Resurgence of Pertussis and Its Laboratory Diagnosis

Clinical Microbiology N E W S L E T

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Vol. 37, No. 9 May 1, 2015 www.cmnewsletter.com I N TH IS

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69 Resurgence of Pertussis and Its Laboratory Diagnosis

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Resurgence of Pertussis and Its Laboratory Diagnosis Xuan Qin, Ph.D., D(ABMM), Department of Laboratory Medicine, University of Washington School of Medicine, and Microbiology Laboratory, Seattle Children’s Hospital, Seattle, Washington

Abstract Prevention of whooping cough by vaccination has been one of the outstanding successes in modern medical history. However, despite the great efforts made to improve vaccine safety and efficacy, as well as campaigns for immunization, there has been a worldwide resurgence of pertussis since the early 1990s. These outbreaks occur independently of the vaccine coverage rates or economic standards of the affected region. The Morbidity and Mortality Weekly Report (MMWR) of the U.S. Centers for Disease Control and Prevention (CDC) has documented statewide outbreaks, such as “Pertussis Outbreak— Vermont, 1996,” “Notes from the Field: Pertussis—California, 2010,” “Pertussis Epidemic—Washington, 2012,” and, most recently, “Pertussis Epidemic—California, 2014.” This review summarizes the pathogenesis of a strict human pathogen, Bordetella pertussis; CDC guidelines for clinical diagnosis, rapid molecular tests, and the test design principles supporting clinical diagnostics; and the importance of culture and submission of isolates to the local public health laboratory. Finally, we discuss the evolution of a host niche-specialized pathogen and the implications of a vaccination that does not attain complete host immunity.

Background

Corresponding author: Xuan Qin, Ph.D., D(ABMM), Department of Laboratory Medicine, Seattle Children’s Hospital, OC8.720, 4800 Sand Point Way, NE, Seattle, WA 98105. Tel.: 206-9872586. E-mail: xuan.qin@ seattlechildrens.org

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Pertussis, also known as whooping cough, is a highly contagious respiratory infection that still carries a significant incidence of fatality among infants <3 months of age (1,2). During the 2010 outbreak in the state of California, 1,337 cases, including 8 infant deaths, were reported during the first 6 months (258 cases were reported 1 year earlier) (3). The 2012 outbreak in Washington State reached 2,520 reported cases before the end of June, a 1,300% increase compared with the same period in the previous year (4). A significantly high incidence of infection was associated with two specific age groups in these recent outbreaks: infants <1 year old and children 10 to 14 years old. An Editorial Note in the MMWR (20 July 2012) further confirmed that “pertussis is endemic in the United States” (4). Although epidemic peaks have historically cycled in a 3- to 5-year pattern, there has been a gradual and sustained increase in both reported pertus-

sis cases and the frequency of regional outbreaks observed in the U.S. The case count in the U.S. alone from 2012 (n = 41,880) was more than double that of 2011 (n = 18,719) and showed an exponential increase compared to the number of cases recorded in the late 1960s to early 1990s (n = 2,000 to 6,000/year) following worldwide pertussis whole-cell vaccination (http://www.cdc. gov/pertussis/surv-reporting/cases-by-year.html). A population serosurvey in Australia using IgG antibody to pertussis toxin (IgG-PT) has shown a steadily increasing prevalence of undetectable IgG-PT, indicating waning immunity since the late 1990s (5). Since the mid-1990s, the resurgence of pertussis has received a definitive and long-awaited acknowledgement that “the safer (acellular) pertussis vaccines … had come at a steep cost: they do not create immune protection as long-lasting as the (whole-cell) vaccine they replaced” (6). The rise in rates of pertussis infection among 10- to 14-year-old children and

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adolescents suggests that early waning of immunity can be attributed to use of acellular vaccines (component or subunit vaccines have been used interchangeably), which have completely replaced whole-cell vaccines since the early 1990s (6-8).

Pathogenesis of Bordetella pertussis Pertussis infection begins with inhalation of the bacterial particles contained in airway secretory droplets, which are effectively transmitted via the characteristic mechanics of whooping cough. Thus, the organisms gain entry to the mucosal surface of the upper airway and exhibit specific ability to adhere to ciliated cells and initiate a series of actions to overcome the host local innate immune defenses. Once it has achieved initial colonization, the bacterium produces a spectrum of virulence factors orchestrated by a wellstudied regulatory system, namely, the BvgAS two-component regulatory (TCR) system. Like other bacterial TCR systems, the BvgAS system is typically composed of a DNA-binding response regulator, BvgA, and a transmembrane sensor kinase, BvgS, that is able to relay environmental signals by phosphorylation of BvgA (9,10). Bacterial TCR systems are known to respond to environmental cues and modulate gene expression. B. pertussis, which is a human host-restricted pathogen, is able to survey and adapt to a wide range of host conditions via the master regulator, BvgAS. Upon adherence to ciliated host cells, B. pertussis displays virulence features commonly referred to as Bvg+ phase bacteria (as opposed to the ex vivo Bvg< phase, which has been identified only in Bordetella bronchiseptica, or an intermediate Bvgi phase for a physiologic state between Bvg+ and Bvg< during transmission) (1). The Bvg+ bacteria are strictly associated with their infective processes in vivo, with gene expression patterns promoting bacterial adherence to host cells. Genes that are activated by BvgAS include those encoding filamentous hemagglutinin (FHA), fimbriae (Fim), and pertactin (Prn) to ensure host cell attachment and avoid expulsion. These early genetic responders are also an integral part of a subsequent cascade of pathogenic events of host cell cytotoxicity and apoptosis known to be the effects of adenylate cyclase toxin (ACT) and pertussis toxins (Ptx). Bordetella ACT is able to enter the host cytoplasmic membrane with pore-forming activity and turns on a signaling pathway by generating “supraphysiologic” levels of cyclic AMP from ATP, which ultimately results in cell death, in addition to anti-inflammatory and anti-phagocytic activities (11-13). Ptx contains two subunits (A and B) that are made from six polypeptides at the following molar ratio: 1 S1:1 S2:1 S3:2 S4:1 S5. The A subunit (or S1 subunit) is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and shows mitogenicity (14). Ptx is produced only by B. pertussis and is able to modulate host cell metabolism by ADP-ribosylation of G proteins, thereby disrupting host cell signaling pathways (15). Ptx has been associated with the systemic presentations of lymphocytosis and insulinemia during infection, for which the underlying mechanism has yet to be determined (16,17). These bacterial virulence factors that are associated with host airway colonization and injury during pertussis infection are familiar to us, as the fac-

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tors or their components are essential for the construction of the acellular vaccines. Without the ability to live ex vivo, the capacity of B. pertussis to circumvent host-acquired immunity is crucial for its persistence in humans and spreading from host to host. Many studies have shown that Ptx is responsible for many of its direct or indirect toxic effects on host local immunity described above, and it is consequently also responsible for the suppression of serum antibody responses and antibody-mediated bacterial clearance (18). Both B. pertussis ACT and the type III secretion system also inhibit maturation of immune cells through interference with the antigen-presenting cells and complex cytokine-mediated inflammatory responses. Therefore, many of the virulence factors studied to date can be linked to their roles in acting against innate or acquired immunity, contributing to either the pathogenesis of B. pertussis natural infection or the consequences of epidemiologic resurgence due to incomplete and short-lived host immunity postvaccination. Without a proper animal model, there has been no evidence to suggest any particular single protein component is the primary adhesin (1). Based on immunization efficacy studies, vaccines containing both Prn and Ptx provide a synergistic host antibody response (19,20). One study has shown that a multicomponent vaccine containing FIM2/3 is able to generate significantly greater efficacy than a vaccine containing just Ptx, FHA, and Prn (21). These findings, in part, explain the variability of vaccine efficacy regardless of how many components are included in vaccines and account for continued re-emergence of pertussis following mass vaccination. The successful colonization of B. pertussis not only involves dodging host defenses, but also requires competition with upper airway symbiotic bacterial flora for the same pool of nutrients. Iron acquisition is an essential prokaryotic cellular process, as the iron concentration is tightly regulated in the human host. In this multipart microbial competition for free iron, B. pertussis is able to gain an advantage over other bacteria by maintaining three distinct iron uptake systems in its genome (22). Selective activation of these different iron systems is postulated to be part of Bordetella adaptation to the dynamic host environment (23). Furthermore, the changing ecology of the nasopharyngeal (NP) microbiota that is eroded during B. pertussis invasion awaits attention and investigation as we better understand the benefits of the human microbiota and its contribution to health.

CDC Guidelines for Clinical Diagnosis and Immunization Pertussis infection may be divided into three clinical stages: catarrhal, paroxysmal, and convalescent. The catarrhal stage is characterized by low-grade fever, with a mild and occasional cough that gradually increases in both frequency and severity. The infection at this stage may be very contagious for 7 to 10 days. The catarrhal stage is very difficult to differentiate from respiratory infections with other causes, but the recognition of this stage of infection for an early and definitive diagnosis is crucial for preventing the spread of infection. The catarrhal stage is characterized by a paroxysmal cough, with rapid coughs followed by a high pitched, inspiratory “whoop” as the patient gasps for air that passes through

the inflamed upper airway and narrowed epiglottis. This stage of illness is the most debilitating and is accompanied by vomiting and exhaustion that can last 1 to 6 weeks. The convalescent stage concludes with gradual recovery and less persistent paroxysmal coughing that tapers off after 2 to 3 weeks. The latest CDC recommendation has provided 3 points to aid in disease recognition: (i) consider a diagnosis of pertussis in close contacts once even a single case is diagnosed in a household; (ii) apnea can be associated with pertussis in very young infants in place of coughing; and (iii) when pertussis is suspected in an older child, adolescent, or adult, inquire about contact with infants and consider prophylaxis, as severity and risk of fatality are associated with infantile pertussis. These suggestions may help to guide clinicians to take note of clinical signs and exposure indicators for early recognition of pertussis infection. This is a critical clinical opportunity for preventing the spread of infection with an emphasis on protection of the very young and vulnerable population. Pertussis infection has traditionally been defined by three types of clinical criteria: clinical diagnosis, laboratory confirmation, and identification of probable cases (24). A clinically defined case of pertussis features a cough *14 days in duration plus either whooping, paroxysms, or post-tussive vomiting. Laboratory confirmation is based on a patient with any cough, in conjunction with either documented isolation of the B. pertussis organism, detection of B. pertussis DNA by PCR, or serological evidence of pertussis. Other cases of clinical cough are classified as probable infections. Severe clinical complications of whooping cough in infants include frequent apnea, vomiting, and other sequelae, such as pneumonia, hypoxia, seizures, encephalopathy, and secondary respiratory infections (25). Viral co-infection may also occur in a significant number of patients (63%) diagnosed with pertussis (X. Qin et al., unpublished data). The characteristic paroxysmal and persistent cough can be common among young children, but a chronic cough with varying degrees of severity and a lower frequency of whooping is often associated with infection in older children, adolescents, and adults, making the distinction from respiratory illnesses due to viral and other bacterial agents difficult (26). Pertussis vaccination consists of a primary phase of at least 5 years for completion of a childhood immunization series of 5 DTaP (a combination vaccine containing a higher dose of. B. pertussis acellular components combined with diphtheria and tetanus toxoids) doses at 2, 4, and 6 months of age; between 15 and 18 months of age; and at 4 to 6 years. The Advisory Committee on Immunization Practices (ACIP) also recommends a booster Tdap (a combination vaccine similar to DTaP with a lower dose of B. pertussis acellular components) for older children, adolescents, and adults. The ACIP has made it very clear that “adolescents and adults need a booster, even if they were completely vaccinated as children.” Moreover, in place of the past recommendation of Td boosters once every 10 years for adults, a dose of Tdap is recommended for those who did not get Tdap as a teenager or pre-teen. Since 2008, a specific Tdap vaccination method called “cocooning” has

been recommended specifically for pregnant women and all family members where a child is expected (24,27,28).

Molecular Tests and Test Design Principles Supporting Clinical Diagnosis The laboratory diagnosis of pertussis in children has traditionally relied on a combination of rapid direct fluorescent antigen (DFA) detection by microscopic examination, culture, and/or bacterialDNA detection by PCR, as well as serology in the setting of confirmatory and epidemiologic investigation (29,30). Molecular tests using real-time PCR for the detection of bacterial DNA have played a significant role in the last 25 years and have entered the mainstream of laboratory diagnosis of infectious diseases in areas where culture is slow and insensitive (31). Until recently, there has been no FDA-cleared molecular test available for B. pertussis, nor have there been standards for laboratories to follow when laboratory-developed tests (LDTs) were designed and validated independently (32-35). Within the last few years, a number of FDA-cleared multiplex PCR panels (BioFire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP FAST) have been widely adopted by clinical laboratories for diagnosis of respiratory viral infections in the U.S. One of these “mega” multiplex devices has included bacterial etiologies with B. pertussis among the expanded panel (FilmArray Respiratory Panel) (36). Although the technical simplicity (<5 min hands-on time) and rapidity (65 min instrument time) of FilmArray is considered a technologic breakthrough for infectious disease diagnosis, it is not without shortcomings (37). FilmArray’s specimen capacity is limited by a “single specimen on a single analyzer” capacity that is unable to keep pace with large specimen surges during outbreaks (36). Moreover, the bundling of tests for both viral and bacterial agents into a single panel not only may complicate test designation, order compliance, and reimbursement, but also represents a significant cultural shift in infectious disease diagnosis and test choices. Test utilization has traditionally been based on clinical guidelines constructed to recognize discrete differences in microbial agents that may produce characteristic clinical symptoms. The all-in-one molecular diagnostic platforms may put this medical tradition and its teaching to an end. As lumping and splitting technologies and clinical traditions collide once more, laboratories today face many challenges both technically and administratively. Laboratory decision making for implementation and utilization of newer technologies is often constrained by (i) financial and personnel resources; (ii) the maturity and applicability of any new technology for clinical diagnosis and its suitability to a particular medical service; (iii) laboratory capability for developing, maintaining, and continued improvement of an LDT that must be held at a high standard of quality for patient care; (iv) laboratory surge capacity during epidemic outbreaks (which have become more frequent); and (v) ultimate demonstration of patient care improvement with cost control. The laboratory diagnosis of B. pertussis by using a real-time PCR test, which is often an LDT, has become common practice in many

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pediatric centers and high-throughput reference laboratories (30). The technology in general is relatively mature, with ample opportunity for automation. The instrumentation investment is no longer cost-prohibitive to many clinical laboratories; often the cost is greatly outweighed by patient safety associated with this clinically significant infection, where medical intervention and infection prevention are highly dependent on rapid diagnosis. In the acute-care setting, routine pertussis diagnosis requires laboratory testing of specimen material from a deep NP swab for B. pertussis by PCR, with or without culture (this is an area that has not been standardized). Molecular real-time PCR testing is far more rapid and sensitive than culture and far more specific than DFA (38). However, in general, the technical complexities of LDTs with regard to specimen preparation, instrumentation, and PCR result interpretation typically require specially trained technologists, making the test result less rapidly available than it might be in an ideal world. When compared to rapid enzyme immunoassay testing for group A streptococci collected on throat swabs, molecular LDTs for B. pertussis have not acquired “stat” status due to specimen batch modality and multistep operation, which is limited to a few skilled laboratory personnel who are typically available only on the laboratory day shift. Real-time PCR LDTs employ PCR primers with or without probes, using fluorescence resonance energy transfer to evaluate the presence or absence of specific DNA materials in the specimen (39,40). Real-time PCR can have variations in probe types, but its sensitivity and specificity do not vary significantly (30,38). Realtime PCR instrumentation is considered to be a closed nucleic acid amplification system, which, in theory, prevents amplicon carryover contamination. However, potential false-positive or falsenegative results continue to be a major concern with all molecular LDTs, especially when dealing with specimen surges during outbreaks (34,35,41). It is common knowledge that false-positive results can be associated with contamination of either specimens or amplified products, and it requires stringent preventative procedures: (i) special specimen collection procedures to minimize contamination during patient visits; (ii) specimen-handling standards in the laboratory with only small batches (no more than 6 to 10 specimens) or, preferably, automation; (iii) physical separation of pre- and post-amplification environments and complete separation of the two environments; and (iv) recognition of potential homologous DNA sequences shared among Bordetella spp. (42,43). Potential factors contributing to false-negative results are (i) insufficient sampling of the nasopharynx; (ii) small-scale specimen sampling (a few microliters of extracted DNA template from a fraction of a milliliter of eluted NP materials), characteristic of all PCRs; (iii) PCR inhibitors present in the specimen material; and (iv) variability of PCR master mix reagents and isothermal cycling dynamics when a single PCR is used for a diagnosis of a single specimen. In order to prevent both false-positive and false-negative PCR results associated with the known variations listed above, molecular test design can employ the same principles that have been

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used in conventional culture-based microbiology, where more than one type of culture medium is used to maximize the recovery and recognition of a wide spectrum of bacteria displaying diverse growth properties and at the same time rule out potential contamination. Molecular detection of Bordetella spp. shows greatly improved test sensitivity when more than one specific sequence target is utilized with built-in redundancies (e.g., duplicated reactions per target) (44). Two critical considerations associated with development of a molecular LDT are DNA sequence specificity and analytical specificity. For DNA sequence specificity, antecedent research data and comparative genomics among closely related species provide critical information for diagnostic PCR test design. Genome-specific DNA insertion (IS) elements are usually multicopy DNA templates, and their use is advantageous for PCR sensitivity; thus, IS elements have been widely used for PCR targets (29,30,38). IS elements, such as IS481 and IS1001, were first thought to be B. pertussis and Bordetella parapertussis specific, respectively, but were later found to cross-react with IS homologues found in Bordetella holmesii or in some strains of B. bronchiseptica (38,42,43,45). Other Bordetella species- or Bordetella subgroup-specific targets were later incorporated into LDTs to improve both genome specificity and analytical specificity. Based on this concept, a molecular diagnostic test for a specific pathogen(s) should involve a set of PCRs. This set of PCRs would include more than one discriminative DNA target, where each target is tested in duplicate (44,46). Once the real-time PCR chemistry and its instrumentation are determined, the common groundbreaking steps are (i) a thorough literature search and independent examination of the performance characteristics of PCR primers and/or probes for the detection of a specific pathogen(s), (ii) sequence verification of the reference primer and/or probes by GenBank database search to ensure there is no cross-match with other organisms, (iii) a pilot study for proof of concept using known organisms from pure culture or organisms mixed with anatomic-site-specific microbial flora commonly seen in patient specimens, (iv) bacterial dilution series mixed with specific type specimens for test optimization (the ratio of input template material to the PCR mixture) and for evaluation of the limit of detection (LOD), and (v) inclusion of a specific control organism(s) (B. pertussis, B. parapertussis, or B. holmesii) at both the high and low ends of the concentration range to ensure that test performances achieve the expected LOD with precision. For analytical specificity, populating PCRs with built-in redundancy to include more than one specific/discriminative genomic target and more than one reaction per target can provide additional in-run controls and reference values for interpretation. Aside from the quality of DNA input materials, individual PCRs utilize many components, i.e., buffer, nucleotides, Taq polymerase, primers, and/or probes, which are assembled in an optimal mixture to ensure successful DNA amplification. Any variation of the contributing reagents or thermodynamics can influence the outcome of a PCR. Therefore, a single PCR with a few microliters

of input DNA material is not sufficient to determine the presence or absence of a pathogen in a patient specimen. The Bordetella multi-target PCR assay modality that was developed and has continued to be used in our laboratory is summarized in Tables 1 and 2. The primary assay panel (Table 1) is designed to test each patient specimen with a total of 8 PCRs, with duplicate tests of 4 specific PCRs. These 4 specific PCRs include 3 reactions targeting Bordetella-specific sequences: (i) IS481; ii) either of the unique B. pertussis sequences, BP283 or BP485 (in monthly rotation); and (iii) a recA*-specific reaction common to B. pertussis, B. parapertussis, and B. bronchiseptica, but not other Bordetella spp. (Tables 1 and 2). The assay also includes a single PCR targeting human DNA (monthly gene target rotation of either human betaactin or ribonuclease p30) for specimen quality control (44,47).

The consensus discretion criteria required to interpret a positive result call for concurrent detection of 2 or 3 Bordetella-specific targets in at least one of the duplicate reactions (Table 1). When only 1 out of the 3 Bordetella-specific sequences is detected, a confirmatory panel is used to repeat (Table 2). On average, the monthly repeat rates are at 4% (Qin et al., unpublished). Additionally, tracking the trends of repeat rates provides quality markers for potential deviations associated with the reagent, the instrument, competency, and carryover contamination. This follow-up speciesconfirmatory panel consists of 4 alternative specific PCRs, and the result interpretation criteria are shown in Table 2 (44). Although testing in this manner may seem redundant and may involve higher cost, confidence in generating analytical reference values for result interpretation is greatly increased, especially during periods of

Table 1. Interpretation criteria for pertussis PCR primary panela B. pertussis VSHFL¿F PCR (BP283/BP485)b

 recA*c

+

+

+

-

-

-

+

+

-

+

-

-

&RQ¿UPDWRU\ testing performed

Reportd

+

No

Positive for B. pertussis

Culture

+

No

Negative

No culture

+

No

Positive for B. pertussis

Culture

+

+

No

Positive for B. pertussis

Culture

+

+

+

No

Positive for B. pertussis

Culture

-

+

+

Yes

Pre indet

No culture

+

-

-

+

Yes

Pre indet

No culture

-

+

-

+

Yes

Pre indet

No culture

-

-

-

-

No

Fin indet

Culture

IS481 (A/B)b

+XPDQ'1$ (BA/RNP)b

Action

a

Both B. pertussis and B. parapertussisDUHLQFOXGHGDVSRVLWLYHFRQWUROVLQWKLVSULPDU\SDQHOSRVLWLYHQHJDWLYH b 7ZRVHWVRI,6481$DQG% WZRVHWVRIB. pertussisVSHFLÀF3&5V%3DQG%3 DQGWZRVHWVRIKXPDQ'1$FRQWUROVKXPDQEHWDDFWLQ>%$@DQGKXPDQULERQXFOHDVHS >513@ ZHUHDOWHUQDWHGPRQWKO\LQRUGHUWRUHGXFHWKHIDOVHSRVLWLYHUDWHGXHWRFRQWDPLQDWLRQ c 7KHSRO\PRUSKLFUHF$FRGLQJVHTXHQFHZDVXVHGWRGHVLJQD3&5SULPHUrecA*) that is common to three related species (B. pertussis, B. parapertussis, and B. bronchiseptica). d 3RVLWLYHLQHLWKHURIWKHGLOXWLRQGXSOLFDWHVZDVDFFHSWHGDVSRVLWLYH3UHLQGHWSUHOLPLQDU\UHSRUW3&5LQGHWHUPLQDWHFRQÀUPDWRU\3&5SHQGLQJ)LQLQGHWÀQDOUHSRUW3&5 indeterminate.

Table 2. Interpretation criteria for Bordetella3&5FRQÀUPDWRU\SDQHOa IS481 (A/B)

ptx-Pr PCRb

recA*c

recABHc

Reportd

Action

+

+ (Tm = BP)

+

-

Positive for B. pertussis

Culture

-

-

-

-

Negative

No culture

+

+ (Tm = BP)

-

-

Positive for B. pertussis

Culture

+

-

+

-

Positive for B. pertussis

Culture

-

+ (Tm = BP)

+

-

Positive for B. pertussis

Culture

-

-

-

Positive for B. pertussis

Culture

+ reproducible

-

-

Positive for B. pertussis

Culture

-

+ reproducible -

-

+ (reproducible)

-

+ (Tm = BPP)

+

+

-

-

+

Negative

Culture

Positive for B. parapertussis

Culture

Positive for B. holmesii

Culture

a

Three control organisms (B. pertussis, B. parapertussis, and B. holmesii DUHLQFOXGHGLQWKLVFRQÀUPDWRU\SDQHOSRVLWLYHQHJDWLYH,QDGGLWLRQWR5HJDQ/RZHDJDUPHGLXP VKHHSEORRGDJDUDQGFKRFRODWHDJDUSODWHVZHUHDGGHGWRFXOWXUHVIRUERWKB. parapertussis and B. holmesii$OOLVRODWHGEDFWHULDZHUHFRQÀUPHGE\3&5SDQHOVDSSURSULDWHIRUWKH VSHFLHVLGHQWLÀFDWLRQ b PCR for the ptx promoter (ptx-Pr) region produced distinctive melting temperature (Tm SHDNVVSHFLÀFIRUB. pertussis (Tm = BP) and B. parapertussis (Tm = BPP). c 7KHSRO\PRUSKLFUHF$FRGLQJVHTXHQFHZDVXVHGWRGHVLJQRQH3&5SULPHUrecABH WKDWLVVSHFLÀFIRUB. holmesii and one (recA*) common to three related species (B. pertussis, B. parapertussis, and B. bronchiseptica). d 3RVLWLYHLQHLWKHURIWKHGLOXWLRQGXSOLFDWHVZDVDFFHSWHGDVSRVLWLYH

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specimen surge. Moreover, multi-target PCR combinations can be designed to include differentiation of Bordetella spp. (Tables 1 and 2). This adds value in epidemiologic investigations during outbreaks, as both B. parapertussis and B. holmesii can cause prolonged cough symptoms and are not vaccine preventable (4,44). There is a golden opportunity for implementation of specimen collection standards during the introduction of a new test or at the time of specimen surge. The laboratory diagnosis of respiratory pathogens in the acutely ill patient has been hampered by the need for the provider to specify which pathogen(s) should be evaluated and which NP specimen (i.e., NP swab, wash, or aspirate) should be collected. Collection devices (such as NP swabs) and the type of material used in these swabs (rayon, Dacron, and flocked swabs), as well as NP aspirate collection methods, have been extensively investigated by the CDC, which has offered recommendations for using these materials (48) (http://www.cdc.gov/pertussis/clinical/ downloads/diagnosis-pcr-bestpractices.pdf). Detailed construction and communication of specimen collection standards and test order guidelines are crucial to the success of rapid laboratory diagnosis. Diagnostic order sets that are made patient specific, service specific, or syndrome specific can be helpful tools to promote the culture of patient safety, antibiotic stewardship, and overall health care cost reduction.

,PSRUWDQFHRI&XOWXUHIRU3XEOLF+HDOWK/DERUDWRU\ Evaluation and Research The importance of isolating an organism by culture from clinical samples cannot be over-emphasized in light of the large-scale, multistate outbreaks that are occurring with increasing frequency. Molecular analysis of currently circulating isolates compared to historical strains provides a great opportunity, not only for assessing antimicrobial susceptibility patterns (although susceptibility to macrolides remains largely unchanged), but more importantly for understanding the worldwide re-emergence of an infection that was once thought to be vaccine preventable (49-51). Worldwide resurgence of pertussis, even in highly vaccinated populations, has led to many studies that have examined the association of bacterial antigenic variations via mutations specifically in genes encoding the antigenic products used in acellular component vaccines (52). In addition to the distinctive mutations described in ptx, prn, and fim, the isolation of pertactin-negative strains in circulating Bordetella isolates is even more startling (50,53,54). In our recent experience with the 2012 outbreak in Washington State, a true surge of >5,000 specimens received in a 6-month period, was a great test of laboratory disaster preparedness at our 23-member microbiology laboratory, which typically serves a 300bed tertiary pediatric hospital with continuous 24-h coverage. All NP specimens were collected on either a flocked swab or a rayontipped swab on a thin and flexible wire shaft (usually a single swab per specimen). The NP swabs were processed by vortexing in 1 ml of saline and then further concentrated for multi-target PCR (44). After saline elution, the swab specimens were refrigerated pending PCR test results. All Bordetella pathogen-positive specimens (including 175 positive for B. pertussis, 11 positive for B. paraper-

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tussis, and 2 positive for B. holmesii) detected during the outbreak period were cultured using the saline-eluted swab. Despite common knowledge that swab specimens post-elution are significantly compromised for culture sensitivity, we were able to achieve a nearly 48% culture-positive rate (including 85 B. pertussis, 6 B. parapertussis, and 1 B. holmesii) (55). Thus, the organisms may be “fastidious” but not as “fragile” as once thought when specimens are delivered promptly without transport medium. This practice of “PCR positive, culture pending” has been our standard laboratory practice since the year 2000, during which time hundreds of isolates have been made available for various molecular investigations that have shown general agreement with worldwide findings (56-58).

Evolution of a Niche-Specialized Pathogen and the ,PSOLFDWLRQVIRU(I¿FDF\RI9DFFLQDWLRQ Why pertussis vaccine fails has become a hot topic of discussion, not only by medical practitioners, but in the scientific community at large (59). The initial conclusions from acellular vaccine trials became questionable due to overly high expectations, inflated initial efficacy bias, and the use of incomplete or unbalanced antigens that did not engender vaccine potency and achieve long-lasting antibody responses. Furthermore, the circulating misconceptions surrounding vaccines have further affected vaccine coverage in communities and reduced the effect of herd immunity (60). B. pertussis has been isolated only from humans, together with the occasional isolation of B. parapertussisHU (a human host-specific lineage as opposed to B. parapertussisOV, found in sheep) and B. holmesii (61,62). B. pertussis is an extremely monomorphic human pathogen, based on strain typing of the worldwide collection of both historic and currently circulating isolates (50,63,64). The genome stasis in both B. pertussis and B. parapertussis is indicative of niche specialization compared to the common ancestral bacterium, B. bronchiseptica. The latest genome-wide comparison of the pre- and post-vaccination strains has revealed no acquisition of novel genes but has identified single nucleotide polymorphisms (SNPs) in currently circulating strains (65,66). These SNPs are allelic changes that are not found in acellular vaccine sequences and are associated with changes in gene regulation. The characteristic SNPs detected are postulated to be a result of the acellular vaccine immune selection (66). The specific SNPs have played a critical role in the enhanced virulence of B. pertussis, due in part to increased strength of the ptxA promoter sites and binding sites for the BvgA regulatory protein (22,65,67). The reemergence of this niche-specialized pathogen in the human population is postulated to interfere with host immune system function post-colonization, such as the inhibition of complement-phagocytic killing and/or the suppression of T-cell and B-cell responses with increased transmissibility among its hosts (68). The pertussis acellular vaccine is known to produce incomplete and short-lived host immunity (6). The highly variable host immunity is further affected by unchecked immunization practices. Under these conditions, the evolutionary selective pressure forces this human host-restricted bacterium to lose species diversity while

maintaining virulence (69). Moreover, the importance of healthy microbiota versus dysbiotic consequences due to the misuse or overuse of antibiotics can not be overstated (70,71). A similar analogy has been used to account for the increasing prevalence of highly resistant and highly virulent clonally distinct “superbugs,” such as methicillin-resistant Staphylococcus aureus, highly invasive Streptococcus pneumoniae, and multidrug-resistant strains of Enterobacteriaceae. These species of bacteria can also exist as members of our microbiota, and they were once clonally highly diverse in host populations (72-74). The clonal explosion of highly virulent and highly transmissible superbugs may be a result of the antibiotic perturbation of our diverse microbiota and of selected vaccines that are unable to eradicate the intended organism from the host population. This may explain the rapid emergence and spread of these organisms, in which the range of host populations affected is no longer limited or typical (71,75,76). It is well documented in modern medical history that vaccination either generates complete immunity and brings about global eradication of agents (such as smallpox and polio in the near future) or may influence bacterial adaptation to incomplete host and herd immunity, as with S. pneumoniae and B. pertussis (51,77,78). Therefore, our emphasis must be placed, not only on rapid laboratory diagnosis as crises arise, but also on better understanding of the combinatorial effects of the microbe-host-antibiotic triad that are narrowly encapsulated by an interconnected world and by the human history of infectious diseases and vaccination. Still, this level of learning and understanding cannot be achieved without laboratory culture and isolation of organisms.

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