In vitro sensitivity of multiresistant Plasmodium falciparum to new candidate antimalarial drugs in western Thailand.

The present study, carried out in 1987 in Thailand, has been designed to validate the in vitro microtest system, standardized by the World Health Organization (WHO), for the new antimalarials pyronaridine and halofantrine. The sensitivity of naturally acquired, multiresistant populations of Plasmodium falciparum has been assessed in order to develop a data base for further longitudinal investigations. For both drugs the in vitro microtest system seems to be suitable. The concentration range of plates can be considered as almost ideal for pyronaridine (0.1-6.40 mumol/l) while for halofantrine (0.002-0.128 mumol/l) an upward extension of the concentration range would be appropriate. Validation studies with artemisinin demonstrated the need for revising the protocol for the production of the dosing solutions. In the light of current knowledge about therapeutic concentration levels it would probably be appropriate to adopt a range of 0.2-12.8 mumol/l. All tested isolates, except possibly three, showed sensitive responses to pyronaridine. The high EC99 value of halofantrine could be indicative of some resistance to this drug. Rank correlation analysis suggested cross-resistance of pyronaridine and chloroquine which could be of consequence for the future introduction of pyronaridine.

Detection of Plasmodium falciparum infection in human patients: a comparison of the DNA probe method to microscopic diagnosis.

We have previously reported the isolation and testing of a DNA probe specific for the detection of Plasmodium falciparum. Field studies to compare the sensitivity and specificity of the DNA probe with that of light microscopy have been performed. In 2 studies in Thailand, 1,397 patients were tested. Microscope slides were prepared in a standard fashion and examined by clinical technicians and expert microscopists. The DNA probe method compares favorably in sensitivity with routine microscopy, detecting parasite densities as low as 40 parasites/microliters blood in the first study and, after modifications, 20-25 parasites/microliters blood in the second. Modifications included the elimination of salt from the lysis buffer, increasing the pH of the lysis buffer, and use of nylon based hybridization membranes instead of nitrocellulose. The DNA probe method offers the advantage of a standardized procedure that can be used in a batchwise fashion on a large number of samples.

Evidence of increased chloroquine sensitivity in Thai isolates of Plasmodium falciparum.

Sensitivity of Thai isolates of Plasmodium falciparum to chloroquine collected over the years 1978-1986 was measured by two methods: (i) by growth of previously cultured isolates for 72 h in presence of drug, and (ii) by the WHO standard in vitro microtest. During this period there were signs of a gradual increase in drug sensitivity, coinciding with the withdrawal of chloroquine for treatment of falciparum malaria in Thailand.

Response of Plasmodium falciparum infections to pyrimethamine-sulfadoxine in Thailand.

The results of this study in Thailand indicate that the early response of falciparum infections to a single dose of pyrimethamine-sulfadoxine is influenced by the developmental stages of the parasite present at the time of treatment. Parasite clearance is slower when young rings predominate at the time of treatment. This should be taken into account when considering the clinical management of patients and the comparative efficacy of antimalarials in clearing parasites from the peripheral blood. The 36-48 hr delay in schizonticidal action observed after treatment of febrile infections and the associated decline in blood concentrations of pyrimethamine suggest that a single dose may not be the ideal way of administering this drug combination and may encourage the emergence of drug-resistant parasites.

Specific DNA probe for the diagnosis of Plasmodium falciparum malaria.

Malaria can be diagnosed either by direct microscopic examination of blood smears, which is time consuming and requires expertise, or by immunological techniques, which are effective but do not distinguish between past and present infections. In this study, a simple procedure was developed for spotting lysed blood from infected patients directly onto nitrocellulose paper and identifying the malaria species on the basis of hybridization of parasite DNA with a species-specific probe. A genomic DNA library of Plasmodium falciparum was screened to detect clones containing DNA sequences that are highly repeated within the parasite genome. Several such clones were further analyzed to identify those that hybridize specifically with P. falciparum DNA but not with DNA from humans, P. vivax, or P. cynomolgi. This technique appears to be sensitive enough to detect 10 picograms of purified P. falciparum DNA (equivalent to 100 parasites) and in field studies is able to detect approximately 40 parasites per microliter of blood.

Sensitivity to quinine and mefloquine of Plasmodium falciparum in Thailand.

Between 1982 and 1984 a regimen consisting of quinine and tetracycline was routinely used in Thailand to treat outpatients with microscopically confirmed falciparum malaria. Due to compliance problems associated with the 7-day multiple-dose regimen, there was a recrudescence rate of approximately 30%. Studies carried out in 1982 and 1984 in four areas of Thailand indicated that there was a significant decrease in the sensitivity of Plasmodium falciparum to quinine. A significant, though less marked, reduction in the sensitivity of P. falciparum to the structurally related drug mefloquine was also observed, although this compound was not operationally deployed in Thailand before 1985. These findings emphasize the need to replace the long multiple-dose quinine regimen by an effective, acceptable, single-dose treatment.

In vitro susceptibility of Plasmodium falciparum collected from pyrimethamine-sulfadoxine sensitive and resistant areas in Thailand.

Seventy Plasmodium falciparum isolates, collected from two geographically separate areas of Thailand, were tested for their in vitro responses to pyrimethamine, sulfadoxine, and a combination of these two drugs. The effects of pyrimethamine and pyrimethamine-sulfadoxine combinations against P. falciparum isolates were found to be significantly greater in a northern area where the combined drug was an effective therapeutic agent than in a south-eastern area, near the Thai-Kampuchean border, where the combined drug was no longer effective. However, the actions of sulfadoxine against parasites obtained from the two areas were not significantly different. There was no significant difference between the mean values of plasma 4-aminobenzoic acid (PABA) in falciparum malaria patients and in healthy controls. The test for PABA determinations used in this study gave positive readings with both PABA and sulfadoxine.