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.

The development of malaria due to Plasmodium falciparum is a complex, multi-stage process. It is usually characterized by an exponential growth in the number of parasite-infected erythrocytes, followed by marked oscillations in this number with a period of 48 h, which are eventually dampened. This course of events has been the subject of various mathematical models. In this paper we propose a new mathematical model for the in-host asexual erythrocytic development of P. falciparum malaria. Synchronicity of the infection is shown to be an inherent feature of infection, irrespective of the duration of merozoite release from the liver. It will, therefore, cause periodic symptoms, as known in malaria patients. We also simulate the effects of an induced host immune response and show how the level of immunity affects the development of disease. The simulations fit well with the clinical observations. We show how infection can become asynchronous and discuss the effect of desynchronization on the circulating and total parasitaemia and demonstrate that synchronized broods will show parasitaemia fluctuations.

The Programme to Eliminate Lymphatic Filariasis (LF), launched in 1997, is a public-sector/private- sector partnership organized as a Global Alliance, with the World Health Organization serving as secretariat. Its principal purpose is to carry out the mandate of the 50th World Health Assembly (1997) to eliminate lymphatic filariasis as a public health problem worldwide, but its tools and strategies for achieving this end also have important additional public health benefits. Foremost among these are the effects the Programme can have on the control of intestinal helminth infections in treated populations, largely because of certain similarities, or overlaps, of the drugs and strategies used in the public health approaches to these parasitic infections. The principal health benefits from treating these intestinal helminth infections are reviewed in detail below, but in addition there might even be an entirely new justification for aggressive treatment and control of these infections if the recently described effects they have on potentiating HIV infections in affected populations can also be further substantiated and extended.

The life-cycle of Plasmodium depends on transmission of the parasite from the vertebrate host into its vector when the insect takes a bloodmeal. Transmission success may depend in part on the parasite's gametocyte density and sex ratio in the blood. P. mexicanum, a parasite of fence lizards in California, USA, exploits the sandfly Lutzomyia vexator as its vector. In experimental transmissions using naturally infected lizards as donors of blood, transmission success (measured as percentage of vectors infected and number of parasite oocysts on the insect's midgut) was positively related to gametocyte density, although density above 20/1000 erythrocytes did not improve transmission. Sex ratio (proportion of microgametocytes in the infection) was positively correlated with gametocyte density. Transmission improved with higher proportion of microgametocytes, but partial correlations revealed that this was a result only of higher gametocyte densities. These results agree with the theory of virulence and sex ratios because single clone infections should produce a more female-biased sex ratio and grow to the minimum parasitaemia that would maximize clonal transmission, whereas multiple clone infections will be closer to a 1[ratio ]1 sex ratio and yield a higher parasitaemia when each clone competes for transmission to the vector.

Asymptomatic malaria parasitaemia is prevalent among schoolchildren in many countries. The relationship between asymptomatic parasitaemia and children's cognitive functions was examined in a case control study and then in a natural experiment. A group (n = 445) of asymptomatic parasitaemic boys were compared with a group of non-parasitaemic boys (n = 142) matched for grade and school on their performance on a battery of cognitive tests. Two weeks later the parasitaemic children were re-screened and 150 children of those who remained parasitaemic were matched for grade and school with 150 children who were no longer parasitaemic. These children were then re-tested and their cognitive function compared. Initially, after controlling for age, socio-economic background and nutritional status the parasitaemic children performed worse than the non-parasitaemic children in fine motor function tests. There was no difference in change in cognitive test scores between those who became non-parasitaemic and those who remained parasitaemic. However, children who initially had the highest parasite density improved the most in 2 fine motor tests and a picture memory test. We were unable to show a benefit from losing parasitaemia over a 2 week period, but it remains possible that parasitaemia may affect cognition and longer term trials should be conducted.

Commitment to the production of female and male gametocytes was studied in the NF54 line of the human malaria parasite Plasmodium falciparum. The development of sibling parasites derived from individual schizonts was followed, and 2 antisera against the female gametocyte-specific protein Pfg377 and the male gametocyte-specific protein α-tubulin II were used to determine the sex of sibling gametocytes. The experiment showed that individual cohorts of sibling gametocytes were stained in a mutually exclusive fashion by only one or the other antiserum, indicating that individual schizonts committed to yield sexual parasite progeny produce gametocytes of the same sex. This work suggests that in P. falciparum commitment to sexual differentiation occurs prior to schizont maturation, at the same moment when the sex of the resulting gametocytes is determined.

To investigate the quantitative distribution of Plasmodium falciparum gametocytes into the vector bloodmeal, Anopheles arabiensis mosquitoes were fed on 3 volunteers naturally infected with gametocytes. The content of each mosquito midgut was smeared on a microscope slide and Giemsa stained. The distribution of gametocytes ingested by mosquitoes followed a negative binomial distribution, with apparently constant overdispersion (parameter k±S.E. = 3·105±0·392) for the 3 series. This aggregation of gametocytes in some midguts probably facilitates the conjunction of gametes and fertilization. This suggests that P. falciparum gametocytes in the peripheral blood flow of infected man do not follow an independent, homogeneous pattern but show a significant aggregation.

The oocysts of Plasmodium falciparum that form on the midgut wall of anopheline vectors represent the meiotic products of the sexual cycle of the parasite and are useful for genetic analyses. Recognizing the limitations of current markers for the analysis of genetic variation, we describe a protocol for the DNA isolation and PCR amplification of microsatellite loci from individual oocysts. We present a simple method for the storage and isolation of individual Plasmodium oocysts from wild-caught mosquitoes and demonstrate that it is possible to extract sufficient oocyst DNA for approximately 50 amplifications. Genotyping was successful for 10 microsatellite loci and, using Genescan technology, we found extensive microsatellite polymorphism in an analysis of a dozen oocysts from each of 2 Anopheles gambiae female specimens collected within 6 months at the same location in northern Tanzania. All of the loci exhibited at least 2 alleles, with a maximum of 5. Homozygous and heterozygous oocysts could easily be discerned, but most of the oocysts were homozygous. The procedure has great potential for shedding light on genetic mechanisms operating during meiosis in mosquito vectors and the influence this may have on the genetic structure of natural populations of P. falciparum.

The parasite, Neospora caninum is an important cause of abortion in cattle. It is transmitted vertically or horizontally and infection may result in abortion or the birth of a live, healthy but infected calf at full-term. Only a proportion of infected cattle abort and the pathogenesis of abortion is not understood. Groups of cattle were infected with 107N. caninum tachyzoites intravenously at different times relative to gestation. Intravenous inoculation was chosen to reproduce the putative haematogenous spread of N. caninum following either recrudescence of endogenous infection or de novo infection. In all cattle, infection was accompanied by high γ-interferon and lymphoproliferative responses, and a biased IgG2 response indicating that N. caninum infection is accompanied by a profound Th1 helper T cell-like response. Infection at 10 weeks gestation resulted in foetopathy and resorption of foetal tissues 3 weeks after infection in 5 out of 6 cows. Infection at 30 weeks gestation resulted in the birth of asymptomatic, congenitally-infected calves at full term in all 6 cows, whereas the 6 cows infected before artificial insemination gave birth to live, uninfected calves. These results suggest that the reason some cows abort is related to the time during gestation when they become infected or an existing infection recrudesces.

Although artesunate, one of the potent derivatives of the qinghaosu family of drugs for treating falciparum malaria, is already in use in the field, its therapeutic protocol has only been developed empirically by hit-or-miss. A pharmacokinetic–pharmacodynamic (PK–PD) model, required for creating such a protocol, is not straightforward. Artesunate presents extremely fast pharmacokinetics. As a result the stage specificity of its action must be treated explicitly. Also, use of standard PK–PD modelling fails to explain the clinical results. Our PK–PD modelling of its activity leads us to the postulation of the existence of a novel effect: a small fraction of the parasites, as a result of chemotherapeutic pressure, become cytostatic, or ‘dormant’. At this stage, the parasite cycle is halted, making them unsusceptible to further dosing until wakening. This slows down the antimalarial activity of the drug, entailing either many frequent doses or an extended period of treatment and surveillance. Based on our modelling, we suggest a method for deciding on rational models of chemotherapy against falciparum malaria.

Isolates of 2 microsporidian species from the genus Encephalitozoon (E. cuniculi and E. hellem) were compared by analysis of DNA amplified from a gene region encoding the repeat domain of a polar tube protein (PTP1). Sequence data obtained for 11 E. cuniculi isolates from 5 different mammalian hosts well support the existence of 3 previously designated strains. Strain type III was characterized by a lack of a 78bp repeat, producing an amplicon of reduced size. Strain type II differed from strain type I by 3 nucleotide substitutions so that AvaII digestion of the corresponding PCR products provided distinct restriction patterns. Surprisingly, the comparison of 2 human isolates of E. hellem belonging to the same rDNA ITS genotype shows a high level of heterogeneity through numerous point mutations and variation in PTP1 repeat number. Further characterization of additional E. hellem isolates based on PTP1 sequence polymorphisms should be of interest for tracing sources of infection.

The purpose of this brief introduction to the supplement that follows is to highlight the current unique opportunity to coordinate intestinal helminth control programmes with the relatively new global initiative to eliminate lymphatic filariasis. Such synergy at global, regional, national and local levels has the potential to greatly enhance the impact, decrease the cost and facilitate sustainability in both of these health programmes.

Members of the phylum Microspora are a group of unusual, obligate intracellular eukaryotic parasites that infect a wide range of hosts. However, there are a limited number of microsporidial infections reported in avian hosts, and no parasite species has been defined as an avian pathogen. A microsporidian organism was recovered from the droppings of a clinically normal peach-faced lovebird (Agapornis roseicollis) and established in in vitro culture. Intermittent parasite spore shedding was documented over a 2-month period using calcofluor M2R staining of cloacal swabs. The organism was identified as Encephalitozoon hellem based on protein and antigenic profiles and molecular sequencing of the small subunit and internal transcribed spacer regions of the ribosomal RNA gene.

IFNγ receptor (IFNγR) deficient mice and IL-4 deficient mice were infected with blood-stage Plasmodium chabaudi AS in order to analyse the role of Th1 (IFNγ) and Th2 (IL-4)-associated cytokines in the development of protective immunity to the parasite. A high mortality rate and failure to reduce the primary parasitaemia to subpatent levels was observed in the IFNγR deficient mice. IL-4 deficient mice controlled a primary P. chabaudi AS infection in a similar manner to control mice and no mortality was observed. IFNγR deficient mice had a reduction in parasite-specific IgG and a significantly increased level of total IgE compared to control mice. There was no reduction in the level of parasite-specific IgG in IL-4 deficient mice. Cytological analysis of the cells present in the spleen and liver during the primary parasitaemia revealed a reduction in the numbers of lymphocytes, monocytes and polymorphonuclear (PMN) cells in the liver at the peak of parasitaemia in both IFNγR deficient mice and IL-4 deficient mice compared to control mice. Adoptive transfer studies demonstrated that cells isolated from the liver at day 11 post-infection could confer some protective immunity to P. chabaudi AS infection.

The North American opossum, Didelphis virginiana, is a definitive host for at least 3 species of Sarcocystis:S. falcatula Stiles 1983, S. neurona Dubey, Davis, Speer, Bowman, de Lahunta, Granstrom, Topper, Hamir, Cummings, Suter 1991, and S. speeri Dubey and Lindsay 1999. In order to identify species of Sarcocystis in the South American opossum, D. marsupialis, Sarcocystis sporocysts from the intestines of a naturally infected opossum (D. marsupialis) from Brazil were fed to 4 gamma-interferon knockout (KO) mice, a nude mouse, and 2 budgerigars (Melopsittacus undulatus). All 4 KO mice became ill and 1 died 42 days post-feeding (p.f.) of sporocysts, 1 was killed 44 days p.f. because of neurological signs, and 2 were killed 52 and 53 days p.f. because of abnormal gaits. Numerous sarcocysts were seen in the skeletal muscles of all 4 KO mice and they were structurally identical to S. speeri seen in KO mice fed sporocysts from D. virginiana from the United States and D. albiventris from Argentina. The nude mouse was killed 41 days p.f. because it appeared weak; schizonts were seen in sections of its liver and sarcocysts were seen in sections of skeletal muscles. Sarcocystis speeri was cultured in bovine turbinate cells inoculated with liver homogenate from this mouse. Sarcocystis neurona was not demonstrable in tissues of mice. The two budgerigars remained asymptomatic and S. falcatula was not found in their tissues when they were killed 29 days p.i. This is the first report of S. speeri from D. marsupialis.

We have studied the activity of the polyether ionophorous antibiotic monensin against the cyst form (bradyzoite) of Toxoplasma gondii. The experiments were conducted in vitro using 2 methods; cysts produced either in mice or in cell culture were exposed to monensin in vitro, and the infectivity of the parasites was then assessed in vivo or in vitro. The data obtained from these 2 systems of evaluation showed that monensin inhibits the infectivity and the viability of the bradyzoites. Its activity was time and concentration dependent. The first effects were observed at very low drug concentrations (i.e. 0·0001 μg/ml). Immunofluorescence and electron microscopy analysis showed significant cytological alterations of the monensin-treated bradyzoites: they were swollen, had a large number of vacuoles in their cytoplasm and were found lysed at higher concentrations in ionophore.

The burden and duration of asymptomatic malaria infections were measured in residents of the malaria endemic village of Gonoa, Madang Province, Papua New Guinea. Plasmodium falciparum, P. vivax and P. malariae infections in people aged 4 years to adulthood were compared. Frequent sampling at 3-day intervals for up to 61 days allowed assessment of individual episodes of infection. Statistical assessment of P. falciparum detection revealed a periodicity consistent with synchronous replication of this species over periods up to 27 days. The duration of P. falciparum episodes was longer across all age groups than that of P. vivax and P. malariae. A trend for decreasing duration with age was also noted in data from each species. This was most prominent in P. falciparum infections: median duration in 4-year-olds was > 48 days compared with a median between 9 and 15 days in older children and adults. The results are consistent with the slow acquisition of immunity to antigenically diverse Plasmodium populations and suggest a faster rate of acquisition to P. vivax and P. malariae than to P. falciparum.

The four most important forms of malnutrition worldwide (protein-energy malnutrition, iron deficiency and anaemias (IDA), vitamin A deficiency (VAD), and iodine deficiency disorders (IDD)) are examined below in terms of their global and regional prevalences, the age and gender groups most affected, their clinical and public health consequences, and, especially, the recent progress in country and regional quantitation and control. Zinc deficiency, with its accompanying diminished host resistance and increased susceptibility to infections, is also reviewed. WHO estimates that malnutrition (underweight) was associated with over half of all child deaths in developing countries in 1995. The prevalence of stunting in developing countries is expected to decline from 36% in 1995 to 32·5% in 2000; the numbers of children affected (excluding China) are expected to decrease from 196·59millions to 181·92millions. Stunting affects 48% of children in South Central Asia, 48% in Eastern Africa, 38% in South Eastern Asia, and 13–24% in Latin America. IDA affects about 43% of women and 34% of men in developing countries and usually is most serious in pregnant women and children, though non-pregnant women, the elderly, and men in hookworm-endemic areas also comprise groups at risk. Clinical VAD affects at least 2·80million preschool children in over 60 countries, and subclinical VAD is considered a problem for at least 251millions; school-age children and pregnant women are also affected. Globally about 740million people are affected by goitre, and over two billions are considered at risk of IDD. However, mandatory salt iodisation in the last decade in many regions has decreased dramatically the percentage of the population at risk. Two recent major advances in understanding the global importance of malnutrition are (1) the data of 53 countries that links protein-energy malnutrition (assessed by underweight) directly to increased child mortality rates, and (2) the outcome in 6 of 8 large vitamin A supplementation trials showing decreases of 20–50% in child mortality.

The differentiation of the two sexes in the gametocytogenesis of Plasmodium falciparum was investigated using a plaque assay and antibodies specific for various stages and sexes of gametocytes. Immunofluorescence assays on plaques of cultured parasites grown in monolayers of erythrocytes revealed that the merozoites released from a single sexually-committed schizont became either all male or all female gametocytes. Thus, the commitment of this species to differentiate into one sex or the other is likely to occur prior to the nuclear division of the sexually-committed schizont. The characteristic female-biased gametocyte sex ratio observed for many Plasmodium species is manifested in P. falciparum by a greater percentage of schizonts that produce female gametocytes (67–71%) than those that yield males. From the plaque assay, it was determined that the number of gametocytes produced per sexually-committed schizont was similar for both sexes, indicating that allocation of parasite resources was equal for each sex of gametocyte. The timing of sexual differentiation and features of the gametocyte sex ratio is discussed in relation to previous observations on P. falciparum and related malaria parasites.

A previous analysis of tick infestation patterns on rodents in Slovakia suggested that the key to the focal distribution of western-type tick-borne encephalitis virus (TBEv) in Europe is the geographically variable degree of synchrony in the seasonal activity of larval and nymphal Ixodes ricinus ticks. This prediction is here tested by examining records, from 7 different countries, of the seasonal variation in the abundance of larvae and nymphs feeding on rodents or questing on the vegetation. Larvae consistently started feeding and questing earlier in the year at sites within TBEv foci than elsewhere, so that they appeared in the spring as soon as nymphs were active. Such larval–nymphal synchrony is associated with a rapid fall in ground-level temperatures from August to October as revealed by the satellite-derived index of Land Surface Temperature (LST). Likewise, of 1992 pixels sampled on a grid across Europe, the 418 that fell within TBEv foci were characterized by a higher than average rate of autumnal cooling relative to the peak midsummer LST. It is proposed that such a seasonal temperature profile may cause unfed larvae to pass the winter in quiescence, from which they emerge synchronously with nymphs in the spring.