Effect of drug dose and timing of treatment on the emergence of drug resistance in vivo in a malaria model.

BACKGROUND AND OBJECTIVES: There is a significant interest in identifying clinically effective drug treatment regimens that minimize the de novo evolution of antimicrobial resistance in pathogen populations. However, in vivo studies that vary treatment regimens and directly measure drug resistance evolution are rare. Here, we experimentally investigate the role of drug dose and treatment timing on resistance evolution in an animal model. METHODOLOGY: In a series of experiments, we measured the emergence of atovaquone-resistant mutants of Plasmodium chabaudi in laboratory mice, as a function of dose or timing of treatment (day post-infection) with the antimalarial drug atovaquone. RESULTS: The likelihood of high-level resistance emergence increased with atovaquone dose. When varying the timing of treatment, treating either very early or late in infection reduced the risk of resistance. When we varied starting inoculum, resistance was more likely at intermediate inoculum sizes, which correlated with the largest population sizes at time of treatment. CONCLUSIONS AND IMPLICATIONS: (i) Higher doses do not always minimize resistance emergence and can promote the emergence of high-level resistance. (ii) Altering treatment timing affects the risk of resistance emergence, likely due to the size of the population at the time of treatment, although we did not test the effect of immunity whose influence may have been important in the case of late treatment. (iii) Finding the 'right' dose and 'right' time to maximize clinical gains and limit resistance emergence can vary depending on biological context and was non-trivial even in our simplified experiments. LAY SUMMARY: In a mouse model of malaria, higher drug doses led to increases in drug resistance. The timing of drug treatment also impacted resistance emergence, likely due to the size of the population at the time of treatment.

The origin and diversification of the merozoite surface protein 3 (msp3) multi-gene family in Plasmodium vivax and related parasites.

The genus Plasmodium is a diversified group of parasites with more than 200 known species that includes those causing malaria in humans. These parasites use numerous proteins in a complex process that allows them to invade the red blood cells of their vertebrate hosts. Many of those proteins are part of multi-gene families; one of which is the merozoite surface protein-3 (msp3) family. The msp3 multi-gene family is considered important in the two main human parasites, Plasmodium vivax and Plasmodium falciparum, as its paralogs are simultaneously expressed in the blood stage (merozoite) and are immunogenic. There are large differences among Plasmodium species in the number of paralogs in this family. Such differences have been previously explained, in part, as adaptations that allow the different Plasmodium species to invade their hosts. To investigate this, we characterized the array containing msp3 genes among several Plasmodium species, including P. falciparum and P. vivax. We first found no evidence indicating that the msp3 family of P. falciparum was homologous to that of P. vivax. Subsequently, by focusing on the diverse clade of nonhuman primate parasites to which P. vivax is closely related, where homology was evident, we found no evidence indicating that the interspecies variation in the number of paralogs was an adaptation related to changes in host range or host switches. Overall, we hypothesize that the evolution of the msp3 family in P. vivax is consistent with a model of multi-allelic diversifying selection where the paralogs may have functionally redundant roles in terms of increasing antigenic diversity. Thus, we suggest that the expressed MSP3 proteins could serve as "decoys", via antigenic diversity, during the critical process of invading the host red blood cells.

Merozoite surface protein-3 alpha as a genetic marker for epidemiologic studies in Plasmodium vivax: a cautionary note.

BACKGROUND: Plasmodium vivax is the most widespread of the human malaria parasites in terms of geography, and is thought to present unique challenges to local efforts aimed at control and elimination. Parasite molecular markers can provide much needed data on P. vivax populations, but few such markers have been critically evaluated. One marker that has seen extensive use is the gene encoding merozoite surface protein 3-alpha (MSP-3α), a blood-stage antigen known to be highly variable among P. vivax isolates. Here, a sample of complete msp-3α gene sequences is analysed in order to assess its utility as a molecular marker for epidemiologic investigations. METHODS: Amplification, cloning and sequencing of additional P. vivax isolates from different geographic locations, including a set of Venezuelan field isolates (n = 10), yielded a sample of 48 complete msp-3α coding sequences. Characterization of standard population genetic measures of diversity, phylogenetic analysis, and tests for recombination were performed. This allowed comparisons to patterns inferred from the in silico simulation of a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) protocol used widely. RESULTS: The larger sample of MSP-3α diversity revealed incongruence between the observed levels of nucleotide polymorphism, which were high in all populations, and the pattern of PCR-RFLP haplotype diversity. Indeed, PCR-RFLP haplotypes were not informative of a population's genetic diversity and identical haplotypes could be produced from analogous bands in the commonly used protocol. Evidence of frequent and variable insertion-deletion mutations and recurrent recombination between MSP-3α haplotypes complicated the inference of genetic diversity patterns and reduced the phylogenetic signal. CONCLUSIONS: The genetic diversity of P. vivax msp-3α involves intragenic recombination events. Whereas the high genetic diversity of msp-3α makes it a promising marker for some epidemiological applications, the ability of msp-3α PCR-RFLP analysis to accurately track parasites is limited. Local studies of the circulating alleles are needed before implementing PCR-RFLP approaches. Furthermore, evidence from the global sample analysed here suggests such msp-3α PCR-RFLP methods are not suitable for broad geographic studies or tracking parasite populations for an extended period of time.