This study reports on practical laboratory aspects of pertussis diagnosis. PCR assays were applied to respiratory specimens obtained during a large study of infants (less than 5 months old) admitted to paediatric intensive care units (n ¼ 122), children (less than 15 years old) admitted to paediatric wards (n ¼ 16) and their household contacts (n ¼ 320). Estimation of antibodies to pertussis toxin and culture for Bordetella pertussis were attempted on specimens from the same patients, where available, and the overall utility of the diagnostic PCR assays was assessed by comparison to these results. A PCR assay for the human mitochondrial cytochrome oxidase (HMCO) gene was used for quality control of the extracted samples and an internal process control (IPC) was included in each sample to test for PCR inhibition. Four of 458 samples were considered unsuitable (three HMCO negative, one IPC negative) and excluded from further analyses. Positive PCR results were considered valid if they were either (i) positive for both of two B. pertussis gene targets (pertussis toxin S1 promoter and the insertion element IS481), i.e. consensus PCR positive, or (ii) repeatably positive in only one assay. Using these criteria, 52 of 454 (11 . 5 %) samples were considered as PCR positive for B. pertussis. Six of 356 samples were culture-positive for B. pertussis, 1/88 infants, 3/14 children and 2/254 contacts, giving an overall isolation rate of 1 . 7 %. Using these data, PCR gave an almost fivefold increase in diagnostic yield compared with culture (McNemar's test; P , 0 . 0001). Sera from 9/111 infants, 5/10 children and 14/210 contacts were positive. Serology and PCR results showed a high level of agreement (113/121) for infants and children. PCR demonstrated a significant improvement in diagnostic yield over culture. Serological testing also resulted in a significant increase in diagnostic yield compared to culture alone. PCR is a useful technique, but validity of results must be assured by careful control. Rapid diagnosis of B. pertussis infection particularly in infants by PCR, together with serological assays, can enhance surveillance systems for pertussis in all age groups.

As part of an enhanced surveillance programme for pertussis in England and Wales, a real-time PCR service for the detection of Bordetella pertussis was introduced for infants aged ¡6 months admitted to a paediatric intensive care unit or paediatric ward with a respiratory illness compatible with pertussis. Two real-time fluorescent resonance energy transfer hybridization probe LightCycler (Roche Diagnostics) PCR assays were used. One (designed in-house) targeted the pertussis toxin S1 promoter (ptxA-pr), and included an internal process control to test for sample inhibition and reagent performance. The other (already published) targeted the insertion element IS481. The analytical sensitivities of the assays were 100 and 10 fg per reaction for the ptxA-pr and IS481 PCRs, respectively. The ptxA-pr assay was specific for B. pertussis, whilst the IS481 PCR also showed some cross-reactivity with Bordetella holmesii and the type strain of Bordetella parapertussis. From April 2002 to March 2007, 848 samples were received from 774 patients and DNA was extracted. Of 824 samples that were suitable for testing, 183 (22.2 %) had evidence of Bordetella infection (18.9 % ptxA-pr and IS481; 3.3 % IS481 only), 621 (75.4 %) were negative and 20 (2.4 %) were inhibitory for the PCR. Within the targeted age group of ¡6 months, most patients (130/138) with evidence of Bordetella spp. by PCR were ¡3 months old. The overall percentage increase in laboratory-confirmed cases due to PCR compared with culture for the 5 year period described ranged from 9 to 26 % per year (mean 19 %). Real-time PCR is an invaluable tool both for enhanced epidemiological surveillance and for the provision of a rapid diagnosis of pertussis where results can affect patient and contact management. INTRODUCTIONInfection with Bordetella pertussis (pertussis or whooping cough) causes significant morbidity and mortality and is one of the least-well-controlled vaccine-preventable diseases (Ward et al., 2005). Pertussis remains an important cause of infant death worldwide and is still of concern, even in countries with high vaccination coverage (WHO, 2005). Mild pertussis may be unrecognized in adults, and such adults may act as a source of infection for those most at risk (infants ,1 year) (Nelson, 1978;Herwaldt, 1991;Crowcroft et al., 2003;Senanayake, 2007). In England and Wales, pertussis is a notifiable disease, and the level of notifications fell from 883 in 2002 to a historic low of 409 in 2003. Since then, numbers have increased to between 500 and 600 per year in -2006 this number rose to 1089(HPA, 2008. However, the main increase in notifications has been in the 15 years and older age group, and is probably due to improved ascertainment due to greater awareness and a consequent increase in laboratory testing (Harnden et al., 2006; Litt et al., 2006;HPA, 2007). Laboratory confirmation of pertussis infection can be made by isolation of the causative organism, B. pertussis; demonstration of the presence of genomic DNA from the organism in clinical samples, ty...