CLINICAL

MICROBIOLOGY
PRESENTED BY GROUP 1

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INTRODUCTION TO CLINICAL
MICROBIOLOGY
OBJECTIVES:
1. Understand The Role Of Clinical
Microbiology In Disease Diagnosis
And Control
2. Understand The Appropriate
Methods Specimen Collection,
Storage, And Transport
3. List Some Conventional Testing
methods Used In The Clinical
Laboratory
4. Understand The Advantages And
Limitations Of PCR
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WHAT IS CLINICAL MICROBIOLOGY?

Clinical microbiology is a branch of microbiology focused on studying

microorganisms that cause infections and diseases in humans. This field
involves identifying, classifying, and understanding these pathogens to
diagnose and manage infectious diseases, guide treatment choices, and
control the spread of diseases in healthcare and community settings.
The study and application of clinical microbiology are essential for public
health, especially given the increasing challenges of antibiotic resistance and
emerging infectious diseases.

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PURPOSE AND IMPORTANCE:
PATHOGEN IDENTIFICATION :Determine the type of microorganism
causing an infection.
EPIDEMIOLOGY: Study the spread of infectious diseases to track
outbreaks and develop prevention strategies.
TREATMENT AND CONTIGENCY MEASURES: guide
antibiotic/antiviral/antifungal therapy and monitor effectiveness and
also Develop and implement infection control policies.
Plays a key role in public health by tracking and controlling outbreaks.

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EPIDEMIOLOGY: STUDIES DISTRIBUTION AND
DETERMINANTS OF DISEASE OVER TIME
ENDEMIC
Always present in the population at a low rate
EPIDEMIC
High frequency over a short period of time
PANDEMIC
Epidemic that occurs over a large geographical area

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TREATMENT AND CONTINGENCY MEASURES
TREATMENT STRATEGIES
 Antimicrobial susceptibility testing
 Patient isolation
 Adjustment of safety measures
DISEASE SURVEILLANCE
 Globalization dangers:
• Air Travel (SARS)
• Overpopulation
• Blood banks and transplantation (HIV, HCV)
Patient tracking:
• Ebola epidemic 2014
• Covid-19 pandemic
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 PATHOGEN IDENTIFICATION
Starting with pathogen identification, we have the specimen
selection and handling. We're going to break this down based on
the type of infection :
parasitic infections : O&P tests, fecal, sputum, blood.
viral infections: depending on suspected virus, swabs,
Blood, tzank smear.

bacterial infections: depending on the site of infection,

Blood ,Swab, spinal taps

psychotic infections :stains, microscopic and

Macroscopic examination.

NB: An ova and parasites (O&P) test checks poop for parasites and their ova (eggs) or cysts, which are
hard shells that protect some parasites at a certain stage in their lifecycle.
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PROCEDURES FOR FECAL, SPUTUM AND
BLOOD SAMPLES.
FECAL SAMPLES:
Collection: Collect fresh stool samples in a clean container.
Microscopic examination:
Direct wet mount: Examine a drop of stool mixed with saline or iodine
solution under a microscope to identify motile parasites, cysts, or eggs.
Concentration techniques: Use methods like centrifugation or flotation to
concentrate parasites for better visualization.
Culture: Incubate stool samples in specific media to allow parasites to
grow and multiply.
Molecular methods: Use techniques like PCR or sequencing to identify
parasite DNA or RNA.
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“CONT.” PROCEDURES FOR FECAL, SPUTUM
AND BLOOD SAMPLES.
SPUTUM SAMPLES:
Collection: Collect expectorated sputum in a sterile container.
Microscopic examination: Examine a stained sputum sample under a microscope
to identify parasites, such as Pneumocystis jirovecii.
Culture: Incubate sputum samples in specific media to allow parasites to grow.
Molecular methods: Use PCR or sequencing to identify parasite DNA or RNA.
BLOOD SAMPLES:
Microscopic examination: Examine a blood smear stained with Giemsa or
Wright's stain to identify parasites, such as malaria or filarial worms.
Thick and thin blood films: Prepare two blood films, one thick for parasite
detection and one thin for species identification.
Molecular methods: Use PCR or sequencing to identify parasite DNA or RNA.

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DISINFECTANT INSTRUMENTS & QUALITY
CONTROL MEASURES
DISINFECTANT INSTRUMENTS
 CHEMICAL DISINFECTION: Use disinfectants like bleach, glutaraldehyde, or alcohol to clean and disinfect
instruments used for sample collection and processing.
 AUTOCLAVING: Sterilize instruments by exposing them to high pressure and temperature in an autoclave.
(121°C, 15 psi, 15-20 minutes)
 UV RADIATION: Use ultraviolet light to disinfect surfaces and instruments.
 Dry heat sterilization (160°C, 2 hours) and Ethylene oxide sterilization

QUALITY CONTROL MEASURES

 Proper sample collection and handling
 Correct staining and microscopy techniques
 Regular instrument maintenance and calibration
 Proficiency testing and certification
 Continuous training and education

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SPECIMEN SELECTION AND HANDLING:
PARASITIC INFECTIONS (0&P)
• Sample Must Be Free Of All Contaminants
• Examined Fresh Or Must Be Preserved
• Lab's Sop For Procedure On Storage
• Formed Stool Must Be Broken Up In Preservative To Allow Proper
Preservation
• 2-3 Stool Specimens Over Several Days
. Intermittent Shedding Nature Of Some Organisms
• Collected Before Treatment Starts
.Mineral Or, Castor Oil →> 7-10 Days To Be Cleared
.Antimicrobial Agents —> 2-3 Weeks
• Labelling With At Least Patient Name And Date Of Collection
• Concentration Techniques
• Wet Preps
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 SPECIMEN SELECTION AND
HANDLING: VIRAL INFECTIONS
Specimens Should Be Collected As Early As Possible Following
Disease Onset:
• Nasopharyngeal Specimens = Influenza
• Urine Specimens = Mumps, rubella
• Fecal Specimens = Rotavirus, Norovirus
• Tzank Smear = Chickenpox
Specimen Depends On:
• The Signs And Symptoms
• Suspected Virus
• Time Of The Year
Processing
• Process Immediately Or Refrigerate At 4°c For Up To 4 Days Or
Freeze At -70°c If Storing For Longer.
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SPECIMEN SELECTION AND
HANDLING: BACTERIAL INFECTIONS
• Collect The Specimen In The Acute Phase Of The Infection And
Before antibiotics Are Administered, If Possible,
• Select The Correct Anatomic Site For Collection Of The Specimen.
• Collect The Specimen Using The Proper Technique And Supplies
With Minimal Contamination From Normal Microbiota (Normal
Flora).
• Collect The Appropriate Quantity Of Specimen.
• Package The Specimen In A Container Or Transport Medium
Designed To Maintain The Viability Of The Organisms And Avoid
Hazards That Result From Leakage.
• Label The Specimen Accurately With The Specific Anatomic Site
And The Patient Information - Patent's Information Name And
Unique Identification Number, As Well As Date And Time Of
Collection.
• Transport The Specimen To The Laboratory Promptly Or Make
Provisions To Store The Specimen In An Environment That
Will Not Degrade The Suspected Organism(s)
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SPECIMEN TESTING
specimen can be tested via the following
techniques:
MOLECULAR TESTING METHOD: PCR
PATHOGEN CULTURE: bacteria culture, Cell culture(cpe)
, mycotic culture.
PROTEIN BASED TEST: Elisa, IFA , mass spectrometry.
BIOCHEMICAL TESTING: catalase test , oxidase test.
STAINING: gram stain, acid fast stain, calcofluor white stain,
India ink stain.

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PATHOGEN IDENTIFICATION:MOLECULAR
METHODS. (POLYMERASE CHAIN REACTION (PCR) )
PCR or Polymerase Chain Reaction is a technique used in molecular biology
to create several copies of a certain DNA segment. This technique was
developed in 1983 by Kary Mullis, an American biochemist. PCR has made it
possible to generate millions of copies of a small segment of DNA. This tool is
commonly used in the molecular biology and biotechnology labs.
Principle of PCR
The PCR technique is based on the enzymatic replication of DNA. In PCR, a
short segment of DNA is amplified using primer mediated enzymes. DNA
Polymerase synthesizes new strands of DNA complementary to the template
DNA. The DNA polymerase can add a nucleotide to the pre-existing 3’-OH
group only. Therefore, a primer is required. Thus, more nucleotides are
added to the 3’ prime end of the DNA polymerase.

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COMPONENTS OF PCR
Components Of PCR constitutes the following:
 DNA Template– The DNA of interest from the
sample.
 DNA Polymerase– Taq Polymerase is used. It is
thermostable and does not denature at very high
temperatures.
 Oligonucleotide Primers- These are the short
stretches of single-stranded DNA complementary
to the 3’ ends of sense and anti-sense strands.
 Deoxyribonucleotide triphosphate– These provide
energy for polymerization and are the building
blocks for the synthesis of DNA. These are single
units of bases.
 Buffer System– Magnesium and Potassium provide
optimum conditions for DNA denaturation and
renaturation. It is also important for fidelity,
polymerase activity, and stability.

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TYPES OF PCR:
PCR is of the following types:
 Real-time PCR
In this type, the DNA amplification is detected in real-time with the help of a fluorescent reporter. The signal
strength of the fluorescent reporter is directly proportional to the number of amplified DNA molecules.
 Nested PCR
This was designed to improve sensitivity and specificity. They reduce the non-specific binding of products due
to the amplification of unexpected primer binding sites.
 Multiplex PCR
This is used for the amplification of multiple targets in a single PCR experiment. It amplifies many different DNA
sequences simultaneously.
 Quantitative PCR
It uses the DNA amplification linearity to detect, characterize and quantify a known sequence in a sample.
 Arbitrary Primed PCR
It is a DNA fingerprinting technique based on PCR. It uses primers the DNA sequence of which is chosen
arbitrarily.

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PCR CYCLES

The PCR involves three major cyclic reactions:

DENATURATION
Denaturation occurs when the reaction mixture is heated to 94℃ for about 0.5 to 2 minutes. This
breaks the hydrogen bonds between the two strands of DNA and converts it into a single-stranded
DNA.
The single strands now act as a template for the production of new strands of DNA. The
temperature should be provided for a longer time to ensure the separation of the two strands.
ANNEALING
The reaction temperature is lowered to 54-60℃ for around 20-40 seconds. Here, the primers bind
to their complementary sequences on the template DNA.
Primers are single-strand sequences of DNA or RNA around 20 to 30 bases in length.
They serve as the starting point for the synthesis of DNA.
The two separated strands run in the opposite direction and consequently there are two primers- a
forward primer and a reverse primer.

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“CONT.” PCR CYCLES
Elongation
At this step, the temperature is raised to 72-
80℃. The bases are added to the 3’ end of the
primer by the Taq polymerase enzyme.
This elongates the DNA in the 5’ to 3’ direction.
The DNA polymerase adds about
1000bp/minute under optimum conditions.
Taq Polymerase can tolerate very high
temperatures. It attaches to the primer and
adds DNA bases to the single strand. As a result,
a double-stranded DNA molecule is obtained.
These three steps are repeated 20-40 times in
order to obtain a number of sequences of DNA
of interest in a very short time period.

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IMPORTANCE/APPLICATION OF PCR:
• The following are the applications of PCR :
 Medicine
• Testing of genetic disease mutations.
• Monitoring the gene in gene therapy.
• Detecting disease-causing genes in the parents.
 Forensic Science
• Used as a tool in genetic fingerprinting.
• Identifying the criminal from millions of people.
• Paternity tests
 Research and Genetics
• Compare the genome of two organisms in genomic studies.
• In the phylogenetic analysis of DNA from any source such as fossils.
• Analysis of gene expression.
• Gene Mapping

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ADVANTAGES & LIMITATION OF PCR
ADVANTAGES OF PCR:
 High sensitivity and specificity
 • Rapid amplification of DNA
 • Allows analysis of small DNA samples
 • Versatile and adaptable to various applications
LIMITATIONS
 Does Not Offer Insight Into The Disease State: Ex
Dead Bacteria Would Still Be Amplified Even
Though The Infection Is No Longer Active.
 False Results May Occur Under Improper Sample
Collection
 Results Must Be Interpreted Carefully Under The
light Of clinical Evidence And Additional Testing
May Be Needed

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PATHOGEN IDENTIFICATION:CULTURE
BACTERIAL CULTURE
 Uses differential and selective media
 SBA and MAC at least in clinical labs
 Pathogen isolation prior to biochemical testing
VIRAL CULTURE•
 Finite, low passage, continuous cell lines
 Used to culture viruses
 Cytopathic effect studies (CPE)
 Viruses have different CPEs in the cells
 Useful for identification
MYCOTIC CULTURE
 May be needed for some specimens
 Cultured fungi are then examined microscopically and macroscopically for
defining features
 Yeasts are cultured similarly to bacteria

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SUMMARY
Endemic, Epidemic, And Pandemic Describe Different Disease
Frequencies In Various Geographical Areas.
Identifying A Pathogen Enables Appropriate Therapy,
Anticipation Of Sequelae, And Disease Tracking
Specimen Collection And Processing Varies Depending On The
Nature Of The Pathogen and The Body Site.
Conventional Methods Used To Identify Pathogens Include:
Staining Procedures, Biochemical Tests, Growth On Selective And
Differential Media
Rapid Techniques For Pathogen Identification Include: Molecular
Techniques Such QRT-PCR And Immunological Techniques Such
As Elisa

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THANK YOU

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