Reemergence of Bordetella parapertussis, United States, 2019-2023.

To determine changes in Bordetella pertussis and B. parapertussis detection rates, we analyzed 1.43 million respiratory multiplex PCR test results from US facilities from 2019 through mid-2023. From mid-2022 through mid-2023, Bordetella spp. detection increased 8.5-fold; 95% of detections were B. parapertussis. While B. parapertussis rates increased, B. pertussis rates decreased.

Pertussis outbreak in children hospitalized in Rabat (Morocco).

INTRODUCTION: Cyclical pertussis epidemics primarily affect young infants. This study aims to estimate pertussis prevalence during the ongoing 2023 outbreak at our institution, focusing on affected age groups and clinical presentations. MATERIEL AND METHODS: This retrospective study includes patients admitted to Rabat University Hospital Center from 1st January 2021 to 30th June 2023. Symptomatic patients underwent Multiplex Respiratory Panel PCR testing for respiratory infections. The analysis included cases where RT-PCR identified Bordetella spp., with data analysed using SPSS 15.0. RESULTS: Pertussis cases sharply increased from December 2022, constituting 85.4 % of positive samples. Most cases (78.2 %) occurred in infants under 3 months, presenting symptoms such as coughing (94.5 %) and dyspnoea (94.5 %). Pertussis was suspected in 60 % of RT-PCR confirmed cases. B. pertussis DNA was identified in 81.8 % of cases and B. parapertussis DNA in 18.2 % of cases. CONCLUSION: The study exposes a significant pertussis outbreak affecting predominantly young infants.

Bordetella parapertussis adenylate cyclase toxin promotes the bacterial survival to the encounter with macrophages.

B. parapertussis is a whooping cough etiological agent, whose incidence in the population has increased remarkably. Virulence factors involved in the bacterial infection, however, remain poorly investigated. We here studied the role of adenylate cyclase (CyaA), the main toxin of B. parapertussis, in the outcome of the bacterial interaction with macrophages. Our results showed that B. parapertussis CyaA intoxicates human macrophages, prevents bacterial phagocytosis and precludes phago-lysosomal fusion eventually promoting the bacterial survival to the encounter with these immune cells. Accordingly, we found that B. parapertussis CyaA induces the transcriptional downregulation of host genes encoding for antimicrobial peptides, proteins involved in bacterial intracellular killing, and the pro-inflammatory cytokine TNF-α, while induces the upregulation of the anti-inflammatory cytokine IL-10. Together with previous reports suggesting a protective role of B. parapertussis CyaA against neutrophils bactericidal activity, the results of this study suggest a central role of CyaA in B. parapertussis immune evasion and persistence.

Genome Characteristic of Bordetella parapertussis Isolated from Iran.

Pertussis also known as whooping cough is a respiratory infection in humans particularly with severe symptoms in infants and usually caused by Bordetella pertussis. However, Bordetella parapertussis can also cause a similar clinical syndrome. During 2012 to 2015, from nasal swabs sent from different provinces to the pertussis reference laboratory of Pasture Institute of Iran for pertussis confirmation, seven B. parapertussis isolates were identified by bacterial culture, biochemical tests, and the presence of IS1001 insertion in the genome. The expression of pertactin (Prn) as one the major virulence factor for bacterial adhesion was investigated using western blot. Moreover, the genomic characteristic of one recently collected isolate, IRBP134, from a seven-month infant was investigated using Illumina NextSeq sequencing protocol. The results revealed the genome with G+C content 65% and genome size 4.7 Mbp. A total of 81 single nucleotide polymorphisms and 13 short insertions and deletions were found in the genome compared to the B. parapertussis 12822 as a reference genome showing ongoing evolutionary changes. A phylogeny relationship of IRBP134 was also investigated using global B. parapertussis available genomes.

Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic.

Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg- mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg- mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.

Multicenter Performance Evaluation of the Simplexa Bordetella Direct Kit in Nasopharyngeal Swab Specimens.

Detection of Bordetella pertussis and Bordetella parapertussis using molecular methods is sensitive and specific with a short turnaround time compared to other diagnostic methods. In this multicenter study, we compared the performance of the Simplexa Bordetella Direct kit to those of other molecular assays in detecting and differentiating B. pertussis and B. parapertussis in nasopharyngeal swab specimens. The limits of detection (LODs) were 150 CFU/ml or 3 fg/µl of DNA for B. pertussis and 1,500 CFU/ml or 10 fg/µl of DNA for B. parapertussis A total of 1,103 fresh and residual frozen specimens from eight clinical sites were tested. Combining the data from individual clinical sites using different comparative assays, the overall positive percent agreement (PPA) and negative percent agreement (NPA) for B. pertussis were 98.7% and 97.3%, respectively. The overall PPA and NPA for B. parapertussis were 96.7% and 100%, respectively. For prospective fresh specimens, the overall PPA and NPA for both targets were 97.7% and 99.3%, respectively. For retrospective frozen specimens, the overall PPA and NPA for both targets were 92.6% and 93.2%, respectively. The percentage of invalid results was 1.0%. A cross-reactivity study using 74 non-Bordetella bacterial species and five yeast species revealed that the Simplexa Bordetella Direct kit was 100% specific. The hands-on time and assay run time of the Simplexa Bordetella Direct kit are favorable compared to those of other commercial and laboratory-developed tests. In summary, the Simplexa Bordetella Direct kit has a performance comparable to those of other molecular assays for the detection of B. pertussis and B. parapertussis.

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.

Multicenter Clinical Evaluation of the Automated Aries Bordetella Assay.

Molecular methods offer superior sensitivity and specificity and reduce testing turnaround time from days to hours for detection of Bordetella pertussis and Bordetella parapertussis In this study, we evaluated the performance of the automated PCR-based Aries Bordetella Assay, which detects both B. pertussis and B. parapertussis directly from nasopharyngeal swab specimens. The limits of detection (LoDs) were 1,800 CFU·ml-1 for B. pertussis and 213 CFU·ml-1 for B. parapertussis The assay detected 16/18 unique B. pertussis/B. parapertussis strains. Of 71 potentially cross-reacting organisms, 5 generated false positives in 1/6 replicates; none of 6 additional Bordetella spp. were erroneously detected. Specimens were stable at 20 to 25°C for at least 10 h, at 4 to 8°C for 10 days, and at temperatures not exceeding -70°C for 6 months. Of 1,052 nasopharyngeal specimens from patients with suspected pertussis, 3.0% (n = 32) were B. pertussis positive and 0.2% (n = 2) were B. parapertussis positive. Combining these data with Aries Bordetella Assay data from 57 nasopharyngeal samples with previously confirmed B. pertussis or B. parapertussis data and with data from 50 contrived B. parapertussis samples, the proportions of positive and negative agreement of the respective Aries assays with the reference assays were 97.1% and 99.0% for B. pertussis and 100% and 99.7% for B. parapertussis The Aries Bordetella Assay provides accurate detection and distinction of B. pertussis and B. parapertussis infections within 2 h. (This study has been registered at ClinicalTrials.gov under registration no. NCT02862262.).

Characterization of Post-Translational Modifications and Cytotoxic Properties of the Adenylate-Cyclase Hemolysin Produced by Various Bordetella pertussis and Bordetella parapertussis Isolates.

Bordetella pertussis and Bordetella parapertussis are the causal agents of whooping cough in humans. They produce diverse virulence factors, including adenylate cyclase-hemolysin (AC-Hly), a secreted toxin of the repeat in toxins (RTX) family with cyclase, pore-forming, and hemolytic activities. Post-translational modifications (PTMs) are essential for the biological activities of the toxin produced by B. pertussis. In this study, we compared AC-Hly toxins from various clinical isolates of B. pertussis and B. parapertussis, focusing on (i) the genomic sequences of cyaA genes, (ii) the PTMs of partially purified AC-Hly, and (iii) the cytotoxic activity of the various AC-Hly toxins. The genes encoding the AC-Hly toxins of B. pertussis and B. parapertussis displayed very limited polymorphism in each species. Most of the sequence differences between the two species were found in the C-terminal part of the protein. Both toxins harbored PTMs, mostly corresponding to palmitoylations of the lysine 860 residue and palmoylations and myristoylations of lysine 983 for B. pertussis and AC-Hly and palmitoylations of lysine 894 and myristoylations of lysine 1017 for B. parapertussis AC-Hly. Purified AC-Hly from B. pertussis was cytotoxic to macrophages, whereas that from B. parapertussis was not.

Nasopharyngeal pertussis toxin IgA antibodies in the diagnosis of pertussis in Australian community patients.

Nasopharyngeal aspirate (NPA) Bordetella pertussis-specific IgA antibody assay using whole-cell (WC) antigen has previously been shown to have promise in the diagnosis of patients with suspected pertussis. Recently, the use of WC assays in serum have been replaced by pertussis toxin (PT) because of specificity concerns. In this study, PT and WC B. pertussis-specific IgA antibody was assayed in 491 NPAs. Specimens also had molecular testing for the presence of B. pertussis and B. parapertussis as per the usual laboratory protocol. Positive concordance of the two serological assays was 51.2%, negative concordance was 67.5% and total concordance was 75.8%. 99 of 119 discordant specimens were resolved by utilising the B. pertussis polymerase chain reaction (PCR) result and clinical status, and yielded a sensitivity of 57.6% and a specificity 97.7% for WC, with 90.2% and 93.1%, respectively, for the PT assay (p < 0.00025 and 0.025-0.01). In contrast, the sensitivity of PCR was only 19.1% in this cohort. We conclude that specificity is not a significant issue for mucosal pertussis-specific IgA assays using WC, but the superior sensitivity of the PT assay favours the latter method. This assay, combined with PCR assays, should significantly improve the diagnosis of pertussis cases.

Do Pertussis Vaccines Protect Against Bordetella parapertussis?

We calculated the effectiveness of pertussis vaccine in preventing parapertussis among Oregon children 2 months to 10 years of age using 2 methods. During 2011-2016, the 2 VE methods found 66% (95% CI, 59-75%) and 82% (95% CI, 69-90%) effectiveness against parapertussis. Pertussis vaccine may induce cross-immunity.

Comparison of molecular detection methods for pertussis in children during a state-wide outbreak.

A state-wide pertussis outbreak occurred in Washington during the winter-spring months of 2012, concurrent with respiratory viral season. We compared performance characteristics of a laboratory-developed pertussis PCR (LD-PCR for Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii) and rapid multiplex PCR (RM-PCR) for respiratory viruses (FilmArray™, BioFire, B. pertussis data unblinded following FDA approval post outbreak). We analyzed three cohorts of patients using physician testing orders as a proxy for clinical suspicion for pertussis or respiratory viruses: Cohort 1, tested by LD-PCR for pertussis pathogens only by nasopharyngeal swab; Cohort 2, by RM-PCR for respiratory viruses only by mid-nasal turbinate swab; and Cohort 3, by both methods. B. pertussis was detected in a total of 25 of the 490 patients in Cohort 3 in which LD-PCR detected 20/25 (80 %) cases and the RM-PCR detected 24/25 (96 %; p = 0.2). Pertussis pathogens were detected in 21/584 (3.6 %) of samples from Cohort 1 where clinicians had a relatively strong suspicion for pertussis. In contrast, B. pertussis was detected in only 4/3071 (0.1 %) specimens from Cohort 2 where suspicion for pertussis was lower (p < 0.001 for comparison with Cohort 1). In summary, the two laboratory methods were comparable for the detection of B. pertussis.

Role of Atypical Pathogens and the Antibiotic Prescription Pattern in Acute Bronchitis: A Multicenter Study in Korea.

The role of atypical bacteria and the effect of antibiotic treatments in acute bronchitis are still not clear. This study was conducted at 22 hospitals (17 primary care clinics and 5 university hospitals) in Korea. Outpatients (aged ≥ 18 yr) who had an acute illness with a new cough and sputum (≤ 30 days) were enrolled in 2013. Multiplex real-time polymerase chain reaction (RT-PCR) was used to detect five atypical bacteria. A total of 435 patients were diagnosed as having acute bronchitis (vs. probable pneumonia, n = 75), and 1.8% (n = 8) were positive for atypical pathogens (Bordetella pertussis, n = 3; B. parapertussis, n = 0; Mycoplasma pneumoniae, n = 1; Chlamydophila pneumoniae, n = 3; Legionella pneumophila, n = 1). Among clinical symptoms and signs, only post-tussive vomiting was more frequent in patients with atypical pathogens than those without (P = 0.024). In all, 72.2% of the enrolled patients received antibiotic treatment at their first visits, and ß-lactams (29.4%) and quinolones (20.5%) were the most commonly prescribed agents. In conclusion, our study demonstrates that the incidence of atypical pathogens is low in patients with acute bronchitis, and the rate of antibiotic prescriptions is high.

Host Specificity of Ovine Bordetella parapertussis and the Role of Complement.

The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, Bordetella parapertussis, is composed of two distinct clades that have specialized to different hosts: one to humans (Bpphu), and the other to sheep (Bppov). While Bpphu and the other classical bordetellae can efficiently colonize mice, Bppov strains are severely defective in their ability to colonize the murine respiratory tract. Bppov genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on B. bronchiseptica and Bpphu strains. Bppov lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing in vitro. Based on these results, we hypothesized that Bppov failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the Bppov defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, Bppov strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of Bppov to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.

Bordetella parapertussis outbreak in Bisham, Pakistan in 2009-2010: fallout of the 9/11 syndrome.

Pertussis or whooping cough is a highly contagious community disease mainly caused by Bordetella pertussis and B. parapertussis. We report a minor outbreak of whooping cough (2009-2010) in symptomatic subjects from Bisham, near Swat, Khyber Pukhtoonkhawa province, Pakistan. Interestingly, our results show that all the culture-positive isolates (n = 21) collected from children (average age 3·46 years), were identified as B. parapertussis after routine identification tests and PCR IS481, IS1001 and IS1002. Furthermore, in the affected patients, none had received immunization with diphtheria-pertussis-tetanus (DTPw) vaccine. Therefore, the possibility of the re-emergence of the disease due to limitation of basic health services as a result of the political unrest due to the 9/11 situation is also examined. Moreover, we discuss the importance of vaccinating both adults and children with DTPwPaw vaccine containing both organisms for better protection.

Bordetella parapertussis PagP mediates the addition of two palmitates to the lipopolysaccharide lipid A.

Bordetella bronchiseptica PagP (PagPBB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection. B. parapertussis contains a putative pagP homolog (encoding B. parapertussis PagP [PagPBPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-type B. parapertussis lipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagPBPa is required for the modification of lipid A with palmitate. While PagPBB transfers a single palmitate to the lipid A C-3' position, PagPBPa transfers palmitates to the lipid A C-2 and C-3' positions. The addition of two palmitate acyl chains is unique to B. parapertussis. Mutation of pagPBPa resulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.

Role of Atypical Pathogens and the Antibiotic Prescription Pattern in Acute Bronchitis: A Multicenter Study in Korea

The role of atypical bacteria and the effect of antibiotic treatments in acute bronchitis are still not clear. This study was conducted at 22 hospitals (17 primary care clinics and 5 university hospitals) in Korea. Outpatients (aged > or = 18 yr) who had an acute illness with a new cough and sputum (< or = 30 days) were enrolled in 2013. Multiplex real-time polymerase chain reaction (RT-PCR) was used to detect five atypical bacteria. A total of 435 patients were diagnosed as having acute bronchitis (vs. probable pneumonia, n = 75), and 1.8% (n = 8) were positive for atypical pathogens (Bordetella pertussis, n = 3; B. parapertussis, n = 0; Mycoplasma pneumoniae, n = 1; Chlamydophila pneumoniae, n = 3; Legionella pneumophila, n = 1). Among clinical symptoms and signs, only post-tussive vomiting was more frequent in patients with atypical pathogens than those without (P = 0.024). In all, 72.2% of the enrolled patients received antibiotic treatment at their first visits, and beta-lactams (29.4%) and quinolones (20.5%) were the most commonly prescribed agents. In conclusion, our study demonstrates that the incidence of atypical pathogens is low in patients with acute bronchitis, and the rate of antibiotic prescriptions is high.