MalHaploFreq: A computer programme for estimating malaria haplotype frequencies from blood samples

<p>Abstract</p> <p>Background</p> <p>Molecular markers, particularly those associated with drug resistance, are important surveillance tools that can inform policy choice. People infected with <it>falciparum </it>malaria often contain several genetically-distinct clones of the parasite; genotyping the patients' blood reveals whether or not the marker is present (i.e. its prevalence), but does not reveal its frequency. For example a person with four malaria clones may contain both mutant and wildtype forms of a marker but it is not possible to distinguish the relative frequencies of the mutant and wildtypes i.e. 1:3, 2:2 or 3:1.</p> <p>Methods</p> <p>An appropriate method for obtaining frequencies from prevalence data is by Maximum Likelihood analysis. A computer programme has been developed that allows the frequency of markers, and haplotypes defined by up to three codons, to be estimated from blood phenotype data.</p> <p>Results</p> <p>The programme has been fully documented [see Additional File <supplr sid="S1">1</supplr>] and provided with a user-friendly interface suitable for large scale analyses. It returns accurate frequencies and 95% confidence intervals from simulated dataset sets and has been extensively tested on field data sets.</p> <suppl id="S1"> <title> <p>Additional File 1</p> </title> <text> <p>User manual for MalHaploFreq.</p> </text> <file name="1475-2875-7-130-S1.pdf"> <p>Click here for file</p> </file> </suppl> <p>Conclusion</p> <p>The programme is included [see Additional File <supplr sid="S2">2</supplr>] and/or may be freely downloaded from <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. It can then be used to extract molecular marker and haplotype frequencies from their prevalence in human blood samples. This should enhance the use of frequency data to inform antimalarial drug policy choice.</p> <suppl id="S2"> <title> <p>Additional File 2</p> </title> <text> <p>executable programme compiled for use on DOS or windows</p> </text> <file name="1475-2875-7-130-S2.exe"> <p>Click here for file</p> </file> </suppl

Comparative population structure of <i>Plasmodium malariae</i> and <i>Plasmodium falciparum</i> under different transmission settings in Malawi

&lt;b&gt;Background:&lt;/b&gt; Described here is the first population genetic study of Plasmodium malariae, the causative agent of quartan malaria. Although not as deadly as Plasmodium falciparum, P. malariae is more common than previously thought, and is frequently in sympatry and co-infection with P. falciparum, making its study increasingly important. This study compares the population parameters of the two species in two districts of Malawi with different malaria transmission patterns - one seasonal, one perennial - to explore the effects of transmission on population structures. &lt;BR/&gt; &lt;b&gt;Methods:&lt;/b&gt; Six species-specific microsatellite markers were used to analyse 257 P. malariae samples and 257 P. falciparum samples matched for age, gender and village of residence. Allele sizes were scored to within 2 bp for each locus and haplotypes were constructed from dominant alleles in multiple infections. Analysis of multiplicity of infection (MOI), population differentiation, clustering of haplotypes and linkage disequilibrium was performed for both species. Regression analyses were used to determine association of MOI measurements with clinical malaria parameters. &lt;BR/&gt; &lt;b&gt;Results:&lt;/b&gt; Multiple-genotype infections within each species were common in both districts, accounting for 86.0% of P. falciparum and 73.2% of P. malariae infections and did not differ significantly with transmission setting. Mean MOI of P. falciparum was increased under perennial transmission compared with seasonal (3.14 vs 2.59, p = 0.008) and was greater in children compared with adults. In contrast, P. malariae mean MOI was similar between transmission settings (2.12 vs 2.11) and there was no difference between children and adults. Population differentiation showed no significant differences between villages or districts for either species. There was no evidence of geographical clustering of haplotypes. Linkage disequilibrium amongst loci was found only for P. falciparum samples from the seasonal transmission setting. &lt;BR/&gt; &lt;b&gt;Conclusions:&lt;/b&gt; The extent of similarity between P. falciparum and P. malariae population structure described by the high level of multiple infection, the lack of significant population differentiation or haplotype clustering and lack of linkage disequilibrium is surprising given the differences in the biological features of these species that suggest a reduced potential for out-crossing and transmission in P. malariae. The absence of a rise in P. malariae MOI with increased transmission or a reduction in MOI with age could be explained by differences in the duration of infection or degree of immunity compared to P. falciparum

Alternative splicing of the Anopheles gambiae Dscam gene in diverse Plasmodium falciparum infections

Background: In insects, including Anopheles mosquitoes, Dscam (Down syndrome cell adhesion molecule) appears to be involved in phagocytosis of pathogens, and shows pathogen-specific splice-form expression between divergent pathogen (or parasite) types (e.g. between bacteria and Plasmodium or between Plasmodium berghei and Plasmodium falciparum). Here, data are presented from the first study of Dscam expression in response to genetic diversity within a parasite species. Methods: In independent field and laboratory studies, a measure of Dscam splice-form diversity was compared between mosquitoes fed on blood that was free of P. falciparum to mosquitoes exposed to either single or mixed genotype infections of P. falciparum. Results: Significant increases in Anopheles gambiae Dscam (AgDscam) receptor diversity were observed in parasite-exposed mosquitoes, but only weak evidence that AgDscam diversity rises further upon exposure to mixed genotype parasite infections was found. Finally, a cluster of AgDscam exon 4 variants that become especially common during Plasmodium invasion was identified. Conclusions: While the data clearly indicate that AgDscam diversity increases with P. falciparum exposure, they do not suggest that AgDscam diversity rises further in response to increased parasite diversit

“Sexual” Population Structure and Genetics of the Malaria Agent P. falciparum

The population genetics and structure of P. falciparum determine the rate at which malaria evolves in response to interventions such as drugs and vaccines. This has been the source of considerable recent controversy, but here we demonstrate the organism to be essentially sexual, in an area of moderately high transmission in the Lower Shire Valley, Malawi. Seven thousand mosquitoes were collected and dissected, and genetic data were obtained on 190 oocysts from 56 infected midguts. The oocysts were genotyped at three microsatellite loci and the MSP1 locus. Selfing rate was estimated as 50% and there was significant genotypic linkage disequilibrium (LD) in the pooled oocysts. A more appropriate analysis searching for genotypic LD in outcrossed oocysts and/or haplotypic LD in the selfed oocysts found no evidence for LD, indicating that the population was effectively sexual. Inbreeding estimates at MSP1 were higher than at the microsatellites, possibly indicative of immune action against MSP1, but the effect was confounded by the probable presence of null mutations. Mating appeared to occur at random in mosquitoes and evidence regarding whether malaria clones in the same host were related (presumably through simultaneous inoculation in the same mosquito bite) was ambiguous. This is the most detailed genetic analysis yet of P. falciparum sexual stages, and shows P. falciparum to be a sexual organism whose genomes are in linkage equilibrium, which acts to slow the emergence of drug resistance and vaccine insensitivity, extending the likely useful therapeutic lifespan of drugs and vaccines

A Molecular Epidemiological Study of var Gene Diversity to Characterize the Reservoir of Plasmodium falciparum in Humans in Africa

BACKGROUND: The reservoir of Plasmodium infection in humans has traditionally been defined by blood slide positivity. This study was designed to characterize the local reservoir of infection in relation to the diverse var genes that encode the major surface antigen of Plasmodium falciparum blood stages and underlie the parasite's ability to establish chronic infection and transmit from human to mosquito. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular epidemiology of the var multigene family at local sites in Gabon, Senegal and Kenya which differ in parasite prevalence and transmission intensity. 1839 distinct var gene types were defined by sequencing DBLα domains in the three sites. Only 76 (4.1%) var types were found in more than one population indicating spatial heterogeneity in var types across the African continent. The majority of var types appeared only once in the population sample. Non-parametric statistical estimators predict in each population at minimum five to seven thousand distinct var types. Similar diversity of var types was seen in sites with different parasite prevalences. CONCLUSIONS/SIGNIFICANCE: Var population genomics provides new insights into the epidemiology of P. falciparum in Africa where malaria has never been conquered. In particular, we have described the extensive reservoir of infection in local African sites and discovered a unique var population structure that can facilitate superinfection through minimal overlap in var repertoires among parasite genomes. Our findings show that var typing as a molecular surveillance system defines the extent of genetic complexity in the reservoir of infection to complement measures of malaria prevalence. The observed small scale spatial diversity of var genes suggests that var genetics could greatly inform current malaria mapping approaches and predict complex malaria population dynamics due to the import of var types to areas where no widespread pre-existing immunity in the population exists

Population Genetics of Schistosoma japonicum within the Philippines Suggest High Levels of Transmission between Humans and Dogs

Schistosomiasis is a disease caused by parasitic worms known as schistosomes, which infect about 200 million people worldwide. In the Philippines, as in China, the species of schistosome (Schistosoma japonicum) which causes the disease infects not only humans, but also many other species of mammals. In China, bovines are thought to be particularly important for harboring and transmitting S. japonicum, whereas in the Philippines infections in bovines are relatively rare. However, dogs, rats and pigs are often infected with S. japonicum in the Philippines, although the extent to which infections in these animals may give rise to human infections is unclear. To help answer this question, we characterized the genetic variation of the parasite in Samar province of the Philippines, and found that S. japonicum samples from humans, dogs, rats and pigs were genetically very similar, with no significant genetic difference between samples from humans and dogs. This suggests that in the Philippines this parasite is frequently transmitted between different mammalian species, particularly between dogs and humans. Reducing levels of infections in dogs may therefore help to reduce infections in humans. The results also suggest high levels of transmission between geographic areas, thus regional co-ordination of treatment programs is recommended

The Mitochondrial Genome of Toxocara canis

Toxocara canis (Ascaridida: Nematoda), which parasitizes (at the adult stage) the small intestine of canids, can be transmitted to a range of other mammals, including humans, and can cause the disease toxocariasis. Despite its significance as a pathogen, the genetics, epidemiology and biology of this parasite remain poorly understood. In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known. Mitochondrial DNA is known to provide genetic markers for investigations in these areas, but complete mitochondrial genomic data have been lacking for T. canis and its congeners. In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy. This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secernentean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida). The mitochondrial genome of T. canis provides genetic markers for studies into the systematics, population genetics and epidemiology of this zoonotic parasite and its congeners. Such markers can now be used in prospecting for cryptic species and for exploring host specificity and zoonotic potential, thus underpinning the prevention and control of toxocariasis in humans and other hosts

Adaptive Radiation within Marine Anisakid Nematodes: A Zoogeographical Modeling of Cosmopolitan, Zoonotic Parasites

Parasites of the nematode genus Anisakis are associated with aquatic organisms. They can be found in a variety of marine hosts including whales, crustaceans, fish and cephalopods and are known to be the cause of the zoonotic disease anisakiasis, a painful inflammation of the gastro-intestinal tract caused by the accidental consumptions of infectious larvae raw or semi-raw fishery products. Since the demand on fish as dietary protein source and the export rates of seafood products in general is rapidly increasing worldwide, the knowledge about the distribution of potential foodborne human pathogens in seafood is of major significance for human health. Studies have provided evidence that a few Anisakis species can cause clinical symptoms in humans. The aim of our study was to interpolate the species range for every described Anisakis species on the basis of the existing occurrence data. We used sequence data of 373 Anisakis larvae from 30 different hosts worldwide and previously published molecular data (n = 584) from 53 field-specific publications to model the species range of Anisakis spp., using a interpolation method that combines aspects of the alpha hull interpolation algorithm as well as the conditional interpolation approach. The results of our approach strongly indicate the existence of species-specific distribution patterns of Anisakis spp. within different climate zones and oceans that are in principle congruent with those of their respective final hosts. Our results support preceding studies that propose anisakid nematodes as useful biological indicators for their final host distribution and abundance as they closely follow the trophic relationships among their successive hosts. The modeling might although be helpful for predicting the likelihood of infection in order to reduce the risk of anisakiasis cases in a given area