Genetic diversity of Plasmodium falciparum parasites from Kenya is not affected by antifolate drug selection.

The genotypes of merozoite surface protein-1, merozoite surface protein-2 and glutamine rich protein are frequently used to distinguish recrudescence from reinfection when parasitaemia reappears after antimalarial drug treatment. However, none of the previous reports has clearly assessed the change of genetic diversity following drug treatment. In the present study, we have assessed the impact of pyrimethamine/sulfadoxine and chlorproguanil/dapsone on the genetic diversity of isolates and the multiplicity of infection in patient isolates from Kilifi, Kenya. We have analysed the length polymorphism of merozoite surface protein-1, merozoite surface protein-2 and glutamine rich protein and the data clearly show that treatment with pyrimethamine/sulfadoxine and chlorproguanil/dapsone did not change the multiplicity of infection found in patients, in contrast to the selection that these drugs exert on the genes encoded by the target enzymes. In addition, we report that children of less than 2 years tend to have fewer numbers of clones per isolate when compared with older children. Overall, this study shows that the selection for genes that confer drug resistance is not a factor in reducing the genetic diversity of parasite clones in a patient.

Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania.

BACKGROUND: Resistance to the affordable malaria treatments chloroquine and pyrimethamine-sulfadoxine is seriously impeding malaria control through treatment in east Africa. We did an open, alternate drug allocation study to assess the efficacy of chlorproguanil-dapsone in the treatment of falciparum malaria clinically resistant to pyrimethamine-sulfadoxine. METHODS: Children younger than 5 years with non-severe falciparum malaria, attending Muheza district hospital in Tanzania, were treated with the standard regimen of pyrimethamine-sulfadoxine. Patients whose clinical symptoms resolved but who remained parasitaemic 7 days after pyrimethamine-sulfadoxine were followed up for 1 month. Clinical malaria episodes were retreated with either single dose pyrimethamine-sulfadoxine or a 3-day regimen of chlorproguanil-dapsone. Those with parasitaemia after 7 days were treated with chlorproguanil-dapsone. Parasite DNA was collected on day 7 after first treatment with pyrimethamine-sulfadoxine and we looked for point mutations in the genes encoding dihydrofolate reductase (dhfr) and dyhydropteroate synthetase (dhps). FINDINGS: 360 children were enrolled and treated with pyrimethamine-sulfadoxine. On day 7, 192 (55%) of 348 had cleared parasitaemia. Of the remaining 156 parasitaemic children, 140 (90%) were followed up to day 28, and 92 (66%) of 140 developed clinical malaria. These 92 patients were alternately retreated with either pyrimethamine-sulfadoxine (46) or chlorproguanil-dapsone (46). 28 (61%) of 46 children retreated with pyrimethamine-sulfadoxine were still parasitaemic at day 7, compared with three (7%) of 44 [corrected] children retreated with chlorproguanil-dapsone. Resistance to pyrimethamine-sulfadoxine increased from 45% (156/348) at the first treatment to 61% (28/46) after retreatment. 83 of 85 parasite isolates collected after the first pyrimethamine-sulfadoxine treatment, and before and after the second treatments with pyrimethamine-sulfadoxine and chlorproguanil-dapsone showed triple-mutant dhfr alleles, associated with a variety of dhps mutations. INTERPRETATION: Most patients treated with pyrimethamine-sulfadoxine, who remain parasitaemic at day 7, develop new malaria symptoms within 1 month. Chlorproguanil-dapsone was a practicable therapy under these circumstances. Analysis of parasite dhfr and dhps before and after treatment supports the view that pyrimethamine-sulfadoxine resistance in this part of Africa is primarily due to parasites with three mutations in the dhfr domain.

Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the world's burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

A new antiplasmodial coumarin from Toddalia asiatica roots.

A new antiplasmodial coumarin, 5,7-dimethoxy-8-(3'-hydroxy-3'methyl-1'-butene)-coumarin (1), has been isolated from the roots of Toddalia asiatica. This finding supports the traditional use of this plant for the treatment of malaria.

The changing in vitro susceptibility pattern to pyrimethamine/sulfadoxine in Plasmodium falciparum field isolates from Kilifi, Kenya.

Two clinical trials that used Falcidin (Cosmos Ltd., Nairobi, Kenya), the antifolate combination of pyrimethamine/sulfadoxine (PM/SD), as treatment for non-severe falciparum malaria in children at Kilifi, Kenya in 1987-1988 and 1993-1995 have presented an opportunity to assess in vitro the susceptibility trend of Plasmodium falciparum to PM and SD over time on the Kenya coast. The first set of isolates was collected prior to the introduction of PM/SD into the Kenya Medical Research Institute/Wellcome Trust Research unit while the second set was taken soon after PM/SD was introduced in the study area as the first-line treatment drug for uncomplicated falciparum malaria. In the first trial, 69 isolates collected before and after treatment of malaria with PM/SD were tested directly in the field for susceptibility to PM and SD using the standard in vitro micro-test technique, with minimal levels of folate. In the second trial, 97 isolates similarly collected were adapted to culture, and tested as described elsewhere. In both studies, PM and SD susceptibility tests were done separately. There was a highly significant decrease (P < 0.01) in the in vitro sensitivity of P. falciparum isolates to PM and SD between the two trials. In the first trial, the isolates were either sensitive to both PM and SD or resistant to PM and sensitive to SD. During the second trial, isolates were either resistant to PM and sensitive to SD or resistant to both drugs. These results are important in estimating the useful therapeutic life (UTL) of PM/SD in this area and in identifying alternative antifolate drugs.

Molecular evidence of greater selective pressure for drug resistance exerted by the long-acting antifolate Pyrimethamine/Sulfadoxine compared with the shorter-acting chlorproguanil/dapsone on Kenyan Plasmodium falciparum.

Pyrimethamine (PM) plus sulfadoxine (SD) is the last remaining affordable drug for treating uncomplicated malaria in Africa. The selective pressure exerted by the slowly eliminated combination PM/SD was compared with that exerted by the more rapidly eliminated combination chlorproguanil/dapsone (CPG/Dap) on Kenyan Plasmodium falciparum. Point mutations were analyzed in dihydrofolate reductase and dihydropteroate synthase and in the genetic diversity of 3 genes in isolates collected before and after CPG/Dap and PM/SD treatments. PM/SD was associated strongly with the disappearance of fully drug-sensitive parasites and with a significant increase in the prevalence of resistant parasites in subsequent parasitemias. However, this was not a characteristic of treatment with CPG/Dap. Moreover, most of the patients who returned with recrudescent infections were in the PM/SD-treated group. The data predict a longer useful therapeutic life for CPG/Dap than for PM/SD, and, thus, CPG/Dap is a preferable alternative for treatment of chloroquine-resistant falciparum malaria in sub-Saharan Africa.

Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites.

The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is the last affordable drug combination available for wide-scale treatment of falciparum malaria in Africa. Wherever this combination has been used, drug-resistant parasites have been selected rapidly. A study of PM-SD effectiveness carried out between 1997 and 1999 at Kilifi on the Kenyan coast has shown the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 days after treatment) in 39 out of 240 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty-one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a further 20 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten additional isolates were obtained from patients who were parasitemic 7 days after treatment but who were not sampled before treatment. More than 65% of the isolates (30 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinations of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mutant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were "pure" triple mutants. In these isolates, the combination of a triple mutant DHFR and wild-type DHPS was detected in 6 of 29 cases (20.7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%), and the combination of a triple mutant DHFR and a double mutant (A437G, L540E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrate that the triply mutated allele of DHFR with or without mutant DHPS alleles is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of PM-SD as a new malaria treatment.

The antimalarial triazine WR99210 and the prodrug PS-15: folate reversal of in vitro activity against Plasmodium falciparum and a non-antifolate mode of action of the prodrug.

We have studied the reversal of activity against Plasmodium falciparum of WR99210, a triazine antimalarial drug, and of the pro-drug PS-15 by folic acid (FA) and folinic acid (FNA). Folic acid and FNA inhibit the growth of P. falciparum in vitro at concentrations > 10(-4.5) and 10(-3.5) mol/L, respectively. The activity of pyrimethamine against Kenyan strains M24 and K39 is reduced 10-12-fold by 10(-5) mol/L of FA, and virtually eliminated by 10(-5) mol/L of FNA. Folates do not antagonise the action of WR99210 against Kenyan strains, and only partially antagonize the action of WR99210 action against the Southeast Asian strains V1/S and W282. Similarly, FA and FNA exerted weak or no antagonism of the action of PS-15. The inability of folates to antagonize the action of WR99210 can be explained in terms of high drug-enzyme affinity, but this does not account for the inability of FA and FNA to antagonize PS-15. These results suggest that action of PS-15 against P. falciparum is primarily due to a non-folate mechanism.

Kenyan Plasmodium falciparum field isolates: correlation between pyrimethamine and chlorcycloguanil activity in vitro and point mutations in the dihydrofolate reductase domain.

Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC50s) for PM and CCG (P < 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC50s of 3.71 +/- 6.94 and 0.24 +/- 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC50s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC50s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).

The disposition of intramuscular artemether in children with cerebral malaria; a preliminary study.

The disposition of intramuscular artemether (AM) was studied in 26 Kenyan children with cerebral malaria. Antimalarial activity determined by bioassay was compared with total plasma AM plus dihydroartemisinin (DHA) determined by high power liquid chromatography (HPLC). Therapeutic levels were achieved in most subjects (21/26) within 1 h of receiving intramuscular AM (3.2 mg/kg), with close correlation between bioassay and HPLC measurements (r = 0.706). However, there was marked inter-individual variation, antimalarial activity was undetectable in 5 subjects ('non-absorbers'), and plasma concentrations were lower in subject with respiratory distress. The 50% parasite clearance time was significantly longer in non-absorbers (mean = 13.1 h, SD = 10.8 vs. mean = 7.8 h, SD = 5.5; P = 0.013). We conclude that the bioavailability of intramuscular AM in children with severe malaria may be highly variable, particularly in the presence of respiratory distress, and may be associated with an inadequate therapeutic response.

The efficacy of antifolate antimalarial combinations in Africa: a predictive model based on pharmacodynamic and pharmacokinetic analyses.

At present, effective treatment for non-severe malaria is the most important malaria control strategy in Africa. Pyrimethamine-sulfadoxine (PSD) is rapidly becoming the first-line treatment in areas of chloroquine resistance, although the parasite chemoresistance factors that dispose towards clinical failure with PSD are still unclear. Here, Bill Watkins and colleagues analyse the relationship between the pharmacokinetic properties of two treatment combinations (PSD and chlorproguanil-dapsone) in vivo and the respective in vitro isobolograms for parasites with specific drug-resistance patterns. From this relationship, they develop a hypothesis that may explain clinical drug failure and differential efficacy between treatments. The deductions can be tested in field studies to validate or refute the model.

Antimalarial activity in crude extracts of Malawian medicinal plants.

Aqueous and organic fractions from Cassia abbreviata, Senna petersiana (both Caesalpiniaceae) and Azanza garckeana (Malvaceae) were tested for in-vitro antimalarial activity against the multi-drug-resistant, Vietnam-Smith strain of Plasmodium falciparum; VI/S. Both roots and leaves from these Malawian medicinal plants were investigated. High activity, with a median inhibitory concentration < 3 micrograms/ml, was seen in the organic fractions of C. abbreviata and S. petersiana, the two species most commonly cited by traditional healers in an ethnobotanical investigation of Malawian antimalarials. Extracts of A. garckeana showed weaker activity. Biologically active compounds have thus been detected within species of the family Caesalpiniaceae. Ethnobotanical investigation appears to be useful in identifying plants with antimalarial activity.

Measurement of physiological concentrations of dapsone and its monoacetyl metabolite: a miniaturised assay for liquid or filter paper-absorbed samples.

A modification of existing HPLC assay methods is described for the measurement of dapsone and monoacetyldapsone in 50-microliter samples of plasma and whole blood. This method, in particular the use of small sample volumes dried onto filter paper strips, is applicable to multi-sample clinical and pharmacokinetic studies in children with malaria, who are often anaemic, and where sample volume must be kept to a minimum. Basified samples were extracted into 5 ml of ethyl acetate-tert.-butylmethyl ether (1:1, v/v), chromatographed on a mu BondapaK C18, 10-micron column with water-acetonitrile-glacial acetic acid (81:17.5:5, v/v) containing 2 g/l l-octanesulphonic acid as the mobile phase and detected at 274 nm.

Cutaneous hypersensitivity reactions to thiacetazone, HIV infection and thiacetazone concentrations in plasma.

We have studied the relationship between the plasma concentration-time profile of thiacetazone over the 24 h between doses [AUC(0.24h)] and the incidence of cutaneous reactions among HIV-infected patients with tuberculosis in Kenya. Cutaneous reactions due to thiacetazone occurred in 4/14 [28.6%] HIV+ve patients compared with 3/47 [6.4%] HIV-ve patients [RR = 4.48, 95% CI-1.1 to 17.7], and all resolved on alternative therapy. Among the HIV+ve patients, those with cutaneous reactions had higher AUC(0.24h) values, although the difference was not significant. These results do not exclude pharmacokinetic change as being at least partly responsible for cutaneous reactions to TCZ in HIV+ve patients, and do not refute an immunological basis for the reaction. With regard to the operational use of TCZ in Africa, there is no indication that a modification of the dose will reduce the frequency of drug reactions.

Potent antimalarial activity of the alkaloid nitidine, isolated from a Kenyan herbal remedy.

Bioassay-guided fractionation of extracts of Toddalia asiatica, a plant used by the Pokot tribe of Kenya to treat fevers, has yielded the alkaloid nitidine as the major antimalarial component. Fractions containing nitidine have in vitro 50% inhibitory concentrations against Plasmodium falciparum in the range of 9 to 108 ng/ml for a range of chloroquine-susceptible and -resistant strains. The results show a lack of cross-resistance between chloroquine and nitidine.

In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs.

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.

Halofantrine pharmacokinetics in Kenyan children with non-severe and severe malaria.

1. Kenyan children with uncomplicated malaria given oral halofantrine (HF; non-micronised suspension; 8 mg base kg-1 body weight 6 hourly for three doses) showed wide variation in the disposition of HF and desbutylhalofantrine (HFm). 2. Eight Kenyan children with severe (prostrate) falciparum malaria who were receiving intravenous quinine, were given the same HF regimen by nasogastric tube. One patient had undetectable HF and two had undetectable HFm at all times after drug administration. 3. The mean AUC(0,24 h) of HF in prostrate children was half (7.54 compared with 13.10 micrograms ml-1 h) (P = 0.06), and that for HFm one-third (0.84 compared with 2.51 micrograms ml-1 h) (P < 0.05) of the value in children with uncomplicated malaria. 4. Oral HF may be appropriate for some cases of uncomplicated falciparum malaria in Africa, but in patients with severe malaria, the bioavailability of HF and HFm may be inadequate.

Differential extraction of artemether and its metabolite dihydroartemisinin from plasma and determination by high-performance liquid chromatography.

A method is described for the separation of artemether (ARM) from its metabolite dihydroartemisinin (DHA) and determination by HPLC. The basis of the separation is differential extraction of the drugs from plasma as a function of plasma pH. Hexane extracted ARM from basiffied plasma and both ARM and DHA from normal plasma. Derivatized extracts were chromatographed on a 5-microns ODS column with water-acetonitrile (40:60) as mobile phase and detected at 254 nm. The method removes the need for expensive absorption cartridges (BondElut). Chromatography has been improved and the elution time shortened in comparison with previous methods.