Testing for Whooping Cough With PCR: Best Practices to Avoid False Results

Why PCR, and its pitfalls

With reported pertussis (whooping cough) cases rising since the early 1990s, health care providers are more likely to see patients who may have it. Polymerase chain reaction (PCR) detects the DNA of the Bordetella pertussis bacterium. It gives timely results and is more sensitive than culture, and unlike culture it doesn't need live bacteria. But PCR can give results that are falsely negative or falsely positive. These practices help avoid the common pitfalls.

Whom to test

Early symptoms are often non-specific, but test only patients whose signs and symptoms fit pertussis. Testing people without symptoms raises the chance of false positives: don't test close contacts of confirmed cases who have no symptoms, and don't use contacts' tests to decide on postexposure prophylaxis. (See CDC's clinical features of pertussis.)

When to test

• Best: in the first 3 weeks of cough, while bacterial DNA is still in the back of the nose and throat.
• Later: after the fourth week, DNA falls off quickly and false negatives become likelier.
• After antibiotics: testing can also give false negatives. How long results stay positive isn't well understood, but testing after 5 days of antibiotics is unlikely to help and isn't generally recommended.

How to collect the specimen

• Where: aspirate or swab the back of the nasopharynx. Swab tips can be polyester (such as Dacron), rayon or nylon-flocked.
• Best: where feasible, a nasopharyngeal aspirate — flushing with saline — which gathers more bacterial DNA than a swab.
• Don't use: throat swabs or front-of-nose swabs, which recover too little DNA, or cotton-tipped or calcium alginate swabs, whose residues block PCR.

See CDC's guidance on laboratory testing and specimen collection.

Avoiding contamination from vaccine DNA

Some pertussis vaccines contain B. pertussis DNA that PCR can detect, and it has been found on clinic surfaces. It doesn't affect the vaccines' safety or how well they work, but DNA carried from a surface into a specimen can cause a false positive. With good practice there's no need to switch to a vaccine without residual DNA:

• prepare and give vaccines in areas separate from where pertussis specimens are collected, taking care not to contaminate surfaces;
• wear gloves just before and during specimen collection, vaccine preparation and vaccine administration, and discard them right after;
• clean surfaces with a 10% bleach solution to reduce nucleic acids in the clinic.

Avoiding contamination from transport media

Liquid transport media probably also cause false positives: DNA carried from hands to the swab shaft can wash into the liquid, which circulates around the tube and is later extracted for testing. To prevent this:

• use a semisolid or non-liquid transport medium, or a dry swab with no medium;
• handle the swab stick only above the mark where the shaft is snapped off;
• consider a nasopharyngeal aspirate — the syringe or bulb kit is a closed system at the point of collection.

Reading the results

PCR tests for pertussis aren't standardized across labs: methods, DNA targets, interpretation criteria and cutoffs for a positive result all differ.

• High Ct values: a high cycle threshold (Ct) means little DNA was amplified. It may still mean infection — or contamination at collection. Labs may report it as positive, detected, indeterminate or equivocal.
• The IS481 target: most labs test for a single target, IS481, which occurs in many copies in B. pertussis and in smaller amounts in other Bordetella species such as B. holmesii and B. bronchiseptica. Its many copies make it especially prone to false positives; multiple targets may improve accuracy.

Ask your lab which target or targets it uses, and interpret results — especially those with high Ct values — alongside the patient's signs and symptoms and the available epidemiological information.

Sources

Based on "Best Practices for Use of Polymerase Chain Reaction for Diagnosing Pertussis," Centers for Disease Control and Prevention; a work of the United States government in the public domain.