ESwab™ System for Bordetella Testing

Abstract

Background: Bordetella pertussis and Bordetella parapertussis cause pertussis. Laboratory detection methods include culture, direct fluorescence assay (DFA), and PCR. Successful detection requires proper specimen collection and transport. We examined the effectiveness of the ESwab™ system (liquid Amies transport medium and a flocked nasopharyngeal swab, Copan Diagnostics, Inc.) for maintenance of culture viability of B. pertussis and B. parapertussis, and preservation of cells for DFA and nuclear material for PCR.

Methods: B. pertussis (ATCC 9340 and 8467 and 3 clinical isolates) and B. parapertussis (ATCC 15237 strain and 2 clinical isolates) were tested. ESwab™ inoculation and culture methods were based on CLSI M40 guidelines. Three saline suspensions (108, 106, and 104 CFU/mL) were tested for each Bordetella strain or isolate. Each suspension was used to inoculate ESwabs™ in triplicate. Inoculated ESwabs™ were stored at 2-8oC before plating, DFA, and PCR testing. At 0, 24, 48, and 96 h of refrigerated storage Amies medium from the inoculated ESwabs™ was cultured onto Regan Lowe plates (Becton Dickinson, Sparks, MD), which were then incubated at 37oC in ambient air for

Results: Bordetella was isolated from all ESwabs™ after 96 h 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 for the three inoculum densities. DFA detected Bordetella in all ESwabs™ at 106 CFU/mL after 96 h. PCR detected B. pertussis and B. parapertussis in all 24- and 96-h ESwabs™, regardless of initial inoculum concentration.

Conclusions: The ESwab™ maintains sufficient B. pertussis and B. parapertussis viability to permit detection in culture and preserves cell and DNA integrity for DFA and PCR detection after 96 h 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 experienced a resurgence of pertussis in 2010, most notably in California where 9273 cases were reported; this represents the highest number of cases reported in 63 years and the highest incidence in 52 years. Part of this increase reflects the natural cyclicity of pertussis, which exhibits incidence peaks approximately every 3 to 5 years. Other contributing factors include waning immunity in older children and adults, low rates of booster shots to maintain immunity, and greater awareness among clinicians.

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 nasopharyngeal 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 cells for DFA as well as nuclear material for 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, B. pertussis ATCC 9340, and B. parapertussis ATCC 15237.

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

Inoculum suspensions of 108, 106, and 104 CFU/mL were prepared and used for testing.
ESwabs™ were rolled into the inoculum, allowed to absorb for 10-15 seconds, and then placed into 1 mL of liquid Amies transport medium.
The 108 and 106 CFU/mL ESwabs™ were processed as follows:
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 seconds.
4- or 2-serial 10-fold dilutions were prepared in 0.9 mL of sterile saline to reach a final concentration of 103 CFU/mL.
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.

DFA Testing

Four recent clinical isolates (Q1, Q2: B. pertussis and Q3, Q4: B. parapertussis) were tested using Becton Dickinson Bordetella DFA reagents according to manufacturer’s instructions.

The 24- and 96-h tubes of the 104 and 106 inoculum suspensions were used with 200

PCR Testing

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

Detection of Bordetella pertussis and B. parapertussis by realtime PCR was accomplished using an in-house developed assay.
Nucleic acids were isolated from patient specimens using the Roche MagNA Pure LC system.
Real-time PCR was accomplished using primers and probes designed to hybridize to sequences found in IS481 and IS1001.