A profile of the Simplexa™ Bordetella Direct assay for the detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs.

INTRODUCTION: Pertussis is a highly contagious respiratory infection caused by Bordetella pertussis and to minor extent B. parapertussis. Despite high vaccination coverage, epidemics persist worldwide. Laboratory testing with the capacity to support increasing demand and generate fast and accurate results is needed to promptly provide treatment to mitigate symptoms, prevent transmission, and thus impact infection control and disease surveillance. AREAS COVERED: This review will describe the features of the Simplexa™ Bordetella Direct Assay and compare this technology with other existing assays. Unmet needs and future directions will be discussed. EXPERT COMMENTARY: Resurgence of pertussis highlights the importance of reliable and accurate diagnosis. The Simplexa™ Bordetella Direct Assay provides an easy workflow, reduced hand-on time, less risk of contamination, and rapid turnaround time. The use of efficient molecular assays in routine clinical laboratory is valuable for increasing demand, improvement of infection control, and surveillance.

A novel multilocus variable-number tandem repeat analysis for Bordetella parapertussis.

Purpose. Human-adapted Bordetella parapertussis is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted B. parapertussis.Methodology. Four highly polymorphic variable number tandem repeat (VNTR) loci in the B. parapertussis genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of B. parapertussis.Results. Multiplex PCR-based MLVA characterized 53 B. parapertussis reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86-0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type.Conclusion. Our novel MLVA method has high discriminatory power for genotyping human B. parapertussis. Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks.

Molecular typing of Bordetella parapertussis isolates circulating in Pakistan.

Although a whole-cell pertussis vaccine was introduced in Pakistan in 1980, little is known about the pertussis prevalence and circulating strains in Pakistan. The aim of this study was to analyze Bordetella parapertussis isolates circulating between 2005 and 2009 in Pakistan and to compare them with those found in other countries during different periods. A total of 59 (7.35%) B. parapertussis isolates from 802 subjects (median age, 3 years) from Pakistan, with pertussis-like symptoms were investigated. We carried out genotyping and DNA microarray analyses on these isolates and compared them with some international isolates of B. parapertussis. We found that the allele for pertactin (prn) found in strains studied from Pakistan was identical to the predominant type found in Europe. We showed that B. parapertussis isolates circulating in Pakistan are part of the same pulsed-field gel electrophoresis group to those circulating in Finland during the period of 1982-2007. Finally, microarray analysis confirmed that the isolates collected in Pakistan, were quite similar to international strains. Overall, these results confirm that B. parapertussis is extremely monomorphic. The high isolation rate of B. parapertussis (7.35%) compared to Bordetella pertussis (0.5%) may suggest that the whole-cell vaccine used in Pakistan is effective against B. pertussis (0.5% infections detected), but much less so against B. parapertussis.

Novel multitarget real-time PCR assay for rapid detection of Bordetella species in clinical specimens.

A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.

Sensitivity and specificity of the World Health Organization pertussis clinical case definition.

OBJECTIVES: Bordetella pertussis continues to circulate even in countries with good childhood vaccination coverage. This study was undertaken to define the relationship between documented disease and the clinical criteria proposed by the World Health Organization (WHO). METHODS: Nasopharyngeal swab samples were collected from previously healthy 6-14-year-old school children in Tehran, presenting with persistent cough of at least 2- week duration. Specimens were examined for Bordetella pertussis and Bordetella parapertussis by culture and polymerase chain reaction (PCR). RESULTS: Out of 6601 students, 328 (5.0%) had been coughing for at least 2 weeks. Of these children with cough, 182 (55.5%) experienced whooping, 194 (59.1%) suffered a paroxysmal cough, and 73 (22.3%) had post-tussive vomiting. Twenty-one (6.4%) samples tested positive for B. pertussis and six (1.8%) for B. parapertussis by PCR. Culture of four (1.2%) specimens was positive for B. pertussis. In comparison to PCR, the sensitivity and the specificity of the WHO clinical criteria (year 2000) were 95.2% and 15.0%, respectively. CONCLUSIONS: Pertussis remains one of the etiologies of prolonged cough, even in communities with high immunization in children. The specificity of the WHO criteria is low in diagnosing pertussis compared with PCR.

Monitoring of Bordetella isolates circulating in Saint Petersburg, Russia between 2001 and 2009.

Bordetella isolates in the Saint Petersburg region have been monitored since 1998. Over the past ten years, concomitant with the increase in pertussis whole-cell vaccine coverage, the incidence of whooping cough has decreased. However, this decrease exists only for Bordetella pertussis infections, as the incidence of Bordetella parapertussis confirmed cases has remained stable, suggesting that pertussis-vaccine-induced immunity is not protective against parapertussis, as expected. B. pertussis and B. parapertussis clinical isolates were analyzed using serotyping, immunoblotting, pulsed-field gel electrophoresis of chromosomal DNA (after digestion with XbaI) and sequencing of virulence genes. The bacterial population is now similar to that observed in other European countries.

Detection and differentiation of Bordetella spp. by real-time PCR.

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/mul, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/mul, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.

Plasmid pBP136 from Bordetella pertussis represents an ancestral form of IncP-1beta plasmids without accessory mobile elements.

The complete 41,268 bp nucleotide sequence of the IncP-1beta plasmid pBP136 from the human pathogen Bordetella pertussis, the primary aetiological agent of whooping cough, was determined and analysed. This plasmid carried a total of 46 ORFs: 44 ORFs corresponding to the genes in the conserved IncP-1beta backbone, and 2 ORFs similar to the XF1596 and XF1597 genes with unknown function of the plant pathogen Xylella fastidiosa. Interestingly, pBP136 had no accessory genes carrying genetic traits such as antibiotic or mercury resistance and/or xenobiotic degradation. Moreover, pBP136 had only two of the kle genes (kleAE) that have been reported to be important for the stability of IncP-1 plasmid in Pseudomonas aeruginosa. Phylogenetic analysis of the Kle proteins revealed that the KleA and KleE of pBP136 were phylogenetically distant from those of the present IncP-1 plasmids. In contrast, IncC1 and KorC, encoded upstream and downstream of the kle genes respectively, and the replication-initiation protein, TrfA, were closely related to those of the IncP-1beta 'R751 group'. These results suggest that (i) pBP136 without any apparent accessory genes diverged early from an ancestor of the present IncP-1beta plasmids, especially those of the R751 group, and (ii) the kle genes might be incorporated independently into the backbone region of the IncP-1 plasmids for their stable maintenance in various host cells.

[Genetic diversity analysis of isolates belonging to Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species]. / Zastosowanie metody aflp do oceny genetycznego podobienistwa szczepow Bordetella pertussis, Bordetella parapertussis oraz Bordetella bronchiseptica.

In this study, Amplified Fragment Length Polymorphism (AFLP) method was used to track differences among human and animal isolates of B. pertussis, B. parapertussis and B. bronchiseptica species. One hundred and sixty representative strains of these species orginated from international and Polish bacterial collections were genotyped according to AFLP involving EcoRI/Msel and SpeI/ApaI restriction/ligation/amplification procedures. This study has confirmed high potential AFLP SpeI/ApaI procedure for intra-species differentiation of B. pertussis and B. bronchiseptica strains. Both AFLP EcoRI/MseI and SpeI/ApaI procedures have been found to be useful for species-specific classification in case of B. pertussis strains. In case of B. bronchiseptica or B. parapertussis species-specific classification, SpeI/ApaI procedure has been found more precise than EcoRI/MseI one.

Differential detection of B. pertussis from B. parapertussis using a polymerase chain reaction (PCR) in presence of SYBR green1 and amplicon melting analysis.

Nucleic acid (DNA) from control stock strains of B. pertussis and B. parapertussis (B. pertussis strain # 9797 and B. parapertussis strain # 15234 from ATCC) was amplified by polymerase chain reaction (PCR) targeting pertussis toxin (PT) promotor region, in presence of SYBR green1 a dye that fluoresces on binding specifically to double stranded DNA; and fluorescent melting profile of amplicon (amplified DNA) was studied. Amplicon of B. pertussis and B. parapertussis generated distinctly different melting bands with melting temperature (Tm) at 89.8 and 91.7 degrees C, respectively. Melting profile and Tm of each randomly selected isolates of B. pertussis and B. parapertussis was identical to that of respective control strains. Distinct difference in Tm between B. pertussis and B. parapertussis specific amplicons allowed differential detection of the two Bordetella species based on a single PCR product. The amplified product of serial diluted control stock of bacteria was analyzed by both agarose gel electrophoresis and melting profile analysis. The analytical sensitivity of detection (1-10 CFU equivalent in the tested volume) by melting profile and Tm analysis was in agreement with that obtained by agarose gel analysis.