Evaluation of four commercial real-time PCR assays for detection of Bordetella spp. in nasopharyngeal aspirates.
ABSTRACT We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the Simplexa Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved unsuitable for routine clinical use.
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ABSTRACT: Pertussis is an infectious respiratory disease caused by the fastidious bacterium Bordetella pertussis, which may infect unvaccinated, previously vaccinated children, and adults in whom immunity has waned. Infants are at particular risk for severe disease and complications. Bordetella parapertussis may cause a similar illness, however the symptoms are less severe and of shorter duration. Pertussis is a highly contagious disease and early diagnosis is essential. Studies have shown that PCR is 2-4 times more likely than culture to detect Bordetella pertussis. We developed a multiplex, real-time PCR assay using analyte-specific reagents (ASR) primers and probes dispensed in a convenient lyophilized bead format that targeted the multi-copy insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively. These specific ASRs were used in conjunction with Cepheid Smartmix. Included in the ASRs is a competitive internal control to evaluate the performance of the PCR reaction. After DNA extraction, amplification and detection were done on the Smart Cycler System, which performs integrated amplification and detection automatically in a single step. Specificity of the assay was confirmed using multiple distinct bacterial strains. Sensitivity of the assay and extraction efficiency were evaluated on DNA isolated from pure bacterial cultures and on spiked respiratory specimens. We also spiked different swab types and transport media to evaluate for interfering substances. To assess accuracy, we studied different patient specimen types received from two outside laboratories that used similar or different methods to detect Bordetella pertussis and Bordetella parapertussis. The sensitivity and the specificity of the assay for B. pertussis was 90% and 96%, respectively, and for B. parapertussis 71% (only 7 positive specimens were available for testing) and 100%, respectively. Our assay was found to be a valid method for the simultaneous detection of Bordetella pertussis and Bordetella parapertussis.Journal of microbiological methods 12/2013; · 2.43 Impact Factor
Article: How to fight pertussis?[Show abstract] [Hide abstract]
ABSTRACT: Universal pertussis vaccination has successfully decreased pertussis mortality and morbidity in childhood. However, despite intensive vaccination of young children, pertussis remains a major public health problem in both developing and industrialized regions. Recent epidemics in California and Australia demonstrated that the agent of the disease is still circulating. They also revealed several aspects that must not be neglected concerning vaccine-preventable diseases. Indeed, pertussis is one of the oldest vaccine-preventable bacterial diseases, so can provide a good illustration of all of the aspects associated with the need for surveillance after the introduction of vaccination. (i) The type of vaccine: two types of pertussis vaccine, whole cell and acellular, inducing different types of immunity are now used around the world. (ii) The vaccine strategy, the vaccine coverage and the duration of vaccine immunity: pertussis epidemics provide evidence that 90% of the infants must be vaccinated, vaccination must be sufficiently early and both vaccine-induced immunity and natural infection-induced immunity to pertussis wane with time indicating that pertussis is not only a pediatric disease. (iii) The agents of the disease, Bordetella pertussis and Bordetella parapertussis: the intensive vaccination of young infants modified the herd immunity, controlled bacteria similar to the vaccine strains but not all, revealing polymorphism of the agents of the disease evidencing the importance of continuing their isolation and their surveillance as well as monitoring their antibiotic resistance. (iv) The diagnosis of the disease: the epidemics showed the importance of specific diagnostic techniques that are easy to use by medical laboratories and the availability of the reagents required. (v) Communication with the public, the health authorities and the health providers: any changes of vaccine type, vaccine strategy, characteristics of the disease, and biological diagnosis must be associated with appropriate communication with the public and training of healthcare workers. Currently, herd immunity needs to be increased by introducing vaccine boosters for adolescents and adults to protect the most vulnerable group: unvaccinated newborns.Therapeutic advances in vaccines. 07/2013; 1(2):59-66.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2011, p. 3943–3946
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 11
Evaluation of Four Commercial Real-Time PCR Assays for Detection
of Bordetella spp. in Nasopharyngeal Aspirates?
Philippe Lanotte,1,2Chloe ´ Plouzeau,3Christophe Burucoa,3,4Carole Gre ´laud,5Sophie Guillot,6
Nicole Guiso,6and Fabien Garnier5*
CHRU de Tours, Service de Bacte ´riologie-Virologie, Ho ˆpital Bretonneau, Tours, France1; Universite ´ Franc ¸ois Rabelais, Tours,
France2; CHU de Poitiers, Laboratoire de Bacte ´riologie, Ho ˆpital La Mile ´trie, Poitiers, France3; Universite ´ de
Poitiers, Poitiers, France4; CHU de Limoges, Laboratoire de Bacte ´riologie-Virologie-Hygie `ne, Limoges,
France5; and Institut Pasteur, Centre National de Re ´fe ´rence de la Coqueluche et
Autres Bordetelloses, Unite ´ de Pre ´vention et The ´rapie Mole ´culaires des
Maladies Humaines, 25 rue du Docteur Roux, 75015 Paris, France6
Received 16 February 2011/Returned for modification 19 May 2011/Accepted 6 September 2011
We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence
detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the
Simplexa Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/
parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved
unsuitable for routine clinical use.
Nucleic acid amplification tests (NAAT), including PCR
and, more recently, real-time PCR, overcome some of the
limitations of culture and serological methods for the diagnosis
of Bordetella infection (1, 6, 12). NAAT targets include IS481
for Bordetella pertussis (this gene is also present in B. holmesii
and sometimes in B. bronchiseptica) (12, 13), the pertussis toxin
promoter or porin gene BP3385 for B. pertussis (10), and
IS1001 for B. parapertussis (5, 13). NAAT methods are highly
sensitive and may be either genus or species specific, depend-
ing on the choice of primers and targets. Real-time PCR ac-
celerates the diagnostic process by combining amplification
and detection (12, 13). These methods have proved more sen-
sitive than the equivalent gel-based system (9, 12), and many
medical laboratories have developed in-house tests. A volun-
tary external molecular quality control procedure for these
in-house methods was set up in France in 2009 by the National
Reference Centre for Whooping Cough and Other Bordetello-
ses (Institut Pasteur, Paris) (3), following a study performed in
eight hospital laboratories throughout France to assess the
performance of in-house methods and adaptations of the tech-
niques developed by Reischl et al. (11), Ko ¨sters et al. (8), Caro
et al. (3), and Templeton et al. (13). Commercial molecular
diagnostic kits are now available but have never been compar-
The aim of this study was to compare the performances of 4
commercial real-time PCR assays for the detection of B. per-
tussis, using as a reference an in-house method evaluated dur-
ing the French external molecular quality control (3).
Eighty-one nonredundant nasopharyngeal aspirates (NPA)
from patients with suspected pertussis were tested in the bac-
teriology laboratories of three French teaching hospitals
(Tours, Poitiers, and Limoges). Sixty-six samples tested posi-
tive with our in-house PCR, while 15 had tested negative but
were from patients with a suspicion of pertussis. The samples
and DNA extracts were stored at ?20°C until use. DNA was
extracted with the Invisorb spin cell minikit (Invitek, Berlin,
Germany) in Tours and with the QIAamp DNA minikit (Qia-
gen) in Poitiers and Limoges by following the manufacturers’
instructions. The principles and procedures of the two extrac-
tion methods are very similar. Tenfold serial dilutions of DNA
from B. pertussis strain Tohama, containing 238 copies of
IS481, were used to determine the analytical sensitivity of each
method. DNA extracted from each of the 81 samples was
tested with the in-house real-time PCR assay using prim-
ers Bp481F (5?-CCGAACCGGATTTGAGAAAC-3?) and
Bp481R (5?-TAGGAAGGTCAATCGGGCAT-3?), which tar-
get a 100-bp fragment of IS481. Detection was based on hy-
bridization of the Bp481S probe (5?–6-carboxyfluorescein
tramethylrhodamine [TAMRA]–3?). Primers were purchased
from Eurogentec (Seraing, Belgium) and were used at a con-
centration of 10 ?mol/liter. Amplification was performed with
a SmartCycler device (Cepheid, Maurens-Scopont, France),
with 5 ?l of DNA, 12.5 ?l of premix Ex Taq (TaKaRa, Foster
City, CA), 4.2 ?l of water, 0.4 ?l of each primer, and 2.5 ?l of
Bp481S probe (1 ?M). The thermal cycling conditions were as
follows: 1 cycle of 15 s at 95°C, followed by 45 cycles of 5 s at
95°C and 10 s at 60°C. Each laboratory tested the specimens
collected in its host hospital and used the same protocols as the
other laboratories. The SmartCycler II apparatus (Cepheid)
was used for all assays.
The four commercial PCR kits, all based on TaqMan tech-
nology, were Bordetella R-gene (Argene, Verniolle, France),
the Simplexa Bordetella pertussis/parapertussis assay (Focus
Diagnostics; distributed in France by Eurobio, Courtabeuf,
France), the Bordetella pertussis real-time PCR kit (catalog no.
RD-0061-02 [Shanghai ZJ Bio-Tech; distributed in France by
* Corresponding author. Mailing address: Laboratoire de Bacte ´ri-
ologie-Virologie-Hygie `ne, Centre Hospitalier Universitaire de Li-
moges, 2 avenue Martin Luther King, 87042 Limoges Cedex, France.
Phone: 33 5 55 05 63 48. Fax: 33 5 55 05 67 22. E-mail: fabien.garnier
?Published ahead of print on 14 September 2011.
TABLE 1. Comparison of main parameters of 4 real-time PCR kits tested
Name of kit
No. of cycles
(no. of cycles
which kit is
Yes (10 ?l IC to
add in sample
ABI Prism, Rotor-Gene
Shanghai ZJ Bio-Tech
Bordetella pertussis real-
time PCR kit
Yes (1 ?l IC to
add in mix
in mix ?20
France by Eurobio)
Yes (5 ?l IC to
add in sample
aIC, internal control.
bAs recommended by the manufacturer of the SmartCycler apparatus.
3944NOTESJ. CLIN. MICROBIOL.
BioAdvance, Bussy-Saint-Martin, France]), and the SmartCy-
cler Bordetella pertussis/parapertussis assay (Cepheid). The dif-
ferent kits were used according to the recommendations of the
manufacturers, including cycle thresholds (CT) of 7 for the
Shanghai ZJ Bio-Tech kit, 15 and 30 for the Focus Diagnostics
kit, and 30 for the other two methods. Differences between the
methods are shown in Table 1.
The analytical sensitivity of each method, tested in duplicate,
is shown in Fig. 1. The CTvalues were similar for the different
methods with up to 104copies of IS481 per reaction, except
that the CTof the Shanghai ZJ Bio-Tech test was about 7
cycles above that of the other methods whatever the number of
copies. Only the in-house method and the Argene, Cepheid,
and Focus Diagnostics kits were positive with the dilution
containing 100 copies of IS481 per reaction.
Each clinical DNA specimen was tested with the in-house
method and with the 4 commercial kits. All the DNA speci-
mens were thawed and tested on the same day (no refreezing
occurred between the different assays). The results are shown
in Table 2. No false positives were noted for any assay. With
the in-house method as the gold standard, clinical sensitivity
was 97% with the Focus Diagnostics kit, 93.9% with the Ceph-
eid kit, 90.9% with the Argene kit, and 51.5% with the Shang-
hai ZJ Bio-Tech kit. The Focus Diagnostics kit had the lowest
mean CT(Table 2), which was not significantly different from
that of the in-house assay. The Argene and Cepheid kits had
slightly but not significantly higher mean CTvalues than the
in-house assay. In contrast, the Shangai ZJ Bio-Tech kit had
significantly higher mean CTvalues than the in-house assay. It
is noteworthy that the manufacturer of the Shanghai ZJ Bio-
Tech kit recommends using only half the amount of DNA used
in the other kits. The performance of the Focus Diagnostics kit
was unaffected when a CTof 15 rather than 30 was used (data
Internal controls are included to detect PCR inhibition. Be-
ta-globin was used for this purpose in the in-house method in
a separate vial. The Argene and Focus Diagnostics kits also
include extraction controls (Table 1). The list of apparatus on
which kits can be used is shown in Table 1. Two kits (the
Cepheid and Focus Diagnostics assays) are claimed to detect
B. pertussis and B. parapertussis in the same tube, while the
other two kits and the in-house method target only IS481. B.
holmesii, which also possesses IS481, cannot be distinguished
from B. pertussis with these kits. This species rarely induces
FIG. 1. Representation of the crossing threshold obtained for each commercial kit and the in-house real-time PCR for the detection of the
Bordetella IS481 sequence.
TABLE 2. Clinical sensitivities and mean CTvalues for real-time PCR kitsa
No. of positive specimens
identified/total no. of
Mean CT? SD
Focus Diagnostics kit
Shanghai ZJ Bio-Tech kit
25.5 ? 8.6
27.1 ? 9.5
25.8 ? 9.2
24.8 ? 8.8b
37.3 ? 8c
aData for the four real-time PCR kits tested are in comparison to results of the in-house real-time PCR assay for detection of the Bordetella IS481 sequence in 66
bAs recommended by the manufacturer, the CTretained for the comparison was 15.
cAs recommended by the manufacturer, the CTretained for the comparison was 7.
dDetermined by paired t test (2). NS, not significant.
VOL. 49, 2011NOTES 3945
pertussis-like symptoms and can be identified by real-time
PCR-based on the recA gene (7).
In conclusion, all four kits tested here were highly specific,
whereas their sensitivities were highly variable. The Argene,
Focus Diagnostics, and Cepheid kits performed as well as the
in-house method in terms of both analytical and clinical sen-
sitivities. These three kits include an internal control to detect
inhibitors, which is important in routine practice (4), but only
two of them, the Argene and Focus Diagnostics tests, validate
the extraction step. The Shanghai ZJ Bio-Tech kit needs to be
improved if it is to be used in routine clinical settings.
We thank Marie Odile Viaud, Lucie Yzon, and Catherine Louin for
their technical assistance.
The different kits were supplied free of charge by the different
corporations, which provided no other financial support for this study.
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