Evaluation of an immunofluorescent-antibody test for rapid identification of Pseudomonas aeruginosa in blood cultures

Abstract

An immunofluorescent-antibody test was developed for rapid detection of Pseudomonas aeruginosa in blood cultures. The test uses a murine monoclonal antibody specific for all strains of P. aeruginosa. In initial tests, bright uniform immunofluorescence signals were seen when each of the 17 international serotypes, as well as 14 additional isolates of P. aeruginosa, were examined. No immunofluorescent staining was observed when 37 other gram-negative and 15 gram-positive species were studied. In a clinical study, the assay was applied to broth smears of 86 gram-negative bacilli isolated from 74 bacteremic patients and 28 additional clinical isolates of Pseudomonas sp. and other oxidase-positive gram-negative bacilli recovered from various other body sites. Smears were made directly from blood cultures which were positive for gram-negative bacilli by Gram staining. Eleven (15%) of 74 patients with gram-negative bacteremia had a positive test for P. aeruginosa. Including the results of these 11 isolates recovered in a prospective study and an additional 10 isolates from a retrospective study, we obtained a sensitivity and specificity of 100% (21 positive specimens and 103 negative specimens, respectively). These preliminary results suggest that this is a useful reagent for rapid presumptive identification of P. aeruginosa in blood cultures. With the immunofluorescent-antibody test, P. aeruginosa could be identified within 1 h of Gram stain evidence of gram-negative bacteremia. Pseudomonas aeruginosa is an important cause of nosocomial bacteremia and pneumonia, especially in immunocompromised patients. In patients receiving organ transplants, this organism was the most common cause of bacteremia among the aerobic gram-negative bacilli and was responsible for 50 (28%) of 180 episodes (4). Case fatality rates ranged from 30 to 50%, despite the use of antibiotics (2, 18; V. T. Andriole, editorial, J. Lab. Clin. Med. 94:196-199), and have consistently remained higher than the mortality associated with bacteremia caused by other gram-negative bacilli (23). Nonetheless, early institution of appropriate antibiotics improves the outcome of P. aeruginosa bacteremia, even in neutropenic patients (3, 23). A review of 410 infections in cancer patients showed that cure rates were reduced from 74 to 46% in patients in whom there was a 1to 2-day delay in the use of appropriate antibiotics (3). Therefore, there is an obvious need for laboratory techniques which permit more rapid identification of P. aeruginosa. The P. aeruginosa immunofluorescent-antibody (IFA) test (Genetic Systems Corp., Seattle, Wash.) was evaluated for use in the clinical laboratory as a rapid presumptive identification method for P. aeruginosa isolated from blood cultures. Production of mouse monoclonal antibodies to porin protein F has been reported by others (9, 17). Previous work has also shown that certain epitopes on this protein may be present on all P. aeruginosa strains but not other gramnegative species. Antibodies to these epitopes are candidates for a...

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection

“…Recombinant protein F puri¢ed from Escherichia coli was shown to retain vaccine e¤cacy in the burned mouse [2] and the rat chronic pulmonary infection [6] models. Protein F is an excellent vaccine candidate in that it is present in all strains of P. aeruginosa [7^9], is antigenically conserved in all strains [8,9], is surface-exposed [10^12], and is essential to the bacterial cell [13]. The problems of obtaining su¤cient quantities of puri¢ed protein F and of having to maintain this integral membrane protein in a detergent environment led us to search for surface-exposed protective epitopes within protein F. Through use of synthetic peptides conjugated to keyhole limpet hemocyanin as a carrier, three surface-exposed linear B-cell epitopes (peptides 9, 10 and 18) [12,14] were identi¢ed.…”

A Chimeric Influenza Virus Expressing an Epitope of Outer Membrane Protein F ofPseudomonas aeruginosaAffords Protection against Challenge withP. aeruginosain a Murine Model of Chronic Pulmonary Infection

“…Protein F is a major OM protein (36) that is surface exposed in wild-type cells (12,18,27). Furthermore, it is present and immunologically crossreactive in all strains of P. aeruginosa (3,12,28). Antibodies elicited by immunization with protein F are opsonic for P. aeruginosa (1, 7) but do not cross-react significantly with cells of other genera of gram-negative bacteria (3,12,28).…”

“…Furthermore, it is present and immunologically crossreactive in all strains of P. aeruginosa (3,12,28). Antibodies elicited by immunization with protein F are opsonic for P. aeruginosa (1, 7) but do not cross-react significantly with cells of other genera of gram-negative bacteria (3,12,28). Purified protein F from P. aeruginosa and recombinant protein F have been shown to provide significant protection in immunized animals against subsequent infection by P. aeruginosa in vari-ous animal models (7,8,11,22,23).…”

Conservation of surface epitopes inPseudomonas aeruginosaouter membrane porin protein OprF

“…In addition, a second function in both outer membrane and cell structure and stability was discovered, in that OprFdeficient mutants have rounded morphology and grow well only in high osmolarity media [10,11]. Studies utilizing mAbs specific for OprF have demonstrated that this protein is potentially useful as a target for diagnostic and immunotherapeutic intervention [12,13]. Indeed, Gilleland and colleagues [14] have presented data to demonstrate that immunization with OprF protects against subsequent Pseudomonas aeruginosa infections.…”