Tag Archives: PCR

“The Molecular Revolution”

Confessions of a Microbiologist, Future of Microbiology, , ,

Time to get back to some writing “post” COVID!

When I started at the laboratory I currently work at in New Zealand in 2007, we only did one molecular assay, a chlamydia PCR, and we did this with separate extraction and amplification platforms on an open bench, with all sorts of potential for contamination. And we were/are not a small lab, a sizeable regional centre, processing well over 1000 microbiology samples a day.

2007, it’s actually not that long ago…

Fast forward 15 years and everything has changed. We now have a very sizeable menu of molecular assays performed on a range of different platforms. CSF, respiratory virus and GI panels, gonorrhoea, trichomonas, HSV/VZV,  HIV, HBV &HCV viral loads, Legionella spp., Mycoplasma pneumoniae, C. pneumoniae, C. difficile to name just a few. We even have a Mpox PCR!

A lot of these assays are now on commercial platforms that perform both the extraction and amplification steps in an automated fashion in a closed environment, essentially allowing placement of the platform anywhere, and can be run by most of our staff. The results are often available within a few hours of the sample being received in the laboratory.

In summary, the clinical service we can now offer is vastly improved from 15 years ago. I suspect it is much the same in many diagnostic labs throughout the world.

The big question is what will happen in the next 15 years? Will high volume sample types such as throat swabs, vaginal swabs, sputum samples, all still culture based at my lab, succumb to the revolution and go molecular? It is entirely possible that this will be the case. It will probably come down to cost first and foremost. Personally I see throat swabs switching to molecular very soon.

And what place will there be for whole genome sequencing in the diagnostic lab? That is a whole other question in itself but there are quite a few labs now in NZ who have acquired Nanopore Minions and are now “playing” with them in the areas of Infection Control and metagenomics.

My prediction is by 2030, for most diagnostic microbiology labs, their molecular department will be bigger than their traditional culture-based bacteriology department…

What do you think?

Michael

“Ageing methodology”

The Science of Microbiology, , , ,

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”

Confessions of a Microbiologist, ,

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