Pertussis continues to present a global public health problem, even in developed countries with good primary vaccination coverage.1 Current evidence confirms that Bordetella pertussis infection still causes fatal illnesses among vulnerable neonates and incompletely immunized infants.2–8 A number of countries have noted an increase in reported pertussis during the past decade.6,9–14 Associated with the increased reporting of pertussis has been a change in the reported frequency by age group. A greater percentage of cases is now noted in adolescents and adults,6,9,15–18 and they represent an underrecognized but major source of infection for neonates and infants.9,16,19–22
Several factors complicate assessment of the true incidence, transmission, and burden of pertussis disease. Underconsulting by adolescents and adults with chronic cough is common,23 yet pertussis is an important cause of prolonged cough in adolescents and adults, including the elderly.15,19,24–29 Underrecognition and underreporting of pertussis are also widespread and are a particular problem for adolescents and adults.30–35 Even when symptoms are typical, pertussis may not be diagnosed36,37 because of the misconception among many physicians that pertussis is a childhood disease.38 Poor surveillance systems, inconsistent case definitions and insensitive, nonstandardized, poorly performed, or a lack of available, laboratory diagnostic techniques are also major contributing factors to underdiagnosis.20,39,40
In 2001, the Global Pertussis Initiative was established as a forum to analyze the status of pertussis disease globally and to evaluate various immunization strategies to improve disease control. The Global Pertussis Initiative recognized that a key part of achieving these objectives would be to review currently used case definitions and laboratory diagnostic techniques. This article examines problems that currently exist in defining pertussis epidemiology from clinical, microbiologic and serologic perspectives.
DIFFICULTIES IN DEFINING PERTUSSIS FROM A CLINICAL PERSPECTIVE
The clinical diagnosis of pertussis is complicated by the wide heterogeneity in disease expression, modification of disease by immunization, mixed infections and a low index of suspicion among many physicians.
Heterogeneous Clinical Spectrum of Pertussis
B. pertussis infections have a wide spectrum of clinical expression and are affected by patient age, previous exposure to the organism (either by vaccination or prior infection), antibiotic administration and the degree of exposure, concomitant infections with other agents and the presence of cross-reacting antibodies.16B. pertussis infection can range from asymptomatic infection in children and adults with strong residual immunity20,32,41 to more severe and life-threatening disease in unprotected newborns and young infants.42
Typical (Classic) Pertussis.
The classic form of pediatric pertussis in the prevaccine era was described as the presence of a paroxysmal cough, posttussive vomiting, inspiratory whoop and duration of cough lasting >28 days and up to 3 months.16,43 Although it is still observed, the classic form of pertussis is seen less often since general immunization began.
Increasing Awareness of Atypical Pertussis.
Routine immunization of infants has resulted in an increased awareness of the reservoir of B. pertussis infections in adolescents and adults.6,9,17,22,44,45 Although some previously immunized individuals do develop typical symptoms, less typical pertussis, characterized by the usual absence of whoop and often a somewhat shorter duration of cough,27,32,41 is more common than typical pertussis among adolescents and adults, making it the main B. pertussis disease in countries with high vaccination coverage. Indeed a significant proportion of prolonged cough illnesses in adolescents and adults are caused by B. pertussis. Studies from Australia, Canada, Denmark, France, Germany and the United States indicate that 12–32% of adolescents with a coughing illness lasting at least 1–2 weeks are infected with B. pertussis.22,24,25,27,31,46–51 In the above studies, most cases were identified by serologic study. In studies in which multiple B. pertussis antigens were used, more cases were identified than when only antibody to pertussis toxin (PT; an antigen specific to B. pertussis) was used. Because other Bordetella species elicit antibodies to filamentous hemagglutinin (FHA) and pertactin (PRN) and some antibodies to FHA can be caused by non-Bordetella agents, it is likely that the true percentage of prolonged cough illnesses caused by B. pertussis infection is in the range of 12%.24
Misdiagnosis of Pertussis: Infection by Other Organisms and Mixed Infections
Misdiagnoses of pertussis as Mycoplasma pneumoniae or Chlamydia pneumoniae infection, laryngitis, upper respiratory tract infection, bronchitis, sinusitis, asthma and chronic bronchitis have been reported.16,27,45
The clinical diagnosis of pertussis can also be complicated by the co-occurrence of other infections. In a study of serum samples from 319 students with cough lasting at least 5 days, 47 (15%) had significant increases in antibody to B. pertussis antigens, 26 (8%) had significant increases to fimbriae-2 or agglutinogens, indicative of B. pertussis infection, and 2 (1%) had evidence of non-B. pertussis Bordetella infections.47 Seventeen (36%) had evidence of mixed infections or cross-reacting antibodies with influenza A or B, adenovirus, C. pneumoniae or M. pneumoniae. Unfortunately in this study, antibody to PT could not be determined because of technical problems with the sera. This led to the use of fimbriae 2 and the presence of agglutinogens for the diagnosis of B. pertussis infection. Single infections with these other pathogens may also present as prolonged paroxysmal coughing52–54 and should therefore be considered in the differential diagnosis, along with conditions such as asthma and airway obstruction. Recent data also highlight the importance of considering infection with Bordetella parapertussis in the differential diagnosis of prolonged cough.55
Low Physician Awareness and Index of Suspicion
Awareness that B. pertussis cough illness affects all ages is low and, in developed countries where pertussis has been well-controlled by immunization, there is widespread belief that B. pertussis infection is not occurring in the population. The diagnosis is often not considered in adolescents and adults, even when symptoms are typical,36,37 because of a widespread lack of physician awareness that these groups experience illness caused by B. pertussis. Typical forms of pertussis may even be misdiagnosed by clinicians in infants and children.56,57
Together the belief that pertussis is a childhood disease, difficulties in differentiating B. pertussis infection from other infections and the failure to recognize less severe and atypical forms of B. pertussis infection in adolescents and adults contribute to low notification rates and underestimation of the disease and of the transmission risks to vulnerable infants.58 There is thus a need to raise awareness of B. pertussis infection so that clinical recognition and diagnosis is enhanced and accurate estimates of the true incidence, transmission and disease burden are obtained.
What is “Pertussis”?
Because of the heterogeneity in B. pertussis illness expression and the prominence of less typical forms, the diagnosis should not rely solely on clinical criteria and should be confirmed by laboratory study. However, laboratory confirmation is secondary to the clinical identification of patients suspected of having pertussis.
Identification of patients with B. pertussis illness based on clinical criteria is fraught with problems of sensitivity and specificity, which can vary depending on the situation. For example, different selection biases may apply in clinical practice, in epidemiologic studies or in vaccine efficacy trials. In each setting, different forces come into play. Whereas clinical suspicion by the treating physician and/or the patient is necessary to prompt laboratory studies for the presence of B. pertussis infection in a given cough illness in clinical practice, diagnostic tests are usually performed more consistently for active case finding in vaccine efficacy trials and prospective epidemiologic studies. Not surprisingly, active surveillance will therefore lead to significantly higher incidence rates that are closer to reality compared with rates from passive surveillance in a general population. However, even under controlled study conditions, subjective, predefined attitudes (so-called “observer bias”) may lead to significant underdiagnosis.59
Identifying Pertussis Patients in Clinical Practice
In clinical practice, diagnosis of B. pertussis illness must occur early to allow prompt therapeutic intervention, to reduce disease severity and ultimately to prevent the spread of the disease. In such a setting, the diagnostic criteria applied should be sensitive, even if specificity is compromised.
Identifying B. pertussis Cough Illnesses in Vaccine Efficacy Trials
In contrast, in vaccine trials, efficacy against mild and severe cough illnesses caused by B. pertussis is of scientific interest. Therefore different levels of sensitivity and specificity by the case definitions are required.
Vaccine efficacy trials have particularly highlighted the difficulties in establishing a suitable clinical case definition of pertussis, given that the case definition used can have a considerable impact on trial outcome. Because specificity is essential in trials, case definitions used in vaccine trials require laboratory confirmation. During the first vaccine efficacy trial with acellular pertussis vaccines in Sweden in 1987, a monovalent vaccine (PT) and a bivalent vaccine (PT plus FHA; PT-FHA) were compared.60 When considering only culture-confirmed cases, irrespective of the duration of cough, efficacy was estimated at 54% for the PT vaccine and 69% for the PT-FHA vaccine. In a second analysis, using a definition corresponding to more severe cases (cough lasting >30 days), improved efficacy was observed (80% for PT and 79% for PT-FHA). This indicates that the pertussis vaccines studied protect reasonably well against classic B. pertussis illness but were less protective against milder symptoms.
World Health Organization (WHO) Case Definition for Vaccine Efficacy Trials.
The differing results yielded by various case definitions noted above underlined the importance of the choice of case definition for vaccine trials and led to a meeting of a WHO Committee in January 1991. Meeting participants agreed that the heterogeneity of case definitions being used in various clinical trials was unsatisfactory and suggested a standardized approach. Recommendations were made and a case definition of pertussis was proposed for future clinical trials (Table 1). 61 This WHO definition of B. pertussis illness required a paroxysmal cough lasting for at least 21 days and laboratory confirmation or contact with a culture-confirmed case (Table 1).
Some committee members were dissatisfied with this case definition, because it discarded legitimate but more mild cases.62 Because these mild cases were more common in vaccinees than in controls, the expressed efficacy was inflated, and clear differences in efficacy among the tested vaccines were obscured. The recognition of this problem by some committee members led the committee to suggest an additional, less stringent clinical criterion to be used in a secondary case definition.
In 3 cohort efficacy trials conducted in the early 1990s, the calculated efficacy decreased by 7–18% when the case definition included mild as well as typical cases.63–65
Identifying Patients with B. pertussis Cough Illnesses for Epidemiologic Studies
In defining B. pertussis infections in epidemiologic studies, a compromise between what is required for clinical practice and what is required for a vaccine trial is necessary. A sensitive method of diagnosis is required to detect all cases, including milder disease, and atypical forms with few symptoms and short duration. Nevertheless good specificity is essential to allow definitive conclusions to be drawn.
During an evaluation of 15 clinical case definitions for pertussis in 1988, Patriarca et al66 concluded that a definition of at least 14 days of cough was both sensitive (84–92%) and specific (63–90%) for monitoring outbreaks and investigating contacts of culture-positive cases. Almost a decade later, the Centers for Disease Control and Prevention (CDC) proposed clinical and laboratory-confirmed case definitions for the purpose of pertussis surveillance (Table 1).67 Although these clinical case definitions are useful in outbreak situations, they are not useful in nonoutbreak illness because of a low level of specificity. For example, only 51% of 216 children with ≥14 days of cough with paroxysms, whoop or posttussive vomiting had laboratory evidence of B. pertussis or B. parapertussis infections.65 Because these children were carefully evaluated as part of a vaccine efficacy trial and laboratory study included culture, frequent polymerase chain reaction (PCR) and serologic study on acute and convalescent-phase sera, it is likely that children without positive laboratory studies did not have pertussis. Interestingly in the same trial, the use of the same clinical case definition by 3 trained investigators resulted in a sensitivity of 55% and a specificity of 99%. When only cough illnesses in nonvaccinated children were considered, the sensitivity increased to 74%. More recently, the WHO proposed 2 similar case definitions (Table 1).68
There is therefore a lack of consistency between case definitions, with different ones being applied in different countries and in different settings. Standard case definitions, such as those by the WHO or CDC, are not universally applied for routine pertussis reporting and surveillance, making intercountry comparisons and global evaluations difficult. If a standard clinical case definition were to be universally used, diagnosis on a purely clinical basis would result in considerable over- or underreporting, depending on the case definition.
DEFINING B. PERTUSSIS COUGH ILLNESS FROM MICROBIOLOGIC AND SEROLOGIC PERSPECTIVES
Laboratory techniques for the identification of B. pertussis have existed for nearly a century. Specific medium for the culture of B. pertussis was originally described in 1906,69 and the first use of serum agglutinating antibody for diagnosis was published in 1916.70
In the present era, extensive acellular pertussis vaccine efficacy trials and other epidemiologic studies have made possible the evaluation of multiple methods for the laboratory diagnosis of B. pertussis infections.63–65,71–80 Common laboratory diagnostic methods currently include culture, direct antigen detection [PCR and direct fluorescent antibody (DFA) testing], and serologic demonstration [enzyme-linked immunosorbent assay (ELISA) or Western blot with various B. pertussis antigens and agglutination] by measuring an increase in titers between acute and convalescence phase serum specimens or high single serum antibody values. When acute phase serum is collected late (3–4 weeks after onset of symptoms in previously vaccinated children and 5–6 weeks after onset of symptoms in previously unvaccinated children), the demonstration of decrease in titer between acute and convalescent phase specimens is also indicative of infection.81 With the proper performance of culture, PCR and ELISA to measure increases or decreases in IgG and IgA antibody titers to PT in paired serum samples, the sensitivity and specificity of the laboratory diagnosis of B. pertussis infection can equal or exceed that for many other bacterial infections.
However, many practical problems affect the sensitivity of the laboratory diagnosis of pertussis. These include delayed specimen collection, poor specimen collection technique, specimen transport problems, laboratory media problems, laboratory inexperience, laboratory contamination, equipment expense and the need for 2 specimen samples for serologic diagnosis. Furthermore exposure to the bacterium, age, stage of disease, antibiotic administration and immunization can all affect the sensitivity of the tests and lead to confusion in the interpretation of serologic results. The greatest sensitivity is obtained when culture, PCR and serologic testing are all performed on individuals with cough illness.65,74,82
The possible inadequacies of current diagnostic laboratory methods, accessibility to appropriate tests in developed and developing countries and the implications of new pertussis variants on the effectiveness of laboratory techniques are reviewed.
Laboratory Confirmation of a Clinical Diagnosis of B. pertussis Illness Culture
B. pertussis is a fastidious, Gram-negative coccobacillus. Suboptimal culture results can occur because of inadequate specimen collection and transport, as well as poor laboratory methods. Methods for specimen collection, transport and culture have been well-described.82–87
Because B. pertussis colonizes the ciliated epithelial cells in the upper and lower respiratory tracts, a specimen for culture must be obtained from the surface of respiratory ciliated epithelial cells of the upper respiratory tract, and not just from the throat or anterior nose.
Two methods have been used satisfactorily in the collection of specimens: nasopharyngeal swab and nasopharyngeal aspiration. In young children, nasal wash specimens may also be satisfactory88; however, dilution may be a problem, and the method has not been adequately evaluated in the diagnosis of B. pertussis infection.
For nasopharyngeal swabs, the shaft should be a fine, flexible wire, and cotton and rayon should not be used because they contain fatty acids that are toxic to B. pertussis. Calcium alginate is the preferred swab material, although Dacron is acceptable. The correct use of a nasopharyngeal swab is an unpleasant experience for the patient and can lead to suboptimal specimen collection. For optimal results, the tip of the swab must come into contact with the ciliated cells of the respiratory epithelium. Failure to achieve this is a major cause of negative cultures.
Although there are data suggesting that nasopharyngeal aspiration leads to a higher isolation rate of B. pertussis,72,89 this method is generally less practical in the clinical setting. To avoid negative cultures, good training is therefore required for adequate specimen collection.
Although immediate culture of a specimen is preferred, this is not always clinically practical.90 For successful transport, the transport medium must prevent the loss of B. pertussis and inhibit the growth of other organisms that will obscure the identification of B. pertussis. The transport medium of choice is modified Regan-Lowe (half-strength charcoal agar supplemented with 10% horse blood and cephalexin 40 mg/l), a nutritive medium that inhibits the growth of normal nasopharyngeal flora. The preincubation of this medium at 36°C overnight before shipment of the specimen might increase the yield of B. pertussis but might also result in a greater growth rate of other bacterial or fungal contaminants. Nonnutritive bacteriologic transport media, such as Amies’ medium, can also be used if they contain charcoal and the period between specimen collection and culture is <24 hours.
Methods for culture of B. pertussis are also well-described.82–86,91 Although many types of culture medium have allowed successful isolation of B. pertussis from clinical specimens, modified Regan-Lowe agar is currently the medium of choice.82 In situations when culture plates are inoculated directly without transport, it is also advisable to inoculate an enrichment medium and then replate after 48 hours of incubation. Recommended enrichment media include Regan-Lowe transport medium and Stainer-Scholte broth. Bordet-Gengou agar is also frequently used for culture, but the need for this medium to be freshly made makes it less practical than charcoal agar (which has an 8-week shelf-life). Because cephalexin inhibits some B. pertussis isolates, some laboratories inoculate specimens onto charcoal agar, both with and without this antibiotic. Agar plates are incubated at 35–36°C in high humidity, ambient air. B. pertussis is catalase- and oxidase-positive and urease-negative and is identified by specific antiserum agglutination or fluorescence.
The main reasons for failure of bacterial growth in culture from correctly collected and transported specimens are bacterial and fungal contamination and the lack of fresh media.
Natural infection with B. pertussis is followed by an increase in serum concentration of IgA, IgG and IgM antibodies to specific antigens, as well as to preparations of the whole organism.76,92,93 Before the development of ELISA techniques, the main serologic test for the diagnosis of pertussis was the demonstration of a 4-fold increase in agglutinating antibody titer. The antibodies measured in this test are primarily IgM directed against fimbriae 2 and 3, PRN and lipooligosaccharide. Experience suggests that this test has reasonably good specificity but lacks sensitivity. The circulation of B. pertussis expressing different PRN variants might also affect this diagnostic test, but this seems improbable since it is the response to the fimbrial antigens, and not PRN, that is most prominent.94,95
During the past 15 years, the mainstay of serologic diagnosis of pertussis has been ELISA, using specific B. pertussis proteins as antigens. Standardized techniques have been developed, and antibodies are quantitated by use of the reference line computer program and reference serum specimens from the Center for Biologics Evaluation and Research, U.S. Food and Drug Administration. The precision of the test is such that intraassay coefficients of variation have been reduced to <10%, making a 2-fold increase in titer significant.
In contrast with natural infection, the primary immunization of children induces mainly IgM and IgG antibodies. Serologic diagnosis of pertussis may be suspected by the demonstration of an increased agglutinin titer or the use of ELISA, showing an increased IgA or IgG antibody titer to PT, FHA, PRN, fimbriae or sonicated whole organism. Antibody responses to FHA and PRN, and to sonicated whole organism, also occur after other Bordetella infections, so that isolated increased antibody titers against these antigens are not specific for B. pertussis infection.65 In addition, high antibody titers to FHA may be the result of cross-reacting epitopes of nonencapsulated Haemophilus influenzae, M. pneumoniae, C. pneumoniae, other bacteria or nonspecific polyclonal stimulation.50 If age-specific control values for a population are determined, the diagnosis of pertussis can be established by high antibody values on single serum samples.19,22,27,28,51,75,96 This method is particularly useful for serologic diagnosis in adolescents and adults. In children, the interpretation of results is less sensitive and specific because of recent immunization. Some culture-positive patients, particularly children younger than 3 months of age, fail to develop measurable antibodies.
A major problem in the serologic diagnosis of B. pertussis infection by ELISA is the delay in obtaining the acute phase specimen. In patients with reinfections, a rapid increase in titer occurs, so that with a delayed acute phase sample, the titer is likely to have already peaked and further titer increases between the acute phase and convalescent phase sera are not observed. Because of this, when serum collection is delayed, decreases in antibody titers are now also taken into account.31,81
The greatest specificity for the serologic diagnosis of B. pertussis infection is achieved by ELISA and measurement of IgG and IgA antibodies to PT. A significant rise in titer (≥2-fold) between acute phase and convalescent phase sera must be demonstrated. In adolescents and adults, single high values of IgG or IgA antibodies to PT also indicate infection. Although IgA antibody to PT is more indicative of a recent antibody response, it is less consistent than a PT IgG response.80 The younger the child, the less consistent is the IgA antibody response.
DFA Testing of Nasopharyngeal Secretions.
The use of DFA for the rapid diagnosis of B. pertussis and B. parapertussis infections has been reviewed by Müller et al.82 This method has been used for 40 years because it is inexpensive, can provide a rapid diagnosis and can yield a positive result when cultures are negative because of antibiotic use. However, DFA is insensitive because amplification is not used and it may lack specificity because of cross-reactions with normal nasopharyngeal flora.71
The use of PCR has made the rapid diagnosis of many infectious diseases possible, and its use in the diagnosis of pertussis infection is rapidly evolving.71,74,77,80,97–99
Key factors for the successful application of PCR in the diagnosis of infection by Bordetella spp. involve sample collection and processing, DNA purification, primer selection and amplification conditions, detection specificity of the amplified product, and internal and external positive and negative controls.100 Calcium alginate swabs should not be used for PCR specimens because of inhibitory factors present in the fiber, and aspirate specimens must be treated with a mucolytic agent to remove PCR-inhibiting substances. Primers have been derived from 4 chromosomal regions (PT promoter region, a region upstream of the porin gene, repeated insertion sequences and the adenylate cyclase toxin gene). All except adenylate cyclase toxin primers are specific for B. pertussis.
During the past 14 years, a number of PCR assays have been evaluated in multiple studies by comparison with culture and clinically typical pertussis. Overall PCR as a diagnostic tool has the advantage of a much higher sensitivity compared with conventional culture. In a prospective study, in which swabs for PCR and culture were obtained simultaneously from 555 individuals with cough illness, the use of PCR increased the identification of B. pertussis infections by almost 4-fold, from 28 to 111.77 In a similar but larger study (n = 7153), Schmidt-Schläpfer et al101 found a 2.6-fold increase in detection of B. pertussis using PCR compared with culture. Only a few studies have been reported in which the sensitivity and specificity of PCR in the diagnosis of B. pertussis infection was determined by comparison with serologically identified cases.74,80,97,98,102 In one study, PCR results were compared with serologic diagnosis; PCR had a sensitivity of 61% and a specificity of 88%.74 In a comparison of the performance of culture, DFA and PCR for the detection of B. pertussis in nasopharyngeal swab specimens, 319 consecutive paired specimens were compared using all 3 tests.103 A total of 59 specimens were positive by 1 or more tests. Of these, 5 were positive by all 3 tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Patients with symptoms meeting the CDC clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true B. pertussis infections. The sensitivity, specificity, positive predictive value and negative predictive value were 15.2, 100, 100 and 87.5%, respectively for culture; 93.5, 97.1, 84.3 and 98.9%, respectively for PCR; and 52.2, 98.2, 82.8 and 92.4%, respectively for DFA, confirming that PCR is a sensitive and specific method for the detection of B. pertussis. However, because serologic evaluation was not part of this study, the high sensitivity percentage for PCR is likely to be artificial.74
One potential caveat for PCR, however, is that it can be associated with false positive results.100 These are commonly caused by contamination at various stages (from sample collection through to the laboratory setting) or laboratory errors, such as unspecific amplification.
Poor Access to Diagnostic or Laboratory Methods.
A major problem in both developed and developing countries is lack of access to diagnostic laboratory methods. Most routine laboratories are not equipped for the diagnosis of B. pertussis infection. This is the result of a general medical misconception that B. pertussis infection does not frequently occur in the population. Few laboratories maintain fresh culture media for diagnosis, and kits for specimen collection and transport are not routinely available. B. pertussis serologic testing with acceptable techniques is rarely available. Commercial laboratories provide an array of B. pertussis ELISA techniques, but in general there is little evidence of either sensitivity or specificity related to patient specimens. However, these tests are particularly useful in adolescents and adults. They are also useful in children who have not been immunized recently (ie, within 2 years). Furthermore PCR is not routinely available.
In countries where pertussis epidemics have occurred in recent years, laboratory facilities for the diagnosis of B. pertussis infection are generally better than in countries where recent outbreaks have not been recognized. However, the mainstay of diagnosis has been culture. Routine services for serologic diagnosis are frequently not available and the use of PCR is variable.
In developing countries, which generally have a higher incidence of pertussis, laboratory services and resources vary markedly from country to country. The facilities for the routine culture of B. pertussis are more readily available, and laboratory workers are more knowledgeable than in many developed countries where pertussis is thought to be controlled. However, routine serologic diagnostic services and PCR are generally not available.
Issues Surrounding New Pertussis Variants and Epidemiologic Typing
B. pertussis strain variation was considered a possible cause of vaccine failure 40 years ago.104 In the early 1960s, pertussis vaccine efficacy in the United Kingdom had decreased, and it was suggested that this decline in efficacy was because the pertussis vaccine in use was devoid of agglutinogen 3 (fimbriae 3) and the most prevalent circulating B. pertussis strains was serotype 1.3. After the use of vaccines that contained agglutinogen 3 as well as agglutinogen 2, an increase in efficacy was observed, thereby supporting the hypothesis. However, the fact that protective unitage of the vaccine also increased at the same time could also have played a part.105
During the past decade, the demonstration of polymorphism in B. pertussis genes encoding the expression of PRN and PT led to the suggestion that vaccine-driven evolution has resulted in decreased vaccine efficacy.12,106–109 However, other factors, such as the use of vaccines with suboptimal efficacy, greater awareness and better laboratory study, are more likely explanations of the increased reporting of pertussis.110
Variation in circulating B. pertussis isolates might affect the results of PCR or serology in the laboratory diagnosis of B. pertussis infection.
Currently B. pertussis polymorphisms have been noted and are related to the S1 subunit of PT and in 2 regions of the PRN gene. However, neither of these gene mutations affects the epitopes used in ELISA, and these regions are not used for PCR diagnosis.
Polymorphism in the PT promoter region can be used to specifically diagnose B. pertussis, B. parapertussis and Bordetella bronchiseptica.111 Nucleotide variations in the primer-annealing region could be a source of false negative PCR results. However, in culture-positive/PCR-negative samples, no nucleotide variation has been noted in the B. pertussis isolates collected.
SUMMARY AND CONCLUSIONS
Although there is evidence to suggest that B. pertussis infection continues to be a serious problem in terms of morbidity and mortality in unimmunized or incompletely immunized neonates and infants, establishing the true incidence of B. pertussis infection and illness is complicated by a number of factors. Underrecognition and underdiagnosis are widespread, particularly among adolescents and adults. Despite increasing awareness that illness with prolonged cough may be the only evidence of B. pertussis infection in adolescents and adults, the diagnosis is usually not considered. When B. pertussis infection is considered a diagnostic possibility, it is often not confirmed because routine laboratory tests are insensitive and tests that may be more sensitive are not all adequately standardized.
Because of the wide variability in disease expression and the increasing awareness of atypical presentations, definition of B. pertussis infection clinically is difficult. However, clinical selection of patients is a prerequisite to laboratory confirmation. Therefore clinical case definitions for B. pertussis cough illness, particularly mild disease, must be developed and universally applied. In the meantime, CDC and WHO definitions should be used.
In addition, rapid and easy-to-use laboratory diagnostic techniques need to be available and criteria for laboratory confirmation of pertussis need to be standardized. Particularly important will be the increased use of PCR and single-serum serology.
To raise awareness of pertussis, education campaigns targeted at healthcare professionals and the public may help to raise awareness of the burden of pertussis.
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