Evaluation of The BIOMIC V3 Microbiology System for Identification of Selected Species on BBL CHROMagar Orientation and CHROMagar MRSA ACCEPTED

Transcription

1 JCM Accepts, published online ahead of print on 13 August 2008 J. Clin. Microbiol. doi: /jcm Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Evaluation of The BIOMIC V3 Microbiology System for Identification of Selected Species on BBL CHROMagar Orientation and CHROMagar MRSA Ellen Jo Baron 1*, Holly D'Souza 1, Andrew Qi Wang 2, David L. Gibbs 2. Stanford University Department of Pathology 1, Stanford,California and Giles Scientific Inc. 2, Santa Barbara, California. *Corresponding author, mailing address: Clinical Microbiology Laboratory, Stanford University Medical Center, Room 1602, 3375 Hillview Ave. Palo Alto, CA Phone , Fax , The BIOMIC V3 microbiology system identifies bacteria by reading the color of colonies selected by users. For CHROMagar Orientation, BIOMIC agreed with conventional methods for 94% of strains assayed. For CHROMagar MRSA, BIOMIC correctly identified 100% of MRSA strains tested and did not misidentify 2 MSSA growing on the plates. Page 1 of 9

2 Use of CHROMagar Orientation agar (BBL, Becton Dickinson, Sparks, MD) for primary isolation enables species identification comparably faster than the rapid abbreviated methods described in CLSI M35 (Abbreviated identification of bacteria and yeast), and 4-24 hours earlier than do traditional methods. (1,6,7,9,10,12) It can also facilitate picking colonies for subsequent conventional identification. Chromogenic agars use proprietary substrates that react with enzymes of specific species to impart colors to colonies. CHROMagar Orientation is designed to allow technologists to recognize primary urinary tract pathogens without further manipulations. Expected colony appearances are: Escherichia coli, dark rose to pink; Enterococcus, small blue-green; Klebsiella/Enterobacter/Serratia group, larger dark blue; Proteus/Providencia/Morganella, pale to beige; Staphylococcus saprophyticus, small light pink to rose; and Streptococcus agalactiae, pinpoint light blue. CHROMagar MRSA is useful for screening for methicillin/oxacillin resistant Staphylococcus aureus (MRSA) colonization. (2-4,8) MRSA appear as medium size mauve colonies. Other organisms are not expected to grow well on this medium. Earlier organism identification may allow faster initiation of decolonization treatment and shorten the transmission opportunity time of a colonized patient. This study was designed primarily to determine if the BIOMIC V3 automated plate reading instrument could interpret the colony color of isolates on primary urine culture plates and thus identify colonies of species for which CHROMagar Orientation has indications as accurately as could an experienced microbiologist. Secondly, the BIOMIC V3 instrument was evaluated for its ability to accurately interpret the colony color of a group of S. aureus subcultured from original cultures to accurately differentiate between MRSA and methicillin-susceptible S. aureus. CHROMagar Orientation plates were inoculated with routine urine specimens sent for culture, yielding 182 isolates from pure cultures and 64 isolates from mixed cultures that included multiple species of pathogens and contaminants (skin or genital microbes) (Table 1). Isolates considered to be contaminants were not included in this study. 246 organism identifications were visually determined on routine CHROMagar Orientation plates and subsequently confirmed (or not) by traditional methods in the Stanford University Medical Center laboratory. The BIOMIC V3 Microbiology System (Giles Scientific Inc, Santa Barbara, CA; was used in parallel to identify semi-automatically, based on color likeness, those same 246 organisms on the CHROMagar Orientation plates. CHROMagar MRSA agar plates were inoculated with 105 previously identified Staphylococcus aureus strains, primarily MRSA, isolated from routine cultures sent to our laboratory (Table 2). Whether plates are inoculated with nares swabs or from colony growth, only isolated colonies chosen by the technologist are evaluated in the BIOMIC system, so use of subcultures from known strains does not negate the results. The user places a culture plate with visible colonies (usually after overnight incubation) onto the pull-out tray of the BIOMIC V3 and allows the instrument to show the image on the screen. The user manipulates the mouse to point to a colony on the plate image to obtain a probable species identification (ID); the ID and plate image are then saved and the next plate is inserted into the instrument. The BIOMIC V3 reports the probable species identification with a color likeness Page 3 of 9

3 percent that is relative to the ideal color profile established for each species. The color likeness percent provides an objective quantitative measurement of colony color. A higher percentage likeness indicates a more typical color for that organism on the specific CHROMagar plate being evaluated. The user chooses the CHROMagar plate type before obtaining a reading, since the instrument has been programmed to interpret colonies on several CHROMagar plate formulas. The overall agreement for the BIOMIC between genus or species identification on CHROMagar Orientation with traditional methods including CLSI M35 Rapid Identification of Bacteria and Yeast, Vitek (Legacy, BioMerieux, Durham, NC) used for gram-negative rods, or MicroScan Walkaway (Dade MicroScan, Sacramento, CA) used for gram-positive cocci, was 94%. Gram positive isolates in general had lower percent agreements with conventional identifications than did gram negative strains. The color likeness of identified colonies ranged from 71 to 95 %. Among 35 strains for which CHROMagar Orientation has no specific identfication claims, there were 4 (11.4%) for which BIOMIC assigned a color that led to an incorrect identification (Table 1). These strains demonstrated colors and morphologies that a technologist would have correctly interpreted as needing further tests for identification. The manufacturer plans to use these results to fine-tune the software. The 31 other strains were not given an identification by the BIOMIC instrument. The overall agreement between identification on CHROMagar MRSA with routine methods, which included CLSI M35 or for inconsistent results API Staph strip (biomerieux, Marcy L Etoile, France); and for susceptibilities MicroScan Walkaway plus oxacillin agar screen, was 100%. The average color likeness of identified MRSA colonies was 92% (Table 2). Two of 15 MSSA organisms grew as clear colonies on CHROMagar MRSA, but were not reported as MRSA. There were no false positives; that is, no MSSA isolates were reported as MRSA. The slightly lower agreement between some species identifications on CHROMagar Orientation and traditional identification methods suggests that selected species with lower color likeness as determined by the BIOMIC V3 may require additional identification methods beyond simple visual or automated color interpretation. However, because CHROMagar Orientation was developed for use with urine cultures, it is expected that laboratories may be willing to sacrifice some accuracy of identification for the increased speed and efficiency gained by using that medium for routine urine specimens, consistent with good patient care practices, as determined with clinician consultation (1). A possible reason for lower agreement for S. saprophyticus could be that some isolates are overlooked on standard media, as suggested by Hengstler et al. (6). The agreement between CHROMagar MRSA and traditional identification methods mirrors those of others and suggests that this medium is useful for screening primary specimens for the presence of MRSA. (2-5,8,11) The BIOMIC instrument did not misidentify the two MSSA (13% of MSSA tested) that yielded atypical colonies on the CHROMagar MRSA. The BIOMIC V3 usually provided accurate color readings of colonies on CHROMagar Orientation and MRSA media that were equivalent to those of an experienced technologist. The BIOMIC V3 System provides the advantage of reporting the objective quantitative % likeness Page 4 of 9

4 1 2 parameter, and provides the potential to reduce technologist training, reduce reading variation between technologists, save time, and save plate images for later review. Page 5 of 9

5 Reference List 1. D'Souza, H. A., M. Campbell, and E. J. Baron Practical bench comparison of BBL CHROMagar Orientation and standard two-plate media for urine cultures. J.Clin.Microbiol. 42: Diederen, B., D. van, I, B. A. van, P. Willemse, K. P. Van, and J. Kluytmans Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus. J.Clin.Microbiol. 43: Flayhart, D., J. F. Hindler, D. A. Bruckner, G. Hall, R. K. Shrestha, S. A. Vogel, S. S. Richter, W. Howard, R. Walther, and K. C. Carroll Multicenter evaluation of BBL CHROMagar MRSA medium for direct detection of methicillin-resistant Staphylococcus aureus from surveillance cultures of the anterior nares. J.Clin.Microbiol. 43: Han, Z., E. Lautenbach, N. Fishman, and I. Nachamkin Evaluation of mannitol salt agar, CHROMagar Staph aureus and CHROMagar MRSA for detection of meticillin-resistant Staphylococcus aureus from nasal swab specimens. J.Med.Microbiol. 56: Hedin, G. and H. Fang Evaluation of two new chromogenic media, CHROMagar MRSA and S. aureus ID, for identifying Staphylococcus aureus and screening methicillinresistant S. aureus. J.Clin.Microbiol. 43: Hengstler, K. A., R. Hammann, and A. M. Fahr Evaluation of BBL CHROMagar orientation medium for detection and presumptive identification of urinary tract pathogens. J.Clin.Microbiol. 35: Houang, E. T., P. C. Tam, S. L. Lui, and A. F. Cheng The use of CHROMagar Orientation as a primary isolation medium with presumptive identification for the routine screening of urine specimens. APMIS 107: Nahimana, I., P. Francioli, and D. S. Blanc Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and CHROMagar MRSA) and ORSAB for surveillance cultures of methicillin-resistant Staphylococcus aureus. Clin.Microbiol.Infect. 12: NCCLS Abbreviated identification of bacteria and yeast; approved guideline. NCCLS, Wayne, PA. 10. Ohkusu, K Cost-effective and rapid presumptive identification of gram-negative bacilli in routine urine, pus, and stool cultures: evaluation of the use of CHROMagar orientation medium in conjunction with simple biochemical tests. J.Clin.Microbiol. 38: Pape, J., J. Wadlin, and I. Nachamkin Use of BBL CHROMagar MRSA medium for identification of methicillin-resistant Staphylococcus aureus directly from blood cultures. J.Clin.Microbiol. 44: Rigby, J. L. and T. J. Neal Evaluation of CHROMagar orientation medium for the identification of surveillance cultures: a novel method. Br.J.Biomed.Sci. 55: Page 7 of 9

6 TABLE 1. CHROMagar Orientation organism identification by BIOMIC V3 compared to experienced technologists using visual and conventional methods. Organisms Isolates from Pure Culture Isolates from Mixed Cultures Agreement Between Methods (%) Average % Color Likeness 1 Range of color Likeness (%) Escherichia coli Enterococcus species Klebsiella, Enterobacter, Serratia Group Proteus, Morganella, Providencia Group Staphylococcus saprophyticus Group B streptococci Other (staphylococci, yeast, Not Pseudomonas aeruginosa, others) applicable Total Isolates Not applicable 1. Color likeness equals the % color that matches the established color profile for each species. A higher percentage indicates a more typical color on CHROMagar Orientation TM plates. A lower % color likeness indicates greater heterogeneity in color reactions of a genus or species (Example: E. coli). 2. A positive PYR test (performed from blood agar) is required to distinguish enterococci from Group B streptococci. Some color overlap is common between these two species, so laboratory protocols should include performance of PYR when such morphologies are seen. Very young (small) colonies of the Enterobacter group could also be mistaken for Enterococcus due to similar color ranges, although additional incubation results in increased colony size for Enterobacter group strains but not for enterococci, which can be distinguished by a microbiologist. 3. Colony morphology, Gram stain, specimen source, and novobiocin 5 mcg disk diffusion (resistance) are required to identify Staphylococcus saprophyticus. BIOMIC misidentified one isolate as S. saprophyticus. Page 8 of 9

7 4. Four of 35 organisms had an incorrect color reading by the instrument which led to wrong ("False Positive") identifications. TABLE 2. CHROMagar MRSA identification by BIOMIC V3 compared to traditional identification methods. Organisms Growth on CHROMagar No growth on CHROMagar Agreement Between BIO MIC V3 and Conventional Methods (%) Average % Color Likeness Range of Color Likeness (%) MRSA MSSA Not applicable 1. Two of 15 MSSA isolates grew on CHROMagar MRSA media but as they were not mauve and not typical MRSA colonies, they were not misidentified as MRSA. Page 9 of 9