Synergistic interaction between atovaquone and retinol in Plasmodium falciparum in vitro.

The study has been conducted with the objective of assessing the blood schizontocidal activities of atovaquone (ATO), retinol (RET) and combinations of both (ATO-RET) at set retinol concentrations corresponding to the 50th, 65th and 80th percentile of the physiological serum retinol levels. The in vitro tests followed the WHO standard protocol Mark II for measuring the inhibition of schizont maturation in Plasmodium falciparum. Valid results for all 5 test lines were obtained with 26 fresh parasite isolates from northwestern Thailand, an area affected by multidrug-resistance. The EC(50) values for atovaquone, retinol and for ATO in ATO-RET low, medium and high were 3.1 nM, 561.8nM, 0.85 nM, 0.73 nM and 0.45 nM, respectively, the EC(90) values 33.7 nM, 9338.6 nM, 25.31 nM, 8.89 nM, and 5.42 nM. The geometric mean cut-off concentrations of schizont maturation of atovaquone alone and for atovaquone in ATO-RET low, medium and high were 282.5 nM, 79.0 nM, 38.7 nM and 23.7 nM, respectively. These results and those of the Berenbaum analysis based on the fractional inhibitory concentrations indicate synergistic pharmacodynamic interaction between atovaquone and retinol, a phenomenon suggesting that the antimalarial activity of atovaquone could be enhanced by supplementation with retinol.

Specific pharmacokinetic interaction between lumefantrine and monodesbutyl-benflumetol in Plasmodium falciparum.

The blood schizontocidal activity of lumefantrine, monodesbutyl-benflumetol (DBB) and a 999:1 combination of both compounds has been investigated in 26 fresh isolates of Plasmodium falciparum from northwestern Thailand, using the WHO standard protocol Mark II for determining the inhibition of schizont maturation. The geometric mean cut-off concentrations of schizont maturation (GMCOC) were 943.2 nM for lumefantrine, 146.3 nM for DBB and 182.2 nM for the 999:1 combination of lumefantrine and DBB. The EC(50) values were 27.3 nM for lumefantrine, 5.7 nM for DBB, and 16.5 nM for the combination, and the EC(90) values 163.1 nM for lumefantrine, 44.1 nM for DBB, and 78.3 nM for the combination. Despite the very low concentration in the combination, DBB exerted significant synergistic activity with lumefantrine that was strongest at the EC(90) and EC(99) levels. Correlation analysis indicates that DBB is the leading determinant for the activity of the combination.

Susceptibility to chloroquine, mefloquine and artemisinin of Plasmodium vivax in northwestern Thailand.

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.

Deployment of early diagnosis and mefloquine-artesunate treatment of falciparum malaria in Thailand: the Tak Malaria Initiative.

BACKGROUND: Early diagnosis and treatment with artesunate-mefloquine combination therapy (MAS) have reduced the transmission of falciparum malaria dramatically and halted the progression of mefloquine resistance in camps for displaced persons along the Thai-Burmese border, an area of low and seasonal transmission of multidrug-resistant Plasmodium falciparum. We extended the same combination drug strategy to all other communities (estimated population 450,000) living in five border districts of Tak province in northwestern Thailand. METHODS AND FINDINGS: Existing health structures were reinforced. Village volunteers were trained to use rapid diagnostic tests and to treat positive cases with MAS. Cases of malaria, hospitalizations, and malaria-related deaths were recorded in the 6 y before, during, and after the Tak Malaria Initiative (TMI) intervention. Cross-sectional surveys were conducted before and during the TMI period. P. falciparum malaria cases fell by 34% (95% confidence interval [CI], 33.5-34.4) and hospitalisations for falciparum malaria fell by 39% (95% CI, 37.0-39.9) during the TMI period, while hospitalisations for P. vivax malaria remained constant. There were 32 deaths attributed to malaria during, and 22 after the TMI, a 51.5% (95% CI, 39.0-63.9) reduction compared to the average of the previous 3 y. Cross-sectional surveys indicated that P. vivax had become the predominant species in Thai villages, but not in populations living on the Myanmar side of the border. In the displaced persons population, where the original deployment took place 7 y before the TMI, the transmission of P. falciparum continued to be suppressed, the incidence of falciparum malaria remained low, and the in vivo efficacy of the 3-d MAS remained high. CONCLUSIONS: In the remote malarious north western border area of Thailand, the early detection of malaria by trained village volunteers, using rapid diagnostic tests and treatment with mefloquine-artesunate was feasible and reduced the morbidity and mortality of multidrug-resistant P. falciparum.

In vitro interaction between artemisinin and chloroquine as well as desbutyl-benflumetol in Plasmodium vivax.

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.

Pharmacodynamic interaction between atovaquone and other antimalarial compounds against Plasmodium falciparum in vitro.

Atovaquone, a 2-hydroxy-1,4-naphthoquinone, was first introduced as a drug against opportunistic infections in immuno-compromised patients. Early clinical-parasitological experiences in the treatment of malaria were disappointing due to highly variable and poor absorption, a phenomenon typical for naphthoquinones. Proguanil was found to potentiate the activity of atovaquone and the combination of the two drugs was introduced as an antimalarial drug with blood schizontocidal and causal prophylactic activity. It is now widely used in therapy and prophylaxis. Despite the enhanced activity, the combination does not always overcome the problem of poor absorption of atovaquone, especially in the presence of gastro-intestinal disorders. Therefore, further combination with a fast-acting blood schizontocide, e.g. one of the artemisinins, could accelerate clinical improvement and normalization of absorption. The interaction between artemisinin and atovaquone and that of artemisinin and atovaquone + proguanil has been investigated in 37 fresh isolates of Plasmodium falciparum from northwestern Thailand, an area with high prevalence of multi-drug resistance. Interaction between atovaquone and artemisinin was synergistic above the EC(30), with mean SigmaFIC (Berenbaum) values of 0.9679 at the EC(50), 0.4014 at the EC(90) and 0.2214 at the EC(99). Synergism was more pronounced with the triple combination, i.e. atovaquone + proguanil and artemisinin, starting at the EC(10) level. The mean SigmaFIC values were 0.7626 at the EC(50), 0.2939 at the EC(90), and 0.1527 at the EC(99). The strong synergism at the therapeutically relevant effective concentrations suggests that the therapeutic efficacy of atovaquone-proguanil can be considerably enhanced by the additional administration of a suitable artemisinin derivative, e.g. artesunate.

Early detection of malaria in an endemic area: model development.

A malaria epidemic warning system was established in Thailand in 1984 using graphs displaying the median or mean incidence of malaria over the previous five years compiled from malaria surveillance data throughout the country. This reporting mechanism is not timely enough to detect the occurrence of a malaria epidemic which usually occurs at the district level over a short period of time. An alternative method for early detection of a malaria epidemic employing the Poisson model has been proposed. The development of this early malaria epidemic detection model involved 3 steps: model specification, model validation and model testing. The model was based on data collected at the Vector Borne Disease Control Unit (VBDU) Level. The results of model testing reveal the model can detect increasing numbers of cases earlier, one to two weeks prior to reaching their highest peak of transmission. The system was tested using data from Kanchanaburi Province during 2000 to 2001. Results from model testing show the model may be used for monitoring the weekly malaria situation at the district level. The Poisson model was able to detect malaria early in a highly endemic province with a satisfactory level of prediction. As the application is essential for the malaria officers in monitoring of malaria epidemics, this early detection system was introduced into malaria epidemiological work. The model may be helpful in the decision making process, planning and budget allocation for the Malaria Control Program. The software for early malaria detection is currently implemented in several endemic areas throughout Thailand.

Strengthening of national capacity in implementation of antimalarial drug quality assurance in Thailand.

Substandard and counterfeit pharmaceutical products, including antimalarial drugs, appear to be widespread internationally and affect both the developing and developed countries. The aim of the study was to investigate the quality of antimalarial drugs, ie, artesunate (ART), chloroquine (CHL), mefloquine (MEF), quinine (QUI), sulfadoxine/pyrimethamine (S/P) and tetracycline (TT) obtained from the government sector and private pharmacies in 4 Thai provinces: Mae Hong Son, Kanchanaburi, Ranong, and Chanthaburi. Three hundred sixty-nine samples of 6 antimalarial drugs from 27 government hospitals, 27 malaria clinics, and 53 drugstores, were collected. Drug quality was assessed by simple disintegration test and semi-quantitative thin-layer chromatography in each province; 10% passed, 100% failed and doubtful samples were sent to be verified by high performance liquid chromatography (HPLC) at the Thai National Drug Analysis Laboratory, (NL). Fifteen point four percent of ART, 11.1% of CHL and 29.4% of QUI were substandard. Based on the finding, drug regulatory authorities in the country took appropriate action against violators to ensure that antimalarial drugs consumed by malaria patients are of good quality.

Synergism of desbutyl-benflumetol and retinol against Plasmodium falciparum in vitro.

This in vitro study was conducted to assess the blood schizontocidal activity of desbutyl-benflumetol (DBB), a new benzindene derivative, retinol and a combination of both compounds. The tests were carried out according to the methodology of the WHO standard test Mark II, measuring the drug-dependent inhibition of schizont maturation, and using 43 fresh isolates of Plasmodium falciparum from northwestern Thailand, an area with established multidrug-resistance. DBB and retinol showed a mean 50% effective concentration (EC-50) of 3.73 nM and 466.86 nM and 90% effective concentration (EC-90) of 19.83 nM and 5531.69 nM respectively. The combination of DBB and 3.50 muM retinol showed strong inhibition of schizont maturation, with an EC-90 for DBB of 0.67 nM. At the therapeutically relevant EC-99, the combination was about 10 times more effective than expected, suggesting that retinol potentiated the effect of DBB. A concentration of 3.50 muM retinol corresponds to the 95th percentile of the physiological serum levels. It is well known that retinol levels are significantly decreased in acute falciparum malaria. Supplementation with retinol during malaria treatment may improve the therapeutic results of blood schizontocides of the fluorene class.

In vitro activity of artemisinin alone and in combination with retinol against Plasmodium falciparum.

Increasing resistance of Plasmodium falciparum to antimalarial drugs is an important public health problem, demanding novel therapeutic approaches. This study had the objective of assessing the in vitro activity of artemisinin and its combination with retinol in fresh isolates of P. falciparum in an area with a high proportion of multidrug-resistant strains. The tests were based on the inhibition of schizont maturation. In 45 successfully tested isolates, the mean effective concentrations (ECs) of artemisinin were 10.29 nM for the EC-50 and 34.86 nM for the EC-90. The EC50 and EC90 for artemisinin in the artemisinin-retinol mixture were 2.71 nM and 13.37 nM, respectively. The combination showed synergistic activity. Retinol appears to be a promising partner for the antimalarial therapy with artemisinins.

Chronicle of malaria epidemics in Thailand, 1980-2000.

The occurrence of malaria epidemics in Thailand was reviewed from the malaria surveillance report of the National Malaria Control Program. The literature review revealed that the four epidemic periods recorded during 1980-2000 almost always occurred in the provinces and districts located along international borders. Malaria epidemics are caused by various factors such as: extensive population movement, multi-drug resistance development, low immune status of the population, lack of knowledge and appropriate personal protection against mosquito biting, and the re-emergence of malaria transmission in low malarious areas. Such factors can lead to changes in the parasite ratio and appearance of malaria epidemics throughout the country. Evidence related to the burden of malaria epidemics was also reviewed to identify causal factors that will be helpful in future research.

Comparative study of the in vitro sensitivity of Plasmodium falciparum to artemisinin in two border areas of Thailand.

Artesunate was introduced in Thailand in 1995 for the treatment of falciparum malaria in areas of multidrug resistance, where it is used in combination with mefloquine. The studies were conducted between May and August 1999, 2000 and 2001 in the provinces Mae Hong Son and Tak (Mae Sot District) in northwestern Thailand, both on the border to Myanmar. The province of Mae Hong Son is still largely unaffected by multidrug resistance and infections with Plasmodium falciparum are treated with mefloquine alone. In the district of Mae Sot, 350 km southwards, more than 60% of the Plasmodium falciparum isolates were found to be resistant to mefloquine. Between 1999 and 2001, a total of 227 fresh isolates of Plasmodium falciparum were successfully tested for their sensitivity to artemisinin using the WHO standard in vitro microtest. The weighted mean EC5o and EC90 values for 1999-2001 were 9.20 nM and 34.37 nM in Mae Hong Son and 11.18nM and 71.63nM in Mae Sot, respectively. The comparison of the sensitivity to artemisinin between Mae Hong Son and Mae Sot showed no significant difference in 1999, but significant differences in 2000 (p<0.05) and in 2001 (p<0.01). This phenomenon could be a consequence of different drug pressure. Furthermore, the lower sensitivity of Plasmodium falciparum to mefloquine in Mae Sot may play a minor (but amplifying) role, as the activities of artemisinin and mefloquine show a significant correlation.

In vitro sensitivity of Plasmodium falciparum to lumefantrine in north-western Thailand.

Lumefantrine (benflumetol) belongs to the class-2 blood schizontocidal compounds. In combination with artemether it serves as an alternative drug for treating chloroquine-resistant infections with Plasmodium falciparum. In view of activity correlations with mefloquine, it is important to monitor the parasite's intrinsic sensitivity to lumefantrine in areas with multi-drug resistant P. falciparum, the objective of this study. The observations were carried out in 2002 at Mae Sot, northwestern Thailand, near the border to Myanmar. The 41 successfully in-vitro tested parasite isolates yielded a geometric mean cut-off concentration of schizont maturation of 237.54 nM, and EC50, EC90 and EC90 values of 15.13 nM, 86.71 nM and 359.97 nM, respectively. As compared to the findings of 1998 and 1999, the susceptibility to lumefantrine has increased, possibly due to the improved therapeutic response of mefloquine-resistant P. falciparum infections to combined treatment with artesunate + mefloquine. The EC9o and EC99 data of 2002 for lumefantrine in the study area suggest fully curative clinical-parasitological efficacy.

Comparative study on the in vitro activity of lumefantrine and desbutyl-benflumetol in fresh isolates of Plasmodium vivax from Thailand.

The occurrence of chloroquine resistance in Plasmodium vivax underlines the need for monitoring the drug response of this important malaria parasite and for the evaluation of alternative therapeutic agents. In-vitro methods facilitate these tasks. This investigation employed a recently developed in-vitro micro-technique and validated it for lumefantrine and desbutyl-benflumetol, a compound that was initially considered a metabolite of lumefantrine. The studies were conducted in 2001 at Mae Sot, a town situated in northwestern Thailand near the border to Myanmar. Parallel in-vitro tests with lumefantrine and desbutyl-benflumetol were carried out with 53 fresh isolates of P. vivax. For both compounds, the parasite showed a homogenous, log-normal inhibition pattern with nearly parallel log-probit regressions. The geometric mean drug concentrations effecting complete growth inhibition were 2361 nM for lumefantrine and 187 nM for desbutyl-benflumetol. With p=3.264 x 10(-18) the difference was highly significant. The EC50 and EC90 values for lumefantrine, 17.6 nM and 448.5 nM, respectively, were much higher as compared to those determined for desbutyl-benflumetol, with 1.5 nM and 39.7 nM. This difference expressed itself in a highly significant Power Ratio (PR) of 11.0. The activity of desbutyl-benflumetol in P. vivax exceeds that of lumefantrine by one order of magnitude, suggesting a high, hitherto unexploited therapeutic potential of desbutyl-benflumetol.

Declining mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border.

Mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border, both in vitro and in vivo, following different first-line treatments for uncomplicated falciparum malaria patients in these areas during the period 1997--2003 were studied. Standard in vitro micro tests and in vivo efficacy according to World Health Organization methodologies were performed. P. falciparum isolates along the Thai-Myanmar border with in vitro sensitivity to mefloquine have had up to a ten-fold decrease in sensitivity compared to a baseline done in 1986, conducted one year after the drug was first introduced to Thailand. The reduction in the mefloquine sensitivity of P. falciparum isolates in Tak Province developed rapidly, with the highest IC50 of 1,254 nM in 1997. The IC50 declined to 1,067 and 737 nM in 1999 and 2001, respectively, but there was no statistically significant difference in the sensitivity. The sensitivity of P. falciparum isolates from Mae Hong Son, Kanchanaburi, and Ranong, where the first line treatment was mefloquine 15 mg/kg single dose, continued to decline, where in 2001 the IC50 were 1,087, 941, and 1,116 nM, respectively, in these provinces. The difference in sensitivities of P. falciparum isolates in Mae Hong Son and Ranong in 2001, compared to 1997, was statistically significant (p<0.05). Good therapeutic efficacy of the artesunate-mefloquine combination in Tak Province was observed. Adequate clinical responses (ACR) were 89.5% and 92.3% in 1997 and 2002, respectively. The efficacy of mefloquine alone in Mae Hong Son, Kanchanaburi, and Ranong has significantly dropped. ACR in 1997 and 2001 in Mae Hong Son were 87.8% and 73.2%, respectively, in Kanchanaburi were 82% and 59.6%, respectively, and in Ranong were 96% and 31.6%, respectively.

Trend of malaria incidence in highly endemic provinces along the Thai borders, 1991-2001.

The intercountry border areas of Thailand have high malaria receptivity and vulnerability that present numerous problems in the control of malaria transmission. This study focused on the 30 provinces of Thailand situated next to neighboring countries, which can be divided into 4 groups: the Thai-Myanmar border (10 provinces), the Thai-Cambodia border (6 provinces), the Thai-Lao border (10 provinces) and the Thai-Malaysia border (4 provinces). The purpose of the present study was to describe the pattern and trend of malaria incidence in the highly endemic provinces along the Thai borders for the 11 years from 1991 to 2001. Analysis of trends showed the distribution of malaria parasites to have shifted from a preponderance of Plasmodium falciparum to Plasmodium vivax along the western border with Myanmar, the northern border with Lao PDR and along the eastern border with Cambodia whereas the southern border with Malaysia the pattern changed from a preponderance of P. vivax to P. falciparum, since 1997. There was a significant difference in annual parasite incidence between borders and non-border districts, especially along the Thai-Myanmar and Thai-Cambodia borders. It is thus evident that all border districts should pay more attention to control of malaria transmission and the activities of the malaria surveillance system, and that monitoring and evaluation of the Thai Malaria Control Program needs to be performed consistently, including some areas where a few malaria cases were found as well as in malaria free areas.

Mekong malaria. II. Update of malaria, multi-drug resistance and economic development in the Mekong region of Southeast Asia.

In an expansion of the first Mekong Malaria monograph published in 1999, this second monograph updates the malaria database in the countries comprising the Mekong region of Southeast Asia. The update adds another 3 years' information to cover cumulative data from the 6 Mekong countries (Cambodia, China/Yunnan, Lao PDR, Myanmar, Thailand, Viet Nam) for the six-year period 1999-2001. The objective is to generate a more comprehensive regional perspective in what is a global epicenter of drug resistant falciparum malaria, in order to improve malaria control on a regional basis in the context of social and economic change. The further application of geographical information systems (GIS) to the analysis has underscored the overall asymmetry of disease patterns in the region, with increased emphasis on population mobility in disease spread. Of great importance is the continuing expansion of resistance of P. falciparum to antimalarial drugs in common use and the increasing employment of differing drug combinations as a result. The variation in drug policy among the 6 countries still represents a major obstacle to the institution of region-wide restrictions on drug misuse. An important step forward has been the establishment of 36 sentinel sites throughout the 6 countries, with the objective of standardizing the drug monitoring process; while not all sentinel sites are fully operational yet, the initial implementation has already given encouraging results in relation to disease monitoring. Some decreases in malaria mortality have been recorded. The disease patterns delineated by GIS are particularly instructive when focused on inter-country distribution, which is where more local collaborative effort can be made to rationalize resource utilization and policy development. Placing disease data in the context of socio-economic trends within and between countries serves to further identify the needs and the potential for placing emphasis on resource rationalization on a regional basis. Despite the difficulties, the 6-year time frame represented in this monograph gives confidence that the now well established collaboration is becoming a major factor in improving malaria control on a regional basis and hopefully redressing to a substantial degree the key problem of spread of drug resistance regionally and eventually globally.