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.