Diagnostic Microbiology Chapter 5

automated systems in the lab

- The MicroScan® System - The Sensititre System - BD PhoenixTM Automated Microbiology System - The VITEK® System - The MicroScan WalkAway - Rapid Negative Identification Panel

VITEK System

1.) Computer based 2.) Uses the Vitek Card 3) Lab evaluation identifies most clinically significant bacteria and yeast and also performs qualitative and quantitative antibiotic susceptibility tests Can also be used for antibiotic susceptibility testing for gram-positive and gram-negative organisms. A gram-positive or gram-negative susceptibility card is inoculated, and the reader measures the amount of light passing through each well hourly for 15 hours. As the organism grows, the amount of light transmitted decreases. Increased turbidity and resulting decreased transmission of light usually indicate the organism is resistant to the antibiotic. A growth curve is developed based on the decline of the percentage of transmission. MICs can be determined based on the growth curve and slope of the curve.

false positive

Antibodies with low specificity may be associated with _______________ _____________ reactions; thus, high specificity is a desired antibody property

Latex

Frequently used carrier molecule for agglutination This is because the molecule is stable and readily covalently bonds with proteins. The latex particle can hold many antibody-binding sites

acute phase specimen

Serum to detect patient antibody production to a particular pathogen should be collected when the disease is first suspected. This sample is known as the _____________ ______________ _________________

Benefits of multitest

Several miniaturized multitest systems have been developed that have converted multistep procedures into single-step procedures, which include multiple reactions Several of these systems have been adapted as a part of semiautomated procedures that may aid in inoculation, incubation, interpretation, and reporting of results. These systems provide for shorter incubation and reaction times and more reliable, reproducible results, with the identification of a greater number of organisms.

epitope

The specific part of the antibody that reacts with the antigen

direct immunofluorescence

also called direct fluorescent antibody (DFA) the specimen is added to a microscopic slide. Next, fluoroscein-conjugated antibody is added to the slide. The specimen is incubated with the labeled antibody, at which time the antigen and antibody form a complex. Washing removes unbound antibody, and the excited fluorochromes emit visible light. DFA techniques detect only antigen. Applications include the detection of chlamydial antigen in urogenital specimens and the detection of Bordetella pertussis antigen in nasopharyngeal specimens.

enzyme linked immunosorbent assays (ELISA)

antibody or antigen is bound to an enzyme that is able to catalyze a reaction. The antibody-binding site remains free to react with antigen, and the enzyme catalyst remains unaltered during the reaction. The colored end product is then measured spectrophotometrically. Antigen can be detected using competitive ELISA techniques or noncompetitive techniques

solid phase immunosorbent assay (SPIA)

antibody or antigen is fixed to a solid phase. The solid phase can be the polystyrene wells of a microtiter tray, plastic beads, filter pads, or ferrous metal beads. Serum or another body fluid is added to the solid phase. If the corresponding antigen or antibody is present, a complex will form. Next, the reaction is washed to remove any unbound, unspecific antibodies. A second antibody that has been complexed to an enzyme is added. The second antibody is usually an antiglobulin conjugate, either horseradish peroxidase or alkaline phosphatase enzyme. If initial binding between the antibody and antigen has occurred, this complex will bind the second antibody, which forms a sandwich with the antigen in the middle. The reaction is washed again to remove any unlabeled antibody, and an enzyme substrate is added. Typical substrates are orthophenylenediamine for horseradish peroxidase and nitrophenyl phosphate for alkaline phosphatase. In a positive reaction the enzyme complex will act on the substrate, converting it to a colored end product. The intensity of the color, which is read spectrophotometrically, is proportional to the amount of antibody or antigen in the original sample.

Sensititre System

can be used as a manual enteric identification system or as an auto-identification system. Each manual plate contains media to perform 23 standard biochemical tests and a control, which are dehydrated in a 96-well microtiter plate. The Sensititre is read and interpreted manually

high volume MicroScan

can incubate up to 96 conventional panels Reagents are added automatically to the panels as needed, and the panels are read and interpreted and results printed. The incorporation of fluorescent labels into some of the substrates provides for bacterial identification within 2 hours. Each fluorescent substrate has a fluorophore attached to a phosphate, sugar, or amino acid compound. In fluorogenic reactions, if the enzyme is produced by the organism, it cleaves the fluorescent compound, releasing the fluorophore, and fluorescent is emitted. In fluorometric reactions, if the organism uses the substrate, the pH drops, and there is a decrease in fluorescence.

Antigens

foreign compounds that stimulate an immune response, leading to the formation of proteins, known as antibodies

BD BBLTM EnterotubeTM II

incorporates 15 standard biochemical tests contained in a compartmented tube designed to permit the simultaneous inoculation of 12 test media. This product system provides rapid, accurate identification of Enterobacteriaceae. This product contains several small pieces of agar substrates in a long plastic tube. The entire tube is inoculated with a long wire, which is enclosed in the system. After incubation and interpretation of results, a profile number is determined, which can be matched to a database. BD BBL Oxi/Ferm TubeTM II provides for the rapid identification of gram-negative oxidative and fermentative bacilli.

MicroScan WalkAway

is a fully automated computer-controlled system that uses both colorimetric and fluorometric principles for the identification and susceptibility testing of microorganisms. This system offers simultaneous automation of overnight, rapid, and specialty panels that test for both gram-negative and gram-positive bacteria. There are two new instrument models—a 40-panel-capacity model for medium-volume laboratories and a 96-panel-capacity model for high-volume laboratories. These instruments have been designed to replace the current WalkAway SI 40 and 96 Systems

VITEK 2

is an automated microbiology system utilizing growth-based technology and is currently available in three formats: - The VITEK® 2 compact is recommended for low- to middle-volume clinical and industrial laboratories, and - VITEK® 2-60 and VITEK® 2 XL are adapted for clinical microbiology laboratories.

Real time PCR

is based on PCR technology and utilizes small automated systems that combine target nucleic acid amplification with the qualitative or quantitative measurement of amplified product. These systems use instruments or platforms that utilize amplification with real-time detection of the product. Real-time PCR has many advantages, which include the ability to detect the amplified target nucleic acid by fluorescent-labeled probes as the hybrids form. Cross-contamination between samples is lessened because amplification and product detection occur in one reaction vessel. Real- time PCR methods also can measure the amount of amplicon and, thus, can quantify the number of copies of target nucleic acid in the original specimen. Real-time PCR requires less time than does conventional PCR because of the use of fluorescent probes and their very rapid thermal cycling.

Nephelometry

is based on the principle of light scattering and has been used for antibiotic susceptibility testing. As an organism grows, the well becomes turbid. Photometers are placed at angles to the turbid suspension, and the scattering of light is measured.

convalescent phase specimen

is collected while the patient is recovering or convalescing. This specimen is usually drawn 2 weeks after the acute phase specimen, although the time may vary depending on the microorganism. In a current infection, the antibody titer in the convalescent phase specimen will show an increase in titer compared with the acute phase. For the test to be diagnostic, a fourfold increase, or two doubling dilutions in the patient's antibody titer in the two specimens, is required

latex agglutination

is dependent on the proper pH, ionic strength of the reaction solution, and osmolarity. There may be false-positive reactions because of nonspecific antibodies or antibody-like compounds. Latex agglutination reactions are performed on cardboard slides or glass surfaces and are graded from 1+ to 4+. In addition to direct specimen testing, latex agglutination may be performed on bacteria isolated from cultures. Latex agglutination methods are rapid and are not labor intensive

BBL Crystal System (BD BBLTM Diagnostics)

is inoculated with one step, which then provides a closed system that is safe and easy to handle. Reagent addition and oil overlay are not needed in this panel design. This miniaturized identification system can identify almost 500 organisms. The Crystal panels may be read manually or with the BBL Crystal Auto Reader, which uses a software program to read and print reports. There are panels for the identification of gram-negative bacilli, enterics, and nonfermenters and for gram-positive aerobic organisms.

Reverse transcriptase PCR (RT-PCR)

is used to amplify RNA targets. In this method, cDNA is formed from RNA targets using reverse transcription followed by amplification of the cDNA using PCR. Thermostable DNA polymerase from thermophilic bacteria can be used as both an RT and a DNA polymerase. Earlier RT-PCR used two separate enzymes, which led to nonspecific primer annealing and poor primer extension.

Counterimmunoelectrophoresis

is useful to detect small amounts of antibody and utilizes the principle of immunoelectrophoresis combined with an electrical current to decrease the reaction time. In this technique, solutions of antibody and sample are placed in small wells cut into agarose. A paper wick connects the two sides of the agarose to trays of buffer. An electrical current is applied through the buffer. The negatively charged bacterial antigens migrate to the positive electrode, while the neutral antibodies are carried by the weakly alkaline buffer to the negative electrode. At the zone of equivalence, antigen and antibody form a complex, and a visible band of precipitin forms. This method also has limited use because of its cost and is somewhat less sensitive than other methods, such as agglutination.

Remel's RapIDTM System

provides a variety of identification products that use enzyme technology; most require 4-hour incubation. Reagents are impregnated in wells in a clear plastic tray. The RapIDTM One uses 19 substrates to identify over 70 oxidase- negative, gram-negative bacilli (Enterobacteriaceae), and the RapIDTM NF Plus is used to identify oxidase-positive enteric organisms as well as nonfermenting gram-negative bacilli. There are also identification systems available for identification ofCorynebacterium and other gram-positive coryneform bacilli (RapIDTM CB Plus) and for Neisseria, Moraxella, Haemophilus, and similar organisms (RapIDTM NH). The RapIDTM Staph Plus provides for the identification of 40 staphylococci and related genera, and the RapIDTM STR System identifies β-hemolytic streptococci and viridans streptococcus. RapIDTM Yeast Plus provides for a 4-hour identification of yeasts. Databases are available for each test system

Specificity

refers to the ability of an antibody to distinguish between antigens that have very small differences

Agglutination

refers to the clumping or aggregation of cells, bacteria, or other particles coated with antigen or antibody with their corresponding antibody or antigen. The antigens or antibodies are attached to the surface of the particle by covalent bonds or electrical forces. In agglutination an aggregation of antibodies and antigens forms a visible framework. There are two stages to agglutination. In the first stage, known as sensitization, antigen combines with antibody. This reaction is not visible. In the second, or visible, reaction, a lattice forms between the antibody-coated particles.

Noncompetitive ELISA

sandwich techniques are used more often than competitive techniques and also can be used to detect antibody. ELISA methods are sensitive and economical, are not labor intensive, and do not require expensive instrumentation

Hemagglutination reaction

use the aggregation of erythrocytes as a positive indicator

Rapid Negative Identification Panel

uses 36 tests and can identify up to 150 species. Currently, identification systems are available for gram-positive bacteria, gram-negative bacteria, yeast, anaerobes, Haemophilus, and Neisseria. Susceptibility testing is available for gram-negative and gram-positive bacteria. The fluorometric system allows for rapid identification results within 2 hours for certain bacteria. The rapid colorimetric system can identify other microorganisms within 4 hours, and susceptibility results are available in 4 to 7 hours. The system uses microtiter trays that contain fluorescent-labeled substrates containing a synthetic and metabolic component. A halogen quartz incandescent lamp is focused on the trays. If the organism produces the enzyme to act on a substrate, the synthetic moiety is released, which enables it to fluoresce. The degree of fluorescence is measured using a fluorometer. Some of the substrates contain fluorescent pH indicators, which emit or fail to emit fluorescence when the pH changes.

Colorimetry

uses a spectrophotometer to measure a color change in the pH indicator or other indicator as an organism metabolizes a particular substrate

MicroScan System

uses plastic 96-well microtiter trays in which up to 32 substrates can be used to identify Enterobacteriaceae. Gram-positive panels, urinary tract panels, and panels for other gram-negative bacteria, as well as for antibiotic susceptibility testing, are also available. The trays are shipped in either a dehydrated or frozen form. The microtubes are inoculated with a heavy suspension of the organism and incubated for 15 to 18 hours at 35°C. The panels may be interpreted manually, and each biochemical result is converted into a seven- or eight-digit code number, which is found in a codebook. Alternatively, an automated tray reader can be used, which will detect bacterial growth or color changes as noted by changes in light transmission. A computer with software analyzes the electronic pulses and compares the reaction patterns with an internal program to identify the organism.

BD Phoenix Automated Microbiology System

uses the principle of nephelometry. It is a fully automated identification instrument that also can provide antimicrobial susceptibility testing. The system consists of disposable panels that combine identification and antimicrobial susceptibility testing. The instrument automatically takes readings every 20 minutes during incubation. The system measures colorimetric changes and changes in fluorescence intensity levels depending on the type of substrate. There are 45 biochemical reactions, which include fluorogenic, fermentative, chromogenic, and carbon source substrates, to identify gram-negative bacilli. Most organisms are identified within 2 to 12 hours; many results are available within 4 hours.

bioMérieux's API systems

which utilize microtubes with dehydrated substrates that are reconstituted by adding a bacterial suspension. After interpretation of reactions, a profile number is determined, which can be matched with that in a profile index from a database. Most of the microorganisms can be identified to the species level using bioMérieux's API® identification products Manual bioMérieux's API® products for gram- negative bacteria include the API® 20E for 18- to 24-hour identification of Enterobacteriaceae and other nonfastidious gram-negative bacilli, the API® Rapid 20E for 4-hour identification of Enterobacteriaceae, the API® 20NE for 24- to 48-hour identification of gram-negative non-Enterobacteriaceae, and the API® NH for 2-hour identification of Neisseria, Haemophilus, and Branhamella catarrhalis