Preclinical drug evaluation system in the Plasmodium knowlesi baboon model of malaria: the methotrexate study.

BACKGROUND: Drug resistance against first-line antimalarials warrants search for new lead compounds and repurposing of drugs such as methotrexate. Animal models are required for preclinical drug development before clinical testing. This study aimed to develop a preclinical drug development system in baboons infected with Plasmodium knowlesi. METHODS: Protocols for drug administration, pharmacokinetics, clinical chemistry and haematology were developed in the baboon model. Baboons were infected with P. knowlesi and methotrexate administered orally for 5 days. Clinical signs, parasitaemia, gross and histopathology examinations were conducted to determine effect of methotrexate in baboons. RESULTS: No major clinical chemistry, haematology and pathological changes attributable to methotrexate were observed. Parasitaemia suppression of 77.67% was achieved at a methotrexate dose of 3.0 mg/kg. CONCLUSIONS: A protocol for preclinical drug development in the baboon was optimized. Methotrexate suppressed P. knowlesi malaria in baboons. These findings warrant further characterization of methotrexate for use in combination therapy.

Plasmodium berghei ANKA: selection of resistance to piperaquine and lumefantrine in a mouse model.

We have selected piperaquine (PQ) and lumefantrine (LM) resistant Plasmodium berghei ANKA parasite lines in mice by drug pressure. Effective doses that reduce parasitaemia by 90% (ED(90)) of PQ and LM against the parent line were 3.52 and 3.93 mg/kg, respectively. After drug pressure (more than 27 passages), the selected parasite lines had PQ and LM resistance indexes (I(90)) [ED(90) of resistant line/ED(90) of parent line] of 68.86 and 63.55, respectively. After growing them in the absence of drug for 10 passages and cryo-preserving them at -80 degrees C for at least 2 months, the resistance phenotypes remained stable. Cross-resistance studies showed that the PQ-resistant line was highly resistant to LM, while the LM-resistant line remained sensitive to PQ. Thus, if the mechanism of resistance is similar in P. berghei and Plasmodium falciparum, the use of LM (as part of Coartem) should not select for PQ resistance.

The role of sequential administration of sulphadoxine/pyrimethamine following quinine in the treatment of severe falciparum malaria in children.

Sulphadoxine/pyrimethamine (SP) is often administered with quinine in the treatment of severe falciparum malaria to shorten the course of quinine. The efficacy of SP alone in the treatment of non-severe malaria has been declining rapidly in East Africa, raising concerns of the usefulness of a shortened course of quinine followed SP. We audited the efficacy of quinine/SP in the treatment of severe malaria in Kenyan children. Children with severe falciparum malaria were treated with parenteral quinine followed by a single oral dose of SP. A clinical evaluation was performed 3 weeks later in which a blood sample was obtained for full haemogram, blood slide and analysis of the parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) codons, mutations of which are associated with resistance to SP. A total of 452 children were enrolled, of whom 374 completed the study. Fifty-two (13.9%) children were parasitaemic by 3 weeks of whom 17 (4.5%) had fever as well. The treatment failure group had a significantly higher parasitaemia (129 061 vs. 43 339; P<0.001) and haemoglobin on admission, but only admission parasitaemia independently predicted treatment failure. Those with treatment failure had a significantly lower rise in haemoglobin at 3 weeks compared with treatment successes (9.0 vs. 10.0 g/dl). Of the 76 parasite isolates collected before treatment, 40 (53%) were triple mutant DHFR-double DHPS (Tp-Db), the genotype most associated with SP resistance. Three weeks after SP treatment, the proportion of Tp-Db increased to 72% (31/43). The high treatment failure rate and proportion of parasites with Tp-Db negate the use of SP to shorten the course of quinine treatment in East Africa.

Chloroquine, sulfadoxine-pyrimethamine and amodiaquine efficacy for the treatment of uncomplicated Plasmodium falciparum malaria in Upper Nile, south Sudan.

The current first-line and second-line drugs for Plasmodium falciparum malaria in South Sudan, chloroquine and sulfadoxine-pyrimethamine (SP), were evaluated and compared with amodiaquine, in an MSF-Holland-run clinic in eastern Upper Nile, South Sudan from June to December 2001. Patients with uncomplicated malaria and fever were stratified by age group and randomly allocated to one of 3 treatment regimes. A total of 342 patients was admitted and followed for 14 d after treatment. The dropout rate was 10.2%. Of those who completed the study, 104 were treated with chloroquine (25 mg/kg, 3 d), 102 with SP (25 mg/kg sulfadoxine and 1.25 mg/kg pyrimethamine, single dose) and 101 with amodiaquine (25 mg/kg, 3 d). Adequate clinical response was observed in 88.5% of patients treated with chloroquine, 100% of patients treated with SP and 94.1% of patients treated with amodiaquine. In children aged < 5 years, the success rate was lower: 83.3% for chloroquine and 93.0% for amodiaquine. In adults no treatment failures were found, but children aged 5-15 years showed intermediate levels. In addition, we determined the initial genotypes of dhfr and dhps of 44 isolates from the SP-treated group and > 80% were found to be wild type for dhfr and 100% for dhps. Two percent of isolates had a single mutation and 16% had double mutations of dhfr. These data are in full agreement with the clinical effectiveness of SP. A change in malaria treatment protocols for South Sudan is recommended.

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.

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.

Mixed populations of Trypanosoma brucei in wild Glossina palpalis palpalis.

In many previous characterization studies of Trypanozoon, isolates have been subpassaged numerous times in laboratory rodents until a quantity of trypanosomes sufficient for analysis has been obtained. In addition to the numerous biochemical effects of such a process on the parasite, it appears probable that adaptation to an unnatural host may also serve to filter out less virulent populations from mixed infections, leading to an underestimate of the true level of genetic diversity. By the early cloning of trypanosomes from susceptible captive flies infected from the primary isolate--the midgut of a wild tsetse--the present study provides evidence of the range of genetically different Trypanosoma brucei populations which may coexist within the midgut of individual tsetse flies in nature. The three primary isolates from tsetse yielded one, five and nine genetically distinct populations. Cloned populations were confirmed as T. brucei using the polymerase chain reaction, and were characterized by karyotype analysis and multilocus isoenzyme electrophoresis. These data allowed a limited assessment of the level of genetic variability in natural populations of T. brucei.