Diagnostics

Year 1 - Semester 2

Process to diagnose a disease

1. identify animal species

2. identify the body system primarily affected

3. identify main symptoms of the disease

4. identify which microorganism is causing the disease

aseptic technique

any method used to sterilise and maintain the sterility of an object or location

What is the main concern when collecting specimens from a patient?

contamination

What are the main principles of sample collection?

specimen must be obtained aseptically from a site that is representative of the disease process, sufficient quantity of material is collected, speciments are collected prior to treatment to maximise pathogen recovery, if cultures are not immediately initiated after collection then specimens must be refrigerated

What must blood cultures NOT contain?

EDTA

Why must blood cultures not contain EDTA?

it is a chelating agent which inhibits the growth of microorganisms, so would prevent a representative culture from forming

What information should be included on a sample submission form?

case history, required tests and sample type

What are the 2 types of swabs?

wet and dry

features of wet swabs

have a transport medium so can transport microorganisms to a lab without desiccation occuring

features of dry swabs

no transport medium so used to transport samples to be tested immediately

function of transport mediums

to prevent microorganisms from dying in transit for a certain time period, suitable for specific microorganisms

What is the ideal temperature for short term storage of microorganisms?

4 degrees

What is the ideal temperature for long term storage of microorganisms?

-80 degrees or below

What are the 2 main methods to identify an unknown pathogen in a specimen?

culture based and non-culture based

What are the non-culture based methods to identify an unknown pathogen in a specimen?

microscope examination, molecular methods, antigen-antibody reactions, point of care tests

What are the 3 ways to directly observe clinical material under a microscope?

make a swab smear, make an aspirate smear, make a tissue impression smear

advantages of direct microscopic observation of a specimen

cost effective, rapid, provides immediate information on presence of bacteria and fungi and the number of organisms present, allows presumptive identification of bacteria using morphological characteristics and gram-staining, provides information on the host cellular response

What are some examples of microorganisms which cannot be observed under a microscope using gram stain?

mycobacterium spp. and leptospira interrogans

How are myobacterium spp. viewed under a microscope?

using Ziehl-Neelsen stain

How are leptospira interrogans observed under a microscope?

dark field microscopy

How are fungal specimens prepared for observation under a microscope?

10% KOH is added to act as a clearing agent, then blue Parker ink is added which is taken up by the fungal spores, making them appear blue-violet

How are viral specimens viewed under a microscope?

H&E stain is used, cannot always see viral particles under a light microscope but can see the effect of the particles on host tissue, can use an electron microscope

What are the aims of bacterial culture?

to isolate the organism in pure culture, to identify the isolate, to enable antimicrobial susceptibility testing

What are the types of media used to culture bacteria?

enrichment, differential, selective

enrichment media

general purpose media supplemented by blood or other special nutrients to encourage the growth of fastidious organisms

differential media

media that allows scientists to distinguish between different groups of bacteria on the basis of their biological characteristics

selective media

media which favour the growth of particular microorganisms and inhibit the growth of others

How can bacteria be isolated in culture?

use of specific media, enrichment and selection protocols, different incubation conditions

Does this show mixed or pure bacterial growth?

mixed

Does this show mixed or pure growth?

pure

phenotypic traits of bacteria used for identification in cultures

oxygen requirements, culture characteristics, staining properties, microscopic morphology, biochemical reactions, MALDI-TOF

What are some examples of biochemical reactions used to identify bacteria from a culture?

catalase, oxidase, urease

What is the effect of beta-haemolytic bacteria on blood agar?

they secrete enzymes/toxins that lyse the erythrocytes in the agar which produces zones of haemolysis

What is a qualitative method for antimicrobial susceptibility testing?

incubation using disc diffusion of antimicrobial agents on an isolated culture

What are the quantitative methods for antimicrobial susceptibility testing?

MIC and E-test

What are the 3 categories of microorganism response to antimicrobial susceptibility testing?

susceptible, resistant, intermediate

antimicrobial susceptible microorganism

a microorganism which is inhibited by a concentration of the anitmicrobial agent that can be attained in blood with the normally recommended dose (suggests infection by this microorganism can be treated with this antimicrobial)

antimicrobial resistant microorganism

a microorganism which is resistant to the concentration of the antimicrobial agent that can be attained with normally recommended doses

antimicrobial intermediate

a buffer zone to avoid misinterpretation of susceptibility testing results which suggests that treatment is possible if infection is in the body sites where the antimicrobial is concentrated

Why can viruses be difficult to culture in a lab?

they are obligate intracellular organisms, specialist labs are required, bacterial growth can occur, not all viruses grow in cell culture, labour intensive process

Which mediums can viruses be preserved in?

tissue cell cultures, embryonated eggs, experimental animals

cytopathic effects

structural and morphological changes in host cells caused by viral invasion, often leading to cell damage or death

examples of cytopathic effects

rounding of cells, fusion, inclusion bodies

How are viruses identified from cultures?

cytopathic effects, morphological characteristics, viral haemagglutination

viral haemagglutination

some virus particles have haemagglutinin particles on their surfaces which bind to red blood cells and cause agglutination

What specimens are taken from a patient for fungal culture?

plucked hairs from lesions, toothbrush, skin scrapings, exudates, biopsies

How is the growth of most bacteria inhibited when culturing fungi?

low pH is used

How are fungal cultures identified?

macroscopic and microscopic morphology

What are some tests that can be used to identify fungal infection?

at-home dermatophyte culture plates, UV fluorescence which identifies fluorescent metabolites produced by some fungi, lab culture

What can detecting a host immune response to an infection indicate?

ongoing infection, past exposure to the pathogen, maternally derived antibodies, vaccination

delayed hypersensitivity reaction

exposure to a pathogen causes a delayed but large immune response which shows that the animal has been exposed to the pathogen before, used to diagnose bovine tuberculosis

How are antigen-antibody reactions used to identify pathogens?

serological tests detect presence of antibodies for a specific pathogen and delayed hypersensitivity reaction tests

advantages of using PCR to identify pathogens

very sensitive, detects microorganisms that are non-viable, uncultivable or slow growing, faster results than culture methods

limitations of using PCR to identify pathogens

reaction is susceptible to inhibitors, contamination and experimental conditions, cannot differentiate between live and dead organisms

What are some examples of microorganisms that are detected using PCR?

mycoplasmas, mycobacteria, spirochaetes, viruses

How are rapid molecular diagnostics performed?

single-step, cartridge-based molecular test devices which target pathogens implicated in clinical syndromes

point of care testing

clinical laboratory testing which is conducted close to the side of the patient where care or treatment is provided, this testing gives rapid results which can improve clinical/economic outcomes

analytic sensitivity of a diagnostic test

the lowest amount a test can detect defined at 95% Cl

analytic specificity of a diagnostic test

the ability of a test to not react with other substances other than the analyte of interest

clinical sensitivity of a diagnostic test

the ability of a test to correctly designate an individual with disease as a positive (true positive)

clinical specificity of a diagnostic test

the ability of a test to correctly designate an individual who does not have a disease as negative to the test (true negative)