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Antimicrobial Agents and Chemotherapy, November 2003, p. 3500-3505, Vol. 47, No. 11
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.11.3500-3505.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

pfcrt Allelic Types with Two Novel Amino Acid Mutations in Chloroquine-Resistant Plasmodium falciparum Isolates from the Philippines

Nanhua Chen,¹ Dennis E. Kyle,^1,2 Cielo Pasay,^3, Elizabeth V. Fowler,⁴ Joanne Baker,¹ Jennifer M. Peters,⁴ and Qin Cheng^1*

Department of Drug Resistance and Diagnostics, Australian Army Malaria Institute, Enoggera,¹ Malaria and Scabies Group, Infectious Diseases Unit, Queensland Institute of Medical Research, Herston, Queensland, Australia,⁴ Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland,² Department of Parasitology, The Research Institute for Tropical Medicine, Manila, Philippines³

Received 17 June 2003/ Returned for modification 22 July 2003/ Accepted 28 July 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutations in the pfcrt and pfmdr1 genes have been associated with chloroquine resistance in Plasmodium falciparum. Ten and five mutations, respectively, have been identified in these genes from chloroquine-resistant parasites worldwide. Mutation patterns in pfcrt revealed that chloroquine resistance evolved independently in southeast Asia, South America, and Papua New Guinea. However, the evolution of chloroquine resistance in the rest of the Pacific region is unclear. In this study, we examined sequence polymorphisms in these genes in isolates from Morong, Philippines, and compared them to known chloroquine resistance sequences. Two novel mutations, A144T and L160Y, were identified outside of the 10 known mutations in pfcrt in Morong isolates. These novel mutations were identified only in parasites with K76T and N326D but without the common A220S mutation found in most chloroquine-resistant isolates. This represents a unique chloroquine resistance allelic type (K76T/A144T/L160Y/N326D) not previously found elsewhere in the world. One Morong isolate also had an additional C72S mutation, whereas only one isolate possessed an allelic type typical of chloroquine resistance in Asia. Parasites with the novel pfcrt allelic types were resistant to chloroquine in vitro and were unresponsive to verapamil (0.9 µM) chemosensitization, similar to chloroquine-resistant parasites from South America and Papua New Guinea. These results suggest that chloroquine resistance evolved independently in the Philippines and represents a second chloroquine resistance founder event in the South Pacific.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies on the molecular mechanism of chloroquine resistance in Plasmodium falciparum have led to the identification and characterization of the pfcrt gene (9). Genetic mutations in pfcrt were reported to be associated with reduced in vitro susceptibilities to chloroquine in laboratory lines and field isolates (1, 5, 9, 12). Mutations in the pfcrt gene has also been shown, by episomal complementation assay (9) and allelic exchange techniques (17), to confer chloroquine resistance in laboratory strains. These studies suggest that the PfCRT protein plays a fundamental role in chloroquine resistance.

In previous studies, point mutations were observed in 10 codons in the pfcrt gene of chloroquine-resistant parasite isolates from various regions. These include mutations at amino acid positions 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371. In general, the chloroquine-resistant parasite isolates from Southeast Asia and Africa have pfcrt genes with seven to nine mutated codons, and their mutated codons are linked into a pattern of CIETH(L)SEST(I)I, from positions 72 to 371 (9, 21). The chloroquine-resistant parasites from South America and Papua New Guinea possess pfcrt genes with four to five mutated codons forming patterns of S(C)MNTHSQDLR (5, 9, 21).

The minimum number of mutations previously reported in pfcrt of chloroquine-resistant parasites was four: C72S, K76T, N326D, and I356L (21). The mutation K76T was found in all chloroquine-resistant parasites and A220S was found in all but two chloroquine-resistant isolates so far sequenced (5, 9, 11, 21), indicating their essential role in chloroquine resistance. The role of the remaining pfcrt mutations in chloroquine resistance remains unclear. The number and pattern of the four to nine mutations in the pfcrt gene were not associated with the levels of chloroquine resistance (5); however, the pfcrt allelic type of the chloroquine-resistant parasites has been shown to be linked with the chloroquine-resistant parasite's response to verapamil chemosensitization (12). Chloroquine-resistant parasites with South American or Papua New Guinea allele of pfcrt are less responsive to verapamil than those with the Southeast Asian allele (12).

Based on the diversity of pfcrt allelic types and flanking microsatellite markers, it has been proposed that chloroquine-resistant parasites originated from at least four independent foci: (i) in Asia spreading to Africa, (ii) in Papua New Guinea, and (iii) two sites in South America (Peru and Colombia) (21). The finding that pfcrt allelic types in Papua New Guinea (5, 12) were different from those found in Asia was a surprise, as previously chloroquine resistance was thought to have migrated south and east from Asia into Oceania (18). Chloroquine resistance has been widely reported in the Philippines (2) and other South Pacific island countries (14); however, in light of the new data, the evolution and spread of chloroquine resistance within the region are not clear.

In this study, we examined the pfcrt gene in 20 P. falciparum isolates collected from Morong, Philippines. Most of these possessed only two of the 10 previously known mutations (K76T and N326D) in pfcrt, yet they also had two novel mutations, A144T and L160Y, not observed previously. The susceptibilities of parasites with the unique allelic types to chloroquine and desethyl chloroquine were investigated, as were their responses to verapamil chemosensitization.

Top Abstract Introduction Materials and Methods Results Discussion References

 
P. falciparum isolates and lines. P. falciparum isolates PH1 to PH10, PH12, and PH16 were collected from Morong, Bataan, Luzon, Philippines, in 1989 to 1991 as part of a baseline malaria epidemiological survey. PH21 to PH28 were collected in the same area 1 to 2 years later (1992 to 1993) in cross-sectional surveys (13). A detailed description of the area has been published (3). FCQ27-D10, K1, and 7G8 are laboratory lines originally from Papua New Guinea, Thailand, and Brazil, respectively. P. falciparum isolates collected from Papua New Guinea (6), Solomon Islands (15), and Bougainville and Thailand (5) were described previously.

PCR amplification of the P. falciparum genes. Genomic DNA was extracted from the cultured parasites with methods described previously (7). P. falciparum gene fragments to be studied were amplified by PCR in 50-µl reaction mixture containing 1.5 µg/ml of each primer, 200 µM deoxynucleoside triphosphates, buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 2.5 mM MgCl₂, and 1.25 units of AmpliTaq Gold DNA polymerase (PE Applied Sciences). msp1, msp2, and glurp were amplified and their fragment sizes were analyzed by agarose gel electrophoresis (16). Three pfcrt fragments were amplified with the following cycle conditions: 94°C 10 min, then 94°C for 50 s, 51°C for 50 s, and 70°C for 1 min for 40 cycles: (i) a fragment flanking codons 72, 74, 75, 76, and 97 with primers D1 (5'-TGT GCT CAT GTG TTT AAA CTT-3') (8) and D3 (5'-AAA GCT TCG GTG TCG TTC-3'); (ii) a fragment flanking codon 220 with primers E3 (5'-CTT ATA CAA TTA TCT CGG AGC AGT-3') and F1 (5'-GTC ATG TTT GAA AAG CAT ACA GG-3'); and (iii) a fragment flanking codon 271 with primers E4 (5'-CCA AGA ATA AAC ATG CGA AAC C-3') and F2 (5'-ATT TCT TAT AGG CTA TGG TAT CC-3').

A fourth fragment of pfcrt flanking codons 326, 356, and 371 was amplified by nested PCR with primers D7 (5'-TTT CTA AGA TAA TAT TTC CTA CAC-3') and F3 (5'-TAG AAA ACC TTC GCA TTG-3') (94°C for 10 min, then 94°C for 50 s, 45°C for 50 s, and 70°C for 1 min for 40 cycles), and then F3 and F4 (5'-ACT TAC CAA AGT TAC GAA ATC-3') (94°C for 10 min, then 94°C for 50 s, 50°C for 50 s, and 70°C for 1 min for 30 cycles). A fifth fragment flanking codons 144 and 160 was amplified with primers 4A (5'-TAGGAACGACACCGAAG-3') and 4B (5'-ATAGTATACTTACCTATATC-3') (94°C for 10 min, then 94°C for 50 s, 45°C for 50 s, and 70°C for 1 min for 45 cycles).

Isolation of total RNA and RT-PCR to amplify a 1,013-bp fragment of the coding sequence of pfcrt (codons 44 to 380). Total RNA was extracted from the cultured parasites (PH1, PH3, PH4, PH5, PH7, PH9, PH10, and FCQ27-D10) with a NucleoSpin RNAII System (Macherey-Nagel GmbH & Co., Germany). cDNA was amplified by reverse transcription (RT)-PCR with a SuperScript One-Step RT-PCR System (Invitrogen Life Technologies) and primers D1 and F4 with the following conditions: 42°C for 30 min, 94°C for 2 min, then 94°C for 50 s, 50°C for 50 s, and 70°C for 1 min for 40 cycles. Fragments of pfmdr1-flanking mutations were also amplified and analyzed (4).

Purification and sequencing of the pfcrt PCR fragments. The PCR products were purified with a NucleoSpin Extract System (Macherey-Nagel GmbH & Co., Germany) following agarose gel electrophoresis, and then sequenced in an ABI Prism 370 sequencer.

In vitro chloroquine susceptibility tests. Parasites were maintained in vitro with conditions modified from those described by Trager and Jensen (19). In vitro susceptibilities to chloroquine and desethyl chloroquine were tested, and 50% inhibitory concentration (IC₅₀) and IC₉₀ values were determined as described previously (12). Verapamil (0.9 µM) was included in one set of susceptibility tests to determine the effect of verapamil on the chloroquine resistance phenotypes of the parasite samples (12).

Nucleotide sequence accession number. The cDNA sequence of PH1 representing the major novel pfcrt allelic type has been deposited in the GenBank database with accession number AY254700.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Novel pfcrt allelic types. Twenty P. falciparum isolates collected from the Morong area of the Philippines were examined in this study. Genotyping of three polymorphic markers, msp1, msp2, and glurp, indicated that 18 out of the 20 isolates were of a single clone of parasite; the remaining 2 isolates (PH6 and PH10) had more than one clone of parasite (data not shown). Sequence polymorphisms in pfcrt, in particular the 10 codons reported to be associated with chloroquine resistance (9), were examined for these isolates, and the results are shown in Table 1.

View this table: [in this window] [in a new window]

TABLE 1. Sequence polymorphisms in PfCRT and PfMDR1a

Compared to the wild-type chloroquine-sensitive PFCRT sequence CMNKHAQNIR in FCQ27-D10, the pfcrt gene of 18 Morong isolates (PH1, PH3, PH5 to PH10, PH12, PH16, and PH21 to PH28) possessed only two mutated codons, K76T and N326D, giving allelic type CMNTHAQDIR (Table 1). One isolate (PH2) had three mutated codons: C72S, K76T and N326D giving an allelic type of SMNTHAQDIR (Table 1). The remaining isolate (PH4) was of the Southeast Asian allelic type (CIETHSESTI) and had eight mutations. These three allelic types are named P2a, P2b, and E1a, respectively (Table 1) according to the nomenclature used by Wellems and Plowe (20). The P allelic type, originally proposed for Papua New Guinea, was extended to include other Pacific region countries where similar allelic types evolved. All 20 Morong isolates were found to carry the K76T mutation, which has been identified as crucial for chloroquine resistance. Interestingly, A220S, a mutation that is almost always linked with the K76T mutation in chloroquine-resistant parasites elsewhere, was present in only one parasite with the Asian allelic type (PH4), but not in the remaining 19 Morong isolates.

Previously it was suggested that a minimum of four mutations were required to confer chloroquine resistance (21). The surprising finding that most of the Morong isolates had mutations in only two of the ten known positions led us to investigate the cDNA sequence for seven of these isolates. Two novel mutations, A144T and L160Y, were detected in each of the isolates that also had K76T and N326D (Table 1). To investigate the frequency of these two novel mutations, primers 4A and 4B were used to amplify the gene fragment containing these positions in the remaining Morong isolates. In total, A144T and L160Y were identified in 19 of the 20 Morong isolates examined, including 11 isolates collected from the pilot study (1989 to 1991) and 8 from two separate surveys (1992 to 1993); the only exception was PH4, which was the only isolate found with an Asian allelic type for pfcrt (Table 1).

The presence of these unique mutations in pfcrt in isolates from Morong led us to investigate their possible occurrence in chloroquine-resistant parasites from other Pacific island countries. In contrast to Morong parasites, no mutations were found at position 144 or 160 in isolates collected from Papua New Guinea (chloroquine sensitive: n = 5, chloroquine resistant: n = 7), Solomon Islands (n = 4), and Thailand (n = 5) (Table 1). A further examination of these two positions in Bougainville (n = 25), Solomon Islands (n = 8), East Timor (n = 23), Thailand (n = 9), Cambodia (n = 3), and Vietnam (n = 10) P. falciparum isolates failed to detect these mutations (data not shown). These data suggest that the two novel pfcrt allelic types, CMNTHTYAQDIR (type P2a) and SMNTHTYAQDIR (type P2b), may be present only in the Philippines.

pfmdr1 types. Sequence polymorphism in pfmdr1 was examined for the Morong isolates. The amino acids encoded at positions 86, 184, 1034, 1042, and 1246 were Y, Y, S, N, and D, respectively, for all isolates except PH4. PH4, the only isolate with the Southeast Asian pfcrt allelic type, also had a different pfmdr1 allelic type than the rest of the isolates (Table 1).

Isolates with novel pfcrt allelic types were resistant to chloroquine. Five Morong isolates with one of the novel allelic types (type P2a), K76T/N326D/A144T/L160Y (PH1, PH3, PH5, PH9, and PH10), and one isolate with the Southeast Asian allelic type (PH4, type E1a) were tested for their susceptibility to chloroquine and desethyl chloroquine, the primary human metabolite of chloroquine. Standard laboratory parasite lines FCQ27-D10 (chloroquine sensitive), K1 (chloroquine resistant, type E1b) and 7G8 (chloroquine resistant, typeW1a) were included as controls. Unfortunately, the other novel allelic type (type P2b), represented by PH2, could not be tested because it failed in adaptation to in vitro cultivation.

Parasites with the novel Morong allelic type (P2a) pfcrt showed a significant increase in chloroquine IC₅₀ (2.76- to 8.33-fold) and IC₉₀ (4.41- to 9.8-fold increase) compared to a known chloroquine-sensitive parasite, FCQ27-D10. This decreased susceptibility to chloroquine was comparable to that of 7G8 but lower than that observed for K1 (Table 2). Similar trends were observed for desethyl chloroquine (Table 2). PH4 parasites (type E1a) (CIETHSESTI) showed IC₅₀ and IC₉₀ values for chloroquine and desethyl chloroquine comparable to those of the other five Morong isolates tested (Table 2).

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TABLE 2. Susceptibility of P. falciparum to chloroquine and desethyl chloroquine in the absence and presence of verapamila

Isolates with novel pfcrt allelic type were not responsive to 0.9 µM verapamil. Verapamil (0.9 µM) was used to assess the degree of potentiation observed in the presence of chloroquine and desethyl chloroquine. Similar to the chloroquine-resistant Brazilian strain 7G8, the Morong isolates with the novel pfcrt genotypes were not responsive to verapamil chemosensitization (Table 2). The resistance modification indices (RMI; IC₅₀ or IC₉₀ in the presence of verapamil/IC₅₀ or IC₉₀ in the absence of verapamil) for the Morong isolates indicated little to no effect of verapamil on the response to chloroquine at either the IC₅₀ or IC₉₀ level. These results were similar to the results obtained for 7G8. In contrast, the RMI for PH4 was 0.37, which was comparable to that of K1 (0.36), indicating a reversal of chloroquine resistance in the presence of verapamil (Table 2). Similarly, verapamil had no effect on the response to desethyl chloroquine in the 7G8 or Morong parasites with the novel allelic types (Table 2). In contrast, resistance to desethyl chloroquine was reversed by verapamil in parasites with an Asian pfcrt allelic type (PH4 and K1).

Responses to quinine and mefloquine. All Morong isolates tested were susceptible to mefloquine; however, the response to quinine differed between the isolates (Table 3). PH4, similar to K1, showed elevated IC₅₀ (57.63 ± 20.01 ng/ml) and IC₉₀ (233.62 ± 75.72 ng/ml) to quinine. The remaining Philippine isolates showed significantly lower IC₅₀ and IC₉₀ for quinine compared to PH4. Interestingly, the quinine response of the only Morong isolate with an Asian pfcrt allelic type (PH4) was not affected by verapamil, whereas K1 resistance to quinine was reduced by 55 to 62% in the presence of verapamil, as previously reported for Asian chloroquine-resistant isolates (10). Verapamil had no effect on the response to mefloquine in any of the parasites tested (Table 3).

View this table: [in this window] [in a new window]

TABLE 3. Susceptibility of P. falciparum to quinine and mefloquine in the absence and presence of verapamila

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the current study, we identified two novel allelic types of the pfcrt gene in chloroquine-resistant P. falciparum isolates collected from Morong, Philippines. These pfcrt allelic types include two novel mutations (A144T and L160Y) in combination with two or three mutated codons (K76T/N326D or C72S/K76T/N326D) from within the previously reported repertoire of 10 codons. Parasites possessing the allelic type K76T/N326D/A144T/L160Y were resistant to chloroquine and desethyl chloroquine in vitro but were susceptible to quinine and mefloquine.

These two novel allelic types found in Morong, Philippines, represent the first indication that mutated codons in pfcrt exist outside the 10 positions found in chloroquine-resistant isolates from Asia, Africa, Papua New Guinea, and South America. The same allelic type was observed in 19 of 20 isolates examined that were collected over a 4-year period, suggesting that these alleles were stably maintained in the area. From our examination of pfcrt sequences in a limited number of isolates from Papua New Guinea, Solomon Islands, East Timor, Indonesia, Vietnam, and Thailand, the A144T and L160Y mutations were identified only in the Philippines. Whether these pfcrt allelic types are unique to the Morong area or common to other islands in the Philippines requires further investigation. Clearly, the occurrence of these novel pfcrt genotypes suggests that chloroquine-resistant parasites evolved independently in the Philippines rather than being spread from Southeast Asia, the Americas, or Papua New Guinea. Therefore, the K76T/N326D/A144T/L160Y allele likely represents the fifth founder event for the evolution of chloroquine resistance, with the other four foci being in Southeast Asia, Papua New Guinea, Colombia, and Peru (21).

One of the 20 Morong isolates studied was of a pfcrt allelic type identical to the Southeast Asia type (type E1a), which suggests that chloroquine resistance is also spreading from Southeast Asia. The difference in its pfmdr1 allelic type compared to the rest of the isolates provides further support that this parasite was introduced into the region from Southeast Asia. The Morong area is effectively isolated on three sides, and contact with other areas occurs only along a narrow coastal strip to the south. It is an area of very low malaria endemicity; however, at the time these isolates were collected, there was a refugee processing center in the area, funded by the United Nations High Commission for Refugees, that housed 11,000 to 16,000 refugees from Southeast Asia (3). It is highly probable that parasites carried by these Asian refugees have spread to local communities.

The tandem occurrence of K76T and A220S, located in predicted transmembrane domains (TMDs) 1 and 6, respectively, in almost all previously examined chloroquine-resistant isolates has suggested that these mutations play an essential role in conferring chloroquine resistance (9, 21). Interestingly, all Morong isolates with novel pfcrt allelic types have the K76T mutation but not the A220S mutation. Instead, these isolates have mutations of A144T and L160Y, which are located in predicted TMDs 3 and 4. Although the relative importance of the A144T and L160Y mutations in conferring chloroquine resistance is not clear, thus far we have not found these mutations coexisting with A220S in any other isolate. Besides the Morong isolates, only two chloroquine-resistant isolates have been reported that have K76T not accompanied by A220S (11, 21). However, the full-length sequence of the gene was not determined in either report, and it is unknown whether additional mutations exist in these isolates.

Therefore, the alternative presence of these two new mutations and their predicted locations in TMDs led us to hypothesize that A144T and L160Y may be compensatory mutations that can confer chloroquine resistance in the absence of A220S. If this hypothesis is correct, the K76T mutation plus one mutation in predicted TMD 6 or two mutations in TMDs 3 and 4 are required to confer chloroquine resistance. However, one possibility that we cannot rule out is that the A144T and L160Y mutations may occur in chloroquine-sensitive parasites in Morong and were inherited by chloroquine-resistant parasites. The full-length sequences of pfcrt genes from chloroquine-sensitive parasites in Morong or allelic replacement studies are required to confirm the role of the new pfcrt mutations (A144T and L160Y) in chloroquine resistance.

Interestingly, the novel Morong pfcrt allelic types P2a and P2b (CMNTHTYAQDIR and SMNTHTYAQDIR) are more closely related to the SMNTHSQDLR types reported for chloroquine-resistant parasites from South America and Papua New Guinea than to the CIETHSESTI type in Southeast Asia and Africa. Similar to parasites with the South American and Papua New Guinea allelic types (12), the chloroquine susceptibility of Morong parasites with the novel pfcrt allele was not affected by verapamil (0.9 µM). These data are consistent with the possibility that amino acids at positions 74 to 76 and/or 271, 326, and 371 are linked with the parasite response to verapamil and that the 74I/75E/76T and/or 271E/326S/371I mutations in pfcrt are positively associated with verapamil reversibility of chloroquine resistance.

The prevalence of two novel pfcrt allelic types in Morong, Philippines, suggests that chloroquine resistance evolved independently in the Philippines and represents the fifth founder event for chloroquine resistance worldwide. The fact that two founder events have occurred in South Pacific island countries may be due to their geographic isolation, especially in the 1960s, when chloroquine resistance first emerged in the region. Additional studies are required to determine the unique diversity of pfcrt alleles in the South Pacific to determine if chloroquine resistance emerged independently in additional loci.

 
This work is published with the approval of the Director-General of Joint Health Services, Australia. The research was funded in part by the U.S. Army Medical Research and Materiel Command, Ft. Detrick, Md.

The views expressed are those of the authors and should not be construed to represent the positions of the Department of the Army or Department of Defense of Australia or the United States.

E. V. Fowler and J. M. Peters were supported by NIH grant RO1 AI047500-3.

We thank Allan Saul from NIH and colleagues from the Malaria study group, the Research Institute for Tropical Medicine, Manila, Philippines, for their contribution in planning and conducting the cross-sectional surveys and collecting samples in Morong, Philippines. We also thank Christine Rzepczyk for providing isolates from the Solomon Islands.

 
* Corresponding author. Mailing address: Department of Drug Resistance and Diagnostics, Australian Army Malaria Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Qld 4051, Australia. Phone: 61-7-3332 4834. Fax: 61-7-3332 4800. E-mail: qin.cheng{at}defence.gov.au.

Present address: Infectious Diseases Unit, Queensland Institute of Medical Research, Herston, Queensland, Australia.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, November 2003, p. 3500-3505, Vol. 47, No. 11
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.11.3500-3505.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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