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Background: Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR. Results: A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%). Conclusions: Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach. [ABSTRACT FROM AUTHOR]

Background: The haemoflagellate Trypanosoma lewisi is a kinetoplastid parasite which, as it has been recently reported to cause human disease, deserves increased attention. Characteristic features of all kinetoplastid flagellates are a uniquely structured mitochondrial DNA or kinetoplast, comprised of a network of catenated DNA circles, and RNA editing of mitochondrial transcripts. The aim of this study was to describe the kinetoplast DNA of T. lewisi. Methods/Results: In this study, purified kinetoplast DNA from T. lewisi was sequenced using high-throughput sequencing in combination with sequencing of PCR amplicons. This allowed the assembly of the T. lewisi kinetoplast maxicircle DNA, which is a homologue of the mitochondrial genome in other eukaryotes. The assembly of 23,745 bp comprises the non-coding and coding regions. Comparative analysis of the maxicircle sequence of T. lewisi with Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma brucei and Leishmania tarentolae revealed that it shares 78 %, 77 %, 74 % and 66 % sequence identity with these parasites, respectively. The high GC content in at least 9 maxicircle genes of T. lewisi (ATPase6; NADH dehydrogenase subunits ND3, ND7, ND8 and ND9; G-rich regions GR3 and GR4; cytochrome oxidase subunit COIII and ribosomal protein RPS12) implies that their products may be extensively edited. A detailed analysis of the non-coding region revealed that it contains numerous repeat motifs and palindromes. Conclusions: We have sequenced and comprehensively annotated the kinetoplast maxicircle of T. lewisi. Our analysis reveals that T. lewisi is closely related to T. cruzi and T. brucei, and may share similar RNA editing patterns with them rather than with L. tarentolae. These findings provide novel insight into the biological features of this emerging human pathogen. [ABSTRACT FROM AUTHOR]

Background: In East Africa, animal trypanosomiasis is caused by many tsetse transmitted protozoan parasites including Trypanosoma vivax, T. congolense and subspecies of T. brucei s.l. (T. b. brucei and zoonotic human infective T. b. rhodesiense) that may co-circulate in domestic and wild animals. Accurate species-specific prevalence measurements of these parasites in animal populations are complicated by mixed infections of trypanosomes within individual hosts, low parasite densities and difficulties in conducting field studies. Many Polymerase Chain Reaction (PCR) based diagnostic tools are available to characterise and quantify infection in animals. These are important for assessing the contribution of infections in animal reservoirs and the risk posed to humans from zoonotic trypanosome species. New matrices for DNA capture have simplified large scale field PCR analyses but few studies have examined the impact of these techniques on prevalence estimations. Results: The Whatman FTA matrix has been evaluated using a random sample of 35 village zebu cattle from a population naturally exposed to trypanosome infection. Using a generic trypanosome-specific PCR, prevalence was systematically evaluated. Multiple PCR samples taken from single FTA cards demonstrated that a single punch from an FTA card is not sufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card. At low parasite densities in the host, this stochastic sampling effect results in underestimation of prevalence based on single punch PCR testing. Repeated testing increased the estimated prevalence of all Trypanosoma spp. from 9.7% to 86%. Using repeat testing, a very high prevalence of pathogenic trypanosomes was detected in these local village cattle: T. brucei (34.3%), T. congolense (42.9%) and T. vivax (22.9%). Conclusions: These results show that, despite the convenience of Whatman FTA cards and specific PCR based detection tools, the chronically low parasitaemias in indigenous African zebu cattle make it difficult to establish true prevalence. Although this study specifically applies to FTA cards, a similar effect would be experienced with other approaches using blood samples containing low parasite densities. For example, using blood film microscopy or PCR detection from liquid samples where the probability of detecting a parasite or DNA molecule, in the required number of fields of view or PCR reaction, is less than one. [ABSTRACT FROM AUTHOR]

Abstract: Direct PCR analysis of trypanosome infected blood samples in the quantities required for large scale epidemiological study has always been problematic. Current methods for identifying and differentiating trypanosomes typically require several species-specific reactions, many of which rely on mouse passaged samples to obtain quality concentrated genomic DNA. As a consequence important epidemiological information may be lost during the sample preparation stage. Here, we report a PCR methodology that reduces processing and improves on the sensitivity of present screening methods. The PCR technique targets the gene encoding the small ribosomal subunit in order to identify and differentiate all clinically important African trypanosome species and some subspecies. The method is more economical, simple, and sensitive than current screening methods, and yields more detailed information, thereby making it a viable tool for large-scale epidemiological studies. [Copyright &y& Elsevier]