Pertussis - Laboratory Testing

Suspect cases of Bordetella pertussis infection can be confirmed by culture or polymerase chain reaction (PCR). Maximum sensitivity and specificity are achieved when both tests are performed.

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Culture
Polymerase Chain Reaction (PCR)
Tests not recommended for confirming pertussis
Submitting isolates and specimens to the Minnesota Department of Health Public Health Laboratory

Culture

Isolation of B. pertussis by culture is the standard and preferred laboratory test for pertussis; however, the organism is difficult to isolate. Culture is less sensitive than PCR, but amplification methods have not been standardized and are not yet recognized as the standard-of-care. A negative culture result does not rule out pertussis infection. B. pertussis is most frequently recovered in the catarrhal or early paroxysmal stage of illness. Once cough has been present for >3 weeks, recovering the organism is unlikely. Pertussis culture and PCR testing are available through a number of laboratories including the Minnesota Department of Health Public Health Laboratory (MDH-PHL).

Specimen Collection: Perform a nasopharyngeal swab in both nares. Gently insert the swab through the nostril to the posterior nasopharynx. Leave swab in place for 15-30 seconds, rotate, and remove.

Swab should be placed into appropriate transport medium or onto agar immediately. Choose transport medium and shipping conditions based on the length of time specimen will be in transit; contact reference laboratory for specific swab and transport medium requirements; cotton swabs are not recommended for culture because cotton is harmful to B. pertussis. Regan-Lowe agar, or fresh Bordet-Gengou agar, casamino acid solution, Amies transport medium with charcoal, and Regan-Lowe transport medium are all common media for the recovery of B. pertussis. Cultures are typically incubated 10-14 days, although results are generally available in 7-10 days.

Additional Testing at MDH: All positive pertussis isolates are to be sent to the Minnesota Department of Health Public Health Laboratory (MDH-PHL) for molecular sub-typing using pulse field gel electrophoresis (PFGE) and drug susceptibility testing, according to the Disease Reporting Rules (Minnesota Rules chapter 4605) on isolate submission.

Molecular Sub-typing: PFGE sub-typing is used to characterize B. pertussis strains present in Minnesota and to determine if certain strains are more likely to be associated with severe disease or vaccine failure. Sub-typing is also used as an adjunct to epidemiology to assess possible transmission patterns.

Drug Susceptibility Testing: Drug susceptibility testing is performed to determine whether erythromycin-resistant strains are present.

Polymerase Chain Reaction (PCR)

PCR is a nucleic acid amplification test, with results typically being available in 2-3 days.

Specimen Collection: Contact reference laboratory for specific swab and transport requirements, calcium alginate swabs should not be used, as they inhibit PCR.

Tests not recommended for confirming pertussis

Direct Fluorescent Antibody (DFA) is a rapid laboratory diagnostic test that provides results within 1-2 days. Because of low sensitivity and variable specificity, DFA is not considered reliable for disease confirmation of B. pertussis.

Serology is not recommended as a diagnostic test for B. pertussis. Serology is not currently standardized in the U.S. and therefore is not considered reliable for laboratory confirmation if performed by a commercial laboratory.

Submitting isolates and specimens to the Minnesota Department of Health Public Health Laboratory

If a rapid, non-culture assay (e.g., PCR) is used for diagnosis, positives should be cultured and isolates submitted. If this is not possible, specimens may be submitted to MDH-PHL. Contact the MDH Special Microbiology Laboratory at 651-201-5073 with any questions.

Protocol for pertussis testing at MDH-PHL:

  1. Standard testing includes both CULTURE and PCR
  2. Collection of 2 separate nasopharyngeal swabs for culture and PCR are required
  3. Specimens for pertussis testing requires special transport conditions (see specifics below)
  4. PCR testing for B. pertussis by MDH-PHL is investigational only and will be reported as such
  5. PCR alone, may be ordered if patient has been symptomatic for more than 4 weeks
  6. There will be no charge to submitters as testing is part of MDH enhanced pertussis surveillance activities funded by the CDC.

CULTURE Requirements:

  1. Nasopharyngeal swabs, washings or aspirates are the specimens of choice. Use flexible Dacron®-tipped swabs; cotton and rayon may inhibit growth of B. pertussis. Throat swabs, nasal swabs, and sputum will not be tested.
  2. In most cases MDH recommends Regan Lowe Transport Medium (RLT) which is available commercially or, by request, through MDH-PHL.
  3. For best recovery of B. pertussis, the specimen should be placed in the RLT at the bedside. Swabs should be left in the transport vial.
  4. Following inoculation of swab onto RLT, incubate the RLT at 35ºC in ambient air for 48 hours.
  5. Ship the pre-incubated RLT at refrigeration temperature. For assistance with transport issues, see “Laboratory Contacts” below.
  6. Specimens may be transported in Amies medium with charcoal if they will arrive at MDH-PHL within 24 of collection.

PCR Requirements:

  1. Acceptable Specimens: Rayon or Dacron® swab either dry or in transport medium; nasopharyngeal swab, washings or aspirates are the preferred specimen. Sputum, BAL, and tracheal aspirate are also accepted.
  2. Unacceptable Specimens: Calcium alginate swab (inhibits PCR); samples received more than four days post-collection
  3. Transport at refrigerator temperature within 4 days of collection. For assistance with transport issues, see "Laboratory Contacts" below.

Transport
To request pre-paid transport labels (both mail and courier) and packaging, call the Public Health Laboratory Specimen Handling Unit at 651-201-4953.

  • If using a courier, use transport packaging appropriate for the specific courier and deliver to:

Specimen Handling
Minnesota Department of Health
Attn: Biological Accessioning
Room 151
601 Robert St. N.
PO Box 64899
St. Paul, MN 55164-0899

  • If sending an isolate or specimen by U.S. mail, use regulatory compliant transport packaging and send to:

Specimen Handling
Send specimens to:
Minnesota Department of Health
Attn: Biological Accessioning
Room 151
601 Robert St. N.
PO Box 64899
St. Paul, MN 55164-0899

 

Updated Monday, 12-Sep-2011 12:10:25 CDT