CASE

A 53-year-old male developed symptoms of malaise, fatigue, and mild cough while on vacation in DELHI with his family. Six days after the onset of symptoms, the patient returned home to BIKANER still ill and complaining of a headache, low grade fever, chest pains, and a cough. Pt treated in line of the viral fever. At day 10, the patient had a cough with the production of purulent sputum. A sputum specimen was collected and sent to the laboratory for culture. The patients symptoms persisted despite treatment with ampicillin-clavulanic acid [Augmentin(R)].

 At day 14 of his illness, developed paroxysmal cough, occasional vomiting after cough, and subconjunctival hemorrhage.

At day 15 his illness was complicated by episodes of seizure, with clonic movements of the arms and legs, brief loss of consciousness, and confusion. The episodes were triggered by mild, unremarkable coughing paroxysms.

 The Gram stain of sputum revealed Gram negative coccobacilli. The nested PCR from two different site of the nasophyrnx and sputum showed Bordetella pertussis. This was supported by culture on LR media and serology.

WHAT THIS CASE INDICATES.

RESURGENCE OF BORDETELLA PERTUSSIS IN ADULTS

PRESENTOR-Dr MAHAVEER SINGH MODERATOR-Dr S.ANURADHA

PERTUSSIS INCIDENCE IN USA

FROM 1990 TO 2011(AGE WISE) PROPORTION OF ADULT CASES

PERTUSSIS INCIDENCE AND GENOME ANALYSIS

BORDETELLA PERUSSIS
Bordetella is a small (approximately 0.8 m by 0.4 m), rodshaped, coccoid, or ovoid gram negative bacterium that is encapsulated and does not produce spores. While nine species of Bordetella have been identified to date, only three additional members, B. bronchiseptica, B. parapertussis, and B. holmesii, have been associated with respiratory infections in humans and other mammals. B. bronchiseptica produces infection in immunocompromised and AIDS.

VIRULENCE FACTOR OF BORDETELLA PERTUSSIS

THE BVG -AS TWO COMPONENT SYSTEM

ADHESIONS
1. FILAMENTOUS HAEMAGGLUTININ 2. FIMBRIAE 3. PERTACTIN

TOXINS
1. ADENYLATE CYCLASE 2. PERTUSSIS TOXIN 3. DERMATONECROTIC TOXIN 4. TRACHEAL CYTOTOXIN LIPOPOLYSACCHARIDE SECRETORY SYSTEMS

Bvg-AS TWO COMPONENT SYSTEM

Bvg locus encodes proteins that transmit extracellular signals to the cellular transcription machinery, causing changes in gene expression. With the exception of tracheal cytotoxin, expression of virulence factors is coordinately regulated by the products ofthe bvg locus. BvgS is a 135 kDa periplasmic sensor histidine kinase. BvgA, the response regulator, is a 23-kDa cytoplasmic protein .

FILAMENTOUS HEAMAGGLUTININ Filamentous hemagglutinin is a large (220 kDa) protein that forms filamentous structures on the cell surface. FHA binds to galactose residues on a sulfated glycolipid called sulfatide which is very common on the surface of ciliated cells(respiratory epithelia).

Mutations in the FHA structural gene reduce the ability of the organism to colonize, and antibodies against FHA provide protection against infection

FIMBRIE
B.pertussis fimbriae are submicroscopic proteinaceous appendages that protrude from the cell surface. They are comprised of a major and a minor subunit.

The minor subunit, named FimD binds to the integrin Vla-5, located on monocytes. The major subunit binds to sulfated sugars like heparan sulfate, chondroitin sulfate and dextran sulfate which are ubiquitous in the respiratory tract

PERTACTIN
Pertactin, a 69 kDa nonfimbrial outer membrane protein, under the control of the bvg locus, is partly responsible for the adhesion of the bacteria to the host cells. The mature protein has two Arginine-Glycine-Aspartic acid (RGD) sequences, at 225-227 and 665-667. These sequences mimics sequences on proteins like fibronectin, vitronectin and fibrinogen.

PERTUSSIS TOXIN
PTx is a two component, A+B bacterial exotoxin. The A subunit (S1) is an ADP ribosyl transferase. The B component, composed of five polypeptide subunits (S2 through S5), binds to specific carbohydrates on cell surfaces. The A subunit gains enzymatic activity and transfers the ADP ribosyl moiety of NAD to the membrane-bound regulatory protein Gi that normally inhibits the eukaryotic adenylate cyclase and intracellular Levels of cAMP increases.

 This has the effect to disrupt cellular function, and in the case of phagocytes, to decrease their phagocytic activities such as chemotaxis, engulfment, the oxidative burst, and bacteridcidal killing Systemic effects of the toxin include lymphocytosis and alteration of hormonal activities that are regulated by cAMP, such as increased insulin production (resulting in hypoglycemia) and increased sensitivity to histamine (resulting in increased capillary permeability, hypotension and shock).

ADENYLATE CYCLASE
This toxin is a 45 kDa protein that may be cell-associated or released into the environment. It is active only in the presence of a eukaryotic regulatory molecule called calmodulin. This toxin acts locally to reduce phagocytic activity and probably helps the organism initiate infection.

TRACHEAL CYTOTOXIN
The tracheal cytotoxin is a peptidoglycan fragment, which appears in the extracellular fluid where the bacteria are actively growing. The toxin kills ciliated cells and causes their extrusion from the mucosa. It also stimulates release of cytokine IL-1, and so causes fever.

DERMATONECROTIC TOXIN
The lethal toxin is a 102 kDa protein composed of four subunits, two with a mw of 24kDa and two with mw of 30 kDa. It causes inflammation and local necrosis adjacent to sites where B. pertussis is located.

LIPOPOLYSACCHARIDE
As a Gram-negative bacterium Bordetella pertussis possesses lipopolysaccharide (endotoxin) in its outer membrane. It is heterogeneous, with two major forms differing in the phosphate content of the lipid moiety. The unfractionated material elicits the usual effects of LPS (i.e., induction of IL-1, activation of complement, fever, hypotension, etc.

PATHOGENESIS OF BORDETELLA PERTUSSIS

Stage 1: Catarrhal

Length Clinical Features Usually 7-10 days; range Characterized by: of 4-21 Coryza

CLINICAL FEATURE
Low-grade fever Mild, occasional cough (which gradually becomes more severe)

Stage 2: Paroxysmal

Usually lasts 1-6 weeks, Characterized by: but may persist for up to Paroxysms of numerous, rapid coughs due to difficulty expelling thick mucus from 10 weeks the tracheobronchial tree.

Long aspiratory effort accompanied by a high-pitched "whoop" at the end of the paroxysms Cyanosis Vomiting and exhaustion

Paroxysmal attacks:

Occur frequently at night, with an average of 15 attacks per 24 hours. Increase in frequency during the first 1-2 weeks, remain at the same frequency for 2-3 weeks, and then gradually decrease.

Stage 3: Convalescent

Usually 7-10 days; range Characterized by: of 4-21 Gradual recovery

Less persistent, paroxysmal coughs that disappear in 2-3 weeks

Paroxysms often recur with subsequent respiratory infections for many months after the onset of pertussis.

VIDEO OF ADULT WHOOPING COUGH

Classification of coughing episodes involving pertussis based on cultures, serology, family exposure, and clinical symptoms
1. B. pertussis isolated from the nasopharynx. 2. At least one of the following criteria fulfilled:

Significant increase in PT IgG. Significant increase in FHA IgG without other criteria for parapertussis. Both PT IgG and FHA IgG 6,000 in the same convalescent serum. Family member with pertussis verified by culture or serology.

3.Clinical pertussis with at least 3 weeks of paroxysmal cough and known exposure to pertussis outside the family; serum samples lacking or suboptimal timing in relation to onset of symptoms.

4.Known exposure to pertussis within or outside the household; clinical symptoms not evaluable because of early erythromycin treatment

BORDETELLA PERTUSSIS IN ADULTS

Most cases of adult pertussis occur in those who have a history of past pertussis vaccination or infection. Pertussis has been found to be the cause in 1320% of adults presenting with prolonged cough.

Adults and adolescents usually present late in the course of the infection, often after 4 or more weeks of coughing

 In a U.S. study of adolescents and adults, the following clinical findings were noted: (i) the median duration of cough was 42 days, with a range from 27 to 66 days. (ii) all subjects had paroxysmal cough. (iii) 26% of the subjects had whooping. (iv) 56% had post-tussive vomiting. (v) 100% had post-tussive gagging

During the paroxysmal stage, adults experience symptoms not described in children, such as a scratchy throat, other pharyngeal symptoms, and episodes of sweating.

COMPLICATIONS
The most frequent complication observed in children is pneumonia, which occurs in 6% of cases. In the first 2 months of life, pneumonia, seizures and encephalopathy have been reported in 25%, 3% and 1% of cases, respectively. After childhood, the risk of complications increases with age. Pneumonia has been observed in 2% of patients less than 30 years of age, as compared with 5%9% of older patients.

 Other complications observed in adults are syncope, urinary incontinence, back pain, rib fracture and hernia.

Severe paroxysm, post-tussive cyanosis, whooping, posttussive vomiting, apnea, pneumonia and seizures are the most frequent reasons patients are admitted to hospital, regardless of age

Complications

Adults
Pneumonia Rib Fracture Weight Loss

Children
Hypoxia Apnea Pneumonia

Hernias Urinary Incontinence

Seizures

DIAGNOSIS OF BORDETELLA PERTUSSIS

CULTURE
Culture is considered to be the gold standard. Its sensitivity decreases steeply if the specimen is taken more than 2 weeks after onset of cough and is reduced by prior immunity from disease or immunisation. Results can be available in 72 h, especially in high titre infection, but 2 weeks are required before culture may be definitively reported as negative.

DNA PCR
Polymerase chain reaction (PCR) methods enhance the probability of identification of B. pertussis.

Conventional-semi nested (CsnPCR) and real-time PCR (RtPCR) are two PCR tools employed for the detection of B. pertussis with the former detecting amplified target gene products visualized by ultra violet (UV) light following agarose gel electrophoresis and the latter monitoring the fluorescence emitted throughout PCR reaction phases indicating gene amplification

 Novel real-time PCR assays targeting the Bordetella pertussis insertion sequence IS481, the toxin promoter region and Bordetella parapertussis insertion sequence IS1001 were designed. PCR assays were capable of detecting 10 copies of target DNA per reaction, with an amplification efficiency of 90%. Since positive results may be obtained even when the organism is no longer culturable. However, the sensitivity of PCR decreases with the duration of symptoms, since the method is based on the detection of the microorganism.

SEROLOGY
Infection of bordetella pertusis is followed by increase in the concentrations in serum of IgA, IgG, and IgM antibodies to specific antigens as well as to preparations of the whole organism.

The antibodies measured to detect infection are directed against FIM2/3, PRN, and LPS In contrast to natural infection, the primary immunization of children induces mainly IgM and IgG antibodies

 Serologic diagnosis of pertussis may be suspected by the demonstration of an increase in agglutinin titer or the use of ELISA, showing an increase in IgA or IgG antibody titer to PT, FHA, PRN, FIM, or to sonicated whole organisms in two serum samples collected 2 to 4 weeks apart.

It is now clear that antibody responses to FHA and PRN also occur following other Bordetella infections, so that isolated increases in titers of antibody against these antigens are not specific for B. pertussis infection..

 In addition, high titers of antibody to FHA may be the result of cross-reacting epitopes of nonencapsulated H.influenzae, M.pneumoniae, C.pneumoniae, and perhaps other bacteria The greatest sensitivity and specificity for the serological diagnosis of B. pertussis infection is achieved by ELISA and measurement of IgG and IgA antibodies to PT. A significant rise in titer (greater than or equal to twofold) between acute-phase and convalescent-phase sera needs to be demonstrated. In adolescents and adults, single high values of IgG or IgA antibodies to PT also indicate infection.

SINGLE SERUM SPECIMEN

Single-sample serology tests for antipertussis toxin IgG must be collected at least two weeks after symptom onset. A high antibody titer more than two years following vaccination supports the diagnosis of pertussis.

A solitary antibody concentration of IgG anti-PT greater than 100-125 EU/mL suggests recent B. pertussis infection or exposure. A study in the Netherlands showed that a titer of 100 EU/mL against this antigen has a sensitivity of 76 percent and specificity of 99 percent for the diagnosis of acute pertussis.

SEROLOGICAL CRIETARIA
Significant increase in ELISA diagnostics 1. Controll serum interassay variation increase in AB Acute to convalascent phase

<10% <15%

>50% >100%

1. MINIMUM LEVEL FOR CONVALESCENT SERUM

A. 4 TIMES MLD FOR ANTIBODIES TO PT B. 32 TIMES MLD FOR ANTIBODIES TO FHA

MANAGEMENT OF BORDETELLA PERTUSSIS

SPECIAL GROUPS
Postexposure prophylaxis
The decision to administer postexposure chemoprophylaxis is made after considering the infectiousness of the patient and the intensity of the exposure, the potential consequences of severe pertussis in the contact, and possibilities for secondary exposure of persons at high risk from the contact (e.g., infants aged <12 months). Administration of postexposure prophylaxis to asymptomatic household contacts within 21 days of onset of cough in the index patient can prevent symptomatic infection.

 Coughing (symptomatic) household members of a pertussis patient should be treated as if they have pertussis. Postexposure prophylaxis should be administered in exposure settings that include infants aged <12 months or women in the third trimester of pregnancy. A macrolide can be administered as prophylaxis for close contacts of a person with pertussis if the person has no contraindication to its use.

CLOSE CONTACT PROPHYLAXIS

A close contact of a patient with pertussis is a person who had face-to-face exposure within 3 feet of a symptomatic patient. Close contacts also can include persons who Have direct contact with respiratory, oral, or nasal secretions from a symptomatic patient. Shared the same confined space in close proximity with a symptomatic patient for >1 hour.

PERTUSSIS VACCINE
The pertussis vaccine is available as: DTaP (Diphtheria Toxoid-Tetanus Toxoid-acellular Pertussis vaccine) DTaP in combination with Haemophilus influenzae type b (Hib) vaccine DTaP in combination with hepatitis B and inactivated polio vaccines DTaP in combination with Hib, hepatitis B and inactivated polio vaccines Tdap (Tetanus Toxoid reduced-Diphtheria-acellular Pertussis vaccine)

VACCINE RECOMMENDATION
Who should receive the vaccine? Most infants and children younger than seven years of age should receive DTaP beginning at two months of age. Children 7-10 years of age who are incompletely immunized against pertussis should receive Tdap.

11-18 year olds should receive a single dose of Tdap instead of a Td booster if they have completed the recommended childhood DTP/DTaP immunization series and have not received Tdap. The preferred age for Tdap vaccination is 11-12 years.

 Adults 19-64 years of age should also receive a single dose of Tdap to replace a single dose of Td for booster immunization if their most recent tetanus toxoid-containing vaccine was 10 or more years earlier.

For adults(>65) who have not received Tdap vaccine and are likely to come in contact with infants suffering from pertussis, a single dose of Tdap vaccine (2 weeks before the contact with the infant) is indicated if 2 years or more have elapsed since the last dose of Td vaccination.

Tdap may be given at an interval shorter than 10 years since the last tetanus toxoid-containing vaccine in order to protect against pertussis in special conditions
Women <65 years of age who might become pregnant.

Women who have not previously received Tdap (including those who are breatfeeding) should receive Tdap as soon after birth as is feasible. Many experts also recommend that Tdap be

considered for pregnant teenagers.

Health-care personnel who have direct patient contact should receive a single dose.

During pertussis outbreaks, the subjects who have not received Tdap vaccine earlier should receive a single dose of Tdap vaccine if 2 years or more have elapsed from the last Td vaccination

Who should not receive the vaccine?

Those with a history of a serious allergic reaction (such as anaphylaxis) to any of the vaccine components. Those with a history of encephalopathy (e.g. coma or prolonged seizures) not attributable to an identifiable cause within 7 days of administration of a vaccine with pertussis components. People with the following conditions should discuss with their health care professional whether they should receive DTaP vaccine.

 Moderate or serious reaction after receiving DTP or DTaP in the past. Seizure or have a parent or sibling who has had a seizure. Brain problem that is unstable or getting worse. People who are moderately or severely ill should consult with their physician before receiving any vaccine.

VACCINATION SCHEDULE
A DTaP vaccine is given to most children at two, four, and six months of age. A fourth dose of DTaP is given between 15 and 18 months, and a fifth dose is given at age four to six years. Children younger than age seven who should not receive the pertussis vaccine should receive the DT (diphtheriatetanus) vaccine.

Between the ages of seven and nine, Tdapwhich contains the same amount of tetanus vaccine as DTaP or DT, but contains much less diphtheria toxoid is given to protect against tetanus, diphtheria and pertussis.

 At age 11-12 years, a booster shot of tetanus-diphtheriaacellular pertussis (Tdap) is needed. It should be given no later than 16 years of age. Every 10 years and thereafter, a booster of Td is needed to maintain protection against diphtheria and tetanus.

One booster dose of Tdap is recommended for adults to replace a Td booster. Every 10 years thereafter, a booster of Td is needed to maintain protection against diphtheria and tetanus

ADVERSE REACTIONS
Mild Problems (Common)
Fever, redness or swelling, soreness or tenderness fussiness, tiredness or poor appetite, vomiting.

Moderate Problems (Uncommon)

Seizure. Non-stop crying, high fever.

Severe Problems (Very Rare)

Serious allergic reaction. Long-term seizures, coma, or lowered consciousness. Permanent brain damage.

RECOMMENDED WHO CASE DEFINATIONS OF PERTUSSIS

Clinical case definition
A case diagnosed as pertussis by a physician or A person with a cough lasting at least two weeks with at least one of the following symptoms: Paroxysms (i.e. fits) of coughing Inspiratory whooping Post-tussive vomiting (i.e. vomiting immediately after coughing) without other apparent cause

 Criteria for laboratory confirmation Isolation of Bordetella pertussis or

Detection of genomic sequences by means of the polymerase chain reaction (PCR) or

Positive paired serology.

 Suspect CaseA clinical syndrome or illness consistent or compatible with pertussis and without other apparent cause such as:
Any acute cough illness with paroxysmal cough or inspiratory whoop. Any acute cough illness in a person who is a close contact to a patient with a confirmed or probable case. Any cough associated with apnea in an infant. Any acute cough illness lasting 7 days when there is a reported outbreak of pertussis in the community. Any acute cough illness with positive PCR results for B. pertussis that does not meet the clinical case definition.

Clinical case definition of pertussis for surveillance purposes

RESURGENCE OF BORDETELLA PERTUSSIS IN ADULTS

CAUSES FOR RESURGENCE

VACCINES OF BORDETELLA PERTUSSIS ?A CAUSE OF RESURGENCE . VNTR CHANGES AND MODIFICATION OF BACTERIAL GENOME. TRANSITION IN VACCINE. ADAPTATION AND WANING IMMUNITY. INCREASED DIAGNOSTIC EFFICACY.

VACCINES OF BORDETELLA PERTUSSIS ?A CAUSE OF RESURGENCE

Is polymorphism in pertactin and pertussis toxin driven by host immunity, or is it the result of random fixation due to genetic drift?

It is conceivable that the increase in fitness associated with nonvaccine types of pertactin and pertussis toxin in vaccinated populations is substantial enough to drive expansion of strains carrying these protein variants.

VNTR CHANGES AND MODIFICATION OF BACTERIAL GENOME

The combined MAST-MLVA profiles were used to perform clustering analyses of the Dutch B. pertussisstrains isolated before the introduction of the pertussis vaccine in 1953 and isolates obtained before and after the pertussis epidemics in the 1990s, a period in which neither vaccine formulation nor vaccine coverage have changed. The analysis showed that the profiles from strains predating the vaccination era were more diverse than those isolated in the 1990s and only distantly related to the recent strains

 There was a strong decrease in diversity in the genotypes of the B. pertussis strains during and after the epidemics in the 1990s, suggesting that these epidemics were caused by a limited number of strains (clonal expansion).

MLVA markers may not reveal causal relationships but can be helpful to signal clonal expansions and thus visualize the spread of a subgroup of the B. pertussis population with increased fitness, e.g., because B. pertussis is able to escape host immunity.

 Previous studies showed that antibodies against Prn1, present in the current vaccine, are less efficient in protecting against pertussis in animals challenged with the other Prn variants. Variation of the prn gene is caused by variation in the number and composition of the repeats in this virulence gene. Hence, this is an example of a VNTR within a virulence gene in which variation results in antigenic change and possible vaccine escape.

In biology, minimum spanning trees can easily be applied to multistate data such as MLVA, MAST, or MLST profiles.

MINIMUM SPANNING TREE

TRANSITION OF THE VACCINE

A Study done in Canada that shows that between July 1997 and April 1998, all provinces and territories in Canada switched from using a combination vaccine of diphtheria and tetanus toxoids, whole-cell pertussis, inactivated poliovirus and Haemophilus influenzae type b conjugate vaccine (DTwPIPVHib) to a combination vaccine containing acellular pertussis (DTaPIPV Hib). In this issue, Vickers and colleagues, using passive surveillance data from the Saskatoon Regional Health Authority in Saskatchewan for the period 19952005, report on their analysis of the effect of the change to the new vaccine on the incidence of pertussis among children less than 10 years of age. They found that, among infants (< 1 year old) and preschool children (aged 14 years), those who had received either the whole-cell pertussis vaccine or a combination of the wholecell and acellular pertussis vaccines had a lower incidence of pertussis than those who had received only the acellular vaccine

ADAPTATION AND WANING IMMUNITY

The Ptx promoter showed a relatively high degree of polymorphism, suggesting that fine tuning of Ptx production has adaptive value. Globally, ptxP1 and ptxP3 were the most prevalent ptxP alleles. The replacement of ptxP1 strains by ptxP3 strains in recent times is a global phenomenon because it has been observed in 11 countries representing 4 continents; Asia, Europe, and North and South America. When compared with ptxP1 strains, ptxP3 strains produced 1.62 times more Ptx.

 First, the high degree of polymorphism in the ptxP promoter indicates positive selection. Second, the increased Ptx production observed by ptxP3 strains provides a rationale for its emergence.

The antigenic divergence observed between vaccine strains and circulating strains may act synergistically. With the ptxP3 polymorphism by enhancing transmission by hosts primed by vaccination.
Pertussis among recently vaccinated children is rare, indicating that pathogen adaptation does not play a role unless immunity has waned

INCREASED DIAGNOSIS AND REPORTING

A recent study in Toronto, Canada, reports that a marked increase in pertussis incidence in 2006 was associated with a markedly increased volume of tests performed, primarily PCRbased assays. Increased press reports and scientific literature on the resurgence of pertussis, as well as reports of pertussis vaccine trials and subsequent licensure of low dose acellular pertussis vaccines for use in adults, may have also led to an increase in clinician awareness and reporting.

TAKE HOME MESSAGE

Pertussis is a respiratory infection which causes whooping cough Pertussis has its resurgence so it should be considered in case of chronic cough of >2 week duration. Typical symptoms are initially cold-like followed by a stage of rapid coughing and finally a recovery stage of coughing which can last for weeks or months. Diagnostics include a nasopharyngeal swab culture, DNA PCR, and ELISA test for antibodies. Macrolides are the primary antibiotic used against an pertussis infection. Vaccination is still the single best way to prevent whooping cough although not everyone will get an immune response and immunity wanes with time. This means that people can still get whooping cough if they have been vaccinated or if they have had previous infection.