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Direct and indirect diagnostic
methods in infectious diseases
Learning Objectives
At the end of this lecture, the student should be able to:
• list the main methods in diagnosis of
different type of microorganisms
• explain the importance of these methods in
diagnosis
• List the main advantages and disadvantages
of each type of test
Case I
• 6 year old child presented with a 24 hour
history of fever, vomiting and complaint of a
sore throat
Etiology
• Strep throat: S. Pyogenes (A group Beta
hemolytic streptococci)
– Mainly viruses
– Rarely:
• Corynebacterium
• Arcanobacterium
• Neisseria gonorrhoeae
Laboratory Diagnosis
Diagnosis
• Rapid antigen test
• Culture and susceptibility test ?
Case II
• Fever
• History
Case II
• Fever
• Has a history to visit to west Africa
• Ebola
• Malaria
Case
•
•
•
•
•
•
Peripheral blood smear:
Plasmodium
Babesia
Trypanosome
Microfilaria
Bacteria and fungi
Case
• 1.5 years old patient presenting to the
emergency room with 2 hour of history of
vomitting, diarrhea, fever
• Stool macroscopy: no mucus no blood
Stool microscopy
• Fecal leucocyte
• Culture: Salmonella, Shigella and
camphylobacter
Fecal leucocyte
Fecal leucocyte absent
Season
• Winter
For rapid diagnosis
• Antigen detection
– Rotavirus
Outbreak!
• Norovirus antigen
Multiplex PCR
case
• A child
• Maculupapular rash
case
• Maculupapular rash
• Viral(measles, rubella, HHV6,parvovirus
B19,EBV, Enterovirus,CMV
• Others
• HIV
Laboratory Diagnosis
Maculopapular rash
•
•
•
•
•
Specific serology:
IgM
Total antibody
Antigen and antibody
NAT
Laboratory diagnosis
• Direct
• Indirect
Laboratory diagnosis
• Direct: -Microscopy
-Culture
-Antigen
-Nucleic acid
• Indirect:
-Specific antibody (Serology)
Laboratory diagnosis
• Direct: -Microscopy
-Culture
-Antigen
-Nucleic acid
• Indirect:
-Specific antibody (IgG, IgM, IgA)
Laboratory diagnosis
• Direct: -Microscopy
-Culture
-Antigen detection
-Nucleic acid detection:
Nucleic acid amplification techniques
(NAT=NAAT)
• Indirect:
-Specific antibody (IgG, IgM, IgA)
• What do you see?
• Which type of microscopy is this?
Microscopic Principles and Applications
• In general, microscopy is used in microbiology
for two basic purposes:
1-the initial detection of microbes
2-the preliminary or definitive identification of
microbes.
Microscopic Principles and Applications
• 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.
- But lacks sensitivity !
Stains
 Because 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,
antibody-conjugated fluorescent stains)
32
Methylene Blue Stain
?
33
Methylene Blue Stain
 Corynebacterium
diphtheriae
34
Direct Examination
The sample:
• can be 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
Lugol iodine :parasitology specimen
Lactophenol Cotton Blue (LCB) Stain
?
36
Lactophenol Cotton Blue (LCB) Stain
 primarily for
observing the
morphology of fungal
molds :
Aspergillus
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Enterobius vermicularis
 Pinworm eggs:
 deposited by adults at night in
the perianal area.
 Eggs are collected by
pressing tape on the anal
surface
 Eggs appear as an embryo
surrounded by a colorless
shell that is characteristically
flattened on one side.
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Iodine Stain
?
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Iodine Stain
 The iodine stain is
a contrast stain
used primarily for
the detection of
intestinal parasites
(Entamoeba coli in
this example).
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Direct Examination
India ink method,
• in which the ink darkens the background rather than
the cell.
• This method is used to detect capsules surrounding
organisms, such as the yeast Cryptococcus (the dye
is excluded by the capsule, creating a clear halo
around the yeast cell), and
• is a rapid method for the preliminary detection and
identification of this important fungus.
•
India Ink Stain
?
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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.
 But now antigen detection
is preferred
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Differential Stains
• Gram stain : -bacteria
-Yeasts (yeasts are grampositive).
Gram staining
• Neisseria gonorrhoae detection in uretral
specimen from males
Differential 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)
Updated Guidelines for the Use of Nucleic
Acid Amplification Tests in the Diagnosis of
Tuberculosis
Conventional tests for laboratory confirmation of TB include
• acid-fast bacilli (AFB) smear microscopy(24 hours)
• culture
Although rapid and inexpensive,
AFB smear microscopy is limited by its poor sensitivity
(45%–80% with culture-confirmed pulmonary TB cases)
Acid-Fast Stains
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Acid-Fast Stains
 Mycobacteria
 If a weak decolorizing solution is
used to remove the primary
stain, then partially acid-fast
organisms such as Nocardia
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• 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).
• These organisms can be stained more efficiently by using
weak decolorizing agent. 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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Differential Stains
• Giemsa stain: blood parasites
Giemsa Stain
 differential stain used
for detection of
parasites in blood
smears
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Giemsa Stain
 Plasmodium
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Darkfield Microscopy
• Treponema pallidum (syphilis): !
used in routine diagnosis
• Leptospira spp. (leptospirosis)
not used
Microscopic Principles and
Applications
• The microscopic detection of organisms
stained with antibodies labeled with
fluorescent dyes or other markers has
proved to be very useful for the specific
identification of many organisms.
Treponema pallidum in the direct fluorescent
antibody test for T. Pallidum:
more sensitive
In Vitro Culture: Principles and
Applications
•
Anton van Leeuwenhoek : Microscobic observation (1676 )
•
Pasteur: culture of bacteria almost 200 years later
• Over the years, microbiologists and cooks have
returned to the kitchen to create hundreds of culture
media that are now routinely used in all clinical
microbiology laboratories.
In Vitro Culture: Principles and
Applications
• Although tests that rapidly detect microbial antigens and
nucleic-acid-based molecular assays have replaced culture
methods for the detection of many organisms,
• 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.
• Antibiotic susceptibility test
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.
Certain bacteria need special
conditions:
• Legionella is an important respiratory pathogen;
media should be supplemented with iron and lcysteine.
• 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.
Types of Culture Media
Culture media can be subdivided into four
general categories:
(1) enriched nonselective media,
(2) selective media,
(3) differential media, and
(4) specialized media
Cell Culture: not routine !
• 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.
Serologic Methods
(Immunologic techniques)
• Detect
• Identify
• Quantitate antigen or antibody
Disadvantage: Cross reaction: False positivity
-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
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:
forms later (immunity)
highest in 4-6 months
usually persists during the whole
• IgG avidity: High: past infection
Low: new infection
life
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…
Quantitative antibody detection:
• Anti-HBs: 10mIU/ml(immune to HBV
infection)
• Rubella IgG: 10-15 IU/ml(immune to Rubella
infection)
Antigen detection
•
•
•
•
•
•
Membrane ELISA
Immunochromatograhic methods
Latex agglutination
Rapid
Less sensitive
Less specific
Antigen detection
•
•
•
•
•
•
•
Strep A
RSV
Influenza
Rota
Norovirus
Legionella pneumophila serogroup I
Malaria
Nucleic acid amplification techniques
(NAT=NAAT)
• Target molecule
– DNA
– RNA
Molecular Diagnosis
•
•
•
•
The advantages of molecular techniques:
their sensitivity
Specificity
safety..
P
C
R
olymerase
hain
eaction
Requirements of PCR:
• Knowing parts of the target DNA sequence to be
amplified
• Two types of synthetic primers, complementary
to the ends of the target sequence
• Large amounts of the four DNA nucleotides
• Taq1, a heat-resistant form of DNA Polymerase
How it works…
Number of amplified pieces = 2n (n = # of cycles)
The Thermocycler
PCR
• The polymerase chain reaction (PCR):
• amplifies single copies of viral DNA millions of times over
• Very sensitive
Danger !:
• Contamination
• False positive
Postamplification detection
• Gel analysis
• Colorimetric microtitre plate system
• Target amplification and detection systems
occur simultaneously in the same tube (RealTime PCR): -sensitive
-specific
-rapid
-easy to use
-expensive
RV12
İnfluenza A
Rapid molecular techniques!!!
Rapid real –time PCR
Direct sequencing
• Combination of PCR with dideoxynucleotide
chain termination methods can be used to
determine sequence of DNA.
• 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
-quantitative detection for followup treatment
-antimicrobial resistance
Laboratory diagnosis
•
•
Direct:
– Microscopy: N.gonorrhoae in male
– Culture:+antibiotic susceptibility
– Antigen detection:rapid, less sensitive(Strep A/RSV/Rota../in urine: L.
pneumophila)
– NAAT: -real-time PCR(M.tuberculosis, Clostridium difficile, MRSA,VRE..)
-multiplex-PCR, quantitation, resistance mutation detection
Indirect:
– spesific IGM: viral
infections(measles,mumps,rubella..
– Quantitattive IgG: immunity:
» Anti-Rubella IgG
» Anti-HBs
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!
Reference:
7th ed 2013
Group A reportable disease
• From every health care centers
Group A reportable disease
Group B reportable disease
• Directly and promtly by phone
• Internationally reported
Group B reportable disease
C Grubu Bildirimi Zorunlu
Hastalıklar
• From selected health care units
Group C reportable disease
Group D reportable disease
• Positivity reported from the lab directly
Group D reportable disease