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Biomedical subjects

D C Warhurst

Publications and source records attributed to D C Warhurst.

At least 37 records · Page 2Linked to original sources
PubMed2000

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.

Animals↗
PubMed1999

In vitro and in vivo evaluation of betulinic acid as an antimalarial.

The lupane-type triterpene betulinic acid was isolated from an ethanol extract of the root bark of the Tanzanian tree Uapaca nitida Müll-Arg. (Euphorbiaceae). The in vitro antiplasmodial IC50 values of betulinic acid against chloroquine resistant (K1) and sensitive (T9-96) Plasmodium falciparum were found to be 19.6 micrograms/mL and 25.9 micrograms/mL, respectively. The in vitro activities of several related triterpenes were also evaluated. Betulin was found to be inactive at 500 micrograms/mL for both K1 and T9-96. Ursolic acid exhibited IC50 values of 36.5 micrograms/mL and 28 micrograms/mL, and oleanolic acid exhibited IC50 values of 88.8 micrograms/mL and 70.6 micrograms/mL against K1 and T9-96, respectively. When betulinic acid was tested for in vivo activity in a murine malaria model (P. berghei) the top dosage employed of 250 mg/kg/day was ineffective at reducing parasitaemia and exhibited some toxicity. Betulinic acid has not previously been evaluated for in vivo activity. This is believed to be the first compound to be isolated from U. nitida.

Animals↗
PubMed1999

Drug resistance in Plasmodium falciparum malaria.

Four classes of drugs are reviewed: blood schizontocides acting only on the hemoglobin-digesting blood stages, the antifolates which attack tetrahydrofolate synthesis in all the growing stages, antimitochondrials affecting synthesis and electron transport, and 8-aminoquinolines which interfere with redox processes. Drug efflux via a multidrug resistance membrane protein, and the production of a protein competing with the drug for the target hemin are thought to be responsible for resistance to blood schizontocides. Structural changes in target enzymes are responsible for easily-developed resistance to antifolates and antimitochondrials. The judicious use of drug combinations can help to avoid development of resistance and combat resistant infections, but new drugs are urgently needed.

Animals↗
PubMed1999

Allele-specific, nested, one tube PCR: application to Pfmdr1 polymorphisms in Plasmodium falciparum.

An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P, falciparum multidrug resistance gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

Alleles↗
PubMed1999

Association of cg2 and pfmdr1 genotype with chloroquine resistance in field samples of Plasmodium falciparum from Nigeria.

This study examines polymorphisms in 2 genes (pfmdr1 and cg2), which have been associated with resistance to chloroquine in Plasmodium falciparum, to determine their value as predictors of resistance status. Among field samples from children in Zaria, northern Nigeria, the Tyr-86 polymorphism in pfmdr1 and Ala-281 and the Dd2 kappa repeat of cg2, were significantly associated. In 8 samples classified resistant by the micro-in vitro test, or, where this failed, by in vivo trial, 7 showed the cg2 Dd2 type kappa repeat, and 6 of these had both the Ala-281 allele and the pfmdr1 Tyr-86 allele. In 26 chloroquine-sensitive samples, none had this combination of 3 polymorphisms (P = 0.00002). This indicates 75% sensitivity and 100% specificity in detection of resistance and shows a positive predictive value for resistant infections of 100%. The negative predictive value, because of sensitivity less than 100%, would depend on the prevalence of resistance. Where prevalence of resistance is approx. 21% as in Zaria, the negative predictive value would be 94%, while in Gabon, with a prevalence of ca 73% it would be 60%. The use of (cg2: Ala-281, Dd2 kappa. pfmdr1: Tyr-86) genotype detection as a predictive epidemiological tool to examine the distribution of chloroquine-resistance in parts of Africa is therefore possible. The sensitivity of detection of resistant strains still requires improvement.

ATP-Binding Cassette Transporters↗
PubMed1999

Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study.

Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites resistant to chloroquine (CQ). As these drugs are introduced it is imperative that resistance is monitored. In this paper we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used in testing with the WHO in vitro microtest. An increase in parasitaemia and/or haematocrit was accompanied by a decrease in the measured sensitivity of 2 laboratory lines. In the context of a field study testing in vitro susceptibility of parasite isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL) and quinine (QUIN). When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and HAL suggesting the presence of a multidrug resistance-like mechanism. These studies underline the importance of inoculum size in in vitro susceptibility testing.

Animals↗
PubMed1999

Evaluation of the anti-plasmodial activity of bisbenzylisoquinoline alkaloids from Abuta grandifolia.

Three alkaloids were isolated from the bark of the traditional medicinal plant Abuta grandifolia (Mart.) Sandw. (Menispermaceae) and tested for in vitro anti-plasmodial activity. Two of them were identified as the Type VIII bisbenzylisoquinoline alkaloids, krukovine (1) and limacine (2), while the least abundant compound (3) could only be characterised to Type I of the same class. Krukovine exhibited potent anti-plasmodial activity with IC50 values of 0.44 microgram/ml and 0.022 microgram/ml against K1 (chloroquine-resistant) and T9-96 (chloroquine-sensitive) Plasmodium falciparum, respectively. Both limacine and compound 3 exhibited moderate anti-plasmodial activity against K1 with IC50 values of 1.35 micrograms/ml and 1.58 micrograms/ml, respectively. Limacine gave an IC50 value of 0.24 microgram/ml against T9-96. Krukovine and limacine showed greater activity against T9-96 than against K1, exhibiting similar activity profiles to that of chloroquine diphosphate (0.187 microgram/ml and 0.013 microgram/ml against K1 and T9-96, respectively). This indicates that krukovine and limacine may be affected by the mechanism of chloroquine resistance present in K1 P. falciparum.

Alkaloids↗
PubMed1999

Plasmodium falciparum: selection of serine 108 of dihydrofolate reductase during treatment of uncomplicated malaria with co-trimoxazole in Ugandan children.

In vivo testing for resistance of Plasmodium falciparum to co-trimoxazole (trimethoprim/sulfamethoxazole) was performed in Uganda in 41 children with uncomplicated malaria, and blood samples were screened before and after treatment for polymorphisms in the antifolate target genes for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Selection towards a specific genotype at some codons of the DHFR and DHPS genes was observed in samples collected after exposure to co-trimoxazole drug pressure. The alleles 51-isoleucine, 59-arginine, and 108-serine of DHFR were significantly associated with clinical resistance, as was allele 581-alanine of DHPS. Resistance against antifolate combinations probably requires resistance-related polymorphisms in both the DHFR and the DHPS genes. In addition, it appears that the trimethoprim-resistant DHFR genotype differs from that for pyrimethamine at residue 108.

Alleles↗
PubMed1998

Pfmdr1 Asn1042Asp and Asp1246Tyr polymorphisms, thought to be associated with chloroquine resistance, are present in chloroquine-resistant and -sensitive Brazilian field isolates of Plasmodium falciparum.

Parasite resistance to antimalarial drugs, particularly chloroquine, is the most disturbing problem of malaria chemotherapy. There is evidence that the codon 86Tyr polymorphism of the Pfmdr1 gene is associated with chloroquine resistance in West African Plasmodium falciparum. The association of this and four other coding alterations of the Pfmdr1 gene with chloroquine resistance has not been extensively investigated in South American isolates. In this study, we examined 51 Brazilian P. falciparum isolates for the presence or absence of Asn86Tyr, Asn1042Asp, and Asp1246Tyr polymorphisms. While these isolates were all sensitive in vitro to mefloquine, amodiaquine, and quinine, only 2 (4%) were chloroquine-sensitive. The findings reported here provide the first observations of this kind on a large number of field parasite samples from South America. We show that in vitro chloroquine-resistant and -sensitive strains carry the Asn1042Asp and Asp1246Tyr polymorphisms and provide support for earlier suggestions that Asn86Tyr may be rare or absent in South American P. falciparum.

Amodiaquine↗
PubMed1998

Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion.

With the spread of resistance to chloroquine, the combination of sulphadoxine and pyrimethamine is growing in importance for the treatment of infection with the malaria parasite Plasmodium falciparum. Mutations in the dhfr gene of P. falciparum have been associated with resistance to pyrimethamine. Recently, several polymorphisms have been identified in the P. falciparum dhps gene which may correlate with sulphadoxine-resistance. Simple and rapid tests have been developed to detect these polymorphisms, using PCR followed by restriction digestion. These tests can accurately identify all the polymorphisms described to date at codons 16, 51, 59, 108, and 164 in the dhfr gene and those at codons 436, 437, 540, 581, and 613 in the dhps gene. A nested system has been developed which allows the accurate detection of these polymorphisms in samples of fingerprick blood collected on glass fiber membranes and filter papers, some with very low parasitaemias.

Animals↗
PubMed1998

Plasmodium falciparum: in vitro chloroquine susceptibility and allele-specific PCR detection of Pfmdr1 Asn86Tyr polymorphism in Lambarene, Gabon.

Plasmodium falciparum resistance to chloroquine has been described in many parts of the world particularly in Africa where malaria is endemic. High levels of chloroquine resistance in our study area, Lambarene-Gabon, has led to the use of an alternative regimen for treatment and prevention of P. falciparum infection. In this study, we examined the in vitro chloroquine sensitivity of 15 isolates from this area and assessed the prevalence of a putative chloroquine resistance associated Pfmdr1 polymorphism (Asn86Tyr) using a novel allele-specific polymerase chain reaction (PCR). Only 4 of the isolates examined were chloroquine sensitive. The allele-specific PCR shows that all 15 isolates carried the variant (86Tyr) codon. Eleven of these were resistant to chloroquine suggesting a 73% agreement between chloroquine resistance phenotype and the point mutation. This molecular marker was examined in a further 73 Gabonese isolates, where 58 (79.5%) showed 86Tyr and 15 (20.5%) showed 86Asn. In all, 4 (4.5%) of the 88 isolates assessed carry both mutant and wild-type codons, suggesting mixed parasite populations. The incomplete agreement found between chloroquine resistance phenotype and Pfmdr1 (86Tyr) polymorphism would support the view that other genetic factors as well as Pfmdr1 may be involved in chloroquine resistance. While our results suggest a high prevalence of 86Tyr polymorphism in Lambarene, the Asp1246Tyr polymorphism (a point mutation which to date has only been associated with South American P. falciparum) seems to be absent in our study area.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania.

The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo. Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.

Animals↗
PubMed1998

In vivo selection for a specific genotype of dihydropteroate synthetase of Plasmodium falciparum by pyrimethamine-sulfadoxine but not chlorproguanil-dapsone treatment.

Plasmodium falciparum present in blood samples collected before and 3 weeks after treatment with either pyrimethamine-sulfadoxine or chlorproguanil-dapsone was analyzed for variants of the genes coding for the target enzymes of antifolate drugs, dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Fragments of the genes were amplified by polymerase chain reactions, and variants were identified by specific restriction endonuclease digestion. Treatment with either drug combination selected for the variants Ile51, Arg59, and Asn108 of DHFR, which have been associated with in vitro resistance to pyrimethamine and cycloguanil. The genotype Ser436, Gly437, and Glu540 of DHPS was selected by pyrimethamine-sulfadoxine but not chlorproguanil-dapsone treatment, showing that a combination of these three variants is important for in vivo resistance to sulfadoxine in the area studied.

Amino Acid Sequence↗
PubMed1998

Giardia lamblia: in-vitro sensitivity to some chemotherapeutic agents.

The susceptibility of Giardia lamblia to eight chemotherapeutic agents was studied in vitro. The criteria of viability of the parasite was the ability of the parasite to attach to the coverslip surface in situ, and to multiply in drug free media on subcultures. The giardicidal and 50% inhibitory concentrations of each drug were measured. The activity of metronidazole in vitro was found to be similar to that of mepacrine, whilst that of chloroquine was inferior. The sensitivity of G. lamblia to mepacrine and chloroquine is markedly greater than that reported for Entamoeba histolytica.

Animals↗
PubMed1997

Guidelines for the prevention of malaria in travellers from the United Kingdom. PHLS Malaria Reference Laboratory, London School of Hygiene and Tropical Medicine.

These guidelines on malaria prevention are an aid to health care workers who advise travellers, particularly those who will be overseas for less than a year. They represent a virtual consensus of the views of 44 doctors, nurses, and pharmacists with special expertise in malariology or travel medicine who met to develop them in 1996 (see list on R152). The guidelines are in three parts. The first part is a summary that emphasises modifications to the advice given in the last set of guidelines, published in 1995. The second part discusses the issues addressed in formulating the guidelines. Doctors, practice nurses, and pharmacists are asked to read this section to avoid doing harm by giving chemoprophylaxis without due attention to the traveller's history or destination and by using oversimplified lists of recommendations by country. The second part also addresses the health care worker's consultation with prospective travellers. The third part gives specific recommendations for travellers to specific destinations and some details of individual drugs. Fuller information on some points was given in earlier versions of the guidelines, which should not be discarded. Meetings of the sort described above have been held since 1980 and the group's membership has included people with varied views and experience. The views expressed in these guidelines reflect experienced professional opinion, since data are inadequate for unequivocal views to be given on several issues. There is often a range of acceptable options, but to meet the requests of general practitioners the guidelines try to give one recommended option and state alternatives, suggesting when and how different regimens can be used to good effect. Decisions on the terms under which different drugs are licensed for use are the responsibility of the Licensing Authority, advised by the Committee on Safety of Medicines (not of these guidelines). The guidelines should be read as a supplement to and not as a substitute for the relevant data sheets. Chemoprophylaxis lies somewhere between vaccination (for which people expect governments to lay down schedules) and treatment of ill people (for which each physician does what seems most appropriate) in concept and practice. The risks of malaria need to be balanced against the risks of the preventive measures, on the basis of the data available. Travellers may ask for an explanation of these risks and doctors and practice nurses need to be well informed and able to present their knowledge to travellers. The second part of these guidelines may be of use to prospective travellers who wish to read about the options themselves. All readers are recommended to read part two in its entirety to get a balanced picture.

Disease Transmission, Infectious↗
PubMed1997

Evidence for selection for the tyrosine-86 allele of the pfmdr 1 gene of Plasmodium falciparum by chloroquine and amodiaquine.

The 4-aminoquinolines chloroquine (CQ) and amodiaquine (AM) were used to treat Gambian children with uncomplicated falciparum malaria in a randomized drug trial. Blood samples were taken immediately before treatment (day 0), and at day 7 and day 28 after treatment. Samples from those parasitologically positive at day 7 following treatment ('early positives') and those positive at day 28 but negative at day 7 ('late positives') have been studied by PCR followed by restriction enzyme digestion to determine the allelic status of the pfmdr 1 locus at the codon-86 position (asparagine or tyrosine), previously associated with resistance to CQ. A significantly higher prevalence of the tyr-86 allele was observed in samples taken immediately before treatment (day 0) in the early positives group when compared with the late positives group. This suggests the tyr-86 allele contributes to drug resistance in the early positives group. This association remained significant for both CQ and AM groups, implying a common genetic basis of resistance. Predominance of the allele at day 7 is consistent with a strong selection in the first week following treatment. In the late positives group, a significantly higher prevalence of the tyr-86 allele was observed in the samples at day 28 when compared with those at day 0, suggestive of selection during the period day 7 to day 28. Differences were observed in the extent of this selection in the CQ and AM groups. The samples were genotyped at 3 unlinked polymorphic loci. These analyses suggested that most parasites observed at day 7 were probably recrudescences whereas most of those at day 28 were reinfections.

ATP-Binding Cassette Transporters↗
PubMed1997

High prevalence of mutations in the dihydrofolate reductase gene of Plasmodium falciparum in isolates from Tanzania without evidence of an association to clinical sulfadoxine/pyrimethamine resistance.

Recently the efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Tanzania has been seriously compromised by the development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequence coding for dihydrofolate reductase (DHFR) is known to confer resistance to pyrimethamine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of one or more DHFR mutations in all the samples, but no relationship was found with the presence of parasites in the blood at day 7. The results suggest that alterations in the coding region for dihydropteroate synthetase (DHPS), the enzyme target for sulfadoxine, should be studied in order to predict resistance to the S/P combination. It has been proposed earlier that sulfadoxine could itself act on DHFR, because of a false dihydrofolate produced by drug metabolism through DHPS and dihydrofolate synthase. The results of this treatment study suggest that such a possibility is unlikely.

Animals↗