Direct Identification of Major Blood Culture Pathogens, Including Pseudomonas aeruginosa and Escherichia coli, by a Panel of Fluorescence In
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微生物临床杂志 2005年第4期
Department of Clinical Microbiology, Aalborg Hospital-Aarhus University Hospital, Aalborg, Denmark
AdvanDx Inc., Woburn, Massachusetts
ABSTRACT
Rapid identification of four major pathogens from 1,231 positive blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes (AdvanDx Inc., Woburn, Mass.) was evaluated. For Escherichia coli, Staphylococcus aureus, and Candida albicans results agreed with conventional identification. The lower sensitivity of the Pseudomonas aeruginosa assay should not compromise the utility of the four assays.
TEXT
Bacteremia is a serious condition, with mortality in the range of 10 to 40% (3, 6, 8). The prime objective of the laboratory is to provide timely reports on blood culture (BC) isolates that may guide antibiotic therapy. However, definite identification and susceptibility testing are time-consuming, and there is a demand for techniques that are directly applicable to positive BCs and thus obviate the need for subculture. Fluorescence in situ hybridization (FISH) with peptide nucleic acid (PNA) probes (AdvanDx Inc., Woburn, Mass.) is a new FISH technique using fluorescein-labeled PNA probes targeting rRNA for rapid and accurate identification of major BC pathogens (1, 4, 5, 9). In this noninterventional cohort study, PNA probes for Pseudomonas aeruginosa and Escherichia coli (7) were evaluated in parallel with Staphylococcus aureus PNA FISH (KT001; AdvanDx Inc.) and Candida albicans PNA FISH (KT002; AdvanDx Inc.) in a routine setting (5, 9).
The study was conducted from May 2003 to April 2004 at the Department of Clinical Microbiology, Aalborg Hospital-Aarhus University Hospital, Aalborg, Denmark.
For adult patients a BC set comprised one standard aerobic (SA) bottle, one FAN aerobic (FA) bottle, and one standard anaerobic (SN) bottle (BacT/Alert3D; bioMerieux Marcy L’Etoile, France). For children, a pediatric aerobic FAN (PF) bottle was used. Positive BCs were examined by direct microscopy and Gram staining, and cultures with either gram-positive cocci in clusters, gram-negative rods, or yeast were included, given that only one morphological type was observed. To obtain a set of independent observations only one bottle per set was analyzed; if two or three bottles in a set were positive the FA bottle was examined; otherwise, the SN or the SA bottle, in that order, was included.
S. aureus PNA FISH and C. albicans PNA FISH were performed according to the manufacturer instructions, and probes for P. aeruginosa and E. coli were applied by the same procedure (5). In brief, 10 μl of BC broth was mixed on one-well Teflon-coated microscope slides with a drop of fixation solution, air dried, and fixed by flame fixation. One drop of hybridization solution containing the fluorescein-labeled PNA probe was added, and a coverslip was applied. For hybridization slides were incubated on a slide warmer (SM30; Grant Boekel) at 55°C for 90 min. Subsequently, coverslips were removed and the slides were submerged for 30 min in a preheated wash solution (55°C) in a water bath. Slides were mounted with mounting fluid, and coverslips were applied before examination with a fluorescence microscope (objective, 100x, Olympus BX40; Osram HBO 100 W/2 Hg lamp) equipped with a fluorescein isothiocyanate-Texas Red double filter (filter no. AC003; AdvanDx).
Two observers blind to the results of conventional identification examined the slides. Distinct green fluorescence in multiple fields of view was scored as a positive result (5, 9) and graded into two categories, faint and bright. Negative slides had a faintly reddish background smear; rarely a few fluorescent bacteria or bacterial clusters were observed, and such slides were also scored as negative.
For conventional identification subculture was performed on 5% horse blood agar, chocolate agar, CPS2 agar (bioMerieux) and vitamin K-enriched chocolate agar for anaerobic culture as deemed appropriate. BC isolates underwent definitive identification as follows: S. aureus by coagulase test with horse citrate plasma and Staph Plus (bioMerieux), C. albicans by chromogenic reaction on Candida ID plates (bioMerieux) and the Vitek2 system (bioMerieux), E. coli by the -glucuronidase reaction on the CPS2 plates provided a spot indole test was positive or, if the isolates were -glucuronidase or indole negative, by Vitek2. Finally P. aeruginosa was identified both by a conventional approach according to the Manual of Clinical Microbiology (2) and by Vitek2.
A total of 1,231 positive BC sets from 946 patients were included. The distribution of the BCs by bottle type was as follows: FA, 737; SA, 257; SN, 172; PF, 65. The distribution of pathogens appears in Table 1. Results obtained by PNA FISH and conventional methods appear in Tables 2 and 3, and the sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of the four assays appear in Table 4. We found close to 100% concordance with conventional identification for S. aureus and C. albicans, corroborating previous reports (1, 4, 5, 9). This study is the first application of PNA FISH for gram-negative rods. For E. coli, the results agreed with those by conventional methods except for one false-positive reaction with 1 of the 63 Klebsiella pneumoniae BCs. Thirty-four BCs grew P. aeruginosa, and identification by PNA FISH was correct for 32. Among 615 BCs with other gram-negative rods 6 were false positive, 4 of which showed a faint staining. These spurious reactions (one isolate each) included reactions for E. coli, Agrobacterium radiobacter, an Achromobacter sp., an Acinetobacter sp., Burkholderia cepacia, and an unidentified Pseudomonas sp. Therefore, sensitivity and PPV for P. aeruginosa PNA FISH were somewhat lower than those for the three other assays.
Polymicrobial growth was detected in 108 BCs. According to the protocol only one bottle per set was examined by PNA FISH, and discordant growth among bottles in some BC sets had an impact on the analysis of results (Table 1). Overall, we did not detect a poorer performance with polymicrobial samples; note, however, that two of five failures with the E. coli probe occurred with such samples (Table 2).
There are some limitations to the present study. It was conducted in a routine setting, and we cannot determine the exact cause of the spurious results. The preparatory steps prior to hybridization could be critical, as we obtained a bright staining of smears prepared from subcultures of one E. coli isolate and two P. aeruginosa isolates undetected by direct examination. Moreover, we cannot exclude the possibility that a Pseudomonas species actually was P. aeruginosa as indicated by PNA FISH.
Faint, but distinct, staining was seen both for P. aeruginosa and some other gram-negative rods belonging to the group of aerobic bacteria. Disregarding such reactions would lead to a lower sensitivity (30 of 34 BCs; 88.2%) and better specificity (613 of 615 BCs; 99.7%), the PPV would be better (30 of 32 BCs; 93.8%), and the NPV would be marginally lower (613 of 617 BCs; 99.4%). From a clinical point of view missing a P. aeruginosa diagnosis is undesirable, and therefore we favor a cutoff point as used in Tables 2 and 3.
Distinguishing E. coli and P. aeruginosa from other gram-negative rods should facilitate early directed antibiotic therapy. Negative results in both assays increase the likelihood of other gram-negative pathogens such as Klebsiella, Citrobacter, and Enterobacter spp., which often express chromosomal -lactamases.
ACKNOWLEDGMENTS
Skillful assistance by biotechnicians Lone Person and Jeanett Srensen is gratefully acknowledged.
This study was supported by grants from Nordjylland Amts Forskningsfond and Aalborg Hospital-Aarhus University Hospital to Mette Sgaard. Research at The Department of Clinical Microbiology, Aalborg Hospital-Aarhus University Hospital, is supported by Det Obelske Familiefond.
REFERENCES
Gonzalez, V., E. Padilla, M. Gimenez, C. Vilaplana, A. Perez, G. Fernandez, M. D. Quesada, M. A. Pallares, and V. Ausina. 2004. Rapid diagnosis of Staphylococcus aureus bacteremia using S. aureus PNA FISH. Eur. J. Clin. Microbiol. Infect. Dis. 23:396-398.
Kiska, D. L., and P. H. Gilligan. 2003. Pseudomonas, p. 719-728. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed., vol. 1. ASM Press, Washington, D.C.
Leibovici, L., I. Shraga, M. Drucker, H. Konigsberger, Z. Samra, and S. D. Pitlik. 1998. The benefit of appropriate empirical antibiotic treatment in patients with bloodstream infection. J. Intern. Med. 244:379-386.
Oliveira, K., S. M. Brecher, A. Durbin, D. S. Shapiro, D. R. Schwartz, P. C. De Girolami, J. Dakos, G. W. Procop, D. Wilson, C. S. Hanna, G. Haase, H. Peltroche-Llacsahuanga, K. C. Chapin, M. C. Musgnug, M. H. Levi, C. Shoemaker, and H. Stender. 2003. Direct identification of Staphylococcus aureus from positive blood culture bottles. J. Clin. Microbiol. 41:889-891.
Oliveira, K., G. W. Procop, D. Wilson, J. Coull, and H. Stender. 2002. Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J. Clin. Microbiol. 40:247-251.
Pedersen, G., H. C. Schnheyder, and H. T. Srensen. 2003. Source of infection and other factors associated with case fatality in community-acquired bacteremia—a Danish population-based cohort study from 1992 to 1997. Clin. Microbiol. Infect. 9:793-802.
Perry-O'Keefe, H., S. Rigby, K. Oliveira, D. Sorensen, H. Stender, J. Coull, and J. J. Hyldig-Nielsen. 2001. Identification of indicator microorganisms using a standardized PNA FISH method. J. Microbiol. Methods 47:281-292.
Pittet, D., and R. P. Wenzel. 1995. Nosocomial bloodstream infections. Secular trends in rates, mortality, and contribution to total hospital deaths. Arch. Intern. Med. 155:1177-1184.
Rigby, S., G. W. Procop, G. Haase, D. Wilson, G. Hall, C. Kurtzman, K. Oliveira, S. Von Oy, J. J. Hyldig-Nielsen, J. Coull, and H. Stender. 2002. Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J. Clin. Microbiol. 40:2182-2186.(M. Sgaard, H. Stender, an)
AdvanDx Inc., Woburn, Massachusetts
ABSTRACT
Rapid identification of four major pathogens from 1,231 positive blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes (AdvanDx Inc., Woburn, Mass.) was evaluated. For Escherichia coli, Staphylococcus aureus, and Candida albicans results agreed with conventional identification. The lower sensitivity of the Pseudomonas aeruginosa assay should not compromise the utility of the four assays.
TEXT
Bacteremia is a serious condition, with mortality in the range of 10 to 40% (3, 6, 8). The prime objective of the laboratory is to provide timely reports on blood culture (BC) isolates that may guide antibiotic therapy. However, definite identification and susceptibility testing are time-consuming, and there is a demand for techniques that are directly applicable to positive BCs and thus obviate the need for subculture. Fluorescence in situ hybridization (FISH) with peptide nucleic acid (PNA) probes (AdvanDx Inc., Woburn, Mass.) is a new FISH technique using fluorescein-labeled PNA probes targeting rRNA for rapid and accurate identification of major BC pathogens (1, 4, 5, 9). In this noninterventional cohort study, PNA probes for Pseudomonas aeruginosa and Escherichia coli (7) were evaluated in parallel with Staphylococcus aureus PNA FISH (KT001; AdvanDx Inc.) and Candida albicans PNA FISH (KT002; AdvanDx Inc.) in a routine setting (5, 9).
The study was conducted from May 2003 to April 2004 at the Department of Clinical Microbiology, Aalborg Hospital-Aarhus University Hospital, Aalborg, Denmark.
For adult patients a BC set comprised one standard aerobic (SA) bottle, one FAN aerobic (FA) bottle, and one standard anaerobic (SN) bottle (BacT/Alert3D; bioMerieux Marcy L’Etoile, France). For children, a pediatric aerobic FAN (PF) bottle was used. Positive BCs were examined by direct microscopy and Gram staining, and cultures with either gram-positive cocci in clusters, gram-negative rods, or yeast were included, given that only one morphological type was observed. To obtain a set of independent observations only one bottle per set was analyzed; if two or three bottles in a set were positive the FA bottle was examined; otherwise, the SN or the SA bottle, in that order, was included.
S. aureus PNA FISH and C. albicans PNA FISH were performed according to the manufacturer instructions, and probes for P. aeruginosa and E. coli were applied by the same procedure (5). In brief, 10 μl of BC broth was mixed on one-well Teflon-coated microscope slides with a drop of fixation solution, air dried, and fixed by flame fixation. One drop of hybridization solution containing the fluorescein-labeled PNA probe was added, and a coverslip was applied. For hybridization slides were incubated on a slide warmer (SM30; Grant Boekel) at 55°C for 90 min. Subsequently, coverslips were removed and the slides were submerged for 30 min in a preheated wash solution (55°C) in a water bath. Slides were mounted with mounting fluid, and coverslips were applied before examination with a fluorescence microscope (objective, 100x, Olympus BX40; Osram HBO 100 W/2 Hg lamp) equipped with a fluorescein isothiocyanate-Texas Red double filter (filter no. AC003; AdvanDx).
Two observers blind to the results of conventional identification examined the slides. Distinct green fluorescence in multiple fields of view was scored as a positive result (5, 9) and graded into two categories, faint and bright. Negative slides had a faintly reddish background smear; rarely a few fluorescent bacteria or bacterial clusters were observed, and such slides were also scored as negative.
For conventional identification subculture was performed on 5% horse blood agar, chocolate agar, CPS2 agar (bioMerieux) and vitamin K-enriched chocolate agar for anaerobic culture as deemed appropriate. BC isolates underwent definitive identification as follows: S. aureus by coagulase test with horse citrate plasma and Staph Plus (bioMerieux), C. albicans by chromogenic reaction on Candida ID plates (bioMerieux) and the Vitek2 system (bioMerieux), E. coli by the -glucuronidase reaction on the CPS2 plates provided a spot indole test was positive or, if the isolates were -glucuronidase or indole negative, by Vitek2. Finally P. aeruginosa was identified both by a conventional approach according to the Manual of Clinical Microbiology (2) and by Vitek2.
A total of 1,231 positive BC sets from 946 patients were included. The distribution of the BCs by bottle type was as follows: FA, 737; SA, 257; SN, 172; PF, 65. The distribution of pathogens appears in Table 1. Results obtained by PNA FISH and conventional methods appear in Tables 2 and 3, and the sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of the four assays appear in Table 4. We found close to 100% concordance with conventional identification for S. aureus and C. albicans, corroborating previous reports (1, 4, 5, 9). This study is the first application of PNA FISH for gram-negative rods. For E. coli, the results agreed with those by conventional methods except for one false-positive reaction with 1 of the 63 Klebsiella pneumoniae BCs. Thirty-four BCs grew P. aeruginosa, and identification by PNA FISH was correct for 32. Among 615 BCs with other gram-negative rods 6 were false positive, 4 of which showed a faint staining. These spurious reactions (one isolate each) included reactions for E. coli, Agrobacterium radiobacter, an Achromobacter sp., an Acinetobacter sp., Burkholderia cepacia, and an unidentified Pseudomonas sp. Therefore, sensitivity and PPV for P. aeruginosa PNA FISH were somewhat lower than those for the three other assays.
Polymicrobial growth was detected in 108 BCs. According to the protocol only one bottle per set was examined by PNA FISH, and discordant growth among bottles in some BC sets had an impact on the analysis of results (Table 1). Overall, we did not detect a poorer performance with polymicrobial samples; note, however, that two of five failures with the E. coli probe occurred with such samples (Table 2).
There are some limitations to the present study. It was conducted in a routine setting, and we cannot determine the exact cause of the spurious results. The preparatory steps prior to hybridization could be critical, as we obtained a bright staining of smears prepared from subcultures of one E. coli isolate and two P. aeruginosa isolates undetected by direct examination. Moreover, we cannot exclude the possibility that a Pseudomonas species actually was P. aeruginosa as indicated by PNA FISH.
Faint, but distinct, staining was seen both for P. aeruginosa and some other gram-negative rods belonging to the group of aerobic bacteria. Disregarding such reactions would lead to a lower sensitivity (30 of 34 BCs; 88.2%) and better specificity (613 of 615 BCs; 99.7%), the PPV would be better (30 of 32 BCs; 93.8%), and the NPV would be marginally lower (613 of 617 BCs; 99.4%). From a clinical point of view missing a P. aeruginosa diagnosis is undesirable, and therefore we favor a cutoff point as used in Tables 2 and 3.
Distinguishing E. coli and P. aeruginosa from other gram-negative rods should facilitate early directed antibiotic therapy. Negative results in both assays increase the likelihood of other gram-negative pathogens such as Klebsiella, Citrobacter, and Enterobacter spp., which often express chromosomal -lactamases.
ACKNOWLEDGMENTS
Skillful assistance by biotechnicians Lone Person and Jeanett Srensen is gratefully acknowledged.
This study was supported by grants from Nordjylland Amts Forskningsfond and Aalborg Hospital-Aarhus University Hospital to Mette Sgaard. Research at The Department of Clinical Microbiology, Aalborg Hospital-Aarhus University Hospital, is supported by Det Obelske Familiefond.
REFERENCES
Gonzalez, V., E. Padilla, M. Gimenez, C. Vilaplana, A. Perez, G. Fernandez, M. D. Quesada, M. A. Pallares, and V. Ausina. 2004. Rapid diagnosis of Staphylococcus aureus bacteremia using S. aureus PNA FISH. Eur. J. Clin. Microbiol. Infect. Dis. 23:396-398.
Kiska, D. L., and P. H. Gilligan. 2003. Pseudomonas, p. 719-728. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed., vol. 1. ASM Press, Washington, D.C.
Leibovici, L., I. Shraga, M. Drucker, H. Konigsberger, Z. Samra, and S. D. Pitlik. 1998. The benefit of appropriate empirical antibiotic treatment in patients with bloodstream infection. J. Intern. Med. 244:379-386.
Oliveira, K., S. M. Brecher, A. Durbin, D. S. Shapiro, D. R. Schwartz, P. C. De Girolami, J. Dakos, G. W. Procop, D. Wilson, C. S. Hanna, G. Haase, H. Peltroche-Llacsahuanga, K. C. Chapin, M. C. Musgnug, M. H. Levi, C. Shoemaker, and H. Stender. 2003. Direct identification of Staphylococcus aureus from positive blood culture bottles. J. Clin. Microbiol. 41:889-891.
Oliveira, K., G. W. Procop, D. Wilson, J. Coull, and H. Stender. 2002. Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J. Clin. Microbiol. 40:247-251.
Pedersen, G., H. C. Schnheyder, and H. T. Srensen. 2003. Source of infection and other factors associated with case fatality in community-acquired bacteremia—a Danish population-based cohort study from 1992 to 1997. Clin. Microbiol. Infect. 9:793-802.
Perry-O'Keefe, H., S. Rigby, K. Oliveira, D. Sorensen, H. Stender, J. Coull, and J. J. Hyldig-Nielsen. 2001. Identification of indicator microorganisms using a standardized PNA FISH method. J. Microbiol. Methods 47:281-292.
Pittet, D., and R. P. Wenzel. 1995. Nosocomial bloodstream infections. Secular trends in rates, mortality, and contribution to total hospital deaths. Arch. Intern. Med. 155:1177-1184.
Rigby, S., G. W. Procop, G. Haase, D. Wilson, G. Hall, C. Kurtzman, K. Oliveira, S. Von Oy, J. J. Hyldig-Nielsen, J. Coull, and H. Stender. 2002. Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J. Clin. Microbiol. 40:2182-2186.(M. Sgaard, H. Stender, an)