Eighty three isolates of P. falciparum were collected from four endemic areas along Thai-Myanmar border (Tak, Kanchanaburi and Ranong) and Thai-Cambodia border (Chantaburi) during 1998 to 2005. These isolates were adapted and cryopreserved in liquid nitrogen before used. Parasites were cultivated continuously in vitro by a modification of Trager and Jensen method 20.

In vitro atovaquone and proguanil susceptibility of these Thai isolates of P. falciparum was determined using modified radioisotopic method of Desjardins et al 21. Both atovaquone and proguanil were kindly provided by Stephen A Ward (Liverpool School of Tropical Medicine, UK). Atovaquone was dissolved in dimethylsulfoxide (DMSO), whereas proguanil was dissolved in 50% ethanol. The IC₅₀ was eventually evaluated using the GRAFIT^® programme (Erithacus Software Ltd., UK). The IC₅₀ value of each isolate was the mean of at least three independent experiments. Comparisons of the IC₅₀ values of parasites collected in different years and from different areas were analyzed using One-way ANOVA. The level of significance was set at p < 0.05.

P. falciparum DNA was isolated using chelex-resin as described by Wooden et al 22. PCR-RFLP was performed to determine the three known polymorphisms at codon 268 of the cytb gene using primers CYTB3/CYTB5, CYTB2/CYTB6 and CYTB2/CYTB7 as previously described 9. For RFLP analysis, the PCR products of each pair of primers were digested with NsiI, AlwNI and SspI, respectively (New England Biolabs, UK). NsiI cut the wild type and asparagine mutation. AlwNI cut the serine mutation while SspI cut wild type and serine mutation but not the asparagine mutation. The digested PCR products were subsequently analyzed by agarose gel electrophoresis.

Since the point mutations in the cytb gene at the codons other than 268, i.e. 133 and 284 were previously reported to be involved with atovaquone sensitivity, DNA sequencing of the cytb gene was also performed. Twenty isolates were randomly selected from 83 isolates collecting from different years and endemic areas. These 20 isolates also represented parasites with the highest and lowest atovaquone IC₅₀ values. The primers pair of CYTB1 and CYTB2 was used for PCR amplification. The PCR products were sequenced by Macrogen Inc, Korea.

The mean atovaquone IC₅₀ values of P. falciparum isolates in this study was 3.4 ± 1.6 (0.83–6.81 nM). While, the mean proguanil IC₅₀ values was 36.5 ± 7.0 (21.2–49.6). The atovaquone and proguanil IC₅₀ values of P. falciparum isolates obtained in various years and from various endemic areas were shown in Figure 1 and 2, respectively. The mean ± SD of atovaquone IC₅₀ values in 1998, 2000, 2002, 2003 and 2005 were 3.4 ± 1.6, 2.8 ± 1.6, 3.8 ± 1.5, 3.2 ± 1.6 and 2.0 ± 0.8 nM, respectively. The mean ± SD of proguanil IC₅₀ values in 1998, 2000, 2002, 2003 and 2005 were 39.9 ± 7.8, 34.8 ± 5.1, 39.2 ± 7.4, 33.8 ± 5.9 and 29.8 ± 7.9 μM, respectively. The mean ± SD of atovaquone IC₅₀ value from Tak, Kanchanaburi, Ranong and Chantaburi were 3.4 ± 1.6, 3.3 ± 1.6, 2.9 ± 1.5 and 2.6 ± 1.6 nM, respectively. The mean ± SD of proguanil IC₅₀ value from Tak, Kanchanaburi, Ranong and Chantaburi were 36.7 ± 7.28, 35.0 ± 9.3, 34.9 ± 4.3 and 31.7 ± 4.7 μM, respectively. The cut-off point used for atovaquone sensitive and atovaquone resistance was the IC₅₀ of <30 nM and >1900 nM, respectively 15. Based on these criteria, all 83 Thai isolates were sensitive to atovaquone. Although the mean atovaquone IC₅₀ value of these Thai isolates was apparently higher than African isolates 1523, this IC₅₀ value (3.2 nM) was still classified in the range of atovaquone sensitive. Futhermore Gay et al determined the correlation of atovaquone and other antimalarial drugs in parasites isolated from the Philippines and found the significant correlations between of the IC₅₀ of atovaquone and chloroquine, quinine, mefloquine, artemisinin and its derivatives 23. The sensitivity of 83 isolates to other drugs was also determined and showed that there was no correlation among atovaquone and chloroquine, quinine, mefloquine and dihydroartemisinin IC₅₀. The absence of the correlation of atovaquone and most available drugs in these isolates is a good evidence for the possibility of using AP as the alternative antimalarial drug in Thailand.

In this study, both atovaquone and proguanil sensitivity of these isolates were gradually decreased over the seven years of collection period. There were significantly differences between the proguanil IC₅₀ values in the years 1998 and 2005 (p = 0.021) and in the year 2002 and 2005 (p = 0.020). There was no difference of the atovaquone and proguanil IC₅₀ values among the isolates adapted from different endemic areas.

Regarding to atovaquone, it has been reported that the mutations in the cytb gene especially at the codon 268 is responsible for atovaquone resistance 8910111213141516. In this study, all isolates were identified as wild-type genotype of the cytb gene at the codon 268. Sequence analysis also confirmed that all selected 20 isolates had a wild-type cytb gene at the codon 268. Recent studies showed a low prevalence of polymorphisms at the codon 268 1324. These point mutations at the codon 268 seems to be sufficient, but not necessary, for AP treatment failure since the point mutations at this position were not identified in every treatment failure cases 13. Other mutations i.e. M133I and V284K have been also linked to atovaquone resistance. From sequence analysis, there was no M133I and V284K mutation in all 20 isolates. The results of DNA sequencing were accorded well with the results of in vitro drug sensitivity assay and suggested that there was no atovaquone-resistant phenotype and genotype in these Thai isolates of P. falciparum. Similar results were previously reported in a study of parasite isolates from Thai-Myanmar border by Naoshima-Ishibashi et al; all samples showed no mutations at the codon 268 of the cytb gene 25.

A recent efficacy study also showed that AP remained highly efficacious for the treatment of multidrug-resistant falciparum malaria in Thailand 18. From both in vitro and in vivo information, AP can be considered as the drug of choice for the treatment and prophylaxis of falciparum malaria in Thailand.