**4.2. PCR assays**

Molecular diagnosis methods based on PCR are 2–6 times more sensitive than culture. The sensitivity of the PCR decreases with the time of evolution of the pathology as ocurrs with the microbiological tests. After the 4th week of cough, the amount of bacterial DNA diminishes,

as the interference of previous vaccinations or previous infections, cross-reactivity with other *Bordetella* species or perhaps other bacteria, and the variable response to *B*. *pertussis* antigens should still be overcome. However, in some countries, pertussis serology is currently used for

Update on Epidemiology, Diagnosis, and Treatment of Pertussis

http://dx.doi.org/10.5772/intechopen.72847

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*Bordetella pertussis*-specific antibodies can be detected by enzyme-linked immunosorbent assays (ELISAs) or multiplex immunoassays. Assays use purified or mixed antigens, and only pertussis toxin (PTx) is specific for *B. pertussis*. Cross-reactivity with other microbial antigens from other *Bordetella* species could be detected when antibodies against filamentous hemagglutinin (anti-FHA), pertactin (anti-PRN), fimbriae (anti-FIM), and adenylate cyclase (anti-ACT) are measured and, for this reason, the measurement of these antibodies is not recommended for the diagnosis of pertussis. The evaluation of the titers of such antibodies may be used in specific studies [52]. For pertussis diagnosis, only IgG anti-PTx antibody titer evaluation is recom-

Dual-sample serology based on ≥100% increase in antibody concentration or on ≥50% decrease in antibody concentration is a sensitive and specific method for serological diagnosis [53]. In clinical practice, diagnosis is mostly based on single-sample serology using a single or a more continuous cutoff. The optimal timing for specimen collection is 2–8 weeks following cough onset. For ELISA assays, it is recommended to use a standard serum from WHO [54]. Due to high levels of vaccine-induced IgG-Ptx, single-serum diagnosis is not reliable for 1–3 years after vaccination with Ptx-containing vaccines. If the IgG-Ptx level is below the chosen cutoff, the diagnosis of pertussis can be neither confirmed nor denied, and a second serum obtained at least 2 weeks later and 4–6 weeks after the onset of disease should be investigated. Increases of threefold in paired sera or any increase to a value above the cutoff or absolute values in single sera can then be considered to confirm the diagnosis

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

In outbreak situations, PCR and culture should be performed from nasopharyngeal 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

diagnostic purposes [50], in particular, during outbreaks [51].

mended. IgA and IgM assays lack adequate sensitivity and specificity.

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

and IgG-anti-PTx should be measured in serum samples.

serological tests are most useful in 2–8 weeks after the onset of cough.

of pertussis.

members.

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

and PCR has optimal sensitivity during the first 3 weeks of cough. As mentioned earlier, the sample of choice is nasopharyngeal aspirate or nasopharyngeal swab.

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

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.
