Tag Archives: PCR

“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.

“Echoes from a Distant Land”

A few years ago, I had never heard of parechovirus. Echovirus yes, but not “parecho”.

I would not have been alone…

However our ability to diagnose such viruses now (using PCR) means that myself and my colleagues now need to go and learn something about such viruses.

And here is a summary of (more or less) all I know about parechoviruses…

  • I know that the “ECHO” bit is an acronym (Enteric, Cytopathogenic, Human, Orphan). That probably tells me as much as I need to know about the background information on the virus. I have no idea what the “Par” stands for. Any help welcome…
  • You can theoretically perform viral culture for parechovirus, if you are happy to wait a couple of weeks for the result. We aren’t, so along with most other diagnostic laboratories in the world now, molecular methodologies have taken over.
  • Parechoviruses are the 2nd most common cause of viral sepsis like illness and meningitis in infants (after enteroviruses). Therefore if you are performing a CSF PCR panel in children, then it must include parechovirus. (Unlike enteroviruses, parechoviruses are a relatively rare cause of infection in older children and adults)
  • In complement to the above, parechovirus infection in the CSF does not give a positive enterovirus PCR test. They are sufficiently different viruses.
  • Unlike most other viruses, parechovirus meningitis does not cause a significant CSF leucocytosis. This has been our personal experience in the laboratory, and also that of other people in the medical literature. This is important because if, like us, you have testing criteria in place for CSF viral PCR based on leucocyte count, then you need to make neonates & young infants an exception to the rule.
  • Parechovirus (like enteroviruses) belongs to the picornaviridae family, and because of this may be susceptible to the viral capsid inhibitor pleconaril. However the jury is still very much out on pleconaril, and as such I would want to be speaking to someone who knows much more about viruses than me before even considering its use in a sick child.

So I now know a little bit about parechovirus, not a lot, but enough to allow me to do my job.

We don’t need to be “walking encyclopaedias”, just knowledgable enough to function in an information overloaded world…

Michael

“What we know is what we get”

Myco_culture

We are all familiar with chlamydia, gonorrhoea, syphilis etc when thinking about STIs.

But when it comes to Mycoplasma genitalium, the knowledge base might be a little more patchy…

Mycoplasma genitalium has been associated with urethritis since the early 80s. However in those days, the only means of diagnosis was culture.

Have you ever tried to culture a Mycoplasma? It is not surprising that Mycoplasma genitalium was kept in the dark as a causative agent of Non-specific Urethritis (NSU) for a couple of decades. Quite simply, it was put in the ‘too difficult basket’ in terms of laboratory diagnosis.

It is only in the past few years that commercial PCR assays have become increasingly available for this pathogen. Consequently clinicians and microbiologists are becoming much more aware of it.

As a cause of urethritis it is more common than Neisseria gonorrhoeae, but less common than Chlamydia trachomatis.

Most labs still do not test for M. genitalium routinely, restricting testing to treatment failures or other special circumstances. However I think this will change in the future, and it may well be included in an NSU panel with C. trachomatis and N. gonorrhoeae.

If I was an examiner, and it is a relief to many that I am not, Mycoplasma genitalium would be one topic that I would ask about. I suspect it would be a good topic to separate the passes from the distinctions…

Michael

Click here for a short CDC review article on Mycoplasma genitalium (about a 5 minute read)