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P111 Interpretation of Gram stains of positive blood cultures – frequency and types of errors
S62
Poster Presentations
spp. and P. aeruginosa showed good concordance with standard method (SM) of testing. Repeat testing from an overnight culture is not necessary for these organisms unless Gram stain, overnight culture or AST results raise doubt about the ID or purity of the culture. This study aimed to further streamline the method of DI from positive BACTEC BC bottles. Methods: From Sept 2005 to Sept 2007, positive BC with GNB on Gram stain were included. BC positive with E. coli, Klebsiella spp., Salmonella spp. and P. aeruginosa had both DI and SM performed only if there were doubts about the ID. Other GNB had automatic DI and SM. Criteria for suspicious ID included atypical motility for the organism ID, <95% identity and resistant susceptibility pattern by DI. ID and AST results from DI and SM were compared. Discrepant ID was resolved either by API 20E or API 20NE or referral to the reference laboratory. Minor errors (mE), major errors (ME) and very major errors (VME) in AST were determined for each drug using SM as the gold standard. Results: Of 100 positive BC included in the study, 95 were identified correctly. 93 had ID agreement to species; all had 95% identity in the PHX by DI. 2 had ID agreement to genus with <95% identity. High concordance between DI and SM occurred for Enterobacter spp. (95%) and S. marcescens (92%); and for E. coli, Salmonella spp. and P. aeruginosa (all 100%) even when doubts were raised about their ID. ID of Klebsiella spp. had agreement of only 86%. All 5 misidentified isolates had either <95% identity, atypical motility or ID of infrequently isolated organism from BC. Overall AST agreement DI:SM = 98.5%. Of 29 AST errors, 18 (1%) were mE, 4 (0.2) were ME, and 5 (0.3%) were VME. 2/5 VME involved piperacillin (not in the current PHX GNB panel). Conclusion: PHX results obtained by DI from BC bottles with E. coli, Salmonella spp, P. aeruginosa, Enterobacter spp. and S. marcescens showed good concordance with SM. Repeat testing is not necessary unless DI produces <95% identity in PHX, atypical motility for organism identified, or an ID that is rarely seen in blood culture isolates.
P111 Interpretation of Gram stains of positive blood cultures frequency and types of errors
with negative Gram stains (acridine orange was also negative) and only one misinterpretation of the morphology (cocci rather than bacilli). Conclusions: Initial Gram stain reports of positive blood cultures are highly accurate, particularly for Gram positive cocci and Gram negative bacilli. When assessing competence of staff or training new staff, emphasis should be focused on areas with higher error rates which in our laboratory are related to polymicrobial cultures or associated with decolorization. P112 Rapid diagnosis of acute meningitis using reagent strips M. Mamani1 *, S. Hashemi1 , A. Niayesh1 , S. Jamal-Omidi1 . 1 Hamedan University of Medical Sciences, Hamedan, Iran Objective: Examination of cerebrospinal fluid (CSF) glucose, protein and cells is necessary for diagnosis of acute meningitis. Reagent strips have been used for rapid CSF analysis with varying results. The aim of this study was to determine the usefulness of reagent strips in the evaluation of pleocytosis, CSF glucose and protein levels for early diagnosis of meningitis. Methods: CSF samples from 79 patients with clinical suspicion of meningitis were tested for protein, glucose and leucocytes with reagent strips. The results were compared with those obtained from the standard laboratory evaluation. Results: CSF analysis by standard laboratory method identified 21 cases of probable bacterial meningitis, 18 of aseptic meningitis and 40 with normal CSF. By comparing the results of reagent strips we obtained that reagent strips leukocyte esterase test effectively distinguished bacterial from aseptic meningitis (sensitivity 95.2%, specificity 100%, P = 0.001) and bacterial meningitis from normal CSF (sensitivity 95%, specificity 82/5% P = 0.001). Conclusion: Reagent strips can be used for the rapid CSF analysis and distinguish normal from infected CSF and are valuable in the diagnosis of meningitis. P113 Diagnostic algorithm using a sensitive broth culture method for detection of Clostridium difficile toxin from stool samples
L. Roy1 *, M. Desjardins, B. Toye1 . 1 The Ottawa Hospital, Ottawa, Canada
P. Bayardelle1 *. 1 University of Montreal, Montreal, Canada
Background: Due to the serious nature of bloodstream infections, one of the most important roles of the microbiology laboratory is the initial notification of the Gram stain of a positive blood culture. This report is frequently used to alter or initiate antimicrobial therapy and is valued by physicians. We determined the frequency and types of errors in the interpretation of positive blood culture Gram stains in our laboratory over a 12 month period. Methods: Our consolidated microbiology laboratory processes blood cultures using the BacT/Alert system (with SA, SN, and PF bottles). All positive blood culture results (initial Gram stain and final culture reports) from Oct 1/07 Sept 30/08 were reviewed. Sources of information used included the lab information system worksheet and the blood culture bench log book. Culture results were used as the reference for comparison. Growing more than one organism group was considered polymicrobial. Discrepancies between initial Gram stain and culture results were reviewed and the cause determined. Results: 27,171 blood cultures were processed and 2,825 were positive (10.4%). There were only 26 discrepancies between the initial Gram stain and the final culture result (0.92%). There was variability between the rates of interpretation errors depending on the organisms cultured: 1/1525 (0.07%) for Gram positive cocci, 4/961 (0.4%) for Gram negative bacilli, 4/149 (2.7%) for Gram positive bacilli, 0/6 (0%) for Gram negative cocci, 2/121 (1.7%) for yeasts, and 15/63 (23.8%) for polymicrobial. Among the errors, the most common cause was failure to detect a Gram negative organism in a mixed culture with a Gram positive organism (10) and errors related to decolorization (8), particularly with reporting Bacillus as Gram-negative (3) or Acinetobacter as Gram-positive (2). There were 3 positive cultures
Objective: The glutamate dehydrogenase (GDH) antigen-cytotoxicity neutralization assay algorithm has been found to be reliable for the diagnosis of toxigenic Clostridium difficile. However, the sensitivity of the method is controversial. The second part of this approach is the direct cytotoxin neutralization assay (DCNA). The objective of this study was to compare the DCNA, using a fecal filtrate, with an indirect cytotoxin neutralization assay (ICNA), using broth culture of stool samples. Methods: During a three-month period all liquid and non formed stool samples submitted to our laboratory for detection of C. difficile toxin were studied. An algorithm was used: detection of GDH in a first step and if positive, the DCNA was performed. MRC-5 cells were used with and without specific C. difficile antitoxin to neutralize cytopathic effect in a 96 well microplate. All positive GDH stool samples and negative controls were inoculated in broth culture using chopped meat broth and tested with the cytotoxin neutralization assay for detection of C. difficile toxin. ICNA was also done on isolates of C. difficile obtained from subculture of broth on cycloserine-cefoxitin fructose agar to confirm broth culture results. The “gold standard” for toxigenic C. difficile was detection of C. difficile-specific GDH followed by toxin production by ICNA. Results: A total of 923 stool specimens from adult patients were tested during a 3 month period between June and August 2008. The prevalence of toxigenic C. difficile was 13.6%. 202 (21.9%) were GDH positive and 721 (71.8%) were GDH negative. 126 (62.4%) GDH positive stool specimens were positive by DCNA and 76 (37.6%) were negative. Of these 76 negative DCNA specimens, 17 were positive by the highly specific (96.45%) ICNA. The sensitivity of the two-step algorithm was
Poster Presentations
spp. and P. aeruginosa showed good concordance with standard method (SM) of testing. Repeat testing from an overnight culture is not necessary for these organisms unless Gram stain, overnight culture or AST results raise doubt about the ID or purity of the culture. This study aimed to further streamline the method of DI from positive BACTEC BC bottles. Methods: From Sept 2005 to Sept 2007, positive BC with GNB on Gram stain were included. BC positive with E. coli, Klebsiella spp., Salmonella spp. and P. aeruginosa had both DI and SM performed only if there were doubts about the ID. Other GNB had automatic DI and SM. Criteria for suspicious ID included atypical motility for the organism ID, <95% identity and resistant susceptibility pattern by DI. ID and AST results from DI and SM were compared. Discrepant ID was resolved either by API 20E or API 20NE or referral to the reference laboratory. Minor errors (mE), major errors (ME) and very major errors (VME) in AST were determined for each drug using SM as the gold standard. Results: Of 100 positive BC included in the study, 95 were identified correctly. 93 had ID agreement to species; all had 95% identity in the PHX by DI. 2 had ID agreement to genus with <95% identity. High concordance between DI and SM occurred for Enterobacter spp. (95%) and S. marcescens (92%); and for E. coli, Salmonella spp. and P. aeruginosa (all 100%) even when doubts were raised about their ID. ID of Klebsiella spp. had agreement of only 86%. All 5 misidentified isolates had either <95% identity, atypical motility or ID of infrequently isolated organism from BC. Overall AST agreement DI:SM = 98.5%. Of 29 AST errors, 18 (1%) were mE, 4 (0.2) were ME, and 5 (0.3%) were VME. 2/5 VME involved piperacillin (not in the current PHX GNB panel). Conclusion: PHX results obtained by DI from BC bottles with E. coli, Salmonella spp, P. aeruginosa, Enterobacter spp. and S. marcescens showed good concordance with SM. Repeat testing is not necessary unless DI produces <95% identity in PHX, atypical motility for organism identified, or an ID that is rarely seen in blood culture isolates.
P111 Interpretation of Gram stains of positive blood cultures frequency and types of errors
with negative Gram stains (acridine orange was also negative) and only one misinterpretation of the morphology (cocci rather than bacilli). Conclusions: Initial Gram stain reports of positive blood cultures are highly accurate, particularly for Gram positive cocci and Gram negative bacilli. When assessing competence of staff or training new staff, emphasis should be focused on areas with higher error rates which in our laboratory are related to polymicrobial cultures or associated with decolorization. P112 Rapid diagnosis of acute meningitis using reagent strips M. Mamani1 *, S. Hashemi1 , A. Niayesh1 , S. Jamal-Omidi1 . 1 Hamedan University of Medical Sciences, Hamedan, Iran Objective: Examination of cerebrospinal fluid (CSF) glucose, protein and cells is necessary for diagnosis of acute meningitis. Reagent strips have been used for rapid CSF analysis with varying results. The aim of this study was to determine the usefulness of reagent strips in the evaluation of pleocytosis, CSF glucose and protein levels for early diagnosis of meningitis. Methods: CSF samples from 79 patients with clinical suspicion of meningitis were tested for protein, glucose and leucocytes with reagent strips. The results were compared with those obtained from the standard laboratory evaluation. Results: CSF analysis by standard laboratory method identified 21 cases of probable bacterial meningitis, 18 of aseptic meningitis and 40 with normal CSF. By comparing the results of reagent strips we obtained that reagent strips leukocyte esterase test effectively distinguished bacterial from aseptic meningitis (sensitivity 95.2%, specificity 100%, P = 0.001) and bacterial meningitis from normal CSF (sensitivity 95%, specificity 82/5% P = 0.001). Conclusion: Reagent strips can be used for the rapid CSF analysis and distinguish normal from infected CSF and are valuable in the diagnosis of meningitis. P113 Diagnostic algorithm using a sensitive broth culture method for detection of Clostridium difficile toxin from stool samples
L. Roy1 *, M. Desjardins, B. Toye1 . 1 The Ottawa Hospital, Ottawa, Canada
P. Bayardelle1 *. 1 University of Montreal, Montreal, Canada
Background: Due to the serious nature of bloodstream infections, one of the most important roles of the microbiology laboratory is the initial notification of the Gram stain of a positive blood culture. This report is frequently used to alter or initiate antimicrobial therapy and is valued by physicians. We determined the frequency and types of errors in the interpretation of positive blood culture Gram stains in our laboratory over a 12 month period. Methods: Our consolidated microbiology laboratory processes blood cultures using the BacT/Alert system (with SA, SN, and PF bottles). All positive blood culture results (initial Gram stain and final culture reports) from Oct 1/07 Sept 30/08 were reviewed. Sources of information used included the lab information system worksheet and the blood culture bench log book. Culture results were used as the reference for comparison. Growing more than one organism group was considered polymicrobial. Discrepancies between initial Gram stain and culture results were reviewed and the cause determined. Results: 27,171 blood cultures were processed and 2,825 were positive (10.4%). There were only 26 discrepancies between the initial Gram stain and the final culture result (0.92%). There was variability between the rates of interpretation errors depending on the organisms cultured: 1/1525 (0.07%) for Gram positive cocci, 4/961 (0.4%) for Gram negative bacilli, 4/149 (2.7%) for Gram positive bacilli, 0/6 (0%) for Gram negative cocci, 2/121 (1.7%) for yeasts, and 15/63 (23.8%) for polymicrobial. Among the errors, the most common cause was failure to detect a Gram negative organism in a mixed culture with a Gram positive organism (10) and errors related to decolorization (8), particularly with reporting Bacillus as Gram-negative (3) or Acinetobacter as Gram-positive (2). There were 3 positive cultures
Objective: The glutamate dehydrogenase (GDH) antigen-cytotoxicity neutralization assay algorithm has been found to be reliable for the diagnosis of toxigenic Clostridium difficile. However, the sensitivity of the method is controversial. The second part of this approach is the direct cytotoxin neutralization assay (DCNA). The objective of this study was to compare the DCNA, using a fecal filtrate, with an indirect cytotoxin neutralization assay (ICNA), using broth culture of stool samples. Methods: During a three-month period all liquid and non formed stool samples submitted to our laboratory for detection of C. difficile toxin were studied. An algorithm was used: detection of GDH in a first step and if positive, the DCNA was performed. MRC-5 cells were used with and without specific C. difficile antitoxin to neutralize cytopathic effect in a 96 well microplate. All positive GDH stool samples and negative controls were inoculated in broth culture using chopped meat broth and tested with the cytotoxin neutralization assay for detection of C. difficile toxin. ICNA was also done on isolates of C. difficile obtained from subculture of broth on cycloserine-cefoxitin fructose agar to confirm broth culture results. The “gold standard” for toxigenic C. difficile was detection of C. difficile-specific GDH followed by toxin production by ICNA. Results: A total of 923 stool specimens from adult patients were tested during a 3 month period between June and August 2008. The prevalence of toxigenic C. difficile was 13.6%. 202 (21.9%) were GDH positive and 721 (71.8%) were GDH negative. 126 (62.4%) GDH positive stool specimens were positive by DCNA and 76 (37.6%) were negative. Of these 76 negative DCNA specimens, 17 were positive by the highly specific (96.45%) ICNA. The sensitivity of the two-step algorithm was