Over the past few years nucleic acid based methods have
revolutionised parasite diagnostics in modern clinical microbiology
(CM) labs. Real-time PCR is really gaining a foothold in CM labs, but
despite the opportunity for plexing, mostly only up to 6 DNA targets
can be included in each assay (due to the number of available
channels).
LUMINEX xMAP technology used for detection of specific nucleic acids (Dunbar, 2006) bypasses this limit, and up to 100 DNA targets can be included in one single assay in a 96-well plate format. You can read about the technology here.
In diagnostic parasitology, this technology is still new, but it has been used to diagnose Entamoeba histolytica, Giardia, Cryptosporidium, Ascaris, Necator, Ancylostoma and Strongyloides (Tainuchi et al., 2011). With relevant DNA extraction methods, such an approach appears very appealing, and can be extended to include more parasites, and it should also be possible to subtype/genotype parasites all along.
A paper is out just now, where the technology is used to differentiate species of Entamoeba (Santos et al., 2013), namely Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovsii, Entamoeba hartmanni and Entamoeba coli (note that these are not the only species infecting humans). And while this is really a great initiative, I think that the paper is an example of one of the pitfalls inherent in diagnostics relying on probe-based detection (Stensvold et al., 2011a). For instance, the two probes that should detect Entamoeba coli only match one of the two subtypes of E. coli (Stensvold et al., 2011b), and there is also evidence of genetic variation in Entamoeba moshkovskii in the E. moshkovskii probe binding sites (unpublished observations), and so the assay would be hampered by sub-optimal sensitivity compared to what's actually possibly at the present stage.
When using probe-based diagnostics, we are first and foremost limited by our own knowledge, and even experts are limited by the data available in GenBank. However, LUMINEX xMAP technology certainly appears to have a huge potential and I'd love to try and use it to develop a Blastocystis 18S allele assay... if only I had the machine!
References:
Dunbar SA (2006). Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clinica chimica acta; international journal of clinical chemistry, 363 (1-2), 71-82 PMID: 16102740
Santos HL, Bandyopadhyay K, Bandea R, Peralta RH, Peralta JM, & Da Silva AJ (2013). LUMINEX(R): a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools. Parasites & vectors, 6 (1) PMID: 23497666
Stensvold CR, Lebbad M, & Verweij JJ (2011a). The impact of genetic diversity in protozoa on molecular diagnostics. Trends in parasitology, 27 (2), 53-8 PMID: 21168365
LUMINEX xMAP technology used for detection of specific nucleic acids (Dunbar, 2006) bypasses this limit, and up to 100 DNA targets can be included in one single assay in a 96-well plate format. You can read about the technology here.
In diagnostic parasitology, this technology is still new, but it has been used to diagnose Entamoeba histolytica, Giardia, Cryptosporidium, Ascaris, Necator, Ancylostoma and Strongyloides (Tainuchi et al., 2011). With relevant DNA extraction methods, such an approach appears very appealing, and can be extended to include more parasites, and it should also be possible to subtype/genotype parasites all along.
A paper is out just now, where the technology is used to differentiate species of Entamoeba (Santos et al., 2013), namely Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovsii, Entamoeba hartmanni and Entamoeba coli (note that these are not the only species infecting humans). And while this is really a great initiative, I think that the paper is an example of one of the pitfalls inherent in diagnostics relying on probe-based detection (Stensvold et al., 2011a). For instance, the two probes that should detect Entamoeba coli only match one of the two subtypes of E. coli (Stensvold et al., 2011b), and there is also evidence of genetic variation in Entamoeba moshkovskii in the E. moshkovskii probe binding sites (unpublished observations), and so the assay would be hampered by sub-optimal sensitivity compared to what's actually possibly at the present stage.
When using probe-based diagnostics, we are first and foremost limited by our own knowledge, and even experts are limited by the data available in GenBank. However, LUMINEX xMAP technology certainly appears to have a huge potential and I'd love to try and use it to develop a Blastocystis 18S allele assay... if only I had the machine!
References:
Dunbar SA (2006). Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clinica chimica acta; international journal of clinical chemistry, 363 (1-2), 71-82 PMID: 16102740
Santos HL, Bandyopadhyay K, Bandea R, Peralta RH, Peralta JM, & Da Silva AJ (2013). LUMINEX(R): a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools. Parasites & vectors, 6 (1) PMID: 23497666
Stensvold CR, Lebbad M, & Verweij JJ (2011a). The impact of genetic diversity in protozoa on molecular diagnostics. Trends in parasitology, 27 (2), 53-8 PMID: 21168365
Stensvold CR, Lebbad M, Victory EL, Verweij JJ, Tannich E, Alfellani M, Legarraga P, & Clark CG (2011b). Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection. Protist, 162 (3), 525-41 PMID: 21295520
Taniuchi M, Verweij JJ, Sethabutr O, Bodhidatta L, Garcia L, Maro A, Kumburu H, Gratz J, Kibiki G, & Houpt ER (2011). Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples. Diagnostic microbiology and infectious disease, 71 (4), 386-90 PMID: 21982218
Luminex Corporation Blog: http://www.luminexcorp.com/blog/
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