Best Practices for Health Care Professionals on the use of
Polymerase Chain Reaction (PCR) for Diagnosing Pertussis
With the continuing resurgence of pertussis, health care professionals will see more patients with
suspected pertussis. Polymerase Chain Reaction (PCR) is an important tool for timely diagnosis of
pertussis and is increasingly available to clinicians. PCR is a molecular technique used to detect DNA
sequences of the
Bordetella pertussis bacterium and unlike culture, does not require viable (live) bacteria
present in the specimen. Despite these advantages, PCR can give results that are falsely-negative or
falsely-positive. The following compilation of best practices is intended to help health care professionals
optimize the use of PCR testing for pertussis by avoiding some of the more common pitfalls leading to
inaccurate results.
Testing Patients with Signs and Symptoms of Pertussis
Early signs and symptoms of pertussis are often non-specific, making it difficult to
determine clinically who has pertussis in the earliest stages.
However, only patients with signs and symptoms consistent with
pertussis should be tested by PCR to confirm the diagnosis. Testing
asymptomatic persons should be avoided as it increases the likelihood
of obtaining falsely-positive results. Asymptomatic close contacts of
confirmed cases should not be tested and testing of contacts should not
be used for post-exposure prophylaxis decisions.
Optimal Timing for PCR Testing for Pertussis
PCR has optimal sensitivity during the first 3 weeks of cough when
bacterial DNA is still present in the nasopharynx. After the fourth
week of cough, the amount of bacterial DNA rapidly diminishes which
increases the risk of obtaining falsely-negative results. For more
information, consult
diagnostic testing for pertussis, including the use of serology for late diagnosis.
PCR testing following antibiotic therapy also can result in
falsely-negative findings. The exact duration of positivity following
antibiotic use is not well understood, but PCR testing after 5 days of
antibiotic use is unlikely to be of benefit and is generally not
recommended.
Optimal Specimen Collection for PCR Testing for Pertussis
Specimens for PCR
testing should be obtained by aspiration or swabbing the posterior
nasopharynx. Throat swabs and anterior nasal swabs have unacceptably
low rates of DNA recovery and should not be used for pertussis
diagnosis. The swab tips may be polyester (such as Dacron®), rayon, or
nylon-flocked. Cotton-tipped or calcium alginate swabs are not
acceptable as residues present in these materials inhibit PCR assays.
If feasible, nasopharyngeal (NP) aspirates that flush the posterior
nasopharynx with a saline wash are preferred over swabs because this
method results in a larger quantity of bacterial DNA in the sample.
Avoiding Contamination of Clinical Specimens with Pertussis DNA
Some pertussis vaccines
[1] have been found to contain PCR-detectable
B. pertussis DNA. Environmental sampling has identified
B. pertussis
DNA from these vaccines in clinic environments. While the presence of
this DNA in the vaccines does not impact the safety or immunogenicity
of these vaccines, accidental transfer of the DNA from environmental
surfaces to a clinical specimen can result in specimen contamination
and falsely-positive results. If health care professionals adhere to
good practices, there is no need to switch vaccines.
Preparation and administration of vaccines in areas separate
from pertussis specimen collection areas may reduce the opportunity for
cross contamination of clinical specimens. Care should be taken when
preparing and administering pertussis vaccines to avoid contamination
of surfaces with vaccine. General adherence to basic
infection-control measures may further prevent contamination of
specimens:
- Wearing gloves immediately before and during specimen
collection or vaccine preparation and administration with immediate
disposal of gloves after the procedure, and
- Cleaning clinic surfaces using a 10% bleach solution[2] to reduce the amount of nucleic acids in the clinic environment.
The use of liquid transport media likely also contributes to
falsely-positive results from contaminant DNA. When using liquid
transport media, DNA that is accidentally transferred from hands to the
swab shaft can be washed off into the liquid medium which freely
circulates around the transport tube; this liquid is later extracted to
obtain DNA for PCR testing. Use of a semisolid or non-liquid transport
media or transport of a dry swab without media should prevent
contaminant DNA on the swab shaft from reaching the part of the specimen
that is later extracted. If using liquid transport medium, the swab
stick should be handled with care and only above the red line or
indentation which marks where the shaft is snapped off after insertion
into the medium. Performing NP aspiration rather than swabbing the NP
may also prevent contamination from occurring as the aspirate kit
(syringe or bulb style) is a closed system at the point of specimen
collection.
Understanding and Interpreting Testing Results
PCR assays for pertussis are not standardized across
clinical laboratories. Testing methods, DNA targets used and result
interpretation criteria vary, and laboratories do not use the same
cutoffs for determining a positive result. With PCR, high cycle
threshold (Ct) values indicate low levels of amplified DNA; for
pertussis, these values may still indicate infection but can also be
the result of specimens contaminated with DNA from the environment at
the time of specimen collection. Clinical laboratories might report
high Ct values as any of the following: positive, detected,
indeterminate, or equivocal. In addition, most clinical laboratories
use a single target PCR for IS
481, which is present in multiple copies in
B. pertussis and in lesser quantities in
B. holmesii and
B. bronchiseptica. Because this DNA sequence is present in multiple copies, IS
481
is especially susceptible to falsely-positive results. Use of
multiple targets may improve specificity of PCR assays for pertussis.
Clinicians are encouraged to inquire about which PCR target or targets
are used by their laboratories. Interpretation of PCR results,
especially those with high Ct values, should be done in conjunction
with an evaluation of signs and symptoms and available epidemiological
information.
Summary
In summary, PCR is an important tool for diagnosis of
pertussis especially in the setting of the current resurgence of
pertussis disease. PCR can provide timely results with improved
sensitivity over culture. Careful specimen collection and transport and
a general understanding of the PCR assays performed will better ensure
that clinicians obtain diagnostic test results that reliably inform
patient diagnosis.
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