Richard Culleton

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites

Artemisinin‐ and artesunate‐resistant Plasmodium chabaudi mutants, AS‐ART and AS‐ATN, were previously selected from chloroquine‐resistant clones AS‐30CQ and AS‐15CQ respectively. Now, a genetic cross between AS‐ART and the artemisinin‐sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS‐30CQ and AS‐ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS‐ATN to artemisinin derivates, the other cannot account solely for the resistance of AS‐ART, relative to the responses of its sensitive progenitor AS‐30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug‐response phenotype was not genetically stable. No mutations in the UBP‐1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.

African origin of the malaria parasite Plasmodium vivax

Plasmodium vivax is the leading cause of human malaria in Asia and Latin America but is absent from most of central Africa due to the near fixation of a mutation that inhibits the expression of its receptor, the Duffy antigen, on human erythrocytes. The emergence of this protective allele is not understood because P. vivax is believed to have originated in Asia. Here we show, using a non-invasive approach, that wild chimpanzees and gorillas throughout central Africa are endemically infected with parasites that are closely related to human P. vivax. Sequence analyses reveal that ape parasites lack host specificity and are much more diverse than human parasites, which form a monophyletic lineage within the ape parasite radiation. These findings indicate that human P. vivax is of African origin and likely selected for the Duffy-negative mutation. All extant human P. vivax parasites are derived from a single ancestor that escaped out of Africa.

Host heterogeneity is a determinant of competitive exclusion or coexistence in genetically diverse malaria infections.

During an infection, malaria parasites compete for limited amounts of food and enemy–free space. Competition affects parasite growth rate, transmission and virulence, and is thus important for parasite evolution. Much evolutionary theory assumes that virulent clones outgrow avirulent ones, favouring the evolution of higher virulence. We infected laboratory mice with a mixture of two Plasmodium chabaudi clones: one virulent, the other avirulent. Using real–time quantitative PCR to track the two parasite clones over the course of the infection, we found that the virulent clone overgrew the avirulent clone. However, host genotype had a major effect on the outcome of competition. In a relatively resistant mouse genotype (C57Bl/6J), the avirulent clone was suppressed below detectable levels after 10 days, and apparently lost from the infection. By contrast, in more susceptible mice (CBA/Ca), the avirulent clone was initially suppressed, but it persisted, and during the chronic phase of infection it did better than it did in single infections. Thus, the qualitative outcome of competition depended on host genotype. We suggest that these differences may be explained by different immune responses in the two mouse strains. Host genotype and resistance could therefore play a key role in the outcome of within–host competition between parasite clones and in the evolution of parasite virulence.

Big Bang in the Evolution of Extant Malaria Parasites

Malaria parasites (genus Plasmodium) infect all classes of terrestrial vertebrates and display host specificity in their infections. It is therefore assumed that malaria parasites coevolved intimately with their hosts. Here, we propose a novel scenario of malaria parasite-host coevolution. A phylogenetic tree constructed using the malaria parasite mitochondrial genome reveals that the extant primate, rodent, bird, and reptile parasite lineages rapidly diverged from a common ancestor during an evolutionary short time period. This rapid diversification occurred long after the establishment of the primate, rodent, bird, and reptile host lineages, which implies that host-switch events contributed to the rapid diversification of extant malaria parasite lineages. Interestingly, the rapid diversification coincides with the radiation of the mammalian genera, suggesting that adaptive radiation to new mammalian hosts triggered the rapid diversification of extant malaria parasite lineages.

Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities

Graphical abstract Research highlights ► We propose that two related species of malaria parasite cause ovale malaria. ► Discriminatory PCR tests identified both species in African surveys. ► Plasmodium ovale curtisi and Plasmodium ovale wallikeri were found together in time and space. ► Lack of recombination between them is not due to geographic or temporal separation. ► Therefore, the species barrier may be maintained by a biological mechanism(s).

Co-infections of Plasmodium knowlesi, P. falciparum, and P. vivax among Humans and Anopheles dirus Mosquitoes, Southern Vietnam

TOC Summary: Forests harboring these mosquitoes may be a reservoir for transmission of P. knowlesi.

Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection

BackgroundLoop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions.MethodsA visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection.ResultsThe wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method.ConclusionsThis novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

Competitive release of drug resistance following drug treatment of mixed Plasmodium chabaudi infections

BackgroundMalaria infections are often genetically diverse, potentially leading to competition between co-infecting strains. Such competition is of key importance in the spread of drug resistance.MethodsThe effects of drug treatment on within-host competition were studied using the rodent malaria Plasmodium chabaudi. Mice were infected simultaneously with a drug-resistant and a drug-sensitive clone and were then either drug-treated or left untreated. Transmission was assessed by feeding mice to Anopheles stephensi mosquitoes.ResultsIn the absence of drugs, the sensitive clone competitively suppressed the resistant clone; this resulted in lower asexual parasite densities and also reduced transmission to the mosquito vector. Drug treatment, however, allowed the resistant clone to fill the ecological space emptied by the removal of the sensitive clone, allowing it to transmit as well as it would have done in the absence of competition.ConclusionThese results show that under drug pressure, resistant strains can have two advantages: (1) they survive better than sensitive strains and (2) they can exploit the opportunities presented by the removal of their competitors. When mixed infections are common, such effects could increase the spread of drug resistance.

Failure to detect Plasmodium vivax in West and Central Africa by PCR species typing

BackgroundPlasmodium vivax is estimated to affect 75 million people annually. It is reportedly absent, however, from west and central Africa due to the high prevalence of the Duffy negative phenotype in the indigenous populations. Despite this, non-African travellers consistently return to their own countries with P. vivax malaria after visiting this region. An attempt was made, therefore, to detect the presence of P. vivax parasites in blood samples collected from the indigenous populations of west and central Africa.MethodsParasite species typing (for all four human malaria parasites) was carried out by PCR on 2,588 blood samples collected from individuals from nine African malaria-endemic countries.ResultsMost infections (98.5%) were Plasmodium falciparum, Plasmodium malariae was identified in 8.5% of all infections, and Plasmodium ovale in 3.9%. The prevalence of both parasites varied greatly by country. Only one case of P. vivax was detected from Sao Tome, an island off the west coast of Africa, confirming the scarcity of this parasite in Africa.ConclusionThe prevalence of P. vivax in local populations in sub-Saharan Africa is very low, despite the frequent identification of this parasite in non-African travellers.

Evidence for the Transmission of Plasmodium vivax in the Republic of the Congo, West Central Africa

Plasmodium vivax is not thought to be transmitted in western and central Africa, because of the very high prevalence of the red blood cell Duffy-negative phenotype in local populations, a condition which is thought to confer complete resistance against blood infection with P. vivax. There are, however, persistent reports of travelers returning from this region with P. vivax infections. To investigate whether transmission occurs in this region, the presence of antibodies specific to P. vivax preerythrocytic-stage antigens was assessed in individuals from the Republic of the Congo. A total of 55 (13%) of 409 samples tested by enzyme-linked immunosorbent assay had antibodies to P. vivax-specific antigens.