Novel Duplex Real-Time PCR Assay Detects Bordetella holmesii in Specimens from Patients with Pertussis-Like Symptoms in Ontario, Canada

Abstract

Bordetella holmesii is a human pathogen found mainly in immunocompromised patients. A specific real-time PCR assay was developed and successfully used to identify specimens from which B. holmesii was misidentified as Bordetella pertussis and to establish the prevalence of B. holmesii in Ontario patients with pertussis-like symptoms.The Gram-negative bacterium Bordetella holmesii was first described in 1995. It is known to cause disease (e.g., septicemia) in patients with serious underlying medical conditions (2,5,7,12,14) and has also been detected in nasopharyngeal specimens from patients with a pertussis-like illness (6, 15). Two insertion sequences, IS481 and IS1001, are commonly used PCR targets for the diagnosis of Bordetella pertussis and Bordetella parapertussis, respectively (4, 11). Since both targets are also present in B. holmesii, disease due to this pathogen may be mistakenly attributed to other Bordetella species (8, 9). The aim of this study was to develop a duplex real-time PCR assay able to detect and discriminate between B. pertussis and B. holmesii. Additionally, 1,775 specimens positive for IS481 and/or IS1001 collected during 2007 and 2008 were retrospectively tested with the new assay to determine the prevalence of B. holmesii in Ontario, Canada.To date, the B. holmesii genome has not been fully sequenced, and thus, it is challenging to identify novel targets for specific PCR identification of this organism. The housekeeping gene recA has been used in previous studies for detection of B. holmesii (1, 13) and was selected as a PCR target for this study. Specific detection of B. holmesii was accomplished by designing a real-time PCR assay targeting a 50-bp segment of the recA gene (GenBank accession no. AF399664) that is polymorphic between B. holmesii, B. pertussis, B. parapertussis, Bordetella bronchiseptica, Bordetella hinzii, and Bordetella avium (Fig. 1).To confirm the conservation of B. holmesii recA (BHrecA) among clinical isolates, the full gene from 8 B. holmesii strains cultured from patients was amplified and sequenced. Sequencing revealed that BHrecA was 100% conserved in the 8 isolates tested.Analytical sensitivity of the assay was determined using a dilution series (10-fold dilutions, 5.5 ng to 5.5 ϫ 10 Ϫ10 ng) of BHrecA cloned into pCR2.1 (Invitrogen, Carlsbad, CA). Each dilution was tested in triplicate, and the experiment was performed 3 separate times to ensure accuracy of the results. The assay found a 100% probability that detection of 55 ag of cloned material (equal to 2 copies of recA) was possible at a threshold cycle (C T ) of 38.3 Ϯ 1.0. Furthermore, specificity of the real-time PCR assay was verified by testing purified genomic DNA from 32 different bacterial, fungal, and viral pathogens, including B. pertussis (n ϭ 54), B. parapertussis (n ϭ 32), B. bronchiseptica (n ϭ 1), B. hinzii (n ϭ 1), Acinetobacter sp., To develop our novel duplex real-time PCR assay for the simultaneous detection of B. pertussis and B. holmesii, the BHrecA primers and probe were combined wit...

Effectiveness of pertussis vaccination and duration of immunity

“…Evaluation of the new assay showed that less than 1% of previously tested pertussis-positive specimens were B. holmesii. 17 Polymerase chain reaction tests were considered positive for pertussis at cycle threshold values of 35 or less, and indeterminate at 36-40. Specimens with no amplification signal were considered negative.…”

“…The laboratory methods have been previously described. [15][16][17] Briefly, until May 28, 2012, primers targeting the insertion sequence IS481 were used to detect B. pertussis. Following this date, primers targeting a 50-base pair segment of the recA gene were also included to distinguish B. pertussis from Bordetella holmesii.…”

The diagnosis and epidemiology of pertussis in Australia

“…Bordetella species have limited genetic diversity, and as such, commercially used PCR assays are often unable to differentiate between Bordetella species. 13,20,179,181,183,184 Furthermore, immunisation against B. pertussis offers little protection against B. holmesii and B. parapertussis, and clinically, infection caused by these species can closely resembles B. pertussis infection. 20 Both B. holmesii and B. parapertussis have been detected in B. pertussis outbreaks and are believed to contribute to pertussis notifications.…”

“…20 Both B. holmesii and B. parapertussis have been detected in B. pertussis outbreaks and are believed to contribute to pertussis notifications. [13][14][15][16][17][18][19][20]103,183 In 2013, only 7% of Australian laboratories reported using multi-target PCR assays that could distinguish between the Bordetella species, with the remaining 93% of Australian laboratories likely to return false positive results for B. pertussis in approximately 90% of cases. 13 As PCR assays are further improved and specialised to detect other species of Bordetella, such as B. holmesii and B. parapertussis, it may be possible to quantify the contribution of these species to pertussis notifications and assessments of vaccine effectiveness.…”

“…13 As PCR assays are further improved and specialised to detect other species of Bordetella, such as B. holmesii and B. parapertussis, it may be possible to quantify the contribution of these species to pertussis notifications and assessments of vaccine effectiveness. 13,179,181,183,184 Improved understanding of the epidemiology and prevalence of other Bordetella species may have implications for Australian immunisation policy and programs, as well as surveillance systems, for example through changes to case definitions or the list of notifiable diseases.…”