Laboratory Diagnosis: An Overview

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 0157H7 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. CDCs PulseNet confirmed
  through laboratory testing that E. coli O157H7
  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 immunological response to pathogen Latex agglutination (LA), complement fixation (CF), enzyme-linked immuno-assay (EIA), fluorescent antibody (FA) assay Safe, does not require further growth of pathogen Routine methods of measurement available Detects past infection - Not all pathogens create immune response - May require sequential specimens
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 O157H7 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 Legionnaires 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 0157H7, 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.ht
  m.
• 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/Micro
  biology/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.o
  rg.

30
References

1. Centers for Disease Control and Prevention.
   Ongoing multistate outbreak of Escherichia coli
   serotype O157H7 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 O157H7
   infections associated with eating ground beef ---
   United States, June--July 2002. MMWR Morb Mort
   Wkly Rep. 200251637639. 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. 200352537-540. Available at
   http//www.cdc.gov/ mmwr/PDF/wk/mm5223.pdf.
   Accessed November 30, 2006.

31
References

1. Martynowicz MA, Prakash, UBS. Pulmonary
   blastomycosis An appraisal of diagnostic
   techniques. Chest. 2002121768-773.
2. 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.edu85/mayer/phage.htm.
   Accessed November 30, 2006.
3. 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.