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Am. J. Trop. Med. Hyg., 70(4), 2004, pp. 395-397
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

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IN VITRO EFFICACY OF ANTIMALARIAL DRUGS AGAINST PLASMODIUM VIVAX ON THE WESTERN BORDER OF THAILAND

KESINEE CHOTIVANICH, RACHANEE UDOMSANGPETCH, WIRONGRONG CHIERAKUL, PAUL N. NEWTON, RONATRAI RUANGVEERAYUTH, SASITHON PUKRITTAYAKAMEE, SORNCHAI LOOAREESUWAN, AND NICHOLAS J. WHITE
Faculty of Tropical Medicine, and Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok. Thailand; Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, United Kingdom; Mae Sot Hospital, Tak, Thailand


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The susceptibility of 20 isolates of Plasmodium vivax on the Thailand-Myanmar border to seven antimalarial drugs was evaluated using the schizont maturation inhibition technique. The geometric mean 50% inhibition concentration (IC50) values were quinine = 308 ng/mL, amodiaquine =14 ng/mL, chloroquine =50 ng/mL, mefloquine = 127 ng/mL, sulfadoxine/pyrimethamine (80:1) = 800/10 ng/mL, pyrimethamine = 8 ng/mL, and artesunate = 0.5 ng/mL. Compared with P. falciparum in this area, P. vivax was more sensitive to chloroquine and artesunate, equally sensitive to quinine, and more resistant to mefloquine.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plamodium vivax is the major cause of human malaria in parts of Central and South America and Asia. On the western border of Thailand, the incidence of P. vivax has recently been reported as 20 per 1,000 population per year, (which is similar to that of P. falciparum1). Although severe complications of vivax malaria are rarely observed, P. vivax causes multiple relapses and therefore considerable morbidity. Vivax malaria in pregnancy is associated with low birth weight.2,3 Choroquine has been the drug of choice for P. vivax malaria for many years. In recent years, resistance to chloroquine in P. vivax has been demonstrated conclusively in vivo in Papua New Guinea,4 different regions of Indonesia,5 and more recently in central America.6 There have also been reports from India7 and Myanmar.8 The susceptibility of P. vivax to antimalarial drugs has not been monitored in vitro because of difficulties in cultivating P. vivax. These difficulties are related to differences in nutrient requirements compared with P. falciparum; the conditions in standard malaria culture medium induced premature rupture of the infected red blood cells, and the limited provision of young red bloods cells or reticulocytes limits P. vivax invasion. Plasmodium vivax invades red blood cells only in the first 14 days after their emergence from the bone marrow.9 In this study, the susceptibility of P. vivax in vitro to different antimalrial drugs has been assessed in short-term culture in an area where multi-drug resistant P. falciparum is prevalent, and where several different antimalarial drugs are available and used for treatment, to set a baseline for P. vivax sensitivity in vitro.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study site. The studies were carried out in Mae Sot Hospital between 1996 and 2001 in Tak Province, NW Thailand. This study was part of clinical studies reviewed and approved by the Ethical and Scientific Review Committee, Ministry of Public Health, Royal Government of Thailand.

Parasites. Two milliliters of blood were collected in heparinized tubes from patients attending the out-patient clinic or admitted to Mae Sot Hospital. Thick and thin blood films were prepared using standard procedures. Parasite species, morphology, and parasitemia were assessed by microscopic examination. Plasma concentrations of quinine and mefloquine were measured by a semi-quantitative dipstick technique.10 Only blood samples from patients with single-species P. vivax malaria with synchronized ring-stage infected red blood cells, parasitemia more than four parasitized red blood cells per 1,000 red blood cells, and no antimalarial drug detected in plasma (and no history of antimalarial drug treatment) were chosen for this study.

Antimalarial drug sensitivity assay. Plasmodium vivax-infected blood was centrifuged at 2,000 rpm at 4°C for five minutes. After the plasma and buffy coat were discarded, the packed red blood cells were washed three times in RPMI 1640 medium, and resuspended to make a 3% cell suspension in the complete medium. A 50-µL cell suspension was added to triplicate wells of a predosed antimalarial microtiter plate. The following drugs were assessed; quinine, amodiaquine, chloroquine, mefloquine, sulfadoxine-pyrimethamine, and pyrimethamine (World Health Organization, Manila, The Philippines). The concentration (two-fold dilutions) ranges for each drug used on the microtiter plates are shown in Table 1Go. Artesunate was prepared in duplicate wells in 96-well plates as described previously11 200 µL of red blood cell suspension was added into each well. Each drug concentration was tested in duplicate. After adding the erythrocytes, the lid was placed over the plate and the plate was then shaken gently to dissolve the drug. The samples were incubated at 37°C in an atmosphere of 5% CO2 for 40–44 hours depending on the stage of the parasite before culturing. At the end of the incubation, thick and thin blood films were prepared from the samples in each well. Wells without drugs were included as controls.


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TABLE 1
Drug concentration range tested
 
Evaluation efficacy of antimalarial drugs. Thick and thin blood films were fixed with methanol, and stained with Field’s stain, and examined under the microscope. The number of schizonts containing more than eight nuclei was counted per 3,000 red blood cells. The percentage of schizonts in the drug wells was compared with the number of schizonts counted in control samples.

Data analysis. Results of parasite schizogony (for 3,000 red blood cells) after 40–48 hours culture at each drug concentration were fitted to a sigmoid curve by using WinNonlinTM computer software version 3.1 (Pharsight Corporation, Mountain View, CA) to determine the 50% inhibitory concentration (IC50) for schizont development. Correlations were assessed by the method of Spearman.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Twenty isolates were obtained. Growth of P. vivax in the absence of antimalarials drugs was observed in all control wells. The parasites developed from ring stage to mature schizonts containing >= 8 nuclei within a mean ± SD 40 ± 3 hours after cultivation. All 20 isolates used in the experiment developed to complete mature schizonts and could be evaluated for drug responsiveness. Tests were run in parallel for the seven antimalarial drugs. Parasitemia before testing varied between 0.4% and 1.9% (geometric mean ± 95% confidence interval [CI] = 0.5 ± 0.2%). After 40 hours of incubation, the number of schizonts in the control wells of the seven parallel series varied between 5 and 19 per 3,000 red blood cells (geometric mean ± 95% CI = 8.6 ± 0.2). The overall coefficient of variation for the counting of the individual isolates’ control well, was 8%, indicating reproducibility of assessment of schizont maturation.

The drug concentrations that gave 50% inhibition of schizont maturation (IC50) were derived from the sigmoid plots and are summarized in Table 2Go. For most (19 of 20) of the isolates, complete inhibition of schizont maturation occurred in the well containing 64 pmol/well of quinine. This concentration is considered to represent susceptibility to quinine for P. falciparum.12 Complete inhibition of schizont maturation (all isolates) occurred in the well containing 16 pmol of chloroquine/well. For mefloquine, complete inhibition of schizont maturation was achieved for all isolates in the well containing 128 pmol/well. Complete inhibition of schizont maturation occurred in the well containing 8 pmol/well for amodiaquine (18 isolates), 10,000 pmol/well for sulfadoxine-pyrimethamine (18 isolates) and pyrimethamine (all isolates), and 8.4 ng/ml for artesunate (all isolates). No significant correlations were found between the susceptibilities to the different antimalarials. As shown in Table 2Go, these data were compared with earlier studies from the same region on P. falciparum in which schizont maturation was used,13 and with contemporary studies using 3H-hypoxanthine uptake inhibition.11


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TABLE 2
Susceptibility of Plasmodium vivax on the western border of Thailand to antimalarial drugs*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The susceptibility to antimalarial drugs of P. vivax was assessed by a rapid, simple, and readily field-adapted method. This method is similar to that used for P. falciparum, although many laboratories now use the radioisotopic hypoxanthine incorporation assay to assess drug susceptibility in this parasite. The two methods give similar results. When compared with P. falciparum isolates from the same area (assessed by both the hypoxanthine incorporation assay and earlier studies from the same area using schizont maturation assessment), P. vivax was as susceptible to quinine, more sensitive to chloroquine, and more resistant to mefloquine. The P. vivax IC50 of artesunate was approximately three times lower than that of P. falciparum, suggesting that artesunate is a potential treatment for chloroquine-resistant P. vivax malaria. This confirms in vivo observations; P. vivax is highly susceptible to the artemisinin derivatives.14 The IC50 values of sulfadoxine-pyrimethamine and pyrimethamine from this study could not be compared with assessments of susceptibility in P. falciparum because of the differences in methodology and the conditions in the culture medium. Folic acid is a direct competitive antagonist of antifolate activity15 and p-aminobenzoic acid (PABA) is a direct competitor for sulfonamides.16 Therefore the susceptibility of P. falciparum to sulfadoxine-pyrimethamine and pyrimethamine are usually conducted in culture medium deficient in folic acid. We have found that P. vivax needs considerably more folic acid for complete schizont maturation than P. falciparum and will not grow in folate-deficient media. Thus, the IC50 values of sulfadoxine-pyrimethamine and pyrimethamine in this study might not reflect in vivo susceptibility, but could be useful for sequential monitoring of the evolution of antifolate resistance.

In Thailand, P. vivax remains sensitive to chloroquine in vitro and in vivo. The low IC50s obtained in this study which in P. falciparum would correspond to chloroquine sensitivity17 confirm extensive clinical series indicating that there is no significant resistance in this region. This contrasts with P. falciparum, which is highly chloroquine resistant in this area. The IC50 values observed for chloroquine are consistent with previous reports.18 Whether the relatively high IC50 value for mefloquine reflect: acquired resistance, as in P. falciparum in this region, or constitutionally reduced susceptibility will require assessment in other areas where mefloquine is not used. These preliminary data suggest that there are similar in vitro "break-points" for antimalarial drug resistance in the two malaria species. These results provide a current base line of sensitivity of P. vivax to antimalarial drugs. The antimalarial drug susceptibility of P. vivax needs to be monitored continuously to anticipate the emergence of resistance.

Received January 21, 2003. Accepted for publication September 17, 2003.

Acknowledgments: We thank the staff and nurses of Mae Sot Hospital for their help and Dr. Jetsumon Prachumsri for her valuable comments and suggestions.

Financial support: This work was a part of the Wellcome Trust-Mahidol University Oxford Tropical Medicine Research Programme funded by the Wellcome Trust of Great Britain.

Authors’ addresses: Kesinee Chotivanich, Wirongrong Chierakul, Sasithon Pukrittayakamee, and Sornchai Looareesuwan, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road. Bangkok, Thailand. Rachanee Udomsangpetch, Department of Pathobiology, Faculty of Science, Mahidol University. Bangkok, Thailand. Ronatrai Ruangveerayuth, Mae Sot Hospital, Tak, Thailand. Paul N. Newton and Nicholas J White, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand, and Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford OX3 7LJ, United Kingdom, Correspondence: Prof. N. J. White, Telephone: 66-2-354-9172, Fax: 66-2-351-9169, E-mail: fnnjw{at}diamond.mahidol.ac.th.


REFERENCES
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