CDC Expert Commentary

Pertussis Diagnosis: Avoid the Pitfalls of PCR

Sema Mandal, MBBS, MPH

Disclosures

February 28, 2011

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Hi, I'm Dr. Sema Mandal. Thanks for tuning into this CDC Expert Video Commentary on Medscape.

Polymerase chain reaction (PCR) is an important tool for diagnosing pertussis. PCR is a rapid test that detects DNA sequences of the Bordetella pertussis bacterium, and unlike culture, does not require viable bacteria. Despite these advantages, PCR can give inaccurate results. Many common pitfalls of PCR can be avoided by following best practices for who to test, when to test, collection techniques, and preventing contamination.

First, who should be tested? Because early signs and symptoms are often nonspecific, pertussis can be difficult to diagnose. Only patients with signs and symptoms consistent with pertussis should be tested by PCR to confirm diagnosis. Testing asymptomatic persons should be avoided, as it increases the likelihood of obtaining falsely-positive results.

When it comes to timing, 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 DNA rapidly diminishes, which increases the risk of falsely-negative results. For more information on diagnostic testing for pertussis, including the use of serology for late diagnosis, please see the resources listed at the end of this transcript.

PCR testing following antibiotic use can also result in falsely-negative findings. After 5 days of antibiotic use, PCR testing is unlikely to be of benefit.

Proper specimen collection is critical. Specimens for PCR testing should be obtained from the posterior nasopharynx by aspiration or swabbing. Throat swabs and anterior nasal swabs are not recommended because they have unacceptably low rates of DNA recovery. Swab tips should be polyester, such as Dacron®, rayon, or nylon-flocked. Cotton-tipped or calcium alginate swabs are not acceptable, because the 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. Aspiration results in a larger quantity of bacterial DNA in the sample.

You can take steps to avoid contaminating the specimen. Some pertussis vaccines have been found to contain PCR-detectable Bordetella pertussis DNA.(Vaccines shown to contain PCR-detectable DNA include Pentacel®, Daptacel®, and Adacel®.[1]) Environmental sampling has identified this DNA from these vaccines in clinic environments. Although the presence of this DNA in vaccines does not affect safety or immunogenicity, accidental transfer from environmental surfaces to a clinical specimen can result in contamination. If healthcare professionals adhere to good practices, there is no need to switch vaccines. To avoid specimen contamination, CDC has 3 recommendations:

  1. Separate the area where vaccines are prepared and administered from the area where pertussis specimens are collected so that the opportunity for cross-contamination is reduced.

  2. Encourage general adherence to basic infection-control measures. This includes wearing gloves immediately before and during specimen collection, as well as during vaccine preparation and administration, and disposing of gloves immediately after the procedure.

  3. Clean clinic surfaces with a 10% bleach solution to reduce the amount of DNA. (A 10% solution corresponds to 1 and a half cups of household bleach per gallon of water, or 1 part bleach to 9 parts water).

The use of liquid transport media likely also contributes to falsely-positive results. Environmental 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. DNA is later extracted from this liquid.

There are 2 measures you can institute to prevent contaminant DNA on the swab shaft from reaching the usable part of the specimen. First, use a semisolid or nonliquid transport medium or transport a dry swab without media. Second, 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 may also prevent contamination from occurring because the aspirate kit is a closed system.

Finally, pertussis PCR methods vary from lab to lab. With PCR, high cycle threshold (Ct) values indicate low levels of DNA; for pertussis, these values may still indicate infection but can also be the result of DNA-contaminated specimens. Different labs may report high Ct values as any of the following: positive, detected, indeterminate, or equivocal. Most clinical laboratories use a single target PCR for insertion sequence 481, which is present in multiple copies in Bordetella pertussis and in lesser quantities in Bordetella holmesii and Bordetella bronchiseptica. Use of multiple targets may improve specificity. Clinicians are encouraged to know which PCR assays 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 epidemiologic information.

In summary, PCR is an important tool for diagnosis of pertussis, especially in the setting of the current resurgence of pertussis disease. Careful specimen collection and transport and a general understanding of the PCR assays will better ensure that clinicians obtain test results that reliably inform patient diagnosis. Please visit www.cdc.gov/pertussis for more information and resources. Thank you very much.

Web Resources

CDC Best Practices for Health Care Professionals on the use of Polymerase Chain Reaction (PCR) for Diagnosing Pertussis

CDC: Pertussis

CDC Pertussis Vaccines - Recommendations and Licensures

CDC Specimen Collection and Diagnosis Confirmation (videos)

Suggested Reading

Clark TA. CDC commentary: pertussis - recognition and treatment. Public Information from the CDC and Medscape. © 2010. Available at: http://www.medscape.com/viewarticle/713708 Accessed February 10, 2011.

Martin SW. CDC commentary: pertussis - with pertussis on the rise, who needs a Tdap vaccination? Public Information from the CDC and Medscape. © 2010. Available at: http://www.medscape.com/viewarticle/724242 Accessed February 10, 2011.

Tatti K, Slade B, Patel M, et al. Real-time polymerase chain reaction detection of Bordetella pertussis DNA in acellular pertussis vaccines. Pediatr Infect Dis J. 2008;27:73-74.
http://www.ncbi.nlm.nih.gov/pubmed/18162945?dopt=AbstractPlus

Sema Mandal, MBBS, MPH, is a medical epidemiologist with the Meningitis and Vaccine Preventable Diseases Branch at the US Centers for Disease Control and Prevention. She works on pertussis and meningitis in the United States and abroad and has participated in several pertussis outbreak investigations.

Prior to joining CDC in 2009, Sema was based in London, UK, working in public health and communicable disease control for the UK National Health Service and Health Protection Agency. Sema also previously worked for Doctors Without Borders in Sudan.

After graduating in medicine from Cambridge University and University College London Medical School in 1998, Sema undertook dual residencies in internal medicine and public health, and gained a Masters in Public Health from London School of Hygiene and Tropical Medicine in 2006.

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