Category Archives: The Science of Microbiology

“Ageing methodology”

The laboratory detection of verotoxin/shigatoxin producing E. coli (sometimes called enterohaemorrhagic E. coli) has caused much grief for diagnostic microbiology laboratories over the decades. It is a relatively nasty infection, and it can cause bloody diarrhoea in a good proportion of patients. In a small minority it can cause severe complications such as Haemolytic Uraemic Syndrome (HUS) or Thrombotic Thrombocytopenic Purpura (TTP).

Diagnosis was initially centred on the culture of E. coli 0157 which can produce verotoxin/shiga toxin.

SMAC (Sorbitol MacConkey) agar plates were all the rage in the 1990s, taking advantage of the fact that E. coli 0157 does not ferment sorbitol. 

How lucky is that?

These were soon replaced by the more selective CTSMAC (Cefixime Tellurite Sorbitol MacConkey), with the cefixime and tellurite inhibiting other annoying non-sorbitol fermenters such as Proteus Spp.

The only problem is that E. coli 0157 is not the only E. coli serotype that can produce verotoxins. Lots of other E. coli serotypes are capable of doing this as well, e.g. 0111, 026, 045, 0145, etc.. As time passed, and our understanding of the infection improved, it became very apparent that a very significant proportion of VTEC induced diarrhoea was actually not due to E. coli 0157.

How inconvenient…

Nevertheless, CTSMAC plates were now entrenched in laboratories. And it was better than nothing.

As the years passed,  alternative methods came onto the scene.

ELISAs used for “direct” VTEC toxin detection in stool were employed in some labs in the early 2000s. At least they detected non-0157 associated disease, but sensitivity remained an issue when used directly on samples. They were not widely adopted by diagnostic laboratories.

Chromagar plates have also been developed to pick up the main VTEC serotypes. A little pricey however, and still need follow-up work for confirmation.

Then came PCR, and more recently multi-plex PCR, not only detecting (the toxins of) VTEC, but all the other common gastrointestinal pathogens as well.

In the molecular age, CTSMAC plates are starting to look a bit dated. What was seen as  modern methodology a generation ago no longer cuts the mustard.

As we move through this transition period for VTEC detection there is a real mish-mash of different VTEC methodologies used in laboratories worldwide. I don’t think this messy situation will last. In a decade or so I suspect 90% or more of microbiology laboratories will be using molecular methods for VTEC detection (and everything else stool related).

However at the moment, there are still plenty of CTSMAC plates being manufactured worldiwde. We still (guiltily) use them at our lab, as we continue to work out how to afford molecular testing for enteric pathogens…

But now they are used in the knowledge that they will clearly not pick up all VTEC strains in the patient samples, or anywhere close.

CTSMAC plates are getting old, and I for one can’t wait to see the back of them…

Michael

Note that the Infectious Diseases Society of America has just brought out updated guidelines on Infectious Diarrhoea, including quite a bit of detail on VTEC/STEC. Apart from the incorrect spelling of diarrhoea, they are very good!

I will add them to the guidelines section of this website also.

“The Swedish Variant: Selection Pressure by Diagnosis”

When we think about selection pressure the first thing that comes to mind are antibiotics that selectively kill susceptible bacteria and thus allow more resistant bacteria to fill the ecological niche.

But fewer people realise that selection pressure can also be caused (indirectly) by laboratory diagnosis. Microbes which are diagnosed in the laboratory often end up getting treated and eradicated. However a microbe which mutates sufficiently to avoid diagnosis will have a selection advantage over its diagnosable counterpart. This concept is particularly applicable to microbes which are diagnosed by molecular techniques such as PCR where only a minor mutation or deletion can potentially create sufficient change in the base sequence to make the microbe undetectable by the original molecular test.

The most classic example of this is the “Swedish Variant”.

In 2006, a drop in Chlamydia trachomatis diagnoses was noticed on a particular molecular platform X, but not on others in use within Sweden. Further analysis revealed that a mutant strain of Chlamydia trachomatis (nvCT) containing a 377 base pair deletion was circulating. This was undetectable on platform X, but detectable on other molecular platforms.

Interestingly the nvCT strain had a much higher prevalence in geographical areas where platform X was used. In areas where other platforms were utilised, it wasn’t so successful as it didn’t have any selection advantage. But this makes perfect sense when you realise that a strain that avoids laboratory detection and consequently destruction is bound to do better than a strain that is easily diagnosed.

So what implications does all this have for laboratory practice?

Centralisation, tendering, and “packaged” contracts means that we are increasingly relying on just the one molecular assay to diagnose a particular pathogen within a large geographical area.

Laboratories or regions, or even countries which just rely on just one molecular test to diagnose a pathogen are always vulnerable to “escape mutants” such as nvCT emerging which escape detection and thus thrive in the population.

Testing a cohort of samples on alternative molecular platforms to validate the results and to look for these escape mutants is an important quality assurance measure.

The story of the Swedish variant also demonstrates the importance of using the percentage positivity rate of a molecular test over time as a Quality Control measure.

Even though the Swedish variant was diagnosed over 10 years ago, the lessons that can be learned from this episode are probably even more important in the large volume, centralised laboratory landscape that we have today.

In summary, one must be careful not to put all their eggs in one basket…

Michael

Check out this article for a more detailed overview of the Swedish variant. (about a 10 minute read)

“Dead Certs and Long Shots”

The more uncertain the result will be, the more useful the laboratory test generally is…

Sounds a little paradoxical, but it is absolutely true.

If we are looking to confirm something that is almost certain before the lab test is performed, then we need a “super-sensitive” test to fulfil this task. Otherwise we run the risk of giving false negative results.

For example, if we have a teenager with a sore throat and lymphadenopathy, a lymphocytosis and atypical lymphocytes on blood film, then the probability of this being EBV infection is about 90%. There is little point then in doing a confirmatory Monospot test with a sensitivity of 80-85%. This will only lead to giving negative results on patients who actually have EBV infection.

And if we are looking to diagnose a long shot (aka a very unlikely diagnosis) then we had better be sure our laboratory test is “super-specific”, otherwise we will run the risk of giving false positive results.

For example if we want to diagnose dengue fever in a patient with “flu like” symptoms returning from Mexico (an area of relatively low Dengue endemicity), then we need to think twice about performing Dengue serology testing which has a specificity rate of about 95%. You are just as likely to report a positive test in someone who doesn’t actually have Dengue.

What we are doing in actual practice here is taking our pre-test probability, and using it to give a prevalence rate (by proxy) in our tested population. Once we know this, then we can use our test sensitivity and specificity to calculate positive and negative predictive values, not always with the results we would like…

Laboratory specialists tend to be more aware of testing limitations such as these. Clinicians, in general,  tend to just take the laboratory results as gospel.

But I believe it is ultimately the laboratory’s responsibility to stress the limitations of using laboratory testing for “Dead Certs or Long Shots”, and either prevent such testing taking place, or put big disclaimers on the results.

Michael