Rapid Presumptive ID

This section will cover the theory, interpretation, and application of several rapid tests commonly used in the clinical microbiology lab to provide presumptive identification of bacterial isolates. These tests are designed to provide quick results, allowing for timely initiation of antimicrobial therapy and infection control measures

Theory: The Science Behind the Quick Tests

What are Rapid Tests?
- Rapid tests are simple, quick assays performed on bacterial isolates to provide presumptive identification results within minutes to a few hours
- They are based on detecting specific enzymatic activities or metabolic products of bacteria
- They provide initial clues about the identity of an organism, reducing the time required to identify a pathogen
Why Use Rapid Tests?
- Speed: Results are available much faster than traditional biochemical tests
- Cost-Effective: Relatively inexpensive to perform
- Convenience: Simple to perform and require minimal equipment
- Clinical Impact: Enable faster initiation of appropriate antibiotic therapy and infection control measures, improving patient outcomes
Limitations of Rapid Tests
- Presumptive, not Definitive: Results are not always definitive and may require confirmation with additional tests
- Variability: Results can be influenced by factors like inoculum size, media, and incubation conditions
- Specificity and Sensitivity: The accuracy of these tests can vary, and it’s important to understand their limitations for each test
- Not a Stand-Alone: Should be used in conjunction with Gram stain, colony morphology, and other clinical information
Common Rapid Tests
- Catalase: Detects the presence of catalase enzyme
- Coagulase: Detects the presence of coagulase enzyme
- Oxidase: Detects the presence of cytochrome c oxidase
- Indole: Detects the production of indole from tryptophan
- PYR (Pyrrolidonyl Arylamidase): Detects the presence of the enzyme pyrrolidonyl arylamidase

Interpretation: Deciphering the Results

0.8.3 Catalase Test

Principle: Catalase enzyme converts hydrogen peroxide (H₂O₂) to water (H₂O) and oxygen (O₂)
Reagent: 3% hydrogen peroxide (H₂O₂) solution
Procedure
1. Place a drop of H₂O₂ on a glass slide
2. Using a sterile loop or sterile stick, touch a colony of the organism and mix it with the H₂O₂
3. Observe for the production of bubbles (oxygen)
Interpretation
- Positive: Rapid bubbling (oxygen production)
- Negative: No bubbling
Clinical Significance
- Differentiates Staphylococcus (catalase positive) from Streptococcus (catalase negative)
- Differentiates Bacillus (catalase positive) from Clostridium (catalase negative)
- Helps to differentiate Listeria monocytogenes (catalase positive) from Streptococcus

0.8.4 Coagulase Test

Principle: Coagulase enzyme clots plasma by converting fibrinogen to fibrin
Reagents: Rabbit plasma with EDTA or citrate
Procedure
1. Emulsify a few colonies of the organism in a small amount of plasma
2. Incubate at 35-37°C for 1-4 hours, or up to 24 hours if needed
3. Observe for clot formation
Interpretation
- Positive: Clot formation
- Negative: No clot formation
Clinical Significance
- Differentiates Staphylococcus aureus (coagulase positive) from coagulase-negative staphylococci (CoNS)
- Used to differentiate Yersinia pestis (coagulase positive) from other Yersinia species
Types
- Slide Coagulase: Detects bound coagulase (clumping factor) - faster result, but may have false positives
- Tube Coagulase: Detects free coagulase - more sensitive, but takes longer

0.8.5 Oxidase Test

Principle: Detects the presence of cytochrome c oxidase, an enzyme involved in the electron transport chain
Reagents
- Tetramethyl-p-phenylenediamine dihydrochloride (TMPD) or other oxidase reagents
Procedure
1. Wet a filter paper with oxidase reagent
2. Using a sterile loop or swab, rub a colony onto the filter paper
3. Observe for a color change (purple/blue) within 10-30 seconds
Interpretation
- Positive: Rapid development of a purple/blue color
- Negative: No color change, or a delayed color change (after 30 seconds)
Clinical Significance
- Differentiates Neisseria and Moraxella (oxidase positive) from Enterobacteriaceae (oxidase negative)
- Differentiates Pseudomonas aeruginosa (oxidase positive) from other Gram-negative rods

0.8.6 Indole Test

Principle: Detects the production of indole from the breakdown of tryptophan by the enzyme tryptophanase
Reagent: Kovac’s reagent (contains p-dimethylaminobenzaldehyde)
Procedure
1. Inoculate the organism into a tryptophan broth
2. Incubate at 35-37°C for 24 hours
3. Add Kovac’s reagent to the broth
4. Observe for a red ring at the top of the broth
Interpretation
- Positive: Red ring at the top of the broth
- Negative: No red ring (yellow ring or no color change)
Clinical Significance
- Differentiates Escherichia coli (indole positive) from other Enterobacteriaceae
- Helps in the identification of Proteus species

0.8.7 PYR Test

Principle: Detects the presence of the enzyme pyrrolidonyl arylamidase (PYR), which hydrolyzes L-pyrrolidonyl-β-naphthylamide
Reagent
- PYR substrate disk or reagent
- Cinnamaldehyde reagent
Procedure
1. Moisten a PYR disk or area of paper with sterile water
2. Rub a colony of the organism onto the disk or paper
3. Add a drop of cinnamaldehyde reagent
4. Observe for a color change (red)
Interpretation
- Positive: Development of a red color
- Negative: No color change (yellow)
Clinical Significance
- Differentiates Streptococcus pyogenes (Group A Strep, PYR positive) from other beta-hemolytic streptococci
- Differentiates Enterococcus spp. (PYR positive) from other Gram-positive cocci
- Aids in the identification of Aerococcus spp

Application: Putting Knowledge into Practice

Quality Control (QC)
- Control Strains: Use known positive and negative control organisms for each test to ensure accuracy
- Frequency: Perform QC at the beginning of each day or whenever a new lot of reagents is used
- Documentation: Record QC results in a logbook or LIS
Procedure
1. Gram Stain and Colony Morphology: Perform a Gram stain and observe colony morphology before performing rapid tests. This provides essential clues and helps guide test selection
2. Reagent Preparation: Prepare reagents according to manufacturer’s instructions
3. Testing: Follow the specific procedure for each test, ensuring proper technique and observation of results within the recommended time frame
4. Interpretation: Interpret the results based on the expected color changes or reactions
5. Documentation: Record the results in a lab notebook or LIS, along with the Gram stain, colony morphology, and any other relevant information
6. Correlation: Correlate the results of the rapid tests with the Gram stain, colony morphology, and other clinical information to provide a presumptive identification
7. Further Testing: Based on the presumptive identification, select appropriate further biochemical tests or automated identification systems for confirmation
Example: Identifying a Gram-positive coccus in clusters
1. Gram Stain: Reveals Gram-positive cocci in clusters
2. Colony Morphology: Large, golden, beta-hemolytic colonies on blood agar
3. Catalase Test: Positive (bubbles present)
4. Coagulase Test: Perform a slide coagulase test
  - Positive: Presumptive identification of Staphylococcus aureus
  - Negative: Presumptive identification of a coagulase-negative staphylococcus (CoNS)
5. Further Testing: If coagulase-negative, perform additional tests for species-level identification
Example: Identifying a Gram-negative rod
1. Gram Stain: Gram-negative rod
2. Colony Morphology: Pink colonies on MacConkey agar (lactose fermenter)
3. Oxidase Test: Negative
4. Indole Test: Perform the indole test
  - Positive: Presumptive identification of Escherichia coli
  - Negative: Further testing is needed to identify other Enterobacteriaceae
5. Further Testing: Proceed with additional biochemical tests or automated identification
Troubleshooting
- False Positives/Negatives
  - Inoculum Size: Use the recommended inoculum size for each test
  - Reagent Quality: Ensure reagents are fresh and stored properly
  - Cross-Contamination: Maintain strict aseptic technique
  - Incubation Time: Follow the recommended incubation times
- Weak Reactions
  - Old Cultures: Use fresh cultures
  - Incorrect Procedure: Review the test procedure
  - Incubation Conditions: Ensure proper incubation temperature

Key Terms

Presumptive Identification: A preliminary identification based on initial tests, which requires confirmation by more definitive tests
Catalase: An enzyme that breaks down hydrogen peroxide into water and oxygen
Coagulase: An enzyme that clots plasma by converting fibrinogen to fibrin
Oxidase: An enzyme involved in the electron transport chain, often present in aerobic bacteria
Indole: A compound produced from the breakdown of tryptophan by the enzyme tryptophanase
PYR (Pyrrolidonyl Arylamidase): An enzyme that hydrolyzes L-pyrrolidonyl-β-naphthylamide
Gram Stain: A differential staining technique used to classify bacteria based on their cell wall structure
Colony Morphology: The visual characteristics of bacterial colonies on solid media
Reagent: A substance used in a chemical reaction to detect or identify another substance
Inoculum: The material used to inoculate a culture medium
Quality Control (QC): Procedures used to monitor and ensure the reliability of laboratory testing
Control Strains: Known organisms used as positive and negative controls in laboratory tests
False Positive: A test result that incorrectly indicates the presence of a substance or organism
False Negative: A test result that incorrectly indicates the absence of a substance or organism
Enzyme: A protein that catalyzes a specific biochemical reaction

Colony Morphology

Conventional Biochemical ID