Instructor: Bertha Escobar-Poni, MD
Chapter 13 Outline
• Introduction
• Clinical Specimens
• Types of Clinical Specimens Usually
Required to Diagnose Infectious
Diseases
–
Role of Healthcare
Professionals in the Submission • The Pathology Department (“The Lab”)
of Clinical Specimens
–
Importance of High-Quality
Clinical Specimens
–
–
Proper Selection, Collection,
and Transport of Clinical
Specimens
Contamination of Clinical
Specimens with Indigenous
Microflora
–
Anatomical Pathology
–
Clinical Pathology
• The Clinical Microbiology Laboratory
–
Organization
–
Responsibilities
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Introduction
The proper diagnosis of an infectious disease
requires:
1. Taking a complete patient history
2. Conducting a thorough physical examination of the
patient
3. Carefully evaluating the patient’s signs and
symptoms
4. Implementing the proper selection, collection,
transport, and processing of appropriate clinical
specimens
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Clinical Specimens
All specimens should be of the highest possible quality!
• Specimens collected from patients such as blood,
urine, feces, and cerebrospinal fluid (CSF), are known
as clinical specimens.
• Specimens commonly submitted to the hospital’s
Clinical Microbiology Laboratory (CML) include:
• blood, bone marrow, bronchial washings, sputum, CSF,
cervical and vaginal swabs, feces, hair and nail clippings,
pus, skin scrapings, sputum, synovial fluid, throat swabs,
tissue specimens, urethral discharge material, urine, and
urogenital secretions.
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Clinical Specimens
All specimens should be of the highest possible quality!
• The Role of Healthcare Professionals in the
Submission of Clinical Specimens:
– There should be a close working relationship among the
members of the healthcare team to ensure a proper
diagnosis of infectious diseases.
– Healthcare professionals who collect and transport
specimens should exercise extreme caution during the
collection and transport of specimens.
– In the laboratory, all specimens are handled carefully,
exercising Standard Precautions.
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Role of Healthcare Professionals in the
Submission of Clinical Specimens
• Although laboratory professionals do not themselves
make diagnoses, they make laboratory observations
and generate test results that assist clinicians to
correctly diagnose infectious diseases and initiate
appropriate therapy.
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Importance of High-Quality
Clinical Specimens
• High-quality clinical specimens are required to achieve
accurate, clinically relevant laboratory results.
• The 3 components of specimen quality are:
• Proper specimen selection
• Proper specimen collection
• Proper transport of the specimen to the laboratory
• The laboratory must provide written guidelines
(“Laboratory Policies and Procedures Manual”).
• The quality of the laboratory work performed in
CML can be only as good as the quality of the
specimens it receives.
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Proper Selection, Collection, and
Transport of Clinical Specimens
• Specimens must be properly
selected.
• Specimesn must be collected
properly.
• Material (i.e., specimens) should
be collected from a site where the
suspected pathogen is most likely
to be found.
• Specimens should be obtained
before antimicrobial therapy, if
possible.
• The acute stage of the disease
is the most appropriate time to
collect a specimen.
• Specimen collection should be
performed with care and tact to
avoid harming the patient.
• A sufficient quantity of the
specimen must be obtained to
provide enough material for all
required diagnostic tests.
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Proper Selection, Collection, and
Transport of Clinical Specimens
• All specimens should be placed or
collected into a sterile container to
prevent contamination.
• Whenever possible, a sterile,
disposable specimen container
should be used.
• Specimens should be protected from
heat and cold and promptly delivered
to the laboratory.
• The specimen container must
be properly labeled and
accompanied by an appropriate
request slip with adequate
instructions.
• Hazardous specimens must be
handled with even greater care to
avoid contamination of couriers,
patients, and healthcare
professionals.
• Specimens should be collected
and delivered to the lab as
early in the day as possible to
allow sufficient processing time.
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• Blood
– Usually sterile
– The presence of bacteria in the bloodstream is
known as bacteremia.
– Septicemia is a serious disease involving chills,
fever, prostration, and the presence of bacteria
or their toxins in the bloodstream.
– To prevent contamination of a blood specimen
with indigenous skin flora, extreme care must be
taken to use aseptic technique.
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Proper Method of Preparing the
Venipuncture Site When Obtaining
Blood for Culture
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• Urine
– Normally sterile in the bladder; becomes
contaminated by indigenous microflora of the
distal urethra during voiding.
– Contamination is reduced by collecting a cleancatch, midstream urine.
– Urine culture involves 3 parts:
• A colony count (using a calibrated loop)
• Isolation and identification of the pathogen
• Antimicrobial susceptibility testing
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Urine Colony Count
• The colony count is a way of estimating the number of
viable bacteria that are present in a urine specimen.
• A calibrated loop, either 0.01 mL or 0.001 mL, is used
to inoculate the entire surface of a blood agar plate.
• After incubation at 37oC overnight, the colonies are
counted and the number is multiplied by the dilution
factor (either 100 for the 0.01 mL loop, or 1000 for the
0.001 mL loop) to determine the number of colonyforming units (CFUs).
– # Colonies x dilution factor = # CFUs/mL
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Obtaining a Urine Colony Count
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• Cerebrospinal Fluid (CSF)
– Meningitis is inflammation or infection of the membranes
(meninges) that surround the brain and spinal column.
– Encephalitis is inflammation or infection of the brain.
– Meningoencephalitis is inflammation or infection of both
the brain and meninges.
– CSF is collected by lumbar puncture into a sterile tube;
this is a surgically aseptic procedure performed by a
physician.
– CSF is considered a STAT (emergency) specimen in the
lab!
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Technique of Lumbar Puncture
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• Sputum
– Sputum is pus that accumulates deep within the lungs of
a patient with pneumonia, tuberculosis, or other lower
respiratory infection.
– Often, specimens labeled “sputum” are actually just
saliva; saliva specimens don’t provide clinically relevant
information.
– If TB is suspected, extreme care should be taken!
– Better specimens can be obtain by bronchial aspiration or
transtracheal aspiration.
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• Throat Swabs
– Routine throat swabs are
used to determine
whether a patient has
strep throat
– Specific cultures may be
necessary when Neisseria
gonorrhoeae or
Corynebacterium
diphtheriae are suspected
• Wound Specimens
– Whenever possible, a
wound specimen should
be an aspirate (i.e., pus
collected by needle and
syringe), rather than a
swab
– Specimens collected by
swab are frequently
contaminated with
indigenous microflora
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• GC Cultures (for Neisseria gonorrhoeae)
– N. gonorrhoeae is a fastidious, microaerophilic, and
capnophilic bacterium.
– Only Dacron, calcium alginate, or nontoxic cotton swabs
should be used to collect GC specimens.
– Specimens (e.g., vaginal, cervical, urethral, throat, and
rectal swabs) are cultured on special medium (e.g.,
Thayer-Martin medium) and incubated in a CO2 incubator.
– Special transport media are available and GC swabs
should never be refrigerated.
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• Fecal Specimens
– Ideally, fecal (stool) specimens should be
collected at the laboratory and processed
immediately to prevent a decrease in
temperature, which would allow the pH to drop
and cause the death of many Shigella and
Salmonella species.
– Bacteria in fecal flora are obligate-, aerotolerant-,
and facultative anaerobes.
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Types of Clinical Specimens Usually
Required to Diagnose Infectious Diseases
• Fecal Specimens (cont’d)
– A combination of direct microscopic
examination, culture, biochemical tests, and
immunologic tests may be performed to
identify Gram-negative and Gram-positive
bacteria, fungi, intestinal protozoa, and
intestinal helminths isolated from fecal
specimens.
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The Pathology Department (“The Lab”)
• Clinical specimens are submitted to the Clinical
Microbiology Laboratory (CML), which is a part of the
Pathology Department.
• The Pathology Department (often referred to as “the
Lab”) is under the direction of a pathologist (a
physician who has specialized training in pathology).
• The pathology department is divided into 2 major
divisions:
– Anatomical Pathology
– Clinical Pathology
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The Pathology Department
• Anatomical Pathology
– Diseased organs, stained tissue sections, and
cytology specimens are examined here.
– Cytogenetic technologists, cytotechnologists,
histologic technicians, histotechnologists, and
pathologist’s assistants are employed in this division.
– In addition, autopsies are performed in the morgue
and some Pathology Departments have an Electron
Microscopy Laboratory.
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The Pathology Department
• Clinical Pathology
– Consists of several laboratories in addition to the
Clinical Microbiology Laboratory: Clinical Chemistry,
Urinalysis, Hematology/Coagulation, Blood Bank,
and Immunology.
– Personnel include pathologists, chemists and
microbiologists, clinical laboratory scientists (also
known as medical technologists - MTs), and clinical
laboratory technicians (also known as medical
laboratory technicians - MLTs).
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The Clinical Microbiology Laboratory
The CML may be under the direction of a pathologist, a microbiologist, or a
senior clinical laboratory scientist.
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The Clinical Microbiology Laboratory
• Responsibilities
Primary mission of the CML is to assist clinicians in
the diagnosis and treatment of infectious
diseases.
The 4 major day-to-day responsibilities are to:
1. Process various clinical specimens that are submitted
to the CML
2. Isolate pathogens from those specimens
3. Identify (speciate) the pathogens
4. Perform antimicrobial susceptibility testing, when
appropriate to do so
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Processing of Clinical Specimens in the CML
1. Examining the specimen macroscopically
2. Examining the specimen microscopically
3. Inoculating the specimen to appropriate
culture media
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Isolation and Identification (Speciation) of
Pathogens
• Bacteriology Section
– Bacterial pathogens are isolated from specimens, tests are
performed to identify the bacterial pathogens, and antimicrobial
susceptibility testing is performed whenever appropriate to do
so.
– CML professionals are very much like detectives and crime
scene investigators, in that they gather clues about a pathogen
until they are able to identify it.
– Numerous phenotypic characteristics are used to identify the
bacteria (e.g., Gram reaction, cell shape, motility, presence and
location of spores, presence or absence of various enzymes,
etc.)
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CML professionals are very much like detectives and crime scene
investigators -- gathering clues about a pathogen until they have enough
information to identify it.
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Minisystems Used to Identify Bacteria
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
API-20E for identification of
Enterobacteriaceae
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
Enterotube II for identification of
Enterobacteriaceae
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Clues
• Gram reaction
• Cell shape (cocci, bacilli,
curved, spiral, etc)
• Morphologic arrangement of
cells
• Growth or no growth on
various types of media
• Colony morphology
• Presence or absence of a
capsule
• Motility
• Number and location of flagella
• Ability to sporulate
• Location of spores
• Presence or absence of
various enzymes (catalase,
coagulase, etc)
• Ability to catabolize various
carbohydrate and aminoacids
• Ability to reduce nitrate
• Ability to produce indole from
tryptophan
• Atmospheric requirements
• Type of hemolysis produced
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Diagram Ilustrating the 3 types of Hemolysis That Can be
Observed on a Blood Agar Plate
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Isolation and Identification (Speciation) of
Pathogens
• Mycology Section
– Responsibility is to assist clinicians in the diagnosis of
fungal infections (mycoses)
– The specimens processed here are the same as those
that are processed in the Bacteriology Section, with the
addition of hair and nail clippings and skin scrapings.
– A variety of procedures are used to identify fungal
pathogens including special media, KOH preps, tease
mounts, biochemical tests (for yeasts), and a combination
of microscopic and macroscopic observations (for
moulds).
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A Colony (Mycelium) of the Mould Aspergillus fumigatus, a Common
Cause of Pulmonary Infections in Immunosuppressed Patients
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
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Colonies (Mycelia) of a Penicillium Species
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Isolation and Identification (Speciation)
of Pathogens
Parasitology Section
Virology Section
–
Assists clinicians in the diagnosis
of parasitic diseases
–
Assists clinicians in the diagnosis
of viral diseases
–
Parasites are identified by
observing and recognizing
various parasite life cycle stages
(e.g., trophozoites, cysts,
microfilariae, eggs, larvae, adult
worms) in specimens – identified
primarily by their physical
appearance (e.g., size, shape,
internal details)
–
Techniques used in the
identification of viruses include
immuno-diagnostic tests,
cytologic or histologic
examination, electron
microscopy, molecular
techniques, virus isolation by cell
cultures, and cytopathic effect
(CPE)
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Isolation and Identification (Speciation) of
Pathogens
• Mycobacteriology Section (also called the TB Lab)
– Assists clinicians in the diagnosis of tuberculosis
(TB)
– Various types of specimens are submitted, but
sputum is the most common type
– Mycobacterium spp. are identified by the acid-fast
staining procedure and by using a combination of
growth characteristics (e.g., growth rate, colony
pigmentation, photoreactivity, and morphology) and
a variety of biochemical tests
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