[Presence of the double pfmdr1 mutation 86Tyr and 1246 Tyr in clones of a chloroquine-resistant west African isolate of Plasmodium falciparum]. / Detecção da mutação dupla 86TYR e 1246TYR no gene pfmdr1 em clones de uma amostra de Plasmodium falciparum da África Ocidental, resistente à cloroquina.

Isolates of Plasmodium falciparum from three areas of West Africa were recovered from cryopreservation and their chloroquine-sensitivity were determined in vitro. Of the 90 samples studied, 60 were from Guinea-Bissau (30Resistant/30Sensitive), 15 were from S. Tomé and Príncipe (11Resistant/4Sensitive) and 15 were from Angola (11Resistant/4Sensitive). All the isolates were sensitive to mefloquine. Using the polymerase chain reaction/restriction fragment length polymorphism technique (PCR/RFLP) it was possible to detect two mutations in the pfmdr1 gene, often associated with chloroquine-resistance. 66% of the samples from Guiné-Bissau showed a correlation with chloroquine-resistance while 73% of the samples from São Tomé and Angola altogether had the 86Tyr mutation. The present study on West African isolates and clones showed, for the first time, the presence of a double point mutation in the pfmdr1 gene one being found, up to now, only in South America isolates of Plasmodium falciparum.

Mutations in dhfr in Plasmodium falciparum infections selected by chlorproguanil-dapsone treatment.

Treatment with the novel antifolate drug combination chlorproguanil-dapsone effectively cleared asymptomatic Plasmodium falciparum infections in 246 (93.5%) of 263 children in the Usambara Mountains of Tanzania during the course of a 2-week follow-up. Samples from 71 recurrent infections, collected over a 9-week follow-up, showed selection for parasites with the triple mutant Ile(51)-Arg(59)-Asn(108) in dihydrofolate reductase. There was no selection for mutations in dihydropteroate synthetase, the target enzyme of dapsone. Search for complete identity in the highly polymorphic genes coding for merozoite surface proteins 1 and 2 in parasite samples collected before and after treatment indicated that the majority of recurrent parasitemias were new infections. These observations on selection in Tanzania and the lack of selection reported from a less endemic area suggest that the active metabolite of chlorproguanil, which has a short half-life in the blood, may persist in the liver, where it exerts selective pressure on growing preerythrocytic stages.

A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2.

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 ('Human') from 2 ('Cattle') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

Restricted genetic and antigenic diversity of Plasmodium falciparum under mesoendemic transmission in the Venezuelan Amazon.

The study of genetic diversity in malaria populations is expected to provide new insights for the deployment of control measures. Plasmodium falciparum diversity in Africa and Asia is thought to reflect endemicity. In comprehensive epidemiological surveys reported here the genetic and antigenic structure of P. falciparum in the Venezuelan Amazon were studied over a 2-year period. DNA polymorphisms in glutamate-rich protein (GLURP), merozoite-surface protein 1 (MSP1) and MSP2 genes, in a multicopy element (PfRRM), all showed low diversity, 1 predominant genotype, and virtually no multi-clonal infections. Moreover, linkage disequilibrium was seen between GLURP, MSP1 and MSP2. Specific antibody responses against MSP1 and MSP2 recombinant antigens reflected the low genetic diversity observed in the parasite population. This is unexpected in a mesoendemic area, and suggests that the low diversity here may not only relate to endemicity but to other influences such as a bottleneck effect. Linkage disequilibrium and a predominant genotype may imply that P. falciparum frequently propagates with an epidemic or clonal population structure in the Venezuelan Amazon.

Two novel assays for the detection of haemin-binding properties of antimalarials evaluated with compounds isolated from medicinal plants.

Forty-two compounds isolated from nine plants used within South America for the treatment of malaria were tested for haemin binding using two novel, rapid screening methods. The data obtained were analysed with respect to IC(50) values for in vitro toxicity to Plasmodium falciparum trophozoites. One method, a multiwell assay based on the inhibition of the interaction of haemin with glutathione (GSH), is sensitive in the 10 microM range, takes c. 1 h and is suitable for either a high throughput screen or rapid assay during natural product isolation. Of 19 compounds showing antiplasmodial activity (IC(50) < 40 microM), 16 (84%) showed >40% inhibition of GSH-haemin reaction. The sensitivity and specificity of the assay were 0.85 and 0.82, respectively. The positive predictive value was 0.81 and the negative predictive value 0.86. A more sensitive assay (0.1 microM range) is based on the reversal by haemin-binding compounds of the haemin inhibition of the L-dopachrome-methyl ester tautomerase activity of human macrophage migration inhibitory factor. This assay gives a better idea of the affinity of interaction and uses very small amounts of test compound. The log[RI(50)] of eight of the compounds that tested positive in the above assays together with those of quinine and chloroquine showed a positive correlation with log[antiplasmodial IC(50)] for strain T9-96 (r = 0.824) and strain K1 (r = 0.904). Several of the antimalarial compounds that bind haemin are isoquinolines, a class not shown previously to interact with haemin.

Rational use of drugs against Plasmodium falciparum.

Recent studies on resistance to blood schizontocides in Plasmodium falciparum give a rational basis for the use of artemisinins combined with arylaminoalcohols for the treatment of uncomplicated chloroquine-resistant malaria in Africa. In areas where such combinations are introduced, there is reason to believe that the continued use of chloroquine in the community will help protect the new drugs from resistance. In view of several laboratory studies, combinations of artemisinins with antifolates or chloroquine pose a risk of antagonistic interaction. This can be avoided by use of the artemisinin and the companion drug sequentially.

Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine.

Novel alleles of the Plasmodium falciparum dhfr highly resistant to pyrimethamine and chlorcycloguanil, but not WR99210.

We have expressed dhfr alleles of Plasmodium falciparum in the budding yeast, Saccharomyces cerevisiae, and used this yeast model to identify single amino acid substitutions that confer high level pyrimethamine resistance on the background of the triple mutant dhfr (I51+R59+N108). Mutations in three clusters were identified: codons 50-57, 187-193, and 213-214. Several mutations previously identified in field samples were also isolated, including codons 50 and 164. The I164L mutation is of particular interest, because the quadruple mutant genotype (N51I+C59R+S108N+I164L) encodes an enzyme that is no longer inhibited by pyrimethamine, rendering sulfadoxine/pyrimethamine (SP; Fansidar) clinically ineffective. Thirty-six novel alleles were tested to determine their sensitivity to chlorcycloguanil and WR99210, two DHFR inhibitors that are in clinical and pre-clinical development, respectively. Chlorcycloguanil is effective against parasites that carry the triple mutant allele, but in vitro analysis has suggested that chlorcycloguanil will be clinically ineffective against parasites that carry the quadruple mutant allele of dhfr. In our screen, 23 of 36 novel strains were as resistant to chlorcycloguanil as the quadruple mutant, and one strain was 10-fold more resistant. WR99210 is still effective in the nM range against parasites that carry the quadruple mutant allele. In the preliminary screen, 31 of 36 novel alleles were as sensitive to WR99210 as the quadruple mutant. Detailed analysis of the remaining five showed that four of the five had IC(50) values in the same range as the quadruple mutant, and one, N51I+C59R+S108N+E192G, had an IC(50) value about fivefold higher. This result suggests that WR99210 and related compounds will be clinically effective against quadruple mutants currently found in Southeast Asia and South America and against most novel alleles that could be selected on the background of the triple mutant genotype now prevalent in East Africa.

Can pretreatment screening for dhps and dhfr point mutations in Plasmodium falciparum infections be used to predict sulfadoxine-pyrimethamine treatment failure?

This study examines the relationship between malaria treatment failure after sulfadoxine-pyrimethamine (S-P) chemotherapy and presence of mutations in the Plasmodium falciparum dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes (associated with resistance in vitro to S and P) before treatment. In Kenya, 38 malaria patients in a holoendemic area, and 21 in an epidemic area, participated in the trial in 1997-98. In the 2 areas, drug failure occurred in 76% and 75% of cases where any mutation in dhfr was seen (positive predictive values 76% and 75%: P = 0.003 and 0.008) and an identical association was seen with dhfr Asn-108. In the holoendemic area all occurrences of > or = 2 mutations in dhfr predicted drug failure. Only 3 instances were seen in the epidemic focus, but treatment failed in all. Only in the epidemic focus, 7 (88%) of 8 occurrences of > or = 1 mutations in dhps, and all occurrences of the Gly-437 allele of dhps, predicted failure. Association between mutations in dhps and mutations in dhfr was noted in the combined sites, irrespective of outcome. Although this makes the relationship of combined dhfr and dhps mutations to failure more difficult to interpret, it nevertheless supports S-P selection acting on both genes. In the holoendemic site, treatment success increased with age. In this location, acquired immunity may mask the impact of mutations in dhps, since sulfadoxine is a less effective treatment than pyrimethamine.

Terpenoids from Guarea rhophalocarpa.

Four new terpenes including, two sandaracopimaradiene diterpenoids, ent-8(14),15-sandaracopimaradiene-2alpha,18-diol, and ent8(14),15-sandaracopimaradiene-2beta,18-diol, and two lanostane triterpenoids, 23-hydroxy-5alpha-lanosta 7,9(11),24-triene-3-one, and 5alpha-lanosta-7,9(11),24-triene-3alpha,23-diol, were isolated from the methanolic extract prepared from the leaves of G. rhopalocarpa together with the known steroid stigmasterol and the coumarin, scopoletin. The isolates showed weak antiprotozoal activity against Leishmania donovani promastigotes, and Trypanosoma brucei brucei blood stream trypomastigotes, and were devoid of interesting activity towards Plasmodium falciparum. The isolates did not show significant cytotoxic activity against KB cells.

Co-trimoxazole compared with sulfadoxine-pyrimethamine in the treatment of uncomplicated malaria in Kenyan children.

Sulfadoxine-pyrimethamine (SP) and co-trimoxazole were both effective in reducing fever, clearing parasitaemia and improving anaemia in children aged < 5 years with uncomplicated malaria in 2 Kenyan endemic sites, Oyugis in the west and Tiwi on the coast. We compared the efficacy of these 2 regimens (in May-July 1998) by evaluating clinical and parasitological responses over 14 days. The combined incidence of parasitological failure for the combined sites for co-trimoxazole was 14/123 (11%) and for SP 23/145 (16%) (RR 0.72, 95% confidence interval [CI] 0.31-1.46, P = 0.289). The 14-day clinical failure rate for the combined sites for co-trimoxazole was 4/123 (3.3%), and for SP 8/145 (5.5%), (RR 1.69, 95% CI 0.91-3.15, P = 0.129). The results indicate that the risk of treatment failure for the 2 regimens was similar. The antimalarial use of co-trimoxazole in uncomplicated malaria needs further investigation, since the 10-12-h elimination half-life of both components should reduce selective pressure for resistance. In addition, use of a 2-day high-dose course, tested previously, requires further study to demonstrate its efficacy.

Plasmodium falciparum in Kenya: high prevalence of drug-resistance-associated polymorphisms in hospital admissions with severe malaria in an epidemic area.

During an epidemic of Plasmodium falciparum malaria in Chogoria, Kenya, P. falciparum DNA was collected from 24 cases of severe malaria admitted to hospital for parenteral quinine treatment. These patients had all failed first- (chloroquine) and second-line (sulfadoxine-pyrimethamine or amodiaquine) drug treatments. Twenty-two (92%) of the 24 patients sampled carried parasites with the (Asn)86(Tyr) point mutation in the pfmdr1 gene (chromosome 5), 20 (83%) had an (Asp)1246(Tyr) mutation and 18 (82%) had both of these mutations. These alleles are both reported to be associated with chloroquine-resistance. Polymorphisms in the cg2 gene (chromosome 7) are also associated with chloroquine resistance, and 18 (75%) of the 24 parasite samples each had the cg2 and pfmdr1 polymorphisms. These 18 samples also had the mutations associated with resistance to pyrimethamine and sulfadoxine: (Asn)51(Ile), (Cys)59(Arg) and (Ser)108(Asn) of gene dhfr (chromosome 4) and (Ala)437(Gly) and (Lys)540(Glu) of dhps (chromosome 8), respectively. Genotyping of the parasites from all 24 patients revealed extensive diversity in the sequences for the merozoite surface antigens (MSA-1 and MSA-2) and the glutamate-rich protein (GLURP) and indicated that each sample contained more than one parasite clone. Although samples from non-admitted malaria cases were not available, it appears that drug resistance may have played an important role in the development of severe malaria in this epidemic.

Linkage disequilibrium between two chromosomally distinct loci associated with increased resistance to chloroquine in Plasmodium falciparum.

Chloroquine-resistance in Plasmodium falciparum is associated with polymorphisms in a locus on or near the cg2 gene on chromosome 7, and in the pfmdr1 gene on chromosome 5. In this study we typed P. falciparum DNA from uncomplicated malaria cases in The Gambia in 1990, 1995 and 1996 for size polymorphism in the omega repeat of cg2, for sequence polymorphisms in pfmdr1 at codons 86 and 184, in dhfr at codon 108 and in the msp2 gene. Chloroquine sensitivity tests were conducted in vitro. A significant but incomplete association was found between the presence of the cg2 Dd2-like omega repeat size polymorphism and in vitro resistance, and between the tyr-86 allele of pfmdr1 and in vitro resistance. Furthermore there was strong linkage disequilibrium between the pfmdr1 asn-86 allele and the cg2 not Dd2-like omega repeat allele located on different chromosomes. In contrast, no linkage disequilibrium was found between these alleles and either the dhfr ser-108 allele or the msp2 IC sequence polymorphism. No significant linkage was measured between pfmdr1 asn-86 and phe-184 although these loci are separated only by 296 base pairs. Our results suggest that genetic elements linked to the cg2 and the pfmdr1 genes are important determinants of chloroquine resistance. It can be concluded that the observed linkage disequilibrium is maintained epistatically through selection by chloroquine.

Antiplasmodial and antiamoebic activities of medicinal plants from Sierra Leone.

Crude ethanol extracts of 18 medicinal plants from Sierra Leone, West Africa were examined for antiplasmodial activity against Plasmodium falciparum, using an in vitro microtest. Eleven of these extracts were also screened for in vitro antiamoebic activity against Entamoeba histolytica. Only one plant extract, Triclisia patens (Menispermaceae) showed significant antiplasmodial activity (IC(50) = 8 microg/mL). None of the plant extracts was effective against Entamoeba histolytica.

Bioactive compounds from Celaenodendron mexicanum.

Bioactivity-directed fractionation of the CHCl3-MeOH extract of the leaves of Celaenodendron mexicanum by means of the brine shrimp lethality test and chromatographic techniques led to the isolation of three carboxylic acid triterpenes, the new tirucalla-type triterpene, 3 alpha-hydroxytirucalla-7,24Z-dien-26-oic acid, 3-oxotirucalla-7,24Z-dien-26-oic acid, and epi-oleanolic acid, and three biflavonoids amentoflavone, podocarpusflavone A, and podocarpusflavone B. Four non-active compounds friedelin, maytensifolin B, 3 beta-hydroxyfriedelan-16-one, and celaenodendrolide were also obtained. epi-Oleanolic acid was the most active against brine shrimps with LC50 value of 23.3 microM. In addition, all isolates were tested for in vitro antiprotozoal and cytotoxic activities. 3-Oxotirucalla-7,24Z-dien-26-oic acid and epi-oleanolic acid showed the highest activity against Leishmania donovani promastigotes with IC50 values of 13.7 and 18.8 microM, respectively. Only 3-oxotirucalla-7,24Z-dien-26-oic acid showed activity against Trypanosoma brucei brucei bloodstream forms with IC50 value of 16.8 microM.

Oxoaporphine alkaloids and quinones from Stephania dinklagei and evaluation of their antiprotozoal activities.

Bioactivity-guided fractionation of Stephania dinklagei yielded six compounds including, two zwitterionic oxoaporphine alkaloids, N-methylliriodendronine, and 2-O,N-dimethylliriodendronine, two oxoaporphine alkaloids, liriodenine, and dicentrinone, one aporphine alkaloid, corydine, and one anthraquinone, aloe-emodin. Apart from corydine, the isolates have not been reported as constituents of S. dinklagei. N-Methylliriodendronine, and 2-O,N-dimethylliriodendronine are reported for the first time as natural products. All isolated compound were tested for antiprotozoal activity and cytotoxic activities in vitro. N-Methylliriodendronine was the most active against L. donovani amastigotes (IC50 = 36.1 microM). Liriodenine showed the highest activity against Leishmania donovani, and Plasmodium falciparum with IC50 values of 26.16 and 15 microM, respectively. Aloe-emodin was the only compound active (IC50 = 14 microM) against T. b. brucei.

Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum.

The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.

The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin.

Although chloroquine-resistance (CQR) in Plasmodium falciparum is increasing and resistance to other blood schizonticidal anti-malarials has been reported, the molecular basis remains unclear. In this study fresh field isolates were obtained from The Gambia, an area of emerging CQR and tested for sensitivity to the anti-malarial drugs mefloquine, halofantrine, artemisinin, dihydroartemisinin, chloroquine and quinine. Sequence polymorphisms in the pfmdr1 gene and size polymorphisms in the cg2 gene were assessed using PCR-based systems. A strong association was observed between the presence of the tyr-86 allele of pfmdr1 and increased sensitivity to mefloquine and halofantrine, as well as the structurally unrelated drugs artemisinin and dihydroartemisinin. A weaker association was found between the presence of tyr-86 and increased resistance to chloroquine and quinine. The cg2 Dd2-like omega repeat size polymorphism was associated with increased resistance to chloroquine and increased sensitivity to mefloquine and halofantrine. An intragenic association was also found between a polymorphism in the polyasparagine linker region of pfmdr1 and the tyr-86 allele, which may be due to genetic hitchhiking, indicative of recent selection by chloroquine. Our data support a hypothesis where the pfmdr1 gene confers a true multidrug resistance phenotype which is lost by mutation.