Monday, October 31, 2011

Commercial Kits for Identification of Gram negative bacilli

All commercial identification systems are based on one of five different technologies or a combination thereof. These include pH-based reactions that require from 15 to 24 h of incubation, enzyme-based reactions that require 2 to 4 h, utilization of carbon sources, visual detection of bacterial growth, or detection of volatile or nonvolatile fatty acids via gas chromatography (O’Hara,et al, 2003).

In pH-based reactions, a positive test is indicated by a change in the color of one or more dyes. When a carbohydrate is utilized, the pH becomes acidic; when protein is utilized or there is release of a nitrogen-containing compound, the pH becomes alkaline. These reactions are influenced by the inoculum size, incubation time, and temperature of the reaction. In enzyme-based system, color changes were due to the hydrolysis of a colorless complex by an appropriate enzyme with the resulting release of a chromogen or fluorogen. Because the incubation times needed for assay of enzymatic activities were shorter than those required for pH-based media, chance of contamination was not a critical factor (Caroline M and O’Hara., 2005).

In the third type of reaction, utilization of carbon sources, there is a transfer of electrons from an organic product to the dye tetrazolium violet, which is incorporated within each test well. That transfer causes a colorimetric change in the dye, signaling the increased cellular respiration that occurs during the oxidation process. These reactions may occur in as little as 4 h. The fourth method is a simple visual detection of growth of the test organism (increased turbidity) in the presence of a substrate. Results are determined by comparing a control well to the test well and may utilize a Wickerham card to read turbidity. This type of reaction may be difficult to read and always involves a minimum of overnight incubation. The last technology involves detecting the end products of cellular fatty acid metabolism. The end products are displayed on chromatographic tracings that are compared to a library of known patterns. This technology is not commonly used as it is more complex (Caroline M and O’Hara., 2005).

Manual Identification System

The studies cited are those which compared product identifications to identifications obtained by using conventional biochemicals. (Caroline M and O’Hara., 2005)

In 1971, Washington et al. published the first evaluation of the API 20E, originally owned by the Analytab Products Division of American Home Products, which has been owned since 1986 by bioMérieux, Inc. (Durham, N.C.). In the evaluation by Washington et al., approximately 93.0% of the 129

Enterobacteriaceae and five Aeromonas strains were correctly identified to species level (Washington, et al ., 1971).

An impermeable plastic backing support 20 cupules that contain pH-based substrates that have not changed since the product was originally designed in 1970.The database has expanded from 87 taxa in 1977 to 102 taxa in 2003 and includes Y. pestis. The current database is version 4.0 (Caroline M and O’Hara., 2005).

Constructed along the same lines as the API 20E, the API 20NE (bioMe´rieux) has 20 cupules that contain 8 conventional substrates and 12 assimilation tests. Suspensions are prepared in 0.85% NaCl for inoculation into the 8 conventional substrates and in AUX medium for inoculation into the 12 assimilation cupules. The database contains 32 genera and 64 species of nonfastidious gram-negative rods not belonging to the Enterobacteriaceae. The seven-digit profile number is converted to an identification by using the APILAB software, version 3.3.3 (Caroline M and O’Hara., 2005).

API RapiD 20E
Originally marketed in 1982 as the Rapid E system (DMS Laboratories, Flemington, N.J.) but owned since 1986 by API and subsequently by bioMe´rieux Inc., the API RapiD 20E system is designed to identify Enterobacteriaceae in 4 h. Similar to the API 20E in its test configuration, this system has 20 microtubes that contain substrates for the demonstration of enzymatic activity or fermentation of carbohydrates. The seven-digit profile number that is compiled from the test reactions is entered into the APILAB software. The current version of the RapiD 20E software is 3.0. The database

contains 26 genera and 65 species. Identifications are also available by using the Analytical Profile Index (Caroline M and O’Hara., 2005).

Crystal E/NF
Introduced in 1993 by Becton Dickinson (Sparks, Md.), the BBL Crystal Enteric/Nonfermenter (E/NF) ID kit is for the identification of Enterobacteriaceae and more commonly isolated glucose-fermenting and nonfermenting gram-negative bacilli. The plastic panels include 30 tests for the fermentation, oxidation, degradation, or hydrolysis of various substrates. Once the panel is inoculated and snapped together with its lid, it becomes a sealed system posing little risk of exposure to the technologist. The current software version is 4.0 and contains 38 genera and 104 species (Caroline M and O’Hara., 2005).

Automated Identification Systems

With the advancement of automated testing in chemistry and hematology laboratories in the mid-1970s, it was only logical that some degree of automation in microbiology would eventually follow. (Caroline M, et al., 2005). From that, in 1973, the automicrobic system (AMS) (McDonnell Douglas Corp., St. Louis, Mo.) was born. It incorporated a disposable miniaturized plastic specimen-handling system, solid state optics for microbial detection, and a minicomputer for control and processing. Now it is recognized as the first generation of the Vitek instruments. (Aldridge, et al., 1977). Within 10 years, Vitek's competitors included the MS-2 (Abbott Diagnostics, Inc., Chicago, Ill.), the Autobac IDX (Pfizer Inc., Groton, Conn. And General Diagnostics, Morris Plains. N.J.), and the Autoscan-3 (Microscan Corp., Hillsdale, N.J.). Technology
had enabled valid results to be obtained in as little as 4 h. Microbiology was definitely on the fast track to rapid testing and shorter turnaround times (Caroline M and O’Hara., 2005).

Dade Behring MicroScan
In 1981, American MicroScan (then located in Hillsdale, N.J.) introduced the autoSCAN-3, a semiautomated instrument that utilized microdilution trays containing frozen conventional substrates for identification of bacterial isolates. An early evaluation, incorporating both Enterobacteriaceae and nonfermenters, by Ellner and Myers in 1981 reported an agreement of 95.0% between visually read and automated identification thus ensuring that machines were capable of accurate interpretations of the reactions in each well (Ellner, P. D., and D. A. Myers 1981). The company then introduced the autoSCAN-4 in 1983, which brought with it improved dry panels that did not require refrigeration and included an updated database. Baxter Healthcare Corporation and subsequently Dade Behring have owned the company since its move to West Sacramento, Calif. In 1986, the auto-SCAN-WalkAway came into the marketplace.

This instrument is a combination incubator-reader that monitors the growth in bacterial identification panels in a completely “hands-off” method. This instrument has become known as the WalkAway. One of MicroScan’s goals was to shorten the turnaround times for test results by using fluorogenic substrates in the panels.These “rapid” panels were first marketed in 1989. The data management system, called LabPro, runs on an adjacent computer (Caroline M and O’Hara., 2005).

Neg ID type 2. The Neg ID type 2 panel was introduced in 1988 for the identification to species level of aerobic and facultatively anaerobic gram-negative bacilli and was designed to be read either manually or on the WalkAway instrument. The clear plastic 96-well tray contains 26 conventional substrates and 6 antimicrobials for inhibition of growth, all in dried form, and requires overnight incubation (Caroline M and O’Hara., 2005).

Rapid Neg ID type 3. The Rapid Neg ID type 3 panel was introduced in approximately 1998 as an update of the Neg ID type 2 panel. The Rapid Neg ID type 3 replaced 10 of the substrates on the Neg ID type 2 panel with newer ones and eliminated the need for the mineral oil overlay on the decarboxylase test. It also increased the shelf life from 6 months to 1 year when stored at 2 to 8°C. The rapid panel utilizes 36 that work by one of the following mechanisms: hydrolysis of fluorogenic substrates, pH changes following substrate utilization, production of specific metabolic by-products, or evaluation of the rate of production of specific metabolic by-products after 2.5 h of incubation (O’Hara, et al, 2000). These panels can be processed only on a WalkAway instrument, as their opaque color prevents a visual read of the wells. The bacterial suspensions must be made from 18 to 24 h colonies grown on Mac-Conkey agar plates with lactose and crystal violet ( Caroline M and O’Hara., 2005).

The current database is LabPro 1.51, which contains 44 genera and 125 species of both Enterobacteriaceae and oxidase-positive glucose-fermenting and non-fermenting gram-negative bacilli. The database includes Y. pestis, V. cholerae, and E. coli O157:H7. There have been several reports indicating the usefulness and accuracy of direct bacterial identification with inocula from positive blood culture bottles. A study by Waites et al. indicated 99%

concordance between gram-negative identifications when blood was concentrated and the bacterial pellet was used to directly inoculate the panels and identifications resulting from standard biochemical methods (Waites, et al, 1998).

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