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Bacteriology-31 Non culture based methods for clinical diagnosis*


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What are some immunologic techniques for identification of infectious agents in patients
Fluorescent antibody test (direct or indirect method)
Immunochromatographic methods (lateral flow or pregnancy test format)
Enzyme immunoassay (EIA) or ELISA
Latex agglutination
When is the Fluorescent antibody (FA) test for cell-associated antigens used
For visualization of agent in clinical material or tissue culture by microscopy. Can use direct (labeled primary antibody) or indirect (labeled secondary antibody, which is more sensitive)
Fluorescent antibody test (FA) is used for detection of what
Agents that are difficult or time-consuming to culture
What are immunochromatographic methods, such as lateral flow or the pregnancy test, use for
Rapid testing for strep throat; Shiga toxin in fecal extracts
What is ELISA used for
If enzyme labeled secondary antibody develops color, we know the antibody has recognized and bound the antigen we are looking for
What is latex agglutination used for
Rapid identification of pathogens in CSF; Group B strep in vaginal secretions; Clostridium difficile toxin in fecal extracts; Rotovirus in feces
What are the two nucleic acid-based techniques for identification of infectious agents in patient material
Nucleic acid probes and Amplification techniques
What are the basic components of probe-based detection systems
A probe or single stranded DNA or RNA (many copies needed)
A target of nucleic acid
Hybridization conditions
A method for detection of hybridized product
What are the advantages of nucleic acid probes
What is the main disadvantage
High specificity (few false positives)
Very rapid (same day results)
Requires little specialized laboratory equipment
The main disadvantage is the low sensitivity
What are some methods for improving the sensitivity of nucleic acid probes
Amply the number of targets
Add enzymes that make more targets
Enhance the identification of pairs with enzyme-linked anti-hybrid antibodies
What are the best applications for nucleic acid probes
Identify organisms that grow very slowly (TB)
For organisms that are expensive or labor intensive to culture (viruses; Chlamydia)
For organisms that are non culturable (leprosy, syphilis)
What are the components needed for PCR
Primers; Target DNA; Temperature-stable DNA polymerase; Thermocycling device
What are some pathogens for which the PCR reaction has been developed
Mycobacterium tuberculosis
Chlamydia trachomatis
Lyme disease (Borrelia burgdorferi)
What are the advantages of PCR
High specificity and sensitivity
Minute sample can be used
Can detect the presence of microorganisms that cannot be cultured
What are the limitations of PCR
Potential for false positives through contamination
Potential for false negatives through amplification inhibitors
No antimicrobial susceptibility patterns provided
Requires special expensive equipment
What is real-time PCR
PCR where the DNA synthesized can be measured during the reaction using SYBRgreen or DNA probes
What are the advantages of Real time PCR
Allows user to quantitate product
Eliminates electrophoresis step
Allows for detection of multiple products
What are the limitations of Real time PCR
Requires expensive instrumentation
Requires synthesis of fluorescent probes
What are the applications of Real time PCR
Can test for multiple targets at one time, and can estimate viral load (HIV)
What are the main characteristics of RT PCR
The target sequence is RNA. Can identify RNA based viruses, determine what genes are being expressed (to determine viral load), and detect expression of genes in latent infections (CMV)
What are some general considerations in detection of immune response against infectious agents
Time to seroconversion
Discrimination of old versus new infections (antibody class)
Four-fold rise between acute and convalescent sera
What are some means of detection of pathogen-specific antibodies
Precipitation reactions (immunodiffusion and CIE)
Agglutination reactions
Western Blot
Complement fixation
What is molecular subtyping used for
Estimating the degree of relatedness of bacterial isolates of the same or closely related species
In pulse field gel electrophoreis (PFGE), what indicates relatedness
Three band differences or less
What species are tested in Pulsenet's database
Shigella species
Salmonella enterica
E.coli O157:H7
Listeria monocytogenes
What is serotyping used for
Distinguishing closely related species or strains of the same species. Includes the O antigen, capsular antigens, flagellar capsular types, and outer membrane proteins

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