Insertion sequences shared by Bordetella species and implications for the biological diagnosis of pertussis syndrome
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).
[Show abstract] [Hide abstract] ABSTRACT: Bordetella pertussis testing performed using real-time polymerase chain reaction (RT-PCR) is interpreted based on a cycle threshold (Ct) value. At Public Health Ontario Laboratories (PHOL), a Ct value <36 is reported as positive, and Ct values ≥36 and <40 are reported as indeterminate. PHOL reported indeterminate results to physicians and public health units until May 2012, after which these results were only reported to physicians. We investigated the association between Ct value and disease symptom and severity to examine the significance of indeterminate results clinically, epidemiologically and for public health reporting. B. pertussis positive and indeterminate RT-PCR results were linked to pertussis cases reported in the provincial Integrated Public Health Information System (iPHIS), using deterministic linkage. Patients with positive RT-PCR results had a lower median age of 10.8 years compared to 12.0 years for patients with indeterminate results (p = 0.24). Hospitalized patients had significantly lower Ct values than non-hospitalized patients (median Ct values of 20.7 vs. 31.6, p<0.001). The proportion of patients reporting the most indicative symptoms of pertussis did not differ between patients with positive vs. indeterminate RT-PCR results. Taking the most indicative symptoms of pertussis as the gold-standard, the positive predictive value of the RT-PCR test was 68.1%. RT-PCR test results should be interpreted in the context of the clinical symptoms, age, vaccination status, prevalence, and other factors. Further information on interpretation of indeterminate RT-PCR results may be needed, and the utility of reporting to public health practitioners should be re-evaluated.0Comments 1Citation
- "Interpretation of high Ct-value RT-PCR test results for B. pertussis can be challenging due to both the sensitivity of the test and the heterogeneous population that is being tested, ranging from highly symptomatic infants to mildly symptomatic adults with an epidemiological link to a pertussis outbreak. Age, vaccination or immunity status , the number of B. pertussis IS481 copies  and the timing of sample collection  are a few of the many factors that affect the test result, making it difficult to conclusively classify patients as infected, and complicating decisions relating to treatment and public health action. Our analysis found that 88.8% of laboratory-linked confirmed or probable pertussis cases were RT-PCR positive, similar or slightly higher than values reported by others with similar RT-PCR cut-off values [21,22]. "
[Show abstract] [Hide abstract] ABSTRACT: Resistance of Bordetella pertussis, the causative agent of pertussis, to erythromycin is rare. Recently, several Chinese isolates were found to be erythromycin-resistant. This study aimed to investigate the occurrence of pertussis in children suffering persistent cough and the prevalence of B. pertussis resistance to erythromycin in Xi'an, China. Three hundreds thirteen patients with suspected pertussis admitted in Xi'an Children's Hospital, from January 2012 through December 2013 were included and their nasopharyngeal (NP) swabs were taken for culture and PCRs (targeting IS481 and ptx-Pr). PCR-based sequencing was used to identify A2047G mutation of B. pertussis 23S rRNA directly from the NP samples. Sixteen (5.1%) and 168 (53.7%) patients were positive for culture and IS481 PCR. Of the 168 samples positive for IS481 PCR, 122 (72.6%) and 100 (59.5%) were positive for ptx-Pr and 23S rRNA PCRs, respectively. All culture-positive samples were also positive for the three PCRs. Fourteen (87.5%) of the 16 B. pertussis isolates were found to be resistant to erythromycin (MICs > 256 mg/L). All the 14 isolates were confirmed to have a homogeneous A2047G mutation of 23S rRNA. Of the 100 samples positive for 23S rRNA PCR, 85 (85.0%) were found to have the A2047G mutation by sequencing. Our results indicate that in Xi'an, China, pertussis remains endemic in young children, and the circulating B. pertussis strains are mostly erythromycin-resistant. This article is protected by copyright. All rights reserved.0Comments 7Citations
- "Indeed in our study, in addition to 122 patients who were confirmed as having pertussis by both PCRs, IS481 PCR detected 46 additional patients. IS481 has been detected in other Bordetella species; whether these 46 patients were infected by B. pertussis, B. bronchiseptica or B. holmesii remains to be shown . CMI It has been shown that some clinical NP samples can contain inhibitory substances and can inhibit PCR amplification . "
[Show abstract] [Hide abstract] 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.0Comments 5Citations
- "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. "