Tag Archives: molecular testing

“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

“Gatecrashing: Neisseria meningitidis as a genital pathogen.”

Confessions of a Microbiologist, ,

Neisseria meningitidis is more classically known as the cause of meningococcal sepsis and meningococcal meningitis. However its role as a cause of urethritis/cervicitis has been the subject of ongoing speculation over the years, and several studies have backed such a link up. For example, check this study out, and this one.

A recent study has added more weight to this hypothesis, backing the assertion up with DNA studies of the N. meningitidis isolates showing adaptation to a genital environment. (loss of outer capsules, and acquisition of enzymes facilitating survival in a low oxygen environment)

So what does this all mean for clinical microbiology laboratories?

I guess it shows the inherent weakness of molecular diagnosis. There could be a widespread outbreak of urethritis due to Neisseria meningitidis in your local area, but the laboratory would be completely naïve to it, if it only performs molecular testing for C. trachomatis and N. gonorrhoeae. Particularly in the Sexual Health Clinic setting, adjunctive culture of STI samples is important, and not just to obtain the N. gonorrhoeae susceptibilities.

It is also possible that the selective molecular diagnosis and treatment of N. gonorrhoeae will therefore create a “niche” for organisms like Neisseria meningitidis to adapt physiologically and “gatecrash” the party. 

And finally on this topic, there is also intriguing data coming out that suggests that some meningococcal vaccines may have a protective effect for N. gonorrhoeae infection. Suspected for some time, this suggestion has been backed up by some observational data in this study. More research is obviously needed. We are still a bit away I suspect from a gonococcal vaccine.

The physiological and evolutionary relationship between Neisseria meningitidis and Neisseria gonorrhoeae is a fascinating one.  We shouldn’t think too much about one without considering the other…

Michael

 

“Molecular diagnosis of wound infections: The Holy Grail.”

Future of Microbiology, The Science of Microbiology,

The slice of pie taken by the molecular department in the microbiology laboratory is increasing, slowly but surely.

On the contrary, the proportion of culture based microbiology is inexorably declining.

Many clinical microbiology laboratories are now switching, or looking at switching over to molecular diagnosis of enteric pathogens.

Molecular diagnosis of pathogens causing vaginitis and pharyngitis will not be far behind, and a few labs have already moved in this direction.

However diagnosis of wound infections remains firmly culture based. There is not even much in the literature with regards to molecular diagnosis of wound infections…

Is it even possible?

So what are the difficulties?:

  • Potential number of pathogens: There are several pathogens or putative pathogens that are able to cause wound infections. This makes PCR based molecular diagnosis more difficult. However, on the flip side, 95% of wound infections in a general clinical setting are caused by two pathogens, Staphylococcus aureus and Streptococcus pyogenes (Group A streptococcus).
  • Lack of susceptibility information: Molecular diagnosis of resistance determinants is still a little behind phenotypic culture based testing. However with regards to the two main pathogens as above, PCR analysis can easily differentiate between MSSA and MRSA, and susceptibility data for Streptococcus pyogenes is only required for a small proportion of patients who have anaphylactic reactions to penicillin.
  • “Over-sensitivity”: It is always nice to know which are the dominant organisms within a wound. Culture is relatively good at this. However molecular methods are starting to be able to quantify to some extent (e.g by playing with the cycle threshold cut-off).
  • Cost: The culture of a standard wound swab might cost $10 or so, even when overheads are included. In my experience molecular tests have to be performed in very large volumes to even get close to this kind of price. However wound swabs do arrive into the laboratory in very large volumes!

If it was easy it would have been done by now… I suspect it is the 4 things above acting together as a “bundle”, as opposed to any one insurmountable barrier which has contributed to the lack of progress up until now.

However it will come in some form or other, you can be sure of that…

Here is a potential solution I have thought of:

  • Any wound swab accompanied with clinical details suggesting an unusual pathogen e.g. animal bites (Pasteurella), water exposure (vibrios, aeromonas), immunocompromise,  would still undergo routine “catch-all” bacterial culture.
  • The rest (the vast majority) would be subjected to a multiplex PCR for the detection of MSSA, MRSA and Streptococcus pyogenes (and possibly Group G streptococcus also). A positive result would be reported routinely. A  negative result would have an accompanying comment to suggest contacting the laboratory if the patient’s symptoms were persisting, or if further susceptibilities are required, so that culture could be set up if necessary.

In this way molecular diagnostics could be performed on (a good proportion of) wound swabs at a relatively low cost.

In addition multiplex PCRs could be developed specifically for infections in specific clinical situations, e.g. post animal bite,  etc., etc.

Automated bacteriology culture systems like Kiestra TLA would not have happened if the industry didn’t think that there was at least another 20 years or so of culture based bacteriology left, and they are probably right. But I don’t think it will be too long before commercial laboratory diagnostics companies start looking closely at “wholesale” molecular options as above.

And who knows, whole genome sequencing might come in and completely disrupt PCR based molecular diagnostics, and the picture might change again…

“The future’s uncertain and the end is alway near“

Michael