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Keywords:

  • Plasmodium vivax;
  • artemisinin;
  • chloroquine;
  • Vietnam;
  • drug-resistance;
  • recrudescence

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Chloroquine-resistant Plasmodium vivax has not yet occurred in Vietnam. The efficacy of artemisinin for P. vivax was not established. We conducted a double-blind randomized study involving 240 inpatients with P. vivax malaria who received artemisinin (40 mg/kg over 3 days) plus placebo chloroquine (Art) or chloroquine (25 mg/kg over 3 days) plus placebo artemisinin (Chl). Patients were followed up with weekly blood smears for 28 days. In each group 113 cases were analysed. All patients recovered rapidly. The median (range) parasite clearance time of regimen Art was 24 h (8–72) and of Chl 24 h (8–64; P = 0.3). Parasites reappeared in two cases in each group on day 14, in eight cases in each group (7%) on day 16 and in 25 (23%) and 18 (16%) cases, respectively, at the end of 4-week follow-up (P = 0.3). The population parasite clearance curve followed a mono-exponential decline. The parasite reduction ratio per 48 h reproduction cycle was 2.3 × 104 for both regimens. We conclude that artemisinin and chloroquine are equally effective in the treatment of P. vivax infections in Vietnam. Reappearance of parasites before day 16 (7%) suggests the emergence of chloroquine resistance. Three days of artemisinin monotherapy does not prevent recrudescence.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

In South-east Asia morbidity attributable to Plasmodium vivax prevails while P. falciparum gradually comes under control by applying artemisinin drugs with other control measures such as insecticide-treated bed nets (Phan et al. 1999; Nosten et al. 2000). This trend was also observed in Binh Thuan, a mountainous province in the south of Vietnam (unpublished information, Binh Thuan Provincial Malaria Station).

Chloroquine is the first-line drug for treatment of P. vivax in most areas of the world (World Health Organization 1997), although artemisinin and derivatives are also used in various regimens and they appear to be effective (Nguyen et al. 1993). It was not yet known whether chloroquine-resistant P. vivax is prevalent in Vietnam. Artemisinin drugs are very active against P. vivax but the efficacy has not been firmly established. Therefore, this study aimed to establish and compare the efficacy of artemisinin and chloroquine in the treatment of blood stage infections of P. vivax in Vietnam. Based on our own experience in pharmacodynamic modelling of the time course of P. falciparum, a 3-day regimen of artemisinin should be effective. Longer regimens were considered to conflict with good patient compliance.

Patient selection and treatment

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

From September 1997 to January 2000, subjects aged 15 years or older with a microscopically confirmed P. vivax (asexual stage) infection, presenting at eight primary health care posts in Binh Thuan province were enrolled. Exclusion criteria included P. falciparum infection, pregnancy, lactation, complications of malaria, underlying disease, inability to take oral medication, known allergy to the study drugs and intake of antimalarial agents in the previous week. After informed consent the treatment regimen was allocated by a computer-generated randomization code with 120 patients per treatment arm, to detect a difference between approximately 30% and 15% response rates (α = 0.05 and β = 0.20). Patients were admitted to the health facility and treated with artemisinin 20 mg/kg on t = 0 (day 1) followed by 10 mg/kg o.d. on days 2 and 3 plus chloroquine placebo (Art) or with chloroquine 15 mg/kg on t = 0 followed by 5 mg/kg o.d. on days 2 and 3 plus artemisinin placebo (Chl). Artemisinin 250 mg capsules, chloroquine diphosphate 250 mg (150 mg base) tablets and placebos were obtained from Mekophar Company, Ho Chi Minh City, Vietnam. Intake of all medication was supervised. Vomiting within 1 h after intake of the trial medication required re-treatment with a full dose. The contents of the medication was analysed in 12 remaining capsules of artemisinin and 10 tablets of choloroquine (Laboratories of ACE Pharmaceuticals BV, Zeewolde, the Netherlands). Primaquine base 0.25 mg/kg daily was administered for 5 days, starting after day 28.

Patient follow-up

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

A full blood count was performed before patient inclusion and on day 3. The parasitaemia was counted every 8 h until three negative smears had been obtained. Blood smears were repeated on days 8, 15, 22 and 29. The parasite density was expressed as the number of parasites per microlitre of blood, calculated from the ratio with the white blood cell count in the thick smear. Gametocytes were recorded but not enumerated and the slides of t = 0 h or t = 8 h were taken as baseline. All blood smears were retained and reviewed by an experienced technician at the Department of Parasitology of Cho Ray Hospital in Ho Chi Minh City. His results were taken as gold standard.

Fever and parasite clearance times were defined as the time from t = 0 to the first of three consecutive normal temperature readings (<37.0 °C axillary) or negative blood smears, respectively. Clinical and parasitological outcome were assessed separately. Clinical cure was defined as a disappearance of symptoms and of parasites. Parasitological cure was assessed according to WHO criteria for assessment of chloroquine efficacy in therapeutic trials of P. falciparum: RI-parasite clearance by day 8 but recrudescence before day 15 (early RI) or from days 15 to 29 (late RI); RII: initial reduction of parasite count (>75%) without clearance at day 8; RIII: no or less response; radical cure: clinical cure without recrudescence (World Health Organization 1973).

Population dynamics of the parasite count and statistics

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

The time course of parasitaemia was fitted to non-linear mixed effect population models (maximum likelihood method) using the statistical package S-Plus (v. 4.5, Math Soft Inc., Seattle, WA, USA) as described previously (de Vries et al. 2000). The variables treatment regimen, age, sex and body weight were allowed to affect the model. The models were compared by the Bayesian Information Criterion (BIC) (Schwarz 1978). The best model was used to estimate (restricted maximum likelihood method) the elimination rate constant (k), the initial parasite count [P(0)] and the time to achieve a reduction of the initial parasitaemia by 50%, (PC50) or by 90%, (PC90). P(0), k and the duration of treatment converge in the parasitaemia at the end of drug exposure P(term). For artemisinin P(term) can be projected assuming that exposure lasted until 12 h after the last dose. The long half time of chloroquine precludes this projection.

Patient data were analysed with the statistical package SPSS (v. 10.05, SPSS Inc. Chicago, IL, USA). Outcome was expressed as the proportion of patients who completed 28 days of follow-up. Extreme case scenarios such as re-allocating patients who were lost to follow-up was used as an alternative of intention to treat analysis. Patients who, upon review of the blood slides, appeared to be erroneously included were excluded from the analysis. Contingency tables and chi-square tests with continuity correction were applied to categorical variables. Numerical variables were tested for normality and Student's t-test or non-parametric tests were applied for comparison. Parasite clearance and recrudescence were analysed with survival analysis (Kaplan–Meier plots and the Cox proportional hazard model). Statistical significance was accepted when P < 0.05.

Ethical approval

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Informed consent was obtained from all patients who participated in the study before randomization. The study protocol was approved by the scientific board and medical ethics committee of Cho Ray Hospital, Ho Chi Minh City.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

A total of 240 patients were recruited. Fourteen erroneously included patients with P. falciparum infection at baseline were excluded from the analysis. Of the remaining 226 patients, seven (five Art, two Chl) were lost to follow-up because they had left the study area. The patient characteristics are shown in Table 1. Except for a slight difference in sex ratio, there were no significant differences between the two groups. On day 14, parasites had recurred in two patients in each group, on day 16 this had increased to eight patients in each group (7%).

Table 1.  Characteristics of patients with P. vivax infections treated with artemisinin or chloroquine
VariableTreatment regimenP-value
Artemisinin (Art)Chloroquine (Chl)
  • Chi-square test;

  • † 

    † Wilcoxon rank sum test;

  • ‡ 

    ‡ P(0): baseline parasite count;

  • § 

    § 95% CI: 95% confidence interval;

  • ¶ 

    Student's t-test;

  • **

    **95% CI of the difference.

Number of patients included113113 
Number lost to follow-up52 
Sex (F/M)26/8713/1000.03*
Median age (years)27270.4
 Range15–6415–59 
Median height (cm)1601610.4
 Range111–175135–175 
Median weight (kg)50490.7
 Range31–6239–69 
Geometric mean P(0) (per μl)4057.133536.700.1
95% CI§3248.44–5067.662872.19–4355.23−660, 1732**

The remaining artemisinin capsules contained 249.0 mg (SD: ±0.7, range: 237.5–252.5), the chloroquine tablets 257.0 mg (±1.8, 237.5–262.5). Tolerance to the study medication was good. Some mild effects of the medication were recorded but these could also be attributed to malaria. Vomiting of trial medication did not occur.

The radical cure and recrudescence rates are shown in Table 2. The radical cure rates were 77% (83 of 108) and 84% (93 of 111). In the best case scenario the cure rate was 78% (88 of 113) in group Art and 84% (95 of 113) in group Chl; in the worst case scenario the ratios were 73% (83 of 113) and 82% (93 of 113), respectively. Although the cure rate in group Chl was slightly higher than in group Art, the differences did not reach statistical significance. All cases with recrudescence were treated successfully according to routine practice, usually with a second course of chloroquine plus primaquine 15 mg base o.d. for 14 days without follow-up after parasite clearance.

Table 2.  Treatment response of P. vivax infections in the south of Vietnam, treated with artemisinin or chloroquine
VariableTreatment regimenP-value; odds ratio
Artemisinin (Art)Chloroquine (Chl)
  1. * Chi-square test; † Wilcoxon rank sum test; ‡ 95% CI: 95% confidence interval; § 95% CI of the difference.

Number of patients included113113 
Radical cure (n)83 (77%)93 (84%)0.3; odds ratio (95% CI):   0.64 (0.31, 1.33)*
Recrudescence (n)25 (23%)18 (16%) 
 Early (<14 days)22 
 Late (>14 days)2316 
Lost to follow-up (n)52 
Median FCT (h)16160.2
 Range0–480–96 
Median PCT (h)24240.3
 Range8, 728, 64 
Mean K (per hour)0.17470.17280.772
 95% CI0.1661, 0.18321629, 1826−0.015, 0.011§
Geometric mean PC50 (h)14.614.70.749
 95% CI13.6, 15.713.6, 15.9−1.58, 1.55§
Geometric mean PC90 (h)24.224.30.749
 95% CI22.7, 25.822.7, 26.1−2.33, 2.17§

Both regimens offered rapid parasite clearance and defervescence. There was no RII or RIII response. The proportional cumulative parasite clearance and recurrence rates are shown in Figure 1. There was no significant difference between the two regimens (P = 0.1729; odds ratio 0.65; 95% CI 0.35–1.21). Initial parasitaemia was a weak but significant predictor of parasite recurrence (P = 0.010; odds ratio 1.0001; 95% CI 1.0000–1.0001). Age, sex and body weight did not affect the hazard function.

image

Figure 1. Kaplan–Meier curve showing the proportion of patients with parasites of P. vivax treated with artemisinin (solid line) or chloroquine (broken line). The crosses indicate sensored data.

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Figure 2 shows the geometric mean of the observed parasite counts and the population elimination model. A mono-exponential elimination model yielded the best fit, described by the mathematical function: P(t) = P(0) e–k.t in which P(t) is the parasitaemia as a function of time. There was no significant increase in the parasitaemia or any lag time before the parasitaemia starts to decline. Entering the variables age, body weight, sex and treatment regimen did not improve the fit. The mean of the estimates is shown in Table 2. The overall mean (95% CI) value of k was 0.175 (0.167–0.180) per hour, corresponding to a parasite reduction ratio of 2.3 × 104 per 48 h development cycle. The estimated model parameters [including P(term)] were not different for the outcome groups radical cure vs. recrudescence.

image

Figure 2. Time course of P. vivax geometric parasitaemia for patients treated with artemisinin (solid line, crosses) and chloroquine (broken line, round dots) and population estimate for all patients (unbroken line, no markers). Negative blood smears were entered as the value 2.5 parasites/μl. The error bars indicate the 95% CI of the geometric mean.

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Gametocytes were detected at baseline in 91% of patients in group Art and 94% of patients in group Chl. In group Art 0%, 5.3%, 7.4% and 4.2% of patients were still positive on days 8, 15, 22 and 28, respectively. In group Chl this was the case for 0%, 4.2%, 6.3% and 3.2%, respectively. Most of the patients who remained positive for gametocytes also had a recurrence of asexual parasites. There was no significant difference between the two groups regarding gametocytes. Among the remainders who had no gametocytes at baseline, none of these had gametocytes during follow-up, including one patient in group Art with recurrence of asexual parasites on day 8.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

This is the first double-blind randomized trial which shows that RI chloroquine resistance of P. vivax is prevalent in the south of Vietnam. The definition of drug resistance can be based on clinical criteria or on blood or plasma concentrations. The study medication contained the correct doses and the bioavailability of chloroquine is so predictable that it was regarded sufficient to supervise intake of medication. The absence of blood concentrations was not considered to affect the conclusions of this study.

The radical cure rate of 84% in this study is not sufficient although RII or RIII responses were not observed. In endemic areas recurrence of parasites may be a recrudescence or a re-infection or, in the case of P. vivax, a relapse. Genotyping could have assisted in distinguishing the two but this was not available. However, some assumptions can be made regarding early reappearance of parasites. Given an incubation time of 12–17 days and the low transmission rates of malaria in Binh Thuan, it is unlikely that first attacks of second infections were frequent. Therefore the early recurrence of parasites in this study is interpreted as a true recrudescence and for chloroquine this means RI resistance. Relapse of P. vivax malaria is also possible but day 16 is regarded as the minimum time after which relapses can occur (Baird et al. 1997). Under this assumption six more cases in each group would be classified as early reappearance and the rate of recrudescence in this study would be 7%, still higher than in a study in Thailand where no early recurrence was found during 4 weeks after treatment with chloroquine (Luxemburger et al. 1999). When parasites reappear after more than 2 weeks, re-infection, relapse and true recrudescence cannot be distinguished. Nevertheless, because of the long half time of chloroquine, all recurrences within the first 4 weeks may point at some level of resistance. After a standard treatment course the total blood concentration of chloroquine plus its metabolite desethylchloroquine of chloroquine exceeds the minimum effective concentration of a sensitive strain for at least 7 days and often much longer (Rombo et al. 1986; de Vries et al. 1994; Tan-ariya et al. 1995). However, late parasite recurrence was also probably not a relapse in most cases because in nearby Thailand almost all relapses occurred more than 4 weeks after chloroquine administration (Tan-ariya et al. 1995; Luxemburger et al. 1999).

Chloroquine-resistant P. vivax was first seen in the Asia Pacific region (Rieckmann et al. 1989; Schuurkamp et al. 1989, 1992; Murphy et al. 1993). Later reports appeared from other regions in Asia, Ethiopia and also from Guyana (Myat-Phone-Kyaw et al. 1993; Marlar et al. 1995; Phillips et al. 1996; Tulu et al. 1996). In Vietnam P. vivax was still considered sensitive to chloroquine but there is no routine surveillance of treatment response of P. vivax. A recent study in Vietnam did not reveal any chloroquine resistance, but because of the small sample size (29 patients) this does not contradict our findings (Taylor et al. 2000). In addition, chloroquine resistance may be masked because it is often combined with primaquine which also has some effect on asexual blood stages of P. vivax (Pukrittayakamee et al. 1994; Baird et al. 1995). It is tempting to speculate that the emergence of resistant parasites is rather recent, because early recrudescence was infrequent and RII and RIII response were not (yet) observed.

In Thailand the response of P. vivax to chloroquine is good, although in vitro studies indicate declining sensitivity (Tan-ariya et al. 1995; Looareesuwan et al. 1999; Luxemburger et al. 1999). In contrast, chloroquine resistance, including RII and RIII response, was reported from Myanmar (Marlar et al. 1995). One explanation for these differences may be that in Thailand ‘drug pressure’ is less than that in surrounding countries where chloroquine is still used for P. falciparum. Whether this also explains the variable response rate in Indonesia and the Philippines remains unclear (Baird et al. 1991, 1996a,b; Fryauff et al. 1997, 1998).

For artemisinin drugs the situation is different. After treatment with artesunate, parasite clearance of P. vivax and P. falciparum was comparable (Batty et al. 1998). Artesunate monotherapy for 5 days induced rapid recovery and parasite clearance, but in 63% of the patients the parasite reappeared before day 28 (Pukrittayakamee et al. 2000). Three days of artesunate combined with primaquine for 14 days showed no recurrence of parasites. There is no information on the intrinsic activity of artemisinin on P. vivax, in terms of minimum effective concentrations, but the parasite reduction rate of P. vivax in this study is greater than that of P. falciparum (de Vries et al. 2000). This and the lower P(0) explain why in this study the recrudescence rate was similar to that of P. falciparum after 5 days of artemisinin monotherapy (Giao et al. 2001). As stated for chloroquine, it is likely that the recurrences after artemisinin are true recrudescences.

The responses to artemisinin and chloroquine in this study were comparable although the cure rate was slightly lower for artemisinin. Interestingly, the parasite clearance rates were also similar. This is in contrast to P. falciparum. In Gambian children with severe malaria the effect of chloroquine was somewhat slower than of the artemisinin derivative artemether (White et al. 1992).

As a 3-day course of artemisinin is not superior to a standard course of chloroquine it is prudent to study how the efficacy of chloroquine can be improved. The combination of chloroquine and primaquine was shown to be very effective in areas with a high degree of chloroquine-resistant P. vivax (Baird et al. 1995; Obaldia et al. 1997). Recently the Vietnamese policy advised to start a 5-day course of primaquine together with chloroquine on t = 0. The risk of G6PD deficiencies is considered to preclude higher dosages or longer treatment courses of primaquine without further testing. Combinations of chloroquine with artemisinin or artemisinin with concomitant primaquine seem attractive options for further study.

In conclusion, artemisinin and chloroquine both offer rapid parasite clearance and recovery of P. vivax infections in Vietnam, but neither can prevent recrudescence; chloroquine probably because of resistance and artemisinin because of its intrinsic characteristics. Chloroquine resistance should be monitored and combinations of drugs should be studied further.

Footnotes
  • *

    Part of these results were presented as a poster at the conference ‘New challenges in tropical medicine and parasitology’ 18–22 September 2000, Oxford, UK, Abstract No. 179.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

This study was part of a programme supported by the Ministry of Development Cooperation of the Netherlands. Mekophar Pharmaceutical Company, Ho Chi Minh City, Vietnam, provided the study medication and placebos and ACE Pharmaceuticals B.V., Zeewolde, the Netherlands, analysed the contents of the study medication.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Patient selection and treatment
  6. Patient follow-up
  7. Population dynamics of the parasite count and statistics
  8. Ethical approval
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References
  • Baird JK, Basri H, Purnomoet al. (1991) Resistance to chloroquine by Plasmodium vivax in Irian Jaya, Indonesia. American Journal of Tropical Medicine and Hygiene 44, 547552.
  • Baird JK, Basri H, Subianto B et al. (1995) Treatment of chloroquine-resistant Plasmodium vivax with chloroquine and primaquine or halofantrine. Journal of Infectious Diseases 171, 16781682.
  • Baird JK, Caneta-Miguel E, Masbar S et al. (1996a) Survey of resistance to chloroquine of falciparum and vivax malaria in Palawan, The Philippines. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 413414.
  • Baird JK, Sustriayu NM, Basri H et al. (1996b) Survey of resistance to chloroquine by Plasmodium vivax in Indonesia. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 409411.
  • Baird JK, Leksana B, Masbar S et al. (1997) Diagnosis of resistance to chloroquine by Plasmodium vivax: timing of recurrence and whole blood chloroquine levels. American Journal of Tropical Medicine and Hygiene 56, 621626.
  • Batty KT, Le AT, Ilett KF et al. (1998) A pharmacokinetic and pharmacodynamic study of artesunate for vivax malaria. American Journal of Tropical Medicine and Hygiene 59, 823827.
  • Fryauff DJ, Baird JK, Candradikusuma D et al. (1997) Survey of in vivo sensitivity to chloroquine by Plasmodium falciparum and P. vivax in Lombok, Indonesia. American Journal of Tropical Medicine and Hygiene 56, 241244.
  • Fryauff DJ, Tuti S, Mardi A et al. (1998) Chloroquine-resistant Plasmodium vivax in transmigration settlements of West Kalimantan, Indonesia. American Journal of Tropical Medicine and Hygiene 59, 513518.
  • Giao PT, Binh TQ, Kager PA et al. (2001) Artemisinin for treatment of uncomplicated falciparum malaria: is there a place for monotherapy? American Journal of Tropical Medicine and Hygiene 65, 690695.
  • Kyaw MP, Oo M, Lwin M et al. (1993) Emergence of chloroquine-resistant Plasmodium vivax in Myanmar (Burma). Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 687687.
  • Looareesuwan S, Wilairatana P, Krudsood S et al. (1999) Chloroquine sensitivity of Plasmodium vivax in Thailand. Annals of Tropical Medicine and Parasitology 93, 225230.
  • Luxemburger C, Van VM, Jonathan S et al. (1999) Treatment of vivax malaria on the western border of Thailand. Transactions of the Royal Society of Tropical Medicine and Hygiene 93, 433438.
  • Marlar T, Myat PK, Aye YS et al. (1995) Development of resistance to chloroquine by Plasmodium vivax in Myanmar. Transactions of the Royal Society of Tropical Medicine and Hygiene 89, 307308.
  • Murphy GS, Basri H, Purnomoet al. (1993) Vivax malaria resistant to treatment and prophylaxis with chloroquine. Lancet 341, 96100.
  • Nguyen DS, Dao BH, Nguyen PD et al. (1993) Treatment of malaria in Vietnam with oral artemisinin. American Journal of Tropical Medicine and Hygiene 48, 398402.
  • Nosten F, Van Vugt M, Price R et al. (2000) Effects of artesunate-mefloquine combination on incidence of Plasmodium falciparum malaria and mefloquine resistance in western Thailand: a prospective study. Lancet 356, 297302.
  • Obaldia N, Rossan RN, Cooper RD et al. (1997) WR 238605, chloroquine, and their combinations as blood schizonticides against a chloroquine-resistant strain of Plasmodium vivax in Aotus monkeys. American Journal of Tropical Medicine and Hygiene 56, 508510.
  • Phan VT, Tuy TQ & Hung LX (1999) Malaria epidemics in Vietnam (1955–97). Mekong Malaria Forum 1, (issue 3) 1216.
  • Phillips EJ, Keystone JS & Kain KC (1996) Failure of combined chloroquine and high-dose primaquine therapy for Plasmodium vivax malaria acquired in Guyana, South America. Clinical Infectious Diseases 23, 11711173.
  • Pukrittayakamee S, Vanijanonta S, Chantra A, Clemens R & White NJ (1994) Blood stage antimalarial efficacy of primaquine in Plasmodium vivax malaria. Journal of Infectious Diseases 169, 932935.
  • Pukrittayakamee S, Chantra A, Simpson JA et al. (2000) Therapeutic responses to different antimalarial drugs in vivax malaria. Antimicrobial Agents and Chemotherapy 44, 16801685.
  • Rieckmann KH, Davis DR & Hutton DC (1989) Plasmodium vivax resistance to chloroquine? Lancet 2, 11831184.
  • Rombo L, Bjorkman A, Sego E & Ericsson O (1986) Whole blood concentrations of chloroquine and desethylchloroquine during and after treatment of adult patients infected with Plasmodium vivax, P. ovale or P. malariae. Transactions of the Royal Society of Tropical Medicine and Hygiene 80, 763766.
  • Schuurkamp GJ, Spicer PE, Kereu RK & Bulungol PK (1989) A mixed infection of vivax and falciparum malaria apparently resistant to 4-aminoquinoline: a case report. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 607608.
  • Schuurkamp GJ, Spicer PE, Kereu RK, Bulungol PK & Rieckmann KH (1992) Chloroquine-resistant Plasmodium vivax in Papua New Guinea. Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 121122.
  • Schwarz G (1978) Estimating the dimension of a model. Annals of Statistics 6, 461464.
  • Tan-ariya P, Na-Bangchang K, Tin T et al. (1995) Clinical response and susceptibility in vitro of Plasmodium vivax to the standard regimen of chloroquine in Thailand. Transactions of the Royal Society of Tropical Medicine and Hygiene 89, 426429.
  • Taylor WR, Doan HN, Nguyen DT et al. (2000) Assessing drug sensitivity of Plasmodium vivax to halofantrine or choroquine in southern, central Vietnam using an extended 28-day in vivo test and polymerase chain reaction genotyping. American Journal of Tropical Medicine and Hygiene 62, 693697.
  • Tulu AN, Webber RH, Schellenberg JA & Bradley DJ (1996) Failure of chloroquine treatment for malaria in the highlands of Ethiopia. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 556557.
  • De Vries PJ, Oosterhuis B & Van Boxtel CJ (1994) Single-dose pharmacokinetics of chloroquine and its main metabolite in healthy volunteers. Drug Investigation 8, 143149.
  • De Vries PJ, Bich NN, Van TH et al. (2000) Combinations of artemisinin and quinine for uncomplicated falciparum malaria: efficacy and pharmacodynamics. Antimicrobial Agents and Chemotherapy 44, 13021308.
  • White NJ, Waller D, Crawley J et al. (1992) Comparison of artemether and chloroquine for severe malaria in Gambian children. Lancet 339, 317321.
  • World Health Organization (1973) Chemotherapy of Malaria and Resistance to Antimalarials. WHO Technical Report 529.
  • World Health Organization (1997) Management of Uncomplicated Malaria and the Use of Antimalarial Drugs for the Protection of Travelers. Report of an informal consultation, Geneva, 18–21 September 1995. WHO/MAL/96.1075 Rev.1, 1997, Geneva.