Showing posts with label sampling. Show all posts
Showing posts with label sampling. Show all posts

Sunday, May 20, 2012

Brave New World

Using Blastocystis as an example, we have only recently realised the fact that conventional diagnostic methods in many cases fail to detect Blastocystis in faecal samples, which is why we have started using molecular diagnostics for Blastocystis. I was also surprised to realise that apparently no single drug can be used to treat Blastocystis, and that in fact we do not know which combo of drugs will actually consistently eradicate Blastocystis (Stensvold et al., 2010).

There will come a time - and it will be soon - where it will be common to use data from genome sequencing of pathogenic micro-organisms to identify unique signatures suitable for molecular diagnostic assays and to predict suitable targets (proteins) for chemotherapeutic intervention; in fact this is already happening (Hung et al., in press). However, despite already harvesting the fruits of recent technological advances, we will have to bear in mind that the genetic diversity seen within groups of micro-organisms infecting humans may be quite extensive. This of course will hugely impact our ablility to detect these organisms by nucleic acid-based techniques. For many of the micro-eukaryotic organisms which are common parasites of our guts, we still have only very little data available. For Blastocystis, data is building up in GenBank and at the Blastocystis Sequence Typing Databases, but for other parasites such as e.g. some Entamoeba species, Endolimax and Iodamoeba, we have very little data available. We only recently managed to sequence the small subunit ribosomal RNA gene of Iodamoeba, and we demonstrated tremendous genetic variation within the genus; it is now clear that Iodamoeba in humans comprises a species complex rather than "just" Iodamoeba b├╝tschlii (Stensvold et al, 2012).

Cysts of Iodamoeba
Ribosomal RNA is present in all living cells and is the RNA component of the ribosome. We often use this gene for infering phylogenetic relationships, i.e. explaining how closely or distantly related one organism is to another. This again assists us in hypothesising on transmission patterns, pathogenicity, evolution, drug susceptibility and other things. Since ribosomal RNA gene data are available for most known parasites, we often base our molecular diagnostics on such data. However, the specificity and sensitivity of our molecular diagnostic assays such as real-time PCRs are of course always limited by the data available at a given point in time (Stensvold et al., 2011). Therefore substantial sampling from many parts of the world is warranted in order to increase the amount of data available for analysis. In terms of intestinal micro-eukaryotes, we have only seen the beginning. It's great to know data are currently builiding up for Blastocystis from many parts of the world, - recently also from South America (Malheiros et al., 2012) - but the genetic diversity and host specificity of many micro-eukaryotes are still to be explored. It may be somewhat tricky to obtain information, since conventional PCR and sequencing offer significant challenges in terms of obtaining sequence data; such challenges can potentially be solved by metagnomic approaches - today's high throughput take on cloning; however, although the current next generation sequencing technology hype makes us feel that we are almost there, it seems we still have a long way to go - extensive sampling is key!

Cited literature:

Hung, G., Nagamine, K., Li, B., & Lo, S. (2012). Identification of DNA Signatures Suitable for Developing into Real-Time PCR assays by Whole Genome Sequence Approaches: Using Streptococcus pyogenes as a pilot study Journal of Clinical Microbiology DOI: 10.1128/JCM.01155-12

Malheiros AF, Stensvold CR, Clark CG, Braga GB, & Shaw JJ (2011). Short report: Molecular characterization of Blastocystis obtained from members of the indigenous Tapirap├ę ethnic group from the Brazilian Amazon region, Brazil. The American journal of tropical medicine and hygiene, 85 (6), 1050-3 PMID: 22144442

Stensvold, C., Lebbad, M., & Clark, C. (2011). Last of the Human Protists: The Phylogeny and Genetic Diversity of Iodamoeba Molecular Biology and Evolution, 29 (1), 39-42 DOI: 10.1093/molbev/msr238  

Stensvold, C., Lebbad, M., & Verweij, J. (2011). The impact of genetic diversity in protozoa on molecular diagnostics Trends in Parasitology, 27 (2), 53-58 DOI: 10.1016/j.pt.2010.11.005

Stensvold, C., Smith, H., Nagel, R., Olsen, K., & Traub, R. (2010). Eradication of Blastocystis Carriage With Antimicrobials: Reality or Delusion? Journal of Clinical Gastroenterology, 44 (2), 85-90 DOI: 10.1097/MCG.0b013e3181bb86ba