CONTINUOUS CULTIVATION, DRUG SUSCEPTIBILITY TESTING AND IMMUNE RESPONSE TO PLASMODIUM FALCIPARUM INFECTION IN IBADAN AND SAGAMU SOUTH WEST NIGERIA.

Abstract

Continuous cultivation of the parasite Plasmodium falciparum, provide essential material for multidisciplinary research in malaria. Unfortunately, requirement for non immune human blood products as supplement in the culture system, limits application of this technique in malaria endemic countries. This thesis describes modification and application of standard techniques of continuous cultivation of P. falciparum for malaria research in a malaria endemic country. Standard techniques were modified to utilize heat inactivated semi-immune plasma and premixed gases. The modification resulted in successful cultivation of P. falciparum using semi immune human plasma and red blood cells. The modified system was used to study drug susceptibility profiles of P. falciparum isolates obtained from patients in Ibadan and for screening potential resistance reversing compounds. Forty seven percent of the isolates had elevated fifty percent inhibitory concentrations (IC₅₀) for chloroquine compared to the chloroquine sensitive P. falciparum clone D6. Twenty to forty percent of the isolates also had elevated IC₅₀ to other standard antimalarial drugs (mefloquine, halofantrine and artemisinin). There was cross resistance between rnefloquine and halofantrine in 20% of the isolates tested. Five of eight compounds screened for resistance reversing activity restored the susceptibility of resistant isolates to chloroquine. Combination of chloroquine with fixed concentrations (5.0 x 10⁻⁷M to 1.0 x 19⁻⁶) of either BL50610, chlorpromazine, chlorpheniramine, diphenhydramine or mepyramine reduced the IC₅₀ of chloroquine against resistant isolates by between 45.2 ± 2.8% and 80.6 ± 9.1%. Combinations of chIoroquine with fixed concentrations (5.0 x 10⁻⁸M to 8.3 x 10⁻⁶M) of either acetylsalicylic acid, acetaminophen or indomethacin did not increase the activity of chloroquine against resistant parasites. The activity of chloroquine against sensitive parasites was not potentiated by any of these compounds. Plasma obtained from certain donors with apparent resistance to malaria infection failed to support parasite growth in the modified culture system. No biochemical or hematological abnormalities were detected to account for their apparent resistance to malaria despite permanent residence in Sagamu or Ibadan. Immunologic analysis (immunoblot) of plasma samples from these individuals revealed a unique antibody reaction with a low molecular weight protein (24,500 MW) isolated from P. falciparum infected red blood cells. The clinical and epidemiological value of those observations in malaria control are promising. These findings underscore a need for judicious utilization of new antimalarial drugs in order to prevent emergence and spread of drug resistant parasites. Identification of new resistance reversing compounds and a putative immuno-protective component in the blood of some individuals also promise a novel approach for developing new chemotherapeutic and immuno-therapeutic techniques for malaria control.