The development and expansion of resistance to the mainstay antimalarials chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) are the major causes for the increased morbidity and mortality of P. falciparum. In response to the increasing resistance, combination therapy, and especially artemisinin-based combination therapy (ACT), is now increasingly advocated as first-line therapy.1 The hypothesis is that a combination of drugs will achieve a more effective clinical and parasitologic cure, protect each other from the development of resistance, and reduce the overall rate of malaria. However, the choice of drugs is critical, especially bearing in mind the diversity and dynamics of P. falciparum and its possibility to develop different mechanisms of resistance.
The 4-aminoquinoline amodiaquine (AQ) is an antimalarial compound structurally and functionally related to CQ. It is more effective against CQ-resistant P. falciparum strains, and it is currently used in combination with artemisinin derivatives or SP for uncomplicated P. falciparum malaria, both in Africa2,3 and South America (Colombia).4 AQ is absorbed quickly in the gastrointestinal tract and metabolized in the liver by the cytochrome P450 isoform CYP2C8.5 Its main metabolite is desethylamodiaquine (DEAQ), which seems to exert the main antimalarial effect because of its significantly longer elimination half-life.6,7
Several polymorphisms in two P. falciparum genes, pfcrt and pfmdr1, have been associated with resistance to 4-aminoquinoline antimalarial drugs.8–10 Mainly pfcrt 76T, but also pfmdr1 86Y, has been involved in the development of CQ resistance and has also been associated with therapeutic failures after AQ treatment in Sudan, Kenya, and Nigeria.11–13 In Colombia, where CQ-resistant P. falciparum is widespread but a more varied response to AQ has been found,14,15 the pfcrt 76T allele seems to be presently fixed and ubiquitous, whereas the pfmdr1 86Y allele seems to be rare.16,17
In vitro studies have shown that probably through the PFCRT protein, the Ca2+-transporter inhibitor drug verapamil (VP) may sensitize P. falciparum to CQ and to some extent to DEAQ but not to AQ.18,19 These differences between CQ and AQ/DEAQ and between AQ and DEAQ raise the hypothesis that other polymorphisms, maybe in the pfcrt or pfmdr1 genes, could mediate a decreased susceptibility to AQ and/or DEAQ.
To address this hypothesis and to further explore the resistance mechanisms against AQ/DEAQ, we analyzed the relationship of pfcrt and pfmdr1 mutations in 15 Colombian P. falciparum isolates with their in vitro susceptibility to CQ, AQ, and DEAQ. Documented in vitro data from four P. falciparum reference strains (Dd2, HB3, 3D7, and 7G8) were also included in the analysis as comparators.10,18,20
The 15 P. falciparum culture adapted isolates, collected between 1999 and 2001 in three endemic areas along the Colombian Pacific Coast, were analyzed in a radioisotopic in vitro susceptibility assay for their response to CQ, AQ, and DEAQ.21 The isolates were defrosted, adapted to continuous culture in supplemented RPMI-1640, and synchronized with sorbitol.22–24 Ninety-six-well plates were coated with 50–3,200 nmol/L CQ and 5–320 nmol/L AQ and DEAQ after a 2-dilution factor (AQ and CQ were provided by the World Health Organization and DEAQ by the Walter Reed Army Institute). The final concentration of [3H] hypoxanthine was 0.5 μCi/well and that of Albumax I was 0.5%.21 The samples were incubated at 37°C for 48 hours, and each assay was done in duplicate. The counts per minute (cpm) were measured in a scintillation counter (LS7500; Beckman Instruments, Palo Alto, CA), and the IC50s were calculated using the PROBIT program in SPSS 7.5 for Windows 98 (SPSS, Chicago, IL).
The capacity of VP (V4629-1G; Sigma, St. Louis, MO) to sensitize P. falciparum to CQ, AQ, and DEAQ was tested in the isolates TA7519, CA2855, and Dd2 strain, according to the recently developed ELISA-histidine rich protein II (HRP2)–based assay.25 In vitro cultured parasites were diluted to an initial parasitemia of 0.05% and aliquoted into microculture 96-well plates pre-dosed with ascending concentrations of 16–2,020 nmol/L CQ, 2–270 nmol/L AQ, and 6–780 nmol/L DEAQ ± VP 0.8 μmol/L. After incubation at 37°C for 72 hours, the samples were freeze-thawed, transferred, and processed in pre-coated ELISA plates (Cellabs, New South Wales, Australia) for spectrophotometric analysis (Multiskan EX; Thermo Labsystems, Helsingfors, Finland) of parasite growth. The IC50 values were determined using HN-NonLin V1.05 Beta H. Noedl 2001 (http://malaria.farch.net). All samples were done in quadruple.
For the full sequencing of the pfcrt gene, total RNA and cDNA were obtained with Trizol LS (Invitrogen, Carlsbad, CA) and SUPERSCRIPT II RT (Invitrogen), respectively. Using the sequence of the HB3 strain (GenBank no. AF233068), primers were designed to amplify three overlapping fragments representing the complete pfcrt sequence (first block of 437 bp, second block of 409 bp, and third block of 593 bp). After a nested polymerase chain reaction (PCR) strategy, all blocks were sequenced (ABI 3700 Capillary DNA Sequencer; Cybergene AB, Huddinge, Sweden) and bioinformatically analyzed (Chromas V2.75, BLAST, Translate, LALING, and Clustal W). For the pfmdr1 single nucleotide polymorphism (SNP) analysis, a PCR-restriction fragment length polimorphism (RFLP) strategy26 was used to identify SNPs in positions N86Y, F184Y, S1034C, D1042N, and D1246Y (more details about these strategies can be requested from the authors). For the quantification of the pfmdr1 gene copy number, a TaqMan real-time PCR-based protocol (Applied Biosystems, Fresno, CA) was used as published elsewhere.27 Copy numbers were rounded to the nearest integer. This study was approved by the Ethical committees of CIDEIM and Universidad del Valle in Cali, Cali, Colombia.
In all 15 Colombian isolates, the IC50 values for CQ (114.58–415.53 nmol/L, data not shown) were above the WHO recommended cut-off for in vitro CQ resistance (IC50 >100 nmol/L). More variations in IC50 values were observed for both DEAQ (38.31–355.8 nmol/L; Figure 1) and AQ (9.63–64.61 nmol/L; Figure 2).
VP was able to considerably decrease the IC50 values in Dd2, CA2855, and TU7519 for CQ from 340.1 to 129.7; 636.9 to 182.3; and 551.7 to 70.9 nmol/L for each sample, respectively. Similarly, VP reduced the IC50 values for DEAQ from 32 to 12.8; 119.7 to 26.6; and 62.9 to 12.2 nmol/L, respectively. In contrast, VP did not modify the in vitro response to AQ: 13.1 to 11.0; 26.5 to 28.5; and 22.5 to 22.2 nmol/L, respectively.
DNA sequencing of the pfcrt gene was completely successful in 12 of 15 samples. Three samples were uncertain in the 371 position. A total of 15 SNPs in 12 positions in the pfcrt gene were identified (Figures 1 and 2). All 15 Colombian isolates carried the 76T and 220S mutant haplotype. In codons 72–76 of the pfcrt gene, the CVMET haplotype was seen in 13 of 15 isolates. The isolate TU741 carried a CVMNT haplotype, which has also been found in Ecuador and Peru.10,28 The isolate CA2855 showed a CVIET Dd2-like haplotype extending the reports of the presence of this Southeast Asian CQ-resistant haplotype in South America.10,28 The SVMNT haplotype characteristic of the 7G8 CQ-resistant strain from Brazil and reported in P. falciparum populations from Papua New Guinea,29 which has been proposed to be associated with DEAQ resistance,30 was not detected in any Colombian sample.
The SNP 97Q that to this date has been only reported in Colombia was found in the majority (13/15) of the parasites.10,17 In the TU741 isolate, a not previously described SNP, 334N, was identified. The SNP 333S was found in two isolates (TA4640 and TA6182); this SNP was previously found in Cambodia.31 In summary, we identified four pfcrt haplotypes among the fully sequenced samples (amino acids 72, 74–76, 97, 220, 271, 326, 333, 334, 356, and 371; SNPs are underlined): CMETQSQNTSIT (8/12), CMETQSQNSSII (2/12; GenBank no. DQ156109), CMNTHSQDTNLR (1/12; GenBank no. DQ156108), and CIETHSESTSTI (1/12).
Pfmdr1 analyses were successful in all Colombian isolates. Three pfmdr1 haplotypes (amino acids 86, 184, 1034, 1042, and 1246) were identified: NFSDD (6/15), NFSDY (8/15), and YYSND (1/15). All tested Colombian isolates had one pfmdr1 gene copy (Figures 1 and 2).
The high IC50 values for CQ and the more varying IC50 values for AQ and DEAQ, as well as the presence of pfcrt 76T and 220S in all Colombian samples, are in line with the high level of CQ resistance and the more various therapeutic response to AQ in Colombia. The VP results with sensitization for CQ and DEAQ but not for AQ confirm previously published data18,19 and suggest a mechanism of response to AQ less dependent on the pfcrt gene compared with DEAQ.
The TU741 isolate showed the highest IC50 value for DEAQ (355.8 nmol/L) and the second highest for AQ (41 nmol/L). TU741 was the only isolate with a pfcrt 72–76 CVMNT haplotype. TU741 also showed several genetic alterations not present in any of the other studied Colombian isolates, namely the pfcrt 326D and 356L SNPs also seen in the clone 7G8 (pfcrt 72–76 SVMNT), with the second highest IC50 value for DEAQ (229,3 nmol/L), as well as pfcrt 334N. This suggests that the haplotypes pfcrt 72–76 SVMNT/ CVMNT, 326D, 334N, and 356L might be associated with DEAQ resistance. The CA2855 isolate showed the highest IC50 for AQ (64.6 nmol/L) and the third highest to DEAQ (198.1 nmol/L). It was the only isolate carrying a Dd2-like SNP haplotype in both pfcrt and pfmdr1. However, CA2855 did have one copy of pfmdr1 compared with three in Dd2 (Figure 2). In addition, CA2855 was identified as FC27 and Dd2 as IC1 families in the msp2 gene (data not shown).
The presence of pfmdr1 86Y, 184Y, and 1042N in this isolate is consistent with recent in vivo data from Kenya where pfmdr1 86Y was selected among recrudescent infections but not among new re-infections after AQ monotherapy,12 suggesting that this haplotype is a potential factor for AQ resistance. However, Dd2 with the same haplotype had a lower IC50 value for AQ. Maybe this difference can be explained by its multicopied pfmdr1 gene or other genes involved in AQ resistance.
A visual analysis of the sequence data along with the IC50 values of DEAQ (Figure 1) and AQ (Figure 2) showed an accumulation of SNPs in the 3′ region of the pfcrt open reading frame (ORF). There was also a tendency of the pfmdr1 1246Y SNP toward higher IC50 values for AQ. A decrease in the hydrophobicity, volume of lateral chains of the amino acids, and changes in the charges of the PFCRT protein have been predicted to explain the mechanism by which these SNPs could decrease the efficacy of AQ.30
This exploratory study supports the hypothesis that AQ and DEAQ resistance might be associated with different combinations of polymorphisms in the pfcrt and pfmdr1 genes. The pfmdr1 copy number seems not be involved in 4-aminoquinolines resistance, which should be explored further.
In vitro susceptibility to desethylamodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNPs are shadowed. NS; no sequence data.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034
In vitro susceptibility to desethylamodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNPs are shadowed. NS; no sequence data.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034
In vitro susceptibility to desethylamodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNPs are shadowed. NS; no sequence data.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034
In vitro susceptibility to amodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNP are shadowed. NS; no sequence data.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034
In vitro susceptibility to amodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNP are shadowed. NS; no sequence data.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034
In vitro susceptibility to amodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNP are shadowed. NS; no sequence data.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034
Address correspondence to Diego F. Echeverry, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Valle, Colombia. E-mail: difereg@cideim.org.co
Authors’ addresses: Diego F. Echeverry, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Colombia, Telephone: 572–6682164, Fax: 572–6672989. Gabrielle Holmgren, Malaria Research Lab, M9/02, Karolinska Hospital, Stockholm 17176, Sweden. Claribel Murillo, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Valle, Colombia, Telephone: 572–6682164, Fax: 572–6672989. Juan C. Higuita, Universidad Nacional de Colombia, Manizales, Departamento de Ingeniería Química. Manizales, Colombia. Anders Bjorkman, Malaria Research Lab, M9/02, Karolinska Hospital, Stockholm 17176, Sweden. Jose P. Gil, Malaria Research Lab, M9/02, Karolinska Hospital, Stockholm 17176, Sweden. Lyda Osorio, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Colombia, Telephone: 572–6682164, Fax: 572–6672989.
Financial support: This work was supported by an anonymous Swiss foundation.
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