• 1

    White NJ, 2004. Antimalarial drug resistance. J Clin Invest 113 :1084–1092.

  • 2

    Ebisawa I, Muto T, Kameko S, Mitsui G, 1970. Response of Laotian malaria strains to chemotherapy. J Exp Med 40 :151–157.

  • 3

    Al-Tawil N, 1977. Response of falciparum malaria to a standard regimen of chloroquine in Vientiane, Lao People’s Democratic Republic. J Trop Med Hyg 80 :230–237.

    • Search Google Scholar
    • Export Citation
  • 4

    Al-Tawil N, 1978. Clearance of falciparum parasitemia with a single dose sulphadoxine-pyrimethamine in Vientiane, Laos. Southeast Asian J Trop Med Public Health 9 :409–413.

    • Search Google Scholar
    • Export Citation
  • 5

    Watson L, 1999. Lao Malaria Review. Unpublished European Union Malaria Project Report. Vientiane, Lao PDR: European Union.

  • 6

    Pillai DR, Labbe AC, Vanisaveth V, Hongvanthong B, Phompida S, Inthakon S, Zhong K, Kain KC, 2001. Plasmodium falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183 :789–795.

    • Search Google Scholar
    • Export Citation
  • 7

    Guthmann JP, Kasparian S, Phetsouvanh R, Nathan N, Garcia M, Phompida S, Brockman A, Gastellu M, Legros D, 2002. The efficacy of chloroquine for the treatment of acute uncomplicated Plasmodium falciparum malaria in the Lao People’s Democratic Republic. Ann Trop Med Parasitol 96 :553–557.

    • Search Google Scholar
    • Export Citation
  • 8

    Mayxay M, Newton PN, Khanthavong M, Pongvongsa T, Phetsouvanh R, Phompida S, Brockman A, White NJ, 2003. Chloroquine versus sulphadoxine-pyrimethamine for treatment of Plasmodium falciparum malaria in Savannakhet Province, Lao People’s Democratic Republic: An assessment of national antimalarial drug recommendations. Clin Infect Dis 37 :1021–1028.

    • Search Google Scholar
    • Export Citation
  • 9

    Mayxay M, Phetsouvanh R, Phompida S, Newton PN, Khanthavong M, Vannachone B, Brockman A, White NJ, 2003. A randomized comparison of oral chloroquine and sulphadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in Laos. Trans R Soc Trop Med Hyg 97 :343–344.

    • Search Google Scholar
    • Export Citation
  • 10

    Schwobel B, Jordan S, Vanisaveth V, Phetsouvanh R, Christophel EM, Phompida S, von Sonnenburg F, Jelinek T, 2003. Therapeutic efficacy of chloroquine plus sulphadoxine/pyrimethamine compared with monotherapy with either chloroquine or pyrimethamine/sulphadoxine in uncomplicated Plasmodium falciparum malaria in Laos. Trop Med Int Health 8 :19–24.

    • Search Google Scholar
    • Export Citation
  • 11

    Mayxay M, Khanthavong M, Lindegårdh N, Keola S, Barends M, Pongvongsa T, Yapom R, Annerberg A, Phompida S, Phetsouvanh R, White NJ, Newton PN, 2004. Randomized comparison of chloroquine plus sulphadoxine-pyrimethamine versus artesunate plus mefloquine versus artemether-lumefantrine in the treatment of uncomplicated falciparum malaria in the Lao PDR (Laos). Clin Infect Dis 39 :1139–1147.

    • Search Google Scholar
    • Export Citation
  • 12

    Stohrer JM, Dittrich S, Thongpaseuth V, Vanisaveth V, Phetsouvanh R, Phompida S, Monti F, Christophel EM, Lindegårdh N, Annerbergerg A, Jelinek T, 2004. Therapeutic efficacy of artemether-lumefantrine and artesunate-mefloquine for treatment of uncomplicated Plasmodium falciparum malaria in Luang Namtha Province, Lao People’s Democratic Republic. Trop Med Int Health 9 :1175–1183.

    • Search Google Scholar
    • Export Citation
  • 13

    Mayxay M, Thongpaseuth V, Khanthavong M, Lindegårdh N, Barend M, Keola S, Pongvongsa T, Phompida S, Phetsouvanh R, White NJ, Newton PN, 2006. An open, randomized comparison of artesunate plus mefloquine versus dihydroartemisinin-piperaquine for the treatment of uncomplicated falciparum malaria in the Lao People’s Democratic Republic (Laos). Trop Med Intl Hlth 11 :1157–1165.

    • Search Google Scholar
    • Export Citation
  • 14

    Dittrich 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 Intl Hlth 10 :1267–1270.

    • Search Google Scholar
    • Export Citation
  • 15

    Berens N, Schwoebel B, Jordan S, Vanisaveth V, Phetsouvanh R, Christophel EM, Phompida S, Jelinek T, 2003. Plasmodium falciparum: correlation of in vivo resistance to chloroquine and antifolates with genetic polymorphisms in isolates from the south of Lao PDR. Trop Med Int Health 8 :775–782.

    • Search Google Scholar
    • Export Citation
  • 16

    Druilhe P, Moreno A, Blanc C, Brasseur PH, Jacquier P, 2001. A colorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay. Am J Trop Med Hyg 64 :233–241.

    • Search Google Scholar
    • Export Citation
  • 17

    Moreno A, Brasseur P, Cuzin-Ouattara N, Blanc C, Druilhe P, 2001. Evaluation under field conditions of the colourimetric DELI-microtest for the assessment of Plasmodium falciparum drug resistance. Trans R Soc Trop Med Hyg 95 :100–103.

    • Search Google Scholar
    • Export Citation
  • 18

    Brockman A, Singlam S, Phiaphun L, Looareesuwan S, White NJ, Nosten F, 2004. Field evaluation of a novel colorimetric method-double-site enzyme-linked lactate dehydrogenase immunodetection assay-to determine drug susceptibilities of Plasmodium falciparum clinical isolates from northwestern Thailand. Antimicrob Agents Chemother 48 :1426–1429.

    • Search Google Scholar
    • Export Citation
  • 19

    Piper P, Lebras J, Wentworth L, Hunt-Cooke A, Houzé S, Chiodini P, Makler M, 1999. Immunocapture diagnostic assays for malaria using Plasmodium lactate dehydrogenase (pLDH). Am J Trop Med Hyg 60 :109–118.

    • Search Google Scholar
    • Export Citation
  • 20

    Anderson TJC, Nair S, Jacobzone C, Zavai A, Balkan S, 2003. Molecular assessment of drug resistance in Plasmodium falciparum from Bahr El Gazal Province, Sudan. Trop Med Intl Hlth 8 :1068–1073.

    • Search Google Scholar
    • Export Citation
  • 21

    Anderson 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.

    • Search Google Scholar
    • Export Citation
  • 22

    Brasseur P, Druilhe P, Kouamouo J, Brandicourt O, Danis M, Moyou SR, 1986. High level of sensitivity to chloroquine of 72 Plasmodium falciparum isolates from southern Cameroon in January 1985. Am J Trop Med Hyg 35 :711–716.

    • Search Google Scholar
    • Export Citation
  • 23

    Brasseur P, Kouamouo J, Brandicourt O, Moyou-Somo R, Druilhe P, 1988. Patterns of in vitro resistance to chloroquine, quinine, and mefloquine of Plasmodium falciparum in Cameroon, 1985–1986. Am J Trop Med Hyg 39 :166–172.

    • Search Google Scholar
    • Export Citation
  • 24

    Basco LK, Le Bras J, 1994. In vitro susceptibility of Cambodian isolates of Plasmodium falciparum to halofantrine, pyronaridine and artemisinin derivatives. Ann Trop Med Parasitol 88 :137–144.

    • Search Google Scholar
    • Export Citation
  • 25

    Ringwald P, Bickki J, Basco LK, 1996. In vitro activity of antimalarials against clinical isolates of Plasmodium falciparum in Yaounde. Am J Trop Med Hyg 55 :254–258.

    • Search Google Scholar
    • Export Citation
  • 26

    Price RN, Cassar C, Brockman A, Duraisingh M, van Vugt M, White NJ, Nosten F, Krishna S, 1999. The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob Agents Chemother 43 :2943–2949.

    • Search Google Scholar
    • Export Citation
  • 27

    Price RN, Uhlemann AC, Brockman A, McGready R, Ashley A, 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.

    • Search Google Scholar
    • Export Citation
  • 28

    Labbe AC, Bualombai P, Pillai DR, Zhong JY, Vanisaveth V, Hongvanthong B, Looareesuwan S, Kain KC, 2001. Molecular markers for chloroquine-resistant Plasmodium falciparum malaria in Thailand and Laos. Ann Trop Med Parasitol 95 :781–788.

    • Search Google Scholar
    • Export Citation
  • 29

    Thomas SM, Ndir O, Dieng T, Mboup S, Wypij D, Maguire JH, Wirth DF, 2002. In vitro chloroquine susceptibility and PCR analysis of pfCRT and pfMDR1 polymorphisms in Plasmodium falciparum isolates from Senegal. Am J Trop Med Hyg 66 :474–480.

    • Search Google Scholar
    • Export Citation
  • 30

    Brockman A, Price RN, van Vugt M, Heppner DG, Walsh D, Sookto P, Wimonwattrawatee T, Looareesuwan S, White NJ, Nosten F, 2000. Plasmodium falciparum antimalarial drug susceptibility on the north-western border of Thailand during five years of extensive use of artesunate-mefloquine. Trans R Soc Trop Med Hyg 94 :537–544.

    • Search Google Scholar
    • Export Citation
  • 31

    Lim P, Chim P, Sem R, Nemh S, Poravuth Y, Lim C, Seila S, Tsuyuoka R, Denis MB, Socheat D, Fandeur T, 2005. In vitro monitoring of Plasmodium falciparum susceptibility to artesunate, mefloquine, quinine, and chloroquine in Cambodia: 2001–2002. Acta Trop 93 :31–40.

    • Search Google Scholar
    • Export Citation
  • 32

    Huong NM, Hewitt S, Davis TME, Dao LD, Toan TQ, Kim TB, Hanh NT, Phuoung VN, Nhan DH, Cong LD, 2001. Resistance of Plasmodium falciparum to antimalarial drugs in a highly endemic area of southern Vietnam: A study in vivo and in vitro. Trans R Soc Trop Med Hyg 95 :325–329.

    • Search Google Scholar
    • Export Citation
  • 33

    Mayxay M, Chotivanich K, Pukrittayakamee S, Newton PN, Looareesuwan S, White NJ, 2001. The contribution of humoral immunity to the therapeutic response in falciparum malaria. Am J Trop Med Hyg 65 :918–923.

    • Search Google Scholar
    • Export Citation
  • 34

    Vythilingam I, Phetsouvanh R, Keokenchanh K, Yengmala V, Vanisaveth V, Phompida S, Hakim SL, 2003. The prevalence of Anopheles (Diptera: Culicidae) mosquitoes in Sekong Province, Lao PDR in relation to malaria transmission. Trop Med Int Health 8 :525–535.

    • Search Google Scholar
    • Export Citation
  • 35

    Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O, 2005. Resistance of Plasmodium falciparum field isolates to in vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 366 :1960–1963.

    • Search Google Scholar
    • Export Citation

 

 

 

 

IN VITRO ANTIMALARIAL DRUG SUSCEPTIBILITY AND PFCRT MUTATION AMONG FRESH PLASMODIUM FALCIPARUM ISOLATES FROM THE LAO PDR (LAOS)

View More View Less
  • 1 Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao PDR; Department of Post Graduate and Research, Faculty of Medical Science, National University of Laos, Lao PDR; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Shoklo Malaria Research Unit, Mae Sot, Tak Province, Thailand; Australian Centre for International Tropical Health and Nutrition, University of Queensland, Brisbane, Australia; Southwest Foundation for Biomedical Research, San Antonio, Texas; Savannakhet Malaria Station, Savannakhet Province, Lao PDR; Centre of Malariology, Parasitology and Entomology, Vientiane, Lao PDR; Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, United Kingdom.

Recent drug trials in Laos have shown high levels of Plasmodium falciparum resistance to chloroquine, but there are no published data on in vitro antimalarial drug susceptibility. We used the double-site enzyme-linked pLDH immunodetection (DELI) assay to estimate the in vitro antimalarial drug susceptibility of 108 fresh P. falciparum isolates from southern Laos. The geometric mean (95% confidence interval) 50% inhibitory concentration values (nmol/L) were 152.4 (123.8–187.6) for chloroquine, 679.8 (533.8–863.0) for quinine, 45.9 (37.9–55.7) for mefloquine, 5.0 (4.4–6.4) for artesunate, 6.3 (4.5–8.9) for dihydroartemisinin, and 59.1 (46.4–75.3) for lumefantrine. The proportion of isolates defined as resistant were 65%, 40%, and 8% for chloroquine, quinine, and mefloquine, respectively. Of 53 isolates genotyped for the pfcrt T76K chloroquine-resistance mutation, 48 (91%) were mutants. P. falciparum in Laos is multi-drug resistant; antimalarial immunity resulting from the use of ineffective chloroquine before 2005 probably contributes significantly to the therapeutic responses in clinical trials.

INTRODUCTION

Antimalarial drug resistance in Plasmodium falciparum is a very difficult problem for malaria control in most of the tropical world. This is particularly serious in Southeast Asia where P. falciparum has developed resistance to almost all antimalarial drugs available.1 In this situation, it is important that monitoring of antimalarial drug efficacies should be carried out regularly so that optimum treatment strategies can be implemented. In the Lao PDR (Laos), P. falciparum malaria remains an important cause of morbidity and mortality, particularly in the southern provinces. Laos and adjacent northeastern Cambodia have been considered to have generally more drug sensitive parasites than elsewhere in the region. Chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) resistance in P. falciparum were first reported in Laos in the late 1960s.25 Clinical trials of oral CQ and SP for the treatment of uncomplicated falciparum malaria at five different locations in Laos between 2000 and 2003 showed in vivo treatment failures of 35–80% for CQ and 18–35% for SP.610 In contrast, recent clinical trials have shown that artesunate + mefloquine, artemether-lumefantrine, and dihydroartemisinin-piperaquine are highly efficacious, with treatment failure rates of < 6%.1113 CQ and SP remained the first- and second-line nationally recommended treatments for uncomplicated falciparum malaria until 2005, when the Lao Government changed treatment policy to artemisinin-based combination therapy (ACT; artemether-lumefantrine). However, there is no published information on the in vitro antimalarial drug susceptibility of P. falciparum in Laos. We therefore measured the in vitro drug sensitivity of P. falciparum in Savannakhet Province and assessed the prevalence of the chloroquine resistance molecular marker pfcrt.14,15

MATERIALS AND METHODS

Study site and sample collection.

The study was conducted at Phalanxay District Clinic, Savannakhet Province (~605 km southeast of Vientiane) in parallel with clinical trials of antimalarial drugs between June 2003 and September 2004.11,13 Febrile patients presented with P. falciparum parasitemia ≥ 0.5% and without a history of antimalarial drug ingestion in the previous 2 weeks were included in the study provided that they (or their guardians) gave fully informed written consent. The study was approved by the Ethical Committee of the Faculty of Medical Sciences, National University of Laos, and the Oxford Tropical Medicine Research Ethics Committee, University of Oxford, UK.

Venous blood (3–5 mL) was collected in sterile heparinized tubes before patients received antimalarial drug treatment according to the protocols of the clinical trials.11,13 For patients not included in the trials, artesunate + mefloquine for 3 days or artesunate + doxycycline for 7 days were given. Blood samples were centrifuged immediately, the plasma and buffy coat were removed, and red blood cells were washed three times in RPMI 1640 (ICN; ICN Biomedicals, Costa Mesa, CA). Thin blood smears, stained with Field’s stain, were examined to determine parasite densities. The infected red blood cells were set up in the pre-dosed drug plates in complete RPMI with 10% heat-inactivated AB sera at a parasitemia of 0.5% parasitized erythrocytes and a hematocrit of 1.5%. If parasite densities exceeded 0.5%, samples were diluted with freshly washed uninfected red cells (group O) to obtain an initial parasitemia of 0.5%. Two hundred microliters of the suspension was distributed in each well in 96-well plates prepared with the antimalarial drugs (see below). Plates were incubated in a candle jar at 37°C for 48 hours before storing at −30°C for a maximum of 3 months and then at −80°C until the double-site enzyme-linked pLDH immunodetection (DELI) assay was performed.

Drug and plate preparation.

Chloroquine diphosphate and quinine citrate (Sigma Chemicals, St. Louis, MO), lumefantrine (Novartis Pharmacia, Basel, Switzerland), sodium artesunate and dihydroartemisinin (Walter Reed Army Institute of Research [WRAIR], Washington, DC; courtesy of Dr D. E. Kyle), and mefloquine hydrochloride (Hoffman-LaRoche, Basel, Switzerland) were used. Stock solutions of quinine, mefloquine, artesunate, and dihydroartemisinin were prepared in 70% ethanol (ETOH). Chloroquine stock solution was prepared in deionized water, and lumefantrine was dissolved in a 1:1:1 (wt/vol) mixture of ETOH, Triton-X (Sigma), and linoleic acid (Sigma). All drugs were dissolved initially at a concentration of 1 mg/mL and sterilized by ultrafiltration. Serial dilutions were made in complete RPMI medium. The solvent in the final concentrations had no significant effect on parasite growth compared with culture media. The drugs and their respective final concentration ranges in cell-medium mixture were as follows: chloroquine, 10.67–683.0 ng/mL (20.68–1,323.9 nmol/L); quinine, 42.34–2,710.0 ng/mL (117.33–7,509.0 nmol/L); mefloquine, 1.95–124.7 ng/mL (4.70–300.63 nmol/L); lumefantrine, 1.95–124.7 ng/mL (3.69–235.73 nmol/L); artesunate, 0.13–8.52 ng/mL (0.35–22.16 nmol/L); dihydroartemisinin (DHA), 0.13–8.25 ng/mL (0.47–29.96 nmol/L). All concentrations, including drug-free controls, were distributed in 25-μL aliquots in duplicate in 96-well tissue culture plates (Falcon; Becton Dickinson, Oxford, UK). The drug plates were made in bulk and stored at −80°C until use (for up to 3 months).

In vitro drug sensitivity assay.

The double-site enzyme-linked parasite lactate dehydrogenase (pLDH) immunodetection (DELI) assay was used to assess P. falciparum antimalarial drug susceptibility.1619 In brief, the culture plates were thawed and frozen three times to lyse the cells. One hundred microliters from each well was transferred into 96-well plates (Nunc-Immuno plate; Maxisorb; Nalge Nunc International, Roskilde, Denmark) pre-coated with a capture monoclonal antibody 17E419, which specifically recognizes the pLDH, and incubated for 1 hour at 37°C. After washing three times with phosphate-buffered saline (PBS)/0.5% bovine serum albumin (BSA Fraction V; Roche Diagnostics, Mannheim, Germany), a second biotinylated anti-pLDH monoclonal antibody 19G719 was added, and the plates were incubated for 1 hour at 37°C. After removal of unbound antibody by washing three times with PBS/0.5% BSA, the plates were incubated at room temperature for 30 minutes with a 1:10,000 solution of streptavidin-POD conjugate (Roche Diagnostics). After washing the plates three times with PBS/0.5% BSA, the plates were incubated for up to 20 minutes at room temperature with a peroxidase substrate solution, 3,3′,5,5′-tetramethyl-benzidine (KPL, Gaithersburg, MD). The reaction was stopped with 1 mol/L phosphoric acid, and color development was quantified immediately using a spectrophotometer (EL800 Universal Microplate Reader; Bio-Tek Instruments, Winooski, VT) to determine the optical density at 450 nm with a reference filter at 690 nm.

pfcrt genotyping.

To study the frequency of the pfcrt mutation, parasites from blood spot filter papers from 53 patients from the clinical trial in 2003 were genotyped for the amino acid 76 mutation in the pfcrt gene (pfcrt K76T) using a polymerase chain reaction (PCR)-restriction digest assay and fluorescent detection of products.20 Briefly, a 132-bp section of pfcrt was amplified by fluorescent end-labeled primers using semi-nested PCR. The fluorescent end-labeled products from the second PCR reaction were digested with Apo1 (New England Biolabs, Ipswich, MA). Finally the digested products were loaded in an ABI 3100 capillary sequencer (Applied Biosystems, Foster City, CA). The pfcrt resistant alleles were uncut, giving a peak at 132 bp, whereas wild-type alleles were cut giving a labeled fragment of 101 bp.

Data analysis.

Dose–response curves, the concentration of the drugs that resulted in a 50% inhibition of parasite growth (IC50 values), and coefficients of variation were calculated by fitting the data to an inhibitory E-max pharmacokinetic model using WINNONLIN Version 4.1 (Pharsight Corp., Mountain View, CA). To ensure data quality, we rejected all IC50 values with coefficients of variation [(SE × 100)/mean] of estimated IC50 values > 30% and those in which the pLDH production in control wells (parasites, no drug) was less than five times background (red blood cells only). One outlier was removed. For curves from highly resistant or sensitive samples, the range of dilutions was insufficiently high to obtain accurate measures of IC50. In these cases, the curves were “forced” by adding an extra data point (0 indicating no growth or 1 indicating 100% growth) at the next or previous doubling concentration, respectively. This procedure results in conservative IC50 values while allowing us to retain data from interesting parasite isolates with unusually high IC50 values.21 The cut-off IC50 values for in vitro resistance to chloroquine, quinine, and mefloquine were defined as > 100, 800, and 108 nmol/L, respectively.2227 IC50 cut-off values for artesunate, DHA, and lumefantrine resistance have not yet been established.

RESULTS

A total of 108 fresh P. falciparum isolates were obtained from symptomatic patients with uncomplicated falciparum malaria (48 women and 60 men). The mean (95% confidence interval [CI]) age (years) of the patients was 13.3 (11.4–15.3; range, 2–50 years), and 81% of them were children ≤ 15 years. The geometric mean (95% CI) parasitemia (parasites per microliter) at admission was 55,719 (44,720–69,438).

Of all isolates, 75 (69%), 68 (63%), 44 (41%), 70 (65%), 65 (60%), and 60 (56%) produced interpretable data by DELI assay for artesunate, chloroquine, DHA, lumefantrine, mefloquine, and quinine, respectively (Table 1). The proportions of isolates resistant to chloroquine, quinine, and mefloquine were 65%, 40%, and 8%, respectively. The geometric mean (95% CI) IC50s (nmol/L) of isolates defined as resistant were 246 (203–298), 1,613 (1,240–2,098), and 146 (105–203) for chloroquine, quinine, and mefloquine, respectively. The geometric mean (95% CI) IC50s (nmol/L) of isolates defined as sensitive were 63 (54–75), 381 (312–465), and 42 (34.54–50.36) for chloroquine, quinine, and mefloquine, respectively. Because the IC50 cut-off values for artesunate, DHA, and lumefantrine resistance have not been established, the percentage of isolates resistant to these drugs cannot be calculated. There were no significant correlations between chloroquine in vitro IC50 values and those for artesunate and DHA (r = 0.10; P = 0.41 and r = 0.13; P = 0.46, respectively). There were significant positive correlations between IC50 values among the other drugs (P ≤ 0.02; Table 2).

Forty (37%) patients were eligible and consented to the clinical trials of antimalarial drugs with 42-day follow-up. Of these, one patient who was treated with artemether-lumefantrine had treatment failure (recurrence of parasitemia at day 21) as confirmed by parasite genotyping.11 The anti-malarial drug IC50s (nmol/L) of the parasite isolate from this patient were 1.45, 639, 1.65, 23.1, 20.3, and 1,340 for artesunate, chloroquine, DHA, lumefantrine, mefloquine, and quinine, respectively.

Fifty-three isolates were available for genotyping of pfcrt K76T, and 20 had chloroquine in vitro drug susceptibility results. Of all samples genotyped, 48 (91%) were found to be pfcrt T76 mutant types. One of the 20 isolates was wild-type at pfcrt and had a chloroquine IC50 of 167 nmol/L.

DISCUSSION

We studied the in vitro susceptibility of P. falciparum to antimalarial drugs to provide information on antimalarial drug resistance patterns at a clinical trial site in Laos and as a necessary baseline to assess future trends in resistance. Recent clinical trials have shown poor efficacy of chloroquine in different areas of Laos, and in the province where this in vitro study was conducted, the chloroquine treatment failure rate after 42-day follow-up was 36%.8 This clinical finding is supported by the high level of in vitro chloroquine resistance and that 95% of the isolates were found to carry pfcrt mutant types. Studies in the north and the far south of Laos have also shown a high prevalence (64–100%) of pfcrt K76T mutants.6,14,15,28 Using the same DELI assay technique as used here, the mean IC50 of chloroquine in our study was comparable with that found in Thailand and Senegal18,29 but higher than that reported in Burkina Faso (Table 3).17 Comparisons between in vitro susceptibility studies, especially those using different methods, are difficult, and there are no other published data from Southeast Asia using the DELI technique. Additional limitations of the study include the absence of quality control data to allow more accurate comparisons between studies, because we could not maintain cultures of control P. falciparum strains in the field. The relatively low recovery of IC50 information, particularly for DHA, probably result from the stringent criteria we used for acceptance of data (coefficient of variation < 30%).

Therapeutic responses to chloroquine are generally better in Laos than in adjacent Thailand, Cambodia, and Viet Nam, but the parasites causing these infections seem to be as resistant or more resistant as those in the adjoining countries.3032 The significantly lower risk of treatment failure among adults compared with children in P. falciparum clinical trials in Laos strongly suggests that patient immunity plays a considerable role in the response to chloroquine and other antimalarial drugs there.33 In higher transmission settings, such a discrepancy between in vitro and in vivo susceptibility is a usual observation, but transmission intensities in southern Laos are low and not significantly higher than other parts of the region.34 In adjacent Thailand, chloroquine was abandoned nearly 30 years ago because of very high treatment failure rates. The acquisition of significant immunity in Laos has resulted presumably from the widespread use of chloroquine and SP as antimalarials until last year and the consequently greater individual exposure to P. falciparum parasites. The mapping of the geographical distribution of molecular markers of resistance suggests that the falciparum parasites in Savannakhet Province are more sensitive to chloroquine and folate drugs than elsewhere in the country (Mayxay and others, unpublished data). Falciparum parasites in northern and southern Laos are therefore likely to be less sensitive in vitro than those described here.

In Laos, quinine is an alternative for the treatment of both severe and uncomplicated malaria. It has been used alone extensively in villages by village health volunteers and in hospitals. However, no studies have been carried out to assess the in vivo or in vitro efficacy of this drug. In this study, 40% of the parasite isolates showed resistance to quinine based on a cut-off of 800 nmol/L. This supports the recommendation that quinine should not be used alone in Laos, but combined with doxycycline. The mean IC50 of quinine in this study was similar to that given in a recent report from the western border of Thailand using the same DELI technique.18

Mefloquine is very rarely used in Laos, except by tourists as prophylaxis, because it is expensive and rarely available. Consistent with this observation, the proportion of mefloquine resistant P. falciparum found in this study was low. The few mefloquine-resistant parasites found in southern Laos may reflect intrinsically resistant parasites in this area or flow of parasites from southern Vietnam or Cambodia/Thailand. The P. falciparum IC50s for artesunate and DHA were higher in Laos than in Thailand (Table 3), although these differences are not clinically significant. The geometric (SD) in vitro artesunate IC50 of P. falciparum in Cambodia was, at 1.25 (2.8) nmol/L,35 slightly lower than those collected in Laos. However, because the 3H-hypoxanthine technique was used, these data cannot be reliably compared with those from Laos. The IC50 of lumefantrine in this study was similar to that from northwestern Thailand using the same DELI technique.18

In conclusion, in vitro data suggest that high levels of resistance to chloroquine and quinine but not to mefloquine, lumefantrine, and the artemisinin derivatives have developed in this area of Laos. More information on the in vivo response to quinine therapy in Laos is needed. Regular monitoring of antimalarial drug efficacy, with mapping of the distribution of molecular makers of drug resistance, needs to be carried out in Laos to monitor the pattern of antimalarial drug resistance and assist in determining the rational antimalarial policy.

Table 1

In vitro sensitivity of Lao P. falciparum isolates, as measured by DELI assay

Geometric mean IC50 (nmol/L)
DrugNMean95% CIRange
Artesunate755.024.44–6.430.84–21.9
Chloroquine68152.4123.8–187.620.0–1,479
Dihydroartemisinin446.294.47–8.900.69–23.2
Lumefantrine7059.0746.4–75.34.4–251
Mefloquine6545.9337.9–55.74.7–223
Quinine60680533–863100–7,058
Table 2

Correlation of the in vitro responses of Lao P. falciparum isolates among antimalarial drugs

Drug 1Drug 2N*rP
* N, number of paired results.
† Correlation coefficient (R) was calculated from linear regression analysis of logarithmic IC50.
‡ Probability (P) refers to the significance level of the test.
ArtesunateChloroquine620.410.10
DHA420.84< 0.001
Lumefantrine610.72< 0.001
Mefloquine630.69< 0.001
Quinine500.53< 0.001
ChloroquineDHA320.130.46
Lumefantrine550.340.01
Mefloquine560.320.02
Quinine490.450.001
DihydroartemisininLumefantrine350.56< 0.001
Mefloquine340.500.003
Quinine280.600.001
LumefantrineMefloquine570.74< 0.001
Quinine500.55< 0.001
MefloquineQuinine460.59< 0.001
Table 3

Summary of P. falciparum in vitro antimalarial drug susceptibility using the DELI technique

CountryNo. of isolatesChloroquineQuinineMefloquineArtesunateDHALumefantrineReference
DHA, dihydroartemisinin.
* Range.
# Mean (range).
Data are shown as geometric mean (95% CI) in nmol/L unless indicated.
Laos108152.4 (123.8–187.6)678.7 (533.8–863.0)45.9 (37.8–55.7)5.0 (4.4–6.4)6.3 (4.5–8.9)59.1 (46.3–75.3)Current study
Thailand80168.2 (155.5–182.0)636.8 (560.0–724.0)54.7 (46.0–65.3)1.6 (1.4–1.8)2.4 (2.0–2.9)49.1 (42.3–51.2)Brockman and others 200418
Senegal44123–941*Thomas and others 200229
Burkina Faso6257.0 (7.0–335.0)#429.0 (22.0–1,725.0)#21.0 (0.1–72.0)Moreno and others 200117

*

Address correspondence to Paul N. Newton, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR. E-mail: paul@tropmedres.ac

Authors’ addresses: Mayfong Mayxay, Nicholas J. White, and Paul N. Newton, Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Mahosot Road, Vientiane, Lao PDR, Telephone: 85621-250752, Fax: 85621-242168, E-mail: paul@tropmedres.ac. Mayfong Mayxay, Department of Medicine, Faculty of Medical Science, National University of Laos, Lao PDR, Telephone: 85621-250752, Fax: 85621-242168, E-mail: mmayxay@yahoo.com. Marion Barends, Alan Brockman, and Anchalee Jaidee, Shoklo Malaria Research Unit, Mae Sot, Tak Province, Thailand. Marion Barends, Nicholas J. White, and Paul N. Newton, Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, UK. Marion Barends, Alan Brockman, and Nicholas J. White, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 420/6 Rajvithi Road, Bangkok, 10400, Thailand, Telephone: +66 23549172; Fax: +66 23549169; E-mail: nickw@tropmedres.ac. Alan Brockman, Australian Centre of International Tropical Health and Nutrition, University of Queensland, Brisbane, Australia. Shalini Nair, Dan Sudimack, and Tim Anderson, Southwest Foundation for Biomedical Research. San Antonio, Texas, Telephone: +210-2589596. Tiengkham Pongvongsa, Savannakhet Provincial Malaria Station, Savannakhet Province, Lao PDR. Samlane Phompida, and Rattanaxay Phetsouvanh, Centre of Malariology, Parasitology and Entomology, Vientiane, Lao PDR, Telephone: 85621-214040, Fax: 85621-218131.

Acknowledgments: The authors thank all the patients involved in this study and Drs. Maniphone Khanthavong, Vonthalom Thongpraseuth, and Siamphay Keola, Manisack Phommasansack, Bounpone Phimphalat, Pitta Sengkeomahavong, Bounmy Syphachanh, Ammala Phomsimone, Vilayphone, Chanthala Vilaihong, and all medical assistants and nurses in Phalanxay District Hospital for their technical help. We are very grateful to Dr. M. Makler for supplying the monoclonal antibody, to Dr. D. E. Kyle for supplying artesunate and DHA, to Drs. Pranom Phongmany and Odai Xaysitthideth and Phomma for their valuable advice and for the support of the Minister of Health, His Excellency Dr. Ponmek Dalaloy, the Directors of Hygiene and Preventive Medicine, Drs. Douangchanh Keo-Asa and Bounlay Phommasack, and the Director of Mahosot Hospital, Professor Chanpheng Thammavong. Special thanks to Dr. Francois Nosten for reviewing the manuscript.

Financial support: This study was supported by The Wellcome Trust of Great Britain. T.J.C.A., S.N., and .D.S are supported by NIH RO1 AI48071. The molecular work at the Southwest Foundation for Biomedical Research was conducted in facilities constructed with support from Research Facilities Improvement Program Grant C06 RR013556 from the National Center for Research Resources, National Institutes of Health.

REFERENCES

  • 1

    White NJ, 2004. Antimalarial drug resistance. J Clin Invest 113 :1084–1092.

  • 2

    Ebisawa I, Muto T, Kameko S, Mitsui G, 1970. Response of Laotian malaria strains to chemotherapy. J Exp Med 40 :151–157.

  • 3

    Al-Tawil N, 1977. Response of falciparum malaria to a standard regimen of chloroquine in Vientiane, Lao People’s Democratic Republic. J Trop Med Hyg 80 :230–237.

    • Search Google Scholar
    • Export Citation
  • 4

    Al-Tawil N, 1978. Clearance of falciparum parasitemia with a single dose sulphadoxine-pyrimethamine in Vientiane, Laos. Southeast Asian J Trop Med Public Health 9 :409–413.

    • Search Google Scholar
    • Export Citation
  • 5

    Watson L, 1999. Lao Malaria Review. Unpublished European Union Malaria Project Report. Vientiane, Lao PDR: European Union.

  • 6

    Pillai DR, Labbe AC, Vanisaveth V, Hongvanthong B, Phompida S, Inthakon S, Zhong K, Kain KC, 2001. Plasmodium falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183 :789–795.

    • Search Google Scholar
    • Export Citation
  • 7

    Guthmann JP, Kasparian S, Phetsouvanh R, Nathan N, Garcia M, Phompida S, Brockman A, Gastellu M, Legros D, 2002. The efficacy of chloroquine for the treatment of acute uncomplicated Plasmodium falciparum malaria in the Lao People’s Democratic Republic. Ann Trop Med Parasitol 96 :553–557.

    • Search Google Scholar
    • Export Citation
  • 8

    Mayxay M, Newton PN, Khanthavong M, Pongvongsa T, Phetsouvanh R, Phompida S, Brockman A, White NJ, 2003. Chloroquine versus sulphadoxine-pyrimethamine for treatment of Plasmodium falciparum malaria in Savannakhet Province, Lao People’s Democratic Republic: An assessment of national antimalarial drug recommendations. Clin Infect Dis 37 :1021–1028.

    • Search Google Scholar
    • Export Citation
  • 9

    Mayxay M, Phetsouvanh R, Phompida S, Newton PN, Khanthavong M, Vannachone B, Brockman A, White NJ, 2003. A randomized comparison of oral chloroquine and sulphadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in Laos. Trans R Soc Trop Med Hyg 97 :343–344.

    • Search Google Scholar
    • Export Citation
  • 10

    Schwobel B, Jordan S, Vanisaveth V, Phetsouvanh R, Christophel EM, Phompida S, von Sonnenburg F, Jelinek T, 2003. Therapeutic efficacy of chloroquine plus sulphadoxine/pyrimethamine compared with monotherapy with either chloroquine or pyrimethamine/sulphadoxine in uncomplicated Plasmodium falciparum malaria in Laos. Trop Med Int Health 8 :19–24.

    • Search Google Scholar
    • Export Citation
  • 11

    Mayxay M, Khanthavong M, Lindegårdh N, Keola S, Barends M, Pongvongsa T, Yapom R, Annerberg A, Phompida S, Phetsouvanh R, White NJ, Newton PN, 2004. Randomized comparison of chloroquine plus sulphadoxine-pyrimethamine versus artesunate plus mefloquine versus artemether-lumefantrine in the treatment of uncomplicated falciparum malaria in the Lao PDR (Laos). Clin Infect Dis 39 :1139–1147.

    • Search Google Scholar
    • Export Citation
  • 12

    Stohrer JM, Dittrich S, Thongpaseuth V, Vanisaveth V, Phetsouvanh R, Phompida S, Monti F, Christophel EM, Lindegårdh N, Annerbergerg A, Jelinek T, 2004. Therapeutic efficacy of artemether-lumefantrine and artesunate-mefloquine for treatment of uncomplicated Plasmodium falciparum malaria in Luang Namtha Province, Lao People’s Democratic Republic. Trop Med Int Health 9 :1175–1183.

    • Search Google Scholar
    • Export Citation
  • 13

    Mayxay M, Thongpaseuth V, Khanthavong M, Lindegårdh N, Barend M, Keola S, Pongvongsa T, Phompida S, Phetsouvanh R, White NJ, Newton PN, 2006. An open, randomized comparison of artesunate plus mefloquine versus dihydroartemisinin-piperaquine for the treatment of uncomplicated falciparum malaria in the Lao People’s Democratic Republic (Laos). Trop Med Intl Hlth 11 :1157–1165.

    • Search Google Scholar
    • Export Citation
  • 14

    Dittrich 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 Intl Hlth 10 :1267–1270.

    • Search Google Scholar
    • Export Citation
  • 15

    Berens N, Schwoebel B, Jordan S, Vanisaveth V, Phetsouvanh R, Christophel EM, Phompida S, Jelinek T, 2003. Plasmodium falciparum: correlation of in vivo resistance to chloroquine and antifolates with genetic polymorphisms in isolates from the south of Lao PDR. Trop Med Int Health 8 :775–782.

    • Search Google Scholar
    • Export Citation
  • 16

    Druilhe P, Moreno A, Blanc C, Brasseur PH, Jacquier P, 2001. A colorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay. Am J Trop Med Hyg 64 :233–241.

    • Search Google Scholar
    • Export Citation
  • 17

    Moreno A, Brasseur P, Cuzin-Ouattara N, Blanc C, Druilhe P, 2001. Evaluation under field conditions of the colourimetric DELI-microtest for the assessment of Plasmodium falciparum drug resistance. Trans R Soc Trop Med Hyg 95 :100–103.

    • Search Google Scholar
    • Export Citation
  • 18

    Brockman A, Singlam S, Phiaphun L, Looareesuwan S, White NJ, Nosten F, 2004. Field evaluation of a novel colorimetric method-double-site enzyme-linked lactate dehydrogenase immunodetection assay-to determine drug susceptibilities of Plasmodium falciparum clinical isolates from northwestern Thailand. Antimicrob Agents Chemother 48 :1426–1429.

    • Search Google Scholar
    • Export Citation
  • 19

    Piper P, Lebras J, Wentworth L, Hunt-Cooke A, Houzé S, Chiodini P, Makler M, 1999. Immunocapture diagnostic assays for malaria using Plasmodium lactate dehydrogenase (pLDH). Am J Trop Med Hyg 60 :109–118.

    • Search Google Scholar
    • Export Citation
  • 20

    Anderson TJC, Nair S, Jacobzone C, Zavai A, Balkan S, 2003. Molecular assessment of drug resistance in Plasmodium falciparum from Bahr El Gazal Province, Sudan. Trop Med Intl Hlth 8 :1068–1073.

    • Search Google Scholar
    • Export Citation
  • 21

    Anderson 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.

    • Search Google Scholar
    • Export Citation
  • 22

    Brasseur P, Druilhe P, Kouamouo J, Brandicourt O, Danis M, Moyou SR, 1986. High level of sensitivity to chloroquine of 72 Plasmodium falciparum isolates from southern Cameroon in January 1985. Am J Trop Med Hyg 35 :711–716.

    • Search Google Scholar
    • Export Citation
  • 23

    Brasseur P, Kouamouo J, Brandicourt O, Moyou-Somo R, Druilhe P, 1988. Patterns of in vitro resistance to chloroquine, quinine, and mefloquine of Plasmodium falciparum in Cameroon, 1985–1986. Am J Trop Med Hyg 39 :166–172.

    • Search Google Scholar
    • Export Citation
  • 24

    Basco LK, Le Bras J, 1994. In vitro susceptibility of Cambodian isolates of Plasmodium falciparum to halofantrine, pyronaridine and artemisinin derivatives. Ann Trop Med Parasitol 88 :137–144.

    • Search Google Scholar
    • Export Citation
  • 25

    Ringwald P, Bickki J, Basco LK, 1996. In vitro activity of antimalarials against clinical isolates of Plasmodium falciparum in Yaounde. Am J Trop Med Hyg 55 :254–258.

    • Search Google Scholar
    • Export Citation
  • 26

    Price RN, Cassar C, Brockman A, Duraisingh M, van Vugt M, White NJ, Nosten F, Krishna S, 1999. The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob Agents Chemother 43 :2943–2949.

    • Search Google Scholar
    • Export Citation
  • 27

    Price RN, Uhlemann AC, Brockman A, McGready R, Ashley A, 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.

    • Search Google Scholar
    • Export Citation
  • 28

    Labbe AC, Bualombai P, Pillai DR, Zhong JY, Vanisaveth V, Hongvanthong B, Looareesuwan S, Kain KC, 2001. Molecular markers for chloroquine-resistant Plasmodium falciparum malaria in Thailand and Laos. Ann Trop Med Parasitol 95 :781–788.

    • Search Google Scholar
    • Export Citation
  • 29

    Thomas SM, Ndir O, Dieng T, Mboup S, Wypij D, Maguire JH, Wirth DF, 2002. In vitro chloroquine susceptibility and PCR analysis of pfCRT and pfMDR1 polymorphisms in Plasmodium falciparum isolates from Senegal. Am J Trop Med Hyg 66 :474–480.

    • Search Google Scholar
    • Export Citation
  • 30

    Brockman A, Price RN, van Vugt M, Heppner DG, Walsh D, Sookto P, Wimonwattrawatee T, Looareesuwan S, White NJ, Nosten F, 2000. Plasmodium falciparum antimalarial drug susceptibility on the north-western border of Thailand during five years of extensive use of artesunate-mefloquine. Trans R Soc Trop Med Hyg 94 :537–544.

    • Search Google Scholar
    • Export Citation
  • 31

    Lim P, Chim P, Sem R, Nemh S, Poravuth Y, Lim C, Seila S, Tsuyuoka R, Denis MB, Socheat D, Fandeur T, 2005. In vitro monitoring of Plasmodium falciparum susceptibility to artesunate, mefloquine, quinine, and chloroquine in Cambodia: 2001–2002. Acta Trop 93 :31–40.

    • Search Google Scholar
    • Export Citation
  • 32

    Huong NM, Hewitt S, Davis TME, Dao LD, Toan TQ, Kim TB, Hanh NT, Phuoung VN, Nhan DH, Cong LD, 2001. Resistance of Plasmodium falciparum to antimalarial drugs in a highly endemic area of southern Vietnam: A study in vivo and in vitro. Trans R Soc Trop Med Hyg 95 :325–329.

    • Search Google Scholar
    • Export Citation
  • 33

    Mayxay M, Chotivanich K, Pukrittayakamee S, Newton PN, Looareesuwan S, White NJ, 2001. The contribution of humoral immunity to the therapeutic response in falciparum malaria. Am J Trop Med Hyg 65 :918–923.

    • Search Google Scholar
    • Export Citation
  • 34

    Vythilingam I, Phetsouvanh R, Keokenchanh K, Yengmala V, Vanisaveth V, Phompida S, Hakim SL, 2003. The prevalence of Anopheles (Diptera: Culicidae) mosquitoes in Sekong Province, Lao PDR in relation to malaria transmission. Trop Med Int Health 8 :525–535.

    • Search Google Scholar
    • Export Citation
  • 35

    Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O, 2005. Resistance of Plasmodium falciparum field isolates to in vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 366 :1960–1963.

    • Search Google Scholar
    • Export Citation
Save