at any of these amino acid positions are considered to be CQR ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ) ( Fig. 2 ). For CQR P. falciparum , two principal haplotypes, with the amino acid sequences C ₇₂ V ₇₃ I ₇₄ E ₇₅ T ₇₆ and S ₇₂ V ₇₃ M ₇₄ N ₇₅ T ₇₆ ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ) ( Fig. 2 ), are widely distributed. Based on nucleotide sequence data, the SVMNT haplotype is further categorised as either S _agt VMNT or S _tct VMNT, a di-nucleotide polymorphism at codon 72, in which the sequence is changed from AGT to TCT. However, this nucleotide change does not lead to an amino acid substitution, as both AGT and TCT code for serine ( Mehlotra et al. 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. ). Due to the widespread yet structured present-day distribution across P. falciparum -endemic zones across the globe, these two haplotypes are hypothetically considered to be CQR mother haplotypes and the 19 minor haplotypes are believed to have been derived from them ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ) ( Table II ). While it has been established that CVIET and SVMNT are widely prevalent, whether all of the other minor haplotypes were derived from these two or evolved independently is still an open question. It appears that these multiple resistant haplotypes may have evolved independently, but that only some of them have been able to selectively sweep through populations. In addition to the accepted five foci of origin for CQR P. falciparum , specifically, CVIET (Southeast Asia and Africa), S _agt VMNT (Asia, South America and Tanzania), S _tct VMNT (South America and Angola), CVMET (Colombia) and CVMNT (South America and the Philippines), a sixth focus has been described in India and Iran ( Mehlotra et al. 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. , Zakeri et al. 2008Zakeri S, Afsharpad M, Kazemzadeh T, Mehdizadeh K, Shabani A, Djadid ND 2008. Association of pfcrt but not pfmdr1 alleles with chloroquine resistance in Iranian isolates of Plasmodium falciparum . Am J Trop Med Hyg 78 : 633-640. , Wellems et al. 2009Wellems TE, Hayton K, Fairhurst RM 2009. The impact of malaria parasitism: from corpuscles to communities. J Clin Invest 119 : 2496-2505. ). An in-depth description of the distribution of the different haplotypes in the three malaria-endemic continents (Asia, Africa and South America) is discussed below.

Distribution of the CQR-Pfcrt haplotypes in Asia - The distribution of the CQR- Pfcrt haplotypes presents a unique pattern in Asia, particularly in Southeast Asia [Cambodia, Thailand, Bangladesh, Laos, Indochina, Indonesia, Philippines, Papua New Guinea (PNG), East Timor Islands, Solomon Islands and Vanuatu] and South Asia (India, Pakistan, Sri Lanka and Iran). The CVIET mother haplotype is proposed to have originated at the Thailand-Cambodia border in Southeast Asia ( Mehlotra et al. 2001Mehlotra RK, Fujioka H, Roepe PD 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with Pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci USA 98 : 12689-12694. , Wootton et al. 2002Plummer WB, Pereira LMP, Carrington CVF 2004. Pfcrt and pfmdr1 alleles associated with chloroquine resistance in Plasmodium falciparum from Guyana, South America. Mem Inst Oswaldo Cruz 99 : 389-392. ). In Thailand and Bangladesh, the CVIET haplotype is the major reported haplotype ( Hatabu et al. 2005Hatabu T, Kawazu S, Kojima S, Sato K, Singhasivanon P, Looareesuwan S, Kano S 2005. In vitro susceptibility and genetic variations for chloroquine and mefloquine in Plasmodium falciparum isolates from Thai-Myanmar border. Southeast Asian J Trop Med Public Health 36 (Suppl. 4): S73-S79. , Takahashi et al. 2012Takahashi N, Tanabe K, Tsukahara T, Dzodzomenyo M, Dysoley L, Khamlome B, Sattabongkot J, Nakamura M, Sakurai M, Kobayashi J, Kaneko A, Endo H, Hombhanje F, Tsuboi T, Mita T 2012. Large scale survey of novel genotypes of Plasmodium falciparum chloroquine resistance gene Pfcrt . Malar J 11 : 92. ) and, very recently, a new haplotype, CVIEA, was also observed in Thailand ( Chaijaroenkul et al. 2011Chaijaroenkul W, Ward SA, Mungthin M, Johnson D, Owen A, Bray PG, Na-Bangchang K 2011. Sequence and gene expression of chloroquine resistance transporter ( pfcrt ) in the association of in vitro drugs resistance of Plasmodium falciparum . Malar J 10 : 42. ). In Cambodia, apart from CVIET, three more derived haplotypes (CVIDT, CVTNT and CVMNT) have also been reported ( Lim et al. 2003Lim P, Chy S, Ariey F, Incardona S, Chim P, Sem R, Denis MB, Hewitt S, Hoyer S, Socheat D, Merecreau-Puijalon O, Fandeur T 2003. Pfcrt polymorphism and chloroquine resistance in Plasmodium falciparum strains isolated in Cambodia. Antimicrob Agents Chemothe r 47 : 87-94. ). In Laos, CVIET and SVMNT haplotypes have been reported, with the latter having a relatively higher frequency ( Dittrich et al. 2005Dittrich S, Alifrangis M, Stohrer JM, Thongpaseuth V, Vanisaveth V, Phetsouvanh R, Phompida S, Khalil IF, Jelinek T 2005. Falciparum malaria in the north of Laos: the occurrence and implications of the Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) gene haplotype SVMNT. Trop Med Int Health 10 : 1267-1270. ). Interestingly, the Philippine Islands were found to be dominated by SVMNT and its derived haplotypes (CVMNT and CVMHT) ( Chen et al. 2003Chen N, Kyle DE, Pasay C 2003. Pfcrt allelic types with two novel amino acid mutations in chloroquine-resistant Plasmodium falciparum isolates from the Philippines. Antimicrob Agents Chemother 47 : 3500-3505. , Yang et al. 2007Yang Z, Zhang Z, Sun X 2007. Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China. Trop Med Int Health 12 : 1051-1060. ), with a recent report indicating the distribution of the CVIET-derived haplotype CVIDT ( Huaman et al. 2004Huaman MC, Yoshinaga K, Suryanatha A, Suarsana N, Kanbara H 2004. Polymorphisms in the chloroquine resistance transporter gene in Plasmodium falciparum isolates from Lombok, Indonesia. Am J Trop Med Hyg 71 : 40-42. ). The Pfcrt haplotypic view of PNG is quite unusual; despite its geographic proximity to the Southeast Asian focus of the resistance-carrying CVIET haplotype, the CQR parasites in this country harbour haplotypes that are similar to the SVMNT haplotype, which originated from South America ( Chan et al. 2012Chan CW, Spathis R, Reiff DM, McGrath SE, Garruto RM, Lum JK 2012. Diversity of Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) exon 2 haplotypes in the Pacific from 1959 to 1979. PLoS ONE 7 : e30213. ). Multi- locus microsatellite studies have also illustrated a greater evolutionary affinity between P. falciparum isolates from PNG and Southeast Asia, as opposed to South America, which further emphasises the unexpected nature of the Pfcrt polymorphism findings ( Mehlotra et al. 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. ). However, apart from the Pfcrt substitutions, S _agt VMNT and S _tct VMNT have been associated with a different genetic background in PNG and South America, respectively and as such, it has been argued that PNG most likely represents another independent focus of CQR ( Chan et al. 2012Chan CW, Spathis R, Reiff DM, McGrath SE, Garruto RM, Lum JK 2012. Diversity of Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) exon 2 haplotypes in the Pacific from 1959 to 1979. PLoS ONE 7 : e30213. ). In PNG, apart from the S _agt VMNT haplotype, which occurs at appreciable frequency, two CVIET-derived haplotypes (SVIET and CVIKT) have also been reported with minor frequency in Indonesian Papua (West New Guinea) ( Nagesha et al. 2003Nagesha HS, Casey GC, Rieckmann KH, Fryauff DJ, Laksana BS, Reeder JC, Maguire JD, Baird JK 2003. New haplotypes of the Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) gene among chloroquine-resistant parasite isolates. Am J Trop Med Hyg 68 : 398-402. , DaRe et al. 2007DaRe JT, Mehlotra RK, Michon P, Mueller I, Reeder J, Sharma YD, Stoneking M, Zimmerman PA 2007. Microsatellite polymorphism within pfcrt provides evidence of continuing evolution of chloroquine-resistant alleles in Papua New Guinea. Malar J 6 : 34. , Takahashi et al. 2012Takahashi N, Tanabe K, Tsukahara T, Dzodzomenyo M, Dysoley L, Khamlome B, Sattabongkot J, Nakamura M, Sakurai M, Kobayashi J, Kaneko A, Endo H, Hombhanje F, Tsuboi T, Mita T 2012. Large scale survey of novel genotypes of Plasmodium falciparum chloroquine resistance gene Pfcrt . Malar J 11 : 92. ). In the East Timor Islands, Solomon Islands and Vanuatu, the SVMNT haplotype is prevalent ( Tanabe et al. 2004Tanabe K, Sakihama N, Kaneko A 2004. Stable SNPs in malaria antigen genes in isolated populations. Science 303 : 493 , Sakihama et al. 2006Sakihama N, Ohmae H, Bakote’e B, Kawabata M, Hirayama K, Tanabe K 2006. Limited allelic diversity of Plasmodium falciparum merozoite surface protein 1 gene from populations in the Solomon Islands. Am J Trop Med Hyg 74 : 31-40. , Almeida et al. 2009Almeida AD, Arez AP, Cravo PVL, do Rosário VE 2009. Analysis of genetic mutations associated with anti-malarial drug resistance in Plasmodium falciparum from the Democratic Republic of East Timor. Malar J 8 : 59. , Mita et al. 2009Mittra P, Vinayak S, Chandawat H, Das MK, Singh N, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD 2006. Progressive increase in point mutations associated with chloroquine resistance in Plasmodium falciparum isolates from India. J Infect Dis 193 : 1304-1312. , Takahashi et al. 2012Takahashi N, Tanabe K, Tsukahara T, Dzodzomenyo M, Dysoley L, Khamlome B, Sattabongkot J, Nakamura M, Sakurai M, Kobayashi J, Kaneko A, Endo H, Hombhanje F, Tsuboi T, Mita T 2012. Large scale survey of novel genotypes of Plasmodium falciparum chloroquine resistance gene Pfcrt . Malar J 11 : 92. ). In Indonesia, apart from the highly frequent SVMNT haplotype, a new haplotype, CVMNN, was found to be frequent in Lombok and Irian Jaya ( Huaman et al. 2004Huaman MC, Yoshinaga K, Suryanatha A, Suarsana N, Kanbara H 2004. Polymorphisms in the chloroquine resistance transporter gene in Plasmodium falciparum isolates from Lombok, Indonesia. Am J Trop Med Hyg 71 : 40-42. ). Most interestingly, India has a mixture of many CQR- Pfcrt haplotypes, which are primarily dominated by SVMNT, but also show appreciable frequencies of CVIET, CVMNT and CVIDT ( Vinayak et al. 2003Vinayak S, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD 2003. Prevalence of the K76T mutation in the pfcrt gene of Plasmodium falciparum among chloroquine responders in India. Acta Trop 87 : 287-293. , 2006Vinayak S, Mittra P, Sharma YD 2006. Wide variation in microsatellite sequences within each Pfcrt mutant haplotype. Mol Biochem Parasitol 147 : 101-108. , Vathsala et al. 2004Vathsala PG, Pramanik A, Dhanasekaran S, Devi CU, Pillai CR, Subbarao SK, Ghosh SK, Tiwari SN, Sathyanarayan TS, Deshpande PR, Mishra GC, Ranjit MR, Dash AP, Rangarajan PN, Padmanaban G 2004. Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter ( Pfcrt ) gene haplotype SVMNT in P. falciparum malaria in India. Am J Trop Med Hyg 70 : 256-259. , Mittra et al. 2006Mittra P, Vinayak S, Chandawat H, Das MK, Singh N, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD 2006. Progressive increase in point mutations associated with chloroquine resistance in Plasmodium falciparum isolates from India. J Infect Dis 193 : 1304-1312. , Keen et al. 2007Keen J, Farcas GA, Zhong K, Yohanna S, Dunne MW, Kain KC 2007. Real-time PCR assay for rapid detection and analysis of PfCRT haplotypes of chloroquine-resistant Plasmodium falciparum isolates from India. J Clin Microbiol 45 : 2889-2893. Pati et al. 2007Pati SS, Mishra S, Mohanty S, Mohapatra DN, Sahu PK, Priyadarshi N, Kumar S, Sharma SK, Tyagi PK, Chitnis CE, Das BS 2007. Pfcrt haplotypes and in-vivo chloroquine response in Sundergarh district, Orissa, India. Trans R Soc Trop Med Hyg 101 : 650-654. , Bharti et al. 2009Bharti PK, Alam MT, Boxer R, Shukla MM, Gautam SP, Sharma YD, Singh N 2009. Therapeutic efficacy of chloroquine and sequence variation in Pfcrt gene among patients with falciparum malaria in central India. Trop Med Int H ealth 15 : 3340. , Mixon-Hayden et al. 2010Mixon-Hayden T, Jain V, McCollum AM, Poe A, Nagpal AC, Dash AP, Stiles JK, Udhayakumar V, Singh N 2010. Evidence of selective sweeps in genes conferring resistance to chloroquine and pyrimethamine in Plasmodium falciparum isolates in India. Antimicrob Agents Chemother 54 : 997-1006. , Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , Sutar et al. 2011Sutar SKD, Gupta B, Ranjit M, Kar SK, Das A 2011. Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India. Mem Inst Oswaldo Cruz 106 : 78-84. , Lumb et al. 2012Lumb V, Madan R, Das MK, Rawat V, Dev V, Khan W, Khan H, Sharma YD 2012. Differential genetic hitchhiking around mutant pfcrt alleles in the Indian Plasmodium falciparum population. J Antimicrob Chemother 7 : 600-608. ). In Pakistan, Iran and Sri Lanka, the S _agt VMNT haplotype is reported at appreciable frequencies and is believed to have been imported from India ( Zakeri et al. 2008Zakeri S, Afsharpad M, Kazemzadeh T, Mehdizadeh K, Shabani A, Djadid ND 2008. Association of pfcrt but not pfmdr1 alleles with chloroquine resistance in Iranian isolates of Plasmodium falciparum . Am J Trop Med Hyg 78 : 633-640. , Zhang et al. 2011Zhang JJ, Senaratne TN, Daniels R, Valim C, Alifrangis M, Amerasinghe P, Konradsen F, Rajakaruna R, Wirth DF, Karunaweera ND 2011. Distribution pattern of Plasmodium falciparum chloroquine transporter ( pfcrt ) gene haplotypes in Sri Lanka 1996-2006. Am J Trop Med Hyg 85 : 811. , Rawasia et al. 2012Rawasia WF, Sridaran S, Patel JC, Abdallah J, Ghanchi NK, Barnwell JW, Escalante AA, Udhayakumar V, Beg MA 2012. Genetic backgrounds of the Plasmodium falciparum chloroquine resistant transporter ( pfcrt ) alleles in Pakistan. Infect Genet Evol 12 : 278-281. ). A very recent study from Yemen and Saudi Arabia confirmed the major presence of the CVIET haplotype ( Al-Hamidhi et al. 2013Al-Hamidhi S, Mahdy MA, Al-Hashami Z, Al-Farsi H, Al-Mekhlafi AM, Idris MA, Beja-Pereira A, Babiker HA 2013. Genetic diversity of Plasmodium falciparum and distribution of drug resistance haplotypes in Yemen. Malar J 12 : 244. ).

Distribution of the CQR-Pfcrt haplotypes in Africa - The Pfcrt CQR haplotypic view in Africa is completely biased towards the CVIET haplotype, owing to the wide usage of CQ and amodiaquine (AQ) drugs in many African countries ( Djimde et al. 2010Djimde AA, Barger B, Kone A, Beavogui AH, Tekete M, Fofana B, Dara A, Maiga H, Dembele D, Toure S, Dama S, Ouologuem D, Sangare CP, Dolo A, Sogoba N, Nimaga K, Kone Y, Doumbo OK 2010. A molecular map of chloroquine resistance in Mali. FEMS Immunol Med Microbiol 58 : 113-118. ). To date, in most sub-Saharan African countries, including Comoros, Senegal, Gabon, Djibouti, Cameroon, Gambia, Niger, Ivory Coast, Ghana, Nigeria, Kenya, Mali, the Dominican Republic of Congo, Guinea Bissau, Mozambique, Benin, Zambia, Rwanda, Burundi, Tanzania, the Republic of South Africa, Sudan, Congo, Madagascar, Malawi and Uganda, the CVIET haplotype is the only CQR- Pfcrt haplotype that has been reported in high frequency ( Cooper et al. 2005Cooper RA, Hartwig CL, Ferdig MT 2005. Pfcrt is more than the Plasmodium falciparum chloroquine resistance gene: a functional and evolutionary perspective. Acta Trop 94 : 170-180. , Ariey et al. 2006Ariey F, Fandeur T, Durand R, Randrianarivelojosia M, Jambou R, Legrand E, Ekala MT, Bouchier C, Cojean S, Duchemin JB, Robert V, Le Bras J, Mercereau-Puijalon O 2006. Invasion of Africa by a single pfcrt allele of South East Asian type. Malar J 5 : 34. , Randrianarivelojosia et al. 2006Randrianarivelojosia M, Fidock DA, Belmonte O, Valderramos SG, Mercereau-Puijalon O, Ariey F 2006. First evidence of pfcrt mutant Plasmodium falciparum in Madagascar. Trans R Soc Trop Med Hyg 100 : 826-830. , Severini et al. 2006Severini C, Menegon M, Sannella AR, Paglia MG, Narciso P, Matteelli A, Gulletta M, Caramello P, Canta F, Xayavong MV, Moura IN, Pieniazek NJ, Taramelli D, Majori G 2006. Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa. Infect Genet Evol 6 : 262-268. , Juliano et al. 2007Juliano JJ, Kwiek JJ, Cappell K 2007. Minority-variant Pfcrt K76T mutations and chloroquine resistance, Malawi. Emerg Infect Dis 13 : 873-877. , Nsobya et al. 2007Nsobya SL, Dokomajilar C, Joloba M, Dorsey G, Rosenthal PJ 2007. Resistance-mediating Plasmodium falciparum pfcrt and pfmdr1 alleles after treatment with artesunate-amodiaquine in Uganda. Antimicrob Agents Chemother 51 : 3023-3025. , Mehlotra et al. 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. , Niang et al. 2008Niang M, Marrama L, Ekala MT 2008. Accumulation of CVIET Pfcrt allele of Plasmodium falciparum in placenta of pregnant women living in an urban area of Dakar, Senegal. J Antimicrob Chemother 62 : 921-928. , Bob et al. 2010Bob NS, Diop BM, Renaud F, Marrama L, Durand P, Tall A, Ka B, Ekala MT, Bouchier C, Mercereau-Puijalon O, Jambou R 2010. Parasite polymorphism and severe malaria in dakar (Senegal): a west African urban area. PLoS ONE 23 : 5. , Gadalla et al. 2010Gadalla NB, Elzaki SE, Mukhtar S, Warhurst DC, El-Sayed B, Sutherland CJ 2010. Dynamics of pfcrt alleles CVMNK and CVIET in chloroquine-treated Sudanese patients infected with Plasmodium falciparum . Malar J 9 : 74. , Takahashi et al. 2012Takahashi N, Tanabe K, Tsukahara T, Dzodzomenyo M, Dysoley L, Khamlome B, Sattabongkot J, Nakamura M, Sakurai M, Kobayashi J, Kaneko A, Endo H, Hombhanje F, Tsuboi T, Mita T 2012. Large scale survey of novel genotypes of Plasmodium falciparum chloroquine resistance gene Pfcrt . Malar J 11 : 92. ). However, in Tanzania, the SVMNT haplotype is present at an appreciable frequency ( Alifrangis et al. 2006Alifrangis M, Dalgaard MB, Lusingu JP, Vestergaard LS, Staalsoe T, Jensen AT, Enevold A, Rønn AM, Khalil IF, Warhurst DC, Lemnge MM, Theander TG, Bygbjerg IC 2006. Occurrence of the Southeast Asian/South American SVMNT haplotype of the chloroquine-resistance transporter gene in Plasmodium falciparum in Tanzania. J Infect Dis 193 : 1738-1741. ). In Congo and Madagascar, two CVIET-derived haplotypes are also present, SVIET in Congo and CVIDT in Madagascar ( Severini et al. 2006Severini C, Menegon M, Sannella AR, Paglia MG, Narciso P, Matteelli A, Gulletta M, Caramello P, Canta F, Xayavong MV, Moura IN, Pieniazek NJ, Taramelli D, Majori G 2006. Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa. Infect Genet Evol 6 : 262-268. , Rason et al. 2007Rason MA, Andrianantenaina HB, Ariey F, Raveloson A, Domarle O, Randrianarivelojosia M 2007. Prevalent Pfmdr1 N86Y mutant Plasmodium falciparum in Madagascar despite absence of Pfcrt mutant strains. Am J Trop Med Hyg 76 : 1079-1083. ). Interestingly, the Central African Republic shows the CVIET haplotype at an appreciable frequency, along with six derived haplotypes (SVIET, SVIEK, CVIEK, CVMNT, SVMET and CVINT) in low frequencies ( Menard et al. 2006Menard D, Djalle D, Yapou F, Manirakiza A, Talarmin A 2006. Frequency distribution of antimalarial drug-resistant alleles among isolates of Plasmodium falciparum in Bangui, Central African Republic. Am J Trop Med Hyg 74 : 205-210. ). Interestingly, a recent study reported the presence of the S _tct VMNT haplotype in a very high frequency in Angola, with a low frequency of CVIET and three derived haplotypes (CVMNT, CVINT and CVMDT) with relatively lower frequencies ( Gama et al. 2010Gama BE, Pereira-Carvalho GAL, Kosi FJIL, de Oliveira NKA, Fortes F, Rosenthal PJ, Daniel-Ribeiro CT, Ferreira-da-Cruz MF 2010. Plasmodium falciparum isolates from Angola show the StctVMNT haplotype in the pfcrt gene. Malar J 9 : 174. ). Furthermore, high CQR- Pfcrt haplotype diversity and the emergence of the S _agt VMNT haplotype in Cameroon have recently been reported ( Mbenda & Das 2013Mbenda HGN, Das A 2013. Occurrence of multiple chloroquine-resistant Pfcrt haplotypes and emergence of the S(agt)VMNT type in Cameroonian Plasmodium falciparum . J Antimicrob Chemother doi: 10.1093/jac/dkt388.
https://doi.org/10.1093/jac/dkt388... ). Thus, in general, while all of the African countries were found to be dominated by the CVIET Pfcrt -CQR haplotype, Angola, Tanzania, Cameroon and the Central Africa Republic were exceptions. It seems probable that the SVMNT haplotype found in Angola, Tanzania, Cameroon and the Central African Republic might have originated in South America and the Western Pacific. Because Angola and Cameroon are located on the Southwest coast of Africa, these countries might have received the CQR Pfcrt migrants of P. falciparum from South America due to frequent travellers between Brazil and Africa ( Gama et al. 2010Gama BE, Pereira-Carvalho GAL, Kosi FJIL, de Oliveira NKA, Fortes F, Rosenthal PJ, Daniel-Ribeiro CT, Ferreira-da-Cruz MF 2010. Plasmodium falciparum isolates from Angola show the StctVMNT haplotype in the pfcrt gene. Malar J 9 : 174. , Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ) and due to an increase in the use of AQ in Africa either alone or in combination with artesunate ( Summers et al. 2012Summers RL, Nash MN, Martin RE 2012. Know your enemy: understanding the role of PfCRT in drug resistance could lead to new antimalarial tactics. Cell Mol Life Sci 69 : 1967-1995. ). Regardless, the presence of seven different haplotypes (CVIET, SVIET, SVIEK, CVIEK, CVMNT, SVMET and CVINT) in the Central African Republic might be explained with a better knowledge of the drug combinations administered in this region to date ( Menard et al. 2006Menard D, Djalle D, Yapou F, Manirakiza A, Talarmin A 2006. Frequency distribution of antimalarial drug-resistant alleles among isolates of Plasmodium falciparum in Bangui, Central African Republic. Am J Trop Med Hyg 74 : 205-210. ), as the increasing use of AQ in Africa poses the threat of a selective sweep of highly AQ and CQ-resistant parasites with Pfcrt and Pfmdr1 mutations that are as advantaged and persistent as in South America ( Sa et al. 2009Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE 2009. Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci USA 106 : 18883-18889. ).

Distribution of the CQR-Pfcrt haplotypes in South America - South America is thought to be one of the six foci of origin of CQR P. falciparum , as the CQR- Pfcrt haplotype S _tct VMNT was first reported at the Colombia-Venezuela border ( Mehlotra et al. 2001Mehlotra RK, Fujioka H, Roepe PD 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with Pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci USA 98 : 12689-12694. , 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. ) and is still highly prevalent across the continent. The high prevalence of the S _tct VMNT haplotype in South American countries is attributed to many factors, such as (i) the absence of CQ pressure, (ii) the wide usage of AQ, (iii) region-specific differences in drug usage, (iv) a reduced rate of polyclonal infections and (v) the absence of competitive wild type parasites ( Sa et al. 2009Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE 2009. Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci USA 106 : 18883-18889. , Sa & Twu 2010Sa JM, Twu O 2010. Protecting the malaria drug arsenal: halting the rise and spread of amodiaquine resistance by monitoring the Pf CRT SVMNT type. Malar J 9 : 374. , Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ). The highly prevalent S _tct VMNT haplotype is reported to be the sole haplotype in Bolivia. In Brazil, Venezuela and Peru, the S _tct VMNT haplotype is present in high frequency, along with the CVIET haplotype, at an appreciable frequency ( Sa & Twu 2010Sa JM, Twu O 2010. Protecting the malaria drug arsenal: halting the rise and spread of amodiaquine resistance by monitoring the Pf CRT SVMNT type. Malar J 9 : 374. ). In contrast, Ecuador and Guyana are completely dominated by the SVMNT-derived haplotype CVMNT, with some incidences of S _tct VMNT ( Griffing et al. 2010Griffing S, Syphard L, Sridaran S, McCollum AM, Mixson-Hayden T, Vinayak S, Villegas L, Barnwell JW, Escalante AA, Udhayakumar V 2010. Pfmdr1 amplification and fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum in Venezuela. Antimicrob Agents Chemother 54 : 1572-1579. ). In Colombia, the S _tct VMNT haplotype was reported initially, but has been replaced by the CVMNT haplotype ( Restrepo et al. 2008Restrepo E, Fonseca JC, Maestre A 2008. Plasmodium falciparum : high frequency of pfcrt point mutations and emergence of new mutant haplotypes in Colombia. Biomedica 28 : 523-530. ). Apart from these frequent CQR Pfcrt haplotypes, three other low-frequency haplotypes, SVMIT and RVMIT in Guyana (Plummer et al. 2004) and CVMET across the Amazon Basin, have also been reported ( Cortese et al. 2002Cortese JF, Caraballo A, Contreras CE, Plowe CV 2002. Origin and dissemination of Plasmodium falciparum drug-resistance mutations in South America. J Infect Dis 186 : 999-1006. , Vieira et al. 2004Vieira PP, Ferreira MU, Alecrim MDAC, Alecrim WD, da Silva LH, Sihuincha MM, Joy DA, Mu J, Su X-z, Zalis MG 2004. Pfcrt polymorphism and the spread of chloroquine resistance in Plasmodium falciparum populations across the Amazon basin. J Infect Dis 190 : 417-424. , Echeverry et al. 2007Echeverry DF, Holmgren G, Murillo C 2007. Polymorphisms in the pfcrt and pfmdr1 genes of Plasmodium falciparum and in vitro susceptibility to amodiaquine and desethylamodiaquine. Am J Trop Med Hyg 77 : 1034-1038. , Pineda et al. 2008Pineda ER, Arango E, do Rosário VEMA, Cravo P 2008. Studies on antimalarial drug susceptibility in Colombia, in relation to Pfmdr1 and Pfcrt . Parasitology 135 : 547-553. ). In general, the haplotypic view in South America suggests that the S _tct VMNT haplotype and its derivatives are predominant, with the CVIET haplotype also being present in Brazil and Venezuela ( Londono et al. 2009Londono BL, Eisele TP, Keating J, Bennett A, Chattopadhyay C, Heyliger G, Mack B, Rawson I, Vely JF, Désinor O, Krogstad DJ 2009. Chloroquine-resistant haplotype Plasmodium falciparum parasites, Haiti. Emerg Infect Dis 15 : 735-740. ). The CVIET haplotype has only been rarely reported in South America and was most likely imported from Africa, as most of the parasites in Brazil have the typical SVMNT allele. In Haiti, most of the parasites have the CVMNK allele and CVIET is rare. In this context, it is important to recognise that in Central American countries, including Haiti, CQ remains as the primary drug for the treatment of P. falciparum malaria ( Londono et al. 2009Londono BL, Eisele TP, Keating J, Bennett A, Chattopadhyay C, Heyliger G, Mack B, Rawson I, Vely JF, Désinor O, Krogstad DJ 2009. Chloroquine-resistant haplotype Plasmodium falciparum parasites, Haiti. Emerg Infect Dis 15 : 735-740. ). In response to the rise in anti-malarial drug resistance in the Amazon and in South American countries, a surveillance network named Amazon Network for the Surveillance of Antimalarial Drug Resistance was created, with the primary responsibilities of formulating drug policies, monitoring drug resistance and promoting the suitable use of drugs within the continent ( Gama et al. 2011Gama BE, Lacerda MVG, Daniel-Ribeiro CT, Ferreira-da-Cruz MF 2011. Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control. Mem Inst Oswaldo Cruz 106 (Suppl. I): 159-166. ).

Pfcrt haplotypes and the origin and spread of CQR: any correlation? - The putative origin and spread of CQR P. falciparum was mainly inferred by epidemiological surveillance data ( Wernsdorfer & Payne 1991Wernsdorfer WH, Payne D 1991. The dynamics of drug resistance in Plasmodium falciparum . Pharmacol Ther 50 : 95-121. , Wernsdorfer 1994Wernsdorfer WH 1994. Epidemiology of drug resistance in malaria. Acta Trop 56 : 143-156. , Anderson 2009Anderson TJ 2009. Mapping the spread of malaria drug resistance. PLoS Med 6 : e1000054. ). Thus, the current distribution patterns of CQR P. falciparum are primarily based on this inference and are dependent on the time of the report of CQR P. falciparum in any endemic country. Accordingly, three different models based on CQR prevalence data in three separate malaria-endemic zones, Southeast Asia, Africa and South America ( Awasthi et al. 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ), have been suggested. According to the first model, CQR P. falciparum possibly originated independently in Southeast Asia (Thailand-Cambodia border) and South America (Venezuela-Colombia border) during 1957 and 1959, respectively. By 1980, CQR P. falciparum populated a maximum number of Asian countries ( Table I ). Similarly, in South America, Peru and Bolivia reported incidences of CQR P. falciparum in 1980 ( Table I ). In Africa, CQR P. falciparum was reported relatively late. The first report came from Kenya in 1978 and by the early 1990s, CQR P. falciparum isolates were found in almost all African countries. Thus, by the end of the 1980s and in the early 1990s, almost all of the malaria-endemic countries worldwide had some form of CQR P. falciparum .

Since the discovery of the distinct genetic lineages of Southeast Asian (CVIET), South American (S _tct VMNT) and Southeast Asian and Asian (S _agt VMNT) Pfcrt , the epidemiological observations of rare origin and contiguous spread have been interpreted as evidence of a rare and complex underlying genetic mechanism of CQR ( Plowe 2009Plowe CV 2009. The evolution of drug-resistant malaria. Trans R Soc Trop Med Hyg 1035 : 11-14. , Awasthi et al. 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ). Some early studies on the molecular epidemiology of CQR suggested that resistant malaria arose both focally and locally in direct response to CQ drug pressure ( Wernsdorfer & Payne 1991Wernsdorfer WH, Payne D 1991. The dynamics of drug resistance in Plasmodium falciparum . Pharmacol Ther 50 : 95-121. , Wernsdorfer 1994Wernsdorfer WH 1994. Epidemiology of drug resistance in malaria. Acta Trop 56 : 143-156. ). Moreover, it has been suggested that CQR Pfcrt haplotypes resulting from amino acid changes at positions 72-76 are strongly associated with the geographic region-restricted evolution of P. falciparum resistance to CQ and that these haplotypes are good estimators for predicting evolution and geographical spread of resistance, as other polymorphisms outside these positions have no clear geographical association with CQR ( Mita et al. 2009Mita T, Tanabe K, Kita K 2009. Spread and evolution of Plasmodium falciparum drug resistance. Parasitol Int 58 : 201-209. , Mita & Tanabe 2012Mita T, Tanabe K 2012. Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin resistance. Jpn J Infect Dis 65 : 465-475. ).

The differential distribution of the most frequently found CQR Pfcrt haplotypes offers opportunities to track the movement of these haplotypes, creating a haplotypic view across continents and to indirectly infer the spread of CQR P. falciparum . Recently conducted studies in both worldwide and Indian populations have clearly revealed that such patterns can be inferred from several CQR Pfcrt haplotypes, thus offering the opportunity to correlate these patterns with the epidemiological surveillance data on CQR P. falciparum parasites ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ). Accordingly, the CVIET haplotype populated all of Southeast Asia by the early 1970s and reached India by 1973. This haplotype moved out of Asia and into Africa and this fact is well correlated with the epidemiological data ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ). Alternatively, the CVIET haplotype might have moved from the Southeast Asian countries across the Pacific to South America, which is reflected by the presence of the CVIET haplotype in Venezuela and Brazil, although it is only present in a small percentage ( Contreras et al. 2002Contreras CE, Cortese JF, Caraballo A, Plowe CV 2002. Genetics of drug-resistant Plasmodium falciparum malaria in the Venezuelan state of Bolivar . Am J Trop Med Hyg 67 : 400-405. , Cortese et al. 2002Cortese JF, Caraballo A, Contreras CE, Plowe CV 2002. Origin and dissemination of Plasmodium falciparum drug-resistance mutations in South America. J Infect Dis 186 : 999-1006. , Griffing et al. 2010Griffing S, Syphard L, Sridaran S, McCollum AM, Mixson-Hayden T, Vinayak S, Villegas L, Barnwell JW, Escalante AA, Udhayakumar V 2010. Pfmdr1 amplification and fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum in Venezuela. Antimicrob Agents Chemother 54 : 1572-1579. ). Alternatively, the presence of the CVIET haplotype in Brazil and Venezuela ( Vieira et al. 2004Vieira PP, Ferreira MU, Alecrim MDAC, Alecrim WD, da Silva LH, Sihuincha MM, Joy DA, Mu J, Su X-z, Zalis MG 2004. Pfcrt polymorphism and the spread of chloroquine resistance in Plasmodium falciparum populations across the Amazon basin. J Infect Dis 190 : 417-424. ) may be due to its movement from geographically close African countries ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ). Very similarly, the S _tct VMNT haplotype originated in South America, while the S _agt VMNT haplotype originated in PNG ( Mehlotra et al. 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. ). These haplotypes first spread locally within the respective continents before migrating to other malaria-endemic regions. As a result, the S _tct VMNT haplotype moved eastward to reach West African countries, evidenced by the fact that the S _tct VMNT haplotype is found at an appreciable frequency in Angola and in low frequency in Tanzania ( Alifrangis et al. 2006Alifrangis M, Dalgaard MB, Lusingu JP, Vestergaard LS, Staalsoe T, Jensen AT, Enevold A, Rønn AM, Khalil IF, Warhurst DC, Lemnge MM, Theander TG, Bygbjerg IC 2006. Occurrence of the Southeast Asian/South American SVMNT haplotype of the chloroquine-resistance transporter gene in Plasmodium falciparum in Tanzania. J Infect Dis 193 : 1738-1741. , Gama et al. 2010Gama BE, Pereira-Carvalho GAL, Kosi FJIL, de Oliveira NKA, Fortes F, Rosenthal PJ, Daniel-Ribeiro CT, Ferreira-da-Cruz MF 2010. Plasmodium falciparum isolates from Angola show the StctVMNT haplotype in the pfcrt gene. Malar J 9 : 174. ). Within Asia, the S _agt VMNT haplotype, which originated in PNG, established itself quite successfully in many places, over-dominating the original haplotype, CVIET, especially in India, Pakistan, Sri Lanka, PNG, the Philippines and Iran. It seems that Iran received this haplotype (and CVIET) from India and Pakistan ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. , Rawasia et al. 2012Rawasia WF, Sridaran S, Patel JC, Abdallah J, Ghanchi NK, Barnwell JW, Escalante AA, Udhayakumar V, Beg MA 2012. Genetic backgrounds of the Plasmodium falciparum chloroquine resistant transporter ( pfcrt ) alleles in Pakistan. Infect Genet Evol 12 : 278-281. ). Additionally, the CVIET haplotype could have spread to Yemen and Saudi Arabia from either Iran or Africa ( Al-Hamidhi et al. 2013Al-Hamidhi S, Mahdy MA, Al-Hashami Z, Al-Farsi H, Al-Mekhlafi AM, Idris MA, Beja-Pereira A, Babiker HA 2013. Genetic diversity of Plasmodium falciparum and distribution of drug resistance haplotypes in Yemen. Malar J 12 : 244. ). However, the global spread of CQR P. falciparum , as inferred from the epidemiological surveillance data, does not completely correlate with the inferred movements of the CQR Pfcrt haplotypes ( Awasthi et al. 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ). In turn, the routes inferred by the CQR Pfcrt haplotype data correlate well with the intercontinental usage of anti-malarials and the migration and successful establishment of CQR P. falciparum in different parts of the world ( Awasthi et al. 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ). For example, in places such as South America, AQ and CQ, which were not in use for the past several years, have resulted in the complete fixation of the S _tct VMNT haplotype ( Sa et al. 2009Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE 2009. Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci USA 106 : 18883-18889. ). On the contrary, dramatic changes have been observed after discontinued drug pressure in certain African countries and Southeast Asia. In the absence of drug pressure, the SVMNT haplotype provides equal fitness to P. falciparum (as in the presence of drug pressure) in comparison to the CVIET haplotype ( Sa et al. 2009Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE 2009. Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci USA 106 : 18883-18889. ). Furthermore, CVIET haplotype-bearing P. falciparum are known to revert back to the CQS (CVMNK) type ( Kublin et al. 2003Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimdé AA, Kouriba B, Taylor TE, Plowe CV 2003. Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis 187 : 1870-1875. , Mita et al. 2003Mita T, Kaneko A, Lum JK, Bwijo B, Takechi M, Zungu IL, Tsukahara T, Tanabe K, Kobayakawa T, Bjorkman A 2003. Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi. Am J Trop Med Hyg 68 : 413-415. , 2004Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ), whereas SVMNT-bearing P. falciparum do not ( Fidock et al. 2000Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su X-z, Wootton JC, Roepe PD, Wellems TE 2000. Mutations in the P. falcipa rum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 : 861-871. ). For example, a region of Malawi that is known for highly prevalent CQR was re-populated with drug-sensitive parasites within 10 years after CQ use was stopped ( Kublin et al. 2003Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimdé AA, Kouriba B, Taylor TE, Plowe CV 2003. Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis 187 : 1870-1875. ). A similar recovery of CQS P. falciparum populations was recently reported in Kenya and has also been observed in China ( Wang et al. 2005Wang X, Mu J, Li G, Chen P, Guo X, Fu L, Chen L, Su X, Wellems TE 2005. Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People’s Republic of China. Am J Trop Med Hyg 72 : 410-414. , Mwai et al. 2009Mwai L, Ochong E, Abdirahman A, Kiara SM, Ward S, Kokwaro G, Sasi P, Marsh K, Borrmann S, Mackinnon M, Nzila A 2009. Chloroquine resistance before and after its withdrawal in Kenya. Malar J 8 : 106. ). These changes in the absence of drug pressure have also been explained by fitness costs that are carried by CQ-resistant mutants ( Laufer et al. 2006Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjalamala FK, Takala SL, Taylor TE, Plowe CV 2006. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med 355 : 1959-1966. ). However, such a selective disadvantage has been less apparent in South America, where CQS parasites have not replaced their CQ-resistant counterparts. A satisfactory explanation for this difference between the Southeast Asian/African and South American forms of CQR has not been proposed ( Sa et al. 2009Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, Wellems TE 2009. Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci USA 106 : 18883-18889. ). This contention also supports the hypothesis that approximately 70% of the total Pfcrt -CQR haplotypes in Southeast Asia and South America are S _agt VMNT and S _tct VMNT, respectively ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. , 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ).

Evolutionary puzzle of the Pfcrt gene in India - India is a country where malaria is highly endemic and where CQR P. falciparum is widely prevalent ( Sharma 2007Sharma VP 2007. Battling the malaria iceberg with chloroquine in India. Malar J 6 : 105. , Singh et al. 2009Singh V, Mishra N, Awasthi G, Dash AP, Das A 2009. Why is it important to study malaria epidemiology in India? Trends Parasitol 24 : 228-235. ). CQR P. falciparum was first detected as early as 1973 in the Asom state of India ( Sehgal et al. 1973Sehgal PN, Sharma MID, Gogai S, 1973. Resistance to chloroquine in falciparum malaria in Assam state, India. J Comm Dis 5 : 175-180. ). Genetic studies of CQR Pfcrt revealed the presence of four major haplotypes (CVIET, SVMNT, CVMNT and CVIDT) in India, with the SVMNT haplotype populating the majority of the Indian states compared to the CVIET haplotype ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. ). In India, the predominant distribution of the SVMNT haplotype, compared to the minimal presence of the CVIET haplotype, is quite puzzling. Because India is geographically closer to Southeast Asia (Thailand, Cambodia, Bangladesh, Laos) than to PNG and Oceania, it is expected that India should share its haplotypic status with Southeast Asia (high frequency of the CVIET haplotype). However, in reality, India shares its CQR- Pfcrt haplotypic status with PNG, Indonesia and Oceania, which harbour the S _agt VMNT haplotype. Furthermore, based on the distribution of the four haplotypes (S _agt VMNT, CVIET, CVMNT and CVIDT), the two following routes of possible migration of Pfcrt haplotypes (SVMNT and CVIET) into India have been hypothesised: (i) while the S _agt VMNT haplotype originated in PNG, it travelled through PNG ↔ Indonesia ↔ the Philippines ↔ Malaysia ↔ Andaman and the Nicobar Islands and then entered mainland India through the east coastal state of Odisha and the S _tct VMNT haplotype originated in South America, travelled through South America ↔ PNG ↔ Indonesia ↔ the Philippines ↔ Malaysia ↔ Andaman and the Nicobar Islands and reached India via Odisha. Similarly, (ii) the CVIET haplotype from the Thailand-Cambodia border travelled from Thailand and Cambodia through Myanmar to populate Mizoram and other Northeastern Indian states and reached as far as Karnataka (Southern India) ( Awasthi et al. 2011Awasthi G, Prasad GB, Das A 2011. Population genetic analyses of Pfcrt haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41 : 705-709. ). A recent microsatellite variation study of the Pfcrt gene and its adjacent sequences in the Indian population suggested that the CQR- Pfcrt haplotypes might have originated in Southeast Asia and spread into Eastern India and other parts of this country through the Northeastern regions ( Mallick et al. 2013Mallick PK, Sutton PL, Singh R, Singh OP, Dash AP, Singh AK, Carlton JM, Bhasin VK 2013. Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum . Infect Genet Evol 19 : 164-175. ). Although these routes were inferred from in-depth population genetic analyses of the currently available data on the CQR Pfcrt haplotypes, the complexity of the prevalence and distribution of the S _agt VMNT haplotype has confounded the overall scenario of the distribution of the CQR Pfcrt haplotypes in India ( Vathsala et al. 2004Vathsala PG, Pramanik A, Dhanasekaran S, Devi CU, Pillai CR, Subbarao SK, Ghosh SK, Tiwari SN, Sathyanarayan TS, Deshpande PR, Mishra GC, Ranjit MR, Dash AP, Rangarajan PN, Padmanaban G 2004. Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter ( Pfcrt ) gene haplotype SVMNT in P. falciparum malaria in India. Am J Trop Med Hyg 70 : 256-259. ), as India and Iran have also been labelled as the sixth focus of origin of CQR P. falciparum parasites ( Mehlotra et al. 2008Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD, Alifrangis M, Dorsey G, Rosenthal PJ, Fryauff DJ, Kazura JW, Stoneking M, Zimmerman PA 2008. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum . Antimicrob Agents Chemother 52 : 2212-2222. , Zakeri et al. 2008Zakeri S, Afsharpad M, Kazemzadeh T, Mehdizadeh K, Shabani A, Djadid ND 2008. Association of pfcrt but not pfmdr1 alleles with chloroquine resistance in Iranian isolates of Plasmodium falciparum . Am J Trop Med Hyg 78 : 633-640. , Wellems et al. 2009Wellems TE, Hayton K, Fairhurst RM 2009. The impact of malaria parasitism: from corpuscles to communities. J Clin Invest 119 : 2496-2505. ).

Another interesting and puzzling issue is the evolutionary course of the Pfcrt gene in India. It is widely known from global genetic diversity studies of CQR isolates that because the Pfcrt gene is responsible for an important function in P. falciparum and is targeted by natural selection, it is described under the “selective sweep” model ( Clark 2002Clark AG 2002. Malaria variorum . Nature 418 : 283-285. , Wootton et al. 2002Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su X-z 2002. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum . Nature 418 : 320-323. ). This model perfectly fits the explanation of the origin and subsequent proliferation of CQR malaria parasites across the globe ( Wootton et al. 2002Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su X-z 2002. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum . Nature 418 : 320-323. , Mu et al. 2010aMu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su X-z 2010a. Plasmodium falciparum genome wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet 42 : 268-271. , Volkman et al. 2012Volkman SK, Neafsey DE, Schaffner SF, Park DJ, Wirth DF 2012. Harnessing genomics and genome biology to understand malaria biology. Nat Rev Genet 13 : 315-328. ). However, genetic diversity data on the Pfcrt gene from CQR P. falciparum in India do not conform to this evolutionary model ( Mittra et al. 2006Mittra P, Vinayak S, Chandawat H, Das MK, Singh N, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD 2006. Progressive increase in point mutations associated with chloroquine resistance in Plasmodium falciparum isolates from India. J Infect Dis 193 : 1304-1312. , Vinayak et al. 2006Vinayak S, Mittra P, Sharma YD 2006. Wide variation in microsatellite sequences within each Pfcrt mutant haplotype. Mol Biochem Parasitol 147 : 101-108. , Das & Dash 2007Das A, Dash AP 2007. Evolutionary paradigm of chloroquine-resistant malaria in India. Trends Parasitol 23 : 132-135. ). Although a very recent study provided evidence on the role of natural selection in the evolution of the Pfcrt gene in India ( Mixon-Hayden et al. 2010Mixon-Hayden T, Jain V, McCollum AM, Poe A, Nagpal AC, Dash AP, Stiles JK, Udhayakumar V, Singh N 2010. Evidence of selective sweeps in genes conferring resistance to chloroquine and pyrimethamine in Plasmodium falciparum isolates in India. Antimicrob Agents Chemother 54 : 997-1006. ), the inferences of this study are unclear for the two following reasons: (i) the aims of the study were to correlate cerebral malaria with drug resistance gene polymorphisms and thus, the study contains sample bias and (ii) the study analysed only a single population from central India ( Mixon-Hayden et al. 2010Mixon-Hayden T, Jain V, McCollum AM, Poe A, Nagpal AC, Dash AP, Stiles JK, Udhayakumar V, Singh N 2010. Evidence of selective sweeps in genes conferring resistance to chloroquine and pyrimethamine in Plasmodium falciparum isolates in India. Antimicrob Agents Chemother 54 : 997-1006. ). Considering that India is a vast country with a variable climate and malaria epidemiology ( Singh et al. 2009Singh V, Mishra N, Awasthi G, Dash AP, Das A 2009. Why is it important to study malaria epidemiology in India? Trends Parasitol 24 : 228-235. ), the mystery of Pfcrt gene evolution needs to be resolved by deep sampling and finer evolutionary analyses.

Is Pfcrt the sole candidate for CQR? - To visualise the relevance of the genetic basis of any drug resistance from a public health perspective, an absolute correlation between genotype and phenotype is essential. In this respect, Pfcrt has not met all of the requirements for determining this gene as the sole agent of CQR. In fact, several studies have indicated that it is unclear if the Pfcrt gene is directly and solely associated with CQR P. falciparum . For example, (i) not all of the CQR P. falciparum isolates were found to bear the K76T mutation in the Pfcrt gene and vice versa ( Vinayak et al. 2003Vinayak S, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD 2003. Prevalence of the K76T mutation in the pfcrt gene of Plasmodium falciparum among chloroquine responders in India. Acta Trop 87 : 287-293. ), (ii) the Pfcrt homologue in P. vivax ( Pvcrt-o ) is not associated with CQR in P. vivax ( Martin & Kirk 2004Martin RE, Kirk K 2004. The malaria parasite’s chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol Biol Evol 21 : 1938-1949. ), (iii) the K76T mutation is not sufficient for the transport of CQ via Pf CRT, which is consistent with the view that one or more other Pf CRT mutations act in concert with K76T to confer CQR ( Summers et al. 2012Summers RL, Nash MN, Martin RE 2012. Know your enemy: understanding the role of PfCRT in drug resistance could lead to new antimalarial tactics. Cell Mol Life Sci 69 : 1967-1995. ), (iv) a strong LD was observed in the ~40-Kb region surrounding the Pfcrt gene in chromosome 7 ( Mu et al. 2010aMu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su X-z 2010a. Plasmodium falciparum genome wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet 42 : 268-271. ), supporting the fact that the observed genetic patterns in the Pfcrt gene could merely reflect the role of evolutionary force in hitherto uncharacterised gene(s) that have a direct association with CQR P. falciparum ( Gupta et al. 2010Gupta B, Awasthi G, Das A 2010. Malaria parasite genome scan: insights into antimalarial resistance. Parasitol Res 107 : 495-499. , Mu et al. 2010aMu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su X-z 2010a. Plasmodium falciparum genome wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet 42 : 268-271. ) and (v) a strong association was observed between Pfcrt and the adjoining var gene in the VarS4 region of the P. falciparum genome ( Fowler et al. 2006Fowler EV, Chavchich M, Chen N, Peters JM, Kyle DE, Gatton ML, Cheng Q 2006. Physical linkage to drug resistance genes results in conservation of var genes among west pacific Plasmodium falciparum isolates. J Infect Dis 194 : 939-948. ). This final observation ( Fowler et al. 2006Fowler EV, Chavchich M, Chen N, Peters JM, Kyle DE, Gatton ML, Cheng Q 2006. Physical linkage to drug resistance genes results in conservation of var genes among west pacific Plasmodium falciparum isolates. J Infect Dis 194 : 939-948. ) corroborates the findings of Mu et al. (2010a)Mu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su X-z 2010a. Plasmodium falciparum genome wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet 42 : 268-271. , clearly reflecting the importance of the ~100-Kb region of chromosome 7 in the P. falciparum genome ( Fig. 2 ) rather than the Pfcrt gene alone. Furthermore, unlike the global pattern depicting the role of natural selection in the evolution of the Pfcrt gene ( Wootton et al. 2002Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su X-z 2002. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum . Nature 418 : 320-323. ), the Pfcrt gene in Indian P. falciparum does not seem to follow the same pattern, which could be due to a shift in the target of selection ( Das & Dash 2007Das A, Dash AP 2007. Evolutionary paradigm of chloroquine-resistant malaria in India. Trends Parasitol 23 : 132-135. ). In addition, the poor correlation between the CQR epidemiological surveillance data and the CQR Pfcrt haplotypes ( Awasthi et al. 2012Awasthi G, Satya GBK, Das A 2012. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum . Mem Inst Oswaldo Cruz 107 : 129-134. ) weakens the contention that Pfcrt is the sole controller of CQR.

Conclusion and future prospects - The current genetic understanding of CQR P. falciparum not only has provided several meaningful insights and enhanced the knowledge pertaining directly to malaria research, but also has advanced the academic understanding of how a single gene and a single amino acid mutation can significantly affect gross phenotypic characteristics. Because human infectious diseases are difficult to control, mainly due to the development of drug-resistant pathogens and successful environmental adaptation, the detailed genetic understanding of CQR P. falciparum may prove to be a model that can be applied to other infectious disease systems. In this regard, enormous amounts of genetic data on the Pfcrt gene in global P. falciparum have been generated and several genetic, epidemiological and evolutionary hypotheses have been proposed and tested. Furthermore, association studies between the drug response (IC ₅₀ values) and SNPs in different candidate genes have identified several associations between the Pfcrt gene and CQR.

However, despite this wealth of knowledge, it is unclear if one can reliably consider Pfcrt to be the sole gene that is responsible for CQR in P. falciparum . Several studies in global P. falciparum have suggested that Pfcrt is the primary determinant of CQR. At the same time, enough empirical evidence has disputed this hypothesis ( Su et al. 1997Su X-z, Kirkman LA, Fujioka H, Wellems TE 1997. Complex polymorphisms in an approximately 330 kDa protein are linked to chloroquine-resistant P. falciparum in Southeast Asia and Africa. Cell 91 : 593-603. , Basco & Ringwald 1999Basco LK, Ringwald P 1999. Chloroquine resistance in Plasmodium falciparum and polymorphism of the cg2 gene. J Infect Dis 180 : 1979-1986. , 2001Basco LK, Ringwald P 2001. Point mutations in the Plasmodium falciparum cg2 gene, polymorphism of the kappa repeat region and their relationship with chloroquine resistance. Trans R Soc Trop Med Hyg 95 : 309-314. , Durand et al. 1999Durand R, Gabbett E, Di Piazza JP, Delabre JF, Le Bras J 1999. Analysis of κ and ω repeats of the cg2 gene and chloroquine susceptibility in isolates of Plasmodium falciparum from sub-Saharan Africa. Mol Biochem Parasitol 101 : 185-197. ), supporting the presence of secondary determinants of CQR ( Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ). While the role of the Pfcrt gene cannot entirely be negated, with increasing bodies of evidence from several genome wide association studies and quantitative trait loci analyses of genetic crosses, it is reasonable to hypothesise a role for other gene(s) in conferring CQR in P. falciparum ( Wootton et al. 2002Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su X-z 2002. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum . Nature 418 : 320-323. , Kidgell et al. 2006Kidgell C, Volkman SK, Daily J, Borevitz JO, Plouffe D, Zhou Y, Johnson JR, Le Roch K, Sarr O, Ndir O, Mboup S, Batalov S, Wirth DF, Winzeler EA 2006. A systematic map of genetic variation in Plasmodium falciparum . PLoS Pathol 2 : e57. , Volkman et al. 2007Volkman SK, Sabeti PC, DeCaprio D, Neafsey DE, Schaffner SF, Milner Jr DA, Daily JP, Sarr O, Ndiaye D, Ndir O, Mboup S, Duraisingh MT, Lukens A, Derr A, Stange-Thomann N, Waggoner S, Onofrio R, Ziaugra L, Mauceli E, Gnerre S, Jaffe DB, Zainoun J, Wiegand RC, Birren BW, Hartl DL, Galagan JE, Lander ES, Wirth DF 2007. A genome-wide map of diversity in Plasmodium falciparum . Nat Genet 39 : 113-119. , Mu et al. 2010aMu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su X-z 2010a. Plasmodium falciparum genome wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet 42 : 268-271. , Ecker et al. 2012Ecker A, Lehane AM, Clain J, Fidock DA 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28 : 504-514. ). For example, a recently conducted genome scan of global P. falciparum isolates reported important genomic information on the genetic basis of antimalarial resistance ( Mu et al. 2010bMu J, Seydel KB, Bates A, Su X-z 2010b. Recent progress in functional genomic research in Plasmodium falciparum . Curr Genomics 11 : 279-286. ). In particular, a 100-Kb region located in chromosome 7 of the P. falciparum genome ( Fig. 2 ) was found to have very low recombination activity (Gupta et al. 2010, Mu et al. 2010aMu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su X-z 2010a. Plasmodium falciparum genome wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet 42 : 268-271. ). This region contains eight transporter genes (CG1, CG2, CG3, CG4, Pfcrt , CG6, CG7, CG8) ( Fig. 2 ). Furthermore, evidence for the tight linkage between genes located in this chromosomal region has also been documented ( Fowler et al. 2006Fowler EV, Chavchich M, Chen N, Peters JM, Kyle DE, Gatton ML, Cheng Q 2006. Physical linkage to drug resistance genes results in conservation of var genes among west pacific Plasmodium falciparum isolates. J Infect Dis 194 : 939-948. ). Taken together, these data support the hypothesis that other gene(s) located within this linked genetic block on chromosome 7 in P. falciparum might also play a role in conferring CQR, either alone or in close functional associations with the Pfcrt gene. Specifically, one of these eight transporter genes ( Fig. 2 ), CG2 , when placed downstream of the Pfcrt , has been shown to be phenotypically associated with CQR (Su et al. 1997, Basco & Ringwald 1999Basco LK, Ringwald P 1999. Chloroquine resistance in Plasmodium falciparum and polymorphism of the cg2 gene. J Infect Dis 180 : 1979-1986. , 2001Basco LK, Ringwald P 2001. Point mutations in the Plasmodium falciparum cg2 gene, polymorphism of the kappa repeat region and their relationship with chloroquine resistance. Trans R Soc Trop Med Hyg 95 : 309-314. , Durand et al. 1999Durand R, Gabbett E, Di Piazza JP, Delabre JF, Le Bras J 1999. Analysis of κ and ω repeats of the cg2 gene and chloroquine susceptibility in isolates of Plasmodium falciparum from sub-Saharan Africa. Mol Biochem Parasitol 101 : 185-197. ). However, the association between CQR and the CG2 genotype is not sufficient to completely justify an exclusive role for the CG2 gene in CQR ( Gupta et al. 2010Gupta B, Awasthi G, Das A 2010. Malaria parasite genome scan: insights into antimalarial resistance. Parasitol Res 107 : 495-499. ).

Based on the currently available data, it seems that the ~100-Kb region in chromosome 7 in the P. falciparum genome holds the key for the determination of CQR ( Gupta et al. 2010Gupta B, Awasthi G, Das A 2010. Malaria parasite genome scan: insights into antimalarial resistance. Parasitol Res 107 : 495-499. , Mu et al. 2010aMu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P, Krudsood S, White NJ, Udomsangpetch R, Cui L, Ho M, Ou F, Li H, Song J, Li G, Wang X, Seila S, Sokunthea S, Socheat D, Sturdevant DE, Porcella SF, Fairhurst RM, Wellems TE, Awadalla P, Su X-z 2010a. Plasmodium falciparum genome wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet 42 : 268-271. ). Considering the dubious role of Pfcrt and the possible involvement of other nearby transporter genes, further evolutionary genetic studies ( Stephan 2010Stephan W 2010. Detecting strong positive selection in the genome. Mol Ecol Resour 10 : 863-872. ) in this 100-Kb region could provide novel insights into the genetic basis of the P. falciparum drug resistance mechanisms ( Gupta et al. 2010Gupta B, Awasthi G, Das A 2010. Malaria parasite genome scan: insights into antimalarial resistance. Parasitol Res 107 : 495-499. ) and identify previously unknown genes that may be involved in determining CQR in P. falciparum . Further functional validation of such novel genes could possibly clarify the genetic determinants of CQR P. falciparum . This clarification will not only provide new directions for malaria research and further our understanding of the molecular epidemiology of P. falciparum malaria, but also contribute to the development of new genetic control measures for malaria. This increased understanding could also improve the management of other human infectious diseases that are dominated by drug-resistant pathogens.

ACKNOWLEDGEMENTS

To Prof Wolfgang Stephan’s Lab, Ludwig’s Maximilians University, Munich, Germany, where GA and AD were academic visitors, for the initiation of writing this paper, to the Journal of Cell Sciences, UK, and Boehringer Ingelheim Fonds, Germany, for providing travel fellowships to GA, to the ICMR, for Senior Research Fellowship, to the Department of Biotechnology, Govt of India, for providing Overseas Associateship to AD, to Prof W Stephan, for providing excellent facilities and support in his lab, to Ms Hueggette Gaelle Ngassa Mbenda, for help in Pfcrt haplotype data collection from Africa, to the anonymous reviewers, for their helpful and critical comments.

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