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Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are [...]

Abstract Background Giardia duodenalis is a common protozoan parasite of humans and animals. Genetic characterization of single loci indicates the existence of eight groups called assemblages, which differ in their host distribution. Molecular analyses challenged the idea that G. duodenalis is a strictly clonal diplomonad by providing evidence of recombination within and between assemblages. Particularly, inter-assemblage recombination events would complicate the interpretation of multi-locus genotyping data from field isolates: where is a host infected with multiple Giardia genotypes or with a single, recombined Giardia genotype. Methods Population genetic analyses on the single and multiple-locus level on an extensive dataset of G. duodenalis isolates from humans and animals were performed. Results Our analyses indicate that recombination between isolates from different assemblages are apparently very rare or absent in the natural population of Giardia duodenalis. At the multi-locus level, our statistical analyses are more congruent with clonal reproduction and can equally well be explained with the presence of multiple G. duodenalis genotypes within one field isolate. Conclusions We conclude that recombination between G. duodenalis assemblages is either very rare or absent. Recombination between genotypes from the same assemblage and genetic exchange between the nuclei of a single cyst needs further investigation.

Abstract Background Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

Abstract Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.

Abstract Background Individuals living in areas of high malaria transmission often have different Plasmodium falciparum clones detected in the peripheral blood over time. The aim of this study was to assess the dynamics of asymptomatic P. falciparum infections in a few hours intervals. Methods Capillary blood samples were collected 6-hourly during five days from asymptomatic children in a highly endemic area in Tanzania. Parasite densities and maturation stages were investigated by light microscopy. Types and number of clones were analysed by PCR based genotyping of the polymorphic merozoite surface proteins 1 and 2 genes. Results: Parasite densities and maturation stages fluctuated 48-hourly with a gradual shift into more mature forms. Various genotyping patterns were observed in repeated samples over five days with only few samples with identical profiles. Up to six alleles differed in samples collected six hours apart in the same individual. Conclusion This detailed assessment highlights the extensive within-host dynamics of P. falciparum populations and the limitations of single blood samples to determine parasite densities, stages and genotyping profiles in a malaria infected individual.