Chloroquine sensitivity of Plasmodium falciparum in vivo in a savanna town in Cameroon.

Chloroquine-resistant Plasmodium falciparum has been reported in the Sahel and forest regions of Cameroon since 1985. In vivo response to chloroquine treatment (25 mg/kg) was assessed in 19 patients with malaria in the savanna North-West province. 58% of the cases showed RII resistance to chloroquine. RIII resistance was suspected in one patient. Only 35% of cases showed complete parasite clearance by day 5 of treatment. Chloroquine reduced parasite counts by at least 87% in all patients. Chloroquine resistance now seems to be well established and widespread in Cameroon. Its rapid spread and the prevalence of resistance suggest the existence of sustained drug pressure resulting in rapid selection of less sensitive strains. Unfortunately, similar pressure is also being exerted with quinine.     

Chloroquine resistant Plasmodium falciparum on the island of Flores, Indonesia

The in vitro sensitivity of Plasmodium falciparum to chloroquine was studied in parasites from 45 children on the island of Flores, Indonesia. The micro in vitro culture technique and a 12-volt battery-operated field incubator were found to be well suited to the field situation encountered. Parasites from seven children (15.6%) were resistant to chloroquine in vitro: two at 8 pico-moles of chloroquine, two at 16 pico-moles, and three at 32 pico-moles. This is the first report of chloroquine resistant from Flores.     

Chloroquine susceptibility of Plasmodium falciparum in Haiti

In January and February 1982, in vivo and in vitro studies of the chloroquine sensitivity of Plasmodium falciparum were conducted in Port-au-Prince, Haiti. Of 19 infections tested in vivo using the WHO extended test, all but one were susceptible to the drug; the remaining case showed a recurrence of parasitaemia on day 28. Of the 19 corresponding 48-hour in vitro tests, 16 provided interpretable results; 12 tests demonstrated sensitivity to the drug, while in the remaining 4, parasite multiplication was inhibited only at drug concentrations higher than that previously accepted as indicative of sensitivity. (These four included the isolate from the patient who had a recurrence of parasitaemia on day 28.) While these results provide no absolute demonstration of chloroquine resistance, they underline the need for close monitoring of the susceptibility to chloroquine of P. falciparum in Haiti.     

Multiple drug resistant Plasmodium falciparum malaria in a pregnant indigenous Zambian woman

A 24 year old Zambian female presented with falciparum malaria when 27 weeks pregnant. She had recently travelled to the copperbelt from Solwezi in the North-West Province of Zambia. Oral chloroquine in a dose of 29 mg/kg failed to clear the parasitaemia. Chloroquine resistance was confirmed by testing in vitro and in vivo. In addition, Fansidar (2 courses), amodiaquine, quinine and quinine plus erythromycin failed to achieve radical cure. Quinine resistance was confirmed in vitro and in vivo. She was eventually cured by 10 d of quinine plus clindamycin, which was greatly assisted by the spontaneous delivery of a live normal infant at 37 weeks gestation. The baby's birth weight was 2.68 kg and its blood slide was negative for malaria.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria

Thirty-five children with Plasmodium falciparum malaria were treated with 25 mg/kg chloroquine over three days and observed for seven days during which blood films were examined daily for malaria parasites.Asexual forms of P. falciparum which were present in the blood films of all the patients before commencing treatment disappeared rapidly from the blood so that by the third day no parasites were seen in the blood film. The blood films remained negative for the rest of the seven-day observation period.Plasma chloroquine determination in eight of the patients showed high blood levels during the first three days.The results do not confirm the suspicion of chloroquine-resistant P. falciparum in the area studied although RI level of resistance by WHO criteria was not excluded.     

Chloroquine resistance of Plasmodium falciparum in semi-immune children in Zaria, northern Nigeria

Fifty-nine children with Plasmodium falciparum malaria were subjected to the World Health Organization (WHO) extended field test to assess the in vivo sensitivity of the parasite to chloroquine in Zaria urban area, Nigeria. The parasites in 53 children (90%) were positive but those in 6 (10%) were resistant at the RI-RII level. 36 isolates from the patients were successfully cultured in vitro for the WHO standard microtest. 13 (37%) of the isolates underwent schizogony at chloroquine concentrations of 1.6 microM/litre and above. Probit analysis showed that the chloroquine concentrations producing 50% (EC50), 90% (EC90) and 99% (EC99) schizont inhibition were 0.4, 1.6 and 4.9 microM/litre, respectively. The results indicate a rapid decline in the sensitivity of P. falciparum to chloroquine in the study area during the past 3 years.     

Chloroquine resistance of Plasmodium falciparum in Brazil detected by a simple in vitro method

The sensitivity of Plasmodium falciparum to antimalarial drugs is primarily determined by observing changes in the level of parasitaemia after administration of drugs to man. Performance of such a study and interpretation of its results may present certain difficulties, especially when conducted among persons living in malarious areas. The present study was conducted to evaluate the usefulness of a simple in vitro method for detecting chloroquine-resistant strains among infected residents of Mato Grosso, Brazil. The findings indicated the participants were infected with chloroquine-resistant P. falciparum. Subsequent administration of a standard regimen of chloroquine (25 mg/kg) to these subjects confirmed the findings observed in vitro by failing to cure 28 out of 30 infections. This in vitro technique seems to provide a useful method of determining the presence or prevalence of chloroquine-resistant strains in a given area.     

Chloroquine resistance of Plasmodium falciparum is associated with severity of disease in Nigerian children

Chloroquine resistance of Plasmodium falciparum in vitro was significantly higher in isolates from patients with severe malaria than those with uncomplicated disease. This association may be due to either progression of uncomplicated to severe disease following chloroquine failure or increased virulence of chloroquine-resistant parasites. The implication of this for antimalarial treatment policy is discussed.     

Chloroquine blood concentrations and molecular markers of chloroquine-resistant Plasmodium falciparum in febrile children in northern Ghana

Plasmodium falciparum malaria is a predominant reason for health care utilization among children in sub-Saharan Africa. Despite the spread of resistance, chloroquine (CQ) is the most commonly used antimalarial. Little is known about the pattern of CQ use and resistance to the drug prior to attendance at a health care facility, and its impact on clinical presentation in children attending health care facilities in endemic regions. In a cross-sectional study among 840 febrile children presenting at a primary health care facility in northern Ghana from September to December 2000, CQ blood levels were measured by enzyme-linked immunosorbent assay and parasite isolates were genotyped for the CQ resistance markers pfcrt T76 and pfmdr1 Y86. Plasmodium falciparum was present in 95% by polymerase chain reaction and CQ was detected in 64% of the children. Concentrations of CQ in blood ranged from 31 to 3897 nmol/L (median 198 nmol/L). The pfcrt T76 and pfmdr1 Y86 resistance markers were detected in 84% and 57% of the isolates, respectively, and were selected by CQ. A significant trend for higher frequencies of the resistance markers with increasing CQ concentrations was observed. In this typical primary health care setting in sub-Saharan Africa, CQ use prior to attendance at a health care facility and CQ-resistant P. falciparum are frequent. As CQ selects resistant P. falciparum genotypes, CQ should be omitted as a first-line drug even in primary health care facilities when self-treatment with CQ is common.     

Amodiaquine fails to cure chloroquine resistant Plasmodium falciparum in the Punjab

We evaluated amodiaquine as a replacement drug for treating falciparum malaria in an area of Pakistani Punjab where chloroquine-resistant Plasmodium falciparum has recently emerged. Amodiaquine appeared to be 4 to 8 times more effective than chloroquine when P. falciparum isolates were tested in vitro. However, the recrudescence rate was greater than 50% after oral treatment with 20 mg/kg amodiaquine given over two successive days. This lack of therapeutic response from amodiaquine may have been due to selection of resistant parasites in the villages where the study was performed through extensive use of chloroquine for presumptive malaria treatment during the preceding 18 months. We conclude that amodiaquine is not a suitable replacement for chloroquine for treating falciparum malaria in our study area despite in vitro sensitivity data suggesting that it would be efficacious. Baseline in vitro sensitivity to mefloquine is also reported.     

Sera from Cameroon induce crisis forms during Plasmodium falciparum growth inhibition studies in vitro

Sera collected from 176 children and adults from 3 different regions of Cameroon were used in inhibition assays against Plasmodium falciparum in vitro. The individuals had been classified clinically as malarious or non-malarious. Of the 58 non-malarious persons questioned, 39 claimed never to have had symptoms of the disease. Sera were tested for inhibitory effects on either merozoite entry and retardation of growth or inhibition of intraerythrocytic growth. The sera from malarious individuals showed only 9% and 29% mean inhibition values at 24 and 48 h, respectively, while those from the non-malarious persons showed 59% and 73% inhibition for the same periods. Of the 58 sera from non-malarious persons, 34 produced typical crisis forms by 24-36 h. Some sera also inhibited merozoite entry into erythrocytes. Our data suggest an important relationship between acquired immunity to P. falciparum in Cameroon and the types of inhibition described here.     

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.     

Sera from Cameroon recognize proteins of Plasmodium falciparum isolates from geographically diverse areas of the world

Immunological reactions of clinically-defined sera collected from 176 children and adults (3 to 63 years of age) living in malaria endemic regions of Cameroon were evaluated by ELISA, growth inhibition studies and immunoprecipitation assays using different parasite isolates from geographically diverse areas. The proportions of sera positive by ELISA and with positive growth inhibitory activity tended to increase with increased age. SDS-PAGE analyses of immunoprecipitates using [35S]methionine labelled parasite polypeptides revealed that a wide range of proteins was recognized by the sera. There were many similarities in the patterns of antigens immunoprecipitated in the different isolates, particularly when immune sera were used. Variability in response was more evident in sera collected from children. These findings suggest that strains may share components which generate protective immunity.     

Source of drug resistant Plasmodium falciparum in a potential malaria elimination site in Saudi Arabia

A major challenge to the success of malaria control program in Saudi Arabia is the high influx of expatriates and holy visitors from malaria endemic countries. In the present study we examined whether drug resistant parasite genotypes reported in Jazan region, southwest of Saudi Arabia are imported or developed locally. We examined 178 Plasmodium falciparum isolates for alleles of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), associated with Sulfadoxine-Pyrimethamine (SP) resistance, and three microsatellites flanking each gene. In addition, we examined a neutral polymorphic gene (Pfg377). We compared the dhfr and dhps haplotypes in Jazan, using network analysis, to an existing similar data set of 94 P. falciparum isolates from eastern Sudan. In Jazan, double mutant dhfr allele (51I, 108N) occurred with a prevalence of 33%. The vast majority (99%) of dhps were wild-type alleles. The mean expected heterozygosity (H_e) of microsatellites around mutant dhfr alleles (H_e = 0.312; n = 60) was lower (P ? 0.05) than that around the wild-type allele (H_e = 0.834; n = 116). Also, the mutant dhfr isolates showed high H_e for dhps (H_e = 0.80) and the non-drug resistance locus Pfg377 (H_e = 0.63) indicative of selection for mutant dhfr only. The predominant double mutant dhfr haplotype in Jazan (73%), was prevalent among P. falciparum in east Africa. Network analysis suggests the mutant haplotype of dhfr gene was possibly introduced into Jazan from East Africa. The absence of mutations in dhps as well as triple mutant dhfr haplotype associated with SP failure support the current use of SP as a partner with artesunate as a first line therapy in Saudi Arabia. However, the close relationship between the major mutant dhfr haplotype in Sudan and Saudi Arabia, favour the hypothesis of recent migration as a source of the major resistant dhfr lineage. Thus, regular monitoring of the dhfr and dhps haplotypes is of high priority to guard possible importation of high level SP resistant lineages.     

A national survey of the prevalence of chloroquine resistant Plasmodium falciparum malaria in The Gambia

In The Gambia, 760 children less than 10 years of age with Plasmodium falciparum malaria were treated with chloroquine (25 mg/kg) and followed-up 2 and 9 d after the start of treatment. 700 children (92.1%) completed the study. The level of in vivo resistance to chloroquine varied by area from 0.4% to 16.4%. Of the 28 children found to have chloroquine resistant malaria, none was ill when seen at the 9 d follow-up and only 3 (10.3%) required further treatment with alternative antimalarials because of persistent high levels of parasitaemia. However, the fact that 30.4% of the children who completed the study had chloroquine in their urine at presentation may have masked the true level of resistance and led to underestimation of the clinical significance of these findings. The blood film at day 2 did not usefully predict resistance. 67 isolates were tested in vitro for chloroquine sensitivity. The mean EC50 was 15.5 nmol/litre, an eight-fold decrease in sensitivity from that of isolates tested in 1982. 8 (11%) of the isolates had EC50s above the WHO reference value for sensitive isolates of 18.3 nmol/litre, with values ranging from 22 to 65 nmol/litre of culture medium. Gambian isolates were sensitive to quinine (mean EC50 = 49.6 nmol/litre).     

Genetic backgrounds of the Plasmodium falciparum chloroquine resistant transporter (pfcrt) alleles in Pakistan

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with point mutations in the P. falciparum CQ resistance transporter gene (pfcrt). Previous studies have shown 4-5 independent origins for CQ resistant pfcrt alleles globally, two in South America, one each in Southeast Asia, Papua New Guinea (PNG) and Philippines. In Asia, at least two different alleles corresponding to amino acids 72-76 (CVIET and SVMNT) have been found. The CVIET allele originated in Southeast Asia and then spread to Asia and Africa as well. The SVMNT allele, originating from PNG, has been found in India. This study was undertaken to investigate the genetic background of the CQ resistant pfcrt haplotypes in Pakistan. We genotyped microsatellite markers surrounding the pfcrt gene (six different markers at -12.3, -4.8, -1, 1.5, 3.9, 18.8 kb) in 114 clinical isolates of P. falciparum collected from different regions in Pakistan. Microsatellite analysis showed a significant reduction in genetic variation among the mutant SVMNT pfcrt alleles when compared to wild type alleles. The predominant SVMNT haplotype found in this study shared the same microsatellite haplotype found in both PNG and India. Two isolates with CVIET haplotypes showed similar microsatellite background to those found in Africa and Asia. In conclusion, this study suggests that CQ resistant SVMNT haplotypes in India and Pakistan have a common ancestral origin similar to that of Papua New Guinean isolates.