different methods for laboratory diagnosis

1. Laboratory Diagnosis:
An Overview

2. Goals
 Provide an overview of pathogens
tested in public health laboratories
 Describe laboratory tests commonly
used in outbreak investigations

3. A Review of Specimens
 Laboratory staff analyze specimen to
determine presence or absence of
suspected pathogens
 Specimens can tell us:
 Whether different individuals are infected
with the same pathogen
 Whether a particular source is causing an
outbreak

4. A Review of Specimens
 Environmental samples include:
 food (items suspected in a foodborne
outbreak)
 water (from a lake, water supply, or
drinking fountain)
 surfaces (medical equipment, countertops,
etc.)

5. A Review of Specimens
 Proper specimen collection is important
(see FOCUS Volume 4, Issue 2)
 Right sample must be collected
 Collected in proper medium for survival
 Transported within proper time frame and
temperature
 Accompanied by appropriate information

6. Microorganisms
 Bacteria: single-celled organisms
 Examples: Salmonella, Streptococcus (“strep”),
Staphylococcus (“staph”), Escherichia Coli (E. coli)
 Viruses: DNA (or RNA) surrounded by
protective coat of proteins
 Examples: Influenza, HIV, West Nile, Noroviruses,
common cold viruses (Coronavirus, Rhinovirus)
 Other pathogens: toxins produced by
bacteria, parasites, fungi, chemicals

7. Why is Lab Diagnosis
Necessary?
 Lab identification of the agent is crucial:
 Because diagnosis should not be based on clinical
symptoms alone
 Many agents cause similar symptoms
 Clinical symptoms may be unclear or too general
 Physicians might not recognize a rare disease
 To connect individual cases in outbreak
 To ensure proper medical treatment for patients
 Norovirus and Shigella infections cause same symptoms:
Norovirus treatment is symptomatic relief;
Shigella can be treated with antibiotic

8. Why is Lab Diagnosis
Necessary?
 Sometimes necessary to conduct further
studies to determine specific strain or
serotype: a.k.a. subtyping
 Dozens of strains of Noroviruses (e.g.,
including Hawaii virus, Snow Mountain
virus, Desert Shield virus, Toronto virus); if
people infected with a Norovirus have
different strains, the infections are
unrelated

9. Why is Lab Diagnosis
Necessary?
 To help identify outbreaks across state lines
 2006: CDC officials notified of several small
clusters of E. coli 0157:H7 infections in Wisconsin
and Oregon, with fresh spinach implicated as the
probable source. The same day, New Mexico
epidemiologists contacted Wisconsin and Oregon
epidemiologists regarding similar cluster of
infections. CDC’s PulseNet confirmed through
laboratory testing that E. coli O157:H7 strains
from infected patients in Wisconsin had same
PFGE pattern and identified that pattern in
patients from several other states.

10. Laboratory Diagnosis and
Surveillance Programs
 Council of State and Territorial Epidemiologists and
CDC recommend surveillance for list of pathogens
 Each state decides which pathogens healthcare
providers and laboratories must report
 Lab reports to the state health department using
disease reporting system
 Guidelines specify which identification methods are used to
ensure that only confirmed cases are reported
 State lab responsible for identification when local labs
do not have necessary expertise
 State lab has final responsibility for reporting cases to
state health department
 If identification not possible at the state level,
CDC may be asked to help

11. Pathogen Identification and
Typing
 Method depends on the type of
organism
 Some methods are well established for
particular organisms
 Guidelines exist for identifying the
organism

12. Pathogen Identification and
Typing
Identification
method
Tests Pros (+) and cons (-)
Microscopy
Examination of
organisms under
magnification
After preparation with various stains
and reagents, samples put onto
glass slides, examined with
microscope
Smaller microorganisms (viruses)
may require use of electron
microscope
+ Relatively quick, may provide immediate answers
- Clinical specimen may not contain sufficient
numbers of microorganisms for visualization without
culture
Culture
Propagation of
microorganisms in a
growth medium
Organism grown in a nutrient
medium (culture plates, stab culture,
slab culture, or liquid culture) OR
Organism is grown in live cells or
tissue (cell culture or tissue culture)
+ Is the “gold standard”: growth of the organism
provides a definitive diagnosis
- Limited by quality of specimen from which
organism is grown
- Not all pathogens can be cultured
- Does not detect past infection
Antigen detection
Uses antibodies to
detect antigens
Latex agglutination (LA),
complement fixation (CF), enzyme-
linked immuno-assay (EIA),
fluorescent antibody (FA) assay
+ Results often discernable by eye (no microscope
needed)
- Does not detect past infection
- Not possible for all pathogens
Serology
Detects any past
Latex agglutination (LA),
complement fixation (CF), enzyme-
+ Safe, does not require further growth of pathogen
+ Routine methods of measurement available

13. Pathogen Identification and
Typing
Typing method Tests Pros (+) and cons (-)
Phage typing
Uses viruses
(phages) that infect
specific bacteria
Tests using lambda phage,
gamma phage, T4 phage,
T7 phage, leviviruses,
microviruses
+ Very useful for particular strains
(Staphylococcus)
- Many organisms are not typeable
by this method
- Not standardized for many
organisms
Identification and
typing method
Tests Pros (+) and cons (-)
Molecular
techniques
Uses nucleic acid
identification
methods
Pulsed field gel
electrophoresis (PFGE),
random fragment length
polymorphism (RFLP),
random amplified
polymorphic DNA (RAPD),
ribotyping
+ Relatively quick
+ High sensitivity
- Often initially expensive (high
start-up costs)

14. Microscopy
 Useful for larger organisms such as bacteria
or fungi
 For standard optical or light microscope
 Small part of specimen smeared onto glass
slide
 Stains applied to help identify cells and
substances within the specimen
 When using Gram stain, “Gram-positive” bacteria
have a cell wall that will stain purple while “Gram-
negative” bacteria stain as red

15. Microscopy
 Common bacteria shapes:
 Round (cocci)
 Rod-shaped (bacilli)
 Bacteria can cluster in pairs, chains, other
arrangements
 E. coli is a Gram-negative rod
 S. pneumoniae or pneumococcus is a Gram-
positive diplococcus, a round bacterium that
clusters in pairs
 Shapes and growth patterns also used to
identify fungi and fungal spores

16. Microscopy
 Viruses are much smaller than bacteria or
fungi, require a very high degree of
magnification
 Electron microscope shoots electrons at virus
(like a camera flash shoots light at an object
to capture the image)
 Many viruses have a characteristic shape,
can be identified from microscope image

17. Culture
 Provide right temperature, moisture, and
nutrients for a pathogen to thrive and
replicate, introduce a sample, wait for growth
 Case definition may require a definite case to
be “culture confirmed”
 Outbreak of E. coli O157:H7 infections among
Colorado residents in June 2002, part of the case
definition was that specimens taken from patients
were culture-positive for E. coli. Contaminated
beef was implicated and over 350,000 pounds of
beef were recalled
 Can increase amount of organism to
perform other types of tests

18. Culture
 Bacteria often grown on a Petri dish
 Plate containing growth medium (gelatin-like
substance called agar, nutrients other materials)
 Bacteria form distinctive-looking colonies
Culture of
Nocardia
asteroids, a
mycobacterium
commonly found
in soils. It causes
illness in people
with defects in
cellular immunity.

19. Culture
 Some bacteria grow inside the culture
nutrients: stab culture
 Test tube filled with agar and nutrients, sterile
wire is dipped into sample and stabbed into tube
Stab culture of
Legionella
pnuemophila, the
agent that
causes
Legionnaire’s
disease. It is
found in aqueous
environments.

20. Culture
 Viruses need living cells to reproduce, so
often grown in tissue culture derived from
growing cells or tissues.
 May be tested by nucleic acid-based methods
or viewed under an electron microscope
 June 2003: Multistate monkeypox outbreak, with
monkeypox virus isolated from multiple patients
and cultured. All case patients found to have links
to prairie dogs. Virus from patients grown in cell
culture and confirmed using electron microscopy.

21. Culture
 Different organisms require different
conditions
 Not all organisms can be grown in culture;
other methods must be used
 Requires considerable amount of time to
grow certain organisms, can slow
investigation
 Pulmonary blastomycosis (fungal infection that
causes severe respiratory symptoms) can require
up to 5 weeks in culture before confirmatory
diagnostic tests can be performed

22. Culturing a Clinical Specimen
 A clinical specimen is cultured for microorganisms
known to thrive in the particular environment and
associated with certain clinical symptoms
 Fecal samples in diarrheal illnesses are cultured for enteric
pathogenic bacteria, including Salmonella serotypes (typhi,
enteritidis, typhimurium, etc.), Shigella, Campylobacter,
Yersinia, Escherichia coli 0157:H7, Vibrio
 Respiratory samples are cultured for pathogens such as
Streptococcus pneumoniae, Bordetella pertussis,
Haemophilus influenzae, Influenza, Legionella,
mycobacterium
 Cervical, vaginal or penile specimens may be cultured for
Neisseria gonorrhoeae, herpes, other organisms
that cause genital infections

23. Serology
 Uses immune response to determine whether
a person has fought off an infection by a
particular pathogen
 Compare blood samples taken at the time of
exposure (or shortly thereafter) and weeks later
 Looks at antibodies, or immunoglobulins
 If no antibodies are present (or present in early
form) at first blood sample and fully mature
antibodies are present at second sample, person
has been recently exposed
 Example: syphillis rapid plasma reagin (RPR)
test detects presence of antibodies
against syphilis in a blood sample

24. Serology
 Limitations:
 Not useful for a rapid intervention
 Often difficult to obtain a blood sample even once,
let alone twice
 May be useful:
 When pathogen is not easily detected in other
types of samples
 When source of exposure has been eliminated
with no remaining sample to test
 For research purposes

25. Antigen Detection
 Small parts of a viral or bacterial pathogen
 Separate antigens from other material, use
antibodies to find a particular antigen
 If antibodies attach to the target antigen,
pathogen has been identified
 If the antibodies do not find anything to attach to,
do not know which organism is causing infection
 Many ways antigens can be separated from
other matter in a specimen, many ways
antigen test can be performed

26. Phage Typing
 Short for bacteriophage, a virus that infects bacteria
 Each type of phage attacks a particular type of bacteria
 Most often used to identify strains of Staphylococcus aureus
 A “known” phage mixed with “unknown” bacterium,
poured onto an agar plate, allowed to grow
 If bacteria are correct strain, a plaque will form
 If no plaques, bacteria can be eliminated as possible pathogen
A “gamma phage” is used to identify Bacillus
anthracis growing on agar plate. Lawn of
bacteria interrupted where the gamma phage
has attacked the bacteria, causing a “plaque,”
or hole in the bacterial growth.

27. Molecular techniques
 Every pathogen has DNA, RNA, or both
 Can test a sample for presence of a bacteria
or virus by looking for the DNA
 Often referred to as “molecular methods”
 Useful for distinguishing between strains
 Can distinguish between strains of E. coli
normally found in the human gut and a
pathogenic strain causing disease
 Identifying exact strain is important for finding
source of an outbreak

28. Summary
 This overview of diagnostic techniques
can give you a better sense of what
happens once you send that specimen
off to the laboratory
 Future issues of FOCUS will delve
further into more advanced laboratory
techniques, such as molecular
identification and typing

29. Additional Resources
 To see examples of microorganisms that can often be identified
with a Gram stain, go to http://www.uphs.upenn.edu/bugdrug/
antibiotic_manual/gram.htm and click on “Typical Gram stains.”
 To see electron micrographs of viruses, go to
http://www.ncbi.nlm.nih.gov/ICTVdb/Images/index.htm.
 To find information on the diseases most often tested at public
health labs, visit the North Carolina State Laboratory of Public
Health Microbiology Web site:
http://204.211.171.13/Microbiology/default.asp.
 To find infectious disease information from the National Center
for Infectious Diseases, go to
http://www.cdc.gov/ncidod/diseases/index.htm.
 To use the American Society for Microbiology Microbe Library,
visit http://www.microbelibrary.org.

30. References
1. Centers for Disease Control and Prevention. Ongoing multistate
outbreak of Escherichia coli serotype O157:H7 infections associated
with consumption of fresh spinach --- United States, September
2006. MMWR Morb Mort Wkly Rep. 2006; 55(Dispatch):1-2.
Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/
mm55d926a1.htm. Accessed December 8, 2006.
2. Centers for Disease Control and Prevention. Multistate outbreak of
Escherichia coli O157:H7 infections associated with eating ground
beef --- United States, June--July 2002. MMWR Morb Mort Wkly Rep.
2002;51:637—639. Available at: http://www.cdc.gov/mmwr/
preview/mmwrhtml/mm5129a1.htm. Accessed November 30, 2006.
3. Centers for Disease Control and Prevention. Multistate outbreak of
monkeypox—-Illinois, Indiana, and Wisconsin, 2003. MMWR Morb
Mort Wkly Rep. 2003;52:537-540. Available at: http://www.cdc.gov/
mmwr/PDF/wk/mm5223.pdf. Accessed November 30, 2006.

31. References
4. Martynowicz MA, Prakash, UBS. Pulmonary blastomycosis: An
appraisal of diagnostic techniques. Chest. 2002;121:768-773.
5. Mayer G. Bacteriology Chapter 7: Bacteriophage. In: University
of South Carolina School of Medicine. Microbiology and
Immunology On-line [Internet]. September 11, 2003. Available
at: http://www.med.sc.edu:85/mayer/phage.htm. Accessed
November 30, 2006.
6. Herwaldt, et al. Microbial Molecular Techniques. In:
Epidemiologic Methods for the Study of Infectious Diseases, JC
Thomas, DJ Weber, eds. Oxford University Press, 2001: 163-
191.