Bordetella pertussis and Bordetella parapertussis - Culture; IgM, IgA and IgG antibodies; Molecular diagnosis (PCR)


Information 22-07-2018.


Whooping cough or pertussis is an acute, highly contagious respiratory disease caused by the gram-negative coccibacillous Bordetella pertussis. It is characterized by a paroxysmal cough, although the symptoms can be atypical, and even fatal in babies. The whooping cough in adolescents and adults tends to be milder and presents less severe symptoms, but it is still of special concern due to the associated risk of infection. Similar respiratory symptoms, although usually milder, can be caused by other members of the genus Bordetella: B. parapertussis, B. holmesii and B. bronchiseptica. Despite the existing vaccination programs, the number of cases of whooping cough continues to increase in Europe and North America, considering it a re-emerging disease.

Bordetella species are classified in the family Alcaligenaceae and comprise 10 genetically distinct species. Although B. pertussis has been classically identified as the sole agent responsible for pertussis, it has been shown that other Bordetella species infect both humans and animals. In humans, B. parapertussis, B. holmesii and, to a lesser degree B. bronchiseptica, can cause respiratory diseases with nonspecific symptoms similar to those caused by B. pertussis, but milder. Among these bacteria, B. parapertussis is the most widespread and the one that has been studied most widely. B. bronchiseptica, B. parapertussis and B. holmesii can infect a wide range of mammals, including humans, while B. pertussis is a human-specific pathogen.

The bacteria of the genus Bordetella are fastidious with special requirements, difficult to isolate in culture. They grow slowly in medium supplemented with blood and in synthetic media containing appropriate growth factors, such as nicotinamide and sulfur derivatives. They are small Gram-negative coccobacilli, strictly aerobic and with an optimum growth temperature of 35 to 37°C. B. pertussis can be differentiated from other Bordetella species according to its growth and biochemical characteristics. B. pertussis is a difficult species to grow, immobile, hemolytic, catalase and positive oxidase. In contrast, B. parapertussis is easier to grow, oxidase negative and urease positive, and produces a brown pigment in media with heart infusion, Regan-Lowe or Mueller-Hinton agar.

Whooping cough is the result of a coordinated interaction of several virulence factors of B. pertussis, including toxins such as pertussis toxin (PT), adenylate cyclase (AC) toxin, dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT). ). Other factors that influence the virulence of B. pertussis are superficial structures, such as filamentous haemagglutinin (FHA), fimbriae (FIM), pertactin (PRN), type III secretion system, lipopolysaccharide (LPS) and metabolic proteins (BrkA, BapC and BatB). All these virulence factors, such as toxins and surface molecules, are under the control of a single genetic locus: Bordetella Virulence Regulon AS (bvgAS). The bvgAS genes positively control the expression of several virulence factors, playing a fundamental role in the pathogenicity of pertussis. Although the other pathogenic Bordetella species also express a large number of proteins responsible for the colonization and virulence of the bacteria, important differences can be observed between them. B. parapertussis does not produce the fimbrial proteins (FIM 2 and 3) nor the pertussis toxin, an important ADP-ribosylase toxin produced by B. pertussis. On the other hand, B. parapertussis produces an LPS with an O antigen, whereas B. pertussis produces an LPS without O antigen due to the presence of elements of insertion sequences (IS) in the corresponding LPS locus.

Whooping cough is a highly contagious infectious disease. B.pertussis is transmitted from person to person through aerosolized droplets and reaches the upper respiratory tract. The virulence mechanisms of B. pertussis consist of a cascade of events initiated by the adherence of bacteria by filamentous hemagglutinin and fimbrias to the tracheal and pulmonary epithelium as an essential primary step. Once adherence takes place, B.pertussis cells multiply locally, resist host defense mechanisms (such as mucociliary clearance, antimicrobial peptides, and inflammatory cells) and cause local damage to the upper and lower airways with systemic manifestations. The severity of the symptoms depends on several factors, including the age of the patient, the intensity of the immune response and the degree of systemic spread. The clinical symptoms of whooping cough due to B. pertussis include four phases: an incubation period lasting 1 week, a second with atypical symptoms (catarrhal phase) that also lasts 1 week, a phase of 3 to 6 weeks where the cough appears typical paroxysm (a series of 5 to 30 coughs during a single expiration) that increases at night and is often associated with lymphocytosis and vomiting and, finally, the convalescent phase that lasts from 3 to 12 weeks. Whooping cough due to B. parapertussis is not clinically distinguishable from disease due to B. pertussis, but has a shorter duration.

The clinical presentation of whooping cough in adolescents and adults previously immunized or infected is atypical and often asymptomatic, with persistent cough being the main symptom. The infection is more severe in babies who are too young to be vaccinated or who are not yet fully immunized. In infants, bacteria descend from the upper respiratory tract to the lower respiratory tract and, through an unclear mechanism, produce necrotizing bronchitis, diffuse alveolar damage, intraalveolar hemorrhage, fibrinous edema, alveolar infiltrates rich in macrophages, lymphangiectasia, neutrophilic bronchopneumonia and fibrin thrombusi. In more severe cases, these pathological events can lead to pulmonary hypertension, respiratory failure and even death. Pulmonary hypertension develops as an indirect effect of PT through the induction of lymphocytosis (hyperleukocytosis), in which the total white blood cell count may exceed 1 × 105 cells/mm3. These extremely high white blood cell counts produce aggregations of lymphocytes in the pulmonary vasculature that result in an increase in pulmonary vascular resistance. Hyperleukocytosis correlates directly with the severity of the disease and with the risk of developing fulminating pertussis, a combination of rapidly evolving pneumonia, severe pulmonary hypertension, cardiopulmonary failure and neurological involvement that is more common in infants under 1 year of age and in 80% of cases lead to death.

Whooping cough is a disease preventable by vaccination. However, despite the massive vaccination campaigns carried out worldwide for more than 50 years, pertussis remains endemic worldwide, its incidence is increasing in developed countries and remains a problem of clinical importance . Current pertussis vaccines include whole cell vaccines and acellular vaccines (in combination with diphtheria and tetanus toxoids). However, pertussis vaccines do not provide cross protection against these related Bordetella species. Several studies show that none of the vaccines available provides protection against B. parapertussis. The lack of cross protection was attributed mainly to the presence of the O antigen on the surface of B. parapertussis, which blocks the access of antibodies to vaccine antigens common to both species. Epidemiological studies revealed that B. parapertussis infections are globally more frequent than expected, being responsible for a significant number of cases, mainly in vaccinated populations.

The diagnosis of whooping cough can be especially difficult in children under 1 year of age during the winter season, when other pathogens circulate. According to the Centers for Disease Control and Prevention (CDC), if a case of whooping cough is suspected that matches clinical symptoms, it must be confirmed by specific laboratory tests. The highest sensitivity is obtained by combining culture, polymerase chain reaction (PCR) and serological tests. In addition, the differentiation of B. pertussis from other species is an important aspect for the treatment of the infection and the evaluation of the efficacy of the vaccines. The isolation of B. pertussis is the reference standard (Gold standard) for the diagnosis of whooping cough. The success of the culture depends largely on the collection of samples and appropriate laboratory techniques, the age and immune status of the patient. The success of the culture is high with unvaccinated infants, but low in older patients, immunized and partially treated. As the culture is difficult and lacks sensitivity, the diagnosis of pertussis involves the use of molecular techniques (PCR), much more sensitive than bacterial culture. Insertion sequence (IS) segments are often used as targets for PCR, particularly IS481 and IS1001 for the detection of B. pertussis and B. parapertussis, respectively. IS are present in numerous copies in bacterial genomes, so they increase the sensitivity of the technique, but they are not species-specific. IS481 is found in B. pertussis, B. holmesii and some B. bronchiseptica, while IS1001 is found in B. parapertussis and some B. bronchiseptica. In IVAMI, a triple PCR is performed by amplifying these two IS sequences, and the promoter region of the pertussis toxin, specific for B. pertussis. Finally, serology is usually used to confirm retrospective infection with B. pertussis or B. parapertussis in adolescents or adults who cough for more than 3 weeks. For the diagnosis of B. pertussis, ELISA techniques are recommended to quantify the levels of antibodies against the pertussis toxin. Serology has some limitations since it cannot distinguish between immunological responses induced by the vaccine and infection, symptomatic or asymptomatic.

Tests offered by IVAMI:

  • Diagnosis of B. pertussis and B.parapertussis by isolation in culture.
  • Diagnosis of B. pertussis and B.parapertussis by molecular methods (PCR).
  • Diagnosis of B. pertussis IgA, IgM and IgG antibodies and B. parapertussis IgM and IgG antibodies.

Recommended sample:

  • Culture and/or molecular diagnosis: swabs and nasopharyngeal aspirates.
  • Antibodies: serum.

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days.
  • Frozen for more than 2 days.

Delivery of results:

  • Diagnosis of B. pertussis and B.parapertussis by isolation in culture: 5 to 7 days.
  • Diagnosis of B. pertussis and B.parapertussis by molecular methods (PCR): 24 to 48 hours.
  • Diagnosis of B. pertussis IgA, IgM and IgG antiboidies and B. parapertussis IgM and IgG antibodies: 5 days.

Cost of the test:

  • Diagnosis of B. pertussis and B.parapertussis by isolation in culture: Consult
  • Diagnosis of B. pertussis and B.parapertussis by molecular methods (PCR): Consult
  • Diagnosis of B. pertussis IgA, IgM and IgG antibodies and B. parapertussis IgM and IgG antbodies: Consult