Bacteriology Lecture 2

why is diagnostic workup important?

• select treatment

• provides specfic pathogen in clinical syndromes

• improves clinical care

  • helps to prevetn transmission, zoonosis, future infection

how are pathogens detected?

detect infrecting agent

detect host response to infection

how are antibodies produced in the body?

primary response - small antibody production

secondary response - body produces substantial antibodies and are detectable

what is the path of infection and how are each stage detected?

entry of pathogen (Agent detection) —> dissemination (agent detection) —> colonization disease (Agent and Antibody detection) —> elimination (antibody detection)

what does the selection of type of diagnostic depend on?

nature of infectious agent suspected, host species, and the availability of tests

what are examples of detection methods used for agents or their components?

• microscopic examination

• bacterial culture

• ELISA (direct detection of antigens)

• PCR (detection of nucleic acids) MOST COMMON

how is the host immune response used to detect agent?

• humoral immune response - antibodies

• cell mediated immune response - cellular response - EX skin test

how are samples examined microscopically

examine wet smears or stained smears using light, phase contrast, or darkfield microscope

what are the denefits of microscopic examination?

• cost effective

• time effective

• show number, morphologic characteristics seen

• info on likelihood of infection and predominant organism

wh

what are the limitations of microscopy

• low sensitivity

• low specificity

• some bact do not stain

what are gram neg

• thin peptidoglycan layer

• stains PINK

what is gram positive bacteria

• thick peptidoglycan layer

• stains PURPLE/BLUE

what type of bacteria is this

positive cocci

what type of bacteria is this?

gram positive rods

what type of bacteria is this

gram negative coccobaccilus

what does the acid FAST stain test for?

presence of mycolic acid

• positive = pink

• negative = blue

what are some limitations of culturing?

some bacteria do not provide timely and valuable info; obligate intracellular bacteria can not be cultivated in lab media

what do the culture methods depend on?

bacterial organisms vary in growth, need for oxygen, and nutrients; this changes the growing conditions

how is bacteria identified on the culture?

• use of biochemical tests

• semiautomated systems

• MALDI-TOF - mass spectrometry

what is the main method of molecular detection

PCR - polymerase chain reaction - allows amplification and detection of DNA

what is the steps of PCR

1. DNA extraction; mix reagents with target DNA

2. thermocycling - denaturation

3. agarose gel electrophoresis

4. visualize under UV light

what is the process of real time PCR

uses labelled probes/fluorescent dyes; able to watch the reaction in real time

1. mix reagents with target DNA

2. thermocycling

3. data analysis

what is the relevance of the CT value in real time PCR

lower the CT value = more DNA present in clinical sample

how are antigen detection tests performed

utilizes specific antibody reagents to detect pathogens in clinical samples

clinical sample + antibody = detectable reaction

serconversion

antibody development after exposure to a pathogen or an antigen; there is a primary response and secondary response

how are antibodies measured?

antibody titer - measure of serum antibody level against an infectious agent

how are titer’s expressed?

expressed as the reciprocal of the highest dilution of serum that positively reacts in a specific test

EX - a titer of 1280 in a test means that the serum sample remains positive when diluted 1:1280

what are paired titers and what is the purpose?

titers determined at 2-4 week intervals during course of infection

2-4 fold increase in paired titer is suggestive of an active infection

which of the following samples has the highest level of antibodies?

highest titer number = most diluted = highest antibody level

what is the role of a clinician for the diagnostic testing?

develop differential diagnosis absed on hx, symptoms, direct exam of samples

select appropriate samples, tests, collection, transport methods, and a quality lab

provide relevant history, signalment, vaccine and treatment

learn how to interpret results

what should occur if there is a doubt about sample collection and transportation?

• call the lab

• be aware of transportation safety and packaging regulations for public transport of dx samples and infectious substances

what is biosafety levels?

a risk assessment base on lab practices/techniques, safety equipment, and lab facilities to be used to minimize potential exposure to a biohazard for personnel

what does BSL 4 indicate?

highest level that deals with very infectious pathogens, requires highest level of biosafety