Stability of Bordetella pertussis and Bordetella parapertussis in the ESwab™ Transport System for Culture and PCR

Revised Abstract

Objectives

Pertussis (whooping cough) is caused by Bordetella pertussis and Bordetella parapertussis, with several states in the USA reporting an increased incidence. Laboratory detection methods include culture, direct fluorescence assay, and PCR methods; successful culture and PCR detection require proper specimen collection and transport. This study examined the effectiveness of the ESwab™ system (consisting of liquid Amies transport medium and a flocked nasopharyngeal swab, Copan Diagnostics, Inc.) for maintenance of viability of B. pertussis and B. parapertussis for culture and preservation of nuclear material for detection by PCR.

Methods

Eight Bordetella isolates were tested, including 5 B. pertussis isolates (ATCC 9340 and 8467 strains, and 3 recent clinical isolates) and 3 B. parapertussis isolates (ATCC 15237 strain and 2 recent clinical isolates). Test methods were based on CLSI M40 guidelines. Three Bordetella saline suspensions (108, 106, and 104 CFU/mL) were prepared. Each suspension was used to inoculate ESwabs™ in triplicate. The inoculated ESwabs™ were stored refrigerated (2-8oC) for up to 96 hours prior to plating. Bacteria from the ESwabs™ were cultured on Regan Lowe agar plates at 0, 24, 48, and 96 hours of refrigerated storage postinoculation; plates were then incubated at 3-7oC in ambient air for a minimum of 4 days. The numbers of colonies at 24, 48, and 96 hours were compared to the 0 hour count to determine percent recovery (viability). Real-time PCR, using primers targeting the IS481 gene of B. pertussis and the IS1001 gene of B. parapertussis, was performed on 24-hour and 96-hour inoculum in the ESwab™.

Results

Bordetella was isolated from all of the ESwabs™ after 96 hours of refrigerated storage. Percent recovery ranged from 27.2% to 96.3% for B. pertussis, and from 32.7% to 74.5% for B. parapertussis, and was similar across the three inoculum densities for all isolates tested. PCR detected B. pertussis and B. parapertussis in all of the 24- and 96-hour ESwabs™, regardless of initial inoculum concentration (104 or 106).

Conclusion

The ESwab™ maintains sufficient viability of B. pertussis and B. parapertussis to permit detection in bacteria cultures and preserves DNA integrity for PCR detection, even after 96 hours of refrigerated storage.

Introduction

Infection with Bordetella pertussis, the bacterium that causes pertussis (whooping cough), is spread by person-to-person transmission via aerosolized respiratory droplets or by direct contact with respiratory secretions. Pertussis manifests with mild upper respiratory tract symptoms that begin 7–10 days (range 6–21 days) after exposure, followed by a severe lingering cough that becomes paroxysmal and can last for weeks or even months. Coughing paroxysms vary in frequency and are often followed by vomiting. A similar, milder disease is caused by B. parapertussis.

The United States is currently experiencing a resurgence of pertussis, most notably in California where a total of 1,337 cases were reported during the period of January 1 to June 30, 2010. This represents a 418% increase from the same period in 2009. Part of this increase may reflect the natural cycle of pertussis, which exhibits incidence peaks approximately every 5 years; 2010 was at the peak of the cycle. Other contributing factors may include a lower degree of immunity from the current DTaP vaccine relative to the older DTP vaccine, waning immunity in older children and adults, and low rates of booster shots to maintain immunity.

According to current CDC guidelines, laboratory methods for confirmation of pertussis cases meeting the clinical case definition include culture and PCR. Specimens for culture and PCR are often collected from the nasophayngeal region by swab. Other swab systems may not maintain viability of Bordetella without added charcoal, and charcoal swabs may not be acceptable for PCR. This study examined the effectiveness of the ESwab™ system (consisting of liquid Amies transport medium and a flocked nasopharyngeal swab, Copan Diagnostics, Inc.) for maintenance of viability of B. pertussis and B. parapertussis for culture and preservation of nuclear material for detection by PCR.

Methods

A total of 8 strains of Bordetella were tested. Five strains (3 B. pertussis and 2 B. parapertussis) were recent clinical isolates obtained from Quest Diagnostics Nichols Institute (San Juan Capistrano, CA) or Focus Diagnostics. Three quality-control strains were tested including B. pertussis ATCC 8467 (Hardy Diagnostics), B. pertussis ATCC 9340 (Focus stock strain), and B. parapertussis ATCC 15237 (Focus stock strain).

Bacteria viability All tests for bacterial viability were modified from the quantitative elution method described in CLSI M40 A. Modifications included:

1. Inoculum suspensions of 108, 106, and 104 CFU/mL were
prepared and used for testing.

2. ESwab™ were rolled into the inoculum, allowed to absorb for
10-15 sec, and then placed into 1 mL of liquid Amies
transport medium.

3. The 108 and 106 CFU/mL ESwab™ were processed as follows:

a. The ESwab™ transport tube was considered the initial
dilution tube (total of 107 CFU/mL) and was vortexed
in the transport tube for 10-15 sec.

b. 10-fold dilutions were prepared in 0.9 mL of sterile saline
to reach a final concentration of 103 CFU/mL.

4. The 104 CFU/mL ESwab™ remained undiluted.

The average CFU for each incubation time was recorded and compared to the average CFU at 0 h. A percent viability (average CFUs at a particular incubation time compared to the CFUs at 0 h) was obtained for each isolate.

PCR Testing

Performed on all isolates at 0 hr and 96 hr with the 106 and 104 CFU/mL ESwabs™. The 108 swabs were not tested.

1. Detection of B. pertussis and B. parapertussis by real-time
PCR was accomplished using an in-house developed assay.

2. Nucleic acids were isolated from patient specimens using the
Roche MagNA Pure LC system.

3. Real-time PCR was accomplished using primers and probes
designed to hybridize to sequences found in IS481 and IS1001.