Thursday, August 25, 2011

Automated Microbiology Identification Systems

The development of the first generation of automated equipment for clinical microbiology involved essentially two approaches. One can be described as the mechanization of existing techniques. The second combined mechanization with other changes, such as miniaturization and/or incorporation of innovative substrates, inhibitors, or indicators. The primary goal was to enhance data acquisition and processing, particularly with regard to decreasing turnaround time.
The systems reviewed here vary considerably in their approach to the identification of microorganisms. The MIS, for example, analyzes cellular material, whereas others use more conventional end points such as increase in cell density or color changes due to shifts in pH. There is also some variability in the degree of automation, spectrum of organisms identified, and turnaround time. The constant is that most of these instruments have proven their applicability in clinical microbiology laboratories throughout the country.
The devices described here are, with the exception of the Vitek urine card, based on pure culture techniques. In other words, the organism must be isolated before the identification process is performed.
There are, however, procedures, both manual and automated, that can identify organisms directly in specimens by using antibodies or nucleic acid probes.

The Vitek system, based on bacterial growth in micro wells of thin plastic cards These 30 microwell cards contained antibiotics or biochemical substrates. Susceptibility test cards are available for both gram-negative bacilli and gram-positive bacteria with 11 antimicrobial agents per card. Results are available in 4 to 8 h. Vitek has a variety of standard test kits, and
custom-defined test kits can be purchased. Over 40 antimicrobial agents are currently available on the cards, and results from each test include an interpolated MIC, as well as the National Committee for Clinical Laboratory Standards categories of susceptible, moderately susceptible, intermediate, and resistant.
Identification cards automatically interpreted by the Vitek system are the Gram-Negative Identification Test Kit (GNI), the Gram-Positive Identification Test Kit (GPI), and the Yeast Biochemical Test Kit (YBC).
Identification cards that require off-line incubation and manual entry of the results into the Vitek computer are the Anaerobe Identification Test Kit, Neisseria/Haemophilus Identification Test Kit, and Enteric Pathogen Screen Test Kit.
The GNI and GPI each contain 29 substrates and a growth control medium. The GNI substrates include 25 conventional biochemical substrates, 3 proprietary substrates, and 1 antibiotic. The GNI must be marked if the organism is oxidase positive. The GNI data base includes information for identification of 46 species of members of the family Enterobactenaceae and 39 species of other gram-negative organisms. The GPI substrates include 26 based on conventional biochemical tests, two antibiotics, and one dye. The
GPI must be marked for catalase-negative, beta-hemolytic organisms or for coagulase-positive organisms that are catalase positive.
The GPI data base includes information for identification of 23 Streptococcus species, 4 Enterococcus species, 16 Staphylococcus species, and 4 Corynebacterium, Aerococcus, Listenia monocytogenes, and Erysipelothrix rhusiopathiae species or groups.
The YBC contains 26 substrates which are based on conventional methods. The YBC data base includes information for identification of 16 Candida species, 6 Cryptococcus species, 3 Rhodotorula species, 2 Tnichosporon species, 3 Geotnichum species, 2 Prototheca species, and single species of four additional genera.
The Vitek system is an integrated modular system consisting of a filling-sealer unit, reader-incubator, computer control module, data terminal, and multicopy printer. The Vitek system can be purchased with a capacity of 30, 60, 120, or 240 cards and can be interfaced with other computers. A
data management center can be added. Inocula for the identification cards are prepared from selective (GNI or GPI) or nonselective Inocula for the GNI, GPI, and YBC are prepared by suspending several colonies in 1.8 ml of 0.45 to 0.5% saline and adjusting the suspension to the equivalent of
a no. 1 (GNI and GPI) or a no. 2 (YBC) McFarland standard.
The inoculum is automatically transferred to the card via a transfer tube during the vacuum cycle of the filling module.
The GNI and GPI are placed in plastic trays, each tray holding up to 30 cards. The tray is placed in the reader incubator at 35°C, and at hourly intervals a digitized analog optical reading, proportional to the light attenuation for each test well, is obtained for each card. The first reading usually establishes a baseline value, and the amount of light reduction
caused by growth or a biochemical reaction in the microwell is determined on subsequent readings. A predetermined minimum change is required to differentiate between positive and negative reactions. Final identification by
the GNI is reported between 4 and 18 h. Most of the non-glucose-fermenting gram-negative bacilli are reported at 18 h. Organisms are identified by the GPI between 4 and 15 h. The YBC is incubated off-line at 30°C for 24 h and then placed in the reader-incubator for a single reading. A message "reincubate for 24 h at 30°C" indicates that a definitive identification requires more incubation time. At 48 h, one must fill in the 48H mark on the card and obtain a second reading. The biochemical test results for all cards are compared with the data base, and the first and second choices, as well as their absolute likelihoods and normalized percent probabilities, are reported. The biochemical test results, as well as supplemental tests if required, are printed.
The Sensititre fluorogenic system (Radiometer America, Inc., Westlake, Ohio) is a modular system composed of a computer and an automated reader. This system identifies gram-negative bacilli and performs susceptibility tests on both gram-positive and gram-negative bacteria in either 5 or 18 h. Breakpoint or MIC capability for gram-negative and gram-positive bacteria is available for 54 antimicrobial agents. The Sensititre AP80 panel data base includes information for the identification of 84 members of the family Enterobacteriaceae, 24 oxidase-positive fermenters, 16 pseudomonads, and 16 other nonfermenters.
The WalkAway-96 (formerly called the autoSCAN-W/A) and WalkAway-40 (Baxter Diagnostics, Inc., MicroScan Division, West Sacramento, Calif.) are computer-controlled systems that will incubate microtiter panels and automatically interpret biochemical or susceptibility results with either a photometric or a fluorogenic reader. MicroScan also manufactures the autoSCAN-4, which requires off-line incubation and, with the exception of fluorogenic panels, will test the same panels as the other two automated systems. All three systems perform susceptibility tests on aerobic gram negative bacilli, gram-positive bacteria, and anaerobes.
Fluorogenic panels provides a 3.5- to 7-h susceptibility result for aerobic gram-negative bacilli and a 3.5- to 15-h susceptibility result for aerobic gram-positive bacteria. Conventional panels provides a 15- to 24-h susceptibility result for aerobic bacteria and a 24- to 48-h susceptibility result for anaerobes.
MicroScan has numerous panel types, including both MIC and breakpoint susceptibility panels. Custom panels are also available. The three systems automatically identify gram-
negative bacilli, gram-positive bacteria, fastidious aerobic bacteria, anaerobes, and yeasts.
The rapid fluorogenic panels for identification of gram negative bacilli (R-GNB) and gram-positive bacteria (RGPB) use fluorogenic substrates (4-methylumbelliferone or 7-amino-4-methylcoumarin attached to a phosphate, sugarmoiety, or amino acid) or fluorometric indicators. Identification is based on hydrolysis of fluorogenic substrates, pH changes following substrate utilization, production of specific metabolic by-products, or the rate of production of specific metabolic by-products after 2 h of incubation. The modified conventional panel for identification of gram-negative bacilli (GNB) has 29 modified conventional or chromogenic tests and six antibiotics, and results are available in 15 to 42 h. Both the R-GNB and GNB data bases include information for identification of 59 groups, genera, or species of members of the Enterobacteriaceae and 57 groups, genera,
or species of nonfermentative and oxidase-positive gram-negative bacilli. The modified conventional panel for identification of gram-positive bacteria (GPB) has 25 modified conventional or chromogenic substrates, one dye, and three antibiotics and yields an identification in 15 to 42 h.
Both the R-GPB and GPB data bases include information for identification of 24 genera or species of the family Micrococcaceae, 18 members of the family Streptococcaceae, 4Enterococcus spp., Aerococcus viridans, and L. monocytogenes.
The MicroScan Rapid Haemophilus and Neisseria Identification Panel (HNID) has 17 modified conventional or chromogenic tests and one antibiotic, and the data base includes information for identification of Haemophilus influenzae (seven biotypes), Haemophilus parainfluenzae (four biotypes), Haemophilus aphrophilus-Haemophilus paraphrophilus,
Haemophilus haemolyticus, four Neisseria species, Moraxella catarrhalis, and Gardnerella vaginalis in 4h. The MicroScan Rapid Anaerobe Identification Panel (AIP) has 24 modified conventional or chromogenic substrates, and the data base includes information for identification of 21 anaerobic gram-negative bacilli, 13 anaerobic non-spore-forming gram-positive bacilli, 8 anaerobic gram positive cocci, and 16 clostridia in 4 h without anaerobic incubation. The MicroScan Rapid Yeast Identification Panel (YIP) has 27 modified conventional or chromogenic substrates, and the data base includes information for identification of 16 Candida spp. or biotypes, 8 Cryptococcus spp., and 11 genera (representing 16 species) of yeasts or yeast like organisms in 4 h.

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