Highly pyrimethamine-resistant alleles of dihydrofolate reductase in isolates of Plasmodium falciparum from Tanzania

In 2000 we used a sensitive technique to examine 9 isolates from malaria patients in Muheza, Tanzania who had failed treatment with sulfadoxine-pyrimethamine (SP). Three isolates carried, at low levels, the leucine to isoleucine change at amino acid 164 that is associated with clinical failure of SP. Numerous other highly resistant alleles were also observed.     

Sensitivity of Plasmodium falciparum to chloroquine in Busia District,Kenya

Individuals infected with Plasmodium falciparum were randomly divided into two groups; one group was treated with a single dose of 10 mg chloroquine base per kg. body-weight and the other with 25 mg base of chloroquine per kg body-weight given over three days, followed by an observation period of seven days. By Day 3 of observation complete parasite clearance had occurred in all the 125 triple dose recipients and 113 of 114 (99·1%) of those who had the single dose.94·4% of 36 isolates tested in vitro by the macrotechnique were sensitive to drug concentration of 0.75 nmol/ml blood or less. One isolate was relatively less sensitive and required a concentration of chloroquine of 1·50 nmol/ml to inhibit schizont growth. However, the same isolate responded well to 25 mg base of chloroquine. These findings have demonstrated that, at present, isolates of P. falciparum in Busia District are sensitive to a standard dose of 10 mg chloroquine base and there is no reason therefore to resort to alternative antimalaria drugs. These should be reserved for special cases only.     

Synergistic antimalarial activity of dapsone/dihydrofolate reductase inhibitors and the interaction of antifol, antipyrimidine and antipurine combinations against Plasmodium falciparum in vitro

Using low folate, low p-aminobenzoic acid medium, 2 isolates of Plasmodium falciparum were tested in vitro against a wide range of antimetabolite compounds with known or potential antimalarial activity. ID50 values (the concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) were determined for each compound against both isolates. The compounds tested may affect folate, pyrimidine or purine metabolism in malaria parasites and various combinations of compounds were examined for further synergistic antimalarial effects. The combination of any of the dihydrofolate reductase inhibitors cycloguanil, pyrimethamine or WR 99210 with the sulphone drug dapsone demonstrated strongly synergistic antimalarial activity. Combinations of dihydrofolate reductase inhibitors with the antipyrimidine compounds pyrazofurin or menoctone, or with the antipurine compounds tubercidin, bredinin or hadacidin, or with primaquine, failed to demonstrate synergistic activity. Most combinations of an antipurine with an antipyrimidine compound also failed to show any synergistic effect. However, weak synergism was consistently seen in the tubercidin/pyrazofurin and tubercidin/menoctone combinations. Over the 48 h intraerythrocytic cycle using tightly synchronized parasites, tubercidin demonstrated both a cytotoxic and a cytostatic effect.     

Plasmodium falciparum spatial analysis, western Kenya highlands

We carried out a population-based study to determine the unbiased, age-specific Plasmodium falciparum prevalence, asexual and sexual parasite density, and spatial distribution to establish rates of infection at a site in western Kenya. Three cross-sectional surveys were carried out in western Kenya highlands. Blood samples were taken from 1,388 persons from 6 months to 75 years of age. Parasite prevalence and densities in the population decreased with age and distance from valley bottoms. Children from 1 to 4 years of age had the highest parasite prevalence (38.8%-62.8%); in adults, prevalence declined to 2.9%-24.1%. Malaria prevalence declined by an average of 19% from July to December 2002 across age groups. These observations suggest that parasite transmission is intense at this altitude. Asexual parasite density indicated clustering near major vector breeding habitats. Variability in seasonal prevalence indicates transmission instability and susceptibility to epidemics.     

Chloroquine-resistant Plasmodium falciparum malaria in Kenya.

A case of chloroquine-resistant Plasmodium falciparum malaria in a non-immune male is reported. Primary attack came 19 days after return to a non-malarious country from a visit to Kenya. Recrudescences occurred three times with intervals of 30 to 33 days after standard chloroquine treatment. The WHO extended field test for sensitivity of falciparum malaria to chloroquine was followed by recrudescence 31 days later. Treatment with Fansidar terminated the infection. If continuous treatment of the patient with lithium does not interfere with the schizontocidal action of chloroquine, this strain shows a resistance pattern of R I delayed recrudescence.     

Activity of dihydrofolate reductase inhibitors on the hepatic stages of Plasmodium yoelii yoelii in vitro.

The effects of the dihydrofolate reductase inhibitors proguanil and chlorproguanil, their active metabolites cycloguanil and chlorcycloguanil, and pyrimethamine, against the hepatic stages of Plasmodium yoelii yoellii were investigated in cultured BALB/c mouse hepatocytes. Proguanil was inactive at concentrations of 10(-8) M, whereas the other compounds were fully active at this and lower concentrations. Chlorcycloguanil was the most active compound and almost completely inhibited schizont development in concentrations as low as 10(-12) M.     

In vitro response of Plasmodium falciparum to chloroquine in the Nandi district, Kenya

Thirty-six isolates of Plasmodium falciparum from Nandi district, Kenya, which were tested for their sensitivity to chloroquine using the WHO in vitro macrotechnique, yielded a total of 29 successful tests, one of which showed overt resistance with schizont maturation at chloroquine levels of > 1.5 × 10^-6 mol/l. The majority of isolates showed reduced sensitivity to chloroquine, and the EC₉₉ was 1.7218 × 10^-6 mol/l. These findings are indicative of widespread in vitro resistance of a low degree which may remain largely unnoticed in immune individuals. However, in nonimmune subjects one may expect also in vivo resistance because the parasites will not be completely cleared after a normally curative dose of chloroquine.     

Novel inhibitors of Trypanosoma cruzi dihydrofolate reductase.

There is an urgent need for the development of new drugs to treat Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi. The enzyme dihydrofolate reductase (DHFR) has been a very successful drug target in a number of diseases and we decided to investigate it as a potential drug target for Chagas' disease. A homology model of the enzyme was used to search the Cambridge Structural Database using the program DOCK 3.5. Compounds were then tested against the enzyme and the whole parasite. Compounds were also screened against the related parasite, Trypanosoma brucei.     

Cortisol and Plasmodium falciparum infection in pregnant women in Kenya

Women living under holoendemic conditions of malaria in Kenya exhibited an increased prevalence of clinical malaria during pregnancy. In addition parasite rate and density were higher in primigravidae compared to multigravidae. Higher serum cortisol concentrations were found in women with patent malaria during pregnancy and the levels were higher before, during and after the malaria episode. A causal relation between serum cortisol levels and suppression of malaria immunity during pregnancy is discussed.     

Failure of erythromycin to improve chloroquine treatment of Plasmodium falciparum malaria in Kenya

58 children aged 1 to 10 years who had pure Plasmodium falciparum infections acquired on the coast of Kenya were treated with chloroquine 25 mg/kg given over 3 d and erythromycin 10 mg/kg 4 times a day given for 7 d. After 4 weeks follow-up, 62% had recurrent infections and 11% failed to clear their parasitaemia (1 had an RIII pattern of resistance). Of 38 children treated with chloroquine 25 mg/kg alone, 55% had recurrences and 21% failed to clear (including 1 RIII). In vitro microtests classified 74% of isolates from initial infections and 91% of isolates from recurrent infections as resistant. Erythromycin does not improve chloroquine treatment in children with infections due to P. falciparum having low to moderate levels of chloroquine resistance.     

In vivo response of Plasmodium falciparum to chloroquine in pregnant and non-pregnant women in Siaya District, Kenya

Chemoprophylaxis using chloroquine (CQ) in suppressive doses has been recommended to protect pregnant women in malarious areas from the adverse effects of malaria during pregnancy. In a malaria-endemic area of western Kenya with CQ-resistant Plasmodium falciparum, we determined the prevalence and density of falciparum infection in gravid and nulligravid women and compared the in-vivo parasite response to CQ using two regimens: 25 mg/kg body weight (CQ25) divided over a period of three days (for high-density parasitaemias) and 5 mg/kg body weight (CQ5) weekly for 4 weeks (for low-density parasitaemias). P. falciparum infections were present in 102 (42%) of 244 pregnant women. A greater proportion of primigravidae were parasitaemic (68%) than nulligravidae (50%, P=0.02) or multigravidae (33%, P <10^-6). Primigravidae showed a higher geometric mean parasite density. In the CQ25 treatment group, failure to clear parasites by day 7 was more common in primigravidae than nulligravidae (P=0.008) or multigravidae (P=0.15). In the CQ5 treatment group, primigravidae were more likely to show increasing parasite density than nulligravidae or multigravidae.     

Competitive inhibition of folate absorption by dihydrofolate reductase inhibitors, trimethoprim and pyrimethamine

Trimethoprim and pyrimethamine, inhibitors of dihydrofolate reductase (DHFR), cause folate deficiency in some patients. We investigated impairment of intestinal folate absorption by these drugs. By use of the in vivo intestinal-loop methods in rats, absorption of [3H] folic acid was significantly decreased in the presence of either drug. Kinetic studies using the influx chamber method demonstrated a pattern of competitive inhibition of folate transport. [3H] folic acid absorption from jejunal loops was determined 3-16 h after IV administration of methotrexate; this treatment abolished DHFR activity in the small intestine. In rats pretreated with methotrexate, luminal disappearance and systemic absorption of folic acid were significantly enhanced with respect to controls. Trimethoprim and pyrimethamine are weak competitive inhibitors of intestinal folate transport and folate absorption inhibition occurs at the site of membrane transport and appears to be unrelated to concurrent inhibition of DHFR activity in enterocytes.     

Meteorologic influences on Plasmodium falciparum malaria in the Highland Tea Estates of Kericho,Western Kenya

Recent epidemics of Plasmodium falciparum malaria have been observed in high-altitude areas of East Africa. Increased malaria incidence in these areas of unstable malaria transmission has been attributed to a variety of changes including global warming. To determine whether the reemergence of malaria in western Kenya could be attributed to changes in meteorologic conditions, we tested for trends in a continuous 30-year monthly malaria incidence dataset (1966-1995) obtained from complete hospital registers at a Kenyan tea plantation. Contemporary monthly meteorologic data (1966-1995) that originated from the tea estate meteorologic station and from global climatology records were also tested for trends. We found that total hospital admissions (malaria and nonmalaria) remained unchanged while malaria admissions increased significantly during the period. We also found that all meteorologic variables showed no trends for significance, even when combined into a monthly suitability index for malaria transmission. We conclude that climate changes have not caused the highland malaria resurgence in western Kenya.     

Response of falciparum malaria in vivo to a standard regimen of chloroquine in Kisumu District, Western Kenya

The sensitivity of Plasmodium falciparum to chloroquine was studied in 140 children in two locations in Western Kenya. The standard WHO in vivo field test was used and chloroquine phosphate 25 mg base/kg administered in divided doses over three days. In one area 13.2% of cases had recrudescent parasitaemias, while in the other area 8.2% of infections were resistant, with 3.5% having an RII pattern. The remaining isolates were sensitive to chloroquine. Further in vivo and in vitro tests in the region are needed to document the extent and level of resistance.     

The reservoir of Plasmodium falciparum malaria in a holoendemic area of western Kenya.

The reservoir of infectious Plasmodium falciparum gametocytes in a population living in an area of holoendemic malaria in western Kenya was estimated by directly feeding mosquitoes on volunteers. Resulting mosquito infections were assessed both by midgut examination for oocysts and by enzyme-linked immunosorbent assay for P. falciparum circumsporozoite antigen. Calculations based on the age structure of the population and the resulting rates of mosquito infections indicated that children under 10 years of age were responsible for 72% of mosquito infections, individuals between 10 and 21 years of age contributed 12%, and those over 21 years of age accounted for 16%. No infection resulted in mosquitoes fed on infants less than 1 year of age.     

An individual-based model of Plasmodium falciparum malaria transmission on the coast of Kenya

Individual-based models provide powerful tools to model complex interactions characterized by individual variability. This paper presents an object-oriented design for individual-based modelling of Plasmodium falciparum malaria transmission. Two kinds of objects, human and mosquito, that exhibit variability among individuals for parameters such as recovery and survival rates are defined. The model tracks the dynamics of human hosts and adult female mosquitoes individually. Immunity, modelled as a function of exposure history, is represented by reduced susceptibility and increased recovery rate. The model was calibrated using epidemiological data collected at 30 sites along the coast of Kenya. The sites were grouped into low, intermediate and high transmission based on mean daily human-biting rates. Simulation results show that malaria transmission was stable even in low transmission areas where the human-biting rate is approximately 0.5 bite per day. The model was used to examine the effect of infection control programmes that aim at interrupting transmission by reducing human-vector contact rates and implementing active case detection and drug treatment of infections. With this intervention, local elimination of malaria is likely with a probability of extinction of approximately 0.8 in low transmission areas. However, a small amount of immigration (> 0.3%) by infected people into the community could prevent local extinction of the parasite. In intermediate and high transmission areas, reduction in prevalence is short-lived and the probability of local elimination is low, even at high coverage levels of the intervention.