A real-time PCR assay with improved specificity for detection and discrimination of all clinically relevant Bordetella species by the presence and distribution of three Insertion Sequence elements

Background

In Dutch laboratories molecular detection of B. pertussis and B. parapertussis is commonly based on insertion sequences IS 481 and IS 1001, respectively. Both IS elements are more widely spread among Bordetella species. Both Bordetella holmesii, and B. bronchiseptica can harbour IS 481. Also, IS 1001 is found among B. bronchiseptica. IS 481, and IS 1001 based PCR thus lacks specificity when used for detection of specific Bordetella spp.

Findings

We designed a PCR based on IS 1002, another IS element that is present among Bordetella species, and exploited it as a template in combination with PCR for IS 481, and IS 1001. In combining the PCRs for IS 481, IS 1001, and IS 1002, and including an inhibition control, we were able to detect and discriminate all clinically relevant Bordetella species.

Conclusions

We developed an improved PCR method for specific detection of B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica.

The genus Bordetella is comprised of 8 species, 4 of which are known to infect humans; B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica. The most important cause for whooping cough is B. pertussis, followed by B. parapertussis. Bordetella holmesii was first described in 1995 [1], and has since been isolated from patients with a serious underlying disease [2–5]. B. bronchiseptica is usually restricted to animals but occasionally is isolated from immunocompromised patients [6, 7].

A large number of PCR (polymerase chain reaction) assays have been described for detection of Bordetella pertussis and B. parapertussis, and more recently B. holmesii[8–14]. Most PCRs are based on detection of the insertion sequence elements IS 481, and IS 1001, because they exist in multiple copies in the chromosome. A high copy number of PCR target, contributes to the sensitivity of detection. IS 481 (1053 bp) shows a high degree of homology between different members of Bordetella species of 96%. All IS 1001 (1306 bp) sequences known in Genbank are 100% homologues, as is IS 1002 (1040 bp). The degree of homology between IS 481, IS 1001, and IS 1002 is less than 5%.

The insertion sequences (IS) that are present in Bordetella spp., are distributed according to species and/or host specificity. For example, IS 1001 is found in all B. parapertussis, but IS 1002 is found only in B. parapertussis that infect humans and is absent from B. parapertussis sheep isolates [15]. B. pertussis harbors both IS 481 and IS 1002 while some B. bronchiseptica strains may have either IS 481 or IS 1001 [15]. B. holmesii only has IS 481.

Due to the distribution of IS 481, and IS 1001, PCRs for B. pertussis and B. parapertussis lack specificity [16]. In recent years many newly developed PCRs were introduced with improved specificity but often with a compromise to sensitivity [11, 13, 14].

The aim of this study was to improve the specificity of PCR by including another IS element, IS 1002, as target in PCR. IS 1002, in addition to IS 481, and IS 1001, which enables discrimination of B. pertussis, B. holmesii, B. parapertussis, and B. bronchiseptica.

IS 1002 has not been exploited before as a template in PCR detection. We developed a specific IS 1002 PCR (Table 1) to improve our ability to recognize the correct Bordetella species, and to combine it with IS 481, and IS 1001 specific PCRs. Addition of Phocine Herpes Virus (PhHV) as internal control acts to monitor the extraction as well as the efficiency of amplification [17].

We investigated standard laboratory strains of Bordetella (kindly provided by Dr. Frits Mooi and Kees Heuvelman, Laboratory for Vaccine Preventable Diseases, National Institute of Public Health and the Environment, Bilthoven, The Netherlands), which are shown in Table 2. Since detection of B. pertussis is of highest concern, we investigated the performance of the newly developed PCR on 100 clinical samples that were previously positive. To verify the specificity of the PCR we investigated 20 clinical respiratory tract samples that were suspect for other pathogens than Bordetella. Prior to PCR, laboratory strains were diluted and boiled to release DNA (equivalent to approximately 5 cells/μl). Clinical samples were extracted using EasyMAG (Biomerieux, Grenoble, France).

PCRs for detection of IS 481, IS 1001, and IS 1002 were performed in a reaction mixture of 25 μl containing 0.5 μM of IS 481 and IS 1001 primers, 0.8 μM and 0.6 μM of IS 1002 Forward and Reverse primer, respectively, and 0.2 μM of PhHV primers. Probes (Table 1) were added with concentrations of 0.14, 0.14, 0.16, and 0.08 μM for respectively IS 481, IS 1001, IS 1002, and the internal control in PCR reaction mix (Sigma -Aldrich (E3004), Munich, Germany). Nine μl of template DNA was added. Amplification was carried out on an ABI 7500 Real-Time PCR system (Applied Biosystems (ABI), Nieuwerkerk a/d IJsel, The Netherlands). The temperature profile included initial denaturation of 4 min. at 94°C, followed by 50 cycles of 94°C for 15 sec., and 60°C for 1 min. Cycle treshold (Ct) values were determined automatically using the ABI SDS software.

Evaluation of sensitivity

In order to evaluate the performance of the combination of all four PCRs in a multiplex format performed on standard laboratory strains (Table 2): IS 481, IS 1001, IS 1002, and the internal control, we compared Ct values to each single PCR. No significant differences were found (not shown). Since IS 481 is present with a much higher copy number in B. pertussis than IS 1002 (approximately 200 and 10 copies, respectively), Ct values of IS 481 were on average 3.7 lower compared to Ct values of IS 1002 detection. Of a 10 CFU/ml dilution of B. pertussis cells both IS 481 and IS 1002 were positive with Ct values of respectively 30.6 and 34.4. The sensitivity of B. parapertussis detection was comparable with that using only IS 1001 as target.

Evaluation of specificity

To evaluate the specificity of each IS-based PCR, laboratory strains of most known Bordetella species (with exception of B. avium and B. trematum; Table 2), were subjected to the IS-based PCRs. Results were as was expected (Table 2).

To investigate whether all targets were amplified with high fidelity, and would not confound the specificity of detection, we made serial dilutions and subjected these to PCR. With limiting concentrations of B. pertussis bacteria, detection of IS 1002 is lost before detection of IS 481, because of the difference in copy number. To ensure specificity and to assess a cut-off Ct value for B. pertussis, 100 previously positive clinical samples were subjected to PCR. Only when Ct values of IS 481 >37, IS 1002 was negative (2% of 100 samples). Thus, a cut-off of Ct 35 should be taken for IS 481. In 20 respiratory tract samples suspect for other pathogens, no Bordetella positives were found. With B. parapertussis PCR detection, similar Ct values of IS 1001 and IS 1002 were found, and hence no cut-off Ct values are necessary for IS 1001, and IS 1002.

Quality assessment

In addition, the multiplex IS-based PCR was evaluated using the External Quality Assessment Programme of Bordetella pertussis from the Quality Control for Molecular Diagnostics (QCMD, Glasgow, UK). The QCMD panel of samples consisted of 5 B. pertussis in a range of concentrations, 1 B. parapertussis, 1 B. holmesii, 2 B. bronchiseptica, 1 B. hinzii, 1 Haemophilus influenza, and 1 negative sample. Using our newly developed PCR we correctly identified B. pertussis even in the lowest concentration of 10 CFU/ml, and B. parapertussis. B. holmesii and one IS 481 containing B. bronchiseptica were recognized correctly and not as B. pertussis. Haemophilus influenza did not cross react with any of the targeted IS elements.

In this study we included IS 1002 as target in PCR based detection of Bordetella, in addition to IS 481 and IS 1001, which is the commonly used PCR in The Netherlands. We aimed to improve the specificity of PCR because the addition of IS 1002 enables the discrimination of B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica.

In the final report of QCMD (Pierard, D., O. Soetens, and G. Ieven. Bordetella pertussis (BPDNA09) EQA Pilot Study. February 2010. QCMD, Glasgow UK.), a high degree of false positivity was observed for detection of B. pertussis. It appeared that more than 80% of laboratories contributing to the study used a PCR that is based on IS 481 and this accounts for the large proportion of false positive results. Indeed, IS 481 positive samples could either be positive for B. pertussis, B. holmesii, or B. bronchiseptica. Here, we have shown that in our PCR assay these organisms can be discriminated from one another. There are however some limitations. In a previous study [15] it was shown that IS 481 is very rarely found among B. bronchiseptica (1% of strains), in contrast to IS 1001 that was found in approximately 50% of the studied strain collection. Consequently, approximately 50% of B. bronchiseptica carry no known IS elements and thus cannot be detected by IS-based PCR. B. bronchiseptica strains that contain a copy of IS 481 cannot be discriminated from B. holmesii using IS-based PCRs. If PCR presents a single IS 481 positive signal, one might re-investigate the sample using the B. holmesii specific PCR targeting the recA gene that was described earlier [18] to discriminate from B. bronchiseptica.

The performance of our assay may fail if clinical samples might contain more than one Bordetella species. However, after more than 15 years of experience with PCR detection of B. pertussis (IS 481) and B. parapertussis (IS 1001), we did not find more than one Bordetella species present in clinical specimens.

During the evaluation, we sometimes observed a weak positive signal for IS 481 (Ct > 37) from B. hinzii, although high concentrations of this organism were negative in PCR. This may indicate that B. hinzii might contain an IS 481-like sequence that does not exactly match our PCR. As B. hinzii is solely confined to birds, this finding will not confound PCR based on IS 481, IS 1001 and IS 1002 for detection and discrimination of the clinically relevant Bordetella species.

In conclusion, we have developed a real time PCR with improved specificity for detection and discrimination of all clinically relevant Bordetella species.

Bordetella strains were kindly provided by Dr Frits Mooi and Kees Heuvelman, Laboratory for Vaccine Preventable Diseases, National Institute of Public Health and the Environment, Bilthoven, The Netherlands.

We thank Quality Control for Molecular Diagnostics, Glasgow, UK, for using their data.

Affiliations

1. Maasstad Laboratory, Molecular Diagnostics Unit, Maasstad Hospital, Olympiaweg 350, 3078HT, Rotterdam, The Netherlands

   • Lieuwe Roorda
   • , Johannes Buitenwerf
   • , Jacobus M Ossewaarde
   •  & Anneke van der Zee

2. Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands

   • Jacobus M Ossewaarde

Corresponding author

Correspondence to Anneke van der Zee.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

LR carried out the molecular genetic studies, participated in the design of PCR and drafted the manuscript. JB and JMO participated in coordination of the study and helped to draft the manuscript. AZ conceived of the study, and participated in the design. All authors read and approved the final manuscript.

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