Lecture 2 - Diagnosis of bacterial infections

What are the two ways to diagnose bacterial and fungal infections? - 1x a wk

1. detection of infecting agent

2. detection of host immune response to the infection

What is the primary response? - 1x a wk

the immune response that results from the first exposure to an antigen. This starts at IgM them changes to IgG at around 10 - 20 days. Until this happens, you can only look for a host response

What is the secondary response? - 1x a wk

the immune response stimulated on the second exposure to the same antigen. IgG compared to first exposure will be significantly higher.

What can you test for at these stages? - EOD

1. Entry of pathogen

2. Dissemination

3. Colonization/Disease

4. Elimination

1. agent detection

2. agent detection

3. agent detection and antibody detection

4. antibody detection

In what order are you likely to find pathogen in samples? - 1x a wk

blood, urine, serum

What determines your choice of diagnostic method? - 1x a wk

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

What methods are used to detect infecting agents or their components? - 1x a wk

1. microscopic exam

2. isolation and ID (culture)

3. direct detection of antigens, toxins (ELISA)

4. detection of nucleic acids (PCR)

What methods are used to detect host immune responses? - 1x a wk

1. humoral immune responses (detects antibodies to an infecting agent)

2. cell mediated response (cellular responses to an infectious agent: IFN y, PPD skin test, TB)

What are microscope examinations examples? - 1x a wk

1. wet smears (light, phase contrast, darkfield)

2. stained smears with light (diff-quick, gram, acid-fast)

What are the benefits and limitations to microscopic exam? - 1x a wk

Most important and cost effective

Benefit - information on the number, morphologic characteristics, and sometimes host cellular response

Limitations - low sensitivity, low specificity, some bacteria do not stain well (Mycoplasma and spirochetes)

What is the color of gram negative bacteria and why? - 1x a wk

pink because the thin cell wall is blocked by a memebrane so the stain cannot detect the peptidoglycan (also not enough)

What color is gram positive bacteria and why? - 1x a wk

purple because the thick cell wall retains the large crystal violet and grams iodine complexes retains the primary stain, the purple is just more visible over the pink staining

How to interpret acid-fast staining? - 1x a wk

positive organisms will the color of Carbol Fuchsin (pink) because of mycolic acid. Negative organisms are not colored and retain the blue counterstain due to the absence of mycolic acid.

What are routine cultures? - EOD

aerobic, anaerobic, microaerophilic, fungal

What are some special culture procedures? - 1x a wk

mycolasma, salmonella, listeria, mycobacterium, campylobacter

What is an example of a bacteria that will not provide you with any valuable information with a cultuture? - 1x a wk

leptospira

What kind of bacteria cannot be cultivated in lab media? - 1x a wk

obligate intracellular

What types of bacteria need oxygen for growth?

aerobic, microaerophilic, capnophilic (carbon dioxide)

What types of bacteria does not need or utilize oxygen for growth? - EOD

obligate anaerobe, aerotolerant anaerobe

What oxygen does not need, but can utilize oxygen for growth? - 1x a wk

faculatative anaerobe

What is the most common molecular method of detection and how does it work? - 1x a wk

PCR allows for the amplification and detection of target DNA

What are the components of PCR? - 1x a wk

1. primer (forward and reverse)

2. DNTP

3. enzyme (Taq polymerase)

4. template DNA

What is a conventional PCR? - 1x a wk

a unique region of any microorganism can be copied a billion fold or more BUT it needs an additional agarose gelectrophoresis step for the detection of amplified DNA

What is real-time PCR? - EOD

a semi-quantitative PCR that has an additonal (2nd) component of labelled probes and fluorescent dyes

How to you analyze a real-time PCR? - 1x a wk

look at the threshold (ct) value. The lower the CT value, the more DNA is present in the sample

What is an antigen detection test and examples? - EOD

uses specific antibody reagents to detect pathogens in clinical samples. Immunofluorescence, agglutination tests, and enzyme immunoassay

What is seroconversion? - EOD

the development of antibodies as a result of infection

What samples are used to detect a humoral response? - 1x a wk

body fluids (mainly serum)

1. agglutination

2. precipitation

3. ELISA

What samples are used to detect a cell-mediated response? - 1x a wk

immune cells or body's reaction to a specific component of a pathogen

1. TB reaction

2. inferon gamma test

How is an antibody titer done and how to you interpret the results?- 1x a wk

measures the serum antibody level against an infectious agent. Expressed as a recipocal of highest dilution of serum that had a positive reaction. Bigger number = higher antibodies against pathogen

What is a paired titer?- 1x a wk

titers are determined at 2-4 week intervals during an infection. If there is a 2-4 fold increase in the paired titer, this is suggestive of an active infection.

What is BSL? - 1x a wk

biosafety level needed for specific microbes

What is BSL-1?- 1x a wk

non pathogenic and minimal risk

What is BSL-2? - 1x a wk

moderate risk and requires a biosafety cabinet with PPE (most vet)

What is BSL-3? - 1x a wk

serious/lethal pathogen through inhalation; requires special vents and restricted access

What is BSL-4? - 1x a wk

high risk/life threatening. No treatment and requires full body positive pressure suit