Genetic complexity and gametocyte production of Plasmodium falciparum in Fulani and Mossi communities in Burkina Faso.

We have examined Plasmodium falciparum gametocyte prevalence, density and their genetic complexity among children of 2 sympatric ethnic groups (Mossi and Fulani) in villages in Burkina Faso. The 2 groups are known to have distinct differences in their susceptibility and immune responses to malaria. We used RT-PCR and sequence-specific probes to detect and type RNA of the gametocyte-specific protein Pfs48/45. There were no differences in detection rates of asexual forms and gametocytes among the 2 groups, using PCR and RT-PCR, respectively. However, there were significant differences in densities of asexual forms and gametocytes, which were both higher among Mossi than Fulani. Both asexual forms and gametocyte densities were influenced by age and ethnicity. Multiple-clone infections with more than 1 gametocyte genotype were equally prevalent among Fulani and Mossi. These differences can most probably be attributed to genetic differences in malaria susceptibility in the 2 ethnic groups.

Local differentiation in Plasmodium falciparum drug resistance genes in Sudan.

Studies of population genetic structure of parasites can be used to infer which parasite genes are under selection. Here, the population structure of 4 genes associated with drug resistance of Plasmodium falciparum (the chloroquine resistance transporter, pfcrt, dihydrofolate reductase, dhfr, dihydropteroate synthase, dhps, and multi-drug resistance, pfmdr-1) were examined in parasite populations in 3 villages in eastern Sudan and in an urban area of Khartoum, the capital. In order to differentiate the effects of drug selection from neutral influences on population structure, parasites were also genotyped for 3 putatively neutral microsatellite loci (polyalpha, TA81 and pfg377), and for 2 antigenic loci that are either under balancing selection or neutral, merozoite surface protein 1 and 2, (MSP-1 and MSP-2). Cross-sectional surveys were carried out during the peak transmission (wet) season and in the ensuing dry season. No significant variation in frequencies of MSP-1 and MSP-2 alleles was seen among villages in the eastern region and between the villages and Khartoum, nor between the wet and dry season. However, the drug resistance genes, pfmdr-1, pfcrt and dhfr and to a lesser extent the microsatellite loci showed high FST values when comparing villages with Khartoum, indicating strong geographical differentiation at these loci. Moreover, variation in frequencies of the drug resistance genes, pfmdr-1, pfcrt and dhfr, was observed between the wet and dry season. These differences most probably reflect the variation in drug pressure between each region, and in drug usage between the wet and dry season in a given region.

Diversity of Plasmodium falciparum clones infecting children living in a holoendemic area in north-eastern Tanzania.

The diversity of Plasmodium falciparum clones and their role in progression from asymptomatic to symptomatic condition in children have been investigated. Attempts to identify whether particular parasite genotypes were associated with the development of clinical symptoms have been made. A cohort of 34 initially asymptomatic parasitaemic children aged 1-5 years were followed daily for 31 days. Clinical examinations were made each day for signs and symptoms of clinical malaria, followed by parasitological investigation. Nineteen children developed symptoms suggestive of clinical malaria during this period. Daily blood parasite samples from 13 children who developed clinical malaria symptoms and 7 who remained asymptomatic were genotyped by PCR-amplification of the polymorphic regions of the merozoite surface proteins 1 and 2 (MSP1 and MSP2) and the glutamate rich protein (GLURP) genes. Infections were found to be highly complex in both groups of children. Every isolate examined from both groups had a mixture of parasite clones. Daily changes were observed in both parasite density and genotypic pattern. The mean number of genotypes per individual was estimated at 4.9 and 2.7 for asymptomatic and symptomatic groups of children, respectively. Analysis of allele frequency distributions showed that these differed significantly for the MSP1 locus only.

Antimalarial drugs clear resistant parasites from partially immune hosts.

Circumstantial evidence in human malaria suggests that elimination of parasites by drug treatment meets higher success rates in individuals having some background immunity. In this study, using the rodent malaria model Plasmodium chabaudi, we show that drug-resistant parasites can be cleared by drugs when the host is partially immune.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

Of mice and malaria mutants: unravelling the genetics of drug resistance using rodent malaria models.

It is well recognized that drug resistance is the most significant obstacle to gaining effective malaria control. Despite the enormous advances in the knowledge of the biochemistry and molecular biology of malaria parasites, only a few genes determining resistance to the commonly used drugs have been identified. The idea that rodent malaria parasites should be exploited more widely for such work, in view of the practical problems of studying this subject experimentally in human malaria, is presented.

High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance Gene pfmdr1.

Polymorphisms were examined in 2 Plasmodium falciparum genes, as were chloroquine responses of clones and isolates from a village in eastern Sudan. There was a significant association between an allele of the P. falciparum chloroquine resistance transporter gene (pfcrt-T76) and both in vitro and in vivo resistance. There was a less significant association with the multidrug resistance gene pfmdr1-Y86 allele. A significant association between pfmdr1-Y86 and pfcrt-T76 was apparent among resistant isolates, which suggests a joint action of the 2 genes in high-level chloroquine resistance.

Insecticide treated curtains and allelic polymorphism of Plasmodium falciparum genes in a rural area of Burkina Faso (west Africa).

To assess the possible impact of insecticide treated curtains (ITC) on the composition of a Plasmodium falciparum population in a rural area of Burkina Faso, blood samples were collected during the rainy season of 1997 from 226 children aged 3-6 years, from 4 villages equipped with ITC and 2 control villages without ITC. The analysis of fragment lengths of 3 highly polymorphic P. falciparum genes (msp-1, msp-2 and glurp) revealed a maximum number of 3 alleles per infected person for each gene. The mean number of clones per infected person was similar in villages with and without ITC.

Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction.

A molecular assay has been developed for the specific detection and genetic characterisation of Plasmodium falciparum gametocytes in the blood of malaria infected individuals. The assay is based on the reverse transcription and polymerase chain reaction (RT-PCR) amplification of the messenger RNA of gene pfg377, a sexual-stage specific transcript abundantly produced in maturing gametocytes. The gene contains four regions of repetitive sequences, of which region 3 was shown to be the most polymorphic in laboratory clones and field isolates of the parasite. Analysis of samples of malaria infected blood by RT-PCR specific for region 3 has enabled identification of multiple gametocyte-producing clones within single infections. The assay is able to detect gametocytes below the threshold of microscopic detection, and is highly specific for its gametocyte targets also in the presence of a vast excess of asexual forms.

Commitment to sexual differentiation in the human malaria parasite, Plasmodium falciparum.

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.

Age- and species-specific duration of infection in asymptomatic malaria infections in Papua New Guinea.

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.

Genetic diversity and dynamics of plasmodium falciparum and P. vivax populations in multiply infected children with asymptomatic malaria infections in Papua New Guinea.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3alpha, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6.6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co-infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5-14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

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

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

Population dynamics of Plasmodium falciparum in an unstable malaria area of eastern Sudan.

The Plasmodium falciparum population in Asar village, eastern Sudan, where malaria transmission is markedly seasonal, was monitored monthly over a period of 15 months. A cohort of infected patients was treated and then followed monthly throughout the dry season until the next transmission season. Parasitaemia detected by microscopy among the cohort reduced dramatically following treatment, but remained sporadic during the dry season, and reappeared following the onset of the next wet season. However between 40 and 50% of the cohort retained a persisting parasitaemia detectable by PCR throughout the dry season. These parasites were genetically complex, consisting of multiple clones with a large repertoire of alleles of the studied genes. While the number of clones per host dropped significantly following treatment of acute cases during the transmission season, drug treated people nevertheless maintained an average of one clone throughout the dry season. Allele frequencies of MSP-1, MSP-2 and GLURP showed slight, statistically insignificant, fluctuations between the dry and wet seasons. A higher frequency of inbreeding was estimated among the parasites that survived the dry season compared to the wet season.

Cross-species interactions between malaria parasites in humans.

The dynamics of multiple Plasmodium infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.

Antigenic and sequence diversity at the C-terminus of the merozoite surface protein-1 from rodent malaria isolates, and the binding of protective monoclonal antibodies.

Merozoite surface protein-1 (MSP-1) is a major candidate in the development of a vaccine against malaria. Immunisation with a recombinant fusion protein containing the two Plasmodium yoelii MSP-1 C-terminal epidermal growth factor-like domains (MSP-1(19)) can protect mice against homologous but not heterologous challenge, and therefore, antigenic differences resulting from sequence diversity in MSP-1(19) may be crucial in determining the potential of this protein as a vaccine. Representative sequence variants from a number of distinct P. yoelii isolates were expressed in Escherichia coli and the resulting recombinant proteins were screened for binding to a panel of monoclonal antibodies (Mabs) capable of suppressing a P. yoelii YM challenge infection in passive immunisation experiments. The sequence polymorphisms affected the binding of the antibodies to the recombinant proteins. None of the Mabs recognised MSP-1(19) of P. yoelii yoelii 2CL or 33X or P. yoelii nigeriensis N67. The epitopes recognised by the Mabs were further distinguished by their reactivity with the other fusion proteins. The extent of sequence variation in MSP-1(19) among the isolates was extensive, with differences detected at 35 out of the 96 positions compared. Using the 3-dimensional structure of the Plasmodium falciparum MSP-1(19) as a model, the locations of the amino acid substitutions that may affect Mab binding were identified. The DNA sequence of MSP-1(19) from two Plasmodium vinckei isolates was also cloned and the deduced amino acid sequence compared with that in other species.

Intragenic recombinants of Plasmodium falciparum identified by in situ polymerase chain reaction.

We report an in situ PCR technique for visualising amplified DNA of blood forms of Plasmodium falciparum on microscope slides by fluorescence microscopy. The method is used to assess the changes in frequency of different alleles of the MSP1 gene in cultures of the progeny of a cross. We show that parasites with a recombinant form of this protein possess an initial growth advantage before declining in numbers over the long-term.

Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania.

Plasmodium falciparum parasites exist as genetically distinct haploid clones in infected people. In the Kilombero valley in south-east Tanzania, at least 85% of the inhabitants of Michenga village harbour more than one clone. Using 2 highly polymorphic unlinked markers, it has been estimated that each infected person harbours between one and 6 P. falciparum clones at any one time, with a mean of 3.5 clones. When mosquitoes acquire gametocytes of 2 different clones in a blood meal, crossing generates recombinant clones differing from their parental genotypes. The inbreeding coefficient of the parasite population has been estimated as 0.33.