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Excepting tropical Africa, where Plasmodium falciparum prevails, Plasmodium vivax is the most frequent cause of malaria in Asia and Latin America. First reliable reports of chloroquine resistance came in 1989 from the area of the distribution of the Chesson-strain of P. vivax. Since then, reports also came from other areas of the world. This study had the objective of measuring the sensitivity of P.vivax to chloroquine and potential alternative compounds in western Thailand. The study was performed in 2008 in Mae Sot, Tak Province, and followed the method of Tasanor. The IC(50) and IC(90) values for chloroquine were 167 nM and 5445 nM, those for mefloquine were 139 nM and 5282 nM, those for artemisinin were 32 nM and 466 nM, and those for atovaquone 30 nM and 650 nM. The values for chloroquine indicate the existing or imminent occurrence of specific resistance. High prevalence of mefloquine resistance precludes its alternative use. However, atovaquone, in combination with proguanil, may be a possible alternative.

The pharmacodynamic interaction between retinol and 4-aminoquinolines has been investigated in 29 fresh isolates of Plasmodium falciparum. Although the parasites were highly resistant against 4-aminoquinolines, significant synergism was observed between chloroquine and retinol as well as amodiaquine and retinol, the latter at physiological concentrations. Combination with retinol reduced the geometric mean concentrations effecting complete inhibition (GMCOC) by chloroquine from 14425 nM to 8943 nM in CHL-RET low, 7042 nM in CHL-RET medium, and 4920 nM in CHL-RET high. Synergism between amodiaquine and retinol was greater, with strong and highly significant reductions of the GMCOC, from 2520 nM for amodiaquine to 1092 nM for AMO-RET low, 800 nM for AMO-RET medium, and 745 nM for AMO-RET high. While it is obviously too late for making practical use of the activity enhancement for chloroquine, the situation is different for amodiaquine, where supplementation with retinol may extend the usefulness of the medicament.

The in vitro study had the objectives of monitoring the sensitivity of Plasmodium vivax to chloroquine and artemisinin, and to assess its baseline sensitivity to mefloquine in northwestern Thailand in an area near the border to Myanmar. The investigations were carried out in 2004 at the malaria clinics of Mae Sot, Chedi Ko and Mae Ka Sa, all in the district of Mae Sot, Province of Tak. The in vitro tests followed the method of Tasanor. Successful tests were obtained with 45 fresh isolates of P. vivax. The EC(50) and EC(90) values for chloroquine were 120.9 nM and 655.7 nM, respectively, the GMCOC was 1699.7 nM. There was a significant decrease of the chloroquine sensitivity since 1998/1999. However, results of parallel investigations continue to indicate clinical-parasitological sensitivity to chloroquine. With mefloquine the EC(50) and EC(90) the (baseline) values were 131.6 nM and 972.6 nM, respectively, the GMCOC was 1987.0 nM. For artemisinin the EC(50) and EC(90) values were 8.7 nM and 105.2 nM, respectively, the GMCOC was 310.5 nM. As compared to 2002, the sensitivity of P. vivax to artemisinin has shown a slight but not significant increase.

This study was conducted during 2002-2004 at Mae Sot District, on the Thai-Myanmar border, an area of multidrug-resistant Plasmodium falciparum malaria. Sixty-two patients with P. vivax malaria were included in the study. All were randomized into two groups to receive a 3-day regimen of chloroquine or a 3-day regimen of quinine. Primaquine was given to patients in both groups for the elimination of hepatic stages. Results from the present study suggest that the standard regimen of chloroquine and a 3-day course of quinine at the dose regimens under investigation were very effective and well tolerated for the treatment of P. vivax malaria in this area. All patients responded well to both drug regimens; the cure rates with chloroquine or quinine, when given concurrently with the tissue schizontocidal drug primaquine, were virtually 100% within 28 days of follow-up. No significant correlations between parasite clearance time (PCT) or fever clearance time (FCT) and inhibitory concentration 50 (IC50) were found. Patients who had PCT < or = 24 h and those with PCT >24 h had comparable IC50 to chloroquine (alone and plus primaquine) and quinine, as well as similar concentrations of chloroquine/desethylchloroquine (in blood) or quinine (in plasma) at the investigated time points.

Malaria resulting from infection with Plasmodium vivax rarely causes death, however, patients usually suffer acute debilitating clinical symptoms and the recovery is slow. This study had the objective of assessing the pharmacodynamic interaction between artimisinin and chloroquine with a view of a potential acceleration of the clinicalparasitological response, and the investigation of therapeutic alternatives in the event of chloroquine resistance in Plasmodium vivax. Tests were based on the growth inhibition of Plasmodium vivax, determined by morphological differential counts of 200 asexual parasites. In total 45 isolates were evaluated successfully with parallel tests for artemisinin, chloroquine and desbutylbenflumetol (DBB) alone and combinations of artemisinin + chloroquine and artemisinin + DBB. Total inhibition was reached at a mean concentration of 1274.8 nM (95% CI 898.5 to 1808.7 nM), and 1852.2 nM (95% CI 1539.5 to 2228.6 nM) for artemisinin, and chloroquine respectively, whilst the 1:1 (m/m) combination of artemisinin and chloroquine was 1860.2 nM (95% CI 1454.4 to 2379.3 nM). EC(50) and EC(90) were 129.9 nM and 1058.5 nM for chloroquine, 32.6 nM and 735.5 nM for artemisinin, and 73.6 nM and 1103.0 nM for the 1:1 combination of both drugs. Interaction analysis according to Berenbaum yielded for the artemisinin + chloroquine combination at the EC(50) a mean SigmaFIC of 1.1126, at the EC(90) a mean SigmaFIC of 1.0331, and at the EC(99) a mean SigmaFIC of 1.1857. These results revealed marked additive interaction. For desbutylbenflumetol (DBB) the EC(50) and EC(90) were 1.5 nM and 28.8 nM, complete growth inhibition was observed at 90.4 nM (95% CI 75.1 to 108.7 nM). Interaction analysis indicated moderate antagonism at the lower concentration ranges, at the EC(90) additive interaction with a mean SigmaFIC of 1.0300, and synergism at the therapeutically most important EC(99) with a mean SigmaFIC of 0.5990.

The blood schizontocidal activity of the four main Cinchona alkaloids against Plasmodium falciparum was compared in 46 fresh parasite isolates, using an in-vitro test measuring the drug-specific inhibition of schizont maturation. The studies were conducted in June-August 2001 at Mae Sot, northwestern Thailand, an area where quinine alone is no longer able to eliminate infections with P. falciparum. Quinidine showed the highest blood schizontocidal activity, followed by cinchonine, cinchonidine and finally quinine, which was identified as the least active compound. The isolates showed marked heterogeneity in their response to the Cinchona alkaloids. There was also high correlation of activity among all four alkaloids. The mean EC50 values for quinine, quinidine, cinchonine and cinchonidine were 144 nM, 80 nM, 104 nM and 225 nM, respectively, and the EC99 values 8040 nM, 861 nM, 1176 nM and 6531 nM. The EC99 values for quinine and cinchonidine are beyond the therapeutic concentration range and those for quinidine within it. For cinchonine, values are likely to be within this range, but toxicological and pharmacokinetic studies on this compound are required for clarifying its potential future role in the treatment of falciparum malaria.

After its rehabilitation for therapeutic use in uncomplicated falciparum malaria, there is renewed interest in amodiaquine. After oral administration, the drug undergoes rapid metabolism to monodesethyl-amodiaquine, and in patients with normal hepatic function the parent drug usually becomes undetectable within a few hours. The main antimalarial activity is therefore mainly due to the metabolite. In a comparative study in northwestern Thailand, 21 fresh isolates of Plasmodium falciparum were tested, in parallel, for their in-vitro sensitivity to both compounds, using the WHO micro-test Mark II, measuring the inhibition of schizont maturation. The geometric mean cut-off concentrations of schizont maturation were 1826 nM (related to blood) for amodiaquine, and 1654 nM for monodesethyl-amodiaquine. The log-probit regressions for both compounds showed good fits to the data points. The EC50 values were 331 nM and 291 nM, and the EC90 values 1337 nM and 993 nM for amodiaquine and monodesethyl-amodiaquine, respectively. Differences between regression slopes and effective concentrations were well below statistical significance. Both compounds showed highly significant activity correlation. These findings suggest that the sensitivity of Plasmodium falciparum to amodiaquine closely reflects its sensitivity to monodesethyl-amodiaquine.

The blood schizontocidal, pharmacodynamic interaction between tafenoquine (WR 238605--a 5-phenoxyprimaquine derivative--and chloroquine was investigated, using an in-vitro test for the inhibition of schizont maturation, in 15 fresh isolates of Plasmodium falciparum that originated from northwestern Thailand and neighbouring Myanmar. In this area the parasite is highly resistant to chloroquine. The geometric mean cut-off concentrations of schizont maturation for tafenoquine and chloroquine were 5261 nM and 7638 nM, respectively. With a mixture of tafenoquine and chloroquine, the mean cut-off concentration was 5252 nM, corresponding to 389 nM tafenoquine + 4863 nM chloroquine. Further analysis showed that the interaction between tafenoquine and chloroquine was additive within the range of EC20 and EC77. At concentrations higher than the EC77, interaction was moderately synergistic. While tafenoquine did not reverse the resistance to chloroquine to the degree of clinically relevant sensitivity, there was evidence that the blood schizontocidal efficacy of tafenoquine would be enhanced in the presence of chloroquine.

Following earlier observations on short-term culture of Plasmodium vivax, an in vitro test system has been developed for assessing the parasite's sensitivity to chloroquine. Fresh isolates with predominantly young trophozoites are diluted 1:19 with a (v/v=1/1) mixture of RPMI 1640 and Waymouth medium. The blood-medium mixture (BMM) is inoculated into the predosed microtitre plates before incubation in a candle jar. Incubation for 30 or 42 h yielded the best results. Incubation for 18 or 24 h was generally insufficient for an adequate development of the parasites. The reading of the test is based on stage-specific differential counts in the Giemsa-stained pre-incubation and post-incubation thick films, the evaluation on log-probit analysis of drug-related inhibition of parasite development. The test system has been evaluated on 200 fresh P. vivax isolates in an area with satisfactory clinical-parasitological response to chloroquine. At 30 or 42 h incubation 121 isolates (61.5%) showed adequate control growth and yielded valid sensitivity tests. Complete inhibition of parasite development occurred within the concentration range of 40-1280 nM. The mean EC50 for 30 h of incubation was 50.3 nM, as compared to 49.7 nM with 42 h of incubation. The geometric mean cut-off concentration of parasite development was 488 nM with 30 h of incubation as against 470 nM with 42 h of incubation.