title: Development of a 4-valent genotyping assay for direct identification of the most frequent Pseudomonas aeruginosa serotypes from respiratory specimens of pneumonia patients.
abstract: Pseudomonas aeruginosa is a common cause of nosocomial infections and is associated with high rates of mortality. In order to facilitate rapid and sensitive identification of the most prevalent serotypes of P. aeruginosa, we have developed a 4-valent real-time PCR-based assay using oligonucleotides specific for open-reading frames constituting the O-antigen-specific lipopolysaccharide loci of P. aeruginosa. The assay simultaneously detects and differentiates between each of the four serotypes IATS-O1, -O6, -O11 and serogroup 2 (IATS-O2, -O5, and -O16) with high sensitivity and specificity in a single-tube reaction. No cross-reactivity was observed with other serotypes of P. aeruginosa or other microbial species, and reproducibility was demonstrated regardless of template, i.e. purified DNA, bacterial culture and clinical specimens (broncho-alveolar lavage). The limit of detection of the assay was approximately 100 copies per reaction for IATS-O1, -O2 and -O11, and 50 copies per reaction for IATS-O6. Comparison of the assay specificity with a commercially available slide agglutination kit showed consistent results; however, the number of non-typable isolates was reduced by 15 % using the genotyping assay. Use of the 4-valent genotyping assay in the context of a clinical trial resulted in identification of pneumonia patients positive for the IATS-O11 serotype, and hence eligible for therapy with panobacumab (an investigational monoclonal antibody against the O-polysaccharide of serotype IATS-O11).
authors.affiliation: Kenta Biotech AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Holger
authors.initials: H
authors.lastname: Koch
authors.affiliation: Kenta Biotech AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Emrich
authors.affiliation: Kenta Biotech AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Jampen
authors.affiliation: Kenta Biotech AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Marianne
authors.initials: M
authors.lastname: Wyss
authors.affiliation: Kenta Biotech AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Verena
authors.initials: V
authors.lastname: Gafner
authors.affiliation: Kenta Biotech AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Hedvika
authors.initials: H
authors.lastname: Lazar
authors.affiliation: Kenta Biotech AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rudolf
mesh_terms: D000328:Adult; D001431:Bacteriological Techniques / Q000379:methods*; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D018410:Pneumonia, Bacterial / Q000175:diagnosis*; D011552:Pseudomonas Infections / Q000175:diagnosis* / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity