Best Practices for Use of Polymerase Chain Reaction for Diagnosing Pertussis

Overview

With the increase in reported pertussis since the early 1990s, healthcare providers will see more patients with suspected pertussis. PCR is an important tool for timely diagnosis of pertussis and is increasingly available to healthcare providers.

PCR is a molecular technique used to detect DNA sequences of the Bordetella pertussis bacterium. Unlike culture, PCR doesn't 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 healthcare providers:

• Optimize the use of PCR testing for pertussis
• Avoid more common pitfalls that lead to inaccurate results

Testing patients with signs and symptoms

Early signs and symptoms of pertussis are often non-specific, making it difficult to determine clinically who has pertussis during the early stages of disease. 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 shouldn't be tested and testing of contacts shouldn't be used for postexposure prophylaxis decisions.

Optimal timing for PCR testing

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.

PCR testing following antibiotic therapy also can result in falsely-negative findings. The exact duration of positivity following antibiotic use isn't well understood. However, PCR testing after 5 days of antibiotic use is unlikely to be of benefit and isn't generally recommended.

Optimal specimen collection for PCR testing

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 shouldn't be used for pertussis diagnosis. The swab tips may be polyester (such as Dacron®), rayon, or nylon-flocked. Cotton-tipped or calcium alginate swabs aren't acceptable as residues present in these materials inhibit PCR assays. If feasible, nasopharyngeal aspirates that flush the posterior nasopharynx with a saline wash are preferred over swabs. This method results in a larger quantity of bacterial DNA in the sample.

Avoiding contamination due to pertussis DNA

Some pertussis vaccinesA have been found to contain PCR-detectable B. pertussis DNA. Environmental sampling has identified B. pertussis DNA from these vaccines in clinic environments. The presence of this DNA in the vaccines doesn't impact the safety or immunogenicity of these vaccines. However, accidental transfer of the DNA from environmental surfaces to a clinical specimen can result in specimen contamination and falsely-positive results.

If healthcare providers adhere to good practices, there's 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. 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 the following procedures with immediate disposal of gloves afterwards:

• Specimen collection
• Vaccine preparation
• Vaccine administration

Cleaning clinic surfaces using a 10% bleach solution to reduce the amount of nucleic acids in the clinic environmentB.

Avoiding contamination due to liquid transport media

The use of liquid transport media likely also contributes to falsely-positive results from contaminant DNA. DNA that's accidentally transferred from hands to the swab shaft can be washed off into the liquid medium. This liquid freely circulates around the transport tube and is later extracted to obtain DNA for PCR testing.

Using one of the following should help prevent contaminant DNA on the swab shaft from reaching the specimen part that's later extracted:

• A semisolid or non-liquid transport media
• Dry swab without transport media

Only handle the swab stick above mark where the shaft is snapped off after insertion into the medium.

Performing nasopharyngeal aspiration rather than swabbing the nasopharynx may also prevent contamination from occurring. 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 aren't standardized across clinical laboratories, such as:

• Testing methods
• DNA targets used
• Varied result interpretation criteria
• Not using 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, and equivocal.

In addition, most clinical laboratories use a single target PCR for IS481. It's 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, IS481 is especially susceptible to falsely-positive results. Use of multiple targets may improve specificity of PCR assays for pertussis.

Healthcare providers 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
• Available epidemiological information