Insertion sequences shared by Bordetella species and implications for the biological diagnosis of pertussis syndrome
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technicka 5, 166 28, Prague 6, Czech Republic. European Journal of Clinical Microbiology
(Impact Factor: 2.67).
08/2012; 32(1). DOI: 10.1007/s10096-012-1718-3
The molecular diagnosis of pertussis and parapertussis syndromes is based on the detection of insertion sequences (IS) 481 and 1001, respectively. However, these IS are also detected in the genomes of various Bordetella species, such that they are not specific for either B. pertussis or B. parapertussis. Therefore, we screened the genome of recently circulating isolates of Bordetella species to compare the prevalence of IS481, IS1001 and, also IS1002 with previously published data and to sequence all IS detected. We also investigated whether the numbers of IS481 and IS1001 copies vary in recently circulating isolates of the different Bordetella species. We used the polymerase chain reaction (PCR) method for screening the genome of circulating isolates and to prepare the fragments for sequencing. We used Southern blotting and quantitative real-time PCR for quantification of the numbers of IS. We found no significant diversity in the sequences of the IS harboured in the genomes of the Bordetella isolates screened, except for a 71-nucleotide deletion from IS1002 in B. bronchiseptica. The IS copy numbers in the genome of recently circulating isolates were similar to those in reference strains. Our results confirm that biological diagnosis targeting the IS481 and IS1001 elements are not specific and detect the species B. pertussis, B. holmesii and B. bronchiseptica (IS481), and B. parapertussis and B. bronchiseptica (IS1001).
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Available from: Nicole Guiso
- "This 2-step diagnostic method is expected to be useful for samples that are positive for IS1001 or positive for IS481 and negative for ptxA-Pr and hIS1001, as it will confirm the presence of either B. parapertussis or B. bronchiseptica DNA (Table 3). However, cases of coinfection with B. parapertussis and B. bronchiseptica isolates not carrying IS in their genome or carrying IS1001 would be problematic (Tizolova et al., 2013): B. bronchiseptica would not be detected in the samples. However, no such case of co-infection has ever been reported. "
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ABSTRACT: Bordetella parapertussis is a causative agent of whooping cough in humans and B. bronchiseptica is causing wide variety of respiratory infections in mammals, including humans. Specific diagnostic tests are not currently available. Our first objective was to develop a real-time PCR test for the specific detection of B. bronchiseptica based on the previously described end-point PCR, targeting an intergenomic sequence of the fla gene locus but it has not been reached. However, there is cross-reactivity between B. parapertussis and B. bronchiseptica. Therefore, the targeted region of several clinical isolates of both species was sequenced and alignment of the sequences allowed the development of a two-step real-time PCR assay. The first PCR assay detected the DNA of all clinical isolates of both B. bronchiseptica and B. parapertussis tested. The second PCR assay detected only the DNA of B. parapertussis clinical isolates, thereby allowing discrimination between B. parapertussis and B. bronchiseptica.
Diagnostic microbiology and infectious disease 04/2014; 78(4). DOI:10.1016/j.diagmicrobio.2013.12.020 · 2.46 Impact Factor
Available from: Cristiana Cerasella Dragomirescu
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ABSTRACT: The incidence of whooping cough in Romania is substantially underestimated, and, as noted by the health authorities, this is mostly due to the lack of both awareness and biological diagnosis. We conducted a one year study in Bucharest in order to assess the circulation of Bordetella pertussis, the main etiological agent of whooping cough. Fifty-one subjects suspected of whooping cough were enrolled. Culture, real-time PCR and ELISA were used for laboratory diagnosis. Whooping cough patients (63%) were distributed among all age groups and most were unvaccinated, incompletely vaccinated or had been vaccinated more than five years previously. Bordetella holmesii DNA was detected in 22% of the bordetellosis cases; these patients included adults, teenagers and surprisingly, young children. B. pertussis isolates were similar to the clinical isolates currently circulating elsewhere in Europe. One isolate does not express pertactin, an antigen included in some acellular pertussis vaccines.
Diagnostic microbiology and infectious disease 01/2013; 78(3). DOI:10.1016/j.diagmicrobio.2013.09.017 · 2.46 Impact Factor
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ABSTRACT: Insertion sequences IS481 and IS1001 are targets for molecular detection of respectively Bordetella pertussis and Bordetella parapertussis. There is a raising concern about specificity of these targets due to sequence similarity with Bordetella holmesii and Bordetella bronchiseptica. The likelihood of false (para)pertussis diagnoses should be correlated with the prevalence of these organisms in the respiratory tract (RT). From October 2010 until September 2011, 2,207 RT samples were submitted to the Belgian reference laboratory for pertussis diagnosis. End-point IS481/IS 1001 PCR and culture were performed for B. pertussis and B. parapertussis. We developed a sensitive culture method followed by screening with matrix-assisted laser desorption/ionisation- time of flight mass spectrometry (MALDI-TOF MS) to look for B. holmesii and B. bronchiseptica in our samples,. Only one B. bronchiseptica and no B. holmesii were detected in RT samples from Belgian patients with pertussis-like symptoms.
Acta clinica Belgica 09/2013; 68(5):341-8. DOI:10.2143/ACB.3341 · 0.59 Impact Factor
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