Differential diagnosis by RT-PCR of Bordetella bronchiseptica in a child without previous pathologic antecedents suffering whooping cough

The genus Bordetella includes various species that can affect human beings.¹ Among them, B. pertussis stands out for its clinical-epidemiological relevance.² However, other species such as B. parapertussis, B. holmesii¹ and, occasionally, B. bronchiseptica^3,4 can cause similar whooping cough symptoms.

The objective of this study was to describe a case of B. bronchiseptica infection and the strategy used for its microbiological diagnosis. A 21-month-old male, with no clinical history of chronic respiratory diseases or immunological abnormalities, who attended the emergency department with a paroxysmal cough accompanied by vomiting, which had started nine days previously, with no apnoea, but accompanied by stridor. The patient was correctly immunised for his age against whooping cough, with three doses of diphtheria vaccine, tetanus vaccine and acellular pertussis vaccine administered at 2, 4 and 11 months. The family lived with a dog that had not shown signs of disease.

In light of these symptoms compatible with whooping cough, nasopharyngeal lavage samples were obtained, and an initial dose of azithromycin at 10 mg/kg/day followed by home treatment with 5 mg/kg/day for four days was prescribed. The patient progressed favourably, with no complications. The sample of nasopharyngeal lavage was studied using a commercial multiplex real-time polymerase chain reaction (RT-PCR) technique (Smart Bp/Bpp, Cepheid AB, Sweden), based on the detection of the insertion sequences IS481 and IS1001. According to the manufacturer's instructions, the results were classified presumptively as positive for both B. pertussis and B. parapertussis. The sample was subsequently processed using five independent RT-PCR assays against different markers: IS481, IS1001, promoter region of the pertussis toxin gene (BPTP), B. pertussis porin protein gene (BPTD_0837) and the insertion sequence similar to IS1001 of B. holmesii (hIS1001). The results obtained (IS481 confirmed as positive, IS1001 confirmed as positive, BPTP positive, BPTD_0837 negative and hIS1001 negative) contributed to an identification as B. bronchiseptica in accordance with the algorithm described in Table 1.

Table 1.

Algorithm of multimarker RT-PCR assays for the identification of the main species of Bordetella spp.

Interpretation	Result of the assays
	IS482	BPTP	BPTD_0837	IS1001	hIS1001
B. pertussis	+	+	+	−	−
B. parapertussis	−	−	−	+	−
B. holmesii	+	−	−	−	+
B. bronchiseptica	±	+	−	±	−

While B. bronchiseptica grows in common media such as MacConkey agar, the culture of B. pertussis is problematic and lacks sensitivity. For this reason, the diagnosis of whooping cough is currently based on PCR techniques on nasopharyngeal samples, using several targets such as the insertion segments IS481 and IS1001, used to identify B. pertussis and B. parapertussis, respectively, as probable.^5,6 Nevertheless, these sequences are not absolutely specific to these species.⁶ The sequence IS481 can be found along with B. pertussis in B. holmesii and in some strains of B. bronchiseptica.⁶ Likewise, the sequence IS1001 can be present, in addition to B. parapertussis, in B. bronchiseptica.⁶ This sequence also shows great similarity with hIS1001, present in B. holmesii.⁵ A simultaneously positive PCR result for IS481 and S1001 could initially be considered a co-infection by different species of Bordetella.⁶ In these situations, the use of other amplification targets may clarify the diagnosis. Both B. pertussis and B. bronchiseptica are positive for BPTP.⁷ However, the gene BPTD_0837 is specific to B. pertussis and does not show cross-reactivity with other Bordetella species.⁸ Other authors have used two structural genes of flagellin as markers, one common for B. bronchiseptica/B. parapertussis (Bb/Bpp-Fla) and another distinctive one for B. parapertussis (Bpp2-Fla).⁹

Among the limitations of this study, it should be noted that a bacterial culture was not carried out, a differential virological diagnosis was not carried out, nor was the sequencing of amplification products carried out to confirm the result definitively.

B. bronchiseptica is considered an opportunistic microorganism which can, in particular, infect the respiratory tract of patients with immunosuppression or cystic fibrosis.^3,4B. bronchiseptica causes infections in different mammals¹ and its transmission has been related to living with pets.⁴ The prevalence of colonisation in healthy dogs seems to be important.¹⁰ Although in this case we assumed that B. bronchiseptica was the probable cause of the symptoms of whooping cough according to its molecular detection, it is not possible to ensure that it was not merely a commensal bacterium. Nevertheless, this microorganism should be taken into account in the differential diagnosis of B. pertussis and B parapertussis when commercial RT-PCR techniques are used.