Small parasites and larval stages pose a problem for molecular analyses because limited amounts of DNA template are available. Isothermal methods for faithfully copying DNA have the potential to revolutionize studies of such organisms. We evaluated the fidelity of multiple displacement amplification (MDA) for amplifying DNA extracted from a single miracidium of Schistosoma mansoni. To do this we genotyped DNA extracted from 28 F1 miracidia following MDA using 56 microsatellite markers. Because these miracidia were obtained from a cross between a male and female worm of known genotypes, we were able to predict the alleles present in the progeny and quantify the genotyping error rate. We found just 8/1568 genotypes deviated from Mendelian expectations. Furthermore, because 1 of these resulted from a genuine mutation, the error rate due to MDA is 7/1568 (0.45%). We conclude that many hundreds of microsatellites or other genetic markers can be accurately genotyped from a single miracidium using this method, greatly expanding the scope of population genetic, epidemiological and evolutionary studies on this parasite.
Publications
2009
Alteration in gene copy number provides a simple way to change expression levels and alter phenotype. This was fully appreciated by bacteriologists more than 25 years ago, but the extent and implications of copy number polymorphism (CNP) have only recently become apparent in other organisms. New methods demonstrate the ubiquity of CNPs in eukaryotes and their medical importance in humans. CNP is also widespread in the Plasmodium falciparum genome and has an important and underappreciated role in determining phenotype. In this review, we summarize the distribution of CNP, its evolutionary dynamics within populations, its functional importance and its mode of evolution.
Tim Anderson discusses a new study of molecular variation in alleles at the dihydropteroate synthase locus, which underlies resistance to sulfadoxine, in over 5,000 parasites from 50 locations.
We genotyped 160 P. falciparum infections from Malawi for pfmdr-1 copy number changes and SNPs associated with in vivo tolerance and poor in vitro sensitivity to the component drugs of Coartem. We also measured in vitro susceptibility of 49 of these isolates to a variety of drugs in clinical use or with a potential for use in Africa. All 160 infections carried a single copy of pfmdr-1 but 34% exhibited sequence variation at 4 of the 5 polymorphic sites in pfmdr-1. Isolates carrying 86-Asn and 184-Tyr pfmdr-1 alleles were significantly less sensitive (p<0.001) to mefloquine, lumefantrine, artemether and dihydroartemisinin compared with those bearing 86-Tyr and 184-Phe polymorphisms. This study provides baseline measures prior to policy change: continued surveillance for changes in baseline drug susceptibility, pfmdr-1 copy number and SNPs, and other putative Coartem resistance loci will be necessary to provide an early warning of emerging Coartem resistance in this setting.
Aggregated distributions of macroparasites within their host populations are characteristic of most natural and experimental infections. We designed this study to measure the amount of variation that is attributable to host genetic factors in a pig-helminth system. In total, 195 piglets were produced after artificial insemination of 19 sows (Danish Landrace-Yorkshire crossbreds) with semen selected from 13 individual Duroc boars (1 or 2 sows per boar; mean litter size: 10.3; 5-14 piglets per litter). Starting at 10 weeks of age, piglets were repeatedly infected with the gastrointestinal helminths Trichuris suis and Ascaris suum by administering eggs in the feed for 14 weeks until necropsy. Faecal egg counts (FECs) were estimated regularly and A. suum worm burden was obtained at necropsy. Heritability calculations for log (FEC+1) at weeks 7-10 post-infection (p.i.) showed that 0.32-0.73 of the phenotypic variation for T. suis could be attributed to genetic factors. For A. suum, heritabilities of 0.29-0.31 were estimated for log (FEC+1) at weeks 7-14 p.i., whereas the heritability of log worm counts was 0.45. Strong positive genetic correlations (0.75-0.89) between T. suis and A. suum FECs suggest that resistance to both infections involves regulation by overlapping genes. Our data demonstrate that there is a strong genetic component in resistance to A. suum and T. suis infections in pigs. Identification of responsible genes would enhance our understanding of the host immune response to these common nematodes and for the closely related species (T. trichiura and A. lumbricoides) in man infecting more than a billion people.
BACKGROUND: Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS(3)), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border.
METHODS AND FINDINGS: 3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS(3). The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p<0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00-2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS(3) efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07-1.19, p<0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0-98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2-5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend).
CONCLUSION: Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage.
The genotypes of both host and parasite may influence the outcome of parasitic infections, but few attempts have been made to quantify the effect of parasite genotype on macroparasite infections of socio-economic importance. We examined variation in particular traits during the infection in pigs with the parasitic nematode Ascaris suum. We infected 26 pigs with mixtures of equal proportions of embryonated eggs from 4 single female worms each with a unique mtDNA haplotype–the eggs from each female worm were a mixture of siblings and half-siblings. Pigs were necropsied on days 14, 17 and 28 following inoculation, which corresponded to time-points before, during and after the main immune responses against the nematode. A total of approximately 11,000 worms were recovered at necropsy. The location in the small intestine was recorded for all worms and the length and mtDNA haplotype were determined for about 4200 individual worms. There were significant differences in the distribution and abundance of the 4 individual haplotypes among individual pigs demonstrating strong interactions between parasite and host. We found significant differences in the abundance and position in the small intestine as well as the size of worms among haplotypes. We conclude that both parasite and host effects as well as the interplay between them play important roles in determining the characteristics and outcome of infection.
Multiple displacement amplification (MDA) using Phi29 has proved to be an efficient, high-fidelity method for whole genome amplification in many organisms. This project was designed to evaluate this approach for use with the malaria parasite Plasmodium falciparum. In particular, we were concerned that the AT richness and presence of contaminating human DNA could limit efficiency of MDA in this system. We amplified 60 DNA samples using phi29 and scored 14 microsatellites, 9 single-nucleotide polymorphisms (SNPs), and gene copy number at GTP-cyclohydrolase I both before and after MDA. We observed 100% concordance in 829 microsatellite genotypes and in 499 SNP genotypes. Furthermore, copy number estimates for the GTP-cyclohydrolase I gene were correlated (r(2) = 0.67) in pre- and postamplification samples. These data confirm that MDA permits scoring of a range of different types of polymorphisms in P. falciparum malaria and can be used to extend the life of valuable DNA stocks.
2008
Plasmodium vivax mdr1 gene amplification, quantified by real-time PCR, was significantly more common on the western Thailand border (6 of 66 samples), where mefloquine pressure has been intense, than elsewhere in southeast Asia (3 of 149; P = 0.02). Five coding mutations in pvmdr1, independent of gene amplification, were also found.