Abstract
Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies—a molecular marker for MQ resistance—in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ.
TEXT
Artemisinin-based combination therapy (ACT) is used worldwide to treat uncomplicated Plasmodium falciparum malaria. Artesunate plus mefloquine (AS+MQ) was adopted as Cambodia's first-line treatment in 2000. AS+MQ treatment failures were first observed in 10 to 20% of patients in Pailin and Battambang Provinces, Western Cambodia, in 2003 to 2004 (1, 2). The presence of multiple (i.e., ≥2) pfmdr1 copies—a genetic marker of MQ resistance—was associated with AS+MQ failures and reduced parasite in vitro susceptibility to MQ not only in Pailin and Battambang but in neighboring Pursat and Kampot Provinces as well (3–5). This development, along with emerging evidence of reduced in vitro susceptibility to MQ in other provinces (6), prompted Cambodia's National Malaria Control Program to adopt dihydroartemisinin-piperaquine (DHA-PPQ) as the first-line ACT in Western Cambodia in 2008 and in the entire country in 2010. Unfortunately, recent clinical studies suggest that the efficacy of DHA-PPQ is rapidly declining in five Western Cambodian provinces: Pursat, Battambang, Pailin, Oddar Meancheay, and Preah Vihear. For example, DHA-PPQ treatment failures were observed in 25% and 11% of patients in Pailin and Pursat in 2010 (7) and in 36% of patients in Oddar Meancheay in 2013 (8). The rapidly increasing prevalence of DHA-PPQ failures in these provinces, likely due to entrenched artemisinin resistance (9, 10) and suspected PPQ resistance (7, 8), demands additional evaluations of newer antimalarial drugs (11, 12), as well as reevaluation of previously used ACTs.
Only a few ACTs, including AS+MQ and artemether-lumefantrine (AL), are presently available for widespread use in Cambodia to replace DHA-PPQ as it fails. Although AS+MQ and AL treatment failures have previously been reported from Battambang and Pursat (2, 13), the clinical efficacy of these regimens in the setting of DHA-PPQ resistance is not known. However, studies have suggested a lack of cross-resistance between PPQ and MQ in vitro; for example, pfmdr1 amplification is associated with decreased susceptibility to MQ but increased susceptibility to PPQ (14). Given these and related findings (15), and the fact that earlier use of DHA-PPQ was associated with decreasing prevalence of multiple pfmdr1 copies in Pailin (from 33% in 2005 to 5% in 2007) (16), we hypothesized that the recent substantially reduced use of AS+MQ in Pursat and Preah Vihear would select for parasites that have regained sensitivity to MQ. To explore this hypothesis, we used real-time PCR methods to quantify the pfmdr1 copy number in 844 P. falciparum clinical isolates collected in 2008 to 2013 from Pursat and Preah Vihear, as well as Ratanakiri Province in Eastern Cambodia, where DHA-PPQ treatment failures have not been documented. pfmdr1 copy numbers for 360 of these isolates were previously reported (17). Parasitized whole-blood samples were collected from patients enrolled in completed parasite clearance rate (10, 18) and ongoing DHA-PPQ efficacy (ClinicalTrials.gov Identifier, NCT01736319) studies. All patients or their parents provided written informed consent. The pfmdr1 copy number in extracted DNA samples was quantified as previously described (17), and the P. falciparum lines 3D7 (copy number normalized to 1) and Dd2 (mean copy number ± SD, 2.05 ± 0.19; n = 29) were used as controls.
The distribution of 844 parasite isolates by province and year, in relation to Cambodia's recommended use of ACTs, is shown in Table 1. The prevalence of parasites with multiple pfmdr1 copies decreased significantly in Pursat during the 2008-to-2013 period (Cochran-Armitage trend test, P < 0.001), decreased in Preah Vihear during the 2011-to-2013 period (P = 0.065), and did not change in Ratanakiri in the 2010-to-2013 period (P = 0.869). The latter finding may be explained by the fact that Cambodia's remaining stocks of AS+MQ were still being distributed to Ratanakiri until mid-2013, which may have maintained the low prevalence of multiple pfmdr1 copies in this province. In 2013, the prevalence of parasites with multiple pfmdr1 copies was estimated to be 10%, 12%, and 3% in Pursat, Preah Vihear, and Ratanakiri, respectively. Together, these data suggest that MQ resistance is presently decreasing in Pursat and Preah Vihear and remains consistently low in Ratanakiri, likely due to the relatively low prevalence of artemisinin resistance in this province (9, 10). To further investigate these possibilities, we measured the in vitro susceptibility of 514/844 (60.9%) parasite isolates to MQ using a SYBR green I-based growth inhibition assay as previously described (17). The 50% inhibitory concentrations (IC50s) of at least four drugs were previously reported for 242 of these isolates (17). Parasites with multiple pfmdr1 copies had higher geometric mean MQ IC50s than those with one pfmdr1 copy in Pursat (28.2 versus 14.2 nM, P < 0.0001, Mann-Whitney test) and Preah Vihear (27.2 versus 18.4 nM, P = 0.0100) (Table 2). Taken together, these genotype and phenotype data suggest that AS+MQ may now be effective for the treatment of malaria in Pursat, Preah Vihear, and Ratanakiri.
TABLE 1.
pfmdr1 copy number in 844 P. falciparum clinical isolates, stratified by Cambodian province and year of collection
| Province | pfmdr1 copy no. | No. (%) of isolates with indicated copy no. where the recommended ACT(s)a was as indicated in: |
P valueb | |||||
|---|---|---|---|---|---|---|---|---|
| 2008 | 2009 | 2010 | 2011 | 2012 | 2013 | |||
| Pursat | AS+MQ, DHA-PPQ | DHA-PPQ | DHA-PPQ | DHA-PPQ | DHA-PPQ | DHA-PPQ | ||
| 1 | 19 (50) | 64 (70) | 40 (67) | 73 (66) | 44 (90) | 64 (90) | <0.001 | |
| 2 | 10 (26) | 9 (10) | 9 (15) | 11 (10) | 3 (6) | 6 (9) | ||
| 3 | 6 (16) | 13 (14) | 8 (13) | 14 (13) | 1 (2) | 1 (1) | ||
| 4 | 2 (5) | 4 (5) | 1 (2) | 7 (6) | 1 (2) | 0 (0) | ||
| 5 | 1 (3) | 1 (1) | 2 (3) | 5 (5) | 0 (0) | 0 (0) | ||
| Preah Vihear | AS+MQ | AS+MQ | AS+MQ | AS+MQ | AS+MQ, DHA-PPQ | AS+MQ, DHA-PPQ | ||
| 1 | NAc | NA | NA | 63 (78) | 67 (93) | 28 (88) | 0.065 | |
| 2 | NA | NA | NA | 10 (12) | 2 (3) | 2 (6) | ||
| 3 | NA | NA | NA | 2 (3) | 3 (4) | 2 (6) | ||
| 4 | NA | NA | NA | 1 (1) | 0 (0) | 0 (0) | ||
| 5 | NA | NA | NA | 5 (6) | 0 (0) | 0 (0) | ||
| Ratanakiri | AS+MQ | AS+MQ | AS+MQ | AS+MQ | AS+MQ, DHA-PPQ | AS+MQ, DHA-PPQ | ||
| 1 | NA | NA | 54 (100) | 91 (91) | 52 (100) | 33 (97) | 0.869 | |
| 2 | NA | NA | 0 (0) | 8 (8) | 0 (0) | 1 (3) | ||
| 3 | NA | NA | 0 (0) | 0 (0) | 0 (0) | 0 (0) | ||
| 4 | NA | NA | 0 (0) | 0 (0) | 0 (0) | 0 (0) | ||
| 5 | NA | NA | 0 (0) | 1 (1) | 0 (0) | 0 (0) | ||
DHA-PPQ, combination therapy of dihydroartemisinin and piperaquine; AS+MQ, combination therapy of artesunate and mefloquine.
P values were calculated using the Cochran-Armitage trend test (exact P values were estimated by Monte Carlo simulation using the coin R package; number of Monte Carlo replicates = 106). Boldface indicates a statistically significant difference.
NA, not available.
TABLE 2.
In vitro susceptibility of P. falciparum clinical isolates to eight antimalarial drugs in 2010 to 2013, stratified by Cambodian province and pfmdr1 copy number
| Province | Drug | Susceptibility of isolates with pfmdr1 copy number of: |
P valueb | |||
|---|---|---|---|---|---|---|
| One |
Multiple |
|||||
| No. of isolates | IC50 (nM) [GMa (range)] | No. of isolates | IC50 (nM) [GMa (range)] | |||
| Pursat | Chloroquine | 133 | 474.4 (103.9–1,084.3) | 52 | 294.1 (27.6–1,312.8) | <0.0001 |
| Mefloquine | 172 | 14.2 (1.7–56.4) | 53 | 28.2 (7.8–71.3) | <0.0001 | |
| Quinine | 174 | 273.2 (65.5–992.0) | 56 | 319.4 (95.5–825.4) | 0.0311 | |
| Piperaquine | 100 | 45.8 (5.6–185.2) | 40 | 24.0 (2.5–125.7) | 0.0002 | |
| Atovaquone | 64 | 0.5 (0.1–8.3) | 6 | 0.8 (0.2–13.8) | 0.7510 | |
| Pyronaridine | 64 | 5.4 (0.2–16.5) | 6 | 3.3 (0.7–10.3) | 0.1726 | |
| Artesunate | 148 | 2.9 (0.6–8.1) | 40 | 4.0 (1.2–8.7) | 0.0003 | |
| DHA | 176 | 2.7 (0.9–9.8) | 56 | 3.0 (0.7–7.9) | 0.1120 | |
| Preah Vihear | Chloroquine | 105 | 318.4 (16.7–978.5) | 17 | 339.4 (108.7–864.2) | 0.5518 |
| Mefloquine | 111 | 18.4 (3.6–46.6) | 18 | 27.2 (4.3–69.7) | 0.0100 | |
| Quinine | 113 | 200.8 (31.4–956.5) | 19 | 297.3 (61.8–933.1) | 0.0231 | |
| Piperaquine | 107 | 27.9 (4.9–130.1) | 19 | 25.8 (7.9–66.2) | 0.6976 | |
| Atovaquone | 27 | 0.6 (0.1–9.3) | 3 | 0.4 (0.2–1.2) | 0.3837 | |
| Pyronaridine | 27 | 4.5 (1.4–9.2) | 3 | 6.6 (3.2–12.8) | 0.4265 | |
| Artesunate | 111 | 2.4 (0.4–9.5) | 19 | 3.8 (1.4–10.0) | 0.0132 | |
| DHA | 111 | 2.1 (0.5–6.7) | 18 | 2.8 (0.9–7.2) | 0.0753 | |
| Ratanakiri | Chloroquine | 155 | 175.8 (11.8–933.1) | 5 | 149.6 (39.3–301.3) | 0.6912 |
| Mefloquine | 155 | 18.9 (4.2–64.7) | 5 | 16.9 (9.7–32.5) | 0.6554 | |
| Quinine | 160 | 131.8 (10.2–695.8) | 4 | 184.4 (72.8–351.3) | 0.3763 | |
| Piperaquine | 119 | 24.1 (5.5–67.1) | 5 | 36.2 (13.6–72.3) | 0.1567 | |
| Atovaquone | 28 | 0.4 (0.1–2.4) | 0 | NAc | NA | |
| Pyronaridine | 29 | 3.6 (1.2–9.8) | 0 | NA | NA | |
| Artesunate | 119 | 2.0 (0.9–5.6) | 4 | 2.7 (2.3–4.7) | 0.1619 | |
| DHA | 161 | 1.8 (0.5–5.8) | 4 | 1.7 (1.0–2.8) | 0.7787 | |
GM, geometric mean.
P values were calculated using the Mann-Whitney test. Boldface indicates a statistically significant difference.
NA, not available.
We also examined the association of pfmdr1 copy number and the in vitro susceptibility of parasite isolates to chloroquine (CQ; n = 467), quinine (QN; n = 526), PPQ (n = 390), atovaquone (ATV; n = 128), pyronaridine (PYN; n = 129), AS (n = 441), and DHA (n = 526). As for MQ, parasites carrying one pfmdr1 copy were significantly more susceptible to QN and AS in Pursat and Preah Vihear. As observed previously (14, 19–21), parasites harboring one pfmdr1 copy were significantly less susceptible to CQ and PPQ in Pursat (Table 2), indicating that the mechanisms of resistance to MQ and QN differ from those mediating resistance to CQ and PPQ in this province. In Ratanakiri, pfmdr1 copy number did not associate with IC50 for any of the antimalarial drugs tested, but the number of samples with multiple pfmdr1 copies were very few (≤5) in this province. These data indicate that pfmdr1 amplification is involved in decreasing parasite susceptibility to MQ and QN but not to CQ and PPQ in Cambodia. Whether the widespread decrease in the use of AS+MQ, increase in the use of DHA-PPQ, or both are driving the deamplification of pfmdr1 requires further investigation. As expected, parasite isolates are highly susceptible to atovaquone and pyronaridine, which have not been extensively used as ACT partner drugs in Cambodia (12, 22).
In summary, our data indicate that P. falciparum is regaining in vitro susceptibility to MQ in Pursat and, possibly, in Preah Vihear as well and suggest that AS+MQ may be an effective first-line treatment for P. falciparum malaria in Cambodian provinces where DHA-PPQ treatment failures have been documented—a possibility we are presently investigating. In other areas of Southeast Asia where DHA-PPQ treatment failures are suspected, early quantification of the pfmdr1 copy number and MQ IC50s of contemporary isolates is a useful approach to rapidly investigate the suitability of AS+MQ as an alternative ACT.
ACKNOWLEDGMENTS
This study was funded by the Intramural Research Program of the NIAID, NIH.
We thank our patients and their families for their gracious participation, the staff of the Pursat, Preah Vihear, and Ratanakiri provincial health departments for their dedicated collaboration, Michael Fay for statistics advice, and Chanaki Amaratunga, Jennifer Anderson, Robert Gwadz, and Thomas Wellems for their support of this work.
None of the authors has competing financial interests.
REFERENCES
- 1.Denis MB, Tsuyuoka R, Poravuth Y, Narann TS, Seila S, Lim C, Incardona S, Lim P, Sem R, Socheat D, Christophel EM, Ringwald P. 2006. Surveillance of the efficacy of artesunate and mefloquine combination for the treatment of uncomplicated falciparum malaria in Cambodia. Trop Med Int Health 11:1360–1366. doi: 10.1111/j.1365-3156.2006.01690.x. [DOI] [PubMed] [Google Scholar]
- 2.Denis MB, Tsuyuoka R, Lim P, Lindegardh N, Yi P, Top SN, Socheat D, Fandeur T, Annerberg A, Christophel EM, Ringwald P. 2006. Efficacy of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in northwest Cambodia. Trop Med Int Health 11:1800–1807. doi: 10.1111/j.1365-3156.2006.01739.x. [DOI] [PubMed] [Google Scholar]
- 3.Lim P, Alker AP, Khim N, Shah NK, Incardona S, Doung S, Yi P, Bouth DM, Bouchier C, Puijalon OM, Meshnick SR, Wongsrichanalai C, Fandeur T, Le Bras J, Ringwald P, Ariey F. 2009. Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia. Malaria J 8:11. doi: 10.1186/1475-2875-8-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Rogers WO, Sem R, Tero T, Chim P, Lim P, Muth S, Socheat D, Ariey F, Wongsrichanalai C. 2009. Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia. Malaria J 8:10. doi: 10.1186/1475-2875-8-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Alker AP, Lim P, Sem R, Shah NK, Yi P, Bouth DM, Tsuyuoka R, Maguire JD, Fandeur T, Ariey F, Wongsrichanalai C, Meshnick SR. 2007. Pfmdr1 and in vivo resistance to artesunate-mefloquine in falciparum malaria on the Cambodian-Thai border. Am J Trop Med Hyg 76:641–647. [PubMed] [Google Scholar]
- 6.Lim P, Wongsrichanalai C, Chim P, Khim N, Kim S, Chy S, Sem R, Nhem S, Yi P, Duong S, Bouth DM, Genton B, Beck HP, Gobert JG, Rogers WO, Coppee JY, Fandeur T, Mercereau-Puijalon O, Ringwald P, Le Bras J, Ariey F. 2010. Decreased in vitro susceptibility of Plasmodium falciparum isolates to artesunate, mefloquine, chloroquine, and quinine in Cambodia from 2001 to 2007. Antimicrob Agents Chemother 54:2135–2142. doi: 10.1128/AAC.01304-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Leang R, Barrette A, Bouth DM, Menard D, Abdur R, Duong S, Ringwald P. 2013. Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010. Antimicrob Agents Chemother 57:818–826. doi: 10.1128/AAC.00686-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Saunders DL, Vanachayangkul P, Lon C, U.S. Army Military Malaria Research Program, National Center for Parasitology, Entomology, and Malaria Control (CNM), Royal Cambodian Armed Forces. 2014. Dihydroartemisinin-piperaquine failure in Cambodia. N Engl J Med 371:484–485. doi: 10.1056/NEJMc1403007. [DOI] [PubMed] [Google Scholar]
- 9.Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, Kim S, Duru V, Bouchier C, Ma L, Lim P, Leang R, Duong S, Sreng S, Suon S, Chuor CM, Bout DM, Menard S, Rogers WO, Genton B, Fandeur T, Miotto O, Ringwald P, Le Bras J, Berry A, Barale JC, Fairhurst RM, Benoit-Vical F, Mercereau-Puijalon O, Menard D. 2014. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505:50–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Mao S, Sam B, Sopha C, Chuor CM, Nguon C, Sovannaroth S, Pukrittayakamee S, Jittamala P, Chotivanich K, Chutasmit K, Suchatsoonthorn C, Runcharoen R, Hien TT, Thuy-Nhien NT, Thanh NV, Phu NH, Htut Y, Han KT, Aye KH, Mokuolu OA, Olaosebikan RR, Folaranmi OO, Mayxay M, Khanthavong M, Hongvanthong B, Newton PN, Onyamboko MA, Fanello CI, Tshefu AK, Mishra N, Valecha N, Phyo AP, Nosten F, Yi P, Tripura R, Borrmann S, Bashraheil M, Peshu J, Faiz MA, Ghose A, Hossain MA, Samad R, Rahman MR, et al. 2014. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 371:411–423. doi: 10.1056/NEJMoa1314981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.White NJ, Pukrittayakamee S, Phyo AP, Rueangweerayut R, Nosten F, Jittamala P, Jeeyapant A, Jain JP, Lefevre G, Li R, Magnusson B, Diagana TT, Leong FJ. 2014. Spiroindolone KAE609 for falciparum and vivax malaria. N Engl J Med 371:403–410. doi: 10.1056/NEJMoa1315860. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Rueangweerayut R, Phyo AP, Uthaisin C, Poravuth Y, Binh TQ, Tinto H, Penali LK, Valecha N, Tien NT, Abdulla S, Borghini-Fuhrer I, Duparc S, Shin CS, Fleckenstein L, Pyronaridine-Artesunate Study Team. 2012. Pyronaridine-artesunate versus mefloquine plus artesunate for malaria. N Engl J Med 366:1298–1309. doi: 10.1056/NEJMoa1007125. [DOI] [PubMed] [Google Scholar]
- 13.Song J, Socheat D, Tan B, Seila S, Xu Y, Ou F, Sokunthea S, Sophorn L, Zhou C, Deng C, Wang Q, Li G. 2011. Randomized trials of artemisinin-piperaquine, dihydroartemisinin-piperaquine phosphate and artemether-lumefantrine for the treatment of multi-drug resistant falciparum malaria in Cambodia-Thailand border area. Malaria J 10:231. doi: 10.1186/1475-2875-10-231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Veiga MI, Ferreira PE, Malmberg M, Jornhagen L, Bjorkman A, Nosten F, Gil JP. 2012. pfmdr1 amplification is related to increased Plasmodium falciparum in vitro sensitivity to the bisquinoline piperaquine. Antimicrob Agents Chemother 56:3615–3619. doi: 10.1128/AAC.06350-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Eastman RT, Dharia NV, Winzeler EA, Fidock DA. 2011. Piperaquine resistance is associated with a copy number variation on chromosome 5 in drug-pressured Plasmodium falciparum parasites. Antimicrob Agents Chemother 55:3908–3916. doi: 10.1128/AAC.01793-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Imwong M, Dondorp AM, Nosten F, Yi P, Mungthin M, Hanchana S, Das D, Phyo AP, Lwin KM, Pukrittayakamee S, Lee SJ, Saisung S, Koecharoen K, Nguon C, Day NP, Socheat D, White NJ. 2010. Exploring the contribution of candidate genes to artemisinin resistance in Plasmodium falciparum. Antimicrob Agents Chemother 54:2886–2892. doi: 10.1128/AAC.00032-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Lim P, Dek D, Try V, Eastman RT, Chy S, Sreng S, Suon S, Mao S, Sopha C, Sam B, Ashley EA, Miotto O, Dondorp AM, White NJ, Su XZ, Char MC, Anderson JM, Amaratunga C, Menard D, Fairhurst RM. 2013. Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in western, northern, and eastern Cambodia, 2011-2012: association with molecular markers. Antimicrob Agents Chemother 57:5277–5283. doi: 10.1128/AAC.00687-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Amaratunga C, Sreng S, Suon S, Phelps ES, Stepniewska K, Lim P, Zhou C, Mao S, Anderson JM, Lindegardh N, Jiang H, Song J, Su XZ, White NJ, Dondorp AM, Anderson TJ, Fay MP, Mu J, Duong S, Fairhurst RM. 2012. Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. Lancet Infect Dis 12:851–858. doi: 10.1016/S1473-3099(12)70181-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Barnes DA, Foote SJ, Galatis D, Kemp DJ, Cowman AF. 1992. Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum. EMBO J 11:3067–3075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Duraisingh 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(Pt 1):1–7. doi: 10.1017/S0031182099006022. [DOI] [PubMed] [Google Scholar]
- 21.Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewski C, Kawamoto F, Miller RS, Meshnick SR. 2003. Resistance to antimalarials in Southeast Asia and genetic polymorphisms in pfmdr1. Antimicrob Agents Chemother 47:2418–2423. doi: 10.1128/AAC.47.8.2418-2423.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Hoyer S, Nguon S, Kim S, Habib N, Khim N, Sum S, Christophel EM, Bjorge S, Thomson A, Kheng S, Chea N, Yok S, Top S, Ros S, Sophal U, Thompson MM, Mellor S, Ariey F, Witkowski B, Yeang C, Yeung S, Duong S, Newman RD, Menard D. 2012. Focused screening and treatment (FSAT): a PCR-based strategy to detect malaria parasite carriers and contain drug resistant P. falciparum, Pailin, Cambodia. PLoS One 7:e45797. doi: 10.1371/journal.pone.0045797. [DOI] [PMC free article] [PubMed] [Google Scholar]