Burton's Microbiology For The Health Sciences: Diagnosing Infectious Diseases

Burton's Microbiology
for the Health Sciences

Chapter 13.
Diagnosing Infectious Diseases
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Chapter 13 Outline
• Introduction • Types of Clinical Specimens
Usually Required to Diagnose
• Clinical Specimens Infectious Diseases
– Role of Healthcare • The Pathology Department
Professionals in the (“The Lab”)
Submission of Clinical
Specimens – Anatomical Pathology
– Importance of High-Quality – Clinical Pathology
Clinical Specimens
• The Clinical Microbiology
– Proper Selection, Laboratory
Collection, and Transport
of Clinical Specimens – Organization
– Contamination of Clinical – Responsibilities

Specimens with Indigenous
Microflora

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
Clinical Specimens
• 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.
• All specimens should be of the highest possible
quality!
Clinical Specimens, cont.
• 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.

Role of Healthcare Professionals in the
Submission of Clinical Specimens, cont.
• 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.

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 person who collects the specimen is ultimately
responsible for its quality.
Proper Selection, Collection, and
Transport of Clinical Specimens
• Specimens must be properly • The acute stage of the
selected. disease is the most
appropriate time to collect a
• Specimesn must be collected specimen.
properly.
• Specimen collection should
• Material (i.e., specimens) be performed with care and
should be collected from a tact to avoid harming the
site where the suspected patient.
pathogen is most likely to be
found. • A sufficient quantity of the
specimen must be obtained
• Specimens should be to provide enough material
obtained before antimicrobial for all required diagnostic
therapy, if possible. tests.
Proper Selection, Collection, and
Transport of Clinical Specimens, cont.
• All specimens should be • Whenever possible, a sterile,
placed or collected into a disposable specimen
sterile container to prevent container should be used.
contamination.
• The specimen container
• Specimens should be must be properly labeled and
protected from heat and cold accompanied by an
and promptly delivered to appropriate request slip with
the laboratory. adequate instructions.
• Hazardous specimens must • Specimens should be
be handled with even greater collected and delivered to
care to avoid contamination the lab as early in the day as
of couriers, patients, and possible to allow sufficient
healthcare professionals. processing time.
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.

Proper Method of Preparing the
Venipuncture Site When Obtaining Blood
for Culture

Required to Diagnose Infectious Diseases,
cont.
• Urine
– Normally sterile in the bladder; becomes
contaminated by indigenous microflora of the distal
urethra during voiding.
– Contamination is reduced by collecting a clean-catch,
midstream urine.
– Urine culture involves 3 parts:
• A colony count (using a calibrated loop)
• Isolation and identification of the pathogen
• Antimicrobial susceptibility testing
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 colony-
forming units (CFUs).
– # Colonies x dilution factor = # CFUs/mL

Obtaining a Urine Colony Count

cont.
• 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!
Technique of Lumbar Puncture

cont.
• 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.

cont.
• Throat Swabs • Wound Specimens
– Routine throat swabs – Whenever possible, a
are used to determine wound specimen should
whether a patient has be an aspirate (i.e., pus
strep throat collected by needle and
syringe), rather than a
– Specific cultures may be swab
necessary when
Neisseria gonorrhoeae – Specimens collected by
or Corynebacterium swab are frequently
diphtheriae are contaminated with
suspected indigenous microflora

cont.
• 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.
cont.
• 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.
– 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.
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

The Pathology Department, cont.
• 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.

The Pathology Department, cont.
• 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).

The Clinical Microbiology Laboratory
The CML may be under the direction of a pathologist, a

microbiologist, or a senior clinical laboratory scientist.
The Clinical Microbiology Laboratory, cont.
• 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:
• Process various clinical specimens that are
submitted to the CML
• Isolate pathogens from those specimens
• Identify (speciate) the pathogens
• Perform antimicrobial susceptibility testing, when
appropriate to do so
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.)

CML professionals are very much like detectives and
crime scene investigators -- gathering clues about a
pathogen until they have enough information to identify it.

Minisystems Used to Identify Bacteria
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins
API-20E for identification of Enterotube II for identification of

Enterobacteriaceae Enterobacteriaceae

Diagram Ilustrating the 3 types of Hemolysis
That Can be Observed on a Blood Agar Plate

of Pathogens, cont.
• 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).

A Colony (Mycelium) of the Mould Aspergillus fumigatus, a
Common Cause of Pulmonary Infections in
Immunosuppressed Patients

Colonies (Mycelia) of a Penicillium Species

of Pathogens, cont.
• Parasitology Section • Virology Section
– Assists clinicians in the – Assists clinicians in the
diagnosis of parasitic diagnosis of viral
diseases diseases
– Parasites are identified – Techniques used in the
by observing and identification of viruses
recognizing various include immuno-
parasite life cycle stages diagnostic tests,
(e.g., trophozoites, cytologic or histologic
cysts, microfilariae, examination, electron
eggs, larvae, adult microscopy, molecular
worms) in specimens – techniques, virus
identified primarily by isolation by cell cultures,
their physical and cytopathic effect
appearance (e.g., size, (CPE)
shape, internal details)
of Pathogens, cont.
• 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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Burton's Microbiology

for the Health Sciences

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

– Contamination of Clinical – Responsibilities

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

1. Taking a complete patient history

2. Conducting a thorough physical examination of the

3. Carefully evaluating the patient’s signs and

4. Implementing the proper selection, collection,

• Specimens collected from patients such as blood, urine,

• The Role of Healthcare Professionals in the Submission of

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Although laboratory professionals do not

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• The colony count is a way of estimating the number of

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Clinical specimens are submitted to the Clinical

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

The CML may be under the direction of a pathologist, a

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

API-20E for identification of Enterotube II for identification of

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

• Mycobacteriology Section (also called the TB Lab)

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

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