Chapter 12

Diagnosing Infections
Tiana Milanda
Diagnosis of Microbial Infection
Patient Clinical Microbiology
diagnosis

Radiology

Haematology
Biochemistry

Sample Take the correct specimen

Take the specimen correctly
Label & package the
specimen up correctly
Appropriate transport &
storage of specimen
 Specimen
Collection
• The success of
identification and
treatment depends on
how specimens are
collected, handled, and
stored
• General aseptic
procedures must be
used
 Diagnosis of Microbial Infection
1. Microscopy unstained or stained with e.g.
Gram stain

Stain Decolorise Counterstain

2. Culture identification by biochemical or

serological tests on pure growth
from single colony

on plates or in broth

5. Sensitivities by disc diffusion

methods,
breakpoints or
3. Imunological 4. Genotypic
MICs
method method
 Microscopy
Stained preparations

• Gram-stain
• Acid-fast stain(Ziehl-
Neelsen)
Medium (liquid,semisolid, solid)
 Culture of Bacteria
• Solid media
– Agar plates
For Identification/
enumeration
– Slopes
For safe long-term culture, Agar darah, medium diferensial
mengandung sel darah.
e.g. Lowenstein-Jensen
media for TB
 Culture of Bacteria

• Liquid media (broth)

– For enrichment or
maximum sensitivity
 Pure culture
• isolation of single 1

5
clonal colonies 2

– get bacterium in
pure culture 4
3
Jar untuk kultivasi bakteri anaerob.
 Culture of Bacteria in Solid
Media

• identify by colonial
morphology
 Microscopy
Stained preparations

• Gram-stain
• Acid-fast stain(Ziehl-
Neelsen)
 Culture : Physiological/Biochemical
Characteristics
• Diagnostic tests for determining the presence
of specific enzymes and assessing nutritional
and metabolic activities
• Examples
– Fermentation of sugars
– Capacity to metabolize complex polymers
– Production of gas
– Presence of enzymes
 Tes Biokimia : TBC

Tes Niacin (+) : M. tbc Reduksi Nitrat (+) : M. tbc

Tes Katalase : (+) →

sensitif INH
Identification of Bacteria
BIOCHEMICAL
PROPERTIES
 ALT/AKK
Quantification by colony-forming units  antimicrobial dosage
Sampel padat
Pengenceran
1 ml 1 ml kocok
Hitung koloni
menggunakan alat
coulter counter dari
1 ml
masing-masing cawan
9 ml petri, kalikan dengan
medium faktor pengenceran,
cair lalu rata-ratakan. Hasil
1 ml 1 ml 1 ml
Pengenceran yang baik 30-300
koloni per cawan petri

Sampel kental
Contoh :
30.101 X 35.102 x 40.103
20 ml medium padat 3
=……bakteri/ml
www.themegallery.com
 Diagnosis of Microbial Infection
1. Microscopy unstained or stained with e.g.
Gram stain

Stain Decolorise Counterstain

2. Culture identification by biochemical or

serological tests on pure growth
from single colony

on plates or in broth

5. Sensitivities by disc diffusion

methods,
breakpoints or
3. Imunological 4. Genotypic
MICs
method method
 3. Immunological Methods
• Characteristics of antibodies can reveal the
history of a patient’s contact with
microorganisms/other antigens
• Serology: the branch of immunology that
traditionally deals with in vitro diagnostic
testing of the serum
• Serological testing
• Serotyping
 General Features of Immune Testing
• Strategies
– Agglutination
– Precipitation
– Immunodiffusion
– Complement fixation
– Fluorescent antibody tests
– Immunoassay tests
• Specificity and sensitivity
 Agglutination Reactions

• Antibodies cross-link the antigens to form

visible clumps
 Agglutination Testing
• Performed routinely to determine ABO and Rh
blood types
• Widal test: tube agglutination test for
diagnosing salmonelloses
• Rapid plasma regain (RPR) test: tests for
antibodies to syphilis
• Weil-Felix reaction: diagnoses ricketsial
infections
• Latex agglutination tests: tiny latex beads with
antigens affixed
Agglutination Reactions
Latex Aglutination
 Precipitation Tests
• The soluble antigen is precipitated by an antibody
• Reaction is observable as a cloudy or opaque zone at the
point of contact
 Immuno-
difussion
• Double diffusion
method
• Immunoelectro
phoresis
 Immunoelectrophoresis :
The Western Blot for Detecting Proteins
• Involves electrophoretic separation of proteins
followed by an immunoassay to detect those
proteins
• Test material is electrophoresed in a gel to
separate out particular bands
• Gel transferred to a special blotter that binds the
reactants in place
• Blot developed by incubating it with a solution of
antigen or antibody labeled with radioactive,
fluorescent, or luminescent labels
 Introduction
• Western blotting, also known as immunoblotting or
protein blotting, is a technique used to detect the
presence of a specific protein in a complex protein
mixture
• It is a core technique in cell biology, molecular biology,
virology and others
• Western blots have become one of the most common
analytical tools for the
– Detection of viral proteins
– Characterization of monoclonal and polyclonal antibody
preparations
– Determining the specificity of the immune response to
viral antigens
31
Western Blot Applications for Medical
Diagnosis
• For HIV
– Confirmatory HIV-test to detect anti-HIV antibody
in a human serum sample
• For HBV
– Confirmatory test for Hepatitis B infection
• For Herpes
– Detection of HSV infections

32
 Procedure
• The Western blotting procedure relies
upon three key elements to accomplish
this task:
– The separation of protein mixtures by size
using gel electrophoresis
– The efficient transfer of separated proteins
to a solid support;
– and the specific detection of a target
protein by appropriately matched
antibodies
• Once detected, the target protein will be
visualized as a band on a blotting
membrane, X-ray film, or an imaging
system
33
 warna ungu
+ Nitro blue tetrazolium (NBT)
Protein ditransfer ke
AP + 5-Bromo-4-Chloro-3-indolyl phospate
membran nitroselulosa SDS-PAGE (BCIP)
Ab 2o
Ab 2o
Ab 1o
Protein target

Membran nitroselulosa

Dilabel dengan Visualisasi

antibodi spesifik
Contoh hasil Western blot
 Complement Fixation
• Lysin or
cytolysin: an
antibody that
requires
complement
to complete
the lysis of its
antigenic
target cell
Flurorescent Antibodies
Test :
Immunofluorescence
Testing
• Direct testing: an
unknown test specimen or
antigen is fixed to a slide
and exposed to a
fluorescent antibody
solution of known
composition
• Indirect testing: the
fluorescent antibodies are
antibodies made to react
with the Fc region of
another antibody
 Microscopy
Stained preparations

Special structures
•Fluorescence
– Direct, e.g. auramine
– Immunofluorescence
 Immunoassays
Extremely sensitive methods that permit rapid
and accurate measurement of trace antigen or
antibody
•Radioactive isotope labels  RIA
•Enzyme labels  ELISA
•Sensitive electronic sensors
 Radioimmunoassay (RIA)
• Antibodies or antigens
labeled with a
radioactive isotope
used to pinpoint
minute amounts of a
corresponding antigen
or antibody
• Compare the amount
of radioactivity
present in a sample
before and after
incubation with a
known, labeled
antigen or antibody
• Large amounts of a
bound radioactive
component indicate
that the unknown test
substance was not
present
 Enzyme-Linked
Immunosorbent
Assay (ELISA)
Enzyme-antibody
complex that can be used
as a color tracer for
antigen-antibody
reactions
•Indirect
•Direct
• ELISA Test for Serologic Diagnosis
 Diagnosis of Microbial Infection
1. Microscopy unstained or stained with e.g.
Gram stain

Stain Decolorise Counterstain

2. Culture identification by biochemical or

serological tests on pure growth
from single colony

on plates or in broth

5. Sensitivities by disc diffusion

methods,
breakpoints or
3. Imunological 4. Genotypic
MICs
method method
 4.Genotypic Methods
• Primary advantage over phenotypic methods:
actually culturing the microorganisms is not always
necessary
• Also are increasingly automated with results
obtained very quickly

PCR : Rapid identification of pathogens

• Developed for a wide variety of bacteria, viruses,
protozoa, and fungi
 Polymerase Chain Reaction 5 3
Target
sequence
APPLICATION With PCR, any specific segment—the target Genomic DNA 3 5
sequence—within a DNA sample can be copied many times
5 3
(amplified) completely in vitro.

3 5
TECHNIQUE The starting materials for PCR are double-
stranded DNA containing the target nucleotide sequence to be Cycle 1
yields
copied, a heat-resistant DNA polymerase, all four nucleotides, 2 Primers
and two short, single-stranded DNA molecules that serve as molecules

primers. One primer is complementary to one strand at one end

of the target sequence; the second is complementary to the
other strand at the other end of the sequence. New
nucleo-
tides

RESULTS During each PCR cycle, the target DNA

sequence is doubled. By the end of the third cycle, one-fourth
of the molecules correspond exactly to the target sequence, Cycle 2
yields
with both strands of the correct length (see white boxes 4
above). After 20 or so cycles, the target sequence molecules molecules

outnumber all others by a billionfold or more.

Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
 Nucleic Acid Sequencing and rRNA
Analysis
Comparison of the
sequence of nitrogen bases
in rRNA (16s rRNA and 18s
rRNA)
•Effective for
differentiating general
group differences
•Can be fine-tuned to
identify at the species level
 Advancements to sequencing
• Fluorescent tagging sequence data
• Computer read & analyzed
• DNA Analysis Using Genetic Probes
– Hybridization-can identify a bacterial species by
analyzing segments of its DNA
– Small fragments of single-stranded DNA or RNA called
probes Known to be complementary to the specific
sequences of DNA from a particular microbe
• Unknown test DNA from cells is bound to blotter paper
• Add probes to blotter
• Observe for signs that the probes have become fixed to the
test DNA
labeled probe
GA T CAG T A G
genomic DNA

C TAGTCA T C
3’ 5’
 Restriction Fragment Length
Polymorphisms (RFLP)
• DNA is isolated from a sample such as
blood, saliva, semen, tissue, or hair
and purified
• The huge genome is cut up with
restriction enzymes to produce short,
manageable DNA fragments
• DNA fragments are then sorted by size
using gel electrophoresis
• DNA is denatured and transferred and
permanently affixed to a nylon
membrane  Southern Blot
• Attach a radioactive, DNA probe that
is complementary to a VNTR locus to
DNA sequences on the membrane
• Take a picture of it using special X-ray
film
 Sensitivities test
• Antimicrobial sensitivity