**4.3. Serodiagnosis**

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

*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 recommended. IgA and IgM assays lack adequate sensitivity and specificity.

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 of pertussis.
