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J Clin Microbiol. May 2002; 40(5): 1719–1722.
PMCID: PMC130942

Real-Time LightCycler PCR for Detection and Discrimination of Bordetella pertussis and Bordetella parapertussis

Abstract

Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of Bordetella pertussis and Bordetella parapertussis in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for B. pertussis and B. parapertussis, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for B. pertussis and/or B. parapertussis. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (P = 0.08) to 2.3-fold (simplex PCR) (P = 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.

Despite vaccination pertussis remains endemic in most areas of the world (9, 10, 27, 28). Reliable diagnosis is required to start appropriate treatment and prophylaxis of contacts if needed. In many cases the clinical symptoms are typical, but in particular neonates and adults who might be the main source of infection for children often show an atypical course of disease which needs confirmation by laboratory methods. Rapid diagnosis is crucial to eradicating the source of infection and treating contacts early, particularly nonvaccinated infants in whom pertussis might present as a life-threatening disease (1).

Culture is still the “gold standard” due to its high specificity (100%), but its sensitivity varies between 6 and 95% (18, 22) depending on the time of sampling. Results are available only after 3 to 12 days (11). Serology can be sensitive and specific, but mostly paired serum specimens have to be analyzed. Single serum serology with age-specific antibody reference values is still not widely used (17, 21, 30). Moreover, some commercial enzyme-linked immunosorbent assay kits are not reliable and do not distinguish between Bordetella pertussis and Bordetella parapertussis infection (12).

To overcome these problems, PCR is being used increasingly for detection of B. pertussis and B. parapertussis DNA (5, 6, 10). Various PCR protocols have been developed that target different regions of the genome, e.g., insertion sequences IS481 and IS1001 (3, 4, 7, 16, 19), the pertussis toxin promoter region (7, 8, 19, 25), the adenylate cyclase gene (2), and the porin gene (5, 14). In a comparison of different PCR assays used in seven pertussis vaccine studies all assays increased sensitivity for detection of pertussis cases by at least 70% compared with culture, and false-positive results were less than 1% of the total (26).

Recently, PCR formats based on real-time detection of the amplification product have been developed. The TaqMan assay has been evaluated elsewhere for the individual diagnosis of B. pertussis and B. parapertussis (13). The LightCycler technology (31), which also allows for real-time detection of the amplicon, adds additional speed to the detection of PCR products. It employs two hybridization probes which allow for sequence-specific detection by using fluorescence energy transfer between the fluorophores conjugated to the probes. In the present study we present a comparison of two real-time PCR methods for the detection and discrimination of B. pertussis and B. parapertussis DNA.

MATERIALS AND METHODS

Bacterial isolates and patient specimens.

For optimization of the PCR protocol, fresh isolates of B. pertussis and B. parapertussis from modified Regan-Lowe medium (Oxoid, Wesel, Germany) were suspended in phosphate-buffered saline (PBS) to an optical density of 0.5 at 600 nm, corresponding to approximately 1.0 × 109 CFU/ml for B. pertussis and B. parapertussis (29), and diluted in 10-fold steps. For preliminary comparison of LightCycler PCR with culture, spiked swabs were produced by immersing Dacron swabs (Hain Diagnostika, Nehren, Germany) into the different dilutions for 10 s. Spiked swabs were held in Amies medium with charcoal (Hain Diagnostika) at room temperature for 48 h to simulate transportation.

As a control for specificity, DNA extracted from Bordetella holmesii, ATCC 51541 was used as well as isolates of Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella avium, Haemophilus influenzae, beta-hemolytic streptococci, Corynebacterium spp., Staphylococcus epidermidis, Neisseria spp., Staphylococcus aureus, Enterococcus spp., Streptococcus pneumoniae, Moraxella catarrhalis, and viridans streptococci, which were suspended in PBS to an optical density of 1.0 at 600 nm.

Nasopharyngeal swabs were obtained from 100 inpatients and outpatients (53 female and 47 male) with symptoms of pertussis, defined according to the Centers for Disease Control and Prevention clinical case definition as a cough illness lasting at least 2 weeks with paroxysms of coughing, inspiratory whoop, or posttussive vomiting. The patients were aged 1 month to 39 years with a mean of 61 months and a median of 36 months. Thirteen swabs were obtained from 13 patients (6 female and 7 male; aged 1 week to 44 years; mean, 11 years; median, 1 week) without symptoms of pertussis. All swabs were immersed into Amies medium with charcoal during transport.

All specimens (spiked swabs and clinical samples) were plated onto Regan-Lowe medium, which was incubated at 37°C for 7 days. Swabs were then immersed in 150 μl of PBS, swirled vigorously, and wrung out to elute any bacteria. If necessary, samples were stored at −20°C.

LightCycler PCR.

DNA extraction of bacterial suspensions was done with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) after proteinase K treatment according to the manufacturer's protocol. Basic reagents for the LightCycler PCR were purchased from Roche Diagnostics, Mannheim, Germany.

Oligonucleotides (TibMolBiol, Berlin, Germany) from IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, were adopted from the work of Reischl et al. (23) (Table (Table1).1). The 20-μl reaction mixture volume in a glass capillary tube contained 4 mM MgCl2, 2 μl of FastStart Reaction Mix Hybridization Probes (component of the FastStart DNA Master Hybridization Probes kit; Roche Diagnostics), 2 μl of DNA extract, and primers and probes depending on the PCR format. For the simplex format 0.5 μM (each) primers BP-1 and BP-2 or BPa-1 and BPa-2 and 0.2 μM (each) matching probes were used; for the duplex format 0.5 μM concentrations (each) of the four primers; 0.2 μM (each) probes BP FLU, BP LCR, and BPa-HP-1; and 0.4 μM probe BPa-HP-2 were used.

TABLE 1.
Primers and probes for LightCycler PCR (simplex and duplex)

Reaction conditions for the three assays were chosen according to a standard LightCycler protocol in our laboratories and were 10 min at 95°C, followed by 40 cycles of 10 s at 95°C, 10 s at 65°C, and 20 s at 72°C. Fluorescence increase, i.e., creation of specific product, was measured during the annealing step at 65°C. A melting curve analysis was performed after the last amplification cycle with 95°C for 0 s, 45°C for 30 s, and 95°C for 0 s. Temperature change rates were 20°C/s for all but the last step, where the rate was 0.1°C/s. Readout of LC-Red 640 values (B. pertussis) was performed in channel F2/Back-F1, and readout of LC-Red 705 values (B. parapertussis) was performed in channel F3/Back-F2.

A sample was regarded as positive when the LightCycler software version 3.5 determined a crossing point in the quantification analysis screen and the PCR product produced a characteristic melting curve with a discernible peak at 63°C for B. pertussis and at 69°C for B. parapertussis in the melting curve screen.

TaqMan PCR was performed as described elsewhere (13).

Statistical analysis.

Differences in positivity rates were tested with the z test for significance (Sigma-Stat; SAS Software, Jandel Scientific, Erkrath, Germany).

RESULTS

PCR conditions were amended from the protocol described before (23) by adjusting MgCl2, primer, and probe concentrations.

Based on extraction and amplification of 1 ml of bacterial suspension, the sensitivity of the LightCycler duplex and simplex assay was 1 to 10 CFU per reaction for B. pertussis and 0.1 to 1 CFU per reaction for B. parapertussis. The TaqMan assay was 1 log dilution step more sensitive than were both LightCycler assays for detection of B. pertussis and B. parapertussis DNA from bacterial suspensions. When spiked swabs were analyzed, the three PCR assays performed similarly. Table Table22 shows that PCRs were generally more sensitive than, but at least as sensitive as, culture. Each PCR assay showed a wide dynamic range over all positive dilution steps starting from 109 CFU/ml.

TABLE 2.
Comparison of analytical sensitivities of culture, TaqMan PCR, and LightCycler PCR in detection of B. pertussis and B. parapertussis

All primer-probe combinations were able to amplify and detect B. parapertussis DNA specifically. As described before (15, 16, 24), positive results for IS481 were obtained with B. pertussis and with B. holmesii ATCC 51541. None of the other bacteria often found in upper respiratory tract specimens tested positive with either PCR format, nor did the other Bordetella spp.

Intra-assay variation for the LightCycler assay based on 10-fold analysis of two different dilutions was found to be between 1.3 and 2.3% for the B. pertussis simplex assay and between 0.6 and 1.6% for the B. pertussis duplex assay. Corresponding values for B. parapertussis were 1.5 to 3.5% and 1.2 to 1.7%, respectively. Interassay variation of one sample assayed on 10 consecutive days was 6.0% for the B. pertussis simplex assay and 5.0% for the duplex assay. Corresponding figures for B. parapertussis were 4.3 and 4.5%, respectively.

Of 100 specimens from patients with clinical symptoms of pertussis, 9 samples were culture positive (4 for B. pertussis, 4 for B. parapertussis, and 1 for both). Table Table33 shows that with both simplex PCRs (LightCycler and TaqMan) 15 specimens were positive for B. pertussis, 4 were positive for B. parapertussis, and 2 were positive for both. The LightCycler duplex assay missed two samples positive for B. pertussis, and one coinfection was positive for B. pertussis only. All three PCR assays identified all culture-positive cases correctly. Compared to culture, the LightCycler simplex PCR and the TaqMan PCR increased the positivity rate 2.3-fold (P = 0.02) whereas the LightCycler duplex assay increased it 2.0-fold (P = 0.08). Differences between the duplex and simplex PCR formats did not reach significance.

TABLE 3.
Numbers of positive and negative samples (total number of samples from patients with symptoms of pertussis [n] = 100)

None of the 13 samples from patients without symptoms of pertussis gave positive signals with either PCR format or culture.

DISCUSSION

In accordance with recently published data (13, 23), we primarily showed that no organism tested except B. pertussis, B. parapertussis, and B. holmesii, which shares the IS481 sequence with B. pertussis (24), was positive with the amended LightCycler protocol.

Similar to other Bordetella PCRs (5, 7), the LightCycler simplex and duplex PCRs achieved an analytical sensitivity of 0.1 to 10 CFU per reaction for the detection of B. pertussis and B. parapertussis from bacterial suspensions. With spiked swabs PCR increased sensitivity by 1 dilution step compared to culture, although the sampling technique even favored culture: swabs were first rolled intensively onto the culture medium before the remaining bacteria were eluted for PCR.

When the same sampling procedure was applied to clinical specimens, both LightCycler assays increased the number of swabs identified as positive for B. pertussis and/or B. parapertussis at least twofold. While none of the samples from patients without symptoms of pertussis gave a positive signal with either PCR format, all PCR-positive specimens were taken from patients with clinical symptoms matching the Centers for Disease Control and Prevention criteria for pertussis. This implies that PCR-positive, culture-negative samples most likely represent true positives and that PCR demonstrated increased sensitivity over the gold standard of culture.

Compared to culture, PCR did better with clinical samples than with spiked swabs. Possibly the number of bacteria in clinical specimens was on average low and close to the detection limit of culture. Clinical samples may also have contained nonviable bacteria, whereas spiked swabs were prepared from fresh suspensions of viable bordetellae. Thus, when PCR is used as the only means of diagnosing pertussis from nasopharyngeal swabs, the increase of positive samples might even be higher because bacteria can be eluted directly without prior plating onto culture medium.

The clinical significance of detecting B. holmesii is not yet fully understood because, so far, B. holmesii has been isolated only infrequently from nasopharyngeal specimens (20, 32)

A comparison of the real-time PCR systems shows that the TaqMan assay and the LightCycler simplex assay were able to detect the same number of positive specimens whereas the LightCycler duplex assay detected fewer positives (P = not significant).

Real-time PCR systems are well suited for the detection of B. pertussis and B. parapertussis from clinical specimens. In contrast to conventional PCR methods they are less prone to carryover contamination because postamplification handling is eliminated. They achieve high specificity and sensitivity by adding specific probes. Product formation can be monitored in real time, making quantification of bacteria possible. The standard LightCycler protocol as described above can complete an analysis of up to 30 samples plus negative and positive controls within a total assay time of 55 min. Although the duplex PCR assay is a very convenient method, allowing for simultaneous detection and discrimination of B. pertussis and B. parapertussis, it has to be kept in mind that it might lead to a clinically relevant loss of sensitivity. Therefore, we suggest using the simplex format for clinical specimens until the duplex format has been shown to be at least as sensitive.

Thus, real-time LightCycler simplex PCR offers a fast tool with high sensitivity and specificity for the diagnosis of B. pertussis and B. parapertussis infections suitable for implementation in a routine diagnostic laboratory.

Acknowledgments

This study was supported by Bundesministerium für Bildung und Forschung (PID-ARI-Net).

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