Molecular techniques to identify Mycobacteria. How good is it?

It seems that molecular techniques (PCR) are over taking the traditional methods for making diagnoses. It can be much more sensitive and have a much quicker turn around time. But, as a trained veterinary pathologist, I would tread with caution. The technique detailed below talks about how they used it to identify Mycobacteria in CLINICAL CASES. This means that they already knew they had the bug and was just testing for the fact. I have no qualms about using PCR to confirm a diagnosis because we regularly use multiple lines of evidence to arrive at our conclusions. But to use it as the sole means to make a diagnosis can be very dangerous.

Recently I’ve had a case where two animals in the collection was destroyed based on PCR detection of the pathogen. A full necropsy and thorough histopathology examination actually showed the animals had a clean bill of health! How could it have gone so wrong? This is because PCR tests only needs to detect a small portion of the DNA of the bug to give a positive result. So here are two simplified scenarios. The first is that the living bug is not present, but only components of the bug is present which is detected by the test. The second scenario is that the bug may be present in whole, but it may not be causing disease. It’s only an incidental findings.

There’s much to be said about studying the whole animal rather than relying on a test on alphabet soup!

The other great advantage of histopathology is that you can spot many other things that might be going on in the animal.

Aquaculture
Volume 411, Number 1-2 (October 2013)
	Use of restriction enzyme fragment length polymorphism (RFLP) of the 16S–23S rRNA internal transcribed spacer region (ITS) for identification of fish mycobacteria
	Authors:	Fazel Pourahmad, Randolph H. Richards
	Author Affiliations:	no affiliations available
	Source:	Aquaculture, Volume 411, Number 1-2 (October 2013)
	Page Numbers:	184 – 189
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	Abstract:	PCR targeting the 16S–23S rRNA gene internally transcribed spacer (ITS) region has been proposed as a rapid and reliable method for the detection of Mycobacteriumspecies in human clinical specimens. Because of variation in ITS sequences amongst Mycobacteriumspecies, a single PCR amplification can be used to differentiate slowly growing and rapidly growing species within this genus. In the present study, analysis by ITS-PCR and ITS-restriction fragment length polymorphism (RFLP) was found to be a useful, simple and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from 13 reference strains and 59 fish isolated mycobacteria using a set of published PCR primers. After PCR, the banding patterns generated allowed slowly growing mycobacteria to be differentiated from all other rapidly growing species, with the exception of Mycobacterium conceptionense. HaeIII was selected as one of two restriction enzymes that, together with the knowledge about amplicon sizes, would produce an acceptable level of discrimination in the resulting RLFP patterns, especially in the rapidly growing group of mycobacteria. After digestion with Sau96I, the amplified products of most isolates of Mycobacterium fortuitum, including subtypes II and V and those 2 isolates with new patterns (220, 100bp), presented identical or very similar patterns as obtained by HaeIII digestion. All isolates of Mycobacterium marinum, Mycobacterium chelonaeand Mycobacterium gordonae, whose PCR products were not digested with HaeIII, produced two well-defined fragments with the Sau96I restriction enzyme.
	Citation:	Fazel Pourahmad, Randolph H. Richards . Use of restriction enzyme fragment length polymorphism (RFLP) of the 16S–23S rRNA internal transcribed spacer region (ITS) for identification of fish mycobacteria. Aquaculture, Volume 411, Numbers 1-2 (October 2013), pp. 184-189, <http://ejournals.ebsco.com/direct.asp?ArticleID=483BA7BA8D76F091EC97&gt;
	URL:	http://ejournals.ebsco.com/direct.asp?ArticleID=483BA7BA8D76F091EC97