Association of HLA alleles with Plasmodium falciparum severity in Malian children.

Pre-erythrocytic immunity to Plasmodium falciparum malaria is likely to be mediated by T-cell recognition of malaria epitopes presented on infected host cells via class I and II major histocompatibility complex (MHC) antigens. To test for associations of human leukocyte antigen (HLA) alleles with disease severity, we performed high-resolution typing of HLA class I and II loci and compared the distributions of alleles of HLA-A, -B, -C and -DRB1 loci in 359 Malian children of Dogon ethnicity with uncomplicated or severe malaria. We observed that alleles A*30:01 and A*33:01 had higher frequency in the group of patients with cerebral disease compared to patients with uncomplicated disease [A*30:01: gf = 0.2031 vs gf = 0.1064, odds ratio (OR) = 3.17, P = 0.004, confidence interval (CI) (1.94-5.19)] and [A*33:01: gf = 0.0781 vs gf = 0.0266, 4.21, P = 0.005, CI (1.89-9.84)], respectively. The A*30:01 and A*33:01 alleles share some sequence motifs and A*30:01 appears to have a unique peptide binding repertoire compared to other A*30 group alleles. Computer algorithms predicted malaria peptides with strong binding affinity for HLA-A*30:01 and HLA-A*33:01 but not to closely related alleles. In conclusion, we identified A*30:01 and A*33:01 as potential susceptibility factors for cerebral malaria, providing further evidence that polymorphism of MHC genes results in altered malaria susceptibility.

Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications.

The first human vaccines against the malaria parasite have been designed to elicit antibodies to the circumsporozoite protein of Plasmodium falciparum. However, it is not known whether any level of naturally acquired antibodies to the circumsporozoite protein can predict resistance to Plasmodium falciparum malaria. In this study, 83 adults in a malaria-endemic region of Kenya were tested for circumsporozoite antibodies and then treated for malaria. They were monitored for the development of new malaria infections for 98 days. Antibody levels, as determined by four assays in vitro, were indistinguishable between the 60 individuals who did and the 23 who did not develop parasitemia during follow-up, and there was no apparent relation between day of onset of parasitemia and level of antibodies to circumsporozoite protein. Unless immunization with sporozoite vaccines induces antibodies that are quantitatively or qualitatively superior to the circumsporozoite antibodies in these adults, it is unlikely that such antibodies will prevent infection in areas with as intense malaria transmission as western Kenya.

Genetic diversity and malaria vaccine design, testing and efficacy: preventing and overcoming 'vaccine resistant malaria'.

The development of effective malaria vaccines may be hindered by extensive genetic diversity in the surface proteins being employed as vaccine antigens. Understanding of the extent and dynamics of genetic diversity in vaccine antigens is needed to guide rational vaccine design and to interpret the results of vaccine efficacy trials conducted in malaria endemic areas. Molecular epidemiological, population genetic, and structural approaches are being employed to try to identify immunologically relevant polymorphism in vaccine antigens. The results of these studies will inform choices of which alleles to include in multivalent or chimeric vaccines; however, additional molecular and immuno-epidemiological studies in a variety of geographic locations will be necessary for these approaches to succeed. Alternative means of overcoming antigenic diversity are also being explored, including boosting responses to critical conserved regions of current vaccine antigens, identifying new, more conserved and less immunodominant antigens, and developing whole-organism vaccines. Continued creative application and integration of tools from multiple disciplines, including epidemiology, immunology, molecular biology, and evolutionary genetics and genomics, will likely be required to develop broadly protective vaccines against Plasmodium and other antigenically complex pathogens.

Unidirectional dominance of cytoplasmic inheritance in two genetic crosses of Plasmodium falciparum.

Malarial parasites have two highly conserved cytoplasmic DNA molecules: a 6-kb tandemly arrayed DNA that has characteristics of a mitochondrial genome, and a 35-kb circular DNA that encodes functions commonly found in chloroplasts. We examined the inheritance pattern of these elements in two genetic crosses of Plasmodium falciparum clones. Parent-specific oligonucleotide probes and single-strand conformation polymorphism analysis identified single nucleotide changes that distinguished the parental 6- and 35-kb DNA molecules in the progeny. In all 16 independent recombinant progeny of a cross between a Central American clone, HB3, and a Southeast Asian clone, Dd2, the 6- and 35-kb DNAs were inherited from the Dd2 parent. In all nine independent recombinant progeny of a cross between clone HB3 and a likely African clone, 3D7, the 6-kb DNA was inherited from the 3D7 parent. Inheritance of cytoplasmic genomes of the Dd2 and 3D7 parents was, therefore, dominant over that of the HB3 parent. Cytoplasmic DNA molecules were found almost exclusively in the female gametes of malarial parasites; hence, clone HB3 did not appear to have served as a maternal parent for the progeny of two crosses. Defective differentiation into male gametes by clone Dd2 is likely to be a reason for the cytoplasmic inheritance pattern seen in the HB3 x Dd2 cross. However, incompetence of male or female gametes is unlikely to explain the uniparental dominance in recombinant progeny of the HB3 x 3D7 cross, since both parents readily self-fertilized and completed the malaria life cycle on their own. Instead, the data suggest unidirectional parental incompatibility in cross-fertilization of these malarial parasites, where a usually cosexual parental clone can participate only as a male or as a female. Such an incompatibility may be speculated as indicating an early phase of reproductive isolation of P. falciparum clones from different geographical regions.

[In vitro cultivation of fields isolates of Plasmodium falciparum in Mali]. / Culture in vitro d'isolats de terrain de Plasmodium falciparum au Mali.

Malaria immunology, molecular biology and pathogenicity studies often require the adaptation of Plasmodium falciparum field isolates to continuous in vitro cultivation. For this purpose we have established propagation protocols of asexual erythrocytic stages of P. falciparum samples from malaria patients or asymptomatic carriers in Mali. The parasites were grown in standard culture medium supplemented by human serum and in a culture medium without human serum but supplemented by AlbuMax 1. The candle jar environment and tissue culture flasks gassed with 5% CO2, 5% O2 and 90% N2 obtained from a portable gas mixer were used. Protocols for parasite cultivation in a resource-poor setting were developed. These protocols were successfully applied to fresh isolates in Mali as well as to blood samples frozen in liquid nitrogen and shipped to a laboratory in U.S.A.

Antimalarial drug resistance in Africa: strategies for monitoring and deterrence.

Despite the initiation in 1998 by the World Health Organization of a campaign to 'Roll Back Malaria', the rates of disease and death caused by Plasmodium falciparum malaria in sub-Saharan Africa are growing. Drug resistance has been implicated as one of the main factors in this disturbing trend. The efforts of international agencies, governments, public health officials, advocacy groups and researchers to devise effective strategies to deter the spread of drug resistant malaria and to ameliorate its heavy burden on the people of Africa have not succeeded. This review will not attempt to describe the regional distribution of drug resistant malaria in Africa in detail, mainly because information on resistance is limited and has been collected using different methods, making it difficult to interpret. Instead, the problems of defining and monitoring resistance and antimalarial drug treatment outcomes will be discussed in hopes of clarifying the issues and identifying ways to move forward in a more coordinated fashion. Strategies to improve measurement of resistance and treatment outcomes, collection and use of information on resistance, and potential approaches to deter and reduce the impact of resistance, will all be considered. The epidemiological setting and the goals of monitoring determine how antimalarial treatment responses should be measured. Longitudinal studies, with incidence of uncomplicated malaria episodes as the primary endpoint, provide the best information on which to base treatment policy changes, while simpler standard in vivo efficacy studies are better suited for ongoing efficacy monitoring. In the absence of an ideal antimalarial combination regimen, different treatment alternatives are appropriate in different settings. But where chloroquine has failed, policy changes are long overdue and action must be taken now.

Conservation of a novel vacuolar transporter in Plasmodium species and its central role in chloroquine resistance of P. falciparum.

Chloroquine resistance in Plasmodium falciparum has recently been shown to result from mutations in the novel vacuolar transporter, PfCRT. Field studies have demonstrated the importance of these mutations in clinical resistance. Although a pfcrt ortholog has been identified in Plasmodiumvivax, there is no association between in vivo chloroquine resistance and codon mutations in the P. vivax gene. This is consistent with lines of evidence that suggest alternative mechanisms of chloroquine resistance among various malaria parasite species.

Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the world's burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

Antifolate resistance due to new and known Plasmodium falciparum dihydrofolate reductase mutations expressed in yeast.

Two new dihydrofolate reductase (DHFR) mutations were recently discovered in Plasmodium falciparum samples from an area of Bolivia with high rates of in vivo resistance to pyrimethamine-sulfadoxine: a Cys-->Arg point mutation in codon 50 and a five amino acid insertion after codon 30, termed the Bolivia repeat. We used a yeast expression system to screen these new DHFR mutants, as well as all of the other known DHFR mutant genotypes, against four antifolates: pyrimethamine, cycloguanil, chlorcycloguanil, and WR99210. The prodrug proguanil was also evaluated. The primary 108-Asn mutation, the known secondary mutations 51-Ile, 59-Arg and 164-Leu, as well as the 50-Arg mutation, all progressively enhanced pyrimethamine resistance in naturally observed combinations with one another, with the presence of 164-Leu most significantly increasing resistance. Cycloguanil and chlorcycloguanil resistance were most impacted by 164-Leu and the paired 16-Val/108-Thr. Proguanil had no effect on malaria DHFR. All DHFRs analyzed were sensitive to WR99210. The Bolivia repeat did not markedly affect drug sensitivity. We conclude that malaria DHFR can be reliably, rapidly and inexpensively analyzed in yeast for activity against a broad spectrum of antifolates. This system may be useful for initially characterizing newly discovered genotypes before proceeding to P. falciparum transfection; for large-scale geographic surveys of drug resistance; and for screening new antifolates or new antifolate combinations for their effectiveness against a large panel of DHFR mutants.

A bifunctional dihydrofolate synthetase--folylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria.

Folate metabolism in the human malaria parasite Plasmodium falciparum is an essential activity for cell growth and replication, and the target of an important class of therapeutic agents in widespread use. However, resistance to antifolate drugs is a major health problem in the developing world. To date, only two activities in this complex pathway have been targeted by antimalarials. To more fully understand the mechanisms of antifolate resistance and to identify promising targets for new chemotherapies, we have cloned genes encoding as yet uncharacterised enzymes in this pathway. By means of complementation experiments using 1-carbon metabolism mutants of both Escherichia coli and Saccharomyces cerevisiae, we demonstrate here that one of these parasite genes encodes both dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) activities, which catalyse the synthesis and polyglutamation of folate derivatives, respectively. The malaria parasite is the first known example of a eukaryote encoding both DHFS and FPGS activities in a single gene. DNA sequencing of this gene in antifolate-resistant strains of P. falciparum, as well as drug-inhibition assays performed on yeast and bacteria expressing PfDHFS--FPGS, indicate that current antifolate regimes do not target this enzyme. As PfDHFS--FPGS harbours two activities critical to folate metabolism, one of which has no human counterpart, this gene product offers a novel chemotherapeutic target with the potential to deliver a powerful blockage to parasite growth.

Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa.

As chloroquine resistance spreads across Africa, the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and proguanil are being used as alternative first-line drugs for the treatment and prevention of Plasmodium falciparum malaria. Resistance to these drugs is conferred by point mutations in parasite DHFR. These point mutations can be detected by polymerase chain reaction (PCR) assays, but better methods for sample collection, DNA extraction, and a diagnostic PCR are needed to make these assays useful in malaria-endemic areas. Here we report methods for collecting fingerstick blood onto filter paper strips that are air-dried, then stored and transported at room temperature. Cell lysis and DNA extraction are accomplished by boiling in Chelex-100. We also report a nested PCR technique that has improved sensitivity and specificity. These procedures readily detect mixed infections of parasites with both sensitive and resistant genotypes (confirmed by direct sequencing) and are reliable at parasite densities less than 250/mm3 in field surveys.

Community pyrimethamine-sulfadoxine use and prevalence of resistant Plasmodium falciparum genotypes in Mali: a model for deterring resistance.

Pyrimethamine-sulfadoxine (PS, Fansidar; Hoffman-LaRoche, Basel, Switzerland) is now the first-line antimalarial therapy in parts of Africa with high rates of chloroquine-resistant Plasmodium falciparum. With PS resistance increasing and no suitably inexpensive and effective third antimalarial drug available, strategies for delaying the spread of PS resistance in Africa are needed. Community PS usage was measured in two Malian villages, one rural and one periurban, and prevalence of pyrimethamine-resistant P. falciparum genotypes was determined at these sites and two urban sites. The prevalence of resistant genotypes was 22.6% (n = 84) in the periurban village where PS was available from multiple sources and large stocks of PS were observed, and 13.5% (n = 89) and 23.4% (n = 77) in a large town and a city, respectively, where PS is widely available. No pyrimethamine-resistant genotypes (n = 58) were detected in Kolle, a rural village with a community-supported dispensary and clinic where PS is used sparingly and no PS was available in pharmacies or markets. The high rates of pyrimethamine resistant genotypes concurrent with higher PS usage argue for a policy of judicious PS use in Mali and in similar settings. A possible model for slowing the spread of drug-resistant malaria is illustrated by the example of the Kolle clinic.

Use of antimalarial drugs in Mali: policy versus reality.

Inappropriate use of antimalarial drugs undermines therapeutic efficacy and promotes the emergence and spread of drug-resistant malaria. Strategies for improving compliance require accurate information about current practices. Here we describe Knowledge-Attitude-Practice surveys conducted among health providers and consumers in two Malian villages, one rural and one periurban. All sanctioned providers limited their first choices of antimalarial drug to those recommended by the national malaria control program and reported using correct dosing regimens. However, the majority of consumers in the two villages chose non-recommended treatments for malaria and reported suboptimal treatment regimens when they did use recommended drugs. Antimalarial drugs were also widely available from unsanctioned sources, often accompanied by erroneous advice on dosing regimens. This study demonstrates that even when the most peripheral health providers are well-trained in correct use of antimalarial drugs, additional measures directly targeting consumers will be required to improve drug use practices.

Pyrimethamine-sulfadoxine efficacy and selection for mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase in Mali.

To assess pyrimethamine-sulfadoxine (PS) efficacy in Mali, and the role of mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in in vivo PS resistance, 190 patients with uncomplicated P. falciparum malaria were treated with PS and monitored for 56 days. Mutation-specific polymerase chain reactions and digestion with restriction endonucleases were used to detect DHFR and DHPS mutations on filter paper blood samples from pretreatment and post-treatment infections. Only one case each of RI and RII level resistance and no cases of RIII resistance or therapeutic failure were observed. Post-PS treatment infections had significantly higher rates of DHFR mutations at codons 108 and 59. No significant selection for DHPS mutations was seen. Pyrimethamine-sulfadoxine is highly efficacious in Mali, and while the low level of resistance precludes assessing the utility of molecular assays for in vivo PS resistance, rapid selection of DHFR mutations supports their role in PS failure.

Chloroquine treatment of uncomplicated Plasmodium falciparum malaria in Mali: parasitologic resistance versus therapeutic efficacy.

Whether and when to replace chloroquine with other antimalarial drugs is an urgent public health question in much of Africa, where Plasmodium falciparum, which is increasingly resistant to chloroquine, continues to kill millions each year. Antimalarial drug efficacy has traditionally been measured as parasitologic resistance, but recent guidelines use both clinical and parasitologic criteria to monitor therapeutic efficacy. To assess the new efficacy protocol, we measured parasitologic and therapeutic outcomes in 514 patients treated with chloroquine for uncomplicated P. falciparum malaria in Mali. There was a general agreement between parasitologic and therapeutic outcomes at two sites, with 13-17% parasitologic resistance rates and 10-15% treatment failure rates. However, the new protocol overestimated early treatment failure rates (21-71% of cases classified as early treatment failure had sensitive or RI parasitologic responses), particularly where resistance was rare, and missed low-level parasitologic resistance. Modifications of the protocol for monitoring antimalarial therapeutic efficacy are recommended.

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Pharmacologic advances in the global control and treatment of malaria: combination therapy and resistance.

Combination drug therapy for malaria is recommended both to prevent and to overcome drug resistance. Drug combinations developed for use in Asia are being deployed in Africa, where higher rates of malaria affect the therapeutic and public health objectives of malaria chemotherapy as well as drug safety. Rational consideration of drug mechanisms, pharmacokinetics (PK), pharmacodynamics (PD), and malaria epidemiology should result in more effective combination regimens that retain therapeutic and prophylactic efficacy in the face of resistance.

Chloroquine-resistant malaria.

The development of chloroquine as an antimalarial drug and the subsequent evolution of drug-resistant Plasmodium strains had major impacts on global public health in the 20th century. In P. falciparum, the cause of the most lethal human malaria, chloroquine resistance is linked to multiple mutations in PfCRT, a protein that likely functions as a transporter in the parasite's digestive vacuole membrane. Rapid diagnostic assays for PfCRT mutations are already employed as surveillance tools for drug resistance. Here, we review recent field studies that support the central role of PfCRT mutations in chloroquine resistance. These studies suggest chloroquine resistance arose in > or = 4 distinct geographic foci and substantiate an important role of immunity in the outcomes of resistant infections after chloroquine treatment. P. vivax, which also causes human malaria, appears to differ from P. falciparum in its mechanism of chloroquine resistance. Investigation of the resistance mechanisms and of the role of immunity in therapeutic outcomes will support new approaches to drugs that can take the place of chloroquine or augment its efficiency.

P. falciparum dihydrofolate reductase and dihydropteroate synthase mutations: epidemiology and role in clinical resistance to antifolates.

Plasmodium falciparum resistance to the antifolates has arisen rapidly in Asia and South America, and threatens the usefulness of these drugs in Africa. In vitro resistance to the antifolates is determined by mutations in parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). The role of DHFR and DHPS mutations in therapeutic failure of antifolate antimalarials is less clear. This review summarizes molecular epidemiological surveys, studies of in vivo selection of mutant alleles by drug treatment, and prospective studies of the ability of mutation-specific assays to predict clinical outcomes, and discusses the potential use of these assays for surveillance of resistance.