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YEDİTEPE
UNIVERSITY
YEDİTEPE
SCHOOL OF
MEDICINE
Laboratory Diagnosis of Infectious Diseases –II
(Examplary Methods and Tests)
(Modified from the lecture given by Prof. Gülden Çelik, 2015-2016)
İ. Çağatay Acuner
M.D., Clinical Microbiologist, Associate Professor
Department of Microbiology
Faculty of Medicine, Yeditepe University, Istanbul
[email protected]
Laboratory Methods in Clinical Microbiology
• Direct:
• -Microscopy
• -Culture
• -Antigen
• -Nucleic acid
• Indirect:
• -Specific antibody (Serology) (IgG, IgM, IgA)
Microscopy
Two basic purposes:
1-the initial detection of microbes
2-the preliminary or definitive identification of microbes.
The microscopic examination of clinical specimens is used to detect:
• bacterial cells,
• fungal elements,
• parasites (eggs, larvae, or adult forms), and
• viral inclusions present in infected cells.
Characteristic morphologic properties can be used for the preliminary identification
of
• most bacteria and
• are used for the definitive identification of many fungi and parasites.
• But lacks sensitivity !
Microscopy
•
•
•
•
•
Brightfield (light) microscopy
Darkfield microscopy
Phase-contrast microscopy
Fluorescent microscopy
Electron microscopy
Microscopy
• Darkfield microscopy (Treponema pallidum)
Treponema pallidum in the direct fluorescent
antibody test : more sensitive and specific
The microscopic detection of organisms stained with antibodies labeled with fluorescent
dyes or other markers:
• very useful for the direct, sensitive and specific identification of many organisms.
Microscopy
Direct Fluorescent Stains (without antibody marker)
• Acridine orange stain:
• Used for detection of bacteria and fungi in clinical specimens.
• Auramine-rhodamine stain:
• Same as acid-fast stains.
• Calcofluor white stain:
• Used to detect fungal elements and Pneumocystis spp.
Direct fluorescent antibody stain
• Antibodies (monoclonal or polyclonal) are complexed with fluorescent molecules.
• Specific binding to an organism is detected by presence of microbial fluorescence.
• Technique has proved useful for detecting or identifying many organisms
• (e.g., Streptococcus pyogenes, Bordetella, Francisella, Legionella, Chlamydia,
Pneumocystis, Cryptosporidium, Giardia, influenza virus, Herpes simplex
virus).
• Sensitivity and specificity of the test are determined by
• the number of organisms present in the test sample and quality of
antibodies used in reagents.
Microscopy
Other Direct Examination Microscopy Methods
• The sample:
• can be suspended in water or saline (wet mount),
• mixed with alkali to dissolve background material (potassium hydroxide [KOH]
method) : fungal elements
• mixed with a combination of alkali and a contrasting dye (e.g., lactophenol
cotton blue: fungal elements
• mixed with Lugol iodine:
• Iodine is added to wet preparations of parasitology specimens to
enhance contrast of internal structures.
• Facilitates differentiation of protozoa and host white blood cells.
Enterobius vermicularis
 Pinworm eggs are deposited
by adults at night in the
perianal area.
 Eggs are collected by pressing
tape on the anal surface and
examining it microscopically.
 Eggs appear as an embryo
surrounded by a colorless shell
that is characteristically
flattened on one side.
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Microscopy
Other Direct Examination Microscopy Methods
• The sample:
• mixed with India ink
• in which the ink darkens the background rather than the cell.
• used to detect capsules surrounding organisms,
• the yeast Cryptococcus (the dye is excluded by the capsule, creating a
clear halo around the yeast cell),
• is a rapid method for the preliminary detection and identification of this
important fungus.
Microscopy
Most organisms are colorless and transparent, various dyes (stains) are used to see
the individual cells
A variety of different types of stains are used in the microbiology lab, including:
• Contrast stains
• (e.g., methylene blue, lactophenol cotton blue, India ink, iodine)
• Differential stains
• (e.g., Gram stain, spore stains, acid-fast stains, Giemsa stain, silver stains,
Trichrome stain)
• Fluorescent stains
• (e.g., acridine orange, auramine-rhodamine, calcofluor white, antibodyconjugated fluorescent stains)
Microscopy
• Differential stains
• (e.g., Gram stain, spore stains, acid-fast stains, Giemsa stain, silver stains,
Trichrome stain)
• Gram stain :
• -bacteria (positive, negative)
• -yeasts (yeasts are gram-positive).
• Iron hematoxylin and trichrome stains:
• protozoan parasites
• Giemsa stain:
• blood parasites and other selected organisms
Methylene Blue Stain
 Corynebacterium
diphtheriae
Lactophenol Cotton Blue (LCB) Stain
 primarily for observing
the morphology of
fungal molds :
Aspergillus
India Ink Stain
 The India ink stain:
 negative contrasting stain
 Cryptococcus neoformans.
 The ink is excluded by the
fungal capsule so the fungi
(arrows) are unstained and
surrounded by a clear halo,
while the ink particles
provide a background
contrast.
Iodine Stain
 The iodine stain is a
contrast stain used
primarily for the
detection of intestinal
parasites
(Entamoeba coli in
this example).
Gram Stain
 gram-positive (purple)

from gram-negative (red) bacteria.
Staphylococcus aureus and Candida
albicans
 S. aureus (black
arrow) and
 yeasts, in this case
Candida albicans (red
arrow).
 Yeast can appear as
gram-positive,
although they tend to
decolorize readily.
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Acid-Fast Stains
 Acid-Fast Stains
 Ziehl-Neelsen stain: Used to
stain mycobacteria and other
acid-fast organisms.
 Kinyoun stain: Cold acid-fast stain
(does not require heating)
 Mycobacteria
 partially acid-fast organisms
such as Nocardia
Microscopy
• Auramine-rhodamine:
• Same principle as other acid-fast stains, except that fluorescent dyes
(auramine and rhodamine) are used for primary stain
• Modified acid-fast stain:
• Weak decolorizing agent is used with any of three acid-fast stains listed.
• Whereas mycobacteria are strongly acid-fast, other organisms stain weaker
(e.g., Nocardia, Rhodococcus, Tsukamurella, Gordonia, Cryptosporidium,
Isospora, Sarcocystis, and Cyclospora).
• Organisms that retain this stain are referred to as partially acid-fast.
Panels A and B, Cryptosporidia. Panel C, Cyclospora. Panel D, Isospora.
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Giemsa Stain
 differential stain used
for detection of
parasites in blood
smears
 Plasmodium
Silver Stain

Silver stains are primarily used in
anatomic pathology labs and not
in microbiology labs.

Fungal elements (hyphae [photo]
and cells) are stained with silver
particles..
Fecal leucocyte negative
Fecal leucocyte positive
Culture
(in vitro; on artificial media or living cells)
Anton van Leeuwenhoek : Microscobic observation (1676 )
Pasteur: culture of bacteria almost 200 years later
• Other rapid tests replaced culture methods
• microbial antigen detection
• nucleic-acid-based assays
• But the ability to grow microbes in the laboratory remains an important procedure
in all clinical labs.
• For many diseases, the ability to grow a specific organism from the site of
infection is the definitive method to identify the cause of the infection.
Culture
(in vitro; on artificial media or living cells=cell culture)
• The success of culture methods is defined by:
•
•
•
•
the biology of the organism
the site of the infection
the patient's immune response to the infection
the quality of the culture media
• Culture media can be subdivided into four general categories:
• enriched nonselective media,
• selective media,
• differential media, and
• specialized media
• Certain bacteria need special conditions:
• Legionella is an important respiratory pathogen; media should be supplemented
with iron and L-cysteine.
• Campylobacter, an important enteric pathogen, highly selective media should
be incubated at 42° C in a microaerophilic atmosphere.
• Chlamydia, an important bacterium responsible for sexually transmitted
diseases, is an obligate intracellular pathogen that must be grown in living cells.
Culture
(in vitro; on artificial media or living cells=cell culture)
Some bacteria and all viruses are strict intracellular microbes
• They can only grow in living cells.
• In 1949, Enders described a technique for cultivating mammalian cells for the
isolation of poliovirus.
• This technique has been expanded for the growth of most strict intracellular
organisms.
• Cell culture is not a routine test!
• The cell cultures can either be cells
• that grow and divide on a surface (i.e., cell monolayer) or
• grow suspended in broth.
• Some cell cultures are well established and can be maintained indefinitely.
• These cultures are commonly commercially available.
• Other cell cultures must be prepared immediately before they are infected with
the bacteria or viruses and cannot be maintained in the laboratory for more than
a few cycles of division (primary cell cultures).
Serologic Methods
(Immunologic techniques)
 Detect
 Identify
 Quantitate antigen or antibody
Disadvantage: Cross reaction
-similar or common epitope
Serologic, Serodiagnosis,
Serology
 Detection of antigen or antibody in serum
 The term serologic is used also for searching antigen or
antibody in mediums other than serum(saliva,urine)
 Serologic assay=immunoassay
Immunoassays
 Antigen or antibody is detected
 In a variety of clinical specimens:
 Mostly sera
 Body fluids(cerebrospinal fluid)
 Tissues
 Environmental substances
Antibodies
Polyclonal:
 Heterogeneous antibody preparations
 Recognizes many epitopes on a single antigen
Monoclonal:
 Recognize individual epitoses on an antigen
Methods of detection
Antibody-antigen complexes can be detected:
 Directly
 Labelling the antibody or the antigen:
-enzyme
-radioactive
-fluorescent dye
Classical serologic methods
 Precipitation
 Immunodiffusion techniques
 Agglutination
Other serologic methods
 Complement fixation
 Hemagglutination inhibition
 Neutralization
Agglutination tests
 Clumping of antigen with its antibody
 Flocculation: similar to agglutination; except that agglutinats
float rather than sediment
 Prozone reaction: high antibody causes false negative. The
sera should be diluted!!
 Antigens passively absorbed on carriers:passive agglutination
Agglutination tests
 Antigens passively absorbed on carriers:passive agglutination
-Red blood cells: passive hemagglutination
-gelatin particles: particle agglutination
Classical agglutination in test tubes:
-Salmonella:Gruber Widal
-Brucella:Wright
-Rickettsiae:Weil-Felix reaction
Agglutination negative
Agglutination positive
Precipitation
 Tubes:solutions
 Gels:
 Double diffusion-Quchterlony
 Radial immunodiffusion
 Countercurrent electrophoresis (pyogenic meningitis and
fungal infections)
Immunoassays
 Immunofluorescence (IFA)
 Enzyme-linked immunosorbant assay (ELISA)
-Western blot
 Radioimmunoassay (RIA)
Serology
 can be used to identify the infecting agent
 evaluate the course of an infection, or determine the nature of the
infection-whether it is a primary infection or a reinfection, and
whether it is acute or chronic.
 Serologic testing is used to identify viruses and other agents that
are difficult to isolate and grow in the laboratory or that cause
diseases that progress slowly
In the diagnosis of infectious diseases
by immunoassays
 Either spesific antigen:
 Directly from specimen
 From the culture for identification
 Specific antibodies are detected:
 IgG
 IgM
 IgA
Specific antibody detection
 Seroconversion occurs when antibody is produced in response
to a primary infection.
 IgM:
early in infection (2-3 weeks)
transient (3-6 months)
*sometimes persists longer
 IgG: later
highest in 4-6 months
usually persists during the whole
 IgG avidity:
High: past infection
Low: new infection
life
Western blot (WB)
Examples of Viruses Diagnosed by
Serology
 Epstein-Barr virus
 Rubella virus, Measles,Mumps
 Hepatitis A, B, C, D, and E viruses
 Human immunodeficiency virus
 Human T-cell leukemia virus
 Arboviruses (encephalitis viruses)
Diagnosis of acute infection
 By specific IgM detection by ELISA:
 HAV
 Measles
 Rubella
 Mumps
 Parvovirus B19
 Varicella zoster…
Rapid antigen assay
 Sensitivity !
 Specificity !
Quantitative antibody detection:
 Anti-HBs: 10mIU/ml
 Rubella IgG: 10-15 IU/ml
Molecular Diagnosis
 Like the evidence left at the scene of a crime, the DNA
(deoxyribonucleic acid), RNA (ribonucleic acid), or proteins of an
infectious agent in a clinical sample can be used to help identify
the agent.
 In many cases the agent can be detected and identified in this way,
even if it cannot be isolated or detected by immunologic means.
New techniques and adaptations of older techniques are being
developed for the analysis of infectious agents.
Molecular methods in infectious diseases
 Target molecule
 DNA
 RNA
Molecular Diagnosis
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
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The advantages of molecular techniques:
their sensitivity
Specificity
safety..
 But expensive for the time being!
P olymerase
C hain
R eaction
PCR
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


The polymerase chain reaction (PCR):
amplifies single copies of viral DNA millions of times over
one of the newest techniques of genetic analysis
a sample is incubated with
- two short DNA oligomers, termed primers, that are
complementary to the ends of a known genetic sequence within
the total DNA
- a heat-stable DNA polymerase (Taq or other polymerase
obtained from thermophilic bacteria)
- nucleotides, and buffers.
PCR
 The oligomers hybridize to the appropriate sequence of DNA and act as




primers for the polymerase, which copies that segment of the DNA.
The sample is then heated to denature the DNA (separating the strands of the
double helix) and cooled to allow hybridization of the primers to the new
DNA.
Each copy of DNA becomes a new template. The process is repeated many (20
to 40) times to amplify the original DNA sequence in an exponential manner.
A target sequence can be amplified 1,000,000-fold in a few hours using this
method.
This technique is especially useful for detecting latent and integrated virus
sequences, such as in retroviruses, herpesviruses, papillomaviruses, and other
DNA viruses.
What is PCR?
 PCR uses the DNA replication ‘machinery’ of
a cell to make multiple copies of a specific
DNA sequence.
 PCR is perhaps the most successful
technique in Biology
 PCR can take a trace amount of DNA and
make enough copies of it for testing
What is it used for?
 PCR is useful in any situation where a small
amount of DNA is insufficient for analysis.
 PCR is used to establish blood relationships,
to identify remains, and to help convict
criminals or exonerate the falsely accused.
 PCR is an essential procedure in any genetics
laboratory.
History
 Discovered in 1983 in California by Kary
Mullis
 Published in a 1985 paper
 Sold by Cetus Corporation for $300 million
 Mullis won the 1993 Nobel Prize in Chemistry
for his discovery
Jonas Salk statement about his Polio vaccine:
“There is no patent. Could you patent the sun?”
How it works…
Number of amplified pieces = 2n (n = # of cycles)
The Thermocycler
Postamplification detection
 Gel analysis
 Colorimetric microtitre plate system
 Target amplification and detection systems occur
simultaneously in the same tube (Real- Time PCR)
RV12
İnfluenza A
Rapid real –time PCR
Other amplification methods
 TAS: transcription-based amplification system
 3SR: self sustained sequence replication
 NASBA: nucleic acid sequence-based amplification
 (very similar)
Other amplification techniques (II)
 LCR : ligase chain reaction
 bDNA: branched DNA
 Qbeta replikase
Molecular Techniques
 Technique Purpose Clinical Examples
 RFLP:




Comparison of DNA Molecular epidemiology, HSV-1
strains
DNA electrophoresis: Comparison of DNA Viral strain
differences (up to 20,000 bases)
Pulsed-field gel electrophoresis: Comparison of DNA (large pieces
of DNA)
Streptococcal strain comparisons
In situ hybridization: Detection and localization of DNA
sequences in tissue Detection of nonreplicating DNA virus
(e.g., cytomegalovirus, human papillomavirus)
Dot blot
Detection of DNA sequences in solution
Detection of viral DNA
 Southern blot: Detection and characterization of DNA
sequences by size Identification of specific viral strains
 Northern blot: Detection and characterization of RNA
sequences by size Identification of specific viral strains
 PCR:
Amplification of very dilute DNA samples
Detection of DNA viruses
 RT-PCR: Amplification of very dilute RNA samples
Detection of RNA viruses
 Real-time PCR:
Quantification of very dilute DNA and RNA
samples Quantitation of HIV genome: virus load
 Branched-chain DNA: Amplification of very dilute DNA or RNA
samples
Quantitation of DNA and RNA viruses
 Antibody capture solution hybridization DNA assay:
Amplification of very dilute DNA or RNA samples
Quantitation of DNA and RNA viruses
SDS-PAGE: Separation of proteins by molecular weight
Molecular epidemiology of HSV
Direct sequencing
 Combination of PCR with dideoxynucleotide chain
termination methods can be used to determine sequence of
DNA: detecting microorganisms
 Genotyping of viruses
 Identification of bacteria and fungi
 Antimicrobial susceptibilty testing to detect mutations
DNA microarrays
 Thousands of oligonucleotides are on a solid support
 A labelled amplification product is hybridized to the probes
Microarray
Multiplex PCR:
Now more often: !!!!
-antigen detection
-rapid real-time PCR
-multiplex PCR
The success of the Microbiology
laboratory
 Quality of the specimen
 The way its sent
 The method used
 The interpretation:
 Do not hesitate to have contact with your microbiology
laboratory!