SPECIMEN COLLECTION, TRANSPORT, AND PROCESSING

Confirming the diagnosis of pertussis is challenging. Culture, which is most sensitive early in the illness, has been the traditional diagnostic standard for pertussis and shows nearly 100% specificity but varied sensitivity. Organisms may become undetectable by culture 2 weeks after the start of paroxysms. Nasopharyngeal aspirates or a nasopharyngeal swab (calcium-alginate or Dacron on a wire handle) are acceptable specimens, because B. pertussis colonizes the ciliated epithelial cells of upper respiratory tract. Calcium-alginate swabs with aluminum shafts are not recommended for PCR, because they may inhibit the polymerase enzyme in PCR detection. In addition, cotton swabs may be inhibitory to specimen growth and are not recommended. Specimens obtained from the throat, sputum, or anterior nose are unacceptable, because these sites are not lined with ciliated epithelium. For collection, the swab is bent to conform to the nasal passage and held against the posterior aspect of the nasopharynx. If coughing does not occur, another swab is inserted into the other nostril to initiate the cough. The swab is left in place during the entire cough, removed, and immediately inoculated onto a selective medium at the bedside (Table 1).

Table1. Examples of Selective Media for Primary Isolation  of B. pertussis and B. parapertussis

Transport time is critical. A fluid transport medium may be used for swabs but must be held for less than 2 hours. Half-strength Regan-Lowe agar enhances recovery when used as a transport and enrichment medium. Cold casein hydrolysate medium and casamino acid broth (available commercially) have proved to be effective transport media, particularly for preparation of slides for direct fluorescent antibody staining. Dry swabs may be transported in ambient air for PCR testing.

DIRECT DETECTION METHODS

A DFA stain using polyclonal antibodies against B. pertussis and B. parapertussis is commercially available for detection of B. pertussis in smears made from nasopharyngeal (NP) material (Becton Dickinson, Sparks, Maryland); an NP specimen that is DFA positive for B. pertussis is shown in Figure 1. Although rapid, this DFA stain has limited sensitivity and variable specificity; therefore, the DFA test should always be used in conjunction with culture. DFA monoclonal reagent is also commercially available with two antisera with different fluorophores to detect B. pertussis and B. parapertussis (Accu-Mab, Altachem Pharma, Edmonton, Canada).

Fig1. Immunofluorescence stains of Bordetella pertussis used for identification. 

Because of the limitations associated with culture and serologic diagnostic methods, significant effort has been put into developing nucleic acid amplification methods. Most diagnostic studies use direct detection of B. pertussis and B. parapertussis by various PCR procedures, including real-time PCR. These assays have a diagnostic sensitivity at least comparable (and in most cases superior) to that of culture. A word of caution: Positive results have been obtained with samples containing B. holmesii and B. bronchiseptica  depending on the sequence targeted in conventional and real-time PCR assays. Most laboratories use transposon insertion sequences IS481 for B. pertussis and IS1001 for B. parapertussis. However, strains of B. holmesii, B. parapertussis, and B. bronchiseptica that carry IS481 have been identified; therefore, careful interpretation of results and correlation with the clinical presentation are required. Additional PCR assays are available for the detection of the pertussis toxin, fimbriae, pertactin and a porin gene. However, because these are single-copy genes and not multicopy insertion sequences, assay sensitivity is reduced. Nasopharyngeal swabs (rayon or Dacron swabs on plastic shafts) and aspirates are the two types of samples primarily used for pertussis PCR; calcium-alginate swabs are unacceptable, as previously mentioned, because these inhibit PCR based detection.

CULTIVATION

Plates are incubated at 35°C in a humidified atmosphere without elevated carbon dioxide for up to 12 days. Most isolates are detected in 3 to 7 days; B. parapertussis appears in 2 to 3 days. Colony morphology is not distinct for the identification of other Bordetella spp.

Regan-Lowe agar, Bordet-Gengou agar, and Stainer Scholte synthetic medium are suitable culture media. Regan-Lowe agar contains beef extract, starch, casein digest, and charcoal supplemented with horse blood. Bordet-Gengou agar is a potato fusion base containing glycerol and either sheep or horse blood. Most media contain cephalexin as an additive for suppression of contaminating organisms. Young colonies of B. pertussis and B. parapertussis are small and shiny, resembling mercury drops; colonies become whitish gray with age (Figure1).

Fig2. Growth of Bordetella pertussis on Regan-Lowe agar. 

Sensitivity of culture approaches 100% in the best of hands and depends on the stage of illness at the time of specimen collection, the technique used for specimen collection, specimen adequacy and transport, and culture conditions.

APPROACH TO IDENTIFICATION

A Gram stain of the organism reveals minute, faintly staining coccobacilli singly or in pairs (Figure 3). Use of a 2-minute safranin “O” counterstain or a 0.2% aqueous basic fuchsin counterstain enhances their visibility. Bordetella spp. characteristics are presented in Table 2. The DFA reagent is used to presumptively identify organisms. Whole-cell agglutination reactions in specific antiserum can be used for species identification.

Table2. Characteristics That Differentiate Bordetella spp.

Fig3. Typical Gram stain appearance of Bordetella pertussis. 

SERODIAGNOSIS

Although several serologic tests are available for the diagnosis of pertussis, including agglutination, complement fixation, and enzyme immunoassay, no single method can be recommended for serologic diagnosis at this time.

The current most reliable serologic test available for diagnosis is an anti-PT (antibody to pertussis toxin) enzyme-linked immunosorbent assay (ELISA) that has been used with acute and paired convalescent sera successfully in older children, adolescents, and adults. A titer greater than 100 to 125 IU/mL has been reported as a reliable indicator of exposure of patients to PT-producing bacteria.

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مواضيع ذات صلة

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