Research brief

Plasmodium falciparum: Hoechst dye 33258-CsCl ultracentrifugation for separating parasite and host DNAs

Broad-range amplification and sequencing of the 16S rRNA gene, directly from clinical samples, is a method that potentially allows detection of any cultivable or non-cultivable bacteria. However, the method is prone to false positive results due to PCR contamination. Another concern is the human DNA abundance compared to bacterial DNA in samples from sterile sites. Those factors may decrease the sensitivity and specificity of the assay and can complicate the analysis and interpretation of the results. The objective of this prospective study was to try to avoid the most common pitfalls, mentioned above, and develop a molecular 16S assay with a high clinical sensitivity and specificity. Fifty-six consecutive tissue samples from patients with suspected deep infections were extracted by 3 different DNA-extraction methods; two based on a principle of bacterial DNA enrichment, and one conventional DNA extraction method. We compared three primer pairs, including both conventional and DPO principle, targeting different variable regions of the 16S rRNA gene. Results from routine tissue culture were used as reference. Clinical data was recorded from patient charts and analyzed in parallel. Of a total of 56 samples, collected from 39 patients, 70% (39 samples) were assessed as true infections by analysis of clinical data. Bacterial enrichment extraction increased sensitivity from 54% to 72%. The 2 sets of primer pairs defining region V1-V3 and V3-V4, showed similar sensitivity, but DPO-primers resulted in better specificity, i.e. less contaminations. The primer pairs covering V1-V8 show significantly lower sensitivity (p < .001) than V1-V3 and V3-V4. Optimizing extraction protocols and choice of primers can increase the sensitivity and specificity of a molecular 16S-analysis, rendering a valuable complement to tissue culture.

We have undertaken the first comparative pilot gene discovery analysis of approximately 25 000 random genomic and expressed sequence tags (ESTs) from three species of Plasmodium, the infectious agent that causes malaria. A total of 5482 genome survey sequences (GSSs) and 5582 ESTs were generated from mung bean nuclease (MBN) and cDNA libraries, respectively, of the ANKA line of the rodent malaria parasite Plasmodium berghei, and 10 874 GSSs generated from MBN libraries of the Salvador I and Belem lines of Plasmodium vivax, the most geographically wide-spread human malaria pathogen. These tags, together with 2438 Plasmodium falciparum sequences present in GenBank, were used to perform first-pass assembly and transcript reconstruction, and non-redundant consensus sequence datasets created. The datasets were compared against public protein databases and more than 1000 putative new Plasmodium proteins identified based on sequence similarity. Homologs of previously characterized Plasmodium genes were also identified, increasing the number of P. vivax and P. berghei sequences in public databases at least 10-fold. Comparative studies with other species of Apicomplexa identified interesting homologs of possible therapeutic or diagnostic value. A gene prediction program, Phat, was used to predict probable open reading frames for proteins in all three datasets. Predicted and non-redundant BLAST-matched proteins were submitted to InterPro, an integrated database of protein domains, signatures and families, for functional classification. Thus a partial predicted proteome was created for each species. This first comparative analysis of Plasmodium protein coding sequences represents a valuable resource for further studies on the biology of this important pathogen.

Carlton, J. M-R., Yowell, C. A., Sturrock, K. A., and Dame, J. B. 2001. Biomagnetic separation of contaminating host leukocytes from Plasmodium-infected erythrocytes. Experimental Parasitology97, 111–114.

Two clones of 1.4 and 4.33 kilobase pairs (kbp) DNA inserts, were selected from a Sarcocystis cruzi sporozoite genomic library constructed in bacteriophage lambda gt10. These clones strongly hybridized with sporozoite and merozoite DNA and were evaluated as probes for detection of merozoite DNA in clinical samples. Of five calves in the experiment, four were each orally dosed with approximately 200 000 S. cruzi sporocysts; one calf served as non-infected control. Subsequently, blood was collected from the calves twice weekly for 3.5 months and fractionated into buffy coats, polymorphonuclear cells, and plasma. Total cellular DNA extracted from these fractions was dot blotted on nylon membranes and hybridized with the probes radiolabeled with [α-³²P]dATP. The probes detected merozoites on Day 22 post infection in the buffy coats and intermittently from Day 25–39 in the granulocyte fraction. Parasitemia (ie. merozoites in blood) was also detected by indirect fluorescent antibody technique (IFAT) and direct microscopy. Diagnosis of sarcocystosis in cattle using genomic DNA probes by dot blot hybridization provides an alternative method of detecting parasitemia that is more rigorous than the other two tests (IFAT, direct microscopy) which rely on morphology of the merozoite and visualization by the examiner. As probes detected merozoite DNA in the granulocyte fraction, polymorphonuclear cells may be involved in the pathogenesis of S. cruzi; however this hypothesis requires further study.

A genomic library of Sarcocystis cruzi sporozoite DNA was constructed in bacteriophage lambda gt10. Recombinant phages containing insert DNA were selected by growth on Escherichia coli strain C600 hflA150. Of 14 clones examined, 11 contained DNA inserts ranging in size from approximately 1.45 kilobase (kb) to 6.18 kb. Insert DNA from four of these clones specifically hybridized to ³²P-labelled S. cruzi merozoite DNA. One of these insert DNA, clone SL41, was selected and labelled with ³²P. This probe did not hybridize with the other ten DNA inserts nor with bovine cellular DNA, but it hybridized with sporozoite, merozoite and bradyzoite DNA preparations. The SL41 probe could detect merozoite DNA in as little as 17 ng total DNA. Genomic probes detecting developmental stages of Sarcocystis spp. could provide an improved means is diagnosis of acute bovine sarcocystosis.