Laboratory Diagnosis of

Bacterial Infections

Qiushui He
Department of Medical Microbiology
Email: qiushui.he@utu.fi
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Advantages in making a specific diagnosis

• Better patient care

• Appropriate use of antibiotic
• Sparing of expenses
• Preventive measures can be initiated

http://www.slideshare.net/drsadhana86/lab-diagnosis-of-bacterial-infections

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 Role of clinical microbiologist

• Identify bacterial, viral, fungal and parasitic

agents that cause human diseases
• Provide diagnostic and therapeutic support for
the clinical management of patients
• Prevent the transmission of infectious diseases
in both the health care system and the
community

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Approach for identification of infectious agents

• Microscopy
• Culture
Direct tests
• Detection of microbial antigens and products
• Detection of microbial nucleic acids
• Serology: detection of specific antibodies Indirect tests

http://www.slideshare.net/drsadhana86/lab-diagnosis-of-bacterial-infections

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 Laboratory procedure used to confirm a clinical diagnosis of infectious
disease with a bacterial etiology

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 Microscopy: Stained preparations

• Gram-stain
• Acid-fast stain
– Ziehl-Neelsen
• Fluorescence
– Direct, e.g. auramine
– Immunofluorescence

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 Gram staining
• Developed by Hans Christian
Gram, a Danish scientist in 1884
• A method to differentiate bacterial
species into two large groups (G+
and G-)
• The differentiation is based on the
chemical and physical properties
of bacterial cell walls,
peptidoglycan, present in a thick
layer in G+ bacteria
• G+ bacteria are stained purple by A Gram stain of mixed Staphylococcus
aureus (Staphylococcus aureus ATCC
crystal violet, whereas G-bacteria 25923, Gram-positive cocci, in purple)
have a thinner layer so do not and Escherichia coli (Escherichia coli
retain the purple stain and are ATCC 11775, gram-negative bacilli, in red)
counter-stained pink by the http://en.wikipedia.org/wiki/Gram_staining
Safranin.

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 Acid-fast stain: Ziehl-Neelsen stain
• First described by two German doctors: the
bacteriologist Franz Ziehl and the
pathologist Friedrich Neelsen
• It is a special bacteriological stain used to
identify acid-fast organisms, mainly
Mycobacteria (M. tuberculosis, M. leprae, M.
kansasii, M. marinum, M bovis, M. afircanum
and members of the M. avium complex).
• Acid fast organisms like Mycobacterium
contain large amounts of lipid substances
within their cell walls called mycolic acids.
These acids resist staining by ordinary
methods such as a Gram stain.
http://en.wikipedia.org/wiki/Gram_staining
• It can also be used to stain a few other
bacteria, such as Nocardia.
• The reagents used are Ziehl–Neelsen
carbol fuchsin, acid alcohol, and methylene
blue.
• Acid-fast bacilli will be bright red after
staining.

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 Bacterial Culture
• Solid media
– Agar plates
• For Identification
• For Enumeration
– Slopes
– For safety
long-term culture,
e.g. Lowenstein-Jensen
media for TB
• Liquid media (broth)
– For enrichment or
maximum sensitivity
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 Advantages of Solid Media

 isolation of single
clonal colonies
– get bacterium in
pure culture
 identify by colonial
morphology
 quantification by
colony-forming units
Adapted from the presentation of Dr. Dou Jianlin
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 Bacterial Identification
 Morphology
 Growth requirements
 Biochemistry
 Enzymes
 Antigens
 Sequence of nucleic acid

Adapted from the presentation

of Dr. Dou Jianlin.

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 Identification of Corynebacterium diphtheria

Pure culture sent to Reference Lab

Reculture Storage tubes to -70oC

Toxin gene PCR Strain Identification

- no product  no gene Toxin production test
- product  gene with ELEK-plates
API Coryne

Biotypes differentiated
- belfanti
- gravis
- mitis 16S sequencing
- intermedius

Reporting the clinical lab

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 Sensitivity test

 on solid media
– disc diffusion technique
– E-test
 in liquid media
– minimum inhibitory
concentration (MIC) test
 To set up breakpoints

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 abome.com/method/Laboratory-Tests-for-Venereal-Diseases.html
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 Sensitivities of immunoassays
Technique Approximate sensitivity
(per ml)
Precipitation, tube 100 mg
Immunodiffusion 1-3 mg
Agglutination 1 µg
Complement fixation (CF) 1 µg
Hemagglutination, passive 50 ng
Particle immunoassay 30-50 ng
ELISA <1 ng
Manual of Clinical Microbiology, 10th Edition, 2011, page 63.

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 Enzyme-linked immunosorbent assay
(ELISA)
• Also known as an enzyme immunoassay (EIA)
• Is a biochemical technique used mainly in immunology to
detect the presence of an antibody or an antigen in a sample.
• The ELISA has been used as a diagnostic tool in infectious
diseases.
• In ELISA, an unknown amount of antigen is affixed to a
surface, and then a specific antibody is applied over the
surface so that it can bind to the antigen. This antibody is
linked to an enzyme, and in the final step a substance is
added that the enzyme can convert to some detectable signal,
most commonly a colour change in a chemical substrate.

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 Enzyme-linked immunosorbent assay
(ELISA)
• Also known as an enzyme immunoassay (EIA)
• Is a biochemical technique used mainly in immunology to
detect the presence of an antibody or an antigen in a sample.
• The ELISA has been used as a diagnostic tool in infectious
diseases.
• In ELISA, an unknown amount of antigen is affixed to a
surface, and then a specific antibody is applied over the
surface so that it can bind to the antigen. This antibody is
linked to an enzyme, and in the final step a substance is
added that the enzyme can convert to some detectable signal,
most commonly a colour change in a chemical substrate.

wikipedia.org/wiki/ELISA

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 ELISA
• Performing an ELISA involves at least one antibody with specificity
for a particular antigen.
• The sample with an unknown amount of antigen is immobilized on a
solid support (usually a polystyrene microtiter plate)
• After the antigen is immobilized, the detection antibody is added,
forming a complex with the antigen. The detection antibody can be
covalently linked to an enzyme, or can itself be detected by a
secondary antibody that is linked to an enzyme through
bioconjugation.
• Between each step, the plate is typically washed with a mild
detergent solution to remove any proteins or antibodies that are
aspecifically bound.
• After the final wash step, the plate is developed by adding an
enzymatic substrate to produce a visible signal, which indicates the
quantity of antigen in the sample.
wikipedia.org/wiki/ELISA

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 Microtiter plate

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 Typical ELISA

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 Principle of the PCR
– The polymerase chain reaction is a molecular
biological method used to exponentially amplify target
DNA sequences (Saiki et al, 1985; Mullis & Faloona,
1987)
– A heat-resistant polymerase enzyme and
oligonucleotide primers complementary to segments
of the target DNA are essential in the reactions
– A common way to evaluate the PCR product is by gel
electrophoresis
– Invented by Dr. Kary Mullis in 1983 who received the
Nobel Prize in Chemistry in 1993.

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 Three major steps in a PCR

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 An exponential increase of the number of
copies of the target sequence

• The cycles are repeated 25-50 times in an automated thermocycler.

• The amount of the amplified genen sequence is increased exponetially.
• In theory, 20 cycles of PCR yield about a million fold (220 ~ 106).
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 Confirmation of PCR product
on agarose gel
• There is a product formed
• The product is of the right size
• Only one band is formed

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 Different PCR formats
• Real-time PCR • Degenerate PCR
• Nested PCR • PCR-ELISA
• Multiplex PCR • Methylation-specific PCR
• Reverse transcription PCR • Quantitative PCR
• Touchdown PCR • Intersequence-specific PCR
• Arbitrarily primed PCR • Ligation-mediated PCR
• Inverse PCR • Solid-phase PCR
• Allele-specific PCR • Long PCR
• Asymmetric PCR • Universial PCR
• Hot start PCR • VNTR
• Digital PCR

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 Different PCR instruments

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 Application of PCR
• Applied research
– Detection of pathogen
– DNA fingerprinting
– Genetic matching
– Pre-natal screening
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• Basic research
– Mutation screening
– Drug discovery
– Classification of organisms
– Genotyping
– Molecular Archaeology
– Molecular Epidemiology
– Molecular Ecology
– Bioinformatics
– Genomic cloning
– Site-directed mutagenesis
– Gene expression studies
– Sequencing
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Microbiological diagnosis of sepsis: polymerase
chain reaction system versus blood cultures

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 Allele specific PCR
J Clin Microbiol 2015;53 (Nov):3418-3422.

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 Assay interpretation
• When chossing an assay for the laboratory and
in-patient diagnosis, it is critical to understand
the concepts of sensitivity, specificity, and
predictive value.
– Sensitivity is the proportion of individuals with a
disease that are correctly identified with a prticular
test.
• Sensitivity defines the true positivies (TP), which are the
patients with disease identified by the assay
• Conversly, false negatives (FN) are the patients with disease
who are not identified by the test.
• Sensitivity= [TP/(TP+FN)] x 100.
Manual of Clinical Microbiology, 10th Edition, 2011, page 61.
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 Assay interpretation
• Specificity is the proportion of those without the disease
that are correctly classified.
– Specificity is a measure of the true negatives (TN), which are the
patients without disease not identified by the assay.
– False positives (FP) are the patients without disease who test
positive.
– Specificity = [TN/(TN+FP)] x 100.
• With a highly sensitive test, the majority of diseased
individuals are picked up, and thus the number of false-
negative results is very low.
• In contrast, with a highly specific test, the majority of
individuals without the disease test negative, so the
number of false-positive results is very low.
Manual of Clinical Microbiology, 10th Edition, 2011, page 61.

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 Assay interpretation
• The probability of having the disease, given the results of a test, is
called the predictive value of the test.
– Positive predictive value (PPV) determines the percentage of patients
with positive results who are diseased: PPV = [TP/(TP+FP)] x 100.
– Negative predictive value (NPV) calculates the percentage of patients
with negative test results who do not have the disease:
NPV = [TN/(TN+FN)] x 100.
• The predictive value of a test combines the prevalence of disease in
a particular population with the sensitivity and specificity.
• Positive and negative predictive values are important because they
assess the ability of a test to predict the presence or absence of
disease in a patient from a particular population.

Manual of Clinical Microbiology, 10th Edition, 2011, page 61.

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 “We ask the lab for a diagnosis,
expecting a yes or no, but often
end up with just a maybe…”

Professor Mark Pallen. University of Birmingham

Adapted from the presentation of Dr. Dou Jianlin.

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The timeline for the processing of clinical
samples with classical pathogens

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 The timeline for the processing of clinical
samples with slow-growing bacteria such as
Mycobacterium tuberculosis

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Laboratory confirmation of pertussis

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 Culture-confirmed cases in Finland

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 Culture and PCR Confirmed Cases in Finland

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 Culture-, PCR- and Serology-Confirmed Cases
in Finland

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 Clin Infect Dis 2013:1-100.

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 Tenets of Specimen Management: Ten points of importance

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 What you should understand:
• Why do we need a specific diagnosis
• What is a PCR
• What is an ELISA
• What is the sensitivity of an assay and how
to calculate it
• What is the specificity of an assay and how
to calculate it
• What are the sensitivities of different
immunoassays

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