Rapid identification of Bordetella pertussis pertactin gene variants using LightCycler real-time polymerase chain reaction combined with melting curve analysis and gel electrophoresis.

Recently, eight allelic variants of the pertactin gene (prn1-8) have been characterized in Bordetella pertussis strains isolated in Europe and the United States. It has been suggested that the divergence of the pertactin types of clinical isolates from those of the B. pertussis vaccine strains is a result of vaccine-driven evolution. Sequencing of the prn, which is relatively time-consuming, has so far been the only method for the differentiation of prn types. We have developed a rapid real-time polymerase chain reaction assay suitable for large-scale screening of the prn type of the circulating strains. This method correctly identified the prn type of all tested 41 clinical isolates and two Finnish vaccine strains. The method is simple and reliable and provides an alternative for sequencing in pertussis research

Antimicrobial susceptibility testing of Finnish Bordetella pertussis isolates collected during 2006-2017

Objectives: Macrolides, such as azithromycin and erythromycin, are first-line drugs for the (prophylactic) treatment of pertussis. This study aimed to screen for macrolide-, quinolone- or trimethoprim/sulfamethoxazole (SXT)-resistant strains among Finnish Bordetella pertussis isolates.Methods: Antimicrobial susceptibility testing was performed on 148 B. pertussis strains isolated during 2006-2017. Isolates were analysed by allele-specific PCR for detection of the macrolide resistance-associated mutation A2047G in the 23S rRNA gene. The gyrA gene was sequenced for detection of the A260G mutation associated with quinolone resistance. For phenotyping, a random selection was made by selecting every third isolate (n=50) to determine the minimum inhibitory concentrations (MICs) for erythromycin and azithromycin by Etest and the inhibition zone size for nalidixic acid (NAL) and SXT by single disk diffusion assay.Results: Neither the macrolide resistance-associated mutation A2047G nor the quinolone resistance-associated mutation A260G was detected in any of the B. pertussis isolates. MICs of azithromycin and erythromycin ranged between 0.016-0.19 mu g/mL and 0.016-0.25 mu g/mL, respectively. The size of the inhibition zone surrounding the NAL disk ranged between 22-27 mm in diameter. The inhibition zone surrounding the SXT disk ranged between 24-37 mm in diameter. No isolates resistant to any of the tested antimicrobials were identified.Conclusions: The allele-specific PCR is a simple and useful tool for screening B. pertussis resistance to macrolides. All Finnish isolates tested were susceptible to macrolides, quinolones and SXT. (C) 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved

Detection of Enterobacterial Lipopolysaccharides and Experimental Endotoxemia by Means of an Immunolimulus Assay Using Both SerotypeSpecific and Cross-Reactive Antibodies

The immunolimulus (IML) assay system uses solid-phase endotoxin antibodies to capture lipopolysaccharide (LPS), which is then quantified by a modification of the chromogenic limulus amebocyte lysate (CLAL) method. Monoclonal antibodies (MAbs) reactive with selected 0 antigen serotypes of Escherichia coli (O18) and Salmonella typhimurium (O-9,12), when used in the IML, were shown to be highly specific in detecting their respective endotoxins in purified form and in plasma samples from experimentally infected animals. A murine MAb that was broadly cross-reactive with E. coli, Salmonella, and Shigella endotoxins also proved to be highly effective in the IML assay for capturing LPS molecules from both E. coli and S. typhimurium strains. These results indicate that IML assays can detect smooth-type enterobacterial endotoxins in plasma and suggest that such assays have potential for use in the rapid diagnosis of sepsis and endotoxemia caused by different enterobacterial specie

SNP-Based Typing: A Useful Tool to Study Bordetella pertussis Populations

To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). In this study, a single nucleotide polymorphism (SNP) typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in the Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis

Rapid detection of functional gene polymorphisms of TLRs and IL-17 using high resolution melting analysis

Genetic variations in toll-like receptors (TLRs) and IL-17A have been widely connected to different diseases. Associations between susceptibility and resistance to different infections and single nucleotide polymorphisms (SNPs) in TLR1 to TLR4 and IL17A have been found. In this study, we aimed to develop a rapid and high throughput method to detect functional SNPs of above mentioned proteins. The following most studied and clinically important SNPs: TLR1 (rs5743618), TLR2 (rs5743708), TLR3 (rs3775291), TLR4 (rs4986790) and IL17 (rs2275913) were tested. High resolution melting analysis (HRMA) based on real-time PCR combined with melting analysis of a saturating double stranded-DNA binding dye was developed and used. The obtained results were compared to the "standard" sequencing method. A total of 113 DNA samples with known genotypes were included. The HRMA method correctly identified all genotypes of these five SNPs. Co-efficient values of variation of intra-and inter-run precision repeatability ranged from 0.04 to 0.23%. The determined limit of qualification for testing samples was from 0.5 to 8.0 ng/mu l. The identical genotyping result was obtained from the same sample with these concentrations. Compared to "standard" sequencing methods HRMA is cost-effective, rapid and simple. All the five SNPs can be analyzed separately or in combination

Respiratory syncytial virus infections in children 0–24 months of age in the community

Objectives: Respiratory syncytial virus (RSV) is a major cause of hospitalization in young children, but there are little data on RSV infections in early childhood in the community. We conducted a prospective population-based birth-cohort study to determine the rates and characteristics of RSV infections in young children.Methods: We followed 923 children for acute respiratory infections (ARIs) from birth to age 24 months with daily diaries and study clinic visits. Nasal swab samples were obtained at the onset of ARIs and analyzed for RSV by RT-PCR and antigen tests. The rates of RSV infections and associated outcomes were estimated.Results: RSV was detected in 289 (6%) of 4728 ARIs with a nasal sample. The mean estimated annual rate of RSV infections was 37 (95% confidence interval [CI], 35–38) per 100 children at age 0–24 months. For RSV-associated outcomes, the estimated annual rates per 100 children were 34 (95% CI, 32–37) physician visits, 16 (95% CI, 15–17) antibiotic treatments, 12 (95% CI, 11–13) acute otitis media, and 6 (95% CI, 4–7) wheezing illnesses. The prevalence of RSV was 0.6% in asymptomatic children.Conclusions: RSV infections impose a high burden of disease in healthy young children in the community.</p

Clinical presentation of pertussis in fully immunized children in Lithuania

BACKGROUND: In Lithuania, the vaccination coverage against pertussis is high. Nevertheless, there is a significant increase in pertussis cases in fully immunized children. The aim of our study was to determine the frequency of classical symptoms of laboratory confirmed pertussis and describe its epidemiology in children fully vaccinated against pertussis. METHODS: From May to December 2001, 70 children aged 1 month to 15 years, suffering from prolonged cough were investigated in the Centre of Paediatrics, Vilnius University Children's Hospital. The collected information included personal data, vaccination history, clinical symptoms of the current illness, and treatment before hospitalization. At the admission to the hospital blood samples were taken from all studied children for Bordetella pertussis IgM and IgA. RESULTS: A total of 53 (75.7%) of the 70 recruited patients with prolonged cough showed laboratory evidence of pertussis. 32 of them were fully vaccinated with whole cell pertussis vaccine (DTP). The age of fully vaccinated patients varied from 4 to 15 years (average 10.9 ± 3.1; median 11). The time period between the last vaccination dose (fourth) and the clinical manifestation of pertussis was 2.6–13 years (average 8.9 ± 3.0; median 9). More than half of the children before the beginning of pertussis were in contact with persons suffering from long lasting cough illness in the family, school or day-care center. The mean duration from onset of pertussis symptoms until hospitalization was 61.4 ± 68.3 days (range, 7 to 270 days; median 30). For 11 patients who had had two episodes (waves) of coughing, the median duration of cough was 90 days, and for 21 with one episode 30 days (p < 0.0002). Most of the children (84.4%) had paroxysmal cough, 31.3% had post-tussive vomiting, 28.1% typical whoop, and 3.1% apnea. Only 15.6% children had atypical symptoms of pertussis. CONCLUSION: Fully vaccinated children fell ill with pertussis at the median of 11 years old, 9 years following pertussis vaccination. More than half of the children could catch pertussis at home, at school or day-care center. Clinical picture of pertussis in previously immunized children is usually characterized by such classical symptoms as prolonged and paroxysmal cough, rarely by whopping and post-tussive vomiting, and very rarely by apnea

Bordetella pertussis Strains with Increased Toxin Production Associated with Pertussis Resurgence

A more virulent strain of the disease is emerging