Tag Archives: PCR

“Ignorance is bliss”

B. hominis
B. hominis

PCR for enteric pathogens is starting to take hold, both locally and internationally.

One area where there is a significant difference between traditional methods and PCR is in the area of enteric parasites.

PCR is much more sensitive than microscopy at picking up faecal parasites. This has shown great benefit when trying to diagnose conditions like Entamoeba histolytica, (not only in faeces, but also in liver abscesses as well)

So far, so good,

but there are downsides as well….

PCR for Blastocystis hominis and Dientamoeba fragilis are generally much more sensitive methods than their microscopical counterparts. This leads to high numbers of “positives” being reported, particularly in children. However these two parasites are putative pathogens at best, potentially causing symptoms in a small and select number of patients only. Most of the time they are probably just there for the ride.

Using such a sensitive assay for parasites of uncertain significance like this can thus lead to misdiagnosis, over treatment and undue anxiety. More is not always better.

Sometimes ignorance is bliss…

Michael

“Ward based PCR”

There are a couple of new systems just on the market which I suspect will revolutionise PCR testing, particularly where an urgent result is desirable. This is often the case when trying to diagnose Influenza and RSV in the acute care setting.

Cepheid and Roche have both released benchtop PCR systems for Influenza and RSV testing which can be placed in the ward/ED environment, requires minimal training and can give you a highly sensitive result for influenza/RSV in well under an hour.

“Highly sensitive” are the key words here. This is in contrast to rapid antigen tests for Influenza and RSV which although can be useful in certain circumstances, are limited in their clinical usefulness by sub-standard sensitivity. RSV and Influenza rapid antigen tests are now living on borrowed time….

The other highlight is “under an hour”. This allows real-time management of the patient in the ED./acute assessment ward (including treatment, isolation, cohorting, discharge etc.) based on the result of the PCR test.

The big downsides of course are the cost and the “one at a time access”. More expensive (the cost will come down)than traditional batched PCRs it might be a while before such testing systems become commonplace. (A lot depends on the funding model of your healthcare system.)

But they will, you can be sure of that.

Michael

“PCR: A Multiplicity of Multiplexes….”

PCR multiplexes seem to be all the rage just now….

Here is just a selection of what is currently available in New Zealand:

  • CSF Multiplex: HSV, VZV, Enterovirus, Parechovirus, N. meningitidis, S. pneumoniae
  • Respiratory Virus Multiplex: Influenza A&B, CMV, Adenovirus, RSV, hMPV, Rhinovirus, Coronavirus, Parainfluenza 1-3, Bocavirus.
  • Atypical Pneumonia Multiplex: Legionella, Mycoplasma pneumoniae, Chlamydia, Pneumocystis, Bordetella pertussis & parapertussis.
  • Enteric Virus Multiplex: Norovirus, Rotavirus, Adenovirus, Astrovirus.

However there are some downsides to multiplex PCRs, both clinical and technical. These are as follows:

  •  Cost: The clinician may not want to test for all the assays within a multiplex PCR, therefore the cost may be more than with other individual assays that are required. For example, it is usually easy to differentiate between a viral and a bacterial meningitis based on initial CSF findings. However if the “CSF multiplex” includes both bacteria and viruses, then it may lead to unnecessary cost as well as problems with positive predictive value as described below.
  • Expertise: Carrying out a multiplex PCR still requires a reasonable amount of expertise, particularly if the reagents are being prepared “in-house” The expertise level increases further when troubleshooting is required.
  • Controls: Controlling each assay within the multiplex.
  • Test Volumes: Because of the amount of controls required per batch, significant numbers of tests are often required to make it cost-effective. Therefore may restrict some multiplexes to the larger centres.
  • Optimisation: Optimising each assay and avoiding competitive inhibition between the different reagents.
  • Positive predictive Value: If you have 5 tests in a multiplex PCR, then it is likely that at least one of these tests has a very low pre-test probability making interpretation of positive results difficult. For example, during the Influenza season, it may be prudent to test for Influenza first and then worry about other diagnoses if this test is negative.
  • More than one positive result: For example if you are doing a multiplex PCR with 7 or 8 respiratory viruses, it is not uncommon for 2 or even 3 assays to be positive. You then need to decide which one is causing the problem….
  • Only diagnoses what is tested for in the multiplex: I.e. It is not a catch-all method.

 Multiplex PCR can clearly be very useful in some situations. However it is important to be aware of the limitations as described above and have other testing options available. Otherwise the skill of utilising laboratory tests in a cost effective and clinically appropriate manner will be lost….

Michael

For a really simple walk through the basics of the PCR reaction, check out this website. I will go into a bit more detail next week on detection of PCR product, Real-Time PCR etc.