The activity of pyrimethamine and sulphadoxine against Plasmodium falciparum determined by the in vitro microtechnique

The reported increase of infections of Plasmodium falciparum which are no longer susceptible to a combination of pyrimethamine and sulphadoxine—Fansidar—emphasizes the need for an in vitro test to determine the presence and prevalence of drug-resistant parasites. Studies with the pyrimethaminesensitive FCB strain and the pyrimethamine-resistant FTA strain showed that the in vitro microtechnique can be used to determine differences in the susceptibility of these two strains to pyrimethamine and to pyrimethamine-sulphadoxine combinations. The FCB strain was almost six times more susceptible to pyrimethamine than the FTA strain. Although relatively high concentrations of sulphadoxine alone exerted no detectable antimalarial effects, the sulphonamide potentiated the activity of pyrimethamine against both strains of P. falciparum. This synergistic activity was relatively more pronounced against the pyrimethamine-resistant strain, particularly at lower concentrations of sulphadoxine. Further studies are indicated to determine to what extent findings obtained with the in vitro microtechnique can be correlated with the response of falciparum infections to treatment with pyrimethamine-sulphadoxine combinations.     

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.     

Comparison of in vitro pyrimethamine assays and in vivo response to sulphadoxine-pyrimethamine in Plasmodium falciparum from Papua New Guinea

The in vitro pyrimethamine sensitivity of 20 Plasmodium falciparum isolates from Papua New Guinea children was determined. The children were treated with sulphadoxine-pyrimethamine and six were found to have clinically resistant malaria. The P. falciparum isolated from these subjects were more resistant to pyrimethamine in vitro than 13 of the isolates from sensitive cases. These results suggest that pyrimethamine sensitivity alone may be a good indicator of in vitro response to sulphadoxine-pyrimethamine.     

Clones of different sensitivities in drug-resistant isolates of Plasmodium falciparum

Clones of two Thai isolates of Plasmodium falciparum were prepared and examined for variations in drug susceptibility and electrophoretic properties of enzymes. Both isolates were found to include mixtures of genetically distinct parasites. Of particular significance was the finding that one isolate (T₉), which on initial testing was resistant to chloroquine, pyrimethamine and sulfadoxine—pyrimethamine, yielded a clone of parasites markedly more sensitive to these three drugs, while five other clones resembled the original isolate in drug susceptibility.     

A new in vitro test for pyrimethamine/sulfadoxine susceptibility of Plasmodium falciparum and its correlation with in vivo resistance in Kenya

A useful in vitro method for field evaluation of Plasmodium falciparum sensitivity to pyrimethamine/sulfadoxine is described. Thirty-five Kenyan schoolchildren infected with P. falciparum were treated with this drug combination and followed up for 5 weeks. In vitro tests for sensitivity to these drugs and to chloroquine were performed before starting treatment. All infections cleared within 7 days of treatment, but 5 children had recurrent parasitaemia within 35 days. The original isolates from 4 of these 5 children had an in vitro response to pyrimethamine/sulfadoxine similar to a known strain that was resistant to these drugs; only 4 of the remaining 30 isolates from patients in whom recurrent parasitaemia did not occur had a resistant in vitro response (P = 0.006). In the patient with recurrent parasitaemia whose initial isolate appeared sensitive to pyrimethamine/sulfadoxine, the recurrent isolate had a resistant pattern in vitro, suggesting either reinfection or selection of a resistant subpopulation following treatment. The in vitro response to this drug combination was correlated with the in vitro response to either drug alone and with the in vitro response to chloroquine. Two of the 5 infections with recurrent parasitaemia after initial pyrimethamine/sulfadoxine treatment were resistant to chloroquine in vivo. The in vitro test for pyrimethamine/sulfadoxine should be useful for mapping the spread of multidrug-resistant P. falciparum.     

Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

In 1978, studies on the chloroquine sensitivity of Plasmodium falciparum were carried out in the district of Sennar, Sudan. The results of the in vivo tests showed parasites resistant at the RI level only, but the mean clearance time of trophozoites from the blood was higher than for strains found in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reported from Ethiopia. The chloroquine sensitivity of P. falciparum from Sennar is close to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generally found to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration of chloroquine was related to the parasite density owing to the selective uptake of the compound by the parasitized cells.     

Chloroquine tolerance of Ethiopian strains of P. falciparum

11 of 41 Ethiopians in hospital with P. falciparum malaria experienced a delayed recrudescence of parasitaemia following treatment with 10 mg. chloroquine base per kg. of body weight. Parasites from 25 of the 41 patients were successfully cultivated in vitro, and 9 isolates showed development in chloroquine concentrations of 0·5 to 1·0 millimicromoles per c.c. of blood. 3 isolates with development at the 1·0 millimicromole level were from patients who experienced a recrudescence of parasitaemia. In vivo and in vitro results suggest a chloroquine responsiveness of some Ethiopian isolates of P. falciparum which is between that of the sensitive Uganda I strain and the resistant Malayan (Camp.) strain; a finding not previously documented in African parasites.     

Evaluation of four therapeutic regimens for falciparum malaria in Mozambique, 1986

A randomized study on the effect of the following four treatment regimens on Plasmodium falciparum parasitaemia was carried out on 200 asymptomatic schoolchildren in Maputo, Mozambique: chloroquine (25 mg/kg body weight), amodiaquine (25 mg/kg), sulfadoxine—pyrimethamine (25 mg/kg and 1.25 mg/kg), or amodiaquine (25 mg/kg) + sulfadoxine—pyrimethamine (25 mg/kg and 1.25 mg/kg) administered on the third day of the study. The results of in vivo tests indicated that 94% of the infections were resistant to chloroquine, 76% to amodiaquine, and 16% to sulfadoxine—pyrimethamine. The cure rate with amodiaquine + sulfadoxine—pyrimethamine was 100%, which was not significantly different from that with sulfadoxine—pyrimethamine alone; the latter regimen was the most rapidly acting of the treatments studied. It is concluded that amodiaquine is not an appropriate substitute for chloroquine, but that the effect of the combination amodiaquine + sulfadoxine—pyrimethamine may be superior to that of sulfadoxine—pyrimethamine alone, although this requires further study.     

In vivo assessment of the sensitivity of Plasmodium falciparum to sulphadoxine/pyrimethamine combination (Fansidar) in six localities in Tanzania where chloroquine-resistant P. falciparum has been detected

The sensitivity of Plasmodium falciparum strains to Fansidar (500 mg sulphadoxine/25 mg pyrimethamine) was tested in vivo in six localities in the United Republic of Tanzania where chloroquine-resistant P. falciparum strains have been demonstrated by both in vivo and in vitro tests. Single doses as recommended by the manufacturers achieved 100% clearance of parasitaemia in five localities with mean clearance period of between 2·2 and 2·9 days. In one locality (Gonja) the recommended dose failed to clear parasitaemia in two of the 38 cases (5·3%) within seven days. The possibility of using this drug combination for the treatment of chloroquine-resistant P. falciparum strains in the United Republic of Tanzania is discussed.     

Plasmodium falciparum sensitivity to chloroquine in the Buyo Region, Ivory Coast

As part of a project, supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, for achieving a reduction of mortality due to Plasmodium falciparum by means of a primary health care programme, in vivo and in vitro sensitivity testing of P. falciparum to chloroquine was carried out in the Buyo Region of the Ivory Coast. Blood samples from a total of 595 children aged 2-13 years from 5 villages were screened microscopically and 32 of these children were selected for in vivo testing and 36 for in vitro testing. All 32 in vivo test patients were treated with 25 mg chloroquine base per kg, given in divided doses over 3 days, and all of them completed the 7-day observation period. Daily blood slides were taken and these were negative for asexual parasites on days 3 to 7. The mean parasite clearance time was 1.8 days. The 36 in vitro tests produced satisfactory results in 19 isolates. Complete inhibition was achieved in all 19 at 5.7 pmol chloroquine base per well (1.14 μmol/l of blood); 7 of these isolates showed complete inhibition at 1 pmol, 15 at 2 pmol and 17 at 4 pmol.     

Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests

In vitro drug resistance tests of ten isolates of Plasmodium falciparum from three different collection points in Central Thailand have been carried out, and the results compared with those of similar tests with a drug-sensitive West African isolate. Judged by concentration of drug tolerated, the Thai isolates appeared to be about 10 times as resistant to chloroquine, and usually about 10⁵ times as resistant to pyrimethamine, as the African isolate. A little variation amonst the Thai isolates was detected.     

Identification of Plasmodium falciparum isolates lacking histidine-rich protein 2 and 3 in Eritrea

The histidine-rich protein 2 of Plasmodium falciparum is the most common malaria antigen targeted by rapid diagnostic tests for the specific diagnosis of P. falciparum. Recently, pfhrp2 gene deletions have been documented in P. falciparum isolates from South America and some multiple endemic countries in Africa and Asia. Parasites with such gene deletions can produce false negative diagnostic results using HRP2-based rapid diagnostic kits. In the present work, the prevalence of P. falciparum parasites lacking pfhrp2, pfhrp3, which produces a second P. falciparum antigen that is recognized by PfHRP2 -based rapid diagnostic tests, and their flanking genes was evaluated in 135 P. falciparum isolates from Gash Barka region and in 9 isolates from Debub region, in Eritrea. In the analyzed samples, 56% (81/144) of isolates were pfhrp2/pfhrp3 positive, while 9.7% (14/144) showed deletion of exon 2 of pfhrp2 gene and 43% (62/144) of isolates lacked the pfhrp3 gene. These results suggest that the pfhrp2 and pfhrp3 deletion phenomenon is present in a considerable proportion in the study areas, thus making the HRP2/3 based rapid diagnostic tests not completely reliable for malaria diagnosis in Eritrea.     

Fansidar resistant falciparum malaria acquired in South East Asia

A case of Plasmodium falciparum malaria resistant to Fansidar (sulphadoxine plus pyrimethamine) at a level corresponding to R III and resistant to chloroquine is reported. The infection was most certainly acquired in Malaysia, but diagnosed and treated in a non-malarious area.Normal resorption and elimination rates of the Fansidar components excludes cure failure due to abnormal drug fate in the host.P. falciparum parasites from the patient have been maintained in in vitro cultures.The patient was permanently cured with mefloquine.     

Sulfalene with pyrimethamine and chloroquine with pyrimethamine in single-dose treatment of Plasmodium falciparum infections. A trial in a rural population in northern Nigeria

A comparative trial was carried out in northern Nigeria of the ability of the drug combinations chloroquine—pyrimethamine and sulfalene—pyrimethamine to clear the peripheral blood stream of asexual forms of P. falciparum within 7 days. The reappearance of asexual P. falciparum forms within the 70-day follow-up period and the occurrence of vomiting during the 2-3 hours following administration of the drugs were also recorded. The purpose of the trial was to choose the more suitable of the two drug combinations for repeated mass administration in the intervention phase of a collaborative field research project in the epidemiology and control of malaria in the African savannah. No differences were observed between the two drug combinations from a parasitological point of view. However, the sulfalene—pyrimethamine combination was found easier to administer and occasioned fewer records of vomiting. It was therefore recommended for use in the project.     

Plasmodium falciparum in Haiti: susceptibility to pyrimethamine and sulfadoxine-pyrimethamine

Eighteen patients with Plasmodium falciparum infection were studied in Port-au-Prince, Haiti, to monitor the response of the malaria parasite to sulfadoxine-pyrimethamine. In all infections the parasitaemia was cleared rapidly following treatment with standard dose of the drug combination; no recrudescence was observed during follow-up periods of 1 week (4 patients) and 4 weeks (14 patients). Parallel in vitro tests indicated that 5 of the 16 isolates successfully tested were resistant to pyrimethamine alone.     

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.     

Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype

Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.     

Pyrimethamine sensitivity in Plasmodium falciparum: determination in vitro by a modified 48-hour test

Four strains of Plasmodium falciparum recently isolated in culture were assessed in vitro for their response to pyrimethamine. A simple modified 48-hour test was used, which showed two strains to be sensitive to the drug in vitro, while the other two were resistant at a very high level. In the two strains for which relevant clinical information was available the in vivo response to pyrimethamine was corroborated by the in vitro findings. This modified 48-hour test is thus useful for determining patterns of drug sensitivity in laboratory-adapted strains, and would be a valuable asset if found to be equally applicable under field conditions.     

Assessment of chloroquine sensitivity of Plasmodium falciparum in Choluteca, Honduras

During an outbreak of urban malaria in Choluteca, Honduras, the response of local isolates of Plasmodium falciparum to chloroquine was assessed. The 7-day WHO alternative standard field test was used together with three in vitro tests: the Rieckmann macro- and micromethods and a new 48-hour test which underwent its first field trial in this study. No chloroquine resistance was found in in vivo tests in 10 patients or in the in vitro tests on blood samples from 6 patients.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria: II. Correlation of in vitrowith in vivo sensitivity

Plasmodium falciparum malaria was treated in 82 children with 25 mg/kg chloroquine orally over three days. They were observed for 28 days during which blood films were examined periodically for malaria parasites. Asexual forms of P. falciparum, present in the blood films of all the patients before commencing treatment, disappeared rapidly and by the third day no parasites were seen in blood films from any of them. Among the patients observed for more than three days, blood films remained negative throughout the observation period. In vitro tests of sensitivity of blood samples from 10 patients showed chloroquine concentrations of 0·5 to 0·8 nmol/ml to inhibit completely maturation from ring forms to schizonts.This suggests that P. falciparum in the Ibadan area is probably still fully sensitive to chloroquine.