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Comparison of real-time PCR and conventional hemi-nested PCR for the detection of Bordetella pertussis in nasopharyngeal samples

2003; Elsevier BV; Volume: 9; Issue: 7 Linguagem: Inglês

10.1046/j.1469-0691.2003.00601.x

1469-0691

Trevor P. Anderson, K.A. Beynon and, David R. Murdoch,

Rabies epidemiology and control

We directly compared a conventional hemi-nested PCR assay with a real-time (LightCycler) PCR assay for the detection of Bordetella pertussis in nasopharyngeal samples. Of the 152 samples tested, 49 (32%) were positive by first-round conventional PCR, 56 (37%) were positive by the hemi-nested PCR assay, and 39 (26%) were positive by the real-time assay. All samples testing positive with the real-time assay were also positive by the hemi-nested assay (both first- and second-round PCR), and all culture-positive samples tested positive by both PCR assays. These results suggest that the hemi-nested assay is more sensitive than the real-time assay for detecting B. pertussis. We directly compared a conventional hemi-nested PCR assay with a real-time (LightCycler) PCR assay for the detection of Bordetella pertussis in nasopharyngeal samples. Of the 152 samples tested, 49 (32%) were positive by first-round conventional PCR, 56 (37%) were positive by the hemi-nested PCR assay, and 39 (26%) were positive by the real-time assay. All samples testing positive with the real-time assay were also positive by the hemi-nested assay (both first- and second-round PCR), and all culture-positive samples tested positive by both PCR assays. These results suggest that the hemi-nested assay is more sensitive than the real-time assay for detecting B. pertussis. Pertussis (whooping cough) is a highly communicable, vaccine-preventable respiratory disease caused by Bordetella pertussis and, occasionally, other Bordetella species [1Yih WK Silva EA Ida J Harrington N Lett SM George H Bordetella holmesii-like organisms isolated from Massachusetts patients with pertussis-like symptoms.Emerg Infect Dis. 1999; 5: 441-443Crossref PubMed Scopus (119) Google Scholar]. Recently, PCR assays have revolutionized the laboratory diagnosis of pertussis [2Farrell DJ McKeon M Daggard G Loeffelholz MJ Thompson CJ Mukkur TKS Rapid-cycle PCR method to detect Bordetella pertussis that fulfills all consensus recommendations for use of PCR in diagnosis of pertussis.J Clin Microbiol. 2000; 38: 4499-4502PubMed Google Scholar, 3Bäckman A Johansson B Olcén P Nested PCR optimized for detection of Bordetella pertussis in clinical nasopharyngeal samples.J Clin Microbiol. 1994; 32: 2544-2548PubMed Google Scholar, 4Farrell DJ Daggard G Mukkur TKS Nested duplex PCR to detect Bordetella pertussis and Bordetella parapertussis and its application in diagnosis of pertussis in nonmetropolitan South East Queensland, Australia.J Clin Microbiol. 1999; 37: 606-610PubMed Google Scholar, 5Glare EM Paton JC Premier RR Lawrence AJ Nisbet IT Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction.J Clin Microbiol. 1990; 28: 1982-1987PubMed Google Scholar, 6Heininger U Schmidt-Schläpfer G Cherry JD Stehr K Clinical validation of a polymerase chain reaction assay for the diagnosis of pertussis by comparison with serology, culture, and symptoms during a large pertussis vaccine efficacy trial.Pediatrics. 2000; 105: e31Crossref PubMed Scopus (63) Google Scholar, 7Schläpfer G Cherry JD Heininger U et al.Polymerase chain reaction identification of Bordetella pertussis infections in vaccinees and family members in a pertussis vaccine efficacy trial in Germany.Pediatr Infect Dis J. 1995; 14: 209-214Crossref PubMed Scopus (57) Google Scholar, 8van der Zee A Agterberg C Peeters M Schellekens J Mooi FR Polymerase chain reaction assay for pertussis. simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis.J Clin Microbiol. 1993; 31: 2134-2140PubMed Google Scholar, 9Wadowsky RM Michaels RH Libert T Kingsley LA Ehrlich GD Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens.J Clin Microbiol. 1996; 34: 2645-2649PubMed Google Scholar, 10Sloan LM Hopkins MK Mitchell PS et al.Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.J Clin Microbiol. 2002; 40: 96-100Crossref PubMed Scopus (74) Google Scholar]. PCR is more sensitive than culture and direct fluorescent antibody testing, and is more rapid than other currently used methods [11Müller FM Hoppe JE Wirsing von König CH Laboratory diagnosis of pertussis: state of the art in 1997.J Clin Microbiol. 1997; 35: 2435-2443PubMed Google Scholar]. Furthermore, the development of real-time PCR assays has reduced assay times to about one hour [10Sloan LM Hopkins MK Mitchell PS et al.Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.J Clin Microbiol. 2002; 40: 96-100Crossref PubMed Scopus (74) Google Scholar, 12Ksters K Riffelmann M Wirsing von Knig CH Evaluation of a real-time PCR assay for detection of Bordetella pertussis and B. parapertussis in clinical samples.J Med Microbiol. 2001; 50: 436-440PubMed Google Scholar, 13Reischl U Lehn N Sanden GN Loeffelholz MJ Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii.J Clin Microbiol. 2001; 39: 1963-1966Crossref PubMed Scopus (130) Google Scholar]. Despite these advantages, a standardized PCR method has not been established. We are aware of only one study that directly compares different PCR methods for detecting B. pertussis, and in this study each PCR assay had a different set of primers [10Sloan LM Hopkins MK Mitchell PS et al.Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.J Clin Microbiol. 2002; 40: 96-100Crossref PubMed Scopus (74) Google Scholar]. Therefore, we directly compared a conventional hemi-nested PCR assay with a real-time PCR assay for the detection of B. pertussis in clinical samples. Both assays targeted the same region of the insertion sequence IS481 of B. pertussis. We tested nasopharyngeal swab samples from 152 consecutive patients by both a conventional hemi-nested PCR assay and a real-time PCR assay using the LightCycler system (Roche Diagnostics, Mannheim, Germany). All patients had suspected B. pertussis infection during a large outbreak of pertussis in New Zealand. Dry Dacron nasopharyngeal swabs were collected from all patients for PCR testing. Seventy-four of 152 patients also had separate nasopharyngeal swabs collected for bacterial culture. The nasopharyngeal swabs sent for bacterial culture were inoculated on charcoal media (Oxoid, Basingstoke, UK) supplemented with 10% sheep blood and cephalexin. Of the 74 swab samples, 51 nasopharyngeal swabs were inoculated on solid media immediately after collection, while 23 swab samples were sent to the laboratory in Amies charcoal transport media. Inoculated samples were incubated at 37 °C in 5% CO2 for up to 14 days, and observed daily for characteristic colonies of B. pertussis. Suspected B. pertussis colonies were verified by Gram-staining and agglutination with specific B. pertussis antiserum (Difco Laboratories, Sparks, MD, USA). Prior to PCR testing, dry nasopharyngeal swabs were placed in 1 mL of molecular-grade phosphate-buffered saline (PBS) and vortexed for 30 s. DNA was extracted using the QIAamp DNA mini kit spin column method (Qiagen, Hilden, Germany), following the manufacturer's protocol for bacteria. The conventional PCR assay was a modification of the hemi-nested PCR assay described by Lichtinghagen et al. [14Lichtinghagen R Diedrich-Glaubitz R Von HB Identification of Bordetella pertussis in nasopharyngeal swabs using the polymerase chain reaction: evaluation of detection methods.Eur J Clin Chem Clin Biochem. 1994; 32: 161-167PubMed Google Scholar]. This assay uses primers Perl (5′-GATTCAATAGGTTGTATGCATGGTT-3′) and Per3-rev (5′-GCTTCAGGCACACAAACTTGATGG-3′) to amplify a 183-bp region of the multicopy repeated insertion sequence IS481. In brief, 10 μL of the DNA eluate was added to the PCR reaction mixture to give a final reaction volume of 50 μL. The reaction mixture contained 10 × PCR buffer without MgCl2 (Roche Diagnostics), 2.0 mm MgCl2, 250 μm of each deoxynucleoside triphosphate (dNTP), 0.3 μm of each primer, and 1.25 U of Taq DNA polymerase (FastStart Roche). The reaction mixture also contained 0.1 μm each of the primers BGF (5′-GCCAGTGCCAGAAGAGCCAA-3′) and BGR (5′-TTAGGGTTGCCCATAACAGC-3′), which amplify a 500-bp region of the human β-globin gene. This gene is found in most human epithelial cells, and inclusion of these primers served as an internal control in each PCR amplification. Amplification was performed on the Mastercycler Gradient Thermocycler (Eppendorf AG, Hamburg, Germany) with the following parameters: 94 °C for 6 min, followed by 35 cycles of 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 1 min, with the last cycle concluding with 72 °C for 5 min, and 4 °C for 5 min. For the second-round (hemi-nested) PCR reaction, 1 μL of reactant mixture from the first-round PCR was added to 24 μL of PCR mix based on the same conditions as above, except with replacement of the primer Perl with Per2-rev (5′-AATTGCTGGACCATTTCGAGTCGACG-3′), and amplification for 20 cycles. The resulting 153-bp PCR product was visualized with use of 2% agarose gel electrophoresis, 0.1 mg/L ethidium bromide, and ultraviolet fluorescence. The LightCycler real-time PCR assay used was a modification of the assay described by Reischl et al. [13Reischl U Lehn N Sanden GN Loeffelholz MJ Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii.J Clin Microbiol. 2001; 39: 1963-1966Crossref PubMed Scopus (130) Google Scholar], using the same primer pair (Perl and Per3-rev) as in the hemi-nested PCR assay. In brief, 5 μL of the DNA eluate was added to 15 μL of Lightcycler-FastStart DNA Master Hybridization Probes mix (Roche Molecular Biochemical, Mannheim, Germany), including 5.0 mm MgCl2, 0.5 μm of each primer, and 0.4 μm of each of a pair of fluorescence-labeled hybridization probes, BPFLU (5′-AATCGCCAACCCCCCAGTTCACTCAFLUOR-3′) and BP-640 (5′-RED640-AGCCCGG CCGGATGAACACCC-P-3′). Amplification parameters were as follows: 95 °C for 10 min, followed by a 50-cycle amplification profile consisting of heating at 20 °C/s to 95 °C with a 10-s hold, cooling at 20 °C/s to 50 °C with a 10-s hold, and heating at 20 °C/s to 72 °C with a 20-s hold. Positive results for the LightCycler assay were determined by the characteristic fluorescence growth curve breaking higher than the background fluorescence. The parameters for all PCR assays were optimized before being used to test study samples. Analytic sensitivities were 10°CFU/mL (equivalent to one organism per reaction) for the first-round conventional PCR, 10–2CFU/mL for the hemi-nested PCR assay, and 101CFU/mL for the LightCycler PCR assay. Each extraction run contained a positive control (B. pertussis ATCC 8467), and a negative control (molecular-grade PBS). The median age of the 152 patients was 3 years (range one week to 79 years), and 87 (57%) were under the age of six years. Table 1 shows the comparative data for the different PCR assays. All discordant PCR results were confirmed by retesting, and all samples contained the human β-globin gene product, indicating that high-level PCR inhibition was not present. Given that the volume of eluted DNA was greater in the hemi-nested assay than in the real-time assay, the real-time assay reactions were repeated with an eluate volume of 10 μL. The results with this larger volume were identical to those obtained with 5 μL. All LightCycler-negative PCR products that were positive by conventional PCR were subjected to gel analysis to ensure that there was no amplified product that had not been detected by the hybridization probe detection system. No additional positive samples were detected. B. pertussis was isolated from only five of the 74 swabs sent for bacterial culture, all of which were positive by both PCR assays.Table 1Results of the B. pertussis PCR assaysReal-time (LightCycler) PCRPositiveNegativeFirst-round PCRPositive3910Negative0103Hemi-nested PCRPositive3917Negative096 Open table in a new tab Our findings show that the hemi-nested PCR assay has a higher detection rate than the LightCycler real-time PCR assay. All samples testing positive by the real-time assay also tested positive by the hemi-nested assay, but the latter assay detected an additional 11% of positive cases. Furthermore, the first-round PCR had a higher detection rate than real-time PCR, indicating that the increased sensitivity of the conventional assay is not only due to the hemi-nested step. The different detection rates may be due to differences in analytic sensitivity between the two assays. The lower limit of detection of the hemi-nested PCR assay was > 100-fold higher than that of the LightCycler PCR assay, and is similar to the analytic sensitivities reported by other investigators [5Glare EM Paton JC Premier RR Lawrence AJ Nisbet IT Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction.J Clin Microbiol. 1990; 28: 1982-1987PubMed Google Scholar, 10Sloan LM Hopkins MK Mitchell PS et al.Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.J Clin Microbiol. 2002; 40: 96-100Crossref PubMed Scopus (74) Google Scholar, 12Ksters K Riffelmann M Wirsing von Knig CH Evaluation of a real-time PCR assay for detection of Bordetella pertussis and B. parapertussis in clinical samples.J Med Microbiol. 2001; 50: 436-440PubMed Google Scholar, 14Lichtinghagen R Diedrich-Glaubitz R Von HB Identification of Bordetella pertussis in nasopharyngeal swabs using the polymerase chain reaction: evaluation of detection methods.Eur J Clin Chem Clin Biochem. 1994; 32: 161-167PubMed Google Scholar]. Unfortunately, we do not have accurate data on symptom duration or antibiotic therapy for the study patients. It is possible that the samples that tested positive by the hemi-nested assay but negative by the real-time assay were from treated patients in the later stages of the disease who had a lower bacterial burden when the samples were collected. The relatively low culture-positivity rate in this study may be due to the fact that some of the samples were inoculated on solid media after a time delay. However, both PCR assays detected all culture-positive samples and identified many additional positive samples that were culture negative, which is a consistent finding of other studies [5Glare EM Paton JC Premier RR Lawrence AJ Nisbet IT Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction.J Clin Microbiol. 1990; 28: 1982-1987PubMed Google Scholar, 10Sloan LM Hopkins MK Mitchell PS et al.Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.J Clin Microbiol. 2002; 40: 96-100Crossref PubMed Scopus (74) Google Scholar, 15van der Zee A Agterberg C Peeters M Mooi F Schellekens J A clinical validation of Bordetella pertussis and Bordetella parapertussis polymerase chain reaction: comparison with culture and serology using samples from patients with suspected whooping cough from a highly immunized population.J Infect Dis. 1996; 174: 89-96Crossref PubMed Scopus (103) Google Scholar, 16Lichtinghagen R Glaubitz R A competitive polymerase chain reaction assay for reliable identification of Bordetella pertussis in nasopharyngeal swabs.Eur J Clin Chem Clin Biochem. 1995; 33: 87-93PubMed Google Scholar, 17Schläpfer G Senn HP Berger R Just M Use of the polymerase chain reaction to detect Bordetella pertussis in patients with mild or atypical symptoms of infection.Eur J Clin Microbiol Infect Dis. 1993; 12: 459-463Crossref PubMed Scopus (38) Google Scholar, 18He Q Mertsola J Soini H Skurnik M Ruuskanen O Viljanen MK Comparison of polymerase chain reaction with culture and enzyme immunoassay for diagnosis of pertussis.J Clin Microbiol. 1993; 31: 642-645PubMed Google Scholar]. Although the interpretation of PCR-positive but culture-negative results can be difficult, we believe that such findings in this study are likely to be true positive results. All patients had symptoms suggestive of pertussis that occurred during a major pertussis epidemic. Despite the reduced sensitivity compared to the hemi-nested PCR assay, the LightCycler system offers many advantages, especially to laboratories where staff are relatively inexperienced in molecular techniques. There is a reduced risk of carry-over contamination due to the closed system, all amplified products are confirmed within the reaction capillary, and diagnostic information can be provided within one hour of sample receipt. Our findings indicate that there are differences in performance between different PCR assays, and should encourage further comparative studies to help determine the optimal PCR method for detecting B. pertussis. We thank other staff members of the Microbiology Unit, Canterbury Health Laboratories for assistance with data collection, and Howard Potter from the Molecular Pathology Laboratory, Canterbury Health Laboratories for providing primer sequences for the internal control.