Laboratory Diagnosis of Infectious
Diseases
Prof Dr Gülden Çelik
gulden.yilmaz@yeditepe.edu.tr
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
Methods in Laboratory diagnosis of
infectious diseases
 Direct
 Indirect
Laboratory diagnosis
 Direct:
-Microscopy
-Culture
-Antigen
-Nucleic acid
 Indirect:
-Specific antibody (Serology)
Laboratory diagnosis
 Direct:
-Microscopy
-Culture
-Antigen detection
-Nucleic acid detection
 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)
Determinating the value of tests
 Sensitivity
 Specificity
 Positive predictive values
 Negative predictive values
Sensitivity
 Analytical or epidemiologic
 Analytical sensitivity refers to the ability of a test to detect
very small quantities of antibodies as occur during
seroconversion
 Epidemiologic sensitivity(clinical sensitivity): refers to
the ability of a test to detect persons with established
infection.
Sensitivity
True positives
 Sensitivity =
X 100
True positives + false negatives
Specificity
True negatives
 Specificity =
X 100
True negatives+ false positives
Positive predictive value
 PPV=
true positives
____________________ x100
true positives+false positives
Negative predictive value
 PPV=
true negatives
____________________ x100
true negatives+false negatives
Presence of
disease
Number of people
with + test
Number of people
with - test
Total
Sick
TP
FN
TP+FN
Healthy
FP
TN
FP+TN
Total
TP+FP
FN + TN
Diagnostic Sensitivity= [TP/(TP+FN)] x 100
Diagnostic spesifity= [TN/(TN+FP)] x 100
Positive predictive value= [TP/(TP+FP)] x 100
Negative predictive value= [TN/(FN+TN)] x 100
TP + FP + FN + TN
(Positivity in sick people)
(Negativity in healty people)
PREVALANCE : % 0.02
Presence of
disease
Number of people
with + test
Total
Number of people
with - test
Sick
199
1
200
Healthy
2000
997.800
999.800
Total
2199
Diagnostic Sensitivity= % 99.8
Diagnostic spesifity= = % 99.8
997.801
1.000.000
(Positivity in sick people)
(Negativity in healty people)
Positive predictive value= [199/(199+2000)] x 100 = % 9
Negative predictive value= [997.800/(1+997.801)] x 100 = %100
TAHMİN ETTİRİCİ DEĞER: PREVALANS :
% 0.02
Hastalık
durumu
Hastalıklı
Pozitif test
sonuçlu kişiler
Negatif test
sonuçlu kişiler
Toplam
199
1
200
Hastalıksız
2000
997.800
999.800
Toplam
2199
997.801
1.000.000
Diyagnostik duyarlılık = % 99.8
(Hastalıkta pozitif)
Diyagnostik özgüllük = % 99.8
(Hastalıkta negatif)
Pozitif test sonucunun tahmin ettirici değeri = [199/(199+2000)] x 100 = % 9
Negatif test sonucunun tahmin ettirici değeri = [997.800/(1+997.801)] x 100 =
%100
Boy with fever and rash
 In early June
 a 15-year old boy comes to your
practice with his mother.
 He had been fine until about five
days ago when he developed a
fever.
 He has a stiff neck and a rash on
his back.
 His mother reports that he was
playing in the woods with some
friends recently.
Which of the following bacteria may
be the agent
 Pseudomonas aeruginosa
 Clostridium perfringens
 Borrelia burgdorferi
 Streptococcus pyogenes
 What do you see?
 Which type of microscopy is this?
Tick-born disease
 Borrelia burgdorferi is the
causative agent of Lyme
disease.
 This bacterium, just like
Treponema pallidum, is a
member of the spirochetes,
the family of spiral-shaped
bacteria.
Boy with fever and rash
 After an incubation period of 3
to 30 days,
 develop at the site of the tick
bite.
 The lesion (erythema
migrans) begins as a small
macule or papule and then
enlarges over the next few
weeks, ultimately covering an
area ranging from 5 cm to more
than 50 cm in diameter
Definition of Lyme Disease
 Lyme disease begins as an early localized infection,
progresses to an early disseminated stage, and if untreated,
can progress to a late manifestation stage.
B. burgdorferi
 present in low numbers in the skin
So:
 culture of the organism from skin lesions
 detection of bacterial nucleic acids by polymerase chain
reaction (PCR) amplification
 is not prefered
Microscopy
 Microscopic examination of blood or tissues from patients
with Lyme disease is not recommended, because B. burgdorferi
is rarely seen in clinical specimens.
Lyme Disease
 Microscopy
 Culture
 Nucleic-Acid-Based Tests :65% to 75% with skin biopsies, 50% to
85% with synovial fluid
 Antibody Detection :
 Spesific IgM: IgM antibodies appear 2 to 4 weeks after the onset of
erythema migrans in untreated patients; the levels peak after 6 to
8 weeks of illness and then decline to a normal range after 4 to 6
months.
 Spesific IgG
In every infectious disease
 The tests to be used
 The clinical material should be properly selected
 should be properly selected !
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.
Microscopic Principles and
Applications
 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 !
Microscopic Methods
 Brightfield (light) microscopy
 Darkfield microscopy
 Phase-contrast microscopy
 Fluorescent microscopy
 Electron microscopy
Darkfield Microscopy
 Treponema pallidum (syphilis): !
used in routine diagnosis
 Leptospira spp. (leptospirosis)
not used
Treponema pallidum in the direct
fluorescent antibody test for T. Pallidum:
more sensitive
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.
 Fluorescent Stains
 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.
Direct Examination
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
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.
 The dyes nonspecifically stain the cellular material, increasing the contrast with
the background, and permit examination of the detailed structures.
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.
39
Direct Examination
 A variation is the 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.
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, acidfast stains, Giemsa stain, silver stains, Trichrome stain)
 Fluorescent stains (e.g., acridine orange, auraminerhodamine, calcofluor white, antibody-conjugated
fluorescent stains)
41
Differential Stains
 Differential stains:
 Gram stain :
-bacteria
-Yeasts (yeasts are gram-Ipositive).
 Iron hematoxylin and trichrome stains:protozoan parasites
 Giemsa stain: blood parasites and other selected organisms
Methylene Blue Stain
?
46
Methylene Blue Stain
 Corynebacterium
diphtheriae
47
Lactophenol Cotton Blue (LCB) Stain
?
48
Lactophenol Cotton Blue (LCB) Stain
 primarily for observing
the morphology of
fungal molds :
Aspergillus
49
India Ink Stain
?
50
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.
51
Iodine Stain
?
52
Iodine Stain
 The iodine stain is a
contrast stain used
primarily for the
detection of intestinal
parasites
(Entamoeba coli in
this example).
53
Gram Stain
 ?
54
Gram Stain
 gram-positive (purple)

from gram-negative (red) bacteria.
55
?
56
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.
57
 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
60
Acid-Fast Stains
 Mycobacteria
 If a weak decolorizing solution is
used to remove the primary
stain, then partially acid-fast
organisms such as Nocardia
61
 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.
63
Giemsa Stain
 differential stain used
for detection of
parasites in blood
smears
64
Giemsa Stain
 Plasmodium
65
Silver Stain

Silver stains are primarily used in
anatomic pathology labs and not
in microbiology labs.
66
Silver Stain

Fungal elements (hyphae [photo]
and cells) are stained with silver
particles..
67
Fecal leucocyte -
Fecal leucocyte +
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.
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 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.
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
 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: not routine !
 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




The advantages of molecular techniques:
their sensitivity
Specificity
safety..
 But expensive for the time being!
P
C
R
olymerase
hain
eaction
PCR




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?”
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
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
Rapid molecular techniques!!!
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
Laboratory diagnosis
 Direct:
 Microscopy: N.gonorrhoae in male,Mycobacteria
 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: acute viral
infections(measles,mumps,rubella..
 Quantitative IgG: immunity:
o Anti-Rubella IgG
o 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