Wednesday, 9 May 2012

"What are you looking at?"

Not just a phrase favoured by self conscious teenagers, “what are you looking at?” is a question that anyone should be asking when trying to detect something. Diagnostics is a classic case.

With the rise of modern molecular approaches, traditional virological assays would appear to now languish in the dark shadows cast by the likes of real-time RT-PCR and next generation sequencing. This is, of course, unsurprising; molecular assays have many advantages such as speed and sensitivity relative to traditional virological methods. Molecular assays have, and continue to, revolutionise diagnostic capabilities in a multitude of ways. One of the biggest impacts is when unknown viruses arrive. The outbreak of SARS was first reported in March 2003; by May that year a virus had been isolated and, via sequencing, had been identified as a coronavirus. Most recently Schmallenberg virus was identified using metagenomic sequencing of a blood sample from a clinically affected cow. In terms of disease though, Koch’s postulates cannot be fulfilled without first isolating the suspected agent...

Pathogen detection in animals can be divided into either direct detection of the pathogen, or detection of evidence that the pathogen has been there. The latter generally refers to the detection of an immune response, most commonly antibodies. Detection of the pathogen can be further divided into detection of genome, protein or, in the case of viruses, by isolation of the virus. They may sound similar, but in reality it’s not. When it’s stated that a virus was detected by PCR, it can be argued that live virus was not being detected, but actually only a fragment. This may sound trivial, and in many cases PCR positive and virus positive equate, but it is of significance if say an unknown species of blood-sucking insect is responsible for spreading a virus from animal to animal. Any insect that has fed on a viraemic host can potentially be positive for the virus according to PCR, regardless of whether it is capable of supporting virus replication, or even if the virus is still ‘live’. We found a similar scenario with Foot and Mouth Disease Virus - some samples which were negative for live virus were positive by ELISA and/or real-time RT-PCR (detecting the proteins or genome respectively of viruses that had fallen apart in acidic conditions and were no longer infectious). What does this mean regarding whether an animal is infectious? Assuming the absence of contamination, and in the case of FMDV, genome in a clinical sample probably means the animal was infectious. No virus, no genome. On the other hand, it is well established for Bluetongue virus that animals can frequently be positive for RNA by RT-PCR, but non-infectious for biting midges.  
And characterisation? Does a PCR assay suggesting a virus is of serotype X categorically mean it is a virus with serotype X? Well, probably. But whilst a PCR assay detects the genomic sequence, a serotype reflects the expressed form of the genome, i.e. the phenotype. Perhaps this is pedantic, but chimpanzees are 95-99% identical to humans – a PCR assay might easily suggest they are the same, but ultimately they are different. Serotype classically refers to protection against an identical serotype, which by definition refers to serum, i.e. antibodies, and antibodies generally recognise protein.  The advent of sequencing has increased the precision of this and now the aim is to combine the two using mathematical modelling such that serotype is accurately predicted by the genomic sequence.

In the future, virus diagnostics may well involve simply sequencing everything in a sample, resulting in the full spectrum of pathogens present – the equivalent of a deep-sea trawler compared to fishing with a rod and some bait. As such metagenomics approaches expand an enticing prospect is the derivation of a prognosis based upon the molecular signatures present.

Ultimately though, if you’re interpreting diagnostics results, you need to know what you’re looking at!

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