California Association for Medical Laboratory Technology

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1 California Association for Medical Laboratory Technology Distance Learning Program WHAT S GOING ON WITH WHOOPING COUGH (PERTUSSIS)? Course # DL-997 by James I. Mangels, MA, CLS, MT(ASCP) Consultant Microbiology Services Santa Rosa, CA Approved for 3.0 CE CAMLT is approved by the California Department of Public Health as a CA CLS Accrediting Agency (#21) Level of Difficulty: Intermediate Phone: Fremont, CA FAX: Notification of Distance Learning Deadline DON T PUT YOUR LICENSE/CERTIFICATE IN JEOPARDY! This is a reminder that all the continuing education units required to renew your license/certificate must be earned no later than the expiration date printed on your license/certificate. If some of your units are made up of Distance Learning courses, please allow yourself enough time to retake the test in the event you do not pass on the first attempt. CAMLT urges you to earn your CE units early!

2 DISTANCE LEARNING ANSWER SHEET Please circle the one best answer for each question. COURSE NAME WHAT S GOING ON WITH WHOOPING COUGH (PERTUSSIS)? COURSE # DL-997 NAME LIC. # DATE SIGNATURE (REQUIRED) ADDRESS STREET CITY STATE/ZIP 3.0 CE FEE: $36.00 (MEMBER) $66.00 (NON-MEMBER) PAYMENT METHOD: [ ] CHECK OR [ ] CREDIT CARD # TYPE VISA OR MC EXP. DATE SECURITY CODE: a b c d 11. a b c d 21. a b c d 2. a b c d 12. a b c d 22. a b c d 3. a b c d 13. a b c d 23. a b c d 4. a b c d 14. a b c d 24. a b c d 5. a b c d 15. a b c d 25. a b c d 6. a b c d 16. a b c d 26. a b c d 7. a b c d 17. a b c d 27. a b c d 8. a b c d 18. a b c d 28. a b c d 9. a b c d 19. a b c d 29. a b c d 10. a b c d 20. a b c d 30. a b c d DISTANCE LEARNING EVALUATION FORM According to state regulations, this form must be completed and returned in order to receive CE hours. Your comments help us to provide you with better continuing education materials in the distance learning format. Please circle the number that agrees with your assessment with, with 5 meaning you strongly agree and 1 meaning you strongly disagree. 1. Overall, I was satisfied with the quality of this Distance Learning course The objectives of this Distance Learning course were met The difficulty of this Distance Learning course was consistent with the number of CE hours I will use what I learned from this Distance Learning course The time to complete this Distance Learning course was: hours 6. Please comment on this Distance Learning course on the back of this sheet. What did you like or dislike?

3 WHAT S GOING ON WITH WHOOPING COUGH (PERTUSSIS)? Course # DL CE Level of Difficulty: Intermediate James I. Mangels, MA, CLS, MT(ASCP) Consultant Microbiology Services Santa Rosa, CA OUTLINE A. Introduction B. History of Whooping Cough Infection C. Transmission of Bordetella pertussis D. Illness and Clinical Symptoms of Whooping Cough E. Microbiology of Bordetella pertussis F. Pathogenic Mechanisms of Bordetella pertussis G. Laboratory Diagnosis of Whooping Cough H. Treatment of Whooping Cough I. Prevention and Control of Bordetella pertussis Infection J. Conclusions K. Case Study L. References COURSE OBJECTIVES Upon completion of this course, the participant will be able to: 1. outline the history of whooping cough infection; 2. discuss the incidence of B. pertussis infection in the U.S. and in California; 3. list factors involved in the increased incidence of whooping cough in California; 4. explain the pathogenic mechanisms of Bordetella pertussis; 5. outline the clinical features of Bordetella pertussis infection; 6. describe how the laboratory identifies B. pertussis; 7. list methods of preventing and treating whooping cough. A. INTRODUCTION Whooping cough, or pertussis, is caused by the organism Bordetella pertussis. Pertussis is an acute bacterial infectious disease, and one of the most important diseases of children throughout the world. Pertussis is a highly communicable infection of the respiratory tract spread by coughing or sneezing. The term whooping cough refers to the intensive cough followed by an inspiratory whoop sound. Bouts of coughing due to B. pertussis can last so long that patients are deprived of oxygen. At the end of a coughing bout, they forcefully draw air back into the lungs producing the characteristic whoop. While the whooping stage is not universal, it is the hallmark of the clinical disease and has given the disease its name. People sick with pertussis have severe coughing attacks that can occur for months. The terms whooping cough and pertussis will be used interchangeably throughout this course. 1 CAMLT Distance Learning Course DL-997

4 The genus Bordetella is named for Jules Bordet along with Octave Gengou, who in 1906 first described and recovered the organism on special media they developed (1). The two microbiologists from Belgium noticed epidemics of severe coughing or whooping associated with a high mortality among infants and young children. The organism was originally called Bordet-Gengou bacillus, then later assigned the name Hemophilus pertussis. Bordetella spp., however, require different extraneous growth factors and have very distinct genetic differences from the genus Hemophilus spp. Thus, the organism causing whooping cough was given in its own genus, Bordetella. There are currently eight species within the genus Bordetella: B. pertussis, B. bronchiseptica, B. parapertussis, B. avium, B. hinzii, B. holmessii, B. trematum, and B. ansorpii (1). B. pertussis and B. papapertussis are strict human pathogens causing respiratory tract infections (1). Although there is some genetic relatedness among B. pertussis, B. parapertussis, B. bronchiseptica, and B. holmesii (1), this course will only discuss B. pertussis because it is the pathogen responsible for the majority of pertussis cases. It also causes respiratory symptoms of greater severity than the other Bordetella species. B. pertussis produces disease only in humans, who serve as the sole reservoir for the organism. Whooping cough occurs throughout the world in immunized and un-immunized populations causing outbreaks or epidemics in cycles of two to five years. In 2005, for example, B. pertussis caused an estimated 16 million cases of pertussis worldwide and 195,000 deaths, primarily among children in countries lacking organized vaccination programs (2,3). According to data from the CDC and WHO, 95% of the infections were in developing countries, and 86% of these infections were in infants <2 yrs of age (2,3). However, the incidence of pertussis worldwide was much lower in 2015 with 145,500 reported cases of pertussis. Although infection may occur at any age, the clinical disease is most frequently recognized in infants and young children and may cause serious pulmonary complications, and even death. Mortality is associated with the severity of coughing, and is especially prevalent in infants under 1 year of age. California experienced a major outbreak of pertussis in 2014, with more than 11,203 infections reported (5). There were more than 5,900 cases of pertussis reported in 2010, and more than 10,831 in 2014 (5). The number of cases in 2014 is the most cases in California reported in 67 years, when 9,394 were reported in Prior to the major outbreak in 2014, there were minor outbreaks of pertussis such as in 2005 with 3,182 cases reported and 2011 with 2,932 (5). While there was a downward trend in the incidence of pertussis cases during the period , the number of pertussis cases in 2014 increased significantly (see Figure 1). It is believed by healthcare authorities that the high number of pertussis cases is due to the decrease in individuals that are being immunized with the pertussis vaccine, or are not receiving the recommended booster shot (3, 4). (See Figure 1.) 2 CAMLT Distance Learning Course DL-997

5 (Note: this figure needs to be replaced with Figure 1 provided by Jim Mangels ) Figure 1. Number of Whooping Cough Cases in California by Year* Number of Cases Year *Adapted from Ref. #5: California Department of Public Health. Pertussis Summary Reports. B. HISTORY OF WHOOPING COUGH INFECTION Outbreaks of whooping cough were described as early as the 16 th century in Europe. One of the first detailed reports of the disease was from Paris, France, in 1578, that described an epidemic among young infants and children that had a whoop sounding cough (1). Throughout the 18 th and 19 th centuries, whooping cough was one of the most common childhood diseases and a major cause of childhood mortality throughout the world. It was not until 1906, however, that the research of the two Belgium microbiologists, Bordet and Gengou, led to the discovery of the causative organism of whooping cough, Bordetella pertussis. Their research further led to the development of a medium (BG plate) that permitted the growth of pertussis (see Section G: Laboratory Diagnosis of Whooping Cough for composition). In 1909, Bordet and Gengou developed a method of inoculation of B. pertussis in 3 CAMLT Distance Learning Course DL-997

6 which the patient would cough directly onto the surface of the BG plating medium. This procedure was called the cough plate technique. It is no longer used because good transport systems are available to maintain the viability of the organism. Prior to the introduction of the pertussis vaccine in the late 1940s, whooping cough was a common cause of infection and death among children. As many as 265,000 cases and 7,500 deaths were attributed yearly to B. pertussis infection in the U.S. during the 1920s and 1930s, more than the incidence of the childhood diseases of diphtheria, scarlet fever, or measles. During the six-year period from 1940 through 1945, for example, more than 1 million cases of whooping cough were reported in the U.S., an average of 200,000 cases per year (1). Since the widespread use of the vaccine, however, the incidence has decreased more than 80% compared with the pre-vaccine era, gradually declining in the U.S. to 15,000 reported cases in 1960 (3). While the incidence of pertussis is cyclical, with peaks occurring every 2-5 years in the United States, there has been a general increase in the number of reported cases of pertussis since the 1980s (3). The number of pertussis cases has fluctuated over the last few years, but has remained relatively high. For example, the number of cases reported to the Centers for Disease Control and Prevention (CDC) in 2012 was 48,277 cases, the highest number since the 1940s. Whereas, data from 2013 revealed 28,639 cases, in 2014 there were 32,971 cases and 2015 there were 20,762 cases of pertussis (3). A breakdown of the cases by age from 2015 showed 1,970 (9.5%) cases in <6 mos of age, from 6-11 mos 739 (3.3%) cases, from 1-6 yrs 3,739 (18%), from 7-10 yrs of age 2,892 (14%) of cases, from yrs of age 6,734 (32%) of cases, and from over 20 yrs of age 4,650 (22%) of cases. There were 6 fatalities in 2015 due to pertussis infection, with most deaths in children, particularly infants under 3 months of age (3); the second highest group of fatalities due to pertussis was in the <1 year of age. These figures are probably lower than the true incidence because the CDC estimates that only 5-10% of all cases of pertussis are recognized and reported to public health agencies (3). During the period of , the highest annual whooping cough incidence occurred in infants younger than 1 year (up to 40% of all cases), particularly in infants younger than 6 months of age (3). However, by 2015, the incidence was also particularly noticeable among children 7-10 years of age with 14% of cases and among adolescents years of age with 32% of cases (3,4). Several factors have likely contributed to the increase in reported cases among adolescents, including increased awareness and improved recognition of pertussis among clinicians; greater access to and use of laboratory diagnostics, especially polymerase chain reaction (PCR) testing; and increased surveillance and reporting of pertussis to public health departments. However, the most likely cause for the increased number of reported cases of whooping cough among year olds is believed to be a decrease in immunization among this group. Prior immunization or infection with B. pertussis does not provide lifelong immunity as earlier thought, so it is likely that adolescents or adults are actually acquiring the infection again (1,3). Public health experts believe that the increase among infants (<1 year) is probably because many infants are not receiving the correct number of doses of the whooping cough vaccine, perhaps because of parents fears regarding the general safety of vaccinations, and because infants are acquiring the organism from an infected adolescent or adult (4). Of the more than 4,707 cases of pertussis reported in California in 2015, the majority were infants <6 months of age, while the second highest number of cases was in older children years of age representing 14% of all cases. The incidence in California was similar to the trend seen nationwide. There were 94 hospitalized cases with 2 deaths (5). Overall pertussis rates were highest for infants <1 year of age and older children and teens years of ages. Rates by race/ethnicity are highest for white and Hispanic infants <6 months of ae and white, non-hispanic adolescents and teenagers aged 11-4 CAMLT Distance Learning Course DL-997

7 17 year of age. Among the pediatric cases (<1 year of age) with vaccination history information, 45% had never received any doses of pertussis-containing vaccine (5). While California had the majority of pertussis cases in 2015 at 4,707 cases among all the U.S. states, significant whooping cough outbreaks also occurred in Texas, Ohio. Florida, Michigan, New York, North Carolina, and Arizona (3). Worldwide, rate of pertussis has decreased in most parts of the world primarily due to the efforts of the World Health Organization. WHO estimates that in 2015, 86% of individuals have had at least three doses of pertussis vaccine (either the whole cell vaccine or the acellular vaccine) (2). Furthermore, WHO data from 2008 estimated that there were 294,000 deaths related to pertussis infection worldwide; however, in 2015 there were 145,500 reported cases and 89,000 estimated deaths due to pertussis infection primarily among young children (2). C. TRANSMISSION OF BORDETELLA PERTUSSIS B. pertussis is a highly contagious infection and is transmitted from person to person via respiratory droplets and secretions. Since B. pertussis is spread by aerosol respiratory droplets that are breathed in by the host, relatively close contact (within several feet) is required for transmission. B. pertussis in respiratory droplets may remain viable for 15 minutes (1,3). Transmission occurs less frequently by contact with freshly contaminated fomites (articles) from an infected person, but is possible, so precautions need to be taken. An asymptomatic carrier state is not recognized for B. pertussis, and the disease occurs only in humans. Due to their lack of or waning immunity from childhood vaccination or previous disease, adolescents and adults with B. pertussis infection are often the source of transmission to infants. Persons with pertussis are most infectious during the catarrhal period, the first 2-3 weeks after the initial onset of symptoms (an average of 21 days). (See Table 1. Stages of Whooping Cough). However, since the disease may develop slowly and secretions are contagious during both the early catarrhal and the paroxysmal phases (see Section D: Illness and Clinical Symptoms of Whooping Cough), an infected person may unknowingly transmit disease for several weeks. During the first 3 weeks of infection, a child s cough contains enough pertussis bacilli to infect many other children, but by 5 weeks most patients are noninfectious (3). B. pertussis has a very high infection rate (the rate of obtaining infection from an individual with the disease); up to 90% of family members, and up to 25 to 50% of schoolroom contacts with unvaccinated children produce clinical disease following exposure (3). This is a high rate of infection for a bacterial disease and is approximately that of viral diseases such as measles and chickenpox. Mortality from pertussis is about 1%. Sixty-four per cent of the deaths are infants under 1 year of age, while 40% of deaths occur during the first 5 months (3). Pertussis is unique in that its incidence and mortality are significantly greater among females (3). The reason for the higher incidence of pertussis in females is not known. D. ILLNESS AND CLINICAL SYMPTOMS OF WHOOPING COUGH Although the infection may occur at any time of the year, most cases of whooping cough are observed during the spring and summer months. The incubation period for whooping cough is commonly 7 10 days, with a range of 4 21 days, and rarely may be as long as 42 days (1). B. pertussis initially enters the nasopharyngeal area in aerosol droplets (exhaled droplets suspended in the air). The organism then attaches to and colonizes the ciliated respiratory epithelial cells in the bronchi and trachea. B. pertussis is the only pathogenic bacterium known to colonize this cell type exclusively. The symptoms of whooping cough can be variable in their course, but generally the illness is divided into 3 5 CAMLT Distance Learning Course DL-997

8 stages that often overlap: 1) the catarrhal, 2) the paroxysmal, and 3) the convalescent. (See Table 1. Stages of Whooping Cough.) Table 1. Stages of Whooping Cough Stage Length Clinical Features Catarrhal Stage Paroxysmal Stage Begins after 7-10 days after exposure, but can range from 4-21 days after exposure. Catarrhal stage lasts 1-2 weeks. Usually lasts 1-6 weeks, but may persist for up to 10 weeks Coryza (runny nose), low-grade fever, mild occasional cough which gradually increases. Sore throat, early symptoms consistent with upper respiratory viral infection, sneezing and nasal congestion. Progressive cough becoming paroxysmal (coughing fits) Long inspiratory effort develops into high-pitched whoop. Cyanosis, vomiting after coughing attack. Paroxysmal attacks occur frequently at night and increase in frequency lasting 4 to 6 weeks. Infants younger than 6 months of age may not have the strength to have a whoop. Convalescent Stage May last up to 4 weeks Slow recovery, with less persistent paroxysmal coughing that may gradually disappear in 3 to 4 weeks or longer. Paroxysms often recur with subsequent respiratory infections for many months. * Adapted from Ref. #1: Jorgensen, JH., Phaller, MA., Carroll, KD., Funke, G., Landry, ML., Rickles, SS., and Warnock, DW. Manual of Clinical Microbiology, 11 th ed. ASM Press, American Society for Microbiology, Washington, DC. 2015, and Ref. #3, Centers for Disease Control and Prevention (CDC). Pertussis (Whooping Cough) Fact Information The first stage, the catarrhal stage, begins after the incubation period, about 7 to 10 days following exposure. The usual early manifestations are similar to those of any early upper respiratory viral infection and are characterized by the insidious onset of nasal congestion, irritation of the mucous membranes, sneezing, runny nose, low-grade fever, and a mild, occasional cough, similar to what one might expect for the common cold (3). Symptoms are nonspecific during the catarrhal period, and pertussis is frequently not suspected and the clinical diagnosis elusive because the typical cough with whoop appears later in the illness. The cough, however, gradually becomes more severe, and after 1 2 weeks, the second, or paroxysmal stage, begins. Fever is generally minimal throughout the course of the illness. The first stage is probably the most communicable period of the disease. Bacterial cultures in infants and children may be useful during the first two weeks of illness for diagnosis, while the diagnosis by bacterial culture in adults is less likely to be helpful. Samples submitted for PCR testing may be useful in infants and children, and are also productive on adults during this period (See Section G. Laboratory Diagnosis of Whooping Cough). The second stage, called the paroxysmal stage, is where the diagnosis of pertussis is usually suspected because of the development of classic symptoms. The paroxysmal stage may last 1 to 6 weeks 6 CAMLT Distance Learning Course DL-997

9 (as long as 10 weeks in some cases). Characteristically, the patient has bursts, or paroxysms, of numerous, rapid coughs because of the difficulty of expelling thick mucus from the tracheobronchial area. The sudden crescendo of severe coughing fits (paroxysms) is usually accompanied by a characteristic high-pitched whoop when the person labors to breathe in (you can listen to the whooping cough sound at: During such an attack the patient may become cyanotic (turn blue) due to a lack of oxygen intake. The patient may vomit after a coughing fit and become exhausted. Infants and children generally have the most severe symptoms and appear very ill and distressed. In children under 6 months of age, the most common symptom is apnea (suspension of breathing) and cyanosis. The whoop is often absent (they may not have the strength to have a whoop), but the choking spells make it hard for infants to eat, drink, or breathe. Posttussive vomiting is often associated with the choking cough, which often leads to dehydration in the infant. Infants have the highest hospitalization and complication rates. Fifty percent or more of infants <6 months of age who contract pertussis are hospitalized. Twenty-five percent develop pneumonia, four percent develop seizures, one percent suffer permanent brain damage and another one percent die (1,2,3). Older children and adults may also lack the whoop, but have a prolonged paroxysmal cough and vomiting as symptoms. The classic signs of pertussis during this stage are: 1) a progressive cough that becomes paroxysmal (bursts of uncontrolled coughing), 2) coughing that often ends in vomiting, and 3) a forced inspiration (the whoop). Generally, bacterial cultures for B. pertussis during this later period are not useful since the organism is no longer present. The most useful laboratory diagnostic test during this late period may be PCR testing. Ultimately, the paroxysms may be induced by any stimulus (e.g., swallowing, movement, noise, exertion), and they occur with increasing frequency, severity, and duration as the paroxysmal stage progresses. Each paroxysm may consist of several bouts separated by momentary intervals insufficient for inspiration, until hypoxia (reduction of oxygen supply to tissues) and partial asphyxia (decrease in oxygen accompanied by increase in CO 2 which can lead to loss of consciousness) occur. The end of the paroxysmal episode may be marked by a massive single inspiratory stroke causing the classic whoop. During severe paroxysms, patients may become very anxious, believing their life is threatened. Paroxysmal coughing may vary from mild, uncomplicated episodes to severe, protracted episodes requiring hospitalization and acute supportive care, particularly for infants. Following the early paroxysmal stage, attacks occur more frequently at night, with an average of 15 attacks per 24 hours. The attacks increase in frequency as the disease progresses, and may continue to occur over a prolonged period of time, often 4-6 weeks, but may persist for up to 10 weeks, and then gradually decrease (1,3). In the third stage, or convalescent stage, the patient gradually recovers from the disease and may have a non-paroxysmal cough for 4 to 6 weeks (sometimes as long as 3-4 months). In the convalescent stage, recovery is gradual and slow. The cough becomes less paroxysmal and less severe. However, for many months after the initial onset of pertussis, paroxysms can often recur with subsequent respiratory infections related to other agents (e.g., colds, flu) (1,3). Before the development of the pertussis vaccine in the late 1940s, the mortality of whooping cough was greater than 50%. Today, the mortality rate for unvaccinated infants is about 2.4%, whereas in vaccinated infant populations it is approximately 1% for infants younger than 2 months of age and less than 0.5% for infants 2-11 months of age (4). Children who are too young to be fully vaccinated and those who have not completed the primary vaccination series are at highest risk for severe illness. Complications of pertussis are steadily declining, but can occur. The most common complication, and the cause of most pertussis-related deaths, is secondary bacterial pneumonia. Other complications can include dehydration, hypoxia seizures, encephalopathy and coma, and hemorrhagic events, particularly 7 CAMLT Distance Learning Course DL-997

10 in the conjunctivae and soft tissues about the eyes, because of the high venous blood pressure exerted during coughing fits. E. MICROBIOLOGY OF BORDETELLA PERTUSSIS The morphology of B. pertussis when first isolated on culture media is a very small, oval bacillus which stains faintly Gram-negative. Its mean length and width is 0.5 micron (E. coli, for example, is commonly 2 to 3 microns in length and 0.6 microns wide). Bacterial cells from original cultures are uniform in size, but on subculture, the organism may become pleomorphic, tending toward coccobaccillary, sometimes exhibiting a bipolar appearance. B. pertussis survives only a short time outside the human host, and is strictly aerobic. The organism has an optimal growth temperature of 35 to 37 o C. Many studies show that the addition of carbon dioxide to the incubation atmosphere may slow or inhibit the growth of B. pertussis (1). B. pertussis oxidizes amino acids as an energy source, but does not utilize any carbohydrates (6). The organism is catalase-positive, urease negative, nitrate reduction negative, and nonmotile. B. pertussis is easily killed by desiccation, heat, and chemical agents, and does not survive on environmental surfaces for any length of time. B. pertussis is the most fastidious species of the Bordetellae, and is inhibited by constituents present in many media, including fatty acids, metal ions, sulfides, and peroxides. The growth of B. pertussis on artificial media is slow, requiring up to 7 to 10 days, rarely up to 14days, for its initial isolation in some cases (1,6). Media for the primary isolation of B. pertussis must contain protective substances, such as charcoal, blood, or starch. Bordet-Gengou, a potato infusion-based blood medium supplemented with charcoal, is one option, while another option is Regan-Lowe, a charcoal-based medium supplemented with glycerol, peptones, and horse or sheep blood. See Section G, Laboratory Diagnosis of Whooping Cough for more information on primary media. B. pertussis will not grow on common blood agar bases or chocolate agar, whereas B. parapertussis will grow on blood agar and sometimes chocolate blood agar. F. PATHOGENIC MECHANISMS OF BORDETELLA PERTUSSIS B. pertussis produces multiple virulence factors that are classified generally as adhesins or toxins. These factors are responsible for the pathogenicity of B. pertussis and contribute to the specific clinical features of the disease. Whooping cough is primarily a toxin-mediated disease. Once aerosols containing B. pertussis enter the nasopharynx, the organism attaches to and then multiplies on the ciliated respiratory epithelial cells, starting first in the nasopharynx and ending in the bronchi and bronchioles in the lungs. One of the major factors that permit B. pertussis to attach and colonize the ciliated respiratory epithelial cells is an adhesin called filamentous hemagglutinin (FHA), a protein that promotes the binding of B. pertussis to specific carbohydrate structures on the ciliated respiratory epithelium, as well as to the host s phagocytic cells. In addition to FHA, B. pertussis has fimbriae or pili (hair-like protein projections on the bacterial cell wall to allow attachment to the respiratory epithelium), that assist in infection. Recent studies indicate that fimbriae play a major role in the attachment of B. pertussis to the laryngeal mucosa, whereas FHA is important for colonization of the entire respiratory tract (1). A third class of adhesin is called pertactin, a highly immunogenic (provoking an immune response) virulence factor on the outer membrane of only B. pertussis that promotes attachment of the organism to tracheal epithelial cells and binding to phagocytes. (See Table 2: Virulence Factors Associated with the Pathogenicity of Bordetella pertussis.) 8 CAMLT Distance Learning Course DL-997

11 Table 2. Virulence Factors Associated with the Pathogenicity of Bordetella pertussis* Adhesins Mode of Action Filamentous hemagglutinin (FHA) Fimbriae Pertactin Toxins Pertussis toxin Adenylate cyclase toxin Tracheal cytotoxin Tracheal cytotoxin This filamentous protein allows B. pertussis to bind to specific carbohydrate structures on respiratory epithelial cells as well as to phagocytic cells. It permits colonization of the entire respiratory tract. Hair-like structures on the surface of B. pertussis that bind to laryngeal mucosa epithelium. Important for colonization. A highly immunogenic virulence factor from only B. pertussis. Pertactin promotes binding to tracheal epithelial cells, and to phagocytes. Mode of Action Pertussis toxin allows the binding of B. pertussis to host epithelial cells, and allows the organism to evade host defenses by altering host cell activity and disrupting host phagocytic activity. Pertussis, cholera and diphtheria toxins act in a similar manner. Adenylate cyclase toxin penetrates phagocytic cells, disrupting their normal function and inhibiting their ability to signal to one another, reducing chemotaxis (the movement of phagocytic cells toward an infection site) in the host. Adenylate cyclase toxin eventually kills the phagocytic cells. This toxin, considered to be a component of the B. pertussis cell wall peptidogylcan (a layer outside the bacterial cell membrane), inhibits ciliary motility, inhibits DNA synthesis, and ultimately causes death of ciliated epithelial cells. This toxin, considered to be a component of the B. pertussis cell wall peptidoglycan (a layer outside the bacterial cell membrane), inhibits ciliary motility, inhibits DNA synthesis, and ultimately causes death of ciliated epithelial cells. * Adapted from Ref. #1: Jorgensen, JH., Phaller, MA., Carroll, KC., Funke, G., Landry, ML., Rickles, SS., and Warnok, DW. Manual of Clinical Microbiology 11 th ed. ASM Press, American Society for Microbiology, Washington, DC and Ref. #7, Melvin, JA., Scheller, EV., and Cotter, PA. Bordetella pertussis: Current and Future Challenges. Nat. Rev. Microbiol. 12: After attachment, B. pertussis multiplies on the respiratory epithelial cells and then begins to produce various toxins that aid in the pathogenesis of disease. These toxins include pertussis toxin, adenylate cyclase toxin, and tracheal cytotoxin. (See Table 2. Virulence Factors Associated with the Pathogenicity of Bordetella pertussis.) These toxins are responsible for the further development of disease and for the specific clinical features of whooping cough. Pertussis toxin is an exotoxin (a soluble toxin excreted by a microorganism that causes damage to the host) associated with the cell wall of B. pertussis. Pertussis toxin allows the binding of B. pertussis to host epithelial cell receptors, and permits 9 CAMLT Distance Learning Course DL-997

12 the colonization of the organism in the respiratory tract. Pertussis toxin further allows B. pertussis to evade host defenses by altering and inhibiting host cell activity and disrupting host phagocytic activity (7). Investigations have shown that pertussis toxin and cholera and diphtheria toxins act in a similar manner. Adenylate cyclase toxin penetrates phagocytic cells, disrupting their normal function and their ability to signal to one another, thus reducing chemotaxis (the ability of phagocytic cells to be drawn to an infection site). Once adenylate cyclase toxin binds to phagocytes, it enters the phagocyte and induces apoptosis (cell death). Tracheal cytotoxin is actually a fragment of peptidoglycan (a polymer layer of the cell wall) secreted by B. pertussis. Tracheal cytotoxin destroys tracheal ciliated epithelial cells, inhibits DNA synthesis and the regeneration of the respiratory tract epithelium, and ultimately causes death of the respiratory epithelial cells. Tracheal toxin prevents the cilia from clearing debris from the lungs, so the body responds by sending the host into a coughing fit. The toxins of B. pertussis not only kill respiratory epithelial cells and phagocytes, but also cause inflammation of the respiratory tract and interfere with the clearing of pulmonary secretions, leading to the classic whoop sound (1,3). It is common to be unable to recover the organism from the patient after several weeks, but still have toxins from B. pertussis continue to cause symptoms (1,7). Many of the adhesin and toxin virulence factors of B. pertussis have been found to elicit a good antibody response in the host, and thereby are important in providing immunity after infection. Consequently, some of these virulence factors have been incorporated in the production of acellular vaccines to improve their activity (3,4). (See Section I. Prevention and Control of Bordetella pertussis Infection.) G. LABORATORY DIAGNOSIS OF WHOOPING COUGH The laboratory diagnosis of whooping cough can be achieved through either bacterial culture, direct fluorescent antibody (DFA) tests, molecular tests such as polymerase chain reaction (PCR), or by serological tests that detect the presence of specific B. pertussis antibodies in the serum of an infected individual (see Table 3. Laboratory Tests for the Identification of B. pertussis). The direct fluorescent antibody (DFA) is no longer a recommended method to detect B. pertussis since the test lacks sensitivity and specificity (6,7). There are positive and negative aspects of each laboratory test, which this section will describe. The choice of which diagnostic method to use depends on the age of the patient, the duration of symptoms, the immunization status of the patient, the availability of adequate transport procedures, and the availability of required laboratory services. For any of the methods that seek to detect B. pertussis directly, such as bacterial culture, or the DNA of B. pertussis by PCR techniques, specimen collection and transport are the most significant variables to the sensitivity (the number of positives correctly identified) of the test. 10 CAMLT Distance Learning Course DL-997

13 Table 3. Laboratory Tests for the Identification of B. pertussis Test Description Manufacturer Transport Media Jones Kendrick Transport Transport medium for B. pertussis Remel, Medium Regan-Lowe Transport Medium Semi-Solid Transport and selective isolation of Bordetella spp. Transport medium may also contain cephalexin. Hardy Diagnostics, Plating Media Bordet Gengou Agar with 15% blood, potatoes, glycerol, and charcoal Regan-Lowe Medium with or without cephalexin. Contains beef extract, starch, charcoal, niacin, and pancreatic digest of casein. Direct Fluorescent Antibody Test (DFA) Serology Various ELISA assays for IgG, IgM and IgA Molecular Testing (PCR) A technique to amplify certain genes, sequences or portions of the DNA from B. pertussis For cultivation and isolation of B. pertussis Requires addition of sheep, rabbit or horse blood. For cultivation of B. pertussis Also known as Charcoal Agar Medium, usually supplemented with 10% horse blood. The DFA test is no longer recommended by the CDC due to sensitivity and specificity issues Antibody detection, which may indicate a current or past exposure/immunization to B. pertussis. Need a comparison of acute and convalescent samples. Helpful in laboratory confirmation in adults, but cannot be used during early catarrhal stage. Sensitivity of 55%, but decreases progressively during the course of the disease. Generally, 95% sensitive and 99% specific. Use on symptomatic patients within 3 weeks of onset of cough. Remel, Hardy Diagnostics, Remel, Oxoid, Various reference laboratories Euroimmun US, Various clinical laboratories, reference laboratories, and public health laboratories have their own primers and methods. A few commercial manufacturers are: Bio-Rad, Operon, Cepheid, Meridian Bioscience, Roche, 11 CAMLT Distance Learning Course DL-997

14 Specimen Collection The preferred specimen type for the laboratory diagnosis of pertussis is the use of a posterior nasopharyngeal (NP) swab to collect nasopharyngeal secretions. When properly collected, this specimen contains the ciliated respiratory epithelial cells for which B. pertussis exhibits tropism (selectivity to certain receptor cells). Nasopharyngeal washings or aspirates are also a useful specimen particularly for PCR testing (3,6,8). You can transport the nasopharyngeal washings to the laboratory in a clean, dry tube. The nasopharyngeal swab is the easiest specimen type to obtain but not always the best (3,6). Throat swabs are an inferior specimen type for culture of B. pertussis and should not be accepted; they do not sample the ciliated respiratory epithelium, and they contain large numbers of the normal respiratory flora that can result in contamination and overgrowth of the requested laboratory test. A flexible wire Dacron-tipped nasopharyngeal swab is the best collection device for NP secretions (1,3). While material collected on a calcium alginate-tipped swab is acceptable for bacterial culture, it is not acceptable for PCR assay, because residues present in the calcium alginate may inhibit the PCR test. Cotton-tipped wire swabs are not acceptable since they contain inhibitors that will decrease the isolation rate or detection of B. pertussis (1,6). The Dacron-tipped wire swab can be bent to conform to the nasal passage. The swab should be inserted deep into the nostril until it touches the pharynx and gently rotate for 5-10 seconds to loosen the ciliated epithelial cells. (See Figure 2. Proper Technique for Obtaining a Nasopharyngeal Specimen for the Isolation of Bordetella pertussis.) Figure 2. Proper Technique for Obtaining a Nasopharyngeal Specimen for Isolation of Bordetella pertussis* 1. Hold the head steady, get help, or have patient sit with head against a wall, as patients have a tendency to pull away during the procedure. 2. Tilt tip of nose upward. 3. Insert swab into one nostril straight back (not upwards) and continue along the floor of the nasal passage for several centimeters until reaching the nasopharynx (resistance will be met). 4. Rotate the swab gently for 5-10 seconds to loosen the ciliated epithelial cells. 5. Remove swab and immediately inoculate plates or insert swab into transport media at least ½ inch below the surface of the media. 6. Transport to the laboratory immediately. *Adapted from Ref. #3: Centers for Disease Control and Prevention (CDC). Pertussis (Whooping Cough) Fact Information CAMLT Distance Learning Course DL-997

15 Transport of Specimens Because of the fragile nature of B. pertussis outside its normal host environment, proper handling and transport conditions are critical for optimal laboratory identification. Once an NP swab has been collected, it should be plated directly onto suitable selective culture media or immediately placed into a suitable transport medium. Direct plating of NP specimens for B. pertussis is not practical for most hospitals or medical clinics. Cough plates are not recommended, because higher rates of B. pertussis isolation are obtained from NP swabs specimens than from cough plates (1). There are two different transport media recommended for the proper transport of B. pertussis. One is Regan-Lowe transport medium, which contains half-strength charcoal agar base, horse blood, beef extract, starch, and tryptose. Regan-Lowe may also be purchased containing the antibiotic cephalexin to suppress growth of the normal respiratory flora. Another transport medium, Jones- Kendrick Transport Medium, contains beef heart extract, soluble starch, charcoal powder, tryptose, and yeast extract; it does not contain blood. Once the NP specimen has been collected using a Dacron tipped wire swab, it should be placed immediately into the transport medium and transported at room temperature (15 30 C) to the laboratory within four hours of collection. Isolation rates decrease when transport occurs at 4 C instead of at room temperature, or when the transport takes longer than 48 hours (1,6). Remember that B. pertussis is a fragile organism. It does not survive desiccation, temperature, or environmental changes well. Once the specimen has reached the laboratory, it should be plated as soon as possible. For PCR testing, NP swabs can be transported dry, in a transport medium, or in saline after specimen collection. Transport in liquid medium provides a specimen from which multiple aliquots can be tested, whereas processing a dry swab for PCR testing leaves no specimen material available for repeat analysis if required (1). Generally, the same NP specimen can be used both for culture and polymerase chain reaction (PCR) if the volume of the specimen allows (8). Bacterial Culture The culture of B. pertussis is considered the gold standard and the preferred laboratory test for pertussis in some cases. However, the sensitivity of B. pertussis culture can vary according to the following factors: 1) collection method used, 2) transportation, 3) timing of specimen collection during illness, 4) lack of fresh media, 5) choice of media and selective agents, 6) incubation conditions and duration, and 7) various host factors. Culture is considered the most specific of the laboratory tests for pertussis, but it may take up to seven days before results are known. Culture is less sensitive than PCR, but is 100% specific (no false positives). A positive culture for B. pertussis confirms the diagnosis of pertussis; however, a negative culture result does not rule out pertussis infection. B. pertussis is most frequently recovered in the catarrhal or early paroxysmal stage of illness (i.e., first 1-2 weeks of cough); sensitivity decreases steeply (<60%) if the specimen is obtained more than 2 weeks from onset of cough. Isolation rates of B. pertussis on culture media are negatively correlated with increasing age (high rate in infants; low rate in adults), with antimicrobial pre-treatment, and with the number of pertussis vaccination doses received by the patient. Most authorities report that culture for B. pertussis is rarely positive in adults, though it is the gold standard test for diagnosing pertussis in infants and children if obtained early in the infection (1). Usually adults may have been coughing for several weeks before they seek medical attention and it is often too late for culture to be useful in this age group. The first culture medium used to isolate B. pertussis, developed in 1906 by Bordet and Gengou, is called BG medium. It was the traditional culture media for pertussis isolation and was used for many years. It consists of potatoes and glycerol, supplemented with 15% defibrinated sheep or horse blood (human blood is not an acceptable substitute). Charcoal was used in later formulations to neutralize toxic fatty acids. A more recent medium used to isolate B. pertussis is called Regan-Lowe (RL), which 13 CAMLT Distance Learning Course DL-997

16 consists of beef heart extract, starch, charcoal, pancreatic digest of casein, niacin, and 15% defibrinated horse blood. These media are available with the addition of cephalexin or with other antibiotics to inhibit the overgrowth of normal respiratory flora. Many authorities suggest that the specimen should be inoculated onto both a non-selective pertussis medium and onto a selective medium, since inhibition of B. pertussis is sometimes observed on media containing antibiotics (1,6). Regan-Lowe medium appears to provide better isolation and faster growth of B. pertussis than does Bordet Gengou medium (1). The shelf life of media ranges from 5 days for BG medium to 4 to 8 weeks for RL medium. Bordet Gengou medium should be freshly made, if possible, for best results. The short shelf-life of media for B. pertussis is a major consideration for the laboratory. Primary culture and presumptive identification All primary plates are incubated in a humidified atmosphere under aerobic conditions without added carbon dioxide for seven days at C and inspected every two days. The humidity must be sufficient to prevent desiccation of agar plates during the long incubation period. Growth usually appears after 4-5 days; however, cultures cannot be considered negative for B. pertussis until after 7, or rarely up to 14, days of incubation. Typical B. pertussis colonies on BG or RL plates are small (1 mm in diameter after four to five days of incubation), round, domed-shaped and glistening, and resemble mercury droplets. On BG plates, B. pertussis appears slightly hemolytic. On colonies that morphologically resemble B. pertussis and that develop after 4 days of incubation, the following steps are recommended: 1) check purity of the growth by performing a Gram stain. B. pertussis should appear microscopically as small Gram-negative coccobacilli or very short oval rods; 2) determine the organism s biochemical characteristics by testing for oxidase, nitrate-reductase, urease, and motility, and 3) confirm the identification using specific anti-b. pertussis immunofluorescence antiserum if available. B. pertussis is catalase positive, oxidase positive, nitrate reduction test negative, urease production test negative, and is negative for motility. Since B. pertussis is catalase positive, the catalase reaction may help to distinguish the organism from catalase-negative commensal organisms. A negative urease reaction distinguishes B. pertussis from B. parapertussis and B. bronchiseptica, which are both urease positive. For differentiation between B. pertussis and B. bronchiseptica, the oxidase reaction is helpful (B. pertussis is positive and B. parapertussis is negative). B. pertussis is non-fermentative (does not utilize carbohydrates as an energy source); it uses proteins. Direct Fluorescent Antibody Test (DFA) The direct fluorescent antibody test (DFA) for the diagnosis of pertussis became available for use in the United States in the early 1960s. It became widely used by clinical and public health laboratories for a period of time because it provided rapid results. Although the direct DFA test for B. pertussis offers a rapid and simple diagnostic approach, the sensitivity of the test (only about 60% sensitive) is limited compared to other methods such as culture and PCR (6). As such, it is no longer recommended as a laboratory test for B. pertussis since it is so much less specific and sensitive than either culture or PCR testing. Detection of Bordetella pertussis by PCR Nucleic acid amplification methods, such as the polymerase chain reaction (PCR) technique, can be a rapid and sensitive method of diagnosing whooping cough. Since its introduction in the late 1990s, PCR testing to identify B. pertussis has become widely used by many clinical and public health laboratories as the primary diagnostic test for whooping cough. Recommendations from the CDC are that the preferred methods for the laboratory diagnosis of pertussis are culture and polymerase chain reaction, and it is suggested that in most cases both tests be performed if possible (3). The CDC currently recommends that the PCR test should be used in addition to, and not as a replacement for, culture (3). The CDC also recommends that PCR testing should be done on symptomatic patients only 14 CAMLT Distance Learning Course DL-997

17 and within 3 weeks of onset of cough, but the test may provide accurate results for up to 4 weeks in infants or unvaccinated persons (3). Keep in mind that some vaccines contain PCR detectable components and may yield false positive results (3). Like bacterial culture, PCR is also affected by specimen collection technique. Clinical specimens used can be posterior nasopharyngeal swabs (See Fig. 2), or the preferred specimen is saline aspiration that flushes the posterior nasopharynx. The CDC and WHO state that a saline wash is preferred over swabs since a larger quantity of bacterial of bacterial DNA is obtained (3,6,8). An inappropriately obtained nasopharyngeal swab will likely be negative by both culture and PCR. PCR is the method of choice in adult patients with persistent cough in whom pertussis is suspected, as well as in infants and children with epidemiological and clinical features of the disease. PCR results have been shown to be unreliable after 5 days of appropriate antibiotic treatment (1,3). Since PCR detects B. pertussis DNA and not viable organisms, the sensitivity can be as low as < 1 cell. In general, the sensitivity, specificity, and rapid turnaround of PCR for B. pertussis have been shown to be very good, which is important for patient management and control of outbreaks (1). Over time, the amount of B. pertussis DNA will diminish, rendering the assay less sensitive and providing inaccurate results. The PCR procedure amplifies certain genes, sequences, or portions of the DNA from B. pertussis using specific primers that target certain areas of the organism s DNA. The choice of primers (a strand of nucleic acid that serves as a starting point for DNA synthesis) and their target region are key components of the sensitivity and specificity of the PCR test. Generally, PCR for B. pertussis has a sensitivity of 95% and a specificity of 99%, and is usually more sensitive than DFA or culture (1,3). Delay in specimen collection is the main reason for a negative PCR test result in a patient with pertussis. Two major shortcomings of PCR testing, however, are: 1) the potential for false-positive results due to contamination, and 2) the interpretation of a positive result for B. pertussis DNA in the absence of a positive bacterial culture if available. A positive PCR result, therefore, must be interpreted in conjunction with patient symptoms, treatment status, epidemiological factors, and a similarly positive bacterial culture. PCR tests are less sensitive in previously immunized individuals, but are more sensitive than cultures in such patients. Other shortcomings of PCR testing are: 1) PCR testing requires specialized training and is technically difficult to perform; 2) PCR requires more expensive equipment and reagents; and 3) the cost of the PCR test is high (6). The charge for B. pertussis PCR testing from commercial reference laboratories is about $100 or more per test. (See Table 3 for manufacturers). PCR testing, however, is the best option in most clinical circumstances. It provides acceptable sensitivity in both children and adults, has a relatively short turnaround time, and is available at most commercial reference laboratories. An excellent synopsis of best practices for specimen collection, transport, and other methods used in the PCR technique for the diagnosis of pertussis can be reviewed in the CDC reference (8). Serological Detection of B. pertussis Serological detection of B. pertussis identifies antibodies in the patient s serum formed in response to infection or prior immunization. Serological tests have limited value for the initial diagnosis of B. pertussis infection because of the need for paired sera before interpreting results. Valid serologic diagnosis requires data from serum collected at the beginning of cough (acute serum), and from serum collected one month later (convalescent serum) to determine a change or rise in antibody level in the serum of infected individuals. Because of the delay in collecting the convalescent sera, serological tests cannot be used as the sole method of diagnosis. The collection of a single serum specimen for diagnostic purposes is not well standardized and usually not of any use (6). Further, the use of serology as a method of diagnosis of pertussis in young infants is not sensitive and should not be used, as their immune system is immature and may not produce adequate levels of antibodies. The serodiagnosis of pertussis in vaccinated persons can also be a problem since there may be an immediate increase in various 15 CAMLT Distance Learning Course DL-997