SPECIAL FOCUS y Pertussis
Review
Pertussis diagnostics: overview and impact of immunization Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Expert Rev. Vaccines Early online, 1–8 (2014)
Carl-Heinz Wirsing von Ko¨nig Labor:Medizin Krefeld MVZ, Lutherplatz 40, D-47805 Krefeld, Germany Tel.: +49 215 132 2466 Fax: +49 215 132 2012 [email protected]
In all vaccinated populations, infections with Bordetella pertussis and Bordetella parapertussis continue to cause infections in unvaccinated infants and children, as well as in adolescents and adults with waning immunity. Thus in patients with longer lasting coughs a diagnosis of pertussis should be entertained irrespective of their vaccination status. Due to the non-specific clinical symptoms, clinically suspected cases of pertussis must be verified by laboratory methods. Hyperleukocytosis may be helpful in diagnosis for young infants, but in most cases, nonspecific laboratory tests have no role in pertussis diagnosis. Specific laboratory tests include direct detection of the bacteria or their DNA by culture or PCR, whereas serology serves as an indirect method to diagnose pertussis in those patients who present late in the development of the disease. Serology results can be interpreted in relation to reference values for different populations, but serology is unable to distinguish between vaccination and infection. KEYWORDS: Bordetella pertussis • culture • ELISA • parapertussis • PCR • serology
Epidemiology & clinical presentation
Bordetella pertussis and Bordetella parapertussis are the causative agents of pertussis or whooping cough, and infections caused by B. parapertussis tend to be somewhat milder [1–4]. The bacteria are transmitted by droplets, and in susceptible contacts the B. pertussis transmission rate is close to 90%. The basic reproduction number (Ro) was estimated to be around 17–21 [1,2], but in highly vaccinated European countries, it is estimated to be around 6 [5]. Ro indicates the number of other persons that one patient infects during the period of infectivity. B. pertussis continues to circulate in populations where high vaccination coverage of infants and children is achieved [4,6], because the protection after natural infection and vaccination wanes after several years [1,6]. In Western countries, cases of pertussis are observed in neonates, unvaccinated young infants, older schoolchildren, adolescents and adults [1,4,6–8]. Transmission occurs from adolescents and adults to infants or among older vaccinated children, adolescents and adults [2,4]. Neonates and young infants are at risk of being infected by their parents, but also from casual contacts [9].
informahealthcare.com
10.1586/14760584.2014.950237
A carrier state does not exist in pertussis; however, in outbreaks Bordetella-DNA may be detected by PCR in asymptomatic patients [10]. After an incubation period of 7–10 days (range: 4–28 days), the infection starts with rhinorrhoea, sneezing and non-specific coughs (catarrhal phase). The typical clinical symptoms of pertussis are found in primary infections of non-vaccinated children, with coughing spasms, whooping and vomiting (paroxysmal phase) [1,11]. Cases in neonates and unvaccinated young infants often present with apnoea as the only symptom [2,6]. In older children, adolescents and adults the symptoms can vary widely. Adult pertussis is associated with a long persistent cough, which is sometimes paroxysmal, usually worsens at night and has a mean duration of approximately 6 weeks. It is frequently accompanied by choking, vomiting and by whooping [4]. Various clinical case definitions for pertussis have been proposed, and they are summarized in TABLE 1. Most hospitalizations and deaths occur in infancy, and in this age group, deaths may not even be diagnosed as pertussis [6,12]. Pertussis-like symptoms although often with shorter duration of coughing are also caused
2014 Informa UK Ltd
ISSN 1476-0584
1
Review
Wirsing von Koenig
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Table 1. Pertussis case definitions. Organization/ country and year
Clinical criteria
Laboratory and epidemiologic criteria
Comment
WHO 2000
A case diagnosed as pertussis by a physician, or A person with a cough lasting at least 2 weeks with at the least one of the following symptoms: • Paroxysms (i.e., fits) of coughing • Inspiratory ‘whooping’ • Post-tussive vomiting (i.e., vomiting immediately after coughing) without other apparent cause
Isolation of Bordetella pertussis, or Detection of genomic sequences by PCR, or Positive paired serology
Case classification: Clinical case: a case that meets the clinical case definition, but is not laboratory confirmed. Laboratory confirmed case: a case that meets the clinical case definition and is laboratory confirmed
CDC 2010
A cough illness lasting at least 2 weeks with one of the following: paroxysms of coughing, inspiratory ‘whoop’, or post-tussive vomiting, without other apparent cause (as reported by a health professional)
Isolation of B. pertussis from clinical specimen PCR positive for pertussis
Case classification Probable: In the absence of a more likely diagnosis, a cough illness lasting ‡2 weeks, with at least one of the following symptoms: • Paroxysms of coughing OR • Inspiratory ‘whoop’ OR • Post-tussive vomiting Confirmed: Acute cough illness of any duration, with isolation of B. pertussis from a clinical specimen, OR Cough illness lasting ‡2 weeks, with at least one of the following symptoms: • Paroxysms of coughing, OR • Inspiratory ‘whoop’, OR • Post-tussive vomiting AND, at least one of the following: • PCR positive for pertussis, OR • Contact with a laboratoryconfirmed case of pertussis
France 2009
Patient coughing since more than 7 days but with no biological confirmation
Patient coughing more than 7 days with • Positive PCR or • >100 IU/ml of anti-PT abs >3 year of a vaccination or 100% change in the abs titer between two serologies at 1 month interval
Epidemiologically confirmed: Patient coughing since more than 7 days and in contact in the last 20 days with a confirmed case
Canada 2009
Suspect case One or more of the following, with no other known cause: • Paroxysmal cough of any duration • Cough with inspiratory ‘whoop’ • Cough ending in vomiting or gagging, or associated with apnea
Confirmed case Laboratory confirmation of infection: • Isolation of B. pertussis from an appropriate clinical specimen OR • Detection of B. pertussis DNA form an appropriate clinical specimen
Adapted with permission from [39] Oxford University Press (2011).
doi: 10.1586/14760584.2014.950237
Expert Rev. Vaccines
Pertussis diagnostics
Review
Table 1. Pertussis case definitions (cont.).
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Organization/ country and year
Clinical criteria Probable case Cough lasting 2 weeks or longer in the absence of appropriate laboratory tests and not epidemiologically linked to a laboratory – confirmed case AND one or more of the following, with no other known cause: • Paroxysmal cough of any duration • Cough with inspiratory ‘whoop’ • Cough ending in vomiting or gagging, or associated with apnea
Laboratory and epidemiologic criteria
Comment
AND one or more of the following: – Cough lasting 2 weeks or longer – Paroxysmal cough of any duration – Cough with inspiratory ‘whoop’ – Cough ending with vomiting or gagging, or associated with apnea OR Epidemiologic link to a laboratoryconfirmed case AND one or more of the following which there is no other known cause: – Paroxysmal cough of any duration – Cough with inspiratory ‘whoop’ – Cough ending in vomiting or gagging, or associated with apnea
EU, 2008
Cough ‡2 weeks with at least one the following: Paroxysms Inspiratory ‘whooping’ Post-tussive vomiting OR Any person diagnosed as pertussis by a physician OR Apnea episodes in infants
Isolation of B. pertussis Nucleic acids of B. pertussis B. pertussis specific antibody response Epidemiological link by human to human transmission
Possible case: Any person with clinical criteria Probable case: Person with clinical criteria and epidemiological link Confirmed case: Person meeting the clinical and laboratory criteria
Australia
Coughing 2 weeks or more OR Paroxysms of coughing OR inspiratory whoop OR post-tussive vomiting
Culture of B. pertussis PCR for B. pertussis Seroconversion or significant increase of antibodies (without recent vaccination) Single IgA titer to whole cells Detection of B. pertussis by DFA
Probable case: Any person with clinical criteria Confirmed case: Person meeting the clinical and laboratory criteria Or epidemiological link
Global pertussis initiative (2012) [39]
0–3 months: cough and coryza with minimal fever PLUS whoop OR apnea OR post-tussive emesis OR cyanosis 4 months – 9 years: paroxysmal cough with no or minimal fever PLUS whoop OR apnea OR posttussive emesis ‡10 years: non-productive parorysmal cough of ‡2 weeks duration without fever PLUS whoop OR apnea OR sweating episodes
Culture of B. pertussis PCR for B. pertussis IgG-anti-PT serology (if older than 1 year)
Adapted with permission from [39] Oxford University Press (2011).
by adenoviruses, respiratory syncytial virus (RSV), human parainfluenza viruses, influenza viruses, Mycoplasma pneumoniae and other agents [6]. Thus, clinical case definitions for pertussis vary greatly in their sensitivity and specificity. In their seminal paper about clinical case definitions, Patriarca et al. [11] found that for informahealthcare.com
defining pertussis, ‘any cough’ had a sensitivity of 96% and a specificity of 28%, while a severe disease with 2 weeks of coughing with paroxysms and sleep disturbance had a specificity of 88%, but the sensitivity was only 42%. Thus, clinical case definitions are intended for different uses, and a more severe disease doi: 10.1586/14760584.2014.950237
Review
Wirsing von Koenig
with higher specificity and a low sensitivity may be suitable for vaccine testing, whereas for monitoring disease, a case definition requiring shorter clinical symptoms with high sensitivity but low specificity may be acceptable. Overall, however, a laboratory confirmation is required for all pertussis cases, and various nonspecific and specific laboratory tests are available.
culture, they are more sensitive than swabs. Nasopharyngeal swabs taken from older children, adolescents and adults by trained personnel are adequate specimens in these age groups and are much better than throat swabs. Nasopharyngeal swabs should be taken by gently inserting the swab into the nasopharynx under the inferior nasal choana, and the nose of the patient should be slightly bent upward such as shown in a video from the Pasteur Institute, Paris, France [13]. If possible, two nasopharyngeal swabs should be taken, one from each nostril. Small dacron or rayon swabs as well as flocked nylon swabs that are more convenient for the patient may be used for B. pertussis PCR or culture [14–16]. Calcium-alginate swabs should not be used for PCR. For culture, samples should be taken before antibiotic treatment is started [16]. Transport time is critical for culture, and a transport medium protecting the bacteria from drying is required. Usual bacteriological transport media, such as Casamino Acids or AMIES medium, with charcoal may be used, for transport times less than 48 h. Half-strength Regan Lowe charcoal–blood medium may also be used for transport, and it serves as enrichment for B. pertussis [6]. For PCR, swabs can be transported dry at ambient temperature. The use of microbiological transport media, such as AMIES medium with charcoal, did not interfere with PCR [16]. Other respiratory samples such as sputum samples or throat washes are less suitable and have not been validated [2].
Laboratory diagnosis: non-specific tests
Direct detection methods: PCR
In non-vaccinated infants, the white blood count often shows a leukocytosis, with numbers ‡100.000 cells/ml, which in conjunction with clinical symptoms may be indicative for pertussis in this age group. The differential blood count shows a relative and absolute lymphocytosis in infants and children, without having sufficient diagnostic specificity. Measuring acute-phase proteins, such as CRP and the ESR, have no diagnostic relevance in pertussis.
PCR is generally more sensitive than culture, and real-time PCR formats offer a result within several hours instead of several days [15–17]. The sensitivity of PCR decreases with the duration of cough, and it also depends on age and vaccination status; however, due to its higher sensitivity, it may be a useful tool for diagnosis up to 4–6 weeks of coughing [15,16]. In outbreak situations and after household contacts, a positive PCR may also be found in patients with very little or no symptoms [18]. DNA extraction is necessary to limit inhibition of PCR [16]. Some commercially available extraction kits are comparable and appropriate [16], but no head-to-head comparison has been done. Some of these kits are US FDA cleared and/or CE marked for this purpose. Multicopy targets for detection are IS481 (copy number ~200 per cell in B. pertussis) [15,16] present in B. pertussis, B. holmesii and some B. bronchiseptica strains; IS1001 (copy number ~20 per cell in B. parapertussis) [19], which is present in B. parapertussis and IS1002, which is present in B. pertussis and B. parapertussis. The number of IS has remained stable as compared to the sequenced type strains [20]. The combined use of three IS’s has been suggested to diagnose clinically relevant Bordetella spp. and to distinguish B. holmesii by using the specific hIS1001 target [21]. Monogenic targets include the PT-promoter (ptxA-Pr), the PT-gene, the specific recA from B. holmesii and the FHA and the porin gene. Tests detecting single-copy genes are generally less sensitive than IS481-based PCRs [15–17,21–24].
20
Incubation (days)
Coughs
DNA detectable Culture Catarrhal
Antibodies detectable Paroxysmal
Convalescent
15 10
Atypical cough Rhinorrhea
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
5
7 to 10d 1 to 2 weeks
Cyanosis Lymphocytosis Vomiting whoops
3 to 6 weeks
1 to 12 weeks
Figure 1. Clinical symptoms and laboratory tests. Reproduced with permission from Nicole Guiso, Institut Pasteur, Paris.
Laboratory diagnosis: specific tests
Infections with B. pertussis and B. parapertussis can be diagnosed either by direct detection methods, such as PCR or culture, or by indirect detection methods, such as serology. It is important to note that as shown in FIGURE 1, direct detection by PCR is most sensitive during the first 2 weeks of clinical illness and is especially well suited for infants and young unvaccinated children. According to clinical experience, schoolchildren (and their mothers) may see a physician after approximately 1 week of coughing, adolescents may take 2 weeks until a medical visit, and adults may cough for a median 3 weeks before seeking medical attention. Thus, in most adolescent and adult cases, serology may be the method of choice for diagnosing the disease. Direct detection methods: preanalytics
Sampling for culture and PCR is difficult, and it influences the sensitivity markedly. Nasopharyngeal aspirates are the most suitable specimens for infants and young children; and for doi: 10.1586/14760584.2014.950237
Expert Rev. Vaccines
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Pertussis diagnostics
Detection can be done sequence specific by fluorescence resonance energy transfer (FRET)-hybridization probes, TaqMan probes and molecular beacons and also by non-sequence-specific formats using SYBR green I [16]. Most laboratories use sequence-specific formats, and duplex or triplex PCRs for clinically relevant Bordetella spp. have been developed. When using the IS481 target, positive PCR results with a high crossing threshold value (>35 cycles) were shown to have a somewhat reduced specificity [15]. Multiplex PCRs on various platforms for the detection of an array of respiratory agents including Bordetella spp. are also available, which for pertussis are mostly based on IS481 as the sole target. External quality control programs have been implemented in the USA and in European countries [25]. False-positive PCR results may occur due to contamination problems in laboratories, but also due to swabs contaminated with Bordetella-DNA from clinic surfaces [26]. Expected results of real-time PCRs for Bordetella spp. are given in TABLE 2. Direct detection methods: fluorescent antibody staining
Direct fluorescent antibody staining on nasopharyngeal swabs or nasopharyngeal aspirates is rapid and simple, but lacks sensitivity and specificity and is not recommended [2]. Direct detection methods: culture
PCR hast mostly replaced culture in routine diagnosis [16]. Various culture media, such as Regan–Lowe medium, Bordet– Gengou medium and Stainer–Scholte medium can be used for growing B. pertussis and B. parapertussis. Most media are supplemented with cephalexin to suppress concomitant bacteria. The sensitivity of culture depends on the duration of symptoms, the age and the vaccination status of the patient, and it varies from approximately 60% in young unvaccinated infants with few days duration of symptoms to 100 IU/ml can be used as an indicator of recent contact with the bacteria [29,35,36]. IgG-anti-PT antibodies are primarily measured, and IgA-anti-PT may serve as an additional test for equivocal results (>40 and
Review
Pertussis diagnostics: overview and impact of immunization Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Expert Rev. Vaccines Early online, 1–8 (2014)
Carl-Heinz Wirsing von Ko¨nig Labor:Medizin Krefeld MVZ, Lutherplatz 40, D-47805 Krefeld, Germany Tel.: +49 215 132 2466 Fax: +49 215 132 2012 [email protected]
In all vaccinated populations, infections with Bordetella pertussis and Bordetella parapertussis continue to cause infections in unvaccinated infants and children, as well as in adolescents and adults with waning immunity. Thus in patients with longer lasting coughs a diagnosis of pertussis should be entertained irrespective of their vaccination status. Due to the non-specific clinical symptoms, clinically suspected cases of pertussis must be verified by laboratory methods. Hyperleukocytosis may be helpful in diagnosis for young infants, but in most cases, nonspecific laboratory tests have no role in pertussis diagnosis. Specific laboratory tests include direct detection of the bacteria or their DNA by culture or PCR, whereas serology serves as an indirect method to diagnose pertussis in those patients who present late in the development of the disease. Serology results can be interpreted in relation to reference values for different populations, but serology is unable to distinguish between vaccination and infection. KEYWORDS: Bordetella pertussis • culture • ELISA • parapertussis • PCR • serology
Epidemiology & clinical presentation
Bordetella pertussis and Bordetella parapertussis are the causative agents of pertussis or whooping cough, and infections caused by B. parapertussis tend to be somewhat milder [1–4]. The bacteria are transmitted by droplets, and in susceptible contacts the B. pertussis transmission rate is close to 90%. The basic reproduction number (Ro) was estimated to be around 17–21 [1,2], but in highly vaccinated European countries, it is estimated to be around 6 [5]. Ro indicates the number of other persons that one patient infects during the period of infectivity. B. pertussis continues to circulate in populations where high vaccination coverage of infants and children is achieved [4,6], because the protection after natural infection and vaccination wanes after several years [1,6]. In Western countries, cases of pertussis are observed in neonates, unvaccinated young infants, older schoolchildren, adolescents and adults [1,4,6–8]. Transmission occurs from adolescents and adults to infants or among older vaccinated children, adolescents and adults [2,4]. Neonates and young infants are at risk of being infected by their parents, but also from casual contacts [9].
informahealthcare.com
10.1586/14760584.2014.950237
A carrier state does not exist in pertussis; however, in outbreaks Bordetella-DNA may be detected by PCR in asymptomatic patients [10]. After an incubation period of 7–10 days (range: 4–28 days), the infection starts with rhinorrhoea, sneezing and non-specific coughs (catarrhal phase). The typical clinical symptoms of pertussis are found in primary infections of non-vaccinated children, with coughing spasms, whooping and vomiting (paroxysmal phase) [1,11]. Cases in neonates and unvaccinated young infants often present with apnoea as the only symptom [2,6]. In older children, adolescents and adults the symptoms can vary widely. Adult pertussis is associated with a long persistent cough, which is sometimes paroxysmal, usually worsens at night and has a mean duration of approximately 6 weeks. It is frequently accompanied by choking, vomiting and by whooping [4]. Various clinical case definitions for pertussis have been proposed, and they are summarized in TABLE 1. Most hospitalizations and deaths occur in infancy, and in this age group, deaths may not even be diagnosed as pertussis [6,12]. Pertussis-like symptoms although often with shorter duration of coughing are also caused
2014 Informa UK Ltd
ISSN 1476-0584
1
Review
Wirsing von Koenig
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Table 1. Pertussis case definitions. Organization/ country and year
Clinical criteria
Laboratory and epidemiologic criteria
Comment
WHO 2000
A case diagnosed as pertussis by a physician, or A person with a cough lasting at least 2 weeks with at the least one of the following symptoms: • Paroxysms (i.e., fits) of coughing • Inspiratory ‘whooping’ • Post-tussive vomiting (i.e., vomiting immediately after coughing) without other apparent cause
Isolation of Bordetella pertussis, or Detection of genomic sequences by PCR, or Positive paired serology
Case classification: Clinical case: a case that meets the clinical case definition, but is not laboratory confirmed. Laboratory confirmed case: a case that meets the clinical case definition and is laboratory confirmed
CDC 2010
A cough illness lasting at least 2 weeks with one of the following: paroxysms of coughing, inspiratory ‘whoop’, or post-tussive vomiting, without other apparent cause (as reported by a health professional)
Isolation of B. pertussis from clinical specimen PCR positive for pertussis
Case classification Probable: In the absence of a more likely diagnosis, a cough illness lasting ‡2 weeks, with at least one of the following symptoms: • Paroxysms of coughing OR • Inspiratory ‘whoop’ OR • Post-tussive vomiting Confirmed: Acute cough illness of any duration, with isolation of B. pertussis from a clinical specimen, OR Cough illness lasting ‡2 weeks, with at least one of the following symptoms: • Paroxysms of coughing, OR • Inspiratory ‘whoop’, OR • Post-tussive vomiting AND, at least one of the following: • PCR positive for pertussis, OR • Contact with a laboratoryconfirmed case of pertussis
France 2009
Patient coughing since more than 7 days but with no biological confirmation
Patient coughing more than 7 days with • Positive PCR or • >100 IU/ml of anti-PT abs >3 year of a vaccination or 100% change in the abs titer between two serologies at 1 month interval
Epidemiologically confirmed: Patient coughing since more than 7 days and in contact in the last 20 days with a confirmed case
Canada 2009
Suspect case One or more of the following, with no other known cause: • Paroxysmal cough of any duration • Cough with inspiratory ‘whoop’ • Cough ending in vomiting or gagging, or associated with apnea
Confirmed case Laboratory confirmation of infection: • Isolation of B. pertussis from an appropriate clinical specimen OR • Detection of B. pertussis DNA form an appropriate clinical specimen
Adapted with permission from [39] Oxford University Press (2011).
doi: 10.1586/14760584.2014.950237
Expert Rev. Vaccines
Pertussis diagnostics
Review
Table 1. Pertussis case definitions (cont.).
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Organization/ country and year
Clinical criteria Probable case Cough lasting 2 weeks or longer in the absence of appropriate laboratory tests and not epidemiologically linked to a laboratory – confirmed case AND one or more of the following, with no other known cause: • Paroxysmal cough of any duration • Cough with inspiratory ‘whoop’ • Cough ending in vomiting or gagging, or associated with apnea
Laboratory and epidemiologic criteria
Comment
AND one or more of the following: – Cough lasting 2 weeks or longer – Paroxysmal cough of any duration – Cough with inspiratory ‘whoop’ – Cough ending with vomiting or gagging, or associated with apnea OR Epidemiologic link to a laboratoryconfirmed case AND one or more of the following which there is no other known cause: – Paroxysmal cough of any duration – Cough with inspiratory ‘whoop’ – Cough ending in vomiting or gagging, or associated with apnea
EU, 2008
Cough ‡2 weeks with at least one the following: Paroxysms Inspiratory ‘whooping’ Post-tussive vomiting OR Any person diagnosed as pertussis by a physician OR Apnea episodes in infants
Isolation of B. pertussis Nucleic acids of B. pertussis B. pertussis specific antibody response Epidemiological link by human to human transmission
Possible case: Any person with clinical criteria Probable case: Person with clinical criteria and epidemiological link Confirmed case: Person meeting the clinical and laboratory criteria
Australia
Coughing 2 weeks or more OR Paroxysms of coughing OR inspiratory whoop OR post-tussive vomiting
Culture of B. pertussis PCR for B. pertussis Seroconversion or significant increase of antibodies (without recent vaccination) Single IgA titer to whole cells Detection of B. pertussis by DFA
Probable case: Any person with clinical criteria Confirmed case: Person meeting the clinical and laboratory criteria Or epidemiological link
Global pertussis initiative (2012) [39]
0–3 months: cough and coryza with minimal fever PLUS whoop OR apnea OR post-tussive emesis OR cyanosis 4 months – 9 years: paroxysmal cough with no or minimal fever PLUS whoop OR apnea OR posttussive emesis ‡10 years: non-productive parorysmal cough of ‡2 weeks duration without fever PLUS whoop OR apnea OR sweating episodes
Culture of B. pertussis PCR for B. pertussis IgG-anti-PT serology (if older than 1 year)
Adapted with permission from [39] Oxford University Press (2011).
by adenoviruses, respiratory syncytial virus (RSV), human parainfluenza viruses, influenza viruses, Mycoplasma pneumoniae and other agents [6]. Thus, clinical case definitions for pertussis vary greatly in their sensitivity and specificity. In their seminal paper about clinical case definitions, Patriarca et al. [11] found that for informahealthcare.com
defining pertussis, ‘any cough’ had a sensitivity of 96% and a specificity of 28%, while a severe disease with 2 weeks of coughing with paroxysms and sleep disturbance had a specificity of 88%, but the sensitivity was only 42%. Thus, clinical case definitions are intended for different uses, and a more severe disease doi: 10.1586/14760584.2014.950237
Review
Wirsing von Koenig
with higher specificity and a low sensitivity may be suitable for vaccine testing, whereas for monitoring disease, a case definition requiring shorter clinical symptoms with high sensitivity but low specificity may be acceptable. Overall, however, a laboratory confirmation is required for all pertussis cases, and various nonspecific and specific laboratory tests are available.
culture, they are more sensitive than swabs. Nasopharyngeal swabs taken from older children, adolescents and adults by trained personnel are adequate specimens in these age groups and are much better than throat swabs. Nasopharyngeal swabs should be taken by gently inserting the swab into the nasopharynx under the inferior nasal choana, and the nose of the patient should be slightly bent upward such as shown in a video from the Pasteur Institute, Paris, France [13]. If possible, two nasopharyngeal swabs should be taken, one from each nostril. Small dacron or rayon swabs as well as flocked nylon swabs that are more convenient for the patient may be used for B. pertussis PCR or culture [14–16]. Calcium-alginate swabs should not be used for PCR. For culture, samples should be taken before antibiotic treatment is started [16]. Transport time is critical for culture, and a transport medium protecting the bacteria from drying is required. Usual bacteriological transport media, such as Casamino Acids or AMIES medium, with charcoal may be used, for transport times less than 48 h. Half-strength Regan Lowe charcoal–blood medium may also be used for transport, and it serves as enrichment for B. pertussis [6]. For PCR, swabs can be transported dry at ambient temperature. The use of microbiological transport media, such as AMIES medium with charcoal, did not interfere with PCR [16]. Other respiratory samples such as sputum samples or throat washes are less suitable and have not been validated [2].
Laboratory diagnosis: non-specific tests
Direct detection methods: PCR
In non-vaccinated infants, the white blood count often shows a leukocytosis, with numbers ‡100.000 cells/ml, which in conjunction with clinical symptoms may be indicative for pertussis in this age group. The differential blood count shows a relative and absolute lymphocytosis in infants and children, without having sufficient diagnostic specificity. Measuring acute-phase proteins, such as CRP and the ESR, have no diagnostic relevance in pertussis.
PCR is generally more sensitive than culture, and real-time PCR formats offer a result within several hours instead of several days [15–17]. The sensitivity of PCR decreases with the duration of cough, and it also depends on age and vaccination status; however, due to its higher sensitivity, it may be a useful tool for diagnosis up to 4–6 weeks of coughing [15,16]. In outbreak situations and after household contacts, a positive PCR may also be found in patients with very little or no symptoms [18]. DNA extraction is necessary to limit inhibition of PCR [16]. Some commercially available extraction kits are comparable and appropriate [16], but no head-to-head comparison has been done. Some of these kits are US FDA cleared and/or CE marked for this purpose. Multicopy targets for detection are IS481 (copy number ~200 per cell in B. pertussis) [15,16] present in B. pertussis, B. holmesii and some B. bronchiseptica strains; IS1001 (copy number ~20 per cell in B. parapertussis) [19], which is present in B. parapertussis and IS1002, which is present in B. pertussis and B. parapertussis. The number of IS has remained stable as compared to the sequenced type strains [20]. The combined use of three IS’s has been suggested to diagnose clinically relevant Bordetella spp. and to distinguish B. holmesii by using the specific hIS1001 target [21]. Monogenic targets include the PT-promoter (ptxA-Pr), the PT-gene, the specific recA from B. holmesii and the FHA and the porin gene. Tests detecting single-copy genes are generally less sensitive than IS481-based PCRs [15–17,21–24].
20
Incubation (days)
Coughs
DNA detectable Culture Catarrhal
Antibodies detectable Paroxysmal
Convalescent
15 10
Atypical cough Rhinorrhea
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
5
7 to 10d 1 to 2 weeks
Cyanosis Lymphocytosis Vomiting whoops
3 to 6 weeks
1 to 12 weeks
Figure 1. Clinical symptoms and laboratory tests. Reproduced with permission from Nicole Guiso, Institut Pasteur, Paris.
Laboratory diagnosis: specific tests
Infections with B. pertussis and B. parapertussis can be diagnosed either by direct detection methods, such as PCR or culture, or by indirect detection methods, such as serology. It is important to note that as shown in FIGURE 1, direct detection by PCR is most sensitive during the first 2 weeks of clinical illness and is especially well suited for infants and young unvaccinated children. According to clinical experience, schoolchildren (and their mothers) may see a physician after approximately 1 week of coughing, adolescents may take 2 weeks until a medical visit, and adults may cough for a median 3 weeks before seeking medical attention. Thus, in most adolescent and adult cases, serology may be the method of choice for diagnosing the disease. Direct detection methods: preanalytics
Sampling for culture and PCR is difficult, and it influences the sensitivity markedly. Nasopharyngeal aspirates are the most suitable specimens for infants and young children; and for doi: 10.1586/14760584.2014.950237
Expert Rev. Vaccines
Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/03/14 For personal use only.
Pertussis diagnostics
Detection can be done sequence specific by fluorescence resonance energy transfer (FRET)-hybridization probes, TaqMan probes and molecular beacons and also by non-sequence-specific formats using SYBR green I [16]. Most laboratories use sequence-specific formats, and duplex or triplex PCRs for clinically relevant Bordetella spp. have been developed. When using the IS481 target, positive PCR results with a high crossing threshold value (>35 cycles) were shown to have a somewhat reduced specificity [15]. Multiplex PCRs on various platforms for the detection of an array of respiratory agents including Bordetella spp. are also available, which for pertussis are mostly based on IS481 as the sole target. External quality control programs have been implemented in the USA and in European countries [25]. False-positive PCR results may occur due to contamination problems in laboratories, but also due to swabs contaminated with Bordetella-DNA from clinic surfaces [26]. Expected results of real-time PCRs for Bordetella spp. are given in TABLE 2. Direct detection methods: fluorescent antibody staining
Direct fluorescent antibody staining on nasopharyngeal swabs or nasopharyngeal aspirates is rapid and simple, but lacks sensitivity and specificity and is not recommended [2]. Direct detection methods: culture
PCR hast mostly replaced culture in routine diagnosis [16]. Various culture media, such as Regan–Lowe medium, Bordet– Gengou medium and Stainer–Scholte medium can be used for growing B. pertussis and B. parapertussis. Most media are supplemented with cephalexin to suppress concomitant bacteria. The sensitivity of culture depends on the duration of symptoms, the age and the vaccination status of the patient, and it varies from approximately 60% in young unvaccinated infants with few days duration of symptoms to 100 IU/ml can be used as an indicator of recent contact with the bacteria [29,35,36]. IgG-anti-PT antibodies are primarily measured, and IgA-anti-PT may serve as an additional test for equivocal results (>40 and
