Polymerase chain reaction (PCR) testing.

 

This diagnostic method stemmed from the technology that enables specific sequences of nucleic acids to be identified and amplified.  This manufactures multiple copies of the sequence if it is present in the clinical sample making it more easy to detect.  If a unique sequence in a particular pathogen is known, this can be used as a template for amplification and if present in the clinical sample signifies that the pathogen is indeed there.  The advantage of the PCR technique over the older DNA probe methodology is that PCR  is capable of detecting much smaller numbers of organisms in the sample.

 

A full description of the methodology is beyond the scope of this explanation but in brief ; Nucleic acid is extracted from the clinical sample, incubated with a specific template primer and then put through a number of amplification cycles.  Following amplification the sample is placed on a gel to separate the amplified nucleic acid sequence.  The whole process from the receipt of the sample in the laboratory to the result being available is about six to eight hours.  While the technique is highly specific and sensitive there are some drawbacks, the most important of which is that the presence of the organisms nucleic acid does not indicate if the organism is living or dead.  This is important to recognise and does have clinical management implications.  E.g.  The presence of genital tract chlamydia is now often done by PCR detection as the diagnostic screening test.  If positive the patient is treated.  In order to determine if the therapy was effective in eradicating the infection a test of cure needs to be done and has to be a cultural method to see if live organisms remain.  The PCR will remain positive for up to six weeks after successful treatment.  The fact that living organisms are not necessary to give a positive result can also be an advantage.  If CNS infection due to herpes virus is suspected and PCR will be used to make a diagnosis, treatment can be started and the sample for PCR can be sent later, in other words a PCR test is not required to be done prior to therapy being started.  Given that the test is technically demanding not all laboratory technologists can perform it so it is always best to discuss the best diagnostic strategies with the lab when thinking of using PCR tests for diagnostic purposes.

 

There are some PCR tests that have been developed to detect mRNA (which will be present if the organism is alive and metabolically active) but these are not available for wide variety of pathogens.

 

A positive PCR are must be interpreted in light of the clinical picture and understanding that the test signifies the presence of pathogen nucleic acid only.  However a negative result is highly significant and means that the organism is not present in that sample.  There is little point in doing the test if the negative result will be ignored or passed off as a laboratory error.

 

Some ways to improve the usefulness of PCR testing.

1.  Call the lab to discuss the testing strategies and alert them to the patient.

2.  Most laboratories, and ours is no exception, extract the samples and then run them on the gel once or twice a week.  If more immediate results are required then arrangements need to be made with the laboratory.  In most cases this means that if samples are to be run in a single day they need to be received first thing in the morning.

3.  Lab staff may not be available to perform this test on the weekend so for best results specimens should be arranged to be sent during the week if possible.

4.  PCR is not a "stat" test.

5.  The test should not be used for a test of cure, culture is needed for this information.

 

PCR can be done in our laboratories for the following pathogens.

 

A. Herpes Group Viruses (Includes HSV,CMV,EBV) – performed on CSF’s and other sterile fluids as well as whole blood or plasma.

B. CMV mRNA Detection – performed only on whole blood (samples >24 hours old will not be tested.

C. Hepatitis C RNA – performed on serum and batched monthly.

D. Bordetella pertussis PCR – Nasopharyngeal aspirates are the best sample, please contact the lab before submitting an alternative sample.         

E. Other molecular assays may be performed on a research and development basis or referred out to other laboratories.  Please contact the lab to discuss individual cases and appropriate sample collection and transport.

 

Note: Sample volumes for most molecular assays require a minimum of 200 ul (other than NPA’s).  Samples of lesser volume may be tested, but negative results may not be accurate.

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Revised: June 6 2002

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