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.

OBJECTIVE: To assess the diagnostic sensitivity and specificity of a Bordetella pertussis polymerase chain reaction (PCR) assay using nasopharyngeal (NP) specimens from subjects with cough illnesses participating in a large pertussis vaccine efficacy trial. DESIGN: From 1991 to 1994, we conducted a large pertussis vaccine efficacy trial in Germany to determine the efficacy of the Lederle/Takeda acellular pertussis component diphtheria-tetanus toxoids in comparison with the Lederle whole-cell component diphtheria-tetanus toxoids vaccine. In the final year of the follow-up period of this trial, a second NP specimen for PCR, in addition to a culture specimen and blood for specific serology (enzyme-linked immunosorbent assay), was collected by use of a Dacron swab in subjects or family members with cough illnesses >/=7 days duration or in subjects with exposure to a cough illness in a household member to establish a diagnosis of B pertussis infection. Oligonucleotide primers (pTp1 and pTp2) that amplify a 191-bp-sized DNA fragment from the pertussis toxin operon, which is specific for B pertussis, were used. The PCR-amplified products were visualized by dot blot analysis followed by hybridization with a digoxigenin labeled probe and rated as 1+, 2+, or 3+ in comparison with positive controls representing approximately 1 to 10, 11 to 50, and >50 B pertussis organisms, respectively. In the present analysis, we compare PCR findings with those of serology, culture, positive household contact, and clinical characteristics of cough illnesses. RESULTS: Of 392 subjects with NP specimens obtained for PCR, 376 also had NP specimens collected for culture and 282 had serum specimens. PCR and culture were positive in 86 (22%) and 23 (6%) subjects, respectively. Of the positive PCR specimens, 40 were rated 3+, 32 were rated 2+, and 14 were rated 1+; 3+ positive specimens were more prevalent among DT recipients compared with pertussis vaccine recipients. Illnesses in subjects with 3+ positive PCR results were more typical of pertussis than were those in subjects with 2+ and 1+ positive results with a mean duration of cough of 48 days versus 43 and 42 days, respectively; presence of paroxysms, whoop or vomiting in 38% versus 17% and 10%, respectively; and a clinical diagnosis of definite or probable pertussis by the investigators of 26% versus 7% and 4%, respectively. Using serologic evidence of infection as the standard, sensitivity of PCR was 61%, and specificity was 88%. For 3+ positive PCR results, the respective values were 42% and 97%. CONCLUSION: Our findings demonstrate that PCR is more sensitive than conventional culture for the diagnosis of pertussis. They also demonstrate a high specificity of PCR when serology with or without other confirmative criteria (culture and household contact) is used as the reference. Analysis of semiquantitative PCR results revealed that subjects with a 3+ PCR more frequently experienced typical illness compared with patients with 1+ or 2+ PCR. Although specific serologic study remains a necessity in pertussis research its modification for diagnosis in the clinical setting results in low sensitivity and specificity. Therefore, because PCR is more sensitive than culture and is easy to perform, it is a useful addition in the clinical setting.

Impact of polymerase chain reaction on clinical pertussis research: Finnish and Swiss experiences.

Since April 1993 in Finland and March 1994 in Switzerland, polymerase chain reaction (PCR) has been used routinely nationwide for the diagnosis of pertussis. Nasopharyngeal specimens from 3794 patients suspected of having pertussis and 1125 controls were tested. Finnish and Swiss assays found 23% and 36% of clinical specimens positive, respectively. PCR showed a higher incidence of pertussis infection among 1- to 6-year-old children in Switzerland than in Finland (P < .001). This difference may be due to the booster dose of vaccine given at 2 years of age in Finland but not in Switzerland. In Finland, PCR-confirmed asymptomatic cases were more common among children <7 years old than in older children (P < .001), whereas older children tended to have symptomatic infection. The use of PCR markedly improves the diagnosis of pertussis and opens new perspectives for epidemiologic and vaccine efficacy studies.

[Monitoring of a whooping cough epidemic 1994/95 in Switzerland using the sentinel notification system. Sentinella Registry]. / Erfassung einer Keuchhusten-epidemie 1994/95 in der Schweiz durch das Sentinella-Meldesystem. Sentinella-Arbeitsgemeinschaft.

Since June 1991 pertussis cases have been reported in the Swiss Sentinel Network (Sentinella). A total of 150-200 general practitioners, physicians specialized in internal medicine, and pediatricians participate in this system on a voluntary basis. Of the three specialties involved, this non-randomized sample represents 3.0%-3.5% of all physicians registered in Switzerland. The objective of this surveillance system is to monitor clinical pertussis over time. The case definition included all patients with a cough illness lasting at least 14 days with one of the following: paroxysms of cough, inspiratory "whoop", post-tussive vomiting (sporadic cases), or an epidemiological link to a pertussis case (epidemic cases). A laboratory diagnosis based on the polymerase chain reaction technique (PCR) was available for 82.7% of cases reported in 1994 and 1995. Of these, 27.7% had a positive PCR result. Reports of epidemic pertussis tested for Bordetella pertussis by PCR were confirmed by the laboratory in 46.5% of cases. The laboratory confirmation rate was more than twice as high among epidemic cases than among sporadic cases (20.7%). The crude incidence rate of whooping cough was 70 cases per 100,000 population per year in 1992 and 1993. Compared to previous years, pertussis incidence was significantly higher in 1994 and 1995 (370 cases per 100,000 population and 280 cases per 100,000 population respectively). The increase in reports was especially marked between July and October 1994 and whooping cough became epidemic in the third trimester of 1994 and at the beginning of 1995. In these 2 years, Switzerland experienced an estimated 40,000 clinical pertussis cases. Based on the proportion of PCR-positive pertussis cases in the sentinel sample, 12,500 of these would have been laboratory-confirmed. Most cases were observed in infants and in children up to 6 years of age. Assuming a vaccination coverage of 90%, the global efficacy of vaccination (3 or more doses versus less than 3) for 1994 and 1995 among children aged 12 to 47 months and not born before 1991 was 0.74 (0.59 and 0.88 for a vaccination coverage of 85% and 95% respectively). Vaccine efficacy was higher in PCR-positive cases (0.87; 0.79; 0.94) than in PCR-negative cases (0.54; 0.27; 0.78). Vaccination efficacy estimates on the basis of surveillance data are certainly less precise than those inferred from clinical trials. However, our results indicate that the efficacy of vaccination in children significantly declined with increasing age. Whooping cough still has the potential to cause epidemics in Switzerland in spite of a high vaccination coverage. With the introduction of acellular pertussis vaccines and new vaccination schemes in Switzerland, the Swiss Sentinel Network fulfills an important task as a monitoring system and contributes to the evaluation of new vaccination strategies.

Bordetella pertussis infections and sudden unexpected deaths in children.

UNLABELLED: From December 1990 to November 1993 nasopharyngeal specimens were obtained for culture from 50 children (mean 4.9 +/- 3.3 months of age) who had died suddenly. Bordetella pertussis was not isolated. Subsequently, nasopharyngeal specimens for polymerase chain reaction (PCR) analysis were obtained from another 51 victims of sudden death (mean 5.4 +/- 4.4 months of age); nine (18%) were B. pertussis positive. CONCLUSION: Our findings support previous epidemiological studies which noted an association between epidemic pertussis and sudden infant death syndrome. Further PCR studies with both internal and external controls should be performed.