Preservation of Wild Isolates of Human Malaria Parasites in Wet Ice and Adaptation Efficacy to In Vitro Culture

Wild isolates of malaria parasites were preserved in wet ice for 2–12 days and cultivated by a candle jar method. In four isolates of <i>Plasmodium falciparum</i> collected from Myanmar and preserved for 12 days, all failed to grow. In 31 isolates preserved for 5–10 days, nine were transformed to young gametocytes, but 22 isolates grew well. From Ranong, Thailand, nine isolates preserved for 7 days were examined, and six grew well. On the other hand, all of the 59 isolates collected from eastern Indonesian islands failed to establish as culture-adapted isolates, even most of them were preserved only for 2–3 days: 10 isolates stopped to grow, and 49 isolates were transformed to sexual stages by Day 10. These results indicated that a great difference in adaptation to in vitro culture may exist between wild isolates distributed in continental Southeast Asia and in eastern Indonesia and that gametocytogenesis might be easily switched on in Indonesian isolates. In wild isolates of <i>P. vivax</i>, <i>P. malariae</i> and <i>P. ovale</i> preserved for 2–9 days, ring forms or young trophozoites survived, but adaptation to in vitro culture failed. These results indicate that wild isolates can be preserved in wet ice for 9–10 days.

Synergistic enhancement of a copper chelator, bathocuproine disulphonate, and cysteine on in vitro growth of Plasmodium falciparum in glucose-6-phosphate dehydrogenase-deficient erythrocytes.

In vitro growth of Plasmodium falciparum is restricted in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes (RBC), as a result of oxidative stress. Bathocuproine disulphonate (BCS), a copper chelator, as well as cysteine have been shown to synergistically stimulate the in vitro growth of various mammalian cells and Trypanosoma under oxygenated conditions. We examined the effects of these two chemicals on the in vitro growth of P. falciparum in G6PD-deficient RBC, and found that addition of BCS and cysteine synergistically enhanced the growth of the P. falciparum FCR-3 strain in these RBC to the same level as in normal RBC. However, BCS or cysteine alone had no stimulatory effect. To explain this synergistic enhancement, changes in thiol, NADPH and glutathione contents were investigated. After addition of cysteine alone, thiol content in the medium decreased rapidly, but when BCS was added, it was maintained at about 35% at 24 hours after incubation, suggesting that BCS stimulates parasite growth in G6PD-deficient RBC by inhibiting copper-mediated oxidation of cysteine in the medium. In these RBC, no increase in NADPH level, but a slight increase in glutathione, was observed in the presence of both BCS and cysteine. The slight increase of glutathione, was probably due to incorporation of cysteine from the medium, although this could not fully explain the synergistic growth enhancement. These findings taken together suggest that cysteine incorporated into G6PD-deficient RBC may help maintain the thiol groups in many proteins, such as membrane proteins, hemoglobin and enzymes, and plays an important role in maintaining an appropriate culture state necessary for parasite growth. We also examined the effects of BCS and cysteine on adaptation of wild isolates of P. falciparum to in vitro cultivation using the candle jar method. Although there was no drastic effect on growth enhancement, the presence of BCS and cysteine accelerated the appearance of schizonts in many isolates.

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.