A simple magnetic method for the purification of malarial pigment.

Using a simple magnetic separation apparatus, Plasmodium berghei malarial pigment has been purified 200-fold in a single step. The technique exploits the paramagnetic properties of malarial pigment (hemozoin) and provides a simple and rapid method for the isolation of this material with high purification and yield.

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.

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.

Antimalarial drugs. An update.

Over the last decade, chloroquine-resistant falciparum malaria has spread to other areas from its original foci in Southeast Asia and South America. Additionally, new knowledge about the life-cycle of the malaria parasite, and about the pharmacokinetic properties of antimalarial drugs, has emerged. It is appropriate to reassess our approach to prevention and management of malaria with these factors in mind. Antimalarial drugs can be classified in two ways: biologically as tissue schizontocides, hypnozoitocides, blood schizontocides, gametocytocides or sporontocides; or by a mixed chemical/biological classification as 8-aminoquinolines, antimetabolites and (again) blood schizontocides. Chloroquine resistance in P. falciparum can now be found in most areas where malaria occurs. Malarial strains moderately resistant to the chloroquine group of drugs (chloroquine and mepacrine) are generally susceptible to the aryl amino alcohols such as quinine. Indeed, quinine is the most widely used drug for treating malaria due to chloroquine-resistant strains, followed by a 7-day course of tetracycline where some resistance to quinine is also found. Alternatively, the course of quinine may be followed by sulfadoxine/pyrimethamine or the newer quinoline derivative, mefloquine. Quinidine has also shown activity against quinine-resistant strains. Prophylaxis of chloroquine-resistant strains is best undertaken with daily proguanil (chloroguanide), and weekly chloroquine. In severe malaria, including cerebral malaria, an intravenous loading dose of quinine should be considered, and plasma concentration monitoring may be advisable to assist with dosage adjustment. In patients with severe renal insufficiency, there is evidence that the elimination of chloroquine is prolonged, and dosage adjustments may be necessary. Other recent findings on the pharmacodynamic properties, mechanisms of action and toxicity of antimalarial drugs are also discussed.

A study of the uptake of chloroquine in malaria-infected erythrocytes. High and low affinity uptake and the influence of glucose and its analogues.

A study of concentration- and substrate-dependence of chloroquine uptake has been carried out on mouse erythrocytes infected with the chloroquine-sensitive NK65 and the chloroquine-resistant RC strains of Plasmodium berghei. The presence of drug binding sites of high and low affinity in such strains of P. berghei was confirmed. High affinity uptake sites in cells parasitized with chloroquine-sensitive and chloroquine-resistant parasites have similar characteristics, but in the sensitive strain the major component of chloroquine-uptake is at high affinity and dependent on the availability of ATP whilst in the resistant strain the major component of uptake is at low affinity and independent of energy. An absolute increase in the quantity of the low affinity site in erythrocytes parasitized with chloroquine-resistant P. berghei was noted, which may be related to an increase in quantity of parasite membrane.

Rapid action of Qinghaosu and related drugs on incorporation of [3H]isoleucine by Plasmodium falciparum in vitro.

Using the incorporation of [3H]isoleucine into acid-insoluble products as an index of protein-synthetic activity, it was shown that Qinghaosu and two related drugs had a rapid effect on this process in human erythrocytes infected with Plasmodium falciparum in vitro. Inhibition could be seen 1 hr or less after addition of the drugs at concentrations from 5 mumole/1. to 50 nmole/1. It is recommended that the effects of these drugs be studied in cell-free protein-synthetic systems.

Antimalarial activity of optical isomers of quinacrine dihydrochloride against chloroquine-sensitive and -resistant Plasmodium falciparum in vitro.

Both enantiomers of quinacrine and the racemic form of the drug showed equal activity in vitro against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, without detectable stereoselectivity. This contrasts with observations on chloroquine, where a similar lack of stereoselectivity in vitro is accompanied by a 10-fold loss of activity against the resistant strain. The observed in vivo differences reported for the enantiomers of chloroquine and the observations on the optically active metabolites of chloroquine and quinacrine may therefore be ascribed to a difference in the pharmacokinetics of their enantiomers.

In vitro studies on the mode of action of quassinoids with activity against chloroquine-resistant Plasmodium falciparum.

Using the incorporation of [3H]isoleucine or [3H]hypoxanthine into acid-insoluble products as indices of protein- and nucleic acid-synthetic activity, respectively, it was shown that seven plant-derived quassinoids with differing chemical substitutions all inhibited protein synthesis more rapidly than nucleic acid synthesis in human erythrocytes infected with Plasmodium falciparum, in vitro. Five quassinoids (ailanthinone, bruceantin, bruceine B, glaucarubinone and holacanthone) were effective within 30 min at doses 10 times their 48 hr in vitro IC50 values. Chaparrin and glaucarubol differed in that they did not inhibit protein synthesis during the time course of these experiments when applied at 10 times their in vitro IC50 values. When these compounds were used at 209 and 114 times their respective IC50 values, their observed effects were identical to those of the other quassinoids studied. The time (t50) at which nucleic acid synthesis was reduced to 50% of control was directly proportional to the t50 for protein synthesis, suggesting that failure of nucleic acid synthesis is a consequence of inhibition of protein synthesis. It is concluded that in the malaria parasite, as in eukaryote models, quassinoids are rapid and potent inhibitors of protein synthesis, and that this is most likely due to effects upon the ribosome, rather than upon nucleic acid metabolism.

Comparison of the in vitro activities of quassinoids with activity against Plasmodium falciparum, anisomycin and some other inhibitors of eukaryotic protein synthesis.

Using the inhibition of incorporation of [3H]hypoxanthine as an index of viability of malaria parasites, it was shown that a chloroquine-sensitive strain of Plasmodium falciparum (T9-96) and a chloroquine-resistant strain (K1) did not differ in their sensitivities to the quassinoids ailanthinone, bruceantin and chaparrin. Similarly, there were no differences between the strains in their sensitivities to the protein synthesis inhibitors anisomycin, deacetylanisomycin, cephalotaxine, homoharringtonine, cycloheximide, puromycin and puromycin aminonucleoside. The IC50 values derived for ailanthinone and bruceantin, cycloheximide, homoharringtonine and puromycin were in the nanomolar range, whereas those for the anisomycins, cephalotaxine and the aminonucleoside of puromycin were micromolar or greater. Those drugs tested which contain an ester moiety (ailanthinone, bruceantin, anisomycin, homoharringtonine) were more active than the related drugs (chaparrin, deacetylanisomycin, cephalotaxine) that do not. Cross-resistance to inhibitors of protein synthesis appeared not to accompany resistance to chloroquine.

In vitro assessment of susceptibility of Acanthamoeba polyphaga to drugs using combined methods of dye-binding assay and uptake of radiolabelled adenosine.

A technique based on binding of eosin dye to cell was applied to quantitate Acanthamoeba trophozoites in culture. Using this technique in combination with the uptake of radiolabelled adenosine, we assessed the activities of triazole (saperconazole), imidazole (ketoconazole, miconazole) and diamidine (berenil, pentamidine, dibromopropamidine) compounds against A. polyphaga trophozoites. The quantity of dye bound to the trophozoites correlated (r = 0.99) with the number of organisms per well. When inhibition of the growth of Acanthamoeba was the only parameter used to measure the effectiveness of these drugs, saperconazole was found to be most active with IC50 (concentration of drug that inhibited by 50%, the growth of A. polyphaga trophozoites incubated at 28 degrees C for 48 h) of 0.95 microM. The IC50s of the other drugs ranged from > or = 1500 microM for micronazole, the least active, to 3.0 microM for berenil. However, when efficacy was assessed by combining inhibition of growth with the uptake of [14C-8]adenosine by the drug treated organism, the diamidines, particularly berenil and pentamidine were considered most potent.

Counter immunoelectrophoresis as a rapid screening test for amoebic liver abscess.

Counter immunoelectrophoresis using cellulose acetate as the supporting medium was used as a rapid screening test for amoebic abscess. All the sera from 40 cases gave positive results. No false positives were obtained, but the results in intestinal amoebiasis were less reliable. An attempt was made to account for discrepancies in previous reports.

Accuracy of routine laboratory diagnosis of malaria in the United Kingdom.

AIMS: To study the accuracy of routine laboratory diagnosis of malaria with the aim of improving accuracy in diagnosis in the future. METHODS: A comparative study was made of all blood films submitted to two laboratories in London providing a slide-diagnostic service for malaria. RESULTS: There were 17 Plasmodium ovale infections, and of these only five (29.4%) were correctly diagnosed by the submitting laboratory; whereas of 210 other single species infections, 162 (77.1%) were correctly diagnosed (chi 2 = 18.4, p < 0.0001). There were six patients with mixed infections; only one (16.7%) was correctly diagnosed, whereas of 227 single species infections, 167 (73.6%) were correctly diagnosed (p = 0.007, using Fisher's exact test). There was no significant association between the presence of technical faults or numerous platelets and incorrect diagnosis. CONCLUSIONS: Plasmodium ovale and mixed infections were diagnosed incorrectly significantly more often than other species. The presence of technical faults or numerous platelets had no significant effect on whether or not submitting laboratories correctly diagnosed malaria.

Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica.

AIMS: To assess the reliability of the detection of erythrophagocytic amoebic trophozoites in stool samples in the diagnosis of dysentery associated with invasive Entamoeba histolytica. METHODS: Amoebic culture was carried out on single stool samples collected from patients from Mexico, Colombia, and Bangladesh. The stools had been examined by light microscopy. Amoebic dysentery was diagnosed when erythrophagocytic E histolytica trophozoites were observed in a case of bloody diarrhoea. E histolytica isolates were characterised by isoenzyme electrophoresis and results correlated with microscopical findings in stools. Statistical analysis was performed using the chi 2 test. RESULTS: Where erythrophagocytic amoebae had been observed in dysenteric stool specimens the E histolytica phenotype was invariably invasive (p < 0.0001). Observation of erythrophagocytic amoebae in dysentery is 100% specific and predictive of infection with invasive E histolytica. When amoebic culture-positive cases only are considered it is 96% sensitive. In this study E histolytica of zymodeme XIV was more commonly associated with amoebic dysentery than zymodeme II. There was no significant difference between the carriage rate of invasive and non-invasive E histolytica in non-dysenteric diarrhoea. Asymptomatic subjects carried non-invasive E histolytica more frequently than invasive E histolytica. Patients with non-amoebic dysentery, when shown to be infected with E histolytica, carried non-invasive strains (12%). CONCLUSIONS: Sensitivity and specificity of microscopical examination of a single stool specimen for diagnosing amoebic dysentery is very high; intestinal carriage of invasive E histolytica detected by culture is not necessarily an indication of active disease as patients with diarrhoea and asymptomatic subjects shed invasive and non-invasive E histolytica. There are possibly two subpopulations of invasive E histolytica with different pathogenic potential which can be differentiated by zymodeme analysis.

Efficient diagnosis of giardiasis among nursery and primary school children in Santiago, Chile by capture ELISA for the detection of fecal Giardia antigens.

Fecal samples were obtained from 722 of 820 children attending 7 nursery schools and 1 primary school in the city of Santiago, Chile. Microscopy of formol-ether concentrates showed that 33% of the children were infected with Giardia lamblia. Prevalences among primary school students (5-10 years of age) of G. lamblia (38%), Endolimax nana (43%), and Entamoeba coli (25%) were overall higher than among nursery school students (3 months-5 years of age; prevalences 29%, 21%, and 16%, respectively). There was no apparent association between socio-economic intake and levels of G. lamblia infection: the private nursery school had the highest recorded level of infection (40%). One hundred sixty-two triplicate stool specimens were used to compare microscopy with an enzyme-linked immunosorbent assay (ELISA) for the detection of Giardia fecal antigens. The ELISA was highly sensitive and specific either visually (95% and 97%, respectively) or by optical density determination (99% and 96%, respectively). Incorporation of non-immune rabbit immunoglobulin-coated control wells did not enhance sensitivity and specificity. The antigen detection ELISA is an extremely effective tool for the epidemiological investigation of giardiasis.

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.

Association of Vibrio cholerae with fresh water amoebae.

An investigation was undertaken to determine whether Acanthamoeba polyphaga SHI and Naegleria gruberi 1518/1e could affect the survival of various strains of Vibrio cholerae in laboratory microcosms. In microcosms pre-inoculated with trophozoites of amoebae, all six strains of V. cholerae tested survived and multiplied during 24 h. In control microcosms without trophozoites of amoebae, survival of the V. cholerae strains was much decreased. Two strains of V. cholerae were used to determine whether V. cholerae might survive ingestion within amoebae and subsequent encystment. Strain 152 was re-isolated from encysting N. gruberi 1518/1e but not from A. polyphaga SHI. Strain 9112 could not be isolated from cysts of either species of amoebae.

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.

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.

Specific anti-amoebic immunoglobulins and the cellulose acetate precipitin test in Entamoeba histolytica infection.

Using the indirect fluorescent antibody test and sera from 10 proved cases of invasive amoebiasis, the effects of absorption of IgG on observed titres of amoeba-specific IgM and IgA were investigated. In addition, results of cellulose acetate precipitin tests were compared with anti-amoebic antibody levels. Antiamoebic IgM was found at titres of 1:14 to 1:112 after IgG absorption in four cases. Anti-amoebic IgA was detected in sera from four cases, but the maximum titre was 1:28. There was no relationship between the presence of amoeba-specific IgM or IgA and the result of the precipitin test, but a raised anti-amoebic IgG level was consistently found where the precipitin test was positive. However, sera with raised amoeba-specific IgG levels did not invariably give a positive precipitin reaction.

Uptake of [3H] dihydroartemisinine by erythrocytes infected with Plasmodium falciparum in vitro.

Artemisinine ( qinghaosu ) was reduced and radio-labelled using tritiated borohydride. The tritiated dihydroartemisinine produced was differentially accumulated from low concentrations in culture medium into erythrocytes infected with Plasmodium falciparum. Uninfected erythrocytes concentrated the drug less than two-fold whereas infected erythrocytes achieved more than 300 times the medium concentration. The uptake process is reversible and saturable, with a dissociation constant (Kd) at the hypothetical receptor of 10.5 nmol.l-1. Competition studies indicate that the receptor is the same as that for artemether , another quinghaosu derivative. Chloroquine showed an interesting partial inhibition of uptake but was unable to release the bound radio-labelled drug from infected cells.