Systems for Detection of Pathogens I

Question 1

What’s in a NAME?

Answer 1

Names provide us with the opportunities to define boundaries
It is up to the test system to define these boundaries
and provide a measure that informs us
“To what extent can any one microbe be able to cause disease”

Question 2

Mycobacterial genus - how many species and obligate human pathogens

Answer 2

147 current species
Only three are obligate human pathogens
M .tu berc u los is
M .leprae
M .u lc erans

Question 3

How can we define a pathogen?

Answer 3

A microbe
CAPABLE of causing
a specific degree of host damage

Question 4

Commensal Non pathogen (in host) - define w/examples

Answer 4

PRESENT but NOT CAPABLE of causing disease in the host
eg. E.c oli
B ac teroid es thetaiotaomic ron ‘good bacteria’

Question 5

Zoonotic Non pathogen (in carrier)

- define w/examples

Answer 5

PRESENT but only CAPABLE of causing disease in ANOTHER host

eg. E.c oliO157:H7 is subclinical in cattle

Question 6

Commensal Opportunist (in host) - define w/examples

Answer 6

PRESENT and CAPABLE of causing disease in the host
but only in certain circumstances
eg. B ac teroid es fragilis
Coagulase Negative Staphylococcus (CNS)

Question 7

Positive samples in relation to diagnosis - always 100%?

Answer 7

Not all positive samples are diagnostic of active disease

Question 8

Sterile sites vs non sterile sites in tests - give examples

Answer 8

Sterile sites must be free from contamination
eg. Skin flora in blood cultures

Non sterile sites require decontamination of normal flora
eg Faeces, Mouth, Skin

Question 9

Which samples require concentration for testing - give examples

Answer 9

Samples with high volume or relatively low infected pathogen load
require concentration (centrifugation, filtering)
eg CSF, Ascites, 24 hr Urine

Question 10

Desribe sequence of events from preparation to identification phase (compare culture vs direct)

Answer 10

PP for culture = Enrichment
Purification
Amplification

PP for direct = Concentration
Sample treatment

ID same for both =

Molecular DNA/RNA
Gross morphology (Microscopy)
Chemical composition (HPLC MassSpec)

Question 11

Microscopy - advantages

Answer 11

Advantages
Easy to perform
Rapid screening
Some parasites have SPECIFIC morphology
eg. S c his tos oma mans onii
Specific Immunoflourescence staining possible

Question 12

Microscopy - disadvantages

Answer 12

Disadvantages
Not Sensitive
eg. M yc obac teriu m tu berc u los is
screening sputum smears requires
at least 10,000 orgs per ml to be visualised
General stains are not specific
Labour intensive (expensive)
Requires specialist interpretive expertise (more expensive)

Question 13

Bacteriology - what does it rely on

Answer 13

Bacteriology
This relies on the ability of the test system
to be able to grow the pathogen

Question 14

Bacteriology - types and examples of media

Answer 14

Media:

Non Selective Media
eg. Blood Agar

Semi Selective Media
eg. MacConkey Agar, DCA, CLED

Selective growth temperatures
eg. Campylobacter species

Question 15

Describe diagnosis for Streptococcus pneumoniae

Answer 15

Optochin sensitivity
with gram stain microscopy
and colony morphology
is DIAGNOSTIC for
S treptoc oc c u s pneu moniae

Question 16

Classical metabolic testing - results for catalase

Answer 16

E.coli = +ve

Clostridium perfringens –ve

Question 17

Classical metabolic testing - results for indole test

Answer 17

Can cleave indole from tryptophan
(Indole test)

E.coli = +ve
Clostridium perfringens –ve

Question 18

Describe the identification of Enterobacteriacae

Answer 18

Metabolic function and sugar utilisation tests for identification of Enterobacteriacae
Eg. Salmonella, Shigella, E.coli

Question 19

Food Poisoning - describe microscopy test

Answer 19

Stool → Direct microscopy for cysts/eggs of amoebae or parasites

Question 20

Food Poisoning - describe gram stain test

Answer 20

incubate
microaerophilically at 42ºC
(85% N2, 10% CO2
, 5% O2
)at 42ºC

Spread on Campy CVA
selective media plate

Positive growth
(silver grey colonies)

Gram stain

Gram negative curved rods

Oxidase positive

Campylobacter jejuni

Question 21

Food Poisoning - describe antibiotic sensitivity testing

Answer 21

incubate
aerobically at 37ºC

M acC onkeyagar &
D esoxycholate agarplates

Identify bacteria by biochemical profiling
(API 20E strip)

Salmonella
typhimurium + Shigella
flexneri

=

Antibiotic sensitivity testing
disc diffusion plates

Question 22

Virology culture requires what and list its effect

Answer 22

Culture
Requires permissive cell lines
eg.Vero cells (Kidney epithelial)
for H erpes s implex

Cytopathic Effect
Immunofluorescent staining of culture
Direct Antigen Detection
ELISA
eg. Influenza Virus

Question 23

ELISA titres to specific antigens can be measured by

Answer 23

Multiple samples single
antigen with signal positive
colour cut off

Question 24

Classical culture and identification - advantages

Answer 24

Advantages
Cheap simple, reliable reagents
Sensitive
eg. Single organisms can be grown and identified
Validated specificity
eg. ‘Gold Standards’ with multiple parameters
Direct in vivo measurement of effectiveness of therapy
eg Antibiotic sensitivity
Easily archived
eg. Epidemiology

Question 25

Classical culture and identification - disadvantages

Answer 25

Disadvantages
Some pathogens cannot be grown eg. M yc obac teriu m leprae
Some pathogens cannot be well differentiated by biochemistry alone
Slow: culture requires at least overnight incubation:
Viral = 3-10 days
Mycobacterial = 6-12 weeks
Some pathogens grow too slowly to aid rapid diagnosis
eg. M yc obac teriu m tu berc u los is
Labour intensive (expensive)
Requires specialist interpretive expertise (more expensive)