Laboratory diagnosis of pertussis: Agglutination is not suitable (2011)

Pertussis, commonly known as whooping cough, is a highly contagious respiratory infection that is apparently on the rise around the globe. Recently, a large epidemic caused the death of 10 infants in California, USA.1 An outbreak is ongoing in New South Wales, Australia,2 and the Netherlands and Norway have both seen tremendous increases in pertussis cases over the last 15 years.3, 4 Pertussis is often perceived as a childhood disease, and many believe that vaccination will provide lifelong immunity. The truth is, however, that vaccination provides protection for just 4–12 years,5 and many countries have reported large increases in the incidence of adult pertussis.6-8 In this issue of Respirology, a study by Miyashita et al. deals with the laboratory diagnosis of adolescent and adult pertussis in Japan.9 The clinical picture of pertussis among adults and adolescents is often milder than the textbook example of frequent paroxysmal coughing fits, whooping and vomiting. A study of 3255 adolescents and adults with confirmed pertussis has shown that just 31% of the patients experienced the ‘whoop’, whereas 44% experienced post-tussive vomiting.10 Although adult pertussis can be very painful and exhausting, it is usually not dangerous in itself. However, as pertussis is highly contagious,11 it is important to diagnose the patient correctly in order to prevent transmission to young infants, as they are at risk of severe complications or even a fatal outcome from the disease.12 Around the world, laboratory diagnosis is mostly carried out by culture, PCR or ELISA.13 However, the paper by Miyashita et al. describes a diagnostic method frequently used in Japan: the bacterial agglutination (BA) assay.9 This assay is fairly easy to carry out and is cheap in comparison with PCR. Miyashita et al. evaluated two different BA tests for diagnosis of adult and adolescent pertussis and conclude that neither of these tests is sufficiently sensitive for use as a reliable diagnostic test.9 The Yamaguchi BA test showed a specificity of 98.1% with a sensitivity of 58.3%, whereas the Tohama BA test showed a specificity of 95.9% with a sensitivity of just 38.8%, using convalescent sera obtained 4–5 weeks after the onset of cough. In contrast, an anti-pertussis toxin (anti-PT) ELISA showed a specificity of 95.9% with a sensitivity of 80.5%, using the same sera. This is not the first time the BA test has shown unsatisfactory results, as studies from 1983 and 1987 showed sensitivities of 18% and 22% with sera from culture-confirmed cases,14, 15 and studies from 2010 and 2011 showed that 30% or 32% of sera from healthy individuals gave positive results in the test.16, 17 As early as 1973, it was known that most of the population will show weak positive results in the BA test,18 making the choice of cut-off crucial. The choice of agglutinating Bordetella pertussis preparation will also have a great impact, as different strains will give widely different results.19 Although the BA test used in recent studies has probably been refined since the early studies, it is still unlikely to provide satisfactory results for the diagnosis of pertussis. Excluding the BA test, the remaining methods for routine laboratory diagnosis of pertussis are culture, PCR and ELISA. Which one of these tests is then the best choice? The answer is that all three methods are useful but should be used for different purposes. Culture is still the gold standard,13 proving beyond doubt that the causative bacterium is present. Furthermore, culturing provides isolates for typing, and molecular typing of clinical B. pertussis isolates is crucial for surveillance of the distribution and evolution of these bacteria.20 Culture is, however, time-consuming compared with PCR, which has also shown significantly higher sensitivity,21 although primers must be carefully designed so as to pinpoint B. pertussis and not other Bordetella species. Recent reports suggest that triplex PCR may be the best option.22, 23 The third method, ELISA, has generated great interest in the last three decades, in particular, because ELISA is superior to both culture and PCR, for diagnosis of adult pertussis.24, 25 Testing for the presence of IgG antibodies to pertussis toxin (IgG-PT) is the recommended ELISA,26 as this shows both high specificity as well as sensitivity.27-29 Other antigens such as filamentous haemagglutinin and pertactin have also been used for diagnostic pertussis ELISA, but these antigens are not as specific as PT.26 Commercial ELISA kits are available but should be chosen with caution.30, 31 Residual antibodies from pertussis vaccination can produce false-positive results in the ELISA, and serological analysis is therefore not recommended for diagnosis of recently vaccinated patients.32, 33 When choosing between the three diagnostic methods, it is important to bear in mind that culture and PCR are only useful in the early stages of the disease,34 whereas ELISA is reliable from 2 to 3 weeks after the onset of symptoms.21 Adults tend to endure a few weeks of symptoms before seeking medical advice, and ELISA is therefore particularly suitable for diagnosis of pertussis in adults. Furthermore, both PCR and culture tend to have lower sensitivity in adults than children.35 Recently, it was reported that a combination of PCR and ELISA may provide both high sensitivity and high specificity for laboratory diagnosis of pertussis.36 Greater awareness of pertussis and the introduction of PCR and ELISA methods are major factors contributing to the increased reporting of pertussis cases in many countries. Thus, the incidence of pertussis in Norway and Estonia in 2009 was 114.3 and 46.9 per 100 000, respectively, whereas the incidence in the respective neighbouring countries, Sweden and Latvia, was just 3.0 and 0.4 per 100 000.37 Seroprevalence studies from around the world also support the theory that there are significantly more cases of pertussis among the (adult) population than what are registered.38-40 Correct laboratory diagnosis of pertussis is important, and methods that have high sensitivity and specificity should be used. Another consideration is, however, that the chosen methods should be feasible and affordable for regional laboratories. Although the BA test is both feasible and affordable and has probably contributed to the identification of a large number of cases in Japan, Miyashita et al. have shown that this test is not suitable for the diagnosis of adolescent and adult pertussis.9 Tine Dalby and Karen A. Krogfelt are members of the EU Pertstrain group26 and appreciate the fruitful discussions and exchange of knowledge within this group.