The science of chemotherapy has been built upon the seminal concepts arising from pioneering studies, notably by Paul Ehrlich, carried out nearly 100 years ago. The investigations conducted in the early decades of this century were of major significance for antiparasite chemotherapy as they resulted in the discovery of a variety of drugs for treating human and animal diseases. Derivatives of many of these drugs are still being used today, which, it could be claimed, reflects the outstanding success of the early approaches. However, it also highlights the relatively poor record of drug discovery in more recent decades. While major advances were taking place in the development of drugs and vaccines for bacterial, fungal and viral diseases, the discovery of new drugs for parasitic infections progressed relatively slowly. It would be wrong, however, to give an impression that there were no advances. The discovery of the avermectins and praziquantel has had an enormous impact on anthelmintic therapy, and new classes of antiprotozoal drugs have been developed for both prophylaxis and treatment of infections. Nevertheless, major problems remain and for some parasitic diseases the situation now is worse than it was 20 years ago. Malaria is perhaps the best example in this respect. The advent of chloroquine resistance in Plasmodium falciparum has resulted in a major public health problem. In addition, the increase in importance of some diseases, notably those associated with immunosuppression in AIDS, has again highlighted the limitations of antiparasite chemotherapy. New drugs are needed urgently for a number of parasitic diseases.

Colchicine, a drug which poisons the polymerization of microtubules, was assayed for effects on the invasion of Plasmodium falciparum merozoites into red cells in order to investigate if merozoite microtubules have a function in invasion. Culture conditions and concentrations of colchicine were established where the maturation and rupture of schizonts was unaffected by the drug. This was judged first by light microscopy, including morphology and counts of nuclear particle numbers, then by ultrastructural studies which excluded deranged organellogenesis as a cause of merozoite failure, and finally by diachronic cultures in which both recruitment and loss of schizonts could be counted. Specific invasion inhibition was seen when 10 μM–1 mM colchicine was present. Red cells pre-incubated in colchicine and then washed showed no reduction in their extent of invasion, and neither red cell lysis, sphering nor blebbing were apparent. We conclude that intact microtubules are necessary for successful merozoite function.

The chronic, recrudescent nature of malaria has been linked to antigenic diversity of the parasite in which protective immunity against Plasmodium falciparum may be obtained after repeated exposure to infection during a long time. In this study we show that parasite populations with decreased sensitivity to antibody-mediated growth inhibition are readily generated in vitro. A laboratory strain, F32, was cultured for long periods (10–12 weeks) in the presence of suboptimal inhibitory antibody concentrations. The antibodies used were the human monoclonal antibody 33G2 reacting with a linear epitope of the P. falciparum blood-stage antigen 332 and rabbit antibodies to repeat sequences of the blood-stage antigen Pf155/RESA. Our data indicate that the P. falciparum parasites adapt to antibody pressure as reflected by their specifically decreased sensitivity to growth inhibition. A relative resistance of the parasite to growth inhibition mediated by the antibodies used in the culture developed successively, while the parasite remained sensitive to growth inhibition by other antibodies. When the antibody pressure was removed a successive return of sensitivity to growth inhibition developed. Immunofluorescence did not reveal any significant difference in antigen expression between the parasite populations. However, PCR analysis showed that a new population appeared in the parasites grown in the presence of mAb 33G2, while no such change was detected in those grown in the presence of the rabbit antibodies. Our data suggest that the specific decrease in sensitivity to growth inhibition may either be due to down-regulation of antigen synthesis or expression by antibody pressure or, that antibody pressure selects for parasites with low expression of a specific antigen from a heterogeneous parasite population.

Virulent strains of Entamoeba histolytica have been reported to produce a mixture of phosphoglycoconjugates that share some structural features with the lipophosphoglycans (LPGs) of Leishmania. Purification of these glycoconjugates is essential to their precise structural characterization. In this study we have extracted ‘LPG-like’ molecules from various virulent E. histolytica strains and purified on the basis of charge differences, 2 apparently related glycoconjugates a ‘LPG’ and a ‘lipophosphopeptidoglycan (LPPG)’. In marked contrast to the abundance of these ‘LPG’ and ‘LPPG’ molecules in the virulent strains, avirulent E. histolytica and E. dispar strains produce either very low, or no detectable levels of LPG, and either low levels or modified forms of ‘LPPG’. Monospecific polyclonal antibodies prepared against that ‘LPG’ of the virulent strain HM-1: IMSS cl6 identified epitopes shared between both the ‘LPG’ and the ‘LPPG’ of this and other virulent strains, using Western blot analysis. Flow cytometric analysis of a range of strains using these antibodies identified a surface distribution of these molecules and confirmed a correlation between surface exposure of epitopes bound by these antibodies and parasite virulence.

A 30-h in vitro susceptibility test of Plasmodium falciparum wild isolates to artemisinin, chloroquine, sulfadoxine/pyrimethamine and mefloquine was performed in Kibaha, Tanzania. A sigmoid Emax model was fitted to all data for each isolate and drug combination. Artemisinin and mefloquine exhibited 100% growth inhibition against all isolates tested (n=69–74). The EC50 values for artemisinin and mefloquine were 44 and 146 nM respectively. Chloroquine and sulfadoxine/pyrimethamine resistance was 30% and 13% respectively. Susceptibility parameters (EC50, 90, 95 and 99 values and s) varied between compounds and isolates indicating the different sensitivity of P. falciparum isolates. No correlation between susceptibility parameters of artemisinin and the other compounds was found. The high in vitro activities of artemisinin and mefloquine indicate their potential role for the treatment of multidrug-resistant malaria in Africa.

An axenic culture of Giardia was established from a sample of infected intestine obtained following autopsy of a sulphur-crested cockatoo (Cacatua galerita). The cockatoo recently captured in the wild and with good muscle tone died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites which established in the traditional, axenic Giardia medium (TYI-S-33 with supplementary bile) were morphologically identical to G. duodenalis. When outbred Quackenbush Swiss neonatal mice were infected with trophozoites a chronic infection was established and parasites were still present at 38 days post-inoculation. Weight gain by infected mice was reduced by 20%, thus mimicking failure-to-thrive syndrome in children, and maximum parasite load was more than 3-fold higher in comparison with other G. duodenalis strains. Analysis of the electrophoretic karyotype, rDNA and hybridization studies together with Giemsa- and trichrome-stained samples, and scanning electron microscopy indicated that the bird-derived Giardia belonged to the duodenalis group. This is the first report of infection of mammals with Giardia isolated from a bird. These data may have potentially serious implications for contamination of watersheds and establishment of zoonotic infections.

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.

A quick and simple method of purifying the histones from Plasmodium falciparum culture supernatant or from infected erythrocytes is described. The proteins were present only in preparations rich in P. falciparum nuclear material and were soluble at acid pH and in strongly anionic detergents. Four proteins, of 14–18 kDa were identified as the P. falciparum core histones. N-terminal sequence analysis of the 16 and 18 kDa proteins and of a tryptic fragment of the protein mixture revealed strong homologies with deduced cDNA or protein sequences of histones H2A, H2B, and H3 of P. falciparum and other species. Antibodies raised against the proteins cross-reacted weakly with histones of other species and, on immunofluorescence, localized the proteins to schizont nuclei. Anti-P. falciparum histone antibodies were detected in sera of semi-immune human adults but these antibodies did not react with human histones on a Western blot. The large quantities of P. falciparum histone released and the chronic nature of malarial infection, together with the unusually high avidity of histones for ligands found in renal and vascular basement membrane, raise the question of a role for histones in the pathogenesis of malarial infection. Wesuggest that histones or histone±antibody complexes may contribute to disease pathology.

The role of T-cells in development of experimental cerebral malaria was analysed in C57B1/6J and C57B1/10 mice infected with Plasmodium berghei K173 or Plasmodium berghei ANKA by treatment with anti-CD4 or anti-CD8 mAbs. Mice were protected against cerebral malaria (CM) when anti-CD4 or anti-CD8 mAbs were injected before or during infection. Even in mice in end-stage disease, i.e. with a body temperature below 35·5 °C, treatment with anti-CD4 or anti-CD8 antibodies or the combination protected against CM, whereas chloroquine treatment was completely ineffective in inhibiting further development of the cerebral syndrome.

Trypanosoma cruzi, the agent of Chagas' disease, exhibits considerable biological variability. Moreover, it has been postulated that populations of this protozoan are subdivided into natural clones, which can be separated from each other by considerable levels of evolutionary divergence. The authors have proposed that this long-term clonal evolution may have a profound impact on Trypanosoma cruzi biological diversity. In order to test this hypothesis, 16 T. cruzi stocks representing 3 major clonal genotypes of the parasite were analysed for 8 different in vitro biological parameters. The overall results show a strong statistical linkage between genetic and biological differences. This is in agreement with the working hypothesis, although a notable biological variability is observable among the stocks of each of the 3 major clonal genotypes. The authors propose that T. cruzi genetic variability must be taken into account in any applied study dealing with this parasite.

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Currently used antiparasitic drugs, including benzimidazoles, nitroimidazoles, avermectins, polyene ionophores, hydroxynaphthoquinones and sesquiterpene lactones, were identified through the empirical route to drug discovery. The modern rational approach to drug design is focused upon the structure and function of biochemical and molecular targets. The requisite pharmacological properties for new anti-parasite drugs should not be ignored in this process.

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many-fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglobin digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.

Rodent and avian malaria parasites have been reported to have an adverse affect upon the reproductive fitness of mosquitoes. In order to determine whether fecundity reduction occurs in Anopheles gambiae s. l. infected with human malaria a study of wild-caught mosquitoes was undertaken in the Muheza district of north east Tanzania. Fully engorged, indoor resting females were collected daily for 4 months and maintained for 5 days. A sporozoite rate of 11·5% was detected for the whole collection and of those females alive on day 6 an additional 17·5% were infected with oocysts alone. Oocyst, but not sporozoite, infection resulted in a 17·5% reduction in egg production. Fecundity reduction was not caused by a reduction in bloodmeal size in infected females and no size difference was detected between oocyst-infected and uninfected females although sporozoite-positive females were significantly larger. Comparisons in parity between uninfected and infected groups indicate that infection does not affect survival beyond the first gonotrophic cycle as no changes in survivorship occurred as a result of sporozoite infection.

Schistosoma mansoni egg counts by faecal examination vary considerably and are not very sensitive, so prevalences are underestimated. The distribution of egg counts can adequately be described by a stochastic model which distinguishes variation in counts between persons and variation in repeated counts within a person. Based on this model a pocket chart has been developed which predicts the proportion of individuals harbouring at least 1 S. mansoni worm pair – the ‘true prevalence’ – from a simple single survey prevalence and geometric mean egg count (using common duplicate 25 mg Kato–Katz smears). The current paper describes the validation of this chart by comparing predicted true prevalences with prevalences observed after 5–7 repeated Kato–Katz faecal examinations (Burundi), by examination of a large quantity of stool using the Visser filter (Brazil) or a selective sedimentation–filtration method (Surinam). Because 5–7 repeated examinations do not suffice to measure all infections, predictions have been made of the cumulative proportion positives over 5–7 surveys – the ‘approximate true prevalence’ – as well. After dividing the data into age groups, 12 different subsets were considered for validation. In all 12 cases, predicted true prevalences (or approximate true prevalences for the Burundi data) agree well with those observed. The overall agreement depends only slightly on the assumed relationship between worm numbers and mean egg counts, with a good fit for a productivity between 0·8 and 4·4 eggs per gramme faeces (EPG) per worm pair (WP). This interval includes the most plausible value from the literature, i.e. 1·0 EPG/WP, which has been applied in the initial pocket chart. These findings support the validity of the chart to predict true prevalences for a wide range of productivity assumptions, and reinforces the applicability of its underlying stochastic model to describe egg count variation. However, as predictions appear to vary importantly when using only part of the data, it is also concluded that the pocket chart never compensates for limited validity of initial single survey prevalences and geometric means in consequence of small sample sizes.

The protozoan parasite Toxoplasma gondii possesses a triple surface membrane called the pellicle. This is made of an outer plasmalemma and an inner membrane complex lying under the plasmalemma. Using a high salt glycerol treatment followed by sonication, we have obtained a partial dissociation of the pellicle. A plasmalemma-enriched fraction was isolated on 0·7M sucrose. It was identified by immunodetection of the tachyzoite major surface antigens. Protein content, resolved by SDS–PAGE, revealed that the surface protein SAG1 is the major component of the plasmalemma. The plasmalemma fraction is made of small vesicles (20–100 nm) which possess a low density (1·085–1·090 g/cm3 in sucrose) contrasting with other eukaryotic plasma membranes (1·12–1·16 g/cm3).

Oocysts of Cryptosporidium parvum showed relatively low levels of SOD activity. The SOD which had a pI of 4.8 and an approximate molecular weight of 35 kDa appeared to be iron dependent. Catalase, glutathione transferase, glutathione reductase and glutathione peroxidase activity could not be detected, nor could trypanothione reductase. No NADH or NADPH oxidase activity could be detected, nor could peroxidase activity be demonstrated using o-dianisidine, guaiacol, NADPH or NADH as co-substrates. However, an NADPH-dependent H2O2 scavenging system was detected in the insoluble fraction.

In areas where malaria is endemic, helminthic infections, caused by intestinal or filarial parasites, commonly coexist with malaria in the same individual. This study investigates the course of Plasmodium berghei malaria infection in CBA/J mice inoculated with irradiated attenuated 3rd-stage larvae (L3) of Brugia pahangi. Peripheral eosinophil counts, serum IgE levels and cytokine production revealed that the filarial antigen induced T-helper type 2 (Th2) cell predominance in these mice, which protected them against the development of cerebral malaria. These mice significantly prolonged their survival, compared with the control mice after P. berghei infection. All of the mice not inoculated with irradiated L3 died within 12 days with acute neurological manifestations unrelated to the level of parasitaemia after infection of P. berghei. Conversely, most of the inoculated mice lived more than 3 weeks following infection with P. berghei, dying in the fourth week of severe anaemia and overwhelming parasitaemia. This suggests that Th2-dominant responses lead to the downregulation of susceptibility to murine cerebral malaria.

Strains of Entamoeba histolytica isolated in Brazil were characterized using the Low-Stringency Single Specific Primer PCR (LSSP–PCR), that detects single or multiple mutations in gene size DNA fragments. Using this technique, a 482-bp genomic DNA fragment from a structural gene in 8 strains and 2 clones of E. histolytica, isolated from symptomatic and asymptomatic patients in Brazil, including pathogenic and non-pathogenic zymodemes were studied. The results obtained indicate that LSSP–PCR is a valuable method for differentiating strains of E. histolytica. Moreover, the results are consistent with the concept that pathogenic and non-pathogenic strains of E. histolytica may represent distinct species or subspecies and are in accord with phenotypically characteristic isoenzyme patterns.

Advances in the life and physical sciences have enabled us to characterize the 3-dimensional structure and the biochemical or biological activity of both small and large molecules. The use of structural chemistry to assist understanding of biological activity provides information relevant to the design, development or identification of new pharmaceuticals. This structure based approach has become an important component of drug research and is in widespread use by the major pharmaceutical companies. A brief historical introduction, to convey how this area of science has reached the present stage, is given. The basis of the structural approach to understanding the chemistry of small and large molecule biological activity is outlined with an emphasis on the use of results derived from X-ray diffraction methods. Developments in other areas are discussed to emphasize the multidisciplinary nature of this research and the benefits of combining different methods. Examples of protein crystallographic studies in the area of molecular parasitology, some of which are directly relevant to antiparasite drug design, are presented. The characterization of the enzyme trypanothione reductase, a project which has benefited from many of the recent developments, is detailed. Future challenges and difficulties, both scientific and economic, are discussed.