Nasopharyngeal aspirates are the specimen of choice for dem-onstration of bacteria by microscopy and culture. These sam-ples should be collected during the first stage or early in the second stage of disease, because the organisms are most abun-dant in the respiratory secretions during these stages of dis-ease. Also, the specimen for culture should be collected before administration of antibiotics. The culture becomes negative 5 days after treatment with antibiotics. The specimens are usu-ally collected by using either calcium alginate or Dacron swabs but not by cotton swabs because the growth of bacteria is inhib-ited by certain fatty acids in the cotton, which are toxic to B. per-tussis. The specimens for culture are collected by the followingmethods: (a) the pernasal swab, (b) cough plate method, and (c) West’s postnasal swab.
The pernasal swab: In this method, a sterile swab on a flexiblewire is passed gently along the floor of the nasal cavity, and the mucus and pus is collected by the swab.
Cough plate method: In this method, specimens are directlycoughed out by the patient on a culture medium, during a bout of spontaneous or induced coughing in an infected child. The culture plate is held 10–15 cm in front of the patient’s mouth. During the process of coughing by children, infected nasopharyngeal secretions are directly deposited on the medium.
West’s postnasal swab: The West’s postnasal swab is usu-ally employed to collect posterior pharyngeal wall secretions through oral cavity. Contamination with saliva should be avoided for better results. The swab containing the mucus and pus is inoculated immediately on freshly prepared medium, such as Bordet–Gengou or charcoal horse blood agar, at the patient’s bedside. In case of delay, they are transported in 0.2–0.5 mL casamino acid solution (pH 7.2) or in modified Stuart’s transport medium or Regan–Lowe transport medium.
The transport media are transported immediately to labora-tory for processing, because even in these media the bacteria cannot survive for long time.
Microscopic diagnosis is made by a direct fluorescent antibody (DFA) technique for demonstration of B. pertussis in respiratory secretions. In this method, a smear is made from a specimen on a glass slide, air dried, and heat fixed. This smear is then stained with fluorescent labeled antibody against B. pertussis. DFA is a rapid and sensitive method. This method is positive in more than 75% of cases of whooping cough. This technique, however, may show occasional positive reaction in other bacterial infections.
The specimens are cultured on freshly prepared Bordet–Gengou medium or charcoal horse blood agar medium and incubated in moist environment at 35ºC. Incubation even up to 7 days is required, because the colonies are observed only after 3 or more days of incubation.
B. pertussis is identified by several characters:
Indirect hemagglutination and ELISA are frequently used to demonstrate IgG and IgA antibodies against FHA and IgG antibodies against pertussis toxin in the patient’s sera. A significant rise in the titer of antibodies between acute and convalescent serum in paired sera samples by these tests is suggestive of recent infection. ELISA for demonstration of specific secretory IgA antibodies in nasopharyngeal secretions is also useful, especially in culture-negative cases.