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- 1. Bordetella pertussis http://www.hhmi.princeton.edu/sw/2002/psidelsk/Microlinks.htm
- 2. Outline BordetellaPertussis microbiology Whooping Cough/Pertussis Vaccine Current problems with B. pertussis
- 3. Bordetella pertussis Basics Aerobic, Gram negative coccobacillus Alcaligenaceae Family Specific to Humans Colonizes the respiratory tract Whooping Cough (Pertussis) http://microvet.arizona.edu/Courses/MIC420/lecture_notes/bordetella_pertussis/ gram_pertussis.html
- 4. Transmission Very Contagious Transmission occurs via respiratory droplets http://www.ratbags.com/rsoles/history/2000/12december.htm http://www.universityscience.ie/imgs/scientists/whoopingcough.gif
- 5. Adhesions Filamentous hemagglutinin Pertactin Fimbriae http://www.rivm.nl/infectieziektenbulletin/bul1306/kinkhoest.jpg http://www.my-pharm.ac.jp/~yishibas/research/Pertussis1.jpg
- 6. Toxins Pertussis Toxin Adenylate Cyclase Toxin Tracheal cytotoxin Dermonecrotic toxin Heat-labile toxin www.ibl.fr/u447/u447.htm
- 7. Whooping Cough Also known as Pertussis Outbreaks first described in the 16 th Century Major cause of childhood fatality prior to vaccination paaap.org/immunize/ course/slide27.html
- 8. Clinical Features Incubation period 4-21 days 3 Stages 1st Stage- Catarrhal Stage 1-2 weeks 2nd Stage- Paroxysmal Stage 1-6 weeks 3rd Stage- Covalescent Stage weeks-months http://www.cdc.gov/nip/publications/pertussis/chapter1.pdf
- 9. Pertussis Infection gsbs.utmb.edu/ microbook/ch031.htm
- 10. Diagnosis Isolation by culture PCR Directfluorescent antibody Serological testing http://medinfo.ufl.edu/year2/mmid/bms5300/images/d7053.jpg
- 11. Treatment Antibiotic therapy Erythromycin Azithromycin and clarithromycin http://www.aboutthatbug.com/AboutThatBug/files/CCLIBRARYFILES/ FILENAME/0000000032/033_lg.jpg http://www.vet.purdue.edu/bms/courses/lcme510/chmrx/macrohd.htm
- 12. Pertussis Vaccine 1stPertussis vaccine- whole cell Acellular vaccine now used Combination vaccines http://www.tdh.state.tx.us/immunize/providers.htm http://www.nfid.org/publications/clinicalupdates/pediatric/pertussis.html
- 13. Strain Variation B.pertussis population has changed significantly since vaccine introduction Adaptation to vaccine Antigenic divergence Mooi et al., 2001
- 14. Vaccine problems Complications/Safety Multipleadministration Waning adolescent and adult immunity Strain Variability http://www.healthcareforhoosiers.com/Member/vaccineschedule.html
- 15. Conclusions Reemerging in adult and adolescent populations as worldwide vaccination rates increase High vaccination rates not enough Better vaccine development needed
Notes de l'éditeur
- Bordetella pertussis is an aerobic, non-spore forming, Gram negative coccobacillus (Shumilla et al., 2004). It has no known reservoir other than humans and is thought to be unable to survive in the environment for prolonged periods of time (Merkel, 1998). The Bordetella genus of the Alcaligenaceae family is comprised of seven different species, four of which cause upper respiratory tract infections in different host organisms (Babu et al., 2001). Bordetella parapertussis is the most closely related to Bordetella pertussis . It can cause a milder pertussis-like disease in humans, but Bordetella pertussis is the most serious human pathogen in this genus (Babu et al., 2001). B. pertussis invades its human host through entry into the respiratory tract where it colonizes to cause whooping cough, also known as pertussis, which was at one time a very common and potentially life threatening infection for children (Steele, 2004). Today, whooping cough still effects 20-40 million people worldwide each year and causes between 200,000-400,000 fatalities (Shumilla et al., 2004). The image on this slide shows the B. pertussis after Gram staining.
- Pertussis is highly contagious, with an 80% secondary attack rate among susceptible persons (CDC, 2005). Pertussis is generally transmitted from person to person via respiratory droplets, but direct contact with respiratory secretions from infected individuals may also lead to the disease (CDC, 2005). Freshly contaminated articles (such as clothing) from the infected person can also contain infectious respiratory secretions, allowing pertussis to be passed indirectly from the infected person to a susceptible host who comes in direct contact with these items. While the most serious infections occur in young children, with most pertussis related deaths occurring in infants too young to be vaccinated, adolescents and adults also experience a health burden from the disease (Forsyth et al., 2004). Pertussis is on the rise in adolescent and adult populations, and while the health of these age groups are important, they also provide a potential source of major pediatric infections (Forsyth et al., 2004). Parents are a common source of B. pertussis infections for infants, while other relatives such as grandparents, uncles and aunts also provide another potential source of infection (Forsyth et al., 2004).
- Adherence to the ciliated epithelial cells and macrophages is very important in colonization of the respiratory tract (Babu et al., 2001). B. pertussis produces several virulence factors that allow the bacterium to bind to cells and colonize the host. Of these adhesions, filamentous hemagglutinin (FHA) is one of the most important. FHA is a filamentous structure that measures about 2 nm wide and 50 nm long (Babu et al., 2001). It is a large, hairpin shaped molecule that is highly immunogenic and is therefore a primary component in acellular pertussis vaccines (Mattoo et al., 2001). FHA secretion requires the presence of the outer membrane protein FhaC; in its absence, FHA will not be secreted and will instead be degraded within the cell (Mattoo et al., 2001). The opsonization of antibodies to FHA can actually decrease phagocytosis of FHA expressing B. pertussis and FHA can affect cell mediated immunity by inhibiting cytokine responses (Veal-Carr and Stibitz, 2005). FHA is thought to be the major colonizing factor for B. pertussis as it promotes attachment to the upper respiratory tract and the trachea (Steele, 2004). It has been found to be both necessary and sufficient by itself to mediate adherence to rat lung epithelium and is absolutely required for trachael colonization in healthy animals, but cannot be the lone factor in this colonization (Mattoo et al., 2001). Pertactin is a surface associated protein that undergoes autoproteolytic processing of its C-terminus (Mattoo et al., 2001). Pertactin is thought to play a role in attachment because all three pertactin proteins contain motifs commonly present on proteins that are responsible for protein-protein interactions in eukaryotic cell binding (Mattoo et al., 2001). Pertactin is expressed after FHA but before the pertussis and adenylate cyclase toxins during infection, further implicating it as a colonizing factor (Merkel et al., 1998). Purified pertactin has also been shown to cause Chinese Hamster Ovary (CHO) cells to bind to tissue culture wells and expression of pertactin in E. coli increases the bacterial adherence and invasiveness to mammalian cell lines (Mattoo et al., 2001). B. pertussis , like most Gram negative pathogens, express fimbriae on their cell surface and Bordetella predominantly expresses Fim2 and Fim3 serotypes (Mattoo et al., 2001). Fimbriae also serve to mediate attachment to the host epithelia during the important first steps in colonization, but it has been difficult to establish a definitive role for fimbriae in this process due to the expression of other adhesions and the difficulty in finding a proper animal model (Mattoo et al., 2001). Fimbriae, however, have been determined to bind sulfated sugars such as heparan sulfate, chondroitin sulfate, and dextran sulfate that are ubiquitously present throughout the mammalian respiratory tract. The fimbriae mimic fibronectin, a host protein found in the extracellular matrix that also exhibits these binding interactions. The two proteins share significant homology that allows the fimbriae to use host ligand-receptor interactions to further infection (Babu et al., 2001). Fimbriae are thought to be the cause of the persistency of B. pertussis infections (Babu et al., 2001) and Fimbriae have also been found to elicit a host immune response that is important in the prevention of superinfection (Mattoo et al., 2001).
- Whooping cough (Pertussis) is primarily a toxin mediated disease (CDC, 2005). After the bacterium adheres to the ciliated epithelium of the respiratory tract and colonizes the host, it secretes toxins that lead to the death of these epithelium cells, a decrease in ciliary beating, and an accumulation of mucus and cell debris that triggers coughing (Ahuja et al., 2004). Pertussis toxin is unique to the Bordetella pertussis species and is important in colonization, the disruption of host cell signaling pathways, and immune evasion (Mattoo et al., 2001). Adenylate cyclase toxin is an invasive toxin that is also important in the disruption of signal transduction pathways; in addition it also plays a role in the disruption of immune effector cells (Ahuja et al., 2004). Tracheal cytotoxin (TCT) is a disaccharide tetrapeptide that is derived from the cell wall (Coote, 2001). This toxin has been observed to cause paralysis of the cilia and extrusion of ciliated cells in hamster tracheal organ cultures and has also been shown to inhibit DNA synthesis in hamster tracheal epithelial cell cultures, all of which could lead to mucus accumulation and coughing. The destruction of the cilated epithelial cells in a B. pertussis infection is thought to be due to the production of nitric oxide by non-ciliated, mucus secreting epithelial cells in response to the combination of TCT and lipopolysaccharide (LPS) (Coote, 2001). TCT is also associated with the characteristic whooping cough of B. pertussis infection and an increase in body temperature through the simulation of interlukin-1 (Babu et al., 2001). Dermonectoric toxin is a heat stable toxin that induces inflammation, vasoconstriction, and dermonecrotic lesions around colonies of B. pertussis in the respiratory tract (Babu et al., 2001). This toxin also affects the regulation of cell growth and division systems (Babu et al., 2001). Heat-labile toxin may also be involved in tissue damage during infection (Steele, 2004).
- Whooping cough (Pertussis) was first described in 1578 as an epidemic of pediatric respiratory disease that began in Paris and spread throughout Europe (Steele, 2004). It is unclear if this is the first emergence of the disease or simply the first time a careful recording of clinical observations related to B. pertussis was made (Steele, 2004). Pertussis is a highly contagious disease and in the pre-vaccination era, nearly every child contracted the disease and pertussis was a major cause of infant death through the world (Mooi et al., 2001). In the six years between 1940-1945 (pre-vaccination era), more than one million cases of pertussis were reported in the United States, averaging 175,000 cases a year (CDC, 2005). After the whole cell pertussis vaccine was introduced to the United States in the mid-1940’s, pertussis sharply declined until cases were reduced more than 90% when compared to pre-vaccination levels (Hardwick et al., 2002). Although effective vaccination campaigns have been well established in developed nations for more than 50 years, pertussis still remains endemic and epidemic peaks occur every three to five years in the United States (Hardwick et al., 2002). Although vaccines are potentially available, they are not adequately used in many developing countries. Approximately 50 million cases of pertussis occur throughout the world each year with 300,000 deaths annually, making pertussis the fifth leading cause of vaccine preventable deaths (Steele, 2004).
- After transmission of B. pertussis to a new host, there is an incubation period that averages 7-10 days, with a range of 4-21 days. In rare cases, incubation periods have been found to occur for as long as 42 days (CDC, 2005). After the incubation period, an infected person can expect the illness to progress through three stages: the catarrhal stage, the paroxysmal stage, and the covalescent stage. The first stage is the catarrhal stage, which is characterized by a runny nose, sneezing, low fever, and a mild cough. These beginning symptoms are similar to a common cold and gradually become more severe (CDC, 2005). After 1-2 weeks, the paroxysmal stage begins. It is at this point that a diagnosis of pertussis is usually suspected. The cough usually progresses to the characteristic whooping cough, which consists of bursts or paroxysms of numerous, rapid coughs. These coughing episodes seem to be due to a difficulty in expelling mucus from the tracheobronchial tree. These attacks usually end with a long inspiratory effort which is usually accompanied by the high pitched whoop from which the disease gets its name. These attacks may also cause the patient to turn blue and appear very ill and distressed, especially when young children and infants are effected. Vomiting may also accompany these attacks, but the patient generally appears normal between such episodes (CDC, 2005). Paroxsymal attacks are more frequent at night and these attacks will increase in frequency during the first two weeks of this stage and then begin to decline after week three. This stage lasts from 1-6 weeks, but may effect the patient for up to 10 weeks. The characteristic whoop may not be observed in young infants due to a lack of strength, though these coughing episodes occur. Individuals with pertussis are most infectious during the catarrhal stage and the first two weeks after cough onset, with this highly infectious period lasting an approximately 21 days (CDC, 2005). The third and final stage of the disease involves the gradual recovery of the patient from the paroxysmal stage. During this convalescent stage, these coughing attacks continue to gradually decrease and usually disappear within 2-3 weeks, though these episodes may recur following subsequent respiratory infections for many months after the onset of the disease. Adults and adolescents usually have milder symptoms that may be indistinguishable from other respiratory infections and the characteristic whoop is uncommon. B. pertussis is estimated to cause up to 7% of coughing illnesses each year in the less susceptible, older population (CDC, 2005).
- B. pertussis enters its human host through inhalation and proceeds to the lungs (Steele, 2004). B. pertussis does not usually spread from the respiratory tract or establish chronic infection; however, there are other risks associated with infection (Merkel et al., 1998). Neurological conditions including seizures and encephalopathy may occur in extreme cases due to the reduction of the oxygen supply to the brain associated with coughing attacks or perhaps a toxin (CDC, 2005). Irreversible brain damage may also occur, though these incidences are infrequent (Babu et al., 2001). Neurological complications due to pertussis infection are most common in infants (CDC, 2005). Secondary infections are the most common cause of pertussis related deaths (Steele, 2004), and lung collapse, increased intrathoracic pressure and hemorrhages due to blood vessel rupture may also occur in extreme pertussis infections (Babu et al., 2001).
- Isolation of B. pertussis in a culture is the standard and preferred method of diagnosis (CDC, 2005). B. pertussis is difficult to isolate, however, as it has particular growth requirements. Isolation from direct plating is most successful during the catarrhal stage and specimens should be collected from the posterior nasopharynx (not the throat) with a Dacron or calcium alginate (not cotton) swab and plated directly on selective media (CDC, 2005). Selective media for B. pertussis includes Regan-Lowe, Bordet-Gengou, or charcoal agar (Steele, 2004). These cultures may require an incubation period as long as two weeks, so more rapid analysis techniques are preferred for initial diagnosis (Steele, 2004). Successful isolation declines with pervious exposure to antibiotic therapy effective against pertussis or if specimens are collected beyond the first two weeks of illness. Isolation is also difficult for vaccinated patients (CDC, 2005). PCR testing of these nasopharyngeal swabs can also be done to obtain a rapid, sensitive, and specific pertussis diagnosis (CDC, 2005). This technique is currently only available in some laboratories and the assays among these laboratories are not standardized. PCR should be done in addition to culture, for the culture may be necessary for further case analysis including evaluation for antibiotic resistance and molecular typing (CDC, 2005). Direct fluorescent antibody tests can also be performed on nasopharyngeal samples and is another pertussis screening method (CDC, 2005). It uses fluorescent antibodies to detect antigen in the sample. This method has been shown to have low sensitivity and is not very specific with nasopharyngeal samples, so it should not be relied upon for laboratory confirmation (CDC, 2005). This low specificity leads to a high percentage of false positive results (Steele, 2004). Serological testing has also been used to diagnosis B. pertussis in some clinical studies, however this method is not currently standardized (CDC, 2005). This test is positive when IgA antibodies are found against whole cell B. pertussis (Poynten et al., 2002). Results of serological tests are difficult to interpret because of a lack of association between antibody levels and immunity to pertussis, making this type of testing not widely available. Without standardization, serological testing cannot be used for case confirmation and when a case is serologically positive but not culture or PCR positive, the case should be reported as probable (CDC, 2005)
- Pertussis is a significant economic burden in the US. The direct costs of pertussis in infants is estimated at $2822, with hospitalization accounting for two thirds of infant medical costs. For children, this cost drops to $308 while the direct cost for adolescents is $254 and $181 for adults (Forsyth et al., 2004). For children, adolescents, and adults, these costs reflect doctors visits, but antibiotics and hospitalization could also contribute. These costs are more substantial in severe cases and when complications arise. The indirect costs may also be substantial, especially for adults whose illness and childcare responsibilities result in missed work and reduced productivity (Forsyth et al., 2004). Erythromycin is highly effective at removing B. pertussis in infected patients, but does not effect the duration or severity of the clinical disease (Steele, 2004). The drug should be administered in four doses per day for 14 days with 40-50 mg/kg total per day. Azithromycin and clarithromycin are equally effective when the patient is given azithromycin at 10-12 mg/kg per day for five days followed by clarithromycin in two doses at 15-20 mg/kg total per day for seven days (Steele, 2004). Erythromycin should not be given to newborns less than 13 days old because it produces increased gastric motility and can lead to hypertrophic pyloric stenosis. Azithromycin and clarithromycin do not produce this change in the GI tract and should be used instead of erythromycin (Steel et al., 2004). Erythromycin, azithromycin and clarithromycin are macrolides that inhibit protein synthesis by binding to the 23S rRNA in the 50S ribosomal subunit. This binding blocks the exit of growing peptide chains, thereby inhibiting protein synthesis. These antibiotics are safe because humans do not have a 50S ribosomal subunit but instead have 40S and 60S subunits (http://en.wikipedia.org).
- The original pertussis vaccine was a whole-cell vaccine developed in the 1930’s and widely used by the mid 1940’s (CDC, 2005). This whole cell vaccine had the potential for adverse reactions which worried many parents and bred non-compliance in children receiving the vaccine (Steele, 2004). While local reactions such as swelling, redness, or pain at the injection site were common following a DTP dose, more severe reactions such as convulsions and hypotonic-hyporesponsiveness occurred in about 1 out of every 1,750 doses administered. Also, acute encephalopathy occurred in approximately 0-10.5 cases per million doses of vaccine administered, and it was not agreed upon as to whether the vaccine could cause permanent brain damage (CDC, 2005). The safety concerns surrounding the whole cell vaccine lead to the development of acellular vaccines with less risk of adverse reactions (CDC, 2005). Acellular pertussis vaccines use specific proteins extracted from B. pertussis to generate protective immunity (Steele, 2004). DTaP vaccines containing the acellular pertussis product first came into use in Japan in 1981 for primary immunization of 2 year old children. Two DTaP vaccines were licensed for use in the US in 1991, but they were only approved for the 4 th and 5 th doses given to children 15 months or older (Steele, 2004). At this time it was shown that DTaP vaccines were effective for young infants at preventing pertussis with significantly fewer adverse reactions, and the FDA approved two vaccines to be administered as the initial four doses of the vaccine and AcelImune was approved for all five doses (Steele, 2004). In 2000, production of AcelImmune, the only vaccine licensed for all five doses, was discontinued due to manufacturing difficulties and only two vaccines were approved for use in young children until Daptacel, a five component DTaP vaccine containing DTaP, hepatitis B, and inactivated polio, was approved for routine primary immunization in 2003 (Steele, 2004). Combination vaccines are advantageous because they allow the infant to receive multiple immunizations with only one shot.
- Strain variation has been postulated to be the cause for increased pertussis incidence in the Netherlands (Mooi et al, 2001 and Schouls et al., 2004) and high strain variation between currently circulating strains and the vaccination strains has also been observed in the US (Hardwick et al., 2002). B. pertussis strains collected in the Netherlands from 1949-1996 were analyzed using DNA fingerprinting and sequencing of surface protein genes (Mooi et al., 2001). Significant differences were found between pre-vaccination isolates and those collected during post vaccination periods, both in the type and frequency of fingerprint types. After the introduction of the vaccine, genotypic diversity decreased notably, but then returned to pre-vaccination levels. In the 1980s, the genotypic diversity of isolates again decreased, which was later attributed to an expansion of antigenically distinct strains (Mooi et al., 2001). Pertussis toxin and pertactin are important proteins in immune development, as antibodies against these proteins corresponds to protection against pertussis; however, these virulence factors have been found to be polymorphic. These polymorphisms are non-conservative, indicating the occurrence of Darwinian selection (Mooi et al., 2001). Pertussis toxin and pertactin variants in the strains from the 1950s were identical to those included in the Dutch B. pertussis vaccine in 100% of the cases. Between 1990-1996, non-vaccine pertussis toxin and pertactin types were observed in 90% of the isolates (Mooi et al., 2001). It seems as though the vaccine shifted the competitive balance of naturally occurring B. pertussis strains, allowing previously less competitive strains to become more common after immunization controlled the most fit strains (Mooi et al., 2001). More recently, another group used multilocus sequence typing (MLST) to study the molecular epidemiology of Dutch B. pertussis isolates (Schouls et al., 2004). Variation in direct repeat regions of B. pertussis were identified using multiple-locus variable-number tandem repeat analysis (MLVA), which can be applied directly to nasopharyngeal swabs and does not require culturing (Schouls et al., 2004). The profiles of strains from isolates before vaccination began were more diverse than those from 1990s isolates, but they were found to be only distantly related to the current strains. The genotypic diversity of B. pertussis decreased during and after epidemics of the 1990s, which suggests that these epidemics were caused by a smaller number of strains due to clonal expansion (Schouls et al., 2004). The results of this study suggest that variable number tandem repeats (VNTR) evolve before virulence genes, making VTNR analysis more suitable to detect short-term changes in the B. pertussis population. Antibodies against pertactin present in the current vaccine are less effective at preventing the disease against strains with different pertactin variants, showing the importance of antigenic divergence in the current B. pertussis epidemiology (Schouls et al., 2004). Antigenic divergence is not limited to the Netherlands. A shift in prevalence profiles in US B. pertussis strains has been shown using pulsed field gel electrophoresis (PFGE) (Hardwick et al., 2002).
- There are several factors that effect the efficiency of the Pertussis vaccine. Although the development of the acellular pertussis vaccine decreased the incidents of adverse side effects associated with pertussis vaccination, there are still problems associated with the current vaccination program. The acellular vaccine is not free of adverse effects, although this vaccine is an improvement from the whole cell vaccine. Adverse effects are most common in the fifth and final dose of pertussis administered to children, so there may be a further increase in side effects for adolescent and adult boosters (Robbins et al., 2005). These side effects would include the local reactions observed with the whole cell vaccine as well as more serious neurological complications. Also, the administration schedule of the vaccine increases the potential for noncompliance, as infants and children must be vaccinated five times before they reach the age of five in order to be properly protected (Steele, 2004). In the US, this potential noncompliance is combated by the requirement for children to be vaccinated fully before they can be admitted to school (Hardwick et al., 2002). Waning immunity in adolescents and adults may be a factor in the increased incidence of pertussis in these populations (Forsyth et al., 2004). Immunity from vaccination with the whole cell pertussis only persists for about 3-5 years before it begins a decline 6-10 years after vaccination. Although data on the persistence of the acellular pertussis vaccine is limited, it is believed that this vaccine will exhibit the same pattern. Currently in the United States, there is no pertussis booster available for adolescent and adult use, so the last scheduled pertussis vaccine is administered around 5 years of age (Forsyth et al., 2004). This waning immunity leave adults and adolescents particularly vulnerable to pertussis infection. Once a suitable booster is developed, it would be easy to switch from diphtheria and tetanus boosters to DTaP boosters, as the DT booster is already recommended for adult use every 10 years. The availability of a pertussis booster would prolong adult immunity and reduce disease incidence (Forsyth et al., 2004). Although strain variability has not been proven to be a factor for pertussis reemergence in the US and other developed countries, the results of the Dutch study suggest that the strain variability observed in the Netherlands is a universal factor in the reemergence of pertussis in highly vaccinated populations (Schouls et al., 2004). Strain differences have been observed in the US and further studies should indicate if this is a substantial factor in US pertussis incidences (Hardwick et al., 2002). Such a study could mandate changes in the current vaccines to make them more effective at providing protective immunity to the population by adding B. pertussis components from different strains to the vaccine.
- Although pertussis has declined dramatically since the pre-vaccination era, there is still a lot of work that must be done before the disease is controlled. The reemergence of pertussis in adult and adolescent in highly vaccinated populations worldwide suggests that the current vaccination program is decreasing in its successfulness and further research into new vaccinations is needed. A high vaccination rate is not enough. A more successful vaccine needs to be developed that contains different components of commonly circulating B. pertussis strains. This vaccine should also have a lower risk of side effects and should have fewer administrations needed for protective immunity to develop. Development of a booster for adolescents and adults is also very important. Two such boosters are currently on the market in other countries; however, there are not approved for use in the United States (Steele, 2004). Many people believe that pertussis is no longer a problem, but without better control measures the incidence rates will continue to increase and a greater number of people will be effected each year.