**4.4. Recommendations for diagnosis testing with suspected pertussis**

and PCR has optimal sensitivity during the first 3 weeks of cough. As mentioned earlier, the

**Figure 1.** A—Regan low and Bordet Gengou culture media supplemented with 10% v/v sheep blood. B—Growth of

Extraction and purification of DNA is necessary to limit the action of inhibitors present in samples. There are home methods for the extraction of DNA, which are gradually being replaced by commercial methods. The latter are based on the use of ion exchange resins or magnetic separation using silica particles [46]. Neither of these methods is validated for the extraction of DNA from respiratory samples [46]. There are studies demonstrating that, in general, the different methods are suitable for the extraction of DNA from these samples. The PCR assays have evolved from conventional assays to real-time PCR and from singleplex to multiplex PCR. Conventional PCR employs two different sets of primers that are visualized on agarose gels. The most commonly used target sequences for *B*. *pertussis* DNA detection are the insertion sequence 481 (*IS*481) and the pertussis toxin promoter region. To detect *B. parapertussis*, primers that hybridize to the insertion sequence *IS*1001 are used. *IS* elements are generally present in multiple copies in genomes, offering excellent targets for highly sensitive PCR detection. *IS*481 and *IS*1001 occur in *B. pertussis* and *B. parapertussis* isolates obtained from humans at copy numbers of 253 and 22, respectively [47, 48]. *IS*481 is also present in *B. holmesii* isolates and in some isolates of *B. bronchiseptica.* These target sequences are also used in real-time PCR assays [49]. In fact, for simultaneous detection of *B*. *pertussis*, *B*. *parapertussis,* and *B*. *holmesii*, a combination of multiplex and singleplex real-time PCR assays targeting IS

It is recommended that PCR (conventional or real-time PCR) be used together with the culture. Cultivation of the etiological agent should be performed, especially when an outbreak is suspected.

Validation and harmonization of serologic methods are still necessary before they can be widely applied as diagnostic tools. Many of the problems associated with serodiagnosis, such

sample of choice is nasopharyngeal aspirate or nasopharyngeal swab.

*B. pertussis* in Regan Lowe and Bordet Gengou media.

16 Pertussis - Disease, Control and Challenges

elements and pertussis toxin sequence has been developed [49].

**4.3. Serodiagnosis**

In neonates and young infants, PCR and/or culture should be performed on nasopharyngeal samples as soon as possible post-onset of symptoms. These methodologies are also recommended in vaccinated children, adolescents, and adults with less than 2 weeks of coughing. For patients older than 11 years with coughing of less than 3 weeks, PCR and IgG-anti-PTx measurement should be performed. The measurement of IgG-anti-PTx is only meaningful for older children/adults, including parents and other household members.

In outbreak situations, PCR and culture should be performed from nasopharyngeal samples and IgG-anti-PTx should be measured in serum samples.

It is important to note here that the microbiological diagnosis for pertussis is more useful during the first 2 weeks of coughing and before starting the antibiotic treatment. PCR assays may effectively be used for pertussis diagnosis from 2–4 weeks of cough. On the other hand, the serological tests are most useful in 2–8 weeks after the onset of cough.
