07 Lab Identification Flashcards
What are the direct detection methods in primary specimens?
Microscopy. Antigen detection. Molecular methods (DNA/RNA probes, PCR). Toxin detection
What is Microscopy?
Direct observation of organisms with or without staining. Gram stain
What is Antigen detection?
Antigens of organisms are released into various body sites and can be detected even when the organism is not present is sufficient quantity to culture. Antigen may be the organism itself or a toxin
What are some common bacteria that antigen detection is used for?
Group A Strep, Chlamydia, Cryptococcus, N. meningitidis, H. influenzae. Think of the ones that vaccines are made for
What are the 2 methods of Nucleic Acid Detection?
1) Patient sample (fluid or tissue) mixed directly with DNA probe. 2) Patient sample undergoes PCR (DNA amplification) then DNA probe will search for specific sequence of genes
What is a DNA probe?
A single stranded DNA molecule used in laboratory experiments to detect the presence of a complementary sequence among a mixture of other single-stranded DNA molecules
What is the incubation time like for bacteria?
2-5 days
What is the incubation time like for Mycobacteria?
42 days
What is the incubation time like for fungi?
28 days
What is the incubation time like for Viruses?
21 days
What can be used with Agar-based medium?
Bacteria, Mycobacteria, Fungi
What can be used with Broth-based medium?
Bacteria, Mycobacteria
What is tissue culture used for?
Viruses
What is Step 1 in identifying bacteria grown in culture?
Organisms inoculated into a wide variety of media types containing biochemical reagents and nutritional supplements
What is Step 2 in identifying bacteria grown in culture?
Incubation or growth phase
What is Step 3 in identifying bacteria grown in culture?
Interpretation of results: growth vs. no growth, colony type, color change, oxidase, catalase, fermenter
What is an alternate process to Step 3?
Automated processors (Vitek, Microscan, Pheonix). Add bacteria from positive culture to automated “cards”. Specific cards for Gram positive and Gram negative pathogens with different antibiotics. Identification and sensitivity results in 4 hours
What are the indications for antimicrobial susceptibility testing?
Potentially pathogenic bacteria based on source of specimen, species isolated, numbers of colonies present, normal flora, underlying host factors (S. viridans in sputum - normal flora –> not tested). Bacteria with unpredictable susceptibility to the drugs of choice that would be used in treating an infection caused by the bacterium (S. pyogenes in wound - always susceptible to PCN –> not tested)
What are the routine antibacterial susceptibility tests performed in most clinical microbiology laboratories?
Disk Diffusion (Kirby Bauer), E-test. Broth microdilution. Automated tests (e.g. MicroScan, Vitek). Beta-lactamase test
What does the Beta-Lactamase test do?
Measures the presence or absence of B-lactamase for Enterococcus, Hemophilus, Moraxella sp., Neisseria gonorrheae
What is Disk Diffusion?
A suspension of the bacterial strain is adjusted to a standard density and the suspension is swabbed evenly on a Mueller-Hinton agar plate. ABX disks are applied to the inoculated surface of the agar plate. ABX is diffused out from the disk onto the agar surface as the bacteria multiply. NO zone of inhibition if bacterial cells are not inhibited by the ABX. The more susceptible, the larger the zone of inhibition. The diameter of the zone of inhibition is indirectly proportional to the minimal inhibitory concentration (MIC)
What is an E-Test?
Antibiotic is impregnated onto the strip in a gradient of doubling concentrations. MIC is where the zone of inhibition and bacteria meet the strip
What is Broth Microdilution?
Each well contains broth media to support growth of organisms. ABX is added at varying concentrations (2-fold dilution) to each well. Standard inoculum of organisms is added to each well; plus positive and negative controls
What is the Minimum Bactericidal Concentration (MBC)?
Measures the lowest concentration of abx that kills a bacterial isolate
What is Serum Bactericidal Titer?
Measures antimicrobial activity of patient’s serum against his/her own infecting bacteria
What is Synergism Study?
Measures susceptibility of a bacterial isolate to a combination of 2 abxs
How is MBC measured?
By inoculating the broths used for MIC onto drug-free agar. Fist dilution at which no growth on agar is MBC. Cidal drugs have MBC values close to the MIC. Static agents the MIC is lower than the MBC
How is reporting susceptibility results done?
Quantitative: minimum inhibitory concentration (most common), broth microdilution; E-test. Qualitative interpretation: susceptible; intermediate; resistant
What are the characteristics of Quantitative Results: MIC?
Activity of an antimicrobial against a bacterial specimen in vitro is quantified and estimated in vivo - MIC (reported as ug/ml with S, I, or R interpretation)
What is the definition of MIC?
Lowest ABX concentration that prevented visible growth of an organism after 24 hours of incubation in a specified growth medium
What are the MIC breakpoints (S, I, R) determined by?
1) MIC frequency distribution that distinguishes wild-type bacterial populations from those that have either selected or acquired resistance mechanisms. 2) Human and animal PK-PD data (peak serum concentration, T 1/2, degree of protein binding). 3) Clinical and bacteriologic outcomes from clinical studies
What does “Susceptible” mean?
Pathogens with lowest MICs and are most likely to be eradicated during therapy of infections using typical drug doses
What does “Resistant” mean?
Organisms with significantly higher MICs that will cause a less-than-optimal clinical response even at the highest doses
What does “Intermediately Susceptible” mean?
Treatment may be successful when maximum doses of a drug are used or when the drug is known to be concentrated in the infected body site
What is a Cumulative Antibiogram?
Cumulative antimicrobial susceptibility statistics. Each statistic represents the % of isolates of a given species that are S to a particular abx over a particular time frame. Specific for particular institutions. Monitor the development of resistance trends. Guide empirical therapy. Anything above 90% is usually a good choice
What are the assumptions of MIC breakpoint?
Concentrations that pathogens will be exposed to at the infection site are always equal to those achieved in the blood. All areas in the body will have equilibrium-based exposure
What are the caveats to MIC testing?
MIC does NOT represent equivalent abx activity against all bacteria present in an infection (some bacteria may be more or less susceptible). Resistance may not be detected using the inocula tested in vitro (a much higher inoculum in vivo increases the chances for spontaneously resistant mutants). Bacterial growth medium used can affect the activity of many drugs. Bacterial inoculum can significantly affect the MIC of most B-lactam agents and gram-negative bacilli. Discordance between in vitro testing and in vivo infection conditions
What is the applicability of MIC data?
NOT all patients infected with susceptible organisms will have an adequate clinical or microbiologic response. In cases of paradoxical antimicrobial treatment failure, consider: caveats to in vitro MIC testing, drug/host/microbial factors
What are the three factors affecting treatment response?
Host, Drug, Bug
What are the “Drug” factors?
Protein binding (only “unbound” drugs exert antibacterial effect). Route of administration. Levels of drug (may be different at various body sites). Half-life. Bactericidal vs. Bacterostatic properties. Combination therapy resulting in synergy or antagonism
For “Drug” factors, when is there a better prediction of therapeutic response?
When in vitro susceptibility parameters AND in vivo antimicrobial PK and PD parameters are considered
What is the drug distribution of B-lactams?
Distribute evenly throughout the body, except the CNS. Serum concentration reflects extracellular space of any perfused organ or tissue site. Do not penetrate host cells to any significant degree
What is the drug distribution of Aminoglycosides?
Similar properties as the B-lactams. Taken up by host cells over time with continued administration. Cellular uptake is associated with toxicity rather than efficacy
What is the drug distribution like for Macrolides and Fluoroquinolones?
Extensive distribution throughout the body. Concentrations at infection sites (tissues) up to a log-fold higher > corresponding serum. Intracellular concentrations in phagocytes even higher than tissues - up to 2-log fold higher than serum
