Documentation:PATH417archive2020W2/Case 4
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Case 4.
A Chronic Cough
36-year-old Joseph has been suffering from multiple spells of violent coughing for 6 weeks. Prior to developing the chronic cough he had, what he describes as, a mild upper respiratory tract irritation, mild fever, runny nose and a cough that lasted about 3 days. More recently, and at the insistence of his spouse, he finally visits a drop-in clinic where the doctor asks him about his childhood vaccinations. Joseph reports that, to the best of his knowledge he had all the required shots but he admits to not having any since becoming an adult. Upon examining Joseph the doctor finds some redness in his throat but his lungs are clear and there are no enlarged lymph nodes in his neck. The doctor collects a throat swab to send to the laboratory, prescribes some codeine syrup and informs Joseph that the cough may last a few more weeks.
The laboratory is unable to detect Bordetella pertussis or any other pathogen from the swab. Despite this, Joseph is diagnosed with pertussis, commonly known as whooping cough, based on his clinical presentation and lack of adult immunization against pertussis. The coughing fits go away after another 4 weeks.
1. The Body System
(i) What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). results reported. Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).
(ii) Which body system is affected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.
(iii) Why were no antibiotics (started or) prescribed and what is the antibacterial treatment of choice for this organism? What is the relevance of the fact that a codeine cough syrup was prescribed?
(iv) Why did the doctor ask about Joseph’s vaccination history? Is this a reportable communicable disease?
2. The Microbiology Laboratory
(i) Including the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.
(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?
(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.
(iv) What are the results expected from these tests that might allow the identification of the bacteria named in this case.
3. Bacterial Pathogenesis
Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.
i. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria
ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.
iii. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.
iv. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?
4. The Immune Response
i. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.
ii. Host damage: what damage ensues to the host from the immune response?
iii. Bacterial evasion: how does the bacteria attempt to evade these host response elements.
iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?
Responses
Q1. The Body System
(i) What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).
Joseph has been diagnosed with pertussis, a respiratory illness that is also known as whooping cough and is caused by Bordetalla pertussis (B. pertussis) (1). B. pertussis is a small, aerobic Gram-negative coccobacilli in the Bordetella genus along with nine other species (1, 2). B. pertussis are approximately 0.8 μm by 0.4 μm, are encapsulated, and non-spore producing (3, 4). Though some of the species in this genus are motile, B. pertussis are not (2). This means they are incapable of propelling themselves in their environment. These other species include B. parapertussis, which similarly causes respiratory illness, and B. holmesii, which causes infection involving pertussis-like symptoms. A notable difference between B. holmesii and B. pertussis infection is that B. pertussis does not cause bacteremia, or pathogen presence in the bloodstream, nor does it cause non-pulmonary infections that can be seen in B. holmesii infection (1). Additionally, though the majority of the Bordetella species cause disease in animals, B. pertussis alongside B. parapertussis are known to develop respiratory diseases in humans (1). B. pertussis are responsible for inflicting whooping cough disease upon their hosts. Whooping cough is a respiratory infection characterized by severe, spasmodic coughing episodes.
B. pertussis infection is spread through droplet transmission and most commonly involves direct person-to-person contact (2). Upon infection, there is a one to two-week incubation period before disease signs and symptoms manifest; however, most individuals begin to experience symptoms seven to 10 days after exposure to B. pertussis. Pertussis infections typically progress through three distinct phases: the catarrhal, or early, phase, the paroxysmal, or late phase, and the convalescent, or recovery phase (6).
Stage I - Catarrhal Stage:
The catarrhal stage lasts for 1-2 weeks and is the most infectious stage of the disease (6). The catarrhal stage presents similarly to other upper respiratory tract infections and involves inflammation of mucosal membranes (2). Therefore, the disease often begins with cold-like symptoms such as rhinorrhea (runny nose), nasal congestion, sore throat, and malaise (general discomfort) (7). Furthermore, a low-grade fever and mild progressive dry cough are seen during this stage, and the dry cough gradually becomes more frequent (7). These symptoms are seen in Joseph’s case as well, as he experienced mild fever, rhinorrhea, and a gradually progressive cough. Since many symptoms of pertussis in the early stage can appear to be similar to a common cold, healthcare professionals often do not suspect or diagnose whooping cough until more severe symptoms appear (7). Near the end of this stage, infected individuals also may begin experiencing leukocytosis (high white blood cell count) alongside absolute and relative lymphocytosis (3). Absolute lymphocytosis refers to an increase in the presence of lymphocytes while relative lymphocytosis refers to the amount of lymphocytes present being relatively higher than that of white blood cells. Children infected by pertussis may also experience non-purulent conjunctivitis with excessive lacrimation (pink eye with excessive tearing) (2). Distinguishing the general signs versus symptoms in the catarrhal stage of infected individuals: the signs include presence of leukocytosis and absolute and relative lymphocytosis while the symptoms include the low-grade fever, malaise, runny nose, cough, and potential pink eye which are also summarized in Table 1.
| Catarrhal Phase | |
| Signs | Symptoms |
| General
· (mild) leukocytosis · (mild) absolute and relative lymphocytosis |
General
· Low-grade fever · Malaise · Rhinorrhea (runny nose) · Mild cough · Pink eye (more common in children) |
Table 1: Summary of Signs and Symptoms during Catarrhal Phase
Stage II - Paroxysmal Stage:
The paroxysmal stage of pertussis is known as the second stage of illness, and typically extends for the next two to four weeks (1). The paroxysmal stage is characterized by recurring intense and violent episodes of coughing that last several minutes (severe coughing spells). A paroxysm is defined as a series of coughs in rapid succession with increasing intensity (7). In one expiration, individuals in this phase may cough up to as many as 10 or 15 times in a row leading to their faces turning red or purple or may lead to them having an inspiratory whoop, which are high-pitched “whoop” sounds caused by inspiring against a partially closed glottis (2). Individuals often find it difficult to perform inspiration (draw air in) between coughs. The extreme coughing fits can lead to exhaustion and vomiting (4). As a result of the excessive coughing and the force behind it, infected patients may produce mucous plugs that leads to them vomiting; this is referred to as posttussive vomiting (2). Though posttussive vomiting is not very suggestive towards B. pertussis when diagnosing children, in adults this symptom strongly suggests a pertussis infection (2). Whooping is reported in 8 to 82% of adult cases of pertussis, while posttussive vomiting is seen in 17-65% of adult cases (2). As mentioned above, near the end of the catarrhal stage there are signs of leukocytosis and absolute and relative lymphocytosis; subsequently, during the paroxysmal phase, these signs reach their peak to the point of the patient having blood leukocyte levels that resemble those of leukemia (≥ 100,000/mm3), with 60-80% being lymphocytes (3). These levels are factors that risk the patient’s clinical outcome for the worst. Additionally, there is also a chance of the patient showing signs of hyperinsulinemia (excessive insulin presence); however, it is not associated with hypoglycemia (decreased levels of glucose) (2). Additionally, individuals with pertussis may present with bulging eyes, tongue protrusion, excessive salivation, engorgement of the veins in the neck, and thick oral mucus production during the coughing spells (7). The general signs and symptoms of the paroxysmal phase are summarized in Table 2.
In cases where an infant is infected with B. pertussis, their symptoms differ in this phase for they are less likely to show the general characteristics of this phase described above, such as paroxysmal coughing, but rather are more likely to present gagging, gasping, or apnea (pauses in breathing pattern) (2, 4). This apnea is associated with cyanosis (turning blue), due to oxygen desaturation from decreased breathing (4, 6). Moreover, during this phase infants may even show signs of poor feeding and have seizures as well (2).
| Paroxysmal Stage | ||
| Signs | Symptoms | |
| General
· (severe) leukocytosis · (severe) absolute and relative lymphocytosis · Hyperinsulinemia |
General
· Paroxysms (with inspiratory whoop) · Red or purple face · Posttusive vomiting |
Infants
· Gagging · Gasping · Cyanosis · Apnea · Poor feeding · Seizures |
Table 2: Summary of Signs and Symptoms during Paroxysmal Phase
Stage III - Convalescent Stage:
The convalescent stage of pertussis is stage three of the illness, and typically lasts from one to three weeks, but can extend longer than six weeks in some cases (2). During these weeks, the coughing paroxysms reduce in frequency, duration, and severity; however, fits of coughing may return occasionally (4). Eventually, near the end of this phase, the patient is able to largely recover from their pertussis symptoms (1). The signs and symptoms described in the first two phases of infection begin to subsidize as well; however, further complications may arise as a result of the individual’s immune system being more susceptible to respiratory illnesses. If the infected patient contracts another respiratory illness during the convalescent stage, then the classic pertussis cough paroxysm patterns can recur, although this is more commonly seen in children (4, 7). Though the average amount of time an individual with this infection has a cough is about 36-48 days, there are even cases where an individual can be seen coughing for up to a year after a B. pertussis infection (1).
Complications
Pertussis or whooping cough caused by B. pertussis bacteria is a spectrum of disease with its presentation varying depending on the infected individuals age, degree of immunity, use of antibiotics, and respiratory coinfection (2). It is noted that while comparing the different age ranges, infants have the highest case-fatality rate of pertussis (2). The complications that arise during a pertussis infection are very serious and can include from urinary incontinence (involuntary urine leakage), subconjunctival hemorrhage (bleeding in the white of the eye), rib fractures due to excessive coughing, bronchopneumonia, acute encephalopathy, lifelong brain damage, or even death (1, 2, 3). The most common complication seen in patients is pneumonia caused by the B. pertussis infection itself or through confection with another respiratory pathogen such as respiratory syncytial virus (RSV) (2). Moreover, though encephalopathy (disease impacting the brain) is a rarer complication of pertussis, it is still a complication seen both in adults and children with an emphasis on younger children who have not been immunized yet (2). This complication occurs when the pertussis-specific antigens travel past the blood-brain barrier, impacting the central nervous system (2). This complication can be seen manifesting between two to four weeks after the onset of coughing and is most commonly characterized by the patients seizing alongside other symptoms such as blindness, deafness, and more (2).
Atypical Presentations
Adolescents and adults who have been infected with pertussis present with the same signs and symptoms as infants or children who have been infected; however, the signs and symptoms seen in adults are usually milder in comparison. As a result, clinicians may overlook the diagnosis of pertussis in adults or adolescents (7). Although classic presentation of pertussis with the “whooping” cough can occur in adult patients, like Joseph, pertussis without classic paroxysms can be the cause of up to one-third of prolonged coughs in adolescents and adults. In this age group, the duration of coughing can range from 3-8 weeks (7). Furthermore, adults who have been fully immunized against pertussis, or have had the infection before, tend to have less dramatic presentation of symptoms in comparison to children or infants; as a result, they may also be asymptomatic when infected with B. pertussis (6, 7).
Often, young infants are at the highest risk for severe outcomes such as respiratory failure and death, when infected with B. pertussis bacteria. This is likely because younger infants have only partial immunity to pertussis, as they have not yet completed the vaccine series (7). The signs and symptoms associated with pertussis are more severe in this younger age group of infants, and the illness is more likely to cause hospitalization due to the presentation of apnea, pneumonia, and convulsions, which are not commonly seen in adults (7).
Laboratory Samples and Results:
The detection of pathogenic B. pertussis through laboratory testing is enhanced by the proper timing, collection, transport, and storage of the sample. Two preferred samples for B. pertussis infection are posterior nasopharyngeal swab and nasopharyngeal aspirate (1). These samples are commonly collected because B. pertussis uses various virulence factors, including fimbriae (FIM), filamentous hemagglutinin (FHA), and pertussis toxin (PT) to adhere to and colonize ciliated respiratory epithelial cells in the host nasopharynx and trachea during infection (1). Nasopharyngeal swab collection involves inserting a swab deep into the nostril, in order to reach the nasopharynx (2). The optimal material of these swabs is either Dacron, rayon, or calcium alginate; cotton is not preferred because it inhibits B. pertussis growth (1). Collection of nasopharyngeal aspirate involves suctioning nasopharyngeal secretions using a catheter and vacuum containing a mucus trap (2). Nasopharyngeal aspirates are advantageous over nasopharyngeal swabs, as aspirates result in more positive cultures and obtains more samples that can be used for additional testing (1). Transport media for nasopharyngeal swabs and aspirates can include Amies media with charcoal or 1% acid-hydrolyzed casein. Additionally, an enrichment media, such as Regan-Lowe (RL) transport media containing charcoal agar, horse blood, and cephalexin can be used. Cephalexin is included because it suppresses the growth of nasopharyngeal commensal bacteria, however, it may also inhibit the growth of certain B. pertussis strains (1).
Culture characteristics and biochemical tests can be used to identify B. pertussis as the causative agent of disease (1). Culture is the gold standard for pertussis diagnosis, but its sensitivity can vary, reported from 15 to 80%, depending on the transport and collection of the sample, as well as patient characteristics such as previous vaccinations, age, antibiotic usage, and the time of symptom onset (1, 2). Specific laboratory conditions are required because the growth of B. pertussis can be inhibited in common lab media, including fatty acids, sulfides, metal ions, and peroxides (1). The optimal media for B. pertussis includes those that contain blood, charcoal or starch, and can include Bordet-Gengou (BG) which has a potato-starch base, or Regan-Lowe medium that has charcoal, with added glycerol, peptones, and sheep or horse blood. An antibiotic, such as cephalexin, methicillin or oxacillin, is also often added to the medium in order to inhibit the growth of any contaminating bacteria (2). Additionally, A (2,6-O-dimethyl)-b-cyclodextrin supplemented stainer-scholte broth is used as an enrichment medium for B. pertussis (2). Once cultured, B. pertussis should take about 3-4 days to grow at 35-37° C and become visible, and are often incubated up to a total of seven days (1, 2). These colonies should appear round, domed, mercury silver in colour, shiny and be able to produce hemolysis on the brilliant green (BG) agar (2). It is important to note that due to the different growth rates of Bordetella species, it is recommended to allow incubation to occur for up to 12 days for optimal sensitivity (2). B. pertussis colonies are often smaller than B. parapertussis colonies; however, they are indistinguishable from B. bronchiseptica colonies until undergoing further biochemical tests. These biochemical results for B. pertussis would include testing negative for motility, growth on blood-free peptone agar, pigment production, nitrate reduction, and urea hydrolysis but positive for oxidase reaction as summarized in Figure 3 (1, 3).
Serology testing to detect the production of pathogen-specific antibodies is also used in laboratory testing (1). Examples of a serological tests are direct Fluorescent-Antibody Assay (DFA) and enzyme-linked immunosorbent assay (ELISA) (2). Direct Fluorescent-Antibody Assay (DFA) on nasopharyngeal samples is a simple and rapid method that visualizes fluorescent antibodies against B. pertussis in order to confirm diagnosis (2). However, due to low sensitivity and specificity, DFA diagnosis of pertussis should be supported by positive culture, PCR or serology tests (2). Additionally, doctors may also collect a blood sample for serologic tests (8). This blood sample can be used in enzyme-linked immunosorbent assay (ELISA) to detect IgG, IgM, IgA and IgE antibodies to whole B. pertussis bacterial cells or isolated B. pertussis antigens (1). IgG is the most commonly used source of detection because it is the most common and standardized (1). During infection, the amount of IgG antibodies should increase at about two to three weeks post-infection, and antibody titers will peak at eight to ten weeks of B. pertussis infection (2). Paired serology tests are the gold standard, where antibody titers are observed and compared at two timepoints throughout the course of the disease; a twofold increase in B. pertussis-specific antibody titers would be indicative of a positive case of pertussis (1). The presence of PT- and FHA-specific IgA antibodies will also be expected after a natural pertussis infection, unless infection occurred in an infants. A positive ELISA test for IgG antibodies to B. pertussis antigens is currently the most sensitive result to indicate B. pertussis infection (2). However, due to low sensitivity and cross reactivity of antibodies between different Bordetella species in ELISA, positive culture or PCR tests results are most often used for B. pertussis diagnosis instead (2).
PCR testing is a newer, sensitive method of diagnostic testing, which detects certain bacterial gene sequences that can be used to identify the species (1). The same nasopharyngeal swab used for the culture can be used for PCR (8). A common target of PCR testing for B. pertussis includes the B. pertussis pertussis toxin (PT) promoter region, a region upstream of the porin gene, the repetitive insertion sequence IS 481, the ACT (cyaA) gene and a region upstream of the flagellin (2). The detection of these genes through PCR would indicate the presence of B. pertussis in the laboratory sample. PCR testing is highly sensitive, however it detects both live and dead bacteria and therefore can introduce the risk of contamination. As a result, any positive result should be confirmed using culture, serologic, or clinical testing (1).
Key History Findings
A key historical finding by the doctor is that although Joseph received his childhood vaccinations, he has not received vaccinations in his adulthood. As further described in following sections, a single vaccine dose for pertussis is recommended during adulthood. This dosage has been shown to effectively protect against pathogenic B. pertussis infection, and since Joseph did not receive this dose, he is likely more susceptible to infection than those who received the full course of immunization.
Joseph described the signs of a mild fever and mild upper respiratory tract infection, and the symptoms of a runny nose and a cough in the first three days of his infection, which aligns with the expected signs and symptoms of the early, catarrhal stage of pertussis infection. It is likely that Joseph is in the end of the paroxysmal phase of his pertussis infection, as he has reported violent coughing spells for the past six weeks. Following his diagnosis, Joseph continues having coughing fits for four weeks, which likely represents his gradual recovery throughout the convalescent phase of pertussis infection. Upon presentation to the drop-in clinic, the doctor assesses for further signs and observes redness in Joseph’s throat, however his lungs are clear and his neck lymph nodes are not enlarged. Additionally, the throat swab is taken but no B. pertussis or other pathogen is detected. The redness in Joseph’s throat may be due to tissue damage in the respiratory tract that is commonly seen in pertussis infection. Tracheal cytotoxin is an example of a toxin produced by pathogenic B. pertussis during infection that can lead to destruction of ciliated cells in the trachea, which may be contributing to the redness in his throat (2). The mechanism by which lymphocytosis is induced in pertussis is unknown, but the lymphocytosis is associated with impaired entry of lymphocytes into lymph nodes (9). As a result, the lymph nodes are not enlarged during pertussis infections, which was a sign present in Joseph’s case, as noted by the physician. The host antibody response will mount at approximately 2 to 3 weeks of infection, which may explain why Joseph’s lungs were clear and B. pertussis was not detected, as the throat swab would likely be more sensitive during early stages of infection (1).
Although B. pertussis was not detected in the collected sample, Joseph could still be diagnosed because his presentations fits the clinical case for pertussis. According to the Centre for Disease Control (CDC), a clinical case of pertussis can be diagnosed with the presence of a cough persisting for over two weeks, in addition to at least one of a paroxysmal cough, inspiratory whoop, or posttussive vomiting (10). Since Joseph reported a paroxysmal cough persisting longer than two weeks, he can be diagnosed with pertussis according to this criterion. Furthermore, his lack of adulthood vaccinations as mentioned provides evidence that Joseph was likely not protected against pathogenic B. pertussis infection. Further laboratory testing can occur to confirm the diagnosis, and may involve culture, PCR, or confirming that he came into contact with a positive case of pertussis (1).
References
1. Bennett, J. E., Dolin, R., & Blaser, M. J. (2020). Mandell, douglas, and bennett's principles and practice of infectious diseases: 2-volume set (Vol. 2). Elsevier Health Sciences.
2. Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 31. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7813/
3. Van der Zee A, Schellekens JFP, Mooi FR. 2016. Laboratory diagnosis of pertussis. Clin. Microbiol. Re, 28(4): 1005-1026https://doi.org/10.1128/CMR.00031-15
4. Signs and Symptoms of Whooping Cough (Pertussis) | CDC [Internet]. Cdc.gov. 2021 [cited 9 April 2021]. Available from: https://www.cdc.gov/pertussis/about/signs-symptoms.html
5. Cherry, J. D. (2013). Pertussis: challenges today and for the future. PLoS Pathog, 9(7), e1003418.
6. Lauria AM, Zabbo CP. 2021. Pertussis. StatPearls Publishing [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK519008/
7. Kilgore PE, Salim AM, Zervos MJ, Schmitt HJ. 2016. Pertussis: Microbiology, Disease, Treatment, and Prevention. Clinical Microbiology Reviews, 29(3):449–486. https://doi.org/10.1128/CMR.00083-15
8. Carbonetti NH. Immunomodulation in the pathogenesis of Bordetella pertussis infection and disease. Current Opinion in Pharmacology. 2007 Jun 1;7(3):272–8.
9. Mu HH, Cooley MA, Sewell WA. 1994. Studies on the lymphocytosis induced by pertussis toxin. Immunology and Cell Biology, 72(3), 267-270. https://doi.org/10.1038/icb.1994.40
10. Surveillance Manual | Pertussis | Vaccine Preventable Diseases | CDC [Internet]. Cdc.gov. 2021 [cited 2 April 2021]. Available from: https://www.cdc.gov/vaccines/pubs/surv-manual/chpt10-pertussis.html
(ii) Which body system is affected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.
B. pertussis has several characteristic virulence factors that are involved in its effects on the host body system, including pertussis toxin (PT), Filamentous hemagglutinin (FHA), adenylate cyclase toxin, lipopolysaccharide (LPS), hemolysin, heat-labile toxin, trachea cytotoxin all described in greater detail below (1, 2).
Respiratory Tract:
Overview of the respiratory tract:
Physiologically, the respiratory system can also be divided into the conducting zone and the respiratory zone. The conducting zone includes all of the structures from the nose to the bronchioles in the respiratory tract, the majority of which are lined with ciliated respiratory epithelium, known as ciliated pseudostratified columnar epithelium (3). This epithelial tissue makes up the respiratory mucosa and is composed of columnar-shaped cells that have 200-300 cilia protruding from their apical surface, as well as goblet cells that are responsible for producing mucus (4,2,5). Cilia are hair-like structures that are 6-7 micrometers tall and are motile due to repetitive episodes of bending, which produces a phenomenon known as ciliary beating (5, 6). Like all epithelium, respiratory epithelium serves as a physical barrier against pathogens and foreign particles for the innate immune system, but due to its ciliation, it also plays a major role in preventing infection and tissue injury via mucociliary clearance (4). The respiratory epithelium acts as a highly effective barrier to pathogens by preventing the passive transport of molecules and microbes through the lining of the airway lumen and into the body, via specialized structures called tight junctions (7). Additionally, the production of mucus by the epithelial goblet cells serves to nonspecifically trap foreign particles. Some of the important components of airway mucus are mucins (sticky, sugar- coated proteins), defense proteins, salt, and water. Together, these components form a gel that traps particles that enter the airway. Mucus is also involved in the hydration of airways, which helps with the proper function of the cilia (5). Once secreted, the mucus can cover the hair-like ciliary structures and help trap and expel potentially dangerous foreign molecules or pathogens, using the mucociliary escalator (5).
Damage pertained to the respiratory tract:
B. pertussis infections are mainly transmitted through droplets which lead to the bacteria colonizing the mucous membranes of the respiratory tract where they rapidly multiply (2). These bacteria are not known to enter the bloodstream and cause bacteremia meaning it is unlikely for B. pertussis bacteria to enter the circulation of the host and disseminate cause systemic manifestations (1,2). Focusing on the respiratory tract, in studies done using electron microscopes, it has been noted that these bacteria only adhere to the tuft of ciliated cells in the mucosa and no other nonciliated cells (2). Refer to Figure 1 for a better visual representation of the bacteria interacting with the ciliated cells of the respiratory tract. The damage caused to the ciliated epithelial cells is due to triggering the production of nitric oxide (NO) synthase by the bacteria’s peptidoglycan derived disaccharide tetrapeptide tracheal cytotoxin (TCT) (1). This results in coliostasis which is characterized by the slowing or halting of the cilia movement (8). However, it is important to note that dermonecrotic toxin (DNT), adenylate cyclase toxin, and pertactin also contribute to ciliostasis and epithelial damage (9,1). Dermonecrotic toxin (DNT) activity causes necrosis and adenylate cyclase toxin activity in local endothelial cells causes edema (2). In the initial stage of this infection, there is necrotizing inflammation and leukocyte infiltration in parts of the larynx, trachea, and bronchi which occurs post local peribronchial lymphoid hyperplasia (2). This is due to the increased number of lymphocytes in peribronchial and tracheobronchial lymph nodes in response to the B. pertussis bacteria. There is also peri-bronchiolitis (inflammation of tissues around bronchioles), variable patterns of atelectasis (lung collapse), and emphysema (damage of lung air sacs) seen in the initial stage of this infection because of the inflammation and damage caused by the bacteria (2). These can hinder gas exchange because there are fewer functioning alveoli and/or less surface area for gas exchange. Moreover, this manifests as shortness of breath, difficulty breathing, and respiratory distress (2). In severe cases, hypoxemia and respiratory failure may also occur (2,9). Overall, these events hinder the pulmonary system’s ability to deliver oxygen to tissues and organs in the body needed for metabolism and proper function.
Recall, the airway forms the first line of defence against invading pathogens (10). The mucociliary mechanism and secreted mucins work with antimicrobial peptides to inhibit bacterial colonization of airway tissue (10). Normally, mucus is secreted to trap pathogens invading the respiratory tract, then cilia projections help move microbes, debris, and mucus up and out of the airways (8,10). However, ciliostasis, caused by the adhesion and colonization of B. pertussis in the ciliated epithelium can hinder the ability of cilia to sweep and clear mucus and pathogens from the respiratory tract (8,10). And mucus hypersecretion can impair gas exchange and further reduce mucociliary clearance (9). Additionally, the excess mucus and shedding of dead cells contributes to severe, chronic, and ineffective coughing in an effort to clear these secretions to maintain airway patency (1,11).
Overall, the loss of ciliated cells and hyper mucus production impairs the mucociliary clearance system, a vital component of the immune system, and allows B. pertussis to colonize and other pathogens to enter and persist (10). This can leave the patient vulnerable to secondary infection and cause complications such as pneumonia, sinusitis, and otitis media (8). For instance, respiratory syncytial virus (RSV), Staphylococcus aureus and Streptococcus pneumoniae are common pathogens that cause pneumonia (9,12).
Immune System:
The immune response comes into play when the body detects danger and results in the recruitment of a variety of immune cells that each play a different role to assist in the clearance of infection. B. pertussis have been noted to inhibit and interfere with the immune system in a multitude of ways when infecting a human body. For example, these bacteria are able to inhibit the migration and activation of phagocytes through the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate(cAMP) (1). This conversion has also been linked to the suppression of T-lymphocyte activation (1). Furthermore, these microorganisms are also known to target the innate immune system of the lungs through the suppression/inactivation of the G protein-coupled signaling pathways (1). It is able to do this using one of the subunits of its PT (B subunit) which binds the cell surface and enables adenosine diphosphate (ADP)–ribosylation of G proteins by the A (active) subunit, leading to the altering of the cell (1). This delays the recruitment of neutrophils to the respiratory tract and targets airway macrophages (1). Consequently, using its BrkA surface-associated protein, B. pertussis is known to be able to avoid-antibody mediated complement killing which is commonly a response induced during the innate immune response to clear pathogens (1). These mechanisms all function to promote the B. pertussis infection and are demonstrated in summarized form in Figure 1.
Other Body Systems:
As noted in the first section, there is leukocytosis and lymphocytosis that can be seen in B. pertussis-infected patients which are a result of the production of pertussis toxin (PT) (1). As mentioned previously, this is characterized by elevated levels of white blood cells and lymphocytes, respectively (1) Leukocytosis may result in increased vascular resistance that leads to pulmonary hypertension also known as high blood pressure (1). Additionally, there are cases where encephalopathy complications arise, as mentioned in the first part of this section, they are rare but do occur in cases where B. pertussis antigens cross the blood-brain barrier and impact the central nervous system (1,2). Moreover, it has been suggested that encephalopathy may be caused by the effects of PT on the CNS via monocyte chemoattractant protein -1 (MCP-1) over-expression (1). Filamentous hemagglutinin (FHA) effects in the CNS may also b able to contribute to pertussis-associated encephalopathy. Encephalopathy, although rare overall, is more common in unimmunized children than adult patients (1). There are is also sensitization of histamine and serotonin and sensitization of the beta-islet cells of the pancreas that occur, which is the culprit for the sign hyperinsulinemia (and resultant hypoglycemia) especially in young infants mentioned in the first section of this question (1). The summary of the body systems involved in the pertussis infection described above can be seen in Figure 2.
References
- Bennett, J. E., Dolin, R., & Blaser, M. J. (2020). Mandell, douglas, and bennett's principles and practice of infectious diseases: 2-volume set (Vol. 2). Elsevier Health Sciences.
- Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology. 4th edition. (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 31. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7813/
- Pertussis (Whooping Cough). 2017. Signs and Symptoms. Centers for Disease Control. https://www.cdc.gov/pertussis/about/signs-symptoms.html
- Wilson R, Read R, Thomas M, Rutman A, Harrison K, Lund V, Cookson B, Goldman W, Lambert H, Cole P.1991. Effects of Bordetella Pertussis Infection on Human
- Weupe M, Peabody Lever JE, Kennemur JP, Bono TR, Phillips SE, Shei RJ, Rowe SM. 2019. Moving Mucus Matters for Lung Health. Frontiers For Young Minds. https://kids.frontiersin.org/article/10.3389/frym.2019.00106
- Lindemann CB, Lesich KA.2010. Flagellar and ciliary beating: The proven and the possible. Journal of Cell Science, (123): 519-528. https://doi.org/10.1242/jcs.051326
- 16. Moraes TJ, Chow CW, Downey G. 2012. Pulmonary Host Defenses, Chapter 22. Science Direct. Clinical Respiratory Medicine, 4:275-287. https://doi.org/10.1016/B978-1-4557-0792-8.00022-2
- Kerr JR, Matthews RC. 2000. Bordetella pertussis Infection: Pathogenesis, Diagnosis, Management, and the Role of Protective Immunity. Euro. jour.of clin. microbiol. infect. diseases. 19(2):77-88.https://doi.org/10.1007/s100960050435
- Long, S. S., Edwards, K. M., & Mertsola, J. (2018). Bordetella pertussis (pertussis) and other bordetella species. In S.S. Long, C.G. Prober & M. Fischer (eds.), Principles and Practice of Pediatric Infectious Diseases, (Fifth Edition). Elsevier US, New York, New York. https://doi.org/10.1016/B978-0-323-40181-4.00162-6
- Kessie DK, Lodes N, Oberwinkler H, Goldman WE, Walles T, Steinke M, Gross R. 2021. Activity of tracheal cytotoxin of bordetella pertussis in a human tracheobronchial 3D tissue model. Front. Cell. Infect. Microbiol., 10:614994-614994. https://doi.org/10.3389/fcimb.2020.614994
- Committee on Drugs. 1997. Use of codeine- and dextromethorphan-containing cough remedies in children. Pediatrics (Evanston), 99(6), 918-920. https://doi.org/10.1542/peds.99.6.918
- Spector TB, & Maziarz EK. 2013. Pertussis. The Medical Clinics of North America, 97(4), 537-552. https://doi.org/10.1016/j.mcna.2013.02.004
(iii) Why were no antibiotics (started or) prescribed and what is the antibacterial treatment of choice for this organism? What is the relevance of the fact that a codeine cough syrup was prescribed?
Antibiotics
There is controversy regarding the efficacy of antibiotic treatment for B. pertussis infections; although it has been shown to effectively eliminate the pathogen from the nasopharynx, there is a lack of evidence that it has a significant influence on the course of illness or clinical outcomes [1]. Antimicrobial therapy is most effective in individuals who receive medical treatment quickly and relatively early in the disease progression [2]. If the antibiotics are given early in the state of infection, the catarrhal stage, the symptoms of the infection may be improved, and the disease may be cleared [3]. However, the common understanding in regard to antibiotic treatment in patients infected with B. pertussis, especially past the catarrhal stage (e.g. paroxysmal phase), is that although the antibiotic may help with the clearance of the microbe, it generally has no impact on the course the disease takes [3,4]. If the patient is already suffering from violent coughing spells, antimicrobial therapy will only help clear the bacteria from the respiratory tract faster and help reduce the contagiousness of the patient [2,5,6]. However, treatment administered after coughing appears or after 3 weeks of illness is unlikely to help with reducing symptom severity or disease progression [5]. This is because although the bacterial infection may be cleared, B. pertussis has already released toxins that have damaged the body and consequently, the patient will continue to experience pertussis symptoms [5]. Thus, ‘late’ treatment will have no influence on the course of the disease or patient symptoms because pathogen mediated damage to the host has already occurred [4]. As a result, antibiotics are not recommended to be started more than three weeks following the onset of coughing symptoms [1]. In cases where antibiotics are used, the type and dosage are dependent on the age and the period the individual has been ill for.
A person experiencing the full progression of whooping cough symptoms in the late stages of infection will only see symptomatic improvement when the old damaged ciliated cells lining the respiratory tract are replaced with new healthy cilia-bearing cells, over time [7]. Joseph has been suffering from a cough for over 6 weeks and is in the late stages of pertussis. As a result, he has passed the recommended timeline for receiving antimicrobial therapy since the B. pertussis bacteria are likely gone from his body, and only his symptoms remain due to the damage to ciliated cells [8]. This is why antibiotics were not prescribed to him. However, antibiotics should be administered to anyone who has come into close contact with an infected individual, within 3 weeks of the exposure [9], to prevent further spread of the illness.
Antimicrobial Treatment of Choice
The recommended antimicrobial agents for treatment of pertussis belong to the family of macrolides, and include erythromycin, clarithromycin, and azithromycin [9]. Once bound, the macrolide drugs prevent the translation of mRNA by preventing the addition of the next amino acid by the tRNA [10]. The action of macrolides are primarily bacteriostatic, which means they work to limit the growth of bacteria by preventing protein synthesis; however, they can be bactericidal (kill the bacteria) at high concentrations [10]. Erythromycin is the main antibiotic used in pertussis treatment and is usually able to clear B. pertussis from the respiratory tract in a few days [4]. Azithromycin and clarithromycin are both alternative antibiotic treatments to erythromycin for adults; however, they have less data on clinical outcomes [1]. Trimethoprim-sulfamethoxazole (TMP/SMX) antibiotic treatment, which is a combination of trimethoprim and sulfamethoxazole antibiotics, has suggested potential use in seven-day treatments; however, there is currently not enough clinical evidence regarding its efficacy for widespread use. Similarly, fluoroquinolone has demonstrated potential efficacy against B. pertussis in in vitro studies, but lacks clinical efficacy data and additionally is associated with resistance in some B. pertussis strains [1]. When tested in vitro, it has been noted that the B. pertussis bacteria are susceptible to tetracycline and chloramphenicol antibiotics as well [4]. For children and adults, the individuals may be treated with one of azithromycin, erythromycin, or clarithromycin when pertussis is suspected or proven within 21 days of onset of symptoms; however, the dosages and frequencies for children and adults differ [3].
Erythromycin, clarithromycin, and azithromycin can all be safely administered to individuals who are one month or older, and are administered orally. However, the use of erythromycin is not recommended for infected individuals who are younger than one month - for these individuals, azithromycin antibiotics are preferred [9]. This is because there is an association between the administration of erythromycin and infantile hypertrophic pyloric stenosis (IHPS), which is characterized by the blockage of a passage exiting the stomach [1], in infants who are less than one month of age. The reasoning behind this association is not known, but studies have found that erythromycin may play a causal role in the development of IHPS for very young infants [11]. Furthermore, although azithromycin is recommended for use in infants younger than 1 month of age, clinicians should monitor infants younger than 1 month of age who receive a macrolide for the development of IHPS and other serious adverse events [9]. This is because pyloric stenosis has occurred after azithromycin use also, but its risk is significantly less than that after erythromycin [12]. When choosing an antimicrobial for treatment or prophylaxis, the following factors should be taken into account: effectiveness, safety, tolerability, ease of adherence, and cost. A detailed description of each macrolide antibiotic used in pertussis is outlined below.
Erythromycin
Although erythromycin is traditionally the antibiotic of choice used in the treatment of pertussis, over the years its usage has reduced [12]. This is because concerns over tolerability and compliance have been noticed with erythromycin, especially because it has a longer dosing protocol than azithromycin or clarithromycin [13]. There are different types of erythromycin that may be used for treatment: erythromycin estolate, erythromycin ethylsuccinate, and erythromycin stearate. However, erythromycin estolate is considered to be the superior treatment drug of choice due to its ability to reach higher concentrations in the serum and respiratory secretions [3]. As mentioned before, erythromycin is not recommended at all for the treatment of pertussis in infants younger than 1 month of age, because of the associated risk of IHPS. For children, the dosing recommendation is 40-60 mg per kg administered per day as four divided doses for 14 days (max 1-2 g/day). For adults, 2 g per day in four divided doses for 14 days are administered [14]. However, in a controlled clinical trial done in Canada, it was noted that 7 days of a maximum of 1 g of erythromycin estolate treatment was as effective for the eradication of the microbe as the 14-day recommendation [3].
The drawback of oral erythromycin is that there are potential gastrointestinal side effects, including nausea, vomiting, and diarrhea that are seen in approximately 30% of cases [1]. Additionally, this antibiotic can act as an inhibitor of the cytochrome P450 enzyme system (CYP3A4 subclass). Therefore, erythromycin cannot be administered with another drug that requires metabolization by the CYP3A4 subclass, as this would increase the elevation of the other drug in the body and cause other adverse effects [14]. In children and adults, there is also a potential for drug interactions and emerging evidence of erythromycin-resistance in some B. pertussis strains. This resistance is facilitated by mutations in the 23S rRNA gene, which is the binding site for erythromycin; however, there is no evidence that this resistance is increasing in prevalence [1]. While erythromycin treatment does not influence the course of the disease, studies suggest that it may be able to alleviate the severity and frequency of coughing when administered in the earlier stages of infection [15]. Additionally, it can decrease the infectious period of the patient from about five weeks to approximately 5-10 days, which is useful to control the spread of infection. This is because erythromycin effectively eliminates B. pertussis from the nasopharynx in a matter of days [4].
Azithromycin
Azithromycin is administered either as oral suspensions, capsules, or tablets [16]. Azithromycin is a newer macrolide antibiotic that is better tolerated than erythromycin, and has demonstrated similar eradication rates [13]. It is also administered fewer times per day and has a shorter treatment course, thus making patient compliance more likely [13]. As mentioned above, azithromycin is the recommended antibiotic for infants younger than 1 month because there is an association between oral erythromycin treatment and IHPS. For these infants, the dosing recommendations are as follows: 10 mg per kg are administered per day as a single dose, for five days [14]. Infants aged 1-5 months follow the same azithromycin dosing protocol as the infants younger than 1 month [14]. For infants over 6 months of age and older children, azithromycin should be administered 10 mg per kg as a single dose on day 1, followed by 5 mg per kg per day on days 2 through 5 [14]. Lastly, adults receiving azithromycin antibiotics are given a dosing regimen of 500 mg on day 1 as a single dose, and then 250 mg per day from days 2-5 [14]. Side effects of azithromycin include abdominal discomfort or pain, diarrhea, nausea, vomiting, headache, and dizziness; these side effects are often fewer and milder than those associated with erythromycin [16]. Furthermore, azithromycin should be cautiously prescribed to patients with impaired hepatic function because this antibiotic can further exacerbate liver injury and damage [17].
Clarithromycin
Clarithromycin is also a newer macrolide drug that is better tolerated and associated with milder side effects than erythromycin; however, its side effects may be more severe in comparison to azithromycin. Furthermore, it is also administered fewer times per day and has a shorter treatment course, which enhances patient compliance [13,14]. Although it is less severe than erythromycin, clarithromycin is not administered to infants younger than one month of age because it is unknown if clarithromycin is associated with IHPS [14]. In children, clarithromycin is administered as 7.5 mg per kg twice daily for 7 days (max 1g/day); in adults, the dosing regimen is 500 mg twice daily for 7 days [13]. Clarithromycin is similar, both chemically and metabolically, to erythromycin, which is why it also acts as an inhibitor of the CYP3A4 subclass and produces the same effects. Furthermore, the common side effects of this antibiotic are related to the gastrointestinal system: epigastric distress, abdominal cramps, nausea, vomiting, and diarrhea [14].
Trimethoprim/Sulfamethoxazole (TMP/SMX)
TMP/SMX is an alternative antibiotic that is administered to patients two months and older who are allergic to macrolides or who cannot tolerate macrolides [13]. TMP/SMX both work as competitive inhibitors: SMX acts as a competitor of p-aminobenzoic acid (PABA) during the synthesis of dihydrofolate to inhibit the enzyme dihydropteroate synthase, whereas TMP directly competes with the enzyme dihydrofolate reductase to stop the production of tetrahydrofolate to its active form of folate. As a result, both TMP and SMX act together to block two steps in the synthesis of essential bacterial nucleic acids and proteins, thus killing the bacteria [15]. The dosing regimen for infants 2 months and older is as follows: TMP 8 mg/kg per day and SMX 40 mg/kg per day in 2 divided doses for 14 days. The dosing regimen for adults is as follows: TMP 320 mg per day and SMX 1600 mg per day in 2 divided doses for 14 days [16]. TMP/SMX should not be given to pregnant women or infants less than 2 months of age because there is a possible risk of kernicterus, which is a type of brain damage that can occur in infants due to high buildup of bilirubin [14,19].
Post Exposure Prophylaxis & Other Treatment
Prophylaxis is defined as the prevention of disease, and antibiotic prophylaxis has been used to prevent transmission to individuals that have come into contact with patients infected with B. pertussis, even if the antibiotic will not help the infected patient (if they are in the later stages of the disease) [13]. Postexposure antibiotic prophylaxis is often administered to anyone who has come into contact with the pertussis-infected patient within 21 days of the original patient's cough onset. Antibiotics can be used in addition to vaccination; in Canada, it is recommended that babies, pregnant women, or anyone who might have come into direct contact with a positive case of pertussis use antibiotics [1]. Individuals at the highest risk of severe and deadly complications from pertussis are infants younger than 12 months (especially those younger than six months) and women in the third trimester of pregnancy, which is why postexposure prophylaxis should be administered to them and in any settings that include the high-risk individuals [13]. The use of antibiotics could potentially be useful for extra protection of infants under six months of age, since they are an extremely high-risk population. The preferred agent for prophylaxis is azithromycin, and the dosing for all agents is the same as for pertussis treatment.
Supportive care can also complement treatment in the care of pertussis patients; intubation and ventilation may be required for infants with apnea and extra care should be made to monitor for any potential co-infections of the respiratory system [1]. Potential adjunctive therapies can also be used to relieve coughing symptoms. Antihistamines and corticosteroids have been reviewed for use; however, they have not shown any benefits in terms of alleviating coughing or decreasing the length of hospital stays [1].
Codeine Cough Syrup
Coughing is often a reflex mediated by the sensory neurons in the airways reflex of the brainstem in response to mechanical, chemical, or inflammatory irritation of the tracheobronchial tree [20]. The purpose of coughing is to help clear airways of obstructive or irritating material or to clear noxious substances in inspired air [20]. After a clinical examination, the physician in Joseph’s case prescribed Joseph with a codeine cough syrup. This is because codeine is known to be effective cough suppressants in adults, capable of suppressing both artificially induced and disease related cough via a central nervous system mechanism (CNS) [20]. Codeine increases the CNS threshold for coughing [20], and can suppress the cough reflex by direct action on the cough centre in the medulla [21]. In clinical trials performed with codeine, there was a linear relationship present between codeine dosage of ranges within 7.5 to 60 mg/day and the decrease in the frequency of chronic coughing [20]. It is important to note, however, that in none of the trials performed the coughs were completely suppressed, even with the highest dosage of codeine [20]. Although codeine acts as a cough suppressant in adults, the efficacy of codeine as a cough suppressant has yet to be shown in children [20], and there is a lack of pharmacokinetic studies surrounding codeine therapy in children [20].
A drawback of codeine cough syrup is that there is a potential for overdose, which can include adverse effects such as respiratory depression and decreased alertness [20]. Despite this, codeine is a more appropriate choice compared to alternatives such as hydrocodone and hydromorphone, which have similar efficacies, the same potential risk of adverse reactions, and are associated with even higher risks of dependency [20]. Over-the-counter (OTC) cough suppressants are usually ineffective in pertussis infections and should not be administered, as even the strongest OTCs cannot relieve the whooping cough spells [22,23].Thus, overall, codeine cough syrup is an effective antitussive treatment used in adults, but not children, suffering from severe or chronic coughing [20]. When speaking to his doctor, Joseph states that he has been experiencing violent, spasmodic coughs for six weeks. Since Joseph was already suffering from coughing spells for 6 weeks and his lab results were clear, it is likely that the causative agent of his pertussis had been cleared and antibiotic treatment would not help to reduce symptoms or contagiousness, as he is in the convalescent phase of the pertussis infection. Thus, codeine syrup is prescribed instead to simply manage his coughing symptoms.
| Antibiotics | Mechanism of Action | Pros | Cons |
|---|---|---|---|
| Erythromycin | Inhibits bacterial biosynthesis: prevents the translation of mRNA by preventing the addition of the next amino acid by the tRNA |
|
|
| Azithromycin | Inhibits bacterial biosynthesis: prevents the translation of mRNA by preventing the addition of the next amino acid by the tRNA |
|
|
| Clarithromycin | Inhibits bacterial biosynthesis: prevents the translation of mRNA by preventing the addition of the next amino acid by the tRNA |
|
|
| TMP/SMX |
|
|
|
Table 1. Summary of mechanisms of action, pros, and cons of B. pertussis antibiotics
References
- Bennett, J. E., Dolin, R., & Blaser, M. J. (2020). Mandell, Douglas, and Bennett's principles and practice of infectious diseases: 2-volume set (Vol. 2). Elsevier Health Sciences.
- Long, S. S., Edwards, K. M., & Mertsola, J. (2018). Bordetella pertussis (pertussis) and other bordetella species. In S.S. Long, C.G. Prober & M. Fischer (eds.), Principles and Practice of Pediatric Infectious Diseases, (Fifth Edition). Elsevier US, New York, New York. https://doi.org/10.1016/B978-0-323-40181-4.00162-6
- Bordetella pertussis - ClinicalKey [Internet]. [cited 2021 Mar 29]. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002307
- Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 31. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7813/
- Pertussis (Whooping Cough) Diagnosis & Treatment [Internet]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention; 2018 [cited 2021Mar29]. Available from: https://www.cdc.gov/pertussis/about/diagnosis-treatment.html
- Spector TB, & Maziarz EK. 2013. Pertussis. The Medical Clinics of North America, 97(4), 537-552. https://doi.org/10.1016/j.mcna.2013.02.004
- Whooping Cough. 2005. Encyclopedia of Children’s Health. http://www.healthofchildren.com/U-Z/Whooping-Cough.html
- Kilgore PE, Salim AM, Zervos MJ, Schmitt HJ. 2016. Pertussis: Microbiology, Disease, Treatment, and Prevention. Clinical Microbiology Reviews, 29(3):449–486. https://doi.org/10.1128/CMR.00083-15
- Pertussis (Whooping Cough). 2019. Treatment. Centers for Disease Control. https://www.cdc.gov/pertussis/clinical/treatment.html
- Patel PH, Hashmi MF.2021 Macrolides. https://www.ncbi.nlm.nih.gov/books/NBK551495/
- Centers for Disease Control and Prevention (CDC). 1999. Hypertrophic pyloric stenosis in infants following pertussis prophylaxis with erythromycin. MMWR. Morbidity and Mortality Weekly Report, 48(49): 1117-1120. https://pubmed/ncbi.nlm.gov/10634345/
- Cherry JD.2017. Treatment of Pertussis, Journal of the Pediatric Infectious Diseases Society, 7(3):e123–e125. https://doi.org/10.1093/jpids/pix044
- Kline JM, Lewis WD, Smith EA, Tracy LR, Moerschel SK. 2013. Pertussis: A Reemerging Infection. American Family Physician, 88(8):507-514. https://pubmed.ncbi.nlm.nih.gov/24364571/
- Graham L.2006.CDC Releases Guidelines on Antimicrobial Agents for the Treatment and Postexposure Prophylaxis of Pertussis. American Family Physician,74(2):333. Available From: https://www.aafp.org/afp/2006/0715/p333.html
- Hoppe JE: Comparison of erythromycin estolate and erythromycin ethylsuccinate for treatment of pertussis. The Erythromycin Study Group. Pediatr Infect Dis J 1992; 11: pp. 189-193.
- Tiwari T, Murphy TV, Moran J. 2005. Recommended Antimicrobial Agents for the Treatment and Postexposure Prophylaxis of Pertussis. 2005 CDC Guidelines. National Immunization Program. Centers for Disease Control. https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5414a1.htm
- LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet]. 2017. National Institute of Diabetes and Digestive and Kidney Diseases. Azithromycin. https://www.ncbi.nlm.nih.gov/books/NBK548434/
- Kemnic TR, Coleman M. 2020. Trimethoprim Sulfamethoxazole. StatPearls [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK513232/
- Kernicterus. 2019. HealthLink BC, British Columbia. https://www.healthlinkbc.ca/health-topics/ue5852
- Committee on Drugs. 1997. Use of codeine- and dextromethorphan-containing cough remedies in children. Pediatrics (Evanston), 99(6), 918-920. https://doi.org/10.1542/peds.99.6.918
- Drug Bank. 2005. Codeine. https://go.drugbank.com/drugs/DB00318
- Ben-Joseph EP. 2016. Whooping Cough (Pertussis). Kids Health from Nemours. https://kidshealth.org/en/parents/whooping-cough.html
- Whooping Cough (Pertussis). 2019. HealthLink BC, British Columbia. https://www.healthlinkbc.ca/health-topics/hw65653
(iv) Why did the doctor ask about Joseph’s vaccination history? Is this a reportable communicable disease?
Overview of Pertussis Vaccinations
Whooping cough is a dangerous, contagious and global infection that has killed many thousands of individuals, especially children, in the past (1). However, widespread vaccination programs have largely decreased pertussis incidence and mortality (1). Immunization is currently the most effective method for preventing B. pertussis infection (1).
There are two main forms of pertussis vaccine - a whole pertussis (wP) and acellular pertussis (aP) vaccine (2). The wP vaccine is an inactivated whole cell vaccine (2). In this vaccine, the B. pertussis bacteria is chemically inactivated and killed using merthiolate (2). Then the whole killed bacteria is added into the vaccine in a merthiolate-killed bacterial cell suspension (2). This wP vaccine is often combined and administered with diphtheria (D) and tetanus (T) vaccines (1,2). This vaccine also commonly contains aluminum salts to serve as an adjuvant to increase vaccine potency and thimerosal to serve as a preservative (2). These vaccines are highly effective but can cause both local and systemic side effects (1,3). Some minor adverse side effects include fever, vomiting, drowsiness, agitation and redness and swelling at the injection site (2,4). However, more severe and rare side effects can include convulsions, brain damage and hypotonic-hyporesponsive episodes (HHE) (2,5,6). HHE manifests in the patient as reduced consciousness, pallor and decreased muscle tone and often occurs within 12 hours of vaccination administration (5,6). For this reason, aP vaccines have been developed and are used more commonly in many countries in North America and Europe today (1). Diphtheria toxoid (DTaP) and tetanus toxoid (Tdap) are two acellular pertussis vaccines that are commonly used (1). The vaccines contain structural components of B. pertussis, such as detoxified PT, filamentous hemagglutinin, pertactin and fimbrial antigens 2 and 3, and produce less side effects (1). These specific antigens stimulate the immune system to produce antibodies and memory cells for protection against these toxins and future infections (1). But, it is important to note that lipopolysaccharide (LPS) is not included in aP vaccines because it is believed to account for many of the adverse side effects seen in wP vaccine administration (3). DTaP vaccines contain a higher concentration of aP antigens and used for primary immunization in children; whereas, Tdap contain lower concentrations of aP antigens and are used as boosters in adolescents and adults (7).
In Canada, primary immunization for all children combination vaccines is recommended at 2, 4 and 6 and 15-18 months of age (4,7,8). Booster doses are also recommended for children at 4-6 years, around the time of school entry (4,7) For these initial doses in children, the pediatric formulation of the DTaP vaccine containing diphtheria toxoid, tetanus toxoid and aP vaccine is administered (4,7). This primary immunization program of repeated acellular pertussis containing vaccine doses is about 90% effective in preventing pertussis in children, when they are considered to be the most vulnerable to severe pertussis (7). However, due to the gradual waning (declining) immunity found after acellular vaccinations, Tdap boosters are recommended (1,7). For instance, in adolescents, a booster is recommended at 11-18 years of age in high school and to all adults at some point over the age of 18 (4,7). Here, the adult formulation of TdaP vaccine, containing tetanus toxoid, diphtheria toxoid and aP, is administered and used in future doses (4,7). After this, Tdap boosters are recommended every 10 years (1). Additionally, extra boosters or single adult doses are recommended for healthcare workers and pregnant women to boost immunity against B. pertussis (4,7).
Importance of Vaccination History
Overall, the best way to prevent whooping cough is through vaccination (1,9). However, the effectiveness of vaccines decreases over time and adults who were vaccinated against pertussis as children can get whooping cough later on in life as their immunity and protection against the disease declines with time (1,9). In a study, 5 doses of DTap was found to provide about 75-89% immediately after (9). But, in the following years, antibody levels and protection declined quite rapidly (9). Thus, each year, individuals are at a greater risk of pertussis even after DTap vaccination (9). Similarly, Tdap vaccination also shows a pattern of declining antibody levels (9). These findings emphasize the importance of routine immunization and boosters in order to provide protection against pertussis disease (9).
Upon asking about Joseph’s vaccination history, the doctor learns that Joseph received all required child vaccinations, but did not receive any immunizations as an adult. Assuming Joseph lives in Canada, this means he likely received the four recommended doses and booster of aP containing vaccines as a child and adolescent. But, he probably did not receive the recommended Tdap booster in adulthood. This information is important to diagnosis because it lets the doctor know that Joseph’s immunity from childhood pertussis immunization has likely waned, leaving him vulnerable to B. pertussis infection. This indicates that his severe, long lasting cough could be associated with pertussis disease and can guide the physicians decision making in diagnosis, ordering lab tests and creating treatment plans.
Pertussis: A Reportable Communicable Disease and Nationally Notifiable Disease
Pertussis is a highly contagious disease that is transmitted via droplets from the respiratory tract of infected individuals (1). In British Columbia, according to the BC Center for Disease Control (BC CDC) pertussis is listed as a reportable communicable disease (10, 11). This means that clinicians and laboratories must report any suspected or confirmed pertussis cases to provincial/ state authorities (12,13). This information is important to update and improve disease prevention and control programs and to detect potential sources of outbreaks (14). Additionally, this information is important for reaching out to close contacts of infected or exposed individuals to ensure they are taking prophylaxis medication and quarantining from others if necessary (14). Overall, this surveillance is needed so action can be taken to control the infection and prevent other cases or an outbreak (15).
Additionally, in Canada, pertussis is listed as a nationally notifiable disease (13). This means that any suspected pertussis cases and positive pertussis laboratory results should voluntarily be reported to the appropriate health departments for monitoring, surveillance and control (13,16). After this, the health departments should report these cases to CDC through the National Notifiable Diseases Surveillance System (NNDSS) (13,15). In Canada, a similar system is set up, where cases are reported to the Public Health Agency of Canada (PHAC) and then to the Canadian Notifiable Disease Surveillance System (CDNSS) (17). These reports are anonymized and use a standardized case definition that includes clinical and laboratory criteria for pertussis (13). Clinical criteria includes a long lasting cough with violent fits, ‘whooping’ upon inspiration, vomiting and/or apnea; laboratory criteria includes isolation of B. pertussis from a sample and/or a positive PCR test for B. pertussis (13). After surveillance information is reported, the NNDSS or CDNSS distributes the information to pertussis specific programs in the CDC or PHAC respectively (15). These programs can provide information to patients and clinicians to guide treatment and recovery (15). They also contribute to providing education on disease prevention and funding for outbreak control to support public health departments (15). Perhaps most importantly, this information is used to update databases used in epidemiological studies to monitor national trends and evaluate prevention and control strategies (19). Overall, this national surveillance allows for large scale public health policies and initiatives for pertussis prevention (19).
References
- Finger H & von Koenig CHW. 1996. Bordetella. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
- WHO | Pertussis [Internet]. WHO. World Health Organization; [cited 2021 Apr 1]. Available from: https://www.who.int/biologicals/vaccines/pertussis/en/
- Waters V, & Halperin SA. 2020. Bordetella pertussis. In J. E. Bennett, R. Dolin & M. J. Blaser (eds.), Mandell, Douglas and Bennett’s Principles of Practice of Infectious Diseases. Elsevier Inc, Philadelphia, PA. https://doi.org/10.1016/B978-0-323-48255-4.00230-7
- Bordetella pertussis - ClinicalKey [Internet]. [cited 2021 Mar 29]. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002307
- Hypotonic-Hyporesponsive Episodes to Immunisation [Internet]. [cited 2021 Apr 1]. Available from: https://www.medsafe.govt.nz/profs/puarticles/8.htm#1
- McPherson P, Powell KR. 2005. Hypotonic-hyporesponsive episode in a 7-month-old infant after receipt of multiple vaccinations. Pediatric Infectious Disease Journal, 24(11), 1010-1011.
- Pertussis vaccine: Canadian Immunization Guide [Internet]. Government of Canada. Public Health; 2021. [cited 2021Mar30]. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-15-pertussis-vaccine.html
- Pertussis (whooping cough): for health professionals [Internet]. Government of Canada. Public Health; 2021. [cited 2021Mar30]. Available from: https://www.canada.ca/en/public-health/services/immunization/vaccine-preventable-diseases/pertussis-whooping-cough/health-professionals.html
- Long, S. S., Edwards, K. M., & Mertsola, J. (2018). Bordetella pertussis (pertussis) and other bordetella species. In S.S. Long, C.G. Prober & M. Fischer (eds.), Principles and Practice of Pediatric Infectious Diseases, (Fifth Edition). Elsevier US, New York, New York. https://doi.org/10.1016/B978-0-323-40181-4.00162-6
- Reportable Diseases Data Dashboard [Internet]. Bccdc.ca. 2021 [cited 2 April 2021]. Available from: http://www.bccdc.ca/health-professionals/data-reports/reportable-diseases-data-dashboard
- BC Center for Disease Control. 2010. Communicable Disease Control. Management of Specific Diseases-Pertussis. http://www.bccdc.ca/resource-gallery/Documents/Guidelines
- Haston JC, Pickering LK. 2019. CDC’s disease surveillance system critical for public health. American Academy of Pediatrics News. https://www.aappublications.org/news/2019/03/08/mmwr030819
- Pertussis (Whooping Cough) Surveillance & Reporting [Internet]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention; 2018 [cited 2021Mar29]. Available from: https://www.cdc.gov/pertussis/surv-reporting.html
- Mandatory Reporting of Infectious Diseases by Clinicians. 1990. MMWR, Recommendations and Reports, 39(RR-9): 1-11,16-17. https://www.cdc.gov/mmwr/preview/mmwrhtml/00001665.htm
- CDC’s Disease Surveillance System Critical for Public Health [Internet]. AAP News. AAP News & Journals Gateway; 2019 [cited 2021Mar29]. Available from: https://www.aappublications.org/news/2019/03/08/mmwr030819
- Notifiable Diseases Online. 2021. Government of Canada. https://diseases.canada.ca/notifiable/
- Pertussis vaccine (whooping cough): surveillance [Internet]. Government of Canada. Public Health; 2021. [cited 2021Mar30]. Available from: https://www.canada.ca/en/public-health/services/immunization/vaccine-preventable-diseases/pertussis-whooping-cough/surveillance.html
- Pertussis (whooping cough): for health professionals [Internet]. Government of Canada. Public Health; 2021. [cited 2021Mar30]. Available from: https://www.canada.ca/en/public-health/services/immunization/vaccine-preventable-diseases/pertussis-whooping-cough/health-professionals.html
- Sockett PN, Garnett MJ, Scott C. 1996. Communicable disease surveillance: Notification of infectious diseases in Canada. The Canadian journal of infectious diseases, 7(5), 293–295. https://doi.org/10.1155/1996/279482
Q2. The Microbiology Laboratory
(i) Including the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.
Joseph presents with a chronic cough, mild fever, runny nose and upper respiratory tract irritation. These symptoms align with his diagnosis of B. pertussis; however, there are a wide range of bacterial pathogens which can cause these clinical manifestations.
Bordetella Pertussis is a small, gram-negative, aerobic, non-motile, cocci shaped bacterium with outer pili (1). B. pertussis infects ciliated human respiratory mucosal cells since the human respiratory mucosa is where B. pertussis usually resides (1). B. pertussis spreads throughout the body and manifests clinical symptoms through the production of several toxins such as the tracheal cytotoxin, adenylate cyclase toxin, heat-liable toxin and the pertussis toxin (PT) (2). Adhesins and other proteins found on B. pertussis aid in infectious spread as well. These include the filamentous hemagglutinin adhesin, fimbriae 2 and 3 adhesins, the pertactin adhesin and lipooligosaccharide (2,3). The tracheal cytotoxin is responsible for killing respiratory ciliated epithelial cells (2). The adenylate cyclase toxin increases the concentration of intracellular cAMP and blocks immune cell responses when it enters host cells (2). The heat-liable toxin induces vasoconstriction, causing damage to the tissue of the respiratory tract (2). The PT is only produced by the B. pertussis species, even though the species B. parapertussis and B. bronchiseptica contain the gene for the PT (2). However, the tracheal cytotoxin, adenylate cyclase toxin, and heat-liable toxin are produced by other Bordetella species like B. parapertussis and B. bronchiseptica (2). PT is able to inhibit G protein coupling in the cell, interfering with the conversion of ATP to cAMP (4). PT aids in modifying a cysteine residue found on the C terminus of the G protein which causes an increase in cAMP concentration (5). As a result, this effects several downstream signalling events with the ability to decrease the activity or presence of phagocytes, neutrophils, monocytes and leukocytes (6). Overall, PT has been associated with several systemic symptoms such as leukocytosis, histamine sensitivity and hypoglycemia (7).
This bacterium causes whooping cough or pertussis which is an acute respiratory infection that is characterized by a debilitating and spastic cough (1). Typically, whooping cough is observed in children; however, it can be seen in unvaccinated adults and immunocompromised individuals (8). It transmits from human to human by respiratory droplets through coughing, sneezing and breathing in close spaces (9). B. pertussis infection has three stages:
1) Catarrhal Stage: During this phase, the patient is considered to be most contagious, and symptoms include cough, sneezing, runny nose and fever (2). This phase lasts from 1 to 2 weeks (10).
2) Paroxysmal Stage: This phase is characterized by a paroxysmal cough which has a whooping sound (1). This stage lasts from 1 to 6 weeks (10).
3) Convalescent Stage: Respiratory symptoms start to slowly subside; yet the cough may remain for weeks to months to come (1). This phase lasts from 2 to 3 weeks (10). These phases can be visualized through Figure 1.
Bordetella Parapertussis is a small, coccobacilli, gram-negative bacteria with structural and genomic similarity to B. pertussis (2). This bacterium produces the tracheal cytotoxin, adenylate cyclase toxin and the heat-liable toxin, but it does not produce the PT (2). It causes a milder form of whooping cough, although B. parapertussis infection is less common (11). B. parapertussis spreads through respiratory droplets, invades ciliated respiratory mucosa cells and induces a paroxysmal cough characteristic of B. pertussis infection (2). Symptoms of B. parapertussis include a cough, fever, and runny nose (2). Given the symptoms of B. pertussis and B. parapertussis are very similar, laboratory testing such as PCR and antibody testing are necessary to determine which species is responsible for infection (2).
Bordetella bronchiseptica is a gram-negative, coccobacillary bacterium which is closely related to B. parapertussis and B. pertussis, although this bacterium mostly infects animals such as dogs, rabbits and cats (12). It typically causes a respiratory disease deemed “kennel cough” in cats and dogs, as well as swine atrophic rhinitis and pneumonia (8,12). It has been reported to infect humans who are immunocompromised or in close contact with animals (8). Like B. pertussis and B. parapertussis, it produces tracheal cytotoxin, adenylate cyclase toxin and heat-liable toxin (2).
Bordetella holmesii is a rod-shaped, gram-negative bacterium which spreads from human to human through respiratory droplets (13). B. holmesii infection causes an illness similar to that of pertussis with a pertussis-like cough and given the lack of species-specific laboratory testing, it is commonly misdiagnosed as B. pertussis (14). This is largely due to the presence of the insertion sequence (IS) 481 in both B. pertussis and B. holmesii which is a typical PCR target sequence for B. pertussis infection (15). Furthermore, B. holmesii is known to cause invasive infections such as meningitis, pericarditis, bacteriemia, endocarditis, arthritis, and pneumonia (15).
Streptococcus pyogenes is a gram-positive, facultative anaerobic bacterium that spreads through respiratory droplets (16). S. pyogenes colonizes the nasopharynx and oropharynx (17). It commonly causes community-acquired pneumonia and acute pharyngitis or strep throat (18). Symptoms include sore throat, upper respiratory tract irritation, malaise and fever (15,16). Given the symptoms of S. pyogenes are similar to that of early pertussis, misdiagnosis can occur.
Chlamydia pneumoniae is a gram-negative, obligate intracellular, enveloped bacterium (19). C. pneumoniae spreads through respiratory droplets and is associated with community-acquired pneumonia (17). This bacterium uses an elementary body to attach to respiratory mucosal epithelial cells which is then endocytosed by the host (19). Once encased in a phagosome within the host cell, the elementary body escapes endosomal maturation and converts to a reticulate body in order to begin binary fission (19). After hours have passed, the reticulate body becomes an elementary body again and the new elementary bodies are released by the host (19). Through this mechanism, symptoms are induced such as hoarseness, sinusitis, pharyngitis and a prolonged cough (20). In addition, C. pneumoniae can cause severe complications like myocarditis and encephalitis (21). Since C. pneumoniae infection presents with a prolonged cough, misdiagnosis for B. pertussis is possible.
Mycoplasma pneumoniae is a gram-negative, extracellular, very small bacterium that infects the upper and lower respiratory tract (22). This bacterium attaches to the sialic acid receptors on respiratory epithelial cells and produces the cytotoxin called community-acquired respiratory distress syndrome (23). This cytotoxin causes inflammation and damage to the respiratory tract by bringing cytokine-producing inflammatory cells to the site of infection (23). Infection can cause symptoms such as sore throat, pharyngitis, dyspnoea fever, and can lead to tracheobronchitis and community-acquired pneumonia (22). In addition, extrapulmonary manifestations can occur, including joint pain, rash and gastrointestinal symptoms (24). Given M. pneumoniae cannot be gram-stained due to its size and its symptoms overlap with those of early pertussis, it is possible that misdiagnosis can occur (22).
References
- Canada PHA of. Pathogen Safety Data Sheets: Infectious Substances – Bordetella pertussis [Internet]. aem. 2011 [cited 2021 Apr 7]. Available from: https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/bordetella-pertussis.html
- Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2021 Mar 27]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7813/
- Kourova N, Caro V, Weber C, Thiberge S, Chuprinina R, Tseneva G, et al. Comparison of the Bordetella pertussis and Bordetella parapertussis Isolates Circulating in Saint Petersburg between 1998 and 2000 with Russian Vaccine Strains. Journal of Clinical Microbiology. 2003 Aug 1;41(8):3706–11.
- Masin J, Osicka R, Bumba L, Sebo P, Locht C. 6 - Bordetella protein toxins. In: Alouf J, Ladant D, Popoff MR, editors. The Comprehensive Sourcebook of Bacterial Protein Toxins (Fourth Edition) [Internet]. Boston: Academic Press; 2015 [cited 2021 Apr 7]. p. 161–94. Available from: https://www.sciencedirect.com/science/article/pii/B9780128001882000069
- Carbonetti NH. Contribution of pertussis toxin to the pathogenesis of pertussis disease. Pathogens and Disease [Internet]. 2015 Nov 1 [cited 2021 Apr 7];73(ftv073). Available from: https://doi.org/10.1093/femspd/ftv073
- Weingart CL, Weiss AA. Bordetella pertussis virulence factors affect phagocytosis by human neutrophils. Infect Immun. 2000 Mar;68(3):1735–9.
- Carbonetti NH. Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools. Future Microbiology. 2010 Mar 1;5(3):455–69.
- Waters V, Halperin SA. Bordetella pertussis. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases [Internet]. Philadelphia, PA: Elsevier; 2020 [cited 2021 Mar 27]. p. 2793-2802. e3. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002307
- Causes and Transmission of Whooping Cough (Pertussis) | CDC [Internet]. 2021 [cited 2021 Apr 7]. Available from: https://www.cdc.gov/pertussis/about/causes-transmission.html
- Signs and Symptoms of Whooping Cough (Pertussis) | CDC [Internet]. 2021 [cited 2021 Apr 7]. Available from: https://www.cdc.gov/pertussis/about/signs-symptoms.html
- Khelef N, Danve B, Quentin-Millet MJ, Guiso N. Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species. Infect Immun. 1993 Feb;61(2):486–90.
- Goodnow RA. Biology of Bordetella bronchiseptica. Microbiol Rev. 1980 Dec;44(4):722–38.
- Kamiya H, Otsuka N, Ando Y, Odaira F, Yoshino S, Kawano K, et al. Transmission of Bordetella holmesii during pertussis outbreak, Japan. Emerg Infect Dis. 2012 Jul;18(7):1166–9.
- Pittet LF, Emonet S, Schrenzel J, Siegrist C-A, Posfay-Barbe KM. Bordetella holmesii: an under-recognised Bordetella species. The Lancet Infectious Diseases. 2014 Jun 1;14(6):510–9.
- Dion CF, Ashurst JV. Streptococcus Pneumoniae. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 [cited 2021 Apr 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK470537/
- Kanwal S, Vaitla P. Streptococcus Pyogenes. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 [cited 2021 Apr 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK554528/
- Gautam J, Krawiec C. Chlamydia Pneumonia. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 [cited 2021 Apr 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK560874/
- Pharyngitis (Strep Throat): Information For Clinicians | CDC [Internet]. 2021 [cited 2021 Apr 7]. Available from: https://www.cdc.gov/groupastrep/diseases-hcp/strep-throat.html
- Hammerschlag MR, Kohlhoff SA, Gaydos CA. Chlamydia pneumoniae. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases [Internet]. Philadelphia, PA: Elsevier; 2020 [cited 2021 Mar 31]. p. 2323-2331.e2. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B978032348255400182X
- Kuo C-C, Jackson LA, Campbell LA, Grayston JT. Chlamydia pneumoniae (TWAR). CLIN MICROBIOL REV. 1995;8:11.
- Pneumonia | Chlamydia pneumoniae Infection | CDC [Internet]. 2021 [cited 2021 Apr 7]. Available from: https://www.cdc.gov/pneumonia/atypical/cpneumoniae/index.html
- Kashyap S, Sarkar M. Mycoplasma pneumonia: Clinical features and management. Lung India. 2010 Jan 4;27(2):75.
- Holzman RS, Simberkoff MS, Leaf HL. Mycoplasma pneumoniae and Atypical Pneumonia. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases [Internet]. Philadelphia, PA: Elsevier; 2020 [cited 2021 Mar 31]. p. 183, 2332-2339.e3. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554001831
- Abdulhadi B, Kiel J. Mycoplasma Pneumonia. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 [cited 2021 Apr 6]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK430780/
(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?
There are two approaches to laboratory diagnosis of pertussis; direct or indirect (24). Direct diagnosis involves identifying the causative pathogen of the disease by targeting the bacteria directly through tests such as bacterial culture or Polymerase Chain Reaction (PCR), whereas indirect diagnosis concerns serology and detection of antibodies (24). For direct diagnosis, the standard for collecting infections of the upper respiratory tract (URT) include sampling using nasopharyngeal swabs, throat swabs, or aspirates. While samples from the URT nasopharynx are preferred as the URT is the primary area of colonization by B. pertussis, it is possible for bacteria to be present in the lower respiratory tract in severe infection (3). When this occurs, sputum samples can also be taken (24). For indirect diagnosis, samples can be obtained through serology testing of a collected blood sample. Blood samples are taken to run antibody tests (ie. IgA, IgG, IgM), however this testing method indirectly identifies infection and thus is less commonly used in clinical practice (22).
As Joseph had mild URT irritation, it was best for the doctor to take one of the URT samples. As such, in the case, it was described that a throat swab was taken from Joseph. However, nasopharyngeal swabs or nasal aspirates are the samples of choice when testing for B. pertussis infection by either culture or PCR (22). In particular, throat swabs have been shown to be less sensitive than nasopharyngeal swabs in terms of culturing and isolating the organism (10). The main reason why nasopharyngeal samples are better than throat swabs is because throat samples are accompanied by more URT normal flora which inhibits B. pertussis growth and the ability to distinguish the bacterium when cultured (10).
Joseph sought medical attention after having a cough for 6 weeks which means he passed the Catarrhal Phase where patients are considered most contagious and is exiting the Paroxysmal Phase to enter the Convalescent Phase as seen in Figure 1 (8). The bacteria can be found within the nasopharynx from weeks 1-2 of cough, however after more than 2 weeks, the bacteria are less prevalent and become sensitive to culture, creating the possibility for false negatives (17). Therefore, this may possibly be why we saw a negative result for B. pertussis in Joseph’s case.
DIRECT DIAGNOSIS:
1. Nasopharyngeal Swabs
Nasopharyngeal swabs are typically the preferred sample to collect when B. pertussis is suspected(1). This procedure is commonly used to detect URT infections and collects mucous and cells from the nasopharynx or the back of the nose. The nasopharyngeal swab has a long plastic or metal shaft and a flocked tip composed of Dacron, rayon or nylon. Cotton or calcium alginate should not be used in this case as these swabs have been demonstrated to contain residues that may inhibit PCR assays (22). With regards to the sampling procedure, the patient should raise their head back to allow for easier passage through the nose to the nasopharynx (11). The swab is then inserted into the nose until it reaches the posterior nares, about 4 cm into the nasopharynx or half of the distance from the corner of the patient’s nose to the front of their ear (1). The healthcare professional turns the swab several times, removes it from the nose, and places it into the transport medium, breaking the shaft of the swab and closing the container (1). This sampling technique is shown in Figure 2. If this sampling method is used, one swab per nostril is recommended (3). This process can induce coughing and gagging and may be uncomfortable for patients (13). Patients may also have minor nosebleeds following the procedure (13).
In addition to the swab technique, the culture sensitivity relies on the length symptoms have been present and the timelines of the transportation of the sample to the laboratory (8). For instance, nasopharyngeal swabs are utilized to collect high numbers of organisms to culture for respiratory illnesses, best performed within 24-48 hours of symptom onset (15). In addition, transport of these samples is equally important as it must be done in a timely manner (under 24 hours) (20). Alternatively, direct plating of the sample should be completed onto, preferentially, Bordet-Gengou medium or Regan-Lowe medium (20). Regan-Lowe medium is comprised of charcoal agar and horse blood containing cephalexin in order to reduce any growth of bacteria associated with the nasopharyngeal normal flora (20).
2. Throat Swabs
Throat swabs can also be taken for a sample to be used for laboratory testing, which was the method used in Joseph’s case. This is done to collect cells that are present within the throat, and this sample can be tested to determine if any microbes are present that may be causing the infectious symptoms the patient has (4). A sterile swab with a plastic or wire shaft and a Dacron or rayon tip is used (4). Again, cotton swabs should not be used as they can interfere with PCR testing (27). The patient opens their mouth while the healthcare professional uses a tongue depressor to press the patient's tongue down (4). They use the swab to rub the tonsils and the back of the throat, being careful to avoid touching the tongue and side of the cheeks which can contaminate the sample (4). The swab must then be quickly placed in the transport medium, breaking off the shaft of the swab so it can fit into the transport medium container (1). While this is the sampling method taken in Joseph’s case, throat swabs have low rates of DNA recovery, and thus are not recommended for use in pertussis diagnosis (19). Therefore, this can also to contribute as to why Joseph's test results came back negative for B. pertussis.
3. Nasal Aspirates
Nasopharyngeal aspirate samples have been shown to improve isolation rates by 15% in comparison to nasopharyngeal swabs; however, because it is only practical in a hospital setting, it is considered more cumbersome (25). This aspirate method uses a suction device to collect secretions from the nasopharynx. The mucous trap, which the secretions are collected in, is connected to the suction outlet and the suction catheter, which is the tube that will be inserted into the patient’s nose (23). The catheter is inserted into the nose in order to reach the nasopharynx. The suction outlet is then turned on, applying suction and pulling mucous into the mucous trap (23). The catheter is then removed from the nose with a rotating motion and flushed with 3 mL of transport medium. The aspirate sample in the mucous trap is stored at room temperature when transferred to the lab (23). It should be transferred so testing in the microbiology lab can begin within 48 hours. A nasopharyngeal sample can also be collected using a syringe, rather than a suction outlet, but otherwise following the same procedure (7).
4. Sputum Sample
The initial onset of whooping cough during the Catarrhal Phase, the bacterium is usually confined to the URT and the cough is usually a dry cough which does not produce any sputum. However, over a few days, the cough can become more productive and produce phlegm/sputum (24). Therefore, depending on the time of the sample, sputum collection may or may not be effective. In terms of the collection procedure, the healthcare professional should instruct the patient to take a deep breath and cough up sputum into a wide mouth container with a resulting minimum volume of about 1 mL. The resulting sample should have no saliva or postnasal discharge. The specimen container should be labeled and sent to the microbiology laboratory (26).
INDIRECT DIAGNOSIS:
1. Serum sample
If a serology test is required, a blood or serum sample is needed from the patient. Serology as a diagnostic tool for B. pertussis is recommended for those over the age of 10 and those who have had a cough for over 2 weeks (6, 27). A paired sample is ideal for better sensitivity and specificity (6). Serology testing can be helpful for patients who have reached an advanced number of weeks with a cough however, it is not widely used, as testing is not standardized and issues can arise in interpreting the results (8). For instance, if a patient had a vaccination for pertussis within 6 months prior to developing pertussis-like symptoms, the antibodies produced in response to the vaccine and those produced in reaction to the infection would be indistinguishable (8). Although Joseph did not get blood testing, this can be done to determine the levels antibodies specific to B. pertussis and the white blood cell count in the patient’s blood. This is collected via venipuncture using a metal needle to collect blood into a glass tube from the vein in the patient’s arm (5).
The nasopharyngeal swabs, throat swabs, aspirates, or sputum sample after collection should be transported at room temperature and quickly to the microbiology laboratory for culture. The swab or the tip of the catheter can also be placed in Reagan Lowe (RL) or Amies medium containing charcoal transport-medium (24). Isolation rates decrease when transport occurs at 4 °C instead of ambient temperature, or takes longer than 48 hours (24).
Why is microbiology testing important?
The microbiology laboratory plays an important role in the diagnosis of this disease. Early identification of the disease is important for giving the correct antibiotics to patients before the illness has progressed, as starting antibiotics later in the course of the illness is typically ineffective at lessening symptoms (18). As pertussis has similar early signs and symptoms to other URT infections, like mild fever, runny nose, and cough, laboratory testing is extremely important to be able to correctly identify a B. pertussis infection. By detecting the exact causative agent causing the disease, the doctor is able to prescribe specific medication (2). This is beneficial to effectively rid the bacteria from the body, as well as to prevent the rise of antibiotic resistant strains, which can occur when patients are prescribed broad-spectrum antibiotics. Therefore, to prevent the rise of more antibiotic resistance strains, doctors must prescribe the existing antibiotics more judiciously (9).
The laboratory diagnosis is also important as individuals in close contact with someone who has a B. pertussis infection, like those who live in the same household, or those at high risk of developing serious illness, including pregnant women, infants, and individuals with pre-existing conditions, who were exposed to someone with pertussis are treated with prophylactic antibiotics to prevent the development of infection (12). Quickly identifying B. pertussis as the cause of illness allows these individuals who were exposed to someone with infectious pertussis to receive treatment before they have serious illness and limits the spread of the bacteria throughout the community (12). Thus, early diagnosis might help limit spread to other susceptible people, but even if a person infected with B. pertussis did already come in contact with a susceptible immunocompromised individual, testing is still important to administer prophylactic antibiotics.
The microbiology laboratory also allows for the public health department to track the epidemiological changes and the rate of disease caused by B. pertussis (16). Pertussis is a reportable communicable disease, so correctly identifying these cases is important to provide the most accurate information regarding the number of B. pertussis infections (16). Testing can also be performed to monitor the evolution of B. pertussis, allowing better understanding of the bacteria on a molecular level, and providing insight on the effectiveness of the pertussis vaccine and changes that may need to be made to make it more effective as the bacteria evolves (16).
Although the microbiological laboratory is very important for the diagnosis of B. pertussis infection, having a negative result like Joseph did does not rule out the possibility of whooping cough (14). The tests that are performed are more likely to be negative as the disease progresses, as the bacteria is no longer present in the body, despite the cough remaining (14). After 2 weeks of having a cough, the level of bacterial DNA decreases, which is likely why Joseph’s culture or PCR tests came back negative, as he has had a cough for 6 weeks (14).
References:
1. Alberta Health Services (2020, May). Collection of a Nasopharyngeal and Throat Swab for Detection of Respiratory Infection. ProvLab Alberta. Available from: https://www.albertahealthservices.ca/assets/wf/plab/wf-provlab-collection-of-nasopharyngeal-and-throat-swab.pdf
2. Faulkner, A., Skoff, T., Cassiday, P., Tondella, M. L. Liang, J. (2017, October). Chapter 10: Pertussis. Centers for Disease Control and Prevention. Available from: https://www.savyondiagnostics.com/wp-content/uploads/2017/05/B.-pertussis-laboratory-testing-by-CDC.pdf
3. H. Finger and C. H. W. von Koenig, “Bordetella,” in Medical Microbiology, 4th ed., S. Baron, Ed. Galveston (TX): University of Texas Medical Branch at Galveston, 1996.
4. How To Swab a Throat for Testing - Ear, Nose, and Throat Disorders [Internet]. Merck Manuals Professional Edition. [cited 2021 Mar 31]. Available from: https://www.merckmanuals.com/en-ca/professional/ear,-nose,-and-throat-disorders/how-to-do-throat-procedures/how-to-swab-a-throat-for-testing
5. Information NC for B, Pike USNL of M 8600 R, MD B, Usa 20894. Best practices in phlebotomy [Internet]. WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy. World Health Organization; 2010 [cited 2021 Apr 2]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK138665/
6. König C-HW von. Pertussis diagnostics: overview and impact of immunization. Expert Review of Vaccines. 2014 Oct 1;13(10):1167–74.
7. Laboratory manual for the diagnosis of whooping cough caused by Bordetella pertussis-Bordetella parapertussis. Update 2014 [Internet]. [cited 2021 Apr 2]. Available from: https://www.who.int/publications/i/item/laboratory-manual-for-the-diagnosis-of-whooping-cough-caused-by-bordetella-pertussis-bordetella-parapertussis.-update-2014
8. Leber AL. Pertussis: Relevant Species and Diagnostic Update. Clinics in Laboratory Medicine. 2014 Jun 1;34(2):237–55.
9. Lee, C. R., Cho, I. H., Jeong, B. C., & Lee, S. H. (2013). Strategies to minimize antibiotic resistance. International journal of environmental research and public health, 10(9), 4274–4305. https://doi.org/10.3390/ijerph10094274
10. Marcon MJ, Hamoudi AC, Cannon HJ, Hribar MM. Comparison of throat and nasopharyngeal swab specimens for culture diagnosis of Bordetella pertussis infection. Journal of Clinical Microbiology. 1987;25(6):1109–10.
11. Marty FM, Chen K, Verrill KA. How to Obtain a Nasopharyngeal Swab Specimen. New England Journal of Medicine. 2020 May 28;382(22):e76.
12. Pertussis | Outbreaks | PEP Postexposure Antimicrobial Prophylaxis | CDC [Internet]. 2021 [cited 2021 Apr 2]. Available from: https://www.cdc.gov/pertussis/pep.html
13. Nasal Swab: MedlinePlus Medical Test [Internet]. [cited 2021 Apr 1]. Available from: https://medlineplus.gov/lab-tests/nasal-swab/
14. National Organization for Rare Diseases (2018). Nontuberculous Mycobacterial Lung Disease. Available from: https://rarediseases.org/rare-diseases/nontuberculous-mycobacterial-lung-disease/
15. Ottawa Public Health. (2020). How to Collect a Nasopharyngeal (NP) swab. https://www.ottawapublichealth.ca/en/professionals-and-partners/how-to-collect-a-nasopharyngeal--np--swab.asp
16. Surveillance Manual | Pertussis | Vaccine Preventable Diseases | CDC [Internet]. 2020 [cited 2021 Apr 2]. Available from: https://www.cdc.gov/vaccines/pubs/surv-manual/chpt10-pertussis.html
17. Pertussis | Whooping Cough | Diagnosis Confirmation | CDC [Internet]. 2021 [cited 2021 Mar 28]. Available from: https://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-confirmation.html
18. Pertussis | Whooping Cough | Clinical | Treatment | CDC [Internet]. 2021 [cited 2021 Apr 2]. Available from: https://www.cdc.gov/pertussis/clinical/treatment.html
19. “Pertussis | Whooping Cough | Use of PCR for Diagnosis | CDC,” Feb. 26, 2021. https://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-pcr-bestpractices.html (accessed Apr. 02, 2021).
20. Waters V, Halperin SA. Bordetella pertussis. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases [Internet]. Philadelphia, PA: Elsevier; 2020 [cited 2021 Mar 27]. p. 2793-2802. e3. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002307
21. “What to expect if you are being tested for the coronavirus.” https://www.usatoday.com/in-depth/news/2020/04/01/coronavirus-testing-what-expect-if-youre-tested/5077039002/ (accessed Apr. 02, 2021).
22. “Whooping Cough (Pertussis) Tests | Lab Tests Online.”https://labtestsonline.org/tests/whooping-cough-pertussis-tests (accessed Apr. 02, 2021).
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24. WHO. (2014). Laboratory Manual for the Diagnosis of Whooping Cough caused by Bordetella pertussis/ Bordetella parapertussis. World Health Organization. Available from:https://apps.who.int/iris/bitstream/handle/10665/127891/WHO_IVB_14.03_eng.pdf;jsessionid=157A59F7E425FB1BB89DBA96B9494616?sequence=1
25. WHO. (2013). Laboratory manual for the diagnosis of whooping couch caused by Bordetella pertussis/ Bordetella parapertussis. World Health Organization. https://www.who.int/immunization/sage/meetings/2014/april/2_Laboratory_manual_WHO_2013_Update.pdf
26. WHO. (2005, January 12). WHO guidelines for the collection of human specimens for laboratory diagnosis of avian influenza infection. World Health Organization. Available from: https://www.who.int/influenza/human_animal_interface/virology_laboratories_and_vaccines/guidelines_collection_h5n1_humans/en/
27. van der Zee A, Schellekens JFP, Mooi FR. Laboratory Diagnosis of Pertussis. Clin Microbiol Rev. 2015 Oct;28(4):1005–26.
(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.
There are two types of approaches that can be taken when undergoing laboratory diagnosis for this pathogen, such as a direct or indirect diagnosis. A direct diagnosis consists of identifying the disease-causing agent either by culture, real-time polymerase chain reaction (RT-PCR), or direct fluorescent antibody (DFA). However, DFA staining of nasopharyngeal secretions is not recommended because of the tendency to show false positive and false negative results (1). Indirect diagnosis techniques involve using serology, such that we are detecting for antibodies in the serum sample instead of detecting the bacterium directly. The figure below shows the optimal timing for taking samples from the patients post coughing onset (2).
Culture:
The gold standard for diagnosing B. pertussis infection is via culture of the organism (3). Although it is utilized in the detection of pertussis, it is only moderately sensitive, as the success rate of bacterial detection is approximately 60% (1). Success of bacterial culture is noted to be higher in infants, and in adults, bacterial culture is typically only successful if collected within the first 2 weeks of infection (4). If collected early in infection, the causative bacteria can be confirmed by isolating the organism in artificial media and conducting a culture (5). This semi-solid media is able to provide isolated, identifiable, and quantifiable colonies (1). If culture testing is unable to be performed right away following sample collection, it is possible to keep the biological samples at -80 °C for storage. These samples should be kept frozen in Bovine Serum Albumin (BSA)/glutamate solution.
The swab or catheter used to collect the nasopharyngeal or throat samples are streaked onto Regan-Lowe (RL) or Bordet Genou (BG) medium (1). RL contains charcoal agar, which neutralizes molecules that are toxic to B. pertussis like fatty acids and peroxides, and horse blood which has nutrients that support the growth of the bacteria (6). BG agar contains glycerol, potato infusion, and animal blood to support the growth of B. pertussis (7). Both media also contain the antibiotic cephalexin that inhibits the growth of other respiratory bacteria to isolate B. pertussis, but may inhibit the growth of certain strains of B. pertussis (8). The plates are inoculated at 35-36 degrees for 7 days (1). The colonies of both B. pertussis and B. parapertussis are small, pearly, and greyish-white, but B. parapertussis is somewhat larger and duller than B. pertussis (9). On BG media, B. parapertussis can appear brown as it expresses tyrosinase, while B. pertussis appears silver or grey, which can help differentiate between the species (1). These bacteria are small coccobacilli and typically appear alone or in pairs, rather than in chains, which can be observed when they are examined under a microscope (9).
Bacterial culture is not typically utilized to diagnose M. pneumoniae, as culturing is difficult and time-consuming, and often results in false negatives (10). Cultures are sometimes performed to test antimicrobial susceptibility. When M. pneumoniae is cultured, SP4 glucose agar or broth is used (11). SP4 medium contains glucose, beef heart infusion, peptone supplemented with yeast extracts, which provides B vitamins, CMRL 1066 medium, and fetal bovine serum, which provides protein and cholesterol (12). A beta-lactam antibiotic and an antifungal medication, like penicillin G and amphotericin B respectively, should be added to selectively grow M. pneumoniae and prevent the growth of other bacteria in the sample (11).
Culturing C. pneumoniae is also challenging, as it is an intracellular bacteria and requires eukaryotic cells to grow (29). It can also be time consuming and has poor sensitivity and specificity, so the results need to be confirmed via PCR (13). C. pneumoniae is typically grown in monolayers of HL or HEp-2 cells, on Eagle’s minimum essential medium (MEM) or Iscove's Modified Dulbecco's Medium (IMDM) with added fetal calf serum, l-Glutamine, and MEM-non-essential amino acids. MEM contains salts, glucose, amino acids, and vitamins, and IMDM has high levels of glucose and sodium pyruvate (14). The antibiotics, vancomycin and gentamicin, and the antifungal, amphotericin, are added to prevent the growth of other microbes. C. pneumoniae is grown at 35°C in the presence of 5% of CO2 (14). The monolayers of the eukaryotic cells are stained with monoclonal antibodies specific to C. pneumoniae, identifying the presence of the bacteria (14).
S. pyogenes is generally grown on agar media supplemented with sheep blood with trypticase soy base, which allows for the detection of β-hemolysis, in the presence of 5% of CO2 and in a temperature of 35-37°C (15). β-hemolysis is important for identification and enhances the growth of S. pyogenes by adding an external source of catalase. Growth is observed after 24 hours of incubation. The typical appearance of S. pyogenes colonies is dome-shaped with a smooth or moist surface and clear margins. They appear white-greyish and have a diameter of ≥ 0.5 mm, and are surrounded by a zone of β-hemolysis. In the event that only a few colonies of S. pyogenes appear, it is difficult to discern whether the individual is infected, as they are most likely to be streptococcal carriers, as opposed to acutely infected individuals (15).
S. pneumoniae, grown best at 35-37°C, is usually cultured on media containing blood, but can also grown on a chocolate agar plate (16). On blood-agar plates, colonies appear as small, grey, moist colonies that produce a zone of α-hemolysis. After 24-48 hours, the colonies flatten and the central region becomes depressed.
Gram-Stain:
A gram-stain can be performed to confirm if the bacteria growing in the culture are diderm or monoderm. Gram staining is a technique used to differentiate bacteria based on their cell wall constituents (17). Gram-positive bacteria have thick cell walls whereas gram-negative bacteria have thinner cell walls with an outer membrane (18). The cells in the sample are stained with crystal violet dye and Gram’s iodine solution is added, allowing the iodine and the crystal violet stain to form a large complex (19). Solution that dehydrates the peptidoglycan is added to the sample, like acetone, resulting in the thick peptidoglycan layer of monoderm, or gram-positive, bacteria shrinking, preventing the iodine-crystal violet complex from washing out of these cells, while it is removed from the diderm, or gram-negative, bacteria that have a thin peptidoglycan layer (19). A counterstain is added to the sample, like safarin, which stains the gram-negative cells that no longer have violet crystal a pink or red colour, and the gram-positive cells remain purple (19).
B. pertussis and B. parapertussis are gram-negative bacteria (5). M. pneumoniae and C. pneumoniae are gram-negative bacteria. S. pyogenes and S. pneumoniae are gram-positive bacteria.
Oxidase Test:
The lab technicians may then perform an oxidase test to help differentiate if the bacteria in the sample is B. pertussis or B. parapertussis. This test identifies if a bacteria produces cytochrome c oxidase, which is the terminal electron acceptor in aerobic respiration. When this enzyme is present, it oxidizes the reagent, tetramethyl-p-phenylenediamine dihydrochloride, turning it a dark purple or blue colour (20). To perform this test, filter paper is soaked in the reagent, dampened with distilled water, and the colony of interest is smeared on the paper (20). If the bacteria is oxidase positive, meaning it possesses cytochrome c oxidase, it will turn the area of the paper dark blue or purple, and if it is oxidase negative, it will remain white (20).
All Bordetella species except B. parapertussis will have a positive oxidase test, which may assist with differentiation between these bacteria (21).
Slide Agglutination:
Another test to identify B. pertussis is slide agglutination. Colonies of the bacteria are suspended in saline, and antisera, serum with antibodies specific to B. pertussis, is added to the suspension (9). If agglutination, the clumping of bacteria to the antibodies in the serum, occurs, the bacteria is present within the sample, and if agglutination does not occur, the bacteria is not present (9).
Slide agglutination can also be performed to serotype the bacteria. B. pertussis can be divided into three types: serotype 2, serotype 3, and serotype 2,3. The serotyping antigens of B. pertussis have been determined to be associated with their fimbrae (22). After sectioning the isolate on the slide, serogroup-specific antisera are added to the slide, and observed after several minutes to determine the microagglutination (23). Hybrid monoclonal antibodies to the serotype 2 and serotype 3 fimbrae antigens have been produced and utilized in clinical practice (22). A positive result is indicated by agglutination with one specific antiserum and no agglutination with saline (24). However, the bacterial agglutination method is subjective and depends on the ability of the bacteria to form a smooth suspension.
PCR:
Overall, PCR is more sensitive and efficient than culture (22). PCR uses the same nasopharyngeal sample that is required for culture and can be transported dry if PCR is the sole test being performed or the sample can be held in molecular grade water or saline which is boiled to isolate the DNA (3). This will denature the DNA and divide the double-stranded DNA into 2 single strands (27). Purification of the DNA is necessary and usually done by automated extraction systems however, other methods include phenol-chloroform extraction or the use of chaotropic agents or resins via column extraction (27). Nucleic acid primers that are specific to the region of DNA that is being amplified are added, associating with their target sequence. PCR then utilizes a DNA polymerase (typically Taq or Pfu) that builds new strands of DNA from the originals, amplifying the strand (28). Each new strand created is able to be denatured in the following cycle and thus used as another template strand. This cycle of denaturing and synthesizing new DNA continues for many rounds, such as up to 40 times, which can lead to more than one billion copies of the original DNA segment (27).
To identify the presence of a particular bacteria, primers are made that pair with sequences in that specific bacterial genome, but not in others (3). For Bordetella, insertion sequences (IS), mobile genetic elements that have many copies in the genome, are typically used to create the primers (3). A number of IS are present at varying copy numbers within different Bordetella genomes, so PCR results must be carefully interpreted to determine which species is present within the sample (1). Insertion sequences (IS) are great targets for PCR as they are repetitive across the genome therefore, B. pertussis IS481 and B. parapertussis IS1001 are commonly utilized as targets (3). Other genes have also been used as targets for PCR, including ptxA, cyaA, prn, recA, carB, and bhur (3).
However, because PCR detects genomic material, this method does not make a distinction between live and dead bacteria. Though contamination is not a major issue with B. pertussis, because it is not an environmental organism, contamination in the laboratory and collection sites can occur (8).
In the clinical microbiology laboratory, nasopharyngeal samples can be tested quickly for many respiratory pathogens using PCR panels, like the Biofire FilmArray respiratory panel (29). These panels include primers specific to the pathogens they are aiming to detect, and using the same amount of sample, time, and cost as performing PCR for a single pathogen (29). This allows for the detection of common bacteria and viruses that infect the respiratory tract, including B. pertussis, B. parapertussis, M. pneumoniae, and C. pneumoniae (29). In Joseph’s case, this would be useful to quickly determine which of the suspected bacteria are causing his illness, prior to performing PCR only for B. pertussis, and may allow for the laboratory technicians to choose the correct medium that will select for the bacteria identified using PCR.
Serology:
Antibodies against B. pertussis can also be detected in a patient’s blood. This is typically done via an immunoglobulin (Ig) G pertussis toxin (PT) enzyme-linked immunosorbent assay (ELISA) (1). An ELISA assay detects and quantifies the number of antibodies present in a sample which are targeted to a specific antigen (3). Serological testing can be useful because pertussis patients often delay seeking medical attention because of the non-specificity of the symptoms, so that an antibody response may be observed by the time of treatment (8). Cultures, on the other hand, are only effective during the early onset of the illness.
During an ELISA, the antigen, in this case the PT, is secured to the bottom of the plate, and the patient serum is added to the plate (30). If antibodies to PT are present within the sample, they will bind to PT (30). The wells are then washed to remove any unbound sample materials. Horseradish peroxidase labelled anti-human IgG is added to the wells, which binds to any antibodies in the wells that are attached to PT (30). The sample is then washed again to remove any unbound antibodies (30). Tetramethylbenzidine, the substrate of peroxidase, is added to the wells, which turns the sample blue if the enzyme is present in the well, indicating that there were antibodies in the original sample that bound to PT (30).
Most commonly, PT is the antigen used for B. pertussis testing, due to its exclusive expression in B. pertussis (8). FHA, PRN, FIM, lipooligosaccharide, and ACT have also been used in development of ELISAs, but are less frequently used due to the possibility of cross-reaction with non-Bordetella species and lack of consistency (8). Specifically, FHA may produce cross-reactive responses with Haemophilus influenzae and Mycoplasma pneumoniae; PRN and FIM antibodies occur less frequently and are thus less consistent, and ELISAs utilizing lipooligosaccharide and ACT are rare (8).
Direct Fluorescent Antibody Testing:
A Direct Fluorescent Antibody (DFA) assay can be used to determine pertussis diagnosis using a nasopharyngeal sample. Direct fluorescent antibody testing is a rapid and inexpensive method to diagnose pertussis infection, but has poor sensitivity and specificity (8). A slide is prepared with fluorochrome-conjugated monoclonal antibodies which bind to the lipooligosaccharide epitope found on B. pertussis (8). These fluorescent antibodies bound to the bacteria can be seen through a microscope to confirm the presence of bacteria (17). Although this test yields quick results, it is not solely used to diagnose B. pertussis infection because it is rated quite low in terms of sensitivity and specificity (3). The sensitivity of DFA compared with culture is reported to be 30-70%, and can cross-react with other bacteria such as B. bronchiseptica and H. influenzae. Because of these factors, DFA testing is not considered to provide laboratory confirmation and has been largely replaced by other diagnostic tests (8).
References:
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- CDC. (2017, August 7). Diagnosis Confirmation of Pertussis (Whooping Cough). Centers for Disease Control and Prevention. Available from: https://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-confirmation.html#:~:text=Clinicians%20commonly%20use%20several%20types,reaction%20(PCR)%20and%20serology.
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- “Whooping Cough (Pertussis) Tests | Lab Tests Online.” https://labtestsonline.org/tests/whooping-cough-pertussis-tests (accessed Apr. 02, 2021).
- H. Finger and C. H. W. von Koenig, “Bordetella,” in Medical Microbiology, 4th ed., S. Baron, Ed. Galveston (TX): University of Texas Medical Branch at Galveston, 1996.
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(iv) What are the results expected from these tests that might allow the identification of the bacteria named in this case.
Given the presentation of Joseph, we would expect the presence of either B. pertussis or B. parapertussis in laboratory testing. Thus, these bacteria will be the focus of the expected results.
Bacterial Culture
Bacterial cultures would first be observed macroscopically for the presence of B. pertussis or B. parapertussis colonies. Both of these Bordetella species produce isolated colonies on Regan-lowe (RL) and Bordet-Gengou (BG) media which are approximately 1 mm in diameter [1]. After incubation for 3 to 4 days, B. pertussis colonies present as rounded and domed glistening mercury-coloured droplets [1]. On BG media, both B. pertussis and B. parapertussis colonies are hemolytic [1]. There is distinction between B. pertussis and B. parapertussis bacterial culture, as B. parapertussis produces a brown pigment on BG media due to the expression of tyrosine kinase, and parapertussis colonies are larger, duller and are visible sooner [2].
Gram staining
If bacterial colonies grow as expected, it is likely that a gram-staining test would be performed to confirm that the bacteria growing is B. pertussis or B. parapertussis [1]. Both of these bacteria are gram-negative, and thus we would expect to see the bacteria stained pink by safranin.
The Oxidase Test
The oxidase test detects the presence of a cytochrome oxidase system, which is present in bacteria that use cytochrome c as a part of respiration [4]. The oxidase test thus may be useful in differentiating between B. pertussis and B parapertussis. A positive oxidase test, as expected in the presence of B. pertussis will result in the reagent turning blue due to oxidation indicating the presence of a cytochrome c system [5]. A negative oxidase test, as expected in the presence of B parapertussis is evident if the reagent remains colourless [5].
Slide Agglutination Serogrouping (SASG)
SASG testing can determine which serogroup of B. pertussis is involved in an infection. A positive SASG test result is indicated when there is agglutination of a sample with specific antiserum, and no agglutination with saline, as shown on the right [6]. In the case of Joseph, we would expect that the serotype assay would be positive for at least one of Fim2 and Fim3. We would see an antigen-antibody complex form when treated with either Fim2 or Fim3 antibodies depending on the serotype of the bacteria, with anti-Fim2 antibodies forming complexes with Fim2 B. pertussis, anti-Fim3 antibodies forming complexes with Fim3 B. pertussis, and both anti-Fim2 and anti-Fim 3 antibodies forming complexes with Fim2,3 B. pertussis [1].
PCR and Gel Electrophoresis
For a PCR assay, we would expect a positive result to be indicated by amplification of the area of interest, which in the case of B. pertussis would be Pertussis Toxin (PT) [1]. Once multiple cycles of PCR have occurred, samples can be analyzed according to the LightCycler® manual (when using this type of technology). Samples are considered positive when the fluorescence signal increases and shows a typical amplification kinetic curve as seen in the figure to the left, and samples are noted as negative when they do not show the kinetic curve [1].
Amplification can also be visualized and identified by conducting gel electrophoresis, wherein the PT gene would be separated based on molecular size [9]. As the PT gene is composed of 5 toxin subunits, it is expected that 5 bands would appear on gel electrophoresis, each band corresponding to a subunit [9]. PCR is favoured over culture as it is a more sensitive laboratory measure, and it is quite rapid to conduct.
Serology (ELISA)
For an ELISA, a positive result indicating the presence of B. pertussis would be demonstrated by binding of Pertussis Toxin antigens to IgG antibodies upon introduction of a serum sample [10]. The PT antigen attached to the reporter enzyme will be immobilized on a solid surface, and upon introduction of the serum sample, a colour change indicating binding of the antigen complex to antibodies will be observed [10].
The criteria for a positive B. pertussis ELISA result would be IgG-anti-pertussis toxin antibodies over 100 IU/ml [11]. Any results which show IgG-anti-pertussis toxin antibodies under 40 IU/ml means it is unlikely that this individual has a pertussis infection or has been in contact with this bacteria [11]. Typically, IgG antibodies are used to determine whether or not a person is infected with B. pertussis because at the point an individual requires a serology test for B. pertussis diagnosis, this individual is producing mainly IgG antibodies in response to infection [12]. However, IgA-anti-pertussis toxin antibodies can be used to confirm results for the IgG antibody tests that are between 40 IU/ml and 100 IU/ml [11]. IgA and IgM antibodies are not used as the primary antibody in ELISA tests because generally they are not always found in patients diagnosed with a disease caused by a specific microbe, in large enough amounts [13]. This is likely due to the fact that IgM and IgA antibodies are produced in smaller amounts at the early stages of the immune response and disease course [13]. An IgG ELISA for PT is reported to be quite sensitive at 92.2% for serodiagnosis of B. pertussis [14].
Direct Fluorescent Antibody (DFA) Test
A positive result in DFA is detected through the visualization of fluorescence underneath the microscope, as shown to the left. This fluorescence is indicative of binding of fluorescent tagged antibodies directly to the antigen located on B. pertussis [7]. Due to poor sensitivity and specificity, it is not recommended for DFA to be the sole laboratory test for B. pertussis.
There are many laboratory methods that can be used to identify B. pertussis bacterium, and as such it is important that the correct test be conducted based on the disease time point to avoid false negatives. It is difficult to know which stage of infection Joseph is at, however his history indicates that he likely has had this infection for at least 6 weeks prior to visiting his doctor, and therefore the laboratory samples collected may not have been appropriate given his disease status. Given the fact that has had coughing spells for 6 weeks, it is unlikely that the direct approach to laboratory diagnosis, wherein bacterial culture or PCR is conducted on samples, would yield positive results for B. pertussis. Additionally, the use of a throat swab to test for pertussis is not recommended due to low rates of DNA recovery. It therefore would be interesting to see if a serological test conducted on Joseph would yield meaningful and significant results.
Summary of Diagnostic Tests and Expected Results
| Diagnostic Test | Purpose | Expected B. pertussis Results |
| Bacterial Cultures: Regan-lowe (RL) or Bordet-Gengou (BG) Medium | Allows confirmation of bacterial presence and bacterial morphology visualization. | Observed growth of round, domed, shiny, mercury silver coloured colonies, about 1 mm in diameter |
| Gram staining | Allows the differentiation of bacterial species based on morphology of the cell wall. | Gram-negative coccobacilli from sample stain red or pink
|
| Oxidase Test | Identifies the presence of cytochrome oxidase, which is present in bacterial species that utilize cytochrome c for respiration. | B. pertussis bacteria turn the reagent blue due to oxidation indicating the presence of a cytochrome c system. |
| Slide Agglutination Serogrouping (SASG) | Determines serogroups of bacterial species. | Observation of strong microagglutination for antisera associated serotypes 2, 3, or 2,3 |
| Direct Fluorescent Antibody (DFA) Test | Rapidly diagnoses bacterial diseases through binding of target antigens. | Fluorescence is visualized, showing the binding of direct antibody to the antigen |
| PCR and Gel Electrophoresis | PCR: Utilizes nucleic acid primers to bind and amplify DNA sequences and subsequently identify bacterial type.
Gel Electrophoresis: Separate mixtures of DNA, RNA or proteins based on molecular size |
Observation of the amplification of ptxP upon PCR assay.
|
| ELISA | Detects and quantifies antibodies related to bacterial infection through recognizing and binding target cellular components. | Using PT antibody, the formation of an antigen-antibody complex is observed at the bottom of the well |
References:
[1] W. H. Organization, “Laboratory Manual for the diagnosis of whooping cough caused by bordetella pertussis/bordetella parapertussis : update 2014,” Art. no. WHO/IVB/14.03, 2014, Accessed: Apr. 02, 2021. [Online]. Available: https://apps.who.int/iris/handle/10665/127891.
[2] “UK SMI ID 5: identification of Bordetella species,” GOV.UK. https://www.gov.uk/government/publications/smi-id-5-identification-of-bordetella-pertussis-and-bordetella-parapertussis-from-selective-agar (accessed Apr. 08, 2021).
[3] E. Team, “Gram Staining : Principle, Procedure, Interpretation and Animation,” LaboratoryInfo.com, Jan. 08, 2020. https://laboratoryinfo.com/gram-staining-principle-procedure-interpretation-and-animation/ (accessed Apr. 02, 2021).
[4] N. Miyashita et al., “Diagnostic value of symptoms and laboratory data for pertussis in adolescent and adult patients,” BMC Infect. Dis., vol. 13, p. 129, Mar. 2013, doi: 10.1186/1471-2334-13-129.
[5] “Oxidase Test- Principle, Uses, Procedure, Types, Result Interpretation...,” Microbiology Info.com, Jul. 01, 2015. https://microbiologyinfo.com/oxidase-test-principle-uses-procedure-types-result-interpretation-examples-and-limitations/ (accessed Apr. 02, 2021).
[6] “Pertussis Diagnosis Confirmation | CDC,” Apr. 02, 2021. https://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-confirmation.html (accessed Apr. 02, 2021).
[7] “Fluorescent Antibody Techniques | Microbiology.” https://courses.lumenlearning.com/microbiology/chapter/fluorescent-antibody-techniques/ (accessed Apr. 02, 2021).
[8] “Fig. 2. Direct detection of nasopharyngeal swab samples and specificity...,” ResearchGate. https://www.researchgate.net/figure/Direct-detection-of-nasopharyngeal-swab-samples-and-specificity-test-a-Fluorescence_fig2_331542191 (accessed Apr. 02, 2021).
[9] F. Mégret and J. E. Alouf, “A simple novel approach for the purification of pertussis toxin,” FEMS Microbiol. Lett., vol. 51, no. 2–3, pp. 159–162, Jun. 1988, doi: 10.1111/j.1574-6968.1988.tb02989.x.
[10] P. Khramtsov, M. Bochkova, V. Timganova, S. Zamorina, and M. Rayev, “Comparison of anti-pertussis toxin ELISA and agglutination assays to assess immune responses to pertussis,” Infect. Dis. Lond. Engl., vol. 49, no. 8, pp. 594–600, Aug. 2017, doi: 10.1080/23744235.2017.1306101.
[11] C.-H. Wirsing von König, “Pertussis diagnostics: overview and impact of immunization,” Expert Rev. Vaccines, vol. 13, no. 10, pp. 1167–1174, Oct. 2014, doi: 10.1586/14760584.2014.950237.
[12] “Bordetella pertussis - ClinicalKey.” https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554002307 (accessed Apr. 08, 2021).
[13] M. K. Viljanen, O. Ruuskanen, C. Granberg, and T. T. Salmi, “Serological diagnosis of pertussis: IgM, IgA and IgG antibodies against Bordetella pertussis measured by enzyme-linked immunosorbent assay (ELISA),” Scand. J. Infect. Dis., vol. 14, no. 2, pp. 117–122, 1982, doi: 10.3109/inf.1982.14.issue-2.08.
[14] M. Watanabe, B. Connelly, and A. A. Weiss, “Characterization of Serological Responses to Pertussis,” Clin. Vaccine Immunol., vol. 13, no. 3, pp. 341–348, Mar. 2006, doi: 10.1128/CVI.13.3.341-348.2006.
Q3. Bacterial Pathogenesis
Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.
i. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria
Bordetella pertussis is a gram-negative, aerobic and encapsulated coccobacillus that is the causative agent of Whooping Cough (1). Pertussis, the contagious respiratory disease caused primarily by B. pertussis, is only found in humans and is transmitted by aerosolized respiratory droplets (2). There are no animal or insect source or vectors that are known reservoirs of this disease (3).
Geographic Location
Bordetella pertussis can be found circulating worldwide, however, rates of disease are the highest among young children and infants in developing and third world countries, which have low vaccination coverage (4). In developed countries, pertussis disease is seen to be the highest in unvaccinated infants and incidence additionally increases in teens (4). In 2019, the WHO Provisional Pertussis Surveillance Report confirmed 15,662 cases of B. pertussis infection nationwide in the United States, with 21% of these cases occurring in children between 1-6 years of age and 30.4% of cases in young adults 11-19 years of age (5). This data demonstrates the alarming trend that has appeared since the introduction of the acellular pertussis (aP) vaccines in the 1990s in developed countries: that cases of pertussis are gradually increasing in number, with the largest increase seen in adolescents and adults, and that disease is more common in infants and older children (aged 9-19 years) (2,6). Teenagers are often more susceptible to pertussis if re-vaccination does not occur or initial vaccination with DTap did not take place during infancy (7). As recommended by the CDC, Tdap should be administered as a single dose between the ages of 11 to 18 but preferably at the ages of 11 to 12 (7). Additionally, rates of pertussis disease can be seen to be the highest in the months of July and August and the lowest can be seen in October and February, thus infection appears to follow a seasonal pattern with the peak of activity occurring in the summer (8). Peak activity has been found to occur in the summer based on a study conducted in the United States (8). However, the temporal pattern of B. pertussis is still poorly understood (8). Possible explanations for the increased rate of incidence in the summer months include changes in environmental conditions such as increased humidity which favor survival of the pathogen outside the host and pathogen transmission (8). Moreover, the changes in host behaviours such as spending more time indoors or outdoors may also explain this increase (8).
Joseph’s Exposure
Joseph’s exposure to B. pertussis can be associated with multiple factors. As stated previously, pertussis spreads through airborne droplets between individuals in the form of coughing, sneezing or being in the same space (4). B. pertussis may also survive outside of the human host and can therefore be transmitted through contaminated objects, although infection is most common after direct contact with an infected individual (9). Therefore, it is highly likely that Joseph encountered an infected individual during the early catarrhal phase (9). This stage is where an individual is the most infectious due to the presence of mild and non characteristic symptoms (9). Additionally, Joseph’s lack of adulthood vaccination also put him at an increased risk of pertussis. As a child, Joseph would have gotten vaccinated with diphtheria, tetanus, and pertussis (DTap) vaccines for protection against whooping cough (10). However, revaccination with tetanus, diphtheria, and pertussis (Tdap) vaccines is recommended by the CDC every 10 years which Joseph did not receive (11). Moreover, Joseph may have recently travelled to a developed country, as exposure is more frequent in these regions (12). Unimmunized adolescents and adults can contract the disease in these areas and often it results in the development of mild and atypical pertussis (12). It is recommended that travellers be immunized prior to travel to these areas if they have not been fully immunized already (12).
Host Residence
Infants are the most vulnerable age category to B. pertussis infection as immunization is extremely important for the prevention of this disease (13). It is recommended that infants are vaccinated at 6 weeks or have the mother vaccinated during pregnancy (14). The presence of IgG antibodies in the fetus at 6 months old or younger are maternal antibodies which have crossed the placenta to provide limited protective immunity to infants (14). In cases where the source of infection can be traced, infection from family members is most common (15). Adult immunity to pertussis lessens over time and therefore re-vaccination is important in both protecting oneself against infection in addition to protecting ones’ child (13).
As a respiratory infection, B. pertussis can be found in the mouth, nose and throat of infected individuals (16). The bacteria itself is small, gram-negative, non-motile and coccobacillary in shape (17). B .pertussis is an aerobic bacterium, meaning that it requires oxygen for survival (10). This characteristic makes it well-suited to the human respiratory tract which receives oxygen upon inhalation. B. pertussis can reside in the respiratory tract because it produces adhesins such that it can survive innate host defences, such as mucociliary clearance and the action of antimicrobial peptides, multiply locally and resist elimination by inflammatory cells (18). Persistence and colonization of B. pertussis in the respiratory tract has been associated with the formation of biofilms (19). BvgAS-activated proteins such as filamentous hemagglutinin (FHA) contribute to biofilm formation through providing structural integrity by mediating cell substrate and interbacterial adhesions leading to the formation of microcolonies (19). Biofilms can be characterized as multicellular-adherent microbial communities within a self-produced or host-derived matrix (19). They are initially seen as cell clusters separated by individual cells before the formation of mature microcolonies which become encased within an opaque matrix-like material, pillar-like structures, water channels, or irregular shapes (19). Additionally, these matrices are composed of different extracellular polymeric substances (EPS) which include polysaccharides, proteins, metabolites, and extracellular DNA (eDNA) (19). Specifically, eDNA has been associated with maintaining the structural integrity and stability of the biofilm (19). Biofilm formation essentially allows for B. pertussis to resist environmental stress, host defence mechanisms and antibacterial components (19).
B. pertussis displays optimal growth at 37 degrees Celsius, the temperature of the normal human body, and when in this optimal temperature environment, the bacteria are known to have an active BvgAS phosphorelay which regulates many genes and it is this regulation that produces at least 3 known distinct phenotypic phases (2). These genes are placed into different classes; Class 1 genes includes the ptx-ptl operon, which encodes for the pertussis toxin and its transport system, the cyaA-E (which encodes for adenylate cyclase toxin) and the bsc operon, which encodes a type III secretion system (2). Class 2 genes include fhaB, which codes for filamentous haemagglutinin, the fim genes that encode for fimbriae and the bvgAS, which allows for positive autoregulation (2). Class 3 genes include bipA gene, encoding a protein of unknown function (2). Differential expression of these different classes of genes is thought to allow the bacterium to adapt its expression of virulence factors to the different stages of pertussis infection or to niches with different environmental signals (i.e. different airway niches), especially for adaptation to temperature changes (20). In the human airway, the 37 degree Celsius temperature induces full activation of the BvgAS and genes of Class 1 and 2 are maximally expressed; a state that is called the Bvg+ state (2). This state is essential for respiratory colonization and infection as its induced expression of important adhesins and virulence determinants such as the filamentous haemagglutinin and fimbriae, which are necessary for tracheal colonization (2,21). Additionally, the adenylate cyclase toxin and pertussis toxin, both products of Class 1 genes, act to allow for evasion of host defenses while in the host respiratory tract (21). As mentioned previously, filamentous haemagglutinin has been shown to be vital for biofilm development by promoting the formation of microcolonies, thus allowing for colonization and survival of B. pertussis in the human respiratory tract (22).
References
- Bordetella pertussis. In: Wikipedia [Internet]. 2021 [cited 2021 Apr 5]. Available from: https://en.wikipedia.org/w/index.php?title=Bordetella_pertussis&oldid=1014183005
- Melvin JA, Scheller EV, Miller JF, Cotter PA. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol. 2014 Apr;12(4):274–88.
- Pinkbook [Internet]. Cdc.gov. 2020 [cited 2021 Apr 3]. Available from: https://www.cdc.gov/vaccines/pubs/pinkbook/pert.html
- Pertussis (Whooping Cough): Cases in Other Countries | CDC [Internet]. Centers for Disease Control and Prevention. 2021 [cited 2021 Apr 5]. Available from: https://www.cdc.gov/pertussis/countries/index.html
- 2019 Provisional Pertussis Surveillance Report [Internet]. National Center for Immunization and Respiratory Diseases; 2020 [cited 2021 Apr 5]. Available from: https://www.cdc.gov/pertussis/downloads/pertuss-surv-report-2019-508.pdf
- Canada PHA of. Pathogen Safety Data Sheets: Infectious Substances – Bordetella pertussis [Internet]. Government of Canada. 2011 [cited 2021 Apr 5]. Available from: https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/bordetella-pertussis.html
- Summary of pertussis vaccination recommendations [Internet]. Cdc.gov. 2020 [cited 2021 Apr 9]. Available from: https://www.cdc.gov/vaccines/vpd/pertussis/recs-summary.html
- Bhatti MM, Rucinski SL, Schwab JJ, Cole NC, Gebrehiwot SA, Patel R. Eight-Year Review of Bordetella pertussis Testing Reveals Seasonal Pattern in the United States. Journal of the Pediatric Infectious Diseases Society. 2017 Mar 1;6(1):91–3.
- Baron S. Medical Microbiology. 4th ed. University of Texas Medical Branch; 1996.
- Whooping Cough Vaccination [Internet]. Cdc.gov. 2020 [cited 2021 Apr 9]. Available from: https://www.cdc.gov/vaccines/vpd/pertussis/index.html
- Diphtheria, Tetanus, and Whooping Cough vaccination: What you should know [Internet]. Cdc.gov. 2021 [cited 2021 Apr 9]. Available from: https://www.cdc.gov/vaccines/vpd/dtap-tdap-td/public/index.html
- WHO | Pertussis [Internet]. WHO. World Health Organization; [cited 2021 Apr 5]. Available from: https://www.who.int/ith/diseases/pertussis/en/
- Ramsay AM, Emond RTD. Whooping cough (pertussis). In: Infectious Diseases. Elsevier; 1967. p. 233–9.
- Wang C, Zhang H, Zhang Y, Xu L, Miao M, Yang H, et al. Analysis of clinical characteristics of severe pertussis in infants and children: a retrospective study. BMC Pediatr. 2021;21(1):65.
- Riffelmann M, Littmann M, Hülße C, Hellenbrand W, Wirsing von König CH. Pertussis: not only a disease of childhood. Dtsch
- Disease factsheet about pertussis [Internet]. Europa.eu. [cited 2021 Apr 3]. Available from: https://www.ecdc.europa.eu/en/pertussis/facts
- Halperin SA, De Serres G. Pertussis. In: Bacterial Infections of Humans. Boston, MA: Springer US; 2009. p. 577–95.
- Melvin JA, Scheller EV, Miller JF, Cotter PA. Bordetella pertussis pathogenesis: current and future challenges. Nature reviews.Microbiology 2014;12(4):274-288.
- Cattelan N, Dubey P, Arnal L, Yantorno OM, Deora R. Bordetella biofilms: a lifestyle leading to persistent infections. Pathog Dis. 2016;74(1):ftv108
- Gouw D de, Diavatopoulos DA, Bootsma HJ, Hermans PWM, Mooi FR. Pertussis: a matter of immune modulation. FEMS Microbiology Reviews. 2011;35(3):441–74.
- Bennett JE, Dolin R, Blaser MJ. Bordetella Pertussis. In: Mandell, Douglas and Bennett’s Principles and practice of infectious diseases, vol 1-2. 9th ed. Philadelphia: Elsevier; 2020. p. 2793–802.
- Serra DO, Conover MS, Arnal L, Sloan GP, Rodriguez ME, Yantorno OM, et al. FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea. PLoS One. 2011;6(12):e28811.
ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.
B. pertussis first enters the host through inhalation of or direct contact with aerosolized respiratory droplets from an infected individual (1). The bacterial cells then adhere to ciliated respiratory epithelial cells using several important factors and adhesins, which allow for colonization of the respiratory mucosa (1,2). The 2 major adhesins and virulence determinants of B. pertussis include fimbriae and filamentous hemagglutinin (FHA) (3).
Fimbriae
B. pertussis is known to express and produce type I pili, which are also called by the name fimbriae (4). Assembly of fimbriae is mediated by a chaperone-usher mechanism (5). This system is composed of the important proteins including FimB (a periplasmic chaperone), FimC (an outer membrane usher) and FimD proteins (tip adhesin of pilus) which are all translocated across the cytoplasmic membrane, which is mediated by the Sec transport system (5). The FimB chaperone acts to deliver pilin subunits (Fim2 or Fim3) to the usher (FimC), which then results in donor strand exchange and the translocation of a growing pilin subunit chain across the outer membrane, forming a pilus strand (5). The FimD tip adhesin proteins of the pilus are first translocated to form the tip complex (5). The helical structure of the formed pilus rod confers an ability to withstand substantial sheer forces whilst still maintaining adhered as although the structure is rigid, it can uncoil when under stress, thus acting like a shock absorber (5). These features of the pilus are thought to be important for adherence to beating cilia of the ciliated epithelial cells in the trachea and bronchi (5). A lot of the function and the host factors these pili bind to remains unclear. It is thought that the Fim2 and Fim3 subunits may each contain two regions that display heparin-binding activity which may be responsible for binding to the extracellular matrix of respiratory epithelial cells (6). Generally, it is believed that the fimbriae may adhere to sulfated sugar-containing molecules to bind to the host respiratory epithelial cells (6). Additionally, it is suggested that it may be the role of bacterial fimbriae to adhere to cilia specifically, with low to moderate affinity, which brings bacterial cells into close proximity to respiratory epithelial cells, allowing for higher affinity binding mediated by FHA that is responsible for resistance to mucociliary clearance mechanisms during early infection (5). The fimbriae of B. pertussis has also been implicated in binding to the very late antigen 5 receptor located on host macrophages and monocytes which leads to internalization of bacterial cells in a nonbactericidal way (7). This strategy, which will be discussed in a later section, is thought to allow for long persistence of pertussis infection in largely immune populations (7).
Filamentous hemagglutinin (FHA) & Lipid Rafts
Filamentous hemagglutinin is a large, 232-kDA protein and is found in both a secreted and surface-associated form in B. pertussis (6). It, together with the FhaC or filamentous haemagglutinin transporter protein, acts as a prototypical member of the two-partner secretion (or TPS) pathway (4). It is initially made as a pre-pro-protein called FhaB which undergoes processing whilst translocating across the inner bacterial membrane (via the Sec translocation system) and the outer membrane (by the outer membrane protein FhaC), to form the mature FHA protein protruding from the surface (4). The mature FHA protein remains noncovalently attached to the bacterial cell surface, potentially by interactions between the protein’s N-terminal domain and the FhaC protein (6).
Different Binding Domains of FHA
FHA is commonly known as a major adhesin of B. pertussis and is both necessary and sufficient to allow for bacterial adhesion to several different eukaryotic cell types (4). The protein contains several different domains which are responsible for binding different host cell-surface receptors on respiratory epithelial cells (6). There is a carbohydrate recognition domain, involved in the binding to host ciliated respiratory cells and macrophages, is located between the residues 1141 and 1279 (8). This binding domain mediates the binding of FHA to glycolipids of human cilia of respiratory epithelial cells and to galactose-containing glycoconjuages (8). Specifically, this domain recognizes and binds to {beta]1-4-linked galactose residues and galactose-N -acetylglucosamine moieties of glycolipids, including lactosylceramide (8). A second domain of the FHA protein is the heparin-binding domain or HBD that is located in between residues 442-863 of the R1 repeat region of the protein (8). The HBD is important for FHA protein binding to sulfated carbohydrates, and a sequence found outside the HBD between the residues 1069-1073, containing an RRARR sequence, comprises the RRAR motif that is found in the consensus heparin-binding sites of fibronectins (8). This domain would thus allow FHA to bind to both sulfated glycolipids and proteoglycans, including heparan and chondroitin sulfate (8). This domain confers the ability for the FHA protein to bind to host epithelial cells (8). Another FHA domain of note is the subdomain containing an Arg-Gly-Asp or RGD motif that is centralized around a glycine 1098 residue (8). It is proposed that a cross-linking interaction between FHA and the eukaryotic integrin [alpha]v [beta]3 LRI and IAP on macrophages allows for upregulation of CR3 (also known as complement receptor 3 or [alpha]M [beta]2 integrin) which serves as a docking site for the RGD motif of FHA protein to macrophages and may contribute to invasion of these immune cells (7,8). In human respiratory epithelial cells, this RGD binding domain of the FHA protein is proposed to, through interaction with the very late antigen-5 (VLA-5) integrin, induce invasion of B. pertussis in these host cells (8).
FHA and Lipid rafts (and cholesterol)
It has been noted that cholesterol-rich domains within lipid rafts of host respiratory epithelial cell plasma membranes is associated with and necessary for binding of B. pertussis to these cells and for host colonization (9). Lipid rafts are groups of clusters of proteins and lipids in the plasma membrane that are held together by cholesterol (6). In various experiments, it was demonstrated that depletion of cholesterol leads to a decrease in the ability of adherence of B. pertussis to host respiratory epithelial cells indicating that FHA binding to cholesterol-containing lipid rafts is important for adherence (6). Additionally, this strategy is employed by many other bacterial pathogens as lipid rafts provide a platform containing clusters of signaling receptor proteins and lipid cofactors (6).
Other bacterial factors such as adenylate cyclase toxin, tracheal cytotoxin, pertussis toxin and BteA (an effector protein of the type III secretion system with cytotoxic activity) support adhesion to the respiratory epithelium indirectly through toxin-induced cytotoxic activities, as this often leads to the exposure of cryptic receptors that are localized on the basement membrane (6). The action of these toxins will be discussed later but these toxins, in synergy with lip-oligosaccharides help with adhesion by establishing a niche that is devoid of ciliary epithelium which prevents any mechanical clearance of bacteria attaching to the mucosa (6). The pathogens can then bind to the exposed cryptic receptors with more efficiency (6).
References
- Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2021 Apr 5]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7813/
- Sandros J, Tuomanen E. Attachment factors of Bordetella pertussis: mimicry of eukaryotic cell recognition molecules. Trends in Microbiology. 1993 Aug;1(5):192–6.
- Bennett JE, Dolin R, Blaser MJ. Bordetella Pertussis. In: Mandell, Douglas and Bennett’s Principles and practice of infectious diseases, vol 1-2. 9th ed. Philadelphia: Elsevier; 2020. p. 2793–802.
- Melvin JA, Scheller EV, Miller JF, Cotter PA. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol. 2014 Apr;12(4):274–88.
- Scheller EV, Cotter PA. Bordetella filamentous hemagglutinin and fimbriae: critical adhesins with unrealized vaccine potential. Carbonetti N, editor. Pathogens and Disease. 2015 Nov;73(8):ftv079.
- Gouw D de, Diavatopoulos DA, Bootsma HJ, Hermans PWM, Mooi FR. Pertussis: a matter of immune modulation. FEMS Microbiology Reviews. 2011;35(3):441–74.
- Lamberti YA, Hayes JA, Vidakovics MLP, Harvill ET, Rodriguez ME. Intracellular Trafficking of Bordetella pertussis in Human Macrophages. Infection and Immunity. 2010 Mar 1;78(3):907–13.
- Villarino Romero R, Osicka R, Sebo P. Filamentous hemagglutinin of Bordetella pertussis: a key adhesin with immunomodulatory properties? Future Microbiology. 2014 Dec;9(12):1339–60.
- Lamberti Y, Alvarez Hayes J, Perez Vidakovics ML, Rodriguez ME. Cholesterol-dependent attachment of human respiratory cells by Bordetella pertussis. FEMS Immunol Med Microbiol. 2009 Jul;56(2):143–50.
- Kajava AV, Cheng N, Cleaver R, Kessel M, Simon MN, Willery E, et al. Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins. Molecular Microbiology. 2001;42(2):279–92.
- Tong Y-C. The role of cholesterol in prostatic diseases. Urological Science. 2011 Sep 1;22.
iii. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.
Multiplication and Spread: General Overview
B. pertussis is a small, aerobic, non-motile, gram-negative bacterium that exclusively affects humans and colonizes the cilia of the upper respiratory tract in the nasopharynx (1). B. pertussis infection generally remains localized to the mucosal surfaces of the respiratory tract through the adherence to ciliated epithelial cells (2, 3). After adhering to the respiratory epithelium, B. pertussis has also been shown to form biofilms within the human respiratory tract, which allows for multiplication and potentially further dissemination in the respiratory tract (4).
Although B. pertussis is generally classified as an extracellular pathogen, an increasing number of in vitro and in vivo studies have shown that it is capable of invading many cell types and replicating in intracellular compartments, indicating that it may be considered a facultative intracellular bacterium (5, 6). B. pertussis has been found to persist within both epithelial cells and macrophages due to its extensive immunomodulatory characteristics (5). The bacterium has been found inside ciliated respiratory epithelial cells and pulmonary alveolar macrophages of infected infants and children (5).
Spread
Pertussis is a localized infection and rarely disseminates from the respiratory tract (7). However, B. pertussis does not remain at the entry site; it will replicate and spread from the upper respiratory tract to the middle to lower respiratory tract (7). Bacteremia and systemic spread does not typically occur except in cases involving severely immune-compromised individuals (2, 9). Secondary infection sites include the ciliated cells of the trachea and bronchi in the lower respiratory tract (7). These are secondary sites of infection because B. pertussis has the necessary adhesins and invasion mechanisms to attach to and invade these ciliated cells. Replication of the bacteria releases toxins that cause damage to the ciliated epithelial cells lining
the upper, middle and lower respiratory tract (7). Dissemination to the lower areas of the respiratory tract may lead to pneumonia (10). Pneumonia is one of the most common complications associated with pertussis and can either be caused by infection by B. pertussis within the lung tissue or from coinfection with other respiratory pathogens (11).
Replication and Survival in Biofilms
Biofilms are multicellular, highly structured communities composed of bacterial cells that are encapsulated in an extracellular polymeric matrix that can either be host or pathogen-derived (12). The formation of biofilms is an important virulence factor of B. pertussis infection as it has been shown to confer higher antibiotic resistance, decreased susceptibility to host immune responses and allows for longer survival within a tightly controlled niche within the human host (12). It has been found that the Bordetella virulence gene (BvgAS) signal transduction system and multiple Bvg-activated proteins are important for efficient biofilm formation on abiotic surfaces (12). The BvgAS system regulates expression of B. pertussis virulence factors; it is a two-component environmental-sensing (regulatory) system, which consists of a BvgS sensor protein and a BvgA transcriptional activator (13).
Formation of biofilms first requires planktonic bacteria to attach to the respiratory tract mucosal (14). Filamentous haemagglutinin has been shown to be vital for biofilm development by promoting the formation of discrete cell clusters called microcolonies (4). Formation of microcolonies arises either from clonal expansion of the attached cells or by the recruitment of additional planktonic cells (14). There is then subsequent multiplication within these microcolonies and they coalesce to form fully mature microcolonies encased in EPS (14). This biofilm allows the organism to replicate and survive anatomical clearance (1).
Internalization, Replication and Survival in Epithelial Cells
B. pertussis has been shown to localize within host epithelial cells (15). Bacterial entry depends on microtubule assembly, the formation of lipid rafts, and the activation of tyrosine kinase-mediated signalling cascades (15). Lipid raft domains on host respiratory epithelial cells serve as docking sites for bacterial FHA to bind (15). Lipid rafts are small heterogeneous, sterol- and sphigo-lipid-enriched domains that are highly dynamic. They facilitate binding and entry of the pathogen (15). When stimulated, these small rafts can coalesce into large signalling platforms to induce signalling and cytoskeletal reorganization for pathogen invasion of the cell (15). Tyrosine kinases are critical in the signal transduction linkage between integrins and lipid rafts (15). The RGD domain on FHA is responsible for epithelial cell invasion by interacting with host cell a5ß1 integrin to facilitate entry into the cell (15). Once localized within respiratory epithelial cells, B. pertussis is able to evade lysosomal fusion and persist in low-pH environments using the protein BP0414 which is a homologue with Mgtc, an intracellular survival protein found in S. enterica (15, 16).
Internalization, Replication and Survival in Macrophages
In addition to survival in respiratory epithelial cells, B. pertussis is also capable of entering and proliferation within human alveolar macrophage which represents an important intracellular niche for prolonged B. pertussis infections (15, 17). Uptake into phagocytes involved various virulence factors such as fimbriae which bind to the very late antigen (VLA-5) receptor on monocytes and macrophages, which induces the upregulation of the complement receptor 3 or CD11b/CD18 (17). CR3 expression on these cells can also be further upregulated by interaction with the pertussis toxin and FHA (17). It is proposed that the CR3 molecule then serves as a docking platform for B. pertussis and binding leads to uptake in a non-bactericidal way (17). This utilization of CR3 for non-opsonized binding and uptake is beneficial for the pathogen as it does not activate the professional phagocyte (17). In addition, internalized B. pertussis was shown to persist in early endosomes and this species can replicate in non-acidic intracellular compartments within macrophages during the first 48 hours post infection (17). Once inside macrophages and epithelial cells, B . pertussis can block fusion of the phagosome with lysosomes, and can survive and multiply in these non-acidic intracellular compartments (15, 18). After internalization and multiplication, intracellular B. pertussis can be released into the extracellular environment, and may contribute to persistence and spread within the host (15).
References
- Melvin J, Scheller E, Miller J et al. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol 2014; 12, 274–288. https://doi-org.ezproxy.library.ubc.ca/10.1038/nrmicro3235
- Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2021 Apr 5]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7813/
- Sandros J, Tuomanen E. Attachment factors of Bordetella pertussis: mimicry of eukaryotic cell recognition molecules. Trends in Microbiology. 1993 Aug;1(5):192–6.
- Serra DO, Conover MS, Arnal L, Sloan GP, Rodriguez ME, Yantorno OM, et al. FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea. PLoS One. 2011;6(12):e28811.
- Lamberti YA, Hayes JA, Vidakovics MLP, Harvill ET, Rodriguez ME. Intracellular Trafficking of Bordetella pertussis in Human Macrophages. Infection and Immunity. 2010 Mar 1;78(3):907–13.
- Martín C, Etxaniz A, Uribe KB, Etxebarria A, González-Bullón D, Arlucea J, et al. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells. Scientific reports 2015;5(1):13774.
- Smith AM, Guzmán CA, Walker MJ. The virulence factors of Bordetella pertussis: a
- matter of control. FEMS Microbiol Rev 2001;25(3):309-333.
- Solans L, Locht C. The role of mucosal immunity in pertussis. Front Immunol. 2018;9:3068.
- Cooke FJ, Slack MPE. Gram-Negative Coccobacilli. In: Infectious Diseases. Elsevier; 2017. p. 1611-1627.e1.
- Bennett JE, Dolin R, Blaser MJ. Bordetella Pertussis. In: Mandell, Douglas and Bennett’s Principles and practice of infectious diseases, vol 1-2. 9th ed. Philadelphia: Elsevier; 2020. p. 2793–802.
- Dorji D, Mooi F, Yantorno O, Deora R, Graham RM, Mukkur TK. Bordetella Pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance. Med Microbiol Immunol. 2018 Feb;207(1):3–26.
- Mattoo S, Cherry JD. Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of Respiratory Infections Due to Bordetella pertussis and Other Bordetella Subspecies. Clin Microbiol Rev 2005;18(2):326-345; 382.
- Gouw D de, Diavatopoulos DA, Bootsma HJ, Hermans PWM, Mooi FR. Pertussis: a matter of immune modulation. FEMS Microbiology Reviews. 2011;35(3):441–74.
- Lamberti Y, Gorgojo J, Massillo C, Rodriguez ME. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival. Pathogens and Disease. 2013;69(3):194–204.
- Cafiero JH, Lamberti YA, Surmann K, Vecerek B, Rodriguez ME. A Bordetella pertussis MgtC homolog plays a role in the intracellular survival. PLOS ONE. 2018 Aug 30;13(8):e0203204.
- Lamberti YA, Hayes JA, Vidakovics MLP, Harvill ET, Rodriguez ME. Intracellular Trafficking of Bordetella pertussis in Human Macrophages. Infection and Immunity. 2010 Mar 1;78(3):907–13.
- Martín C, Etxaniz A, Uribe KB, Etxebarria A, González-Bullón D, Arlucea J, et al.Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells. Scientific reports 2015;5(1):13774.
iv. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?
Experiments with human nasal epithelial biopsy specimens infected with B. pertussis showed a reduction in the number of ciliated cells, an increase in the number of cells with sparse ciliation, an increase in the number of dead cells, and extrusion of cells from the epithelial surface (1). One culprit is tracheal cytotoxin (TCT), a disaccharide–tetrapeptide monomer of peptidoglycan that is produced during cell wall remodelling. Although most Gram-negative bacteria recycle this molecule, B. pertussis does so inefficiently and releases a large amount of TCT into the extracellular environment (2).
The mechanisms through which TCT causes damage is unclear, however in hamster tracheal rings, TCT functions synergistically with lipo-oligosaccharide to stimulate the production of pro-inflammatory cytokines (such as tumour necrosis factor α (TNFα), IL-1α, IL-1β and IL-6) and inducible nitric oxide synthase. The increased level of nitric oxide radicals will destroy iron dependent enzymes which are essential for mitochondrial function and DNA synthesis in host cells, resulting in the destruction and extrusion of ciliated cells from the epithelial surface (2). Thus, this is an example of damage initiated by bacterial toxins but mediated through the host immune response.
Furthermore, subcutaneous injection of B. pertussis or B. bronchiseptica cells into mice results in the formation of necrotic lesions, owing to the activity of dermonecrotic toxin (DNT) (2). Consistent with a role in infection, there is evidence that DNT contributes to the ability of B. bronchiseptica to induce turbinate atrophy and lung pathology in swine (2).
The hallmark of B. pertussis infection is paroxysmal cough, also known as frequent and violent coughing (1). It seems likely that local cilia damage can result in impaired mucociliary clearance and thus is responsible for this cough (3). However, the total duration of the cough in typical pertussis is longer than the period during which local damage would be expected to last (3). Therefore, it seems possible that there is another, unidentified toxin that contributes to the continued paroxysmal cough (3).
PT is assumed to be the essential protective immunogen in pertussis infection. The pertussis toxin attaches to cilia on respiratory epithelial cells and the cilia become paralyzed (ciliostasis) and inflammation of the respiratory tract occurs, resulting in impaired ability to clear respiratory secretions (2). However, numerous findings indicate that other components, such as FHA, heat-labile toxin, agglutinogens, outer membrane proteins, and ACT may also contribute to the immune response (3). The recruitment of immune cells resulting in inflammation is likely to be responsible for the minor flu-symptoms we see in this illness such as fever and runny nose (3).
Further damage in severe cases of B. pertussis due to an impaired immune response or large doses of the bacteria may result in complications such as pneumonia, seizures, and encephalopathy (3). The pneumonia may be a primary event in response to B. pertussis infection or may be due to a secondary infection with other pathogens (3). Seizures and encephalopathy are most probably due to cerebral hypoxia related to severe paroxysms (3).
References
- Melvin J, Scheller E, Miller J et al. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol 2014; 12, 274–288. https://doi-org.ezproxy.library.ubc.ca/10.1038/nrmicro3235
- Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2021 Apr 5]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7813/
- Sandros J, Tuomanen E. Attachment factors of Bordetella pertussis: mimicry of eukaryotic cell recognition molecules. Trends in Microbiology. 1993 Aug;1(5):192–6.
- Serra DO, Conover MS, Arnal L, Sloan GP, Rodriguez ME, Yantorno OM, et al. FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea. PLoS One. 2011;6(12):e28811.
- Lamberti YA, Hayes JA, Vidakovics MLP, Harvill ET, Rodriguez ME. Intracellular Trafficking of Bordetella pertussis in Human Macrophages. Infection and Immunity. 2010 Mar 1;78(3):907–13.
- Martín C, Etxaniz A, Uribe KB, Etxebarria A, González-Bullón D, Arlucea J, et al. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells. Scientific reports 2015;5(1):13774.
- Smith AM, Guzmán CA, Walker MJ. The virulence factors of Bordetella pertussis: a
- matter of control. FEMS Microbiol Rev 2001;25(3):309-333.
- Solans L, Locht C. The role of mucosal immunity in pertussis. Front Immunol. 2018;9:3068.
- Cooke FJ, Slack MPE. Gram-Negative Coccobacilli. In: Infectious Diseases. Elsevier; 2017. p. 1611-1627.e1.
- Bennett JE, Dolin R, Blaser MJ. Bordetella Pertussis. In: Mandell, Douglas and Bennett’s Principles and practice of infectious diseases, vol 1-2. 9th ed. Philadelphia: Elsevier; 2020. p. 2793–802.
- Dorji D, Mooi F, Yantorno O, Deora R, Graham RM, Mukkur TK. Bordetella Pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance. Med Microbiol Immunol. 2018 Feb;207(1):3–26.
- Mattoo S, Cherry JD. Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of Respiratory Infections Due to Bordetella pertussis and Other Bordetella Subspecies. Clin Microbiol Rev 2005;18(2):326-345; 382.
- Gouw D de, Diavatopoulos DA, Bootsma HJ, Hermans PWM, Mooi FR. Pertussis: a matter of immune modulation. FEMS Microbiology Reviews. 2011;35(3):441–74.
- Lamberti Y, Gorgojo J, Massillo C, Rodriguez ME. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival. Pathogens and Disease. 2013;69(3):194–204.
- Cafiero JH, Lamberti YA, Surmann K, Vecerek B, Rodriguez ME. A Bordetella pertussis MgtC homolog plays a role in the intracellular survival. PLOS ONE. 2018 Aug 30;13(8):e0203204.
- Lamberti YA, Hayes JA, Vidakovics MLP, Harvill ET, Rodriguez ME. Intracellular Trafficking of Bordetella pertussis in Human Macrophages. Infection and Immunity. 2010 Mar 1;78(3):907–13.
- Martín C, Etxaniz A, Uribe KB, Etxebarria A, González-Bullón D, Arlucea J, et al.Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells. Scientific reports 2015;5(1):13774.
4. The Immune Response
i. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection?
Bordetella pertussis is a gram-negative coccobacillus encapsulated bacteria. It is the primary causative agent of pertussis, a highly contagious respiratory disease that is directly transmitted from human to human via aerosolized respiratory droplets (1).
Innate: Physical Barriers. B. pertussis is transmitted by aerosols and infects the ciliated epithelium of the airways. Although there is no further dissemination of infection, the toxins produced in the respiratory tract contribute to pathogenesis.
Innate-Physical Barriers. Physical barriers that prevent infection of the bacteria include the airway epithelium, mucociliary system and tight junctions of epithelial cells (2). The airway epithelium is a sealed and self-cleaning barrier that prevents penetration of inhaled pathogens into the body (2). The barrier is covered with mucus that contains antimicrobial peptides (and antibodies in the adaptive response) that trap pathogens for removal via the mucociliary clearance mechanism (2). This mechanism involves the production and transport of a glycoprotein-rich mucus hydrogel by polarized epithelia that prevent drying of airway surfaces, traps harmful particles like bacteria and mediates elimination of exogenous and endogenous debris (3). Once the first line of physical defense is overcome, airway epithelial cells get into contact with pathogens and release cytokines and chemokines to induce an acute inflammatory reaction and start the immune reaction (2).
There are three intracellular structures of the cytoskeleton of one epithelial cell to that of its neighbors: adherence junctions, hemidesmosomes, and tight junctions (2). Adhesion junctions interconnect the actin filaments of the adherent cells via transmembrane E-cadherin adhesions and anchor proteins (2). Hemidesmosomes are structures that form adhesive bonds between the cytoskeleton of epithelial cells and the lamina lucida in the extracellular matrix (2). Tight junctions form a multiprotein junctional complex that is the main regulation of paracellular permeability (2). The tight junctions and adhesion junctions create dense protein networks that interconnect basolateral sides of epithelial cells in a way that prevents paracellular passage of all molecules such as water, ions, proteins, pathogens and other particulate matter (2).
Innate-Antimicrobial components. There are a large number of host defense proteins that respond to bacterial infection such as lysozyme, lactoferrin, secretory leukoproteinase inhibitor, cathelicidin and defensins (4). Lysozyme kills bacteria through the hydrolysis of cell wall peptidoglycan, but can also kill bacteria independently of peptidoglycan hydrolysis through its positive charge disrupting the negative charge of the bacterial envelope (5). The degradation and lysis of bacteria lead to bacterial contents being released, leading to activation of pattern recognition receptors in host cells (5). Defensins, specifically, β-defensin are selectively protective and promote resistance to B. pertussis infection (4). While B. pertussis can use the type 3 secretion system to block the expression of defensins, it still stands as a potent defender (4). Cationic cathelicidins can bind and disrupt negatively charged membranes, leading to cell death (6). These peptides can also cross membranes and target intracellular processes like RNA and DNA synthesis, impair functions of enzymes and chaperones, and can stimulate protein degradation (6). In addition, cathelicidins have wide variety in immunomodulatory functions, both boosting and inhibiting inflammation, directing chemotaxis, and affecting cell differentiation, primarily towards Th1 immune responses (6).
Innate-Complement. There are three distinct pathways for complement: the classical pathway, the lectin pathway, and the alternative pathway. Activation results in antibacterial mechanisms such as agglutination, opsonization and phagocytosis, recruitment of inflammatory cells and direct killing of the bacteria by formation of the membrane attack complex. B. pertussis has several mechanisms in place to prevent activation of the classical pathway of complement such as the BrkA protein which interferes with complement and by recruiting human C1 esterase inhibitor and C4b-binding protein. This will be expanded further in Section iii. The lectin and alternative pathway have been shown to be less relevant to B. pertussis as well.
Innate-Detection and Phagocytosis. Bacteria often contain pattern associated molecular markers (PAMP) which are invariant structures associated with pathogens such as peptidoglycan, lipopolysaccharide, flagella, outer membrane proteins and others. PAMPS are recognized by pattern recognition receptors (PRR) on host cells for which there are many different types, including surface-expressed Toll like receptors (TLRs), cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors, C-type lectin receptors, and retinoic acid-inducible gene-I-like receptors (7). There are two PRR-PAMP interactions specific to B. pertussis: TLR4-lipooligosaccharide and TLR2-pertussis toxin (7). TLR4 is involved with recognition of lipopolysaccharide, but B. pertussis produces a lipo-oligosaccharide that has a different structure. Instead of having a long-repeating O-side chain, it has a nonrepetitive trisaccharide. Binding results in nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-kB) production and interferon regulatory factor 3 signaling, leading to the production of proinflammatory cytokines including type 1 interferons and IL-6 (7). It should be noted however that this immune response is less severe compared to traditional binding of TLR4 and lipopolysaccharide. The reason for variability in structure might be an evolutionary mechanism that may depend on location of the respiratory tract or it may assist in altering the outcome of bacterial competition by directing the innate immune response to competing flora(7). B. pertussis also induces TLR2 signaling, and pertussis toxin has recently been established to be the agonist triggering these TLR2(7). Lastly, intracellular sensor Nucleotide-binding oligomerization domain-containing protein 1 (NOD1), has been shown to be activated by tracheal cytotoxin (TCT) which is secreted by the bacterium (7). However, this interaction was shown to be mouse specific and further research needs to be done to fully elucidate the relationship in humans (7).
Resident innate immune cells, specifically airway mucosal dendritic cells and alveolar macrophages mediate the initial cellular response to B. pertussis (8). The dendritic cells are positioned between epithelial cells to take up the antigen from the airway lumen and prime T cells soon after, whereas macrophages reside in the mucous layer (8). Macrophages are able to ingest and destroy bacteria within acidic compartments, but a proportion of the bacteria can evade destruction and can replicate in macrophages by residing in the non-acidic compartments (9). Following attachment, there is secretion of cytokines and chemokines, specifically IL-17 and CXCL2 secreted by the macrophages and epithelial cells to promote neutrophil recruitment (8). Activated neutrophils and macrophages participate in antibody mediated phagocytosis and intracellular killing of B.pertussis. Natural killer (NK) cells may also play a protective role through secretion of interferon gamma (IFN-γ) which enhances the antimicrobial activity of macrophages and induces T helper 1 (Th1) cells (8). Depletion of NK cells also resulted in a reduced Th1 and an elevated T helper 2 (Th2) response, highlighting a role for NK cells in regulating the development of T-cell responses during infection (4).
Adaptive Immune Response. Dendritic cells migrate to the lymph nodes to present the antigen to naïve T cells. This results in differentiation of T cells into Th1 and Th17 subtypes that migrate to the lungs for increased activation of neutrophils and macrophages by producing IFN-γ and IL-17, respectively (8). Th1 cells are primarily involved in immunity to intracellular pathogens. They are stimulated by IL-12, which is secreted by innate immune cells in response to virulence factors (4). Th17 cells on the other hand, are activated by IL-6, IL-1 and IL-23 which are secreted by the interaction of DC and naïve T cells (4). There is decreased secretion of IL-4 and IL-5 suggesting that Th2 responses are not created (4), which are primarily for extracellular pathogens. However, some reports suggest that vaccines may create Th2 cell
Part of the immune response also includes promotion of IL-10 secretion and inhibition of IL-12p70 secretion, and maturation of DC and macrophages leading to Treg cell induction (4). Although Th1 and Th17 responses are detected, IL-10 secreting Tr1 type cells are also detected, which secrete high levels of IL-10 and low levels of IL-5, IFN-γ and directly suppress inflammatory Th1 responses (4). Therefore, there is decreased bacterial clearance due to the T regulatory (Treg) cells involved in the adaptive immune response. However, this may ultimately be a benefit for the host by limiting inflammatory pathology.
B cells, after being activated by IFN-γ secreted by Th1 cells, differentiate into plasma cells that produce IgA and IgG antibodies specific to B. pertussis (4,8). However, it has been suggested that presence of IgG antibodies in serum give little information on the induction of immunological memory (4). Regardless, a small population of T and B cells become memory cells to provide effective protection against reinfection (8). But there is still risk of infection, as will be described later.
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References (4.i)
- Melvin JA, Scheller EV, Miller JF, Cotter PA. Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol. 2014 Apr;12(4):274–88.
- Frey A, Lunding LP, Ehlers JC, Weckmann M, Zissler UM, Wegmann M. More Than Just a Barrier: The Immune Functions of the Airway Epithelium in Asthma Pathogenesis. Front Immunol. 2020 Apr 28;11:761.
- Benam KH, Vladar EK, Janssen WJ, Evans CM. Mucociliary Defense: Emerging Cellular, Molecular, and Animal Models. Annals ATS. 2018 Nov;15(Supplement_3):S210–5.
- Higgs R, Higgins SC, Ross PJ, Mills KHG. Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol. 2012 Sep;5(5):485–500.
- Ragland SA, Criss AK. From bacterial killing to immune modulation: Recent insights into the functions of lysozyme. Bliska JB, editor. PLoS Pathog. 2017 Sep 21;13(9):e1006512.
- van Harten R, van Woudenbergh E, van Dijk A, Haagsman H. Cathelicidins: Immunomodulatory Antimicrobials. Vaccines. 2018 Sep 14;6(3):63.
- de Gouw D, Diavatopoulos DA, Bootsma HJ, Hermans PWM, Mooi FR. Pertussis: a matter of immune modulation. FEMS Microbiol Rev. 2011 May;35(3):441–74.
- Brummelman J, Wilk MM, Han WGH, van Els CACM, Mills KHG. Roads to the development of improved pertussis vaccines paved by immunology. Carbonetti N, editor. Pathogens and Disease. 2015 Nov;73(8):ftv067.
ii. Host damage: what damage ensues to the host from the immune response?
The host immune response to B. pertussis is local as this bacterium causes local infection of the upper and lower airway, and does not enter the circulation (1, 2). Therefore, damage to the host from the immune response occurs in the respiratory system (1). In severe cases there may be systemic manifestations of the disease (1).
Although a majority of the damage to the host respiratory system is mediated by B. pertussis toxins, the host immune response may contribute to some host damage (2). In general, pro-inflammatory cytokines, such as IL-6, TNF-a and IL-1 ß, produced by macrophages and DCs during infection with B. pertussis cause damage to the bronchial epithelium (3). A considerable amount of damage to airway epithelial cells is caused by TCT; TCT induces the secretion of IL-10 by the host immune response which results in the production of NO (2). NO damages or kills ciliated epithelial cells in the respiratory tract (2). Under normal circumstances the synthesis of NO is highly regulated, as too much free NO leads to the production of free radicals such as reactive oxygen or reactive nitrogen species (4). The induction of the NO synthase is likely caused by IL-1, generated in response to TCT (5). In addition, the induction of Th1 and Th17 cells by the host immune response may result in chronic inflammation which is initiated by extensive activation of macrophages and neutrophils (2). Th1 and Th17 cells activate and recruit neutrophils and macrophages to the site of infection, which in the case of B. pertussis is the respiratory tract (2). The activation of neutrophils and macrophages results in the widespread release of pro-inflammatory cytokines and chronic inflammation within the respiratory tract (2). Chronic inflammation in the lungs may result in airflow obstruction, increased mucus secretion and destruction of the lung surface (6).
B. pertussis invades the mucosa, which leads to an increase in mucous secretion, and at later stages becomes viscid and tenacious. After 10 to 14 days, mucus secretion enters the paroxysmal stage that is associated with an expulsion of copious viscid mucus (7). The toxin PT induces increased expression of an epithelial anion transporter called pendrin. Pendrin is responsible for the regulation of airway surface liquid volume and mucus viscosity, which contributes to respiratory pathology (8).
It has also been hypothesized that chronic autoimmune inflammation caused by the Th17 response may be responsible for the chronic cough observed in B. pertussis patients (2). However, other studies hypothesize that the release of bradykinin, a host response to tissue damage, may be the cause of the chronic cough associated with B. pertussis (2). Bradykinin is a mediator of vasodilation and, therefore, plays an important role in inflammation in situations such as bacterial infection (9). However, the possible mechanism of bradykinin resulting in a chronic cough is unknown (10).
References (4.ii)
- Kilgore PE, Salim AM, Zervos MJ, Schmitt H-J. Pertussis: Microbiology, Disease, Treatment, and Prevention. Clin Microbiol Rev. 2016;29(3):449–86.
- Waters V, Halperin SA. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Bordetella pertussis.
- Fedele G, Bianco M, Ausiello CM. The Virulence Factors of Bordetella pertussis: Talented Modulators of Host Immune Response. Arch Immunol Ther Exp. 2013;61(6):445–57.
- de Gouw D, Diavatopoulos DA, Bootsma HJ, Hermans PWM, Mooi FR. Pertussis: a matter of immune modulation. FEMS Microbiol Rev. 2011 May;35(3):441–74.
- Flak TA, Goldman WE. Autotoxicity of Nitric Oxide in Airway Disease. Am J Respir Crit Care Med. 1996 Oct;154(4_pt_2):S202–6.
- Moldoveanu B, Otmishi P, Jani P, Walker J, Sarmiento X, Guardiola J, Saad M, Yu J. Inflammatory mechanisms in the lung. J Inflamm Res. 2009;2:1-11.
- Pertussis - Infectious Diseases [Internet]. Merck Manuals Professional Edition. [cited 2021 Apr 2]. Available from: https://www.merckmanuals.com/en-ca/professional/infectious-diseases/gram-negative-bacilli/pertussis
- Carbonetti N. Exacerbation of pertussis airway inflammation and pathology by pertussis toxin. [cited 2021 Apr 2]; Available from: https://grantome.com/grant/NIH/R01-AI101055-02
- Pirahanchi Y, Sharma S. Physiology, Bradykinin. [Updated 2020 Aug 31]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK537187/
- Hewitt M, Canning BJ. Coughing Precipitated by Bordetella pertussis Infection. Lung. 2009;188(S1):73–9.
- Mills K. Immunity to Bordetella pertussis. Microbes and Infection. 2001;3(8):655–77.
iii. Bacterial evasion: how does the bacteria attempt to evade these host response elements?
B. pertussis is able to first overcome the physical barrier of the epithelium and “ciliary escalator” which clears particulate materials by directly binding to the cilia of the respiratory epithelium (1). Otherwise, they would be swept away.
Recognition of a pathogen, such as B. pertussis, is critical for the activation of the innate and adaptive immune response (2). Therefore, preventing the recognition of B. pertussis is an efficient survival strategy for the bacteria (2). Bacterial LPS structures typically have three structural components: lipid A, the core oligosaccharide and O-antigenic repeats (2). Recognition of bacterial LPS by TLR4 requires the LPS-binding protein to form a complex with CD14 on an immune cell (2). Then, LPS is transferred to the TLR4-MD-2 complex, and MyD88 and TRIF are recruited to TLR4 (2). This allows for NFkB and interferon regulatory factor 3 (IRF3) signaling activation which results in the secretion of pro-inflammatory cytokines important for the innate immune response (2). B. pertussis contains a lipo-oligosaccharide that has a branched core structure with a non-repetitive trisaccharide, instead of a long repeating O-side chain (2). The lipid A component of the bacterial lipo-oligosaccharide can also activate TLR4 signalling, however, it binds less efficiently than the typical LPS structure on gram-negative bacteria (2).
In addition, LPS components lipid A and the oligosaccharide domain can also be recognized by surfactant proteins A and D (SP-A and SP-D) expressed in the lower respiratory tract (2). Binding of LPS to SPA activates agglutination, disrupts the bacterial membrane and assists in phagocytosis (2). Studies have demonstrated that the terminal trisaccharide of B. pertussis lipo-oligosaccharide prevents contact of SP-A and SP-D to the lipid A component by steric hindrance, therefore, the bacteria avoids surfactant-mediated killing (2).
B. pertussis that is phagocytosed by macrophages will be degraded within acidic compartments, however a small proportion of the bacterium will evade destruction and multiple within non-acidic compartments (3). They are able to avoid phagolysosome fusion and can survive within these macrophages, evading immune cells. B. pertussis is able to subvert the immune response by inducing immunosuppressive cytokines by innate cells, as well as through the generation of Treg cells to dampen the immune response (3). The residual bacterium survive and replicate inside nonacidic early endosomal compartments (2).
FHA is expressed on the cell surface and is also secreted. FHA promotes phagocytosis of B. pertussis by binding to CR3 on macrophages, and toxin-mediate suppression of intracellular killing will allow the bacterium to reside within the macrophage and evade the host response. Secreted FHA also has immunomodulatory functions, by inducing IL-6 and IL-10 to suppress IL-12 production by macrophages and DCs. This will lead to IL-10 producing Tregs that further suppress IFNg production, stopping the production of Th1 cells and creating a more favorable environment for bacterial survival (3). FHA is also able to induce apoptosis within phagocytic and epithelial cells (2). The formation of B. pertussis biofilms allows evasion of host immune responses as well (4). FHA contributes to formation of a biofilm by promoting cell-substrate and inter-bacterial adhesions.
Secreted PT also contributes to the immunomodulatory effect on the host cells. It inactivates cytoplasmic G proteins to inhibit chemokine signaling pathways, preventing chemotaxis of immune cells such as macrophages, neutrophils, and lymphocytes. PT has two factors: the A subunit and the B subunit (5). The B subunit interacts with the cell surface to activate adenosine diphosphate (ADP)-ribosylation of G proteins by the A subunit, thus disrupting the cell (5). Through ADP-ribosylation, PT delays the recruitment of immune cells to the site of B. pertussis infection, possibly by suppressing the secretion of chemokines from cells within the respiratory tract (5, 2). It is also able to prevent phagocytosis (3). Studies have shown that PT intoxicates and suppresses the phagocytic ability of monocytes through the ADP ribosylation of macrophage G-proteins (2). PT mediates an immune evasion mechanism that interferes with the ability of DCs to migrate to secondary lymph nodes as well (6).
Adenylate cyclase toxin (ACT) can inhibit anti-bacterial functions of innate cells through the induction of apoptosis and cell cycle arrest, preventing their ability to phagocytose, induce chemotaxis or produce superoxides (3). ACT rapidly induces cellular cAMP levels in CR3 phagocytes (2). Production of cAMP leads to the inactivation of RhoA, which suppresses the bactericidal activity of the phagocytes (2). cAMP also suppresses secretion of inflammatory cytokines while promoting secretion of IL-10. IL-10 production once again will promote the development of Treg cells to subvert the immune response (3). Another way that B. pertussis avoids the immune system via ACT is by driving apoptosis in the phagocytes. When ACT enters the cell via a vesicular transport pathway, apoptotic signals will reduce mitochondrial functioning (2). ACT also induces the activity of caspase 3 and 7 in macrophages, leading into the programmed cell death pathway (2). ACT is also found be involved in the suppression of IL-12 in order to prevent Th17 expansion (2). LOS through its aberrant structure and lipid A modifications can also skew the cytokine production of DCs towards a Th2 phenotype, rather than a Th1/Th17 one. ACT also promotes inflammatory responses by acting on TLR2 and TLR4 to increase COX-2 expression (7). The terminal of ACT forms pores in the target cell membrane causing K+ efflux which results in activation of the NLRP3 inflammasome complex (8, 9). This results in activation of caspase-1 and release of IL-1B from dendritic cells (10). IL-1β is critical for host protection and clearance of B. pertussis as it activates Th17 cells and recruits neutrophils to fight infection (8, 10).
B. pertussis has also evolved the Bordetella resistance to killing A (BrkA) protein which confers resistance to classical complement-dependent killing (11). Resistance occurs through the inhibition of neutrophil phagocytosis by interfering with complement deposition onto B. pertussis surfaces (11). Typically, the membrane bound C3b functions as an opsonin for phagocytosis as they are recognized by the CR3 receptors on phagocytes, but BrkA interferes with deposition (2). It has also been shown that B. pertussis is not susceptible to the alternative of mannose-binding lectin pathways either (3). B. pertussis can also evade the classical complement pathway via BrkA-independent mechanisms (2). For example, studies have demonstrated that B. pertussis can bind and recruit the C4b-binding protein (C4BP) and the C1 esterase inhibitor (C1INH) (2). Both C4BP and C1INH are inhibitors of the classical complement pathway (2). C4BP prevents the killing of cells by binding C4b, resulting in C4b becoming more vulnerable to destruction by factor I, a C3b inactivator (2). C1INH disrupts the production of the C1 complex by inhibiting the function of the C1s and C1r proteases (2).
Pertacin (PRN). PRN is an outer membrane autotransporter protein that has a role in adherence of B.pertussis to monocytes and epithelial cells (8, 12). Although it does not appear to have a major role in persistence of B. pertussis, antigen variation in PRN has been implicated in immune escape (8, 13).
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1. Edwards JA, Groathouse NA, Boitano S. Bordetella bronchiseptica Adherence to Cilia Is Mediated by Multiple Adhesin Factors and Blocked by Surfactant Protein A. Infect Immun. 2005 Jun;73(6):3618–26.
2. de Gouw D, Diavatopoulos DA, Bootsma HJ, Hermans PWM, Mooi FR. Pertussis: a matter of immune modulation. FEMS Microbiology Reviews. 2011;35(3):441–74.
3. Higgs R, Higgins SC, Ross PJ, Mills KHG. Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunology. 2012 Sep;5(5):485–500.
4. Dorji D, Mooi F, Yantorno O, Deora R, Graham RM, Mukkur TK. Bordetella Pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance. Med Microbiol Immunol. 2018 Feb 1;207(1):3–26.
5. Waters V, Halperin SA. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Bordetella pertussis.
6. Fedele G, Bianco M, Ausiello CM. The Virulence Factors of Bordetella pertussis: Talented Modulators of Host Immune Response. Arch Immunol Ther Exp. 2013 Dec;61(6):445–57.
7. Perkins DJ, Gray MC, Hewlett EL, Vogel SN. Bordetella pertussis adenylate cyclase toxin (ACT) induces cyclooxygenase-2 (COX-2) in murine macrophages and is facilitated by ACT interaction with CD11b/CD18 (Mac-1). Mol Microbiol. 2007 Nov;66(4):1003–15.
8. Higgs R, Higgins SC, Ross PJ, Mills KHG. Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol. 2012 Sep;5(5):485–500.
9. Gray M, Szabo G, Otero AS, Gray L, Hewlett E. Distinct Mechanisms for K+ Efflux, Intoxication, and Hemolysis by Bordetella pertussis AC Toxin. Journal of Biological Chemistry. 1998 Jul;273(29):18260–7.
10. Dunne A, Ross PJ, Pospisilova E, Masin J, Meaney A, Sutton CE, et al. Inflammasome Activation by Adenylate Cyclase Toxin Directs Th17 Responses and Protection against Bordetella pertussis. JI. 2010 Aug 1;185(3):1711–9.
11. Mills KHG. Immunity to Bordetella pertussis. Microbes and Infection. 2001 Jul 1;3(8):655–77.
12. Everest P, Li J, Douce G, Charles I, De Azavedo J, Chatfield S, et al. Role of the Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells. Microbiology. 1996 Nov 1;142(11):3261–8.
13. Hijnen M, Mooi FR, van Gageldonk PGM, Hoogerhout P, King AJ, Berbers GAM. Epitope Structure of the Bordetella pertussis Protein P.69 Pertactin, a Major Vaccine Component and Protective Antigen. IAI. 2004 Jul;72(7):3716–23.
iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria?
Following infection, the Bordetella organism is successfully cleared from the body upon induction of the adaptive immune response (1). In severe cases, the patient may be treated with antibiotics which also eliminate the bacterial pathogen. However, this is not usually recommended since treatment with antibiotics has minimal effect at reducing disease and symptom progression (1,2). Bacterial clearance is not well-correlated with cessation of symptoms (1). The cough and sore throat usually resolve within four weeks, or sometimes can persist for much longer; this is referred to as the convalescent phase of infection (3). Healthy individuals typically make a full recovery after infection with B. pertussis, however, immunocompromised individuals, the elderly and infants are at a higher risk for complications (4). The most common complication of B. pertussis is bacterial bronchopneumonia (2, 5). Although rare, severe complications may include encephalopathy, seizures, paresis, ataxia, aphasia, blindness, deafness, or death (4). Complications caused by the severe and persistent cough caused by pertussis include subconjunctival hemorrhages, syncope, and rib fractures (4). In most instances, the patient recovers and returns to normal, however severe complications can arise at an approximate rate of 0.001 - 0.003% (2, 4).
Healthy individuals may be asymptomatic carriers of B. pertussis (2, 6) and are of particular concern for the potential to infect unvaccinated or partially vaccinated infants (2). This is also thought to be true amongst vaccinated individuals (6), as cases of pertussis continue to persist in vaccinated populations (6, 7, 8). A case of natural pertussis infection confers immunity to future infections for many years (2, 8, 9) highlighting that long-term immunity in humans is achievable. The specific mechanisms of this immunity have yet to be fully elucidated, although evidence indicates that both a humoral and cell-mediated response is required (7, 8, 9). Evidence obtained from animal models is well supported by what is observed in humans: serum antibodies decrease shortly after infection or immunization, yet individuals remain resistant to pertussis infection for many years (2, 8, 9). In terms of reinfection following natural immunity, one case study followed 4 individuals who were reinfected at 3.5, 7, 12 and 12 years respectively following initial reinfection (10). Interestingly, it seems that the longer the interval between infections, the more severe the complaints (10). IgG antibodies following infection seem to decrease as time increases, which may explain increase susceptibility (11) . Most cases of whooping cough specifically have not been confirmed by the laboratory to have been caused by B. pertussis. Therefore, it is possible that whooping cough could have been caused by other pathogens, as in Joseph’s case (10). Infection following vaccination has also been documented in the literature. One report described reinfection after vaccination to B. pertussis in children (11). For this reason, B and T lymphocytes are thought to play central roles in achieving long-term immunological memory to B. pertussis. A 2019 publication reported the estimated duration of long term protection after a natural infection can range from 7-10 years and upwards of 20 - 30 years (12). In some situations however, protection can even be as short as 3.5 years. This can vary significantly, as around 10% of individuals will lose protection within 10 years post-infection (12).
This is important when considering our current vaccination strategies, particularly the change from whole-cell pertussis (wP) to the new-generation acellular (aP) vaccines given in Canada and the United States (8, 9, 13). While effective at preventing serious disease, the new-generation aP vaccines are speculated as being less effective at preventing the transmission of B. pertussis (8, 9). This is perhaps due to a difference in TH1/TH2 induction, as the ‘less-effective’ aP vaccines are observed to stimulate a TH2 (humoral) response while wP vaccination stimulates a TH1 (cell-mediated) and TH17 immune responses (7, 8, 9). It is thought that the increased efficacy observed from the previous wP vaccine can be attributed to the same reason it was replaced: residually active pertussis toxins present in the vaccine contributed to an increased incidence of adverse reactions, but also an increased immune cell recruitment and subsequent immunological memory (9). These are points to consider when developing future vaccination strategies against pertussis (8, 9).
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1. Melvin, J. A., Scheller, E. V., Miller, J. F., & Cotter, P. A. (2014). Bordetella pertussis pathogenesis: current and future challenges. Nature reviews. Microbiology, 12(4), 274–288. https://doi.org/10.1038/nrmicro3235
2. Finger H, von Koenig CHW. Bordetella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 31. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7813/
3. Kimberlin, D. W. (2018). Red Book: 2018-2021 report of the committee on infectious diseases (No. Ed. 31). American academy of pediatrics.
4. Waters V, Halperin SA. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Bordetella pertussis.
5. Minnesota Dept. of Health. Pertussis (Whooping Cough) [Internet]. [cited 2021Apr3]. Available from: https://www.health.state.mn.us/diseases/pertussis/pfacts.html
6. Althouse, B. M., & Scarpino, S. V. (2015). Asymptomatic transmission and the resurgence of Bordetella pertussis. BMC medicine, 13, 146. https://doi.org/10.1186/s12916-015-0382-8
7. Kapil, P., & Merkel, T. J. (2019). Pertussis vaccines and protective immunity. Current opinion in immunology, 59, 72–78. https://doi.org/10.1016/j.coi.2019.03.006
8. Higgs, R., Higgins, S., Ross, P. et al. Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol 5, 485–500 (2012). https://doi.org/10.1038/mi.2012.54
9. Mills KH. Immunity to Bordetella pertussis. Microbes Infect. 2001 Jul;3(8):655-77. doi: 10.1016/s1286-4579(01)01421-6. PMID: 11445452.
10. Versteegh F, Schellekens J, Nagelkerke A, Roord J. Laboratory-confirmed reinfections with Bordetella pertussis. Acta Paediatrica. 2007 Jan 2;91(1):95–7.
11. de Melker HE, Versteegh FGA, Conyn-van Spaendonck MAE, Elvers LH, Berbers GAM, van der Zee A, et al. Specificity and Sensitivity of High Levels of Immunoglobulin G Antibodies against Pertussis Toxin in a Single Serum Sample for Diagnosis of Infection with Bordetella pertussis. Journal of Clinical Microbiology. 2000;38(2):800–6.
12. Esposito S, Stefanelli P, Fry NK, Fedele G, He Q, Paterson P, et al. Pertussis Prevention: Reasons for Resurgence, and Differences in the Current Acellular Pertussis Vaccines. Front Immunol [Internet]. 2019 Jul 3 [cited 2021 Apr 2];10. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616129/
13. Government of Canada. Canadian Immunization Guide: Part 4 - Active Vaccines. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-15-pertussis-vaccine.html