Section VI. Microbiology Within Healthcare Facilities
CHAPTER 13: DIAGNOSING INFECTIOUS DISEASES
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 SPECIMEN


The various kinds of specimens, including
blood, urine, stools, as well as CSF fluid,
that the patients provide and applied to
diagnose or adhere to clinical specimens
are the developments of infectious
diseases.
The most common types of clinical
specimens that are sent to the hospital’s
microbiology
laboratory
(Clinical
Microbiology Laboratory or CML) listed in
Table13-1:
ROLE OF HEALTHCARE PROFESSIONALS IN
THE SUBMISSION OF CLINICAL SPECIMENS
 A close working relationship among the
members of the healthcare team is
essential for the proper diagnosis of
infectious diseases.
 The doctor, nurse, medical technologist, or
other qualified healthcare professional
must select the appropriate specimen,
collect it properly, and then properly
transport it to the CML for processing (Fig.
13-1).
 Laboratory findings must then be conveyed
to the attending clinician as quickly as
possible to facilitate the prompt diagnosis
and treatment of the infectious disease.
 Healthcare professionals who collect and
transport clinical specimens should
exercise extreme caution during the
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collection
and
transport of clinical
specimens to avoid
sticking
themselves
with needles, cutting
themselves with other
types of sharps, or
coming in contact
with any type of
specimen.
Within the laboratory,
all specimens are
handled
carefully
following
Standard
Precautions
and
ultimately disposed of
as infectious waste.
IMPORTANCE
OF
HIGH-QUALITY
CLINICAL
SPECIMENS
 High-quality
clinical
specimens
are
required to achieve accurate, clinically
relevant
laboratory results.
 It has often been stated that the quality of
the laboratory work performed in a CML
can be only as good as the quality of the
specimens it receives.
 3 components of specimen quality:
(a) Proper specimen selection
(b) Proper specimen collection,
(c) Proper transport of the specimen to
the laboratory
 The laboratory must provide written
instructions for the proper selection,
collection, and transport of clinical
specimens (“Laboratory Policies and
Procedures Manual”).
 The person who collects the specimen is
ultimately responsible for its quality.
 When clinical specimens are improperly
collected and handled:
(a) the etiologic agent (causative agent)
not be found or may be destroyed
(b)
overgrowth
by
indigenous
microflora may mask the pathogen
(c) contaminants may interfere with the
identification of pathogens and the diagnosis of
the patient’s infectious disease.
Section VI. Microbiology Within Healthcare Facilities
CHAPTER 13: DIAGNOSING INFECTIOUS DISEASES
PROPER SELECTION, COLLECTION, AND
TRANSPORT OF CLINICAL SPECIMENS.
When
collecting
clinical
specimens
for
microbiology,
these general precautions should be taken:
 The specimen must be properly selected.
 The specimen must be properly and
carefully collected.
 The material should be collected from a site
where the suspected pathogen is most
likely to be found and where the least
contamination is likely to occur.
 Whenever possible, specimens should be
obtained before antimicrobial therapy has
begun.
 The acute stage of the disease—when the
patient is experiencing the symptoms of the
disease—is the appropriate time to collect
most specimens.
 Specimen collection should be performed
with care and tact to avoid harming the
patient, causing discomfort, or causing
undue embarrassment.
 A sufficient quantity of the specimen must
be obtained to provide enough material for
all required diagnostic tests.
 All specimens should be placed or
collected into a sterile container to prevent
contamination of the specimen by
indigenous microflora and airborne
microbes.
 Specimens should be protected from heat
and cold and promptly delivered to the
laboratory so that the results of the
analyses will validly represent the number
and
types of organisms present at the time of
collection.
 Specimens must be handled with great
care to avoid contamination of the patients,
couriers, and healthcare professionals.
 The specimen container must be properly
labeled and accompanied by an
appropriate laboratory test requisition
containing adequate instructions.
 Ideally, specimens should be collected and
delivered to the laboratory as early in the
day as possible to give CML professionals
sufficient time to process the material,
especially when the hospital or clinic does
not have 24-hour laboratory service.
CONTAMINATION OF CLINICAL SPECIMENS
WITH INDIGENOUS MICROBIOTA
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• Clinical specimens must be collected in a manner
that eliminates, or at least reduces, contamination
of the specimens with members of the indigenous
microflora.

Contamination
can
lead
to
misinterpretation of diagnostic results, as
indigenous microflora may be mistaken for
pathogens causing infection.
TYPES
OF
CLINICAL
SPECIMENS
USUALLY REQUIRED TO DIAGNOSE
INFECTIOUS DISEASES
BLOOD




Usually sterile
Bacteremia, the presence of bacteria in
the bloodstream, may indicate disease.
Septicemia,
a
serious
disease
characterized by chills, fever, and
bacteria/toxins in the bloodstream.
To prevent contamination of the blood
specimen with indigenous skin microbes,
extreme care must be taken to use aseptic
technique when collecting blood for culture.
URINE


Normally sterile in the urinary bladder, but
becomes contaminated during urination by
indigenous microflora from the distal
urethra.
Clean-catch, midstream urine (CCMS
urine) minimizes contamination by
cleansing the area around the urethral
opening before collection.
Section VI. Microbiology Within Healthcare Facilities
CHAPTER 13: DIAGNOSING INFECTIOUS DISEASES
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





"Clean-catch" refers to the fact that the area
around the external opening of the urethra is
cleansed by an antiseptic towelette or washing
with soap and rinsing with water before
urinating.
"Midstream" refers to the fact that the initial
portion of the urine stream is directed into a
toilet or bedpan, and then the urine stream is
directed into a sterile container.
Urine specimens must be processed within
30 minutes of collection or refrigerated at
4°C to prevent bacterial growth.
A urine culture consists of 3 parts:
1. A colony count (using a calibrated
loop)
2. Isolation and identification of the
pathogen
3. Antimicrobial susceptibility testing
Colony count is a way of estimating the
number of viable bacteria that are present
in the urine specimen.
Calibrated loop - bacteriologic loop; either
0.01 mL or 0.001 mL of fluid is used to
inoculate the entire surface of blood agar
plate.
After incubation at 37°, the colonies are
counted, and this number is then multiplied
by the dilution factor (either 100 or 1,000)
to obtain the number of colony-forming
units (CFU) per milliliter of urine.
#Colonies
CFUs/mL
*
dilution
CEREBROSPINAL FLUID (CSF)
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factor
=
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inflammation/infection
of
Meningitis:
meninges
Encephalitis: inflammation/infection of
brain
Meningoencephalitis:
inflammation/infection of both the brain and
meninges
CSF collected via lumbar puncture (spinal
tap) under sterile conditions; performed by
a physician, requiring urgent transport to
the laboratory without refrigeration to
preserve
fragile
pathogens,
thus
considered STAT (emergency) specimen in
the lab.
SPUTUM
AND
OTHER
RESPIRATORY
SPECIMENS
 Sputum is pus that accumulates deep
within the lungs of a patient with
pneumonia, tuberculosis, or other lower
respiratory infection.
 If tuberculosis is suspected, extreme care
in collecting and handling the specimen
should be exercised.
 Clinicians may opt for bronchial or
transtracheal aspiration for better quality
specimens, especially for diagnosing
specific pathogens like Pneumocystis
jiroveci (now classified as a fungus,
although
previously
considered
a
protozoan) pneumonia in AIDS patients.
THROAT SWABS
 Routine throat swabs are collected to
determine whether a patient has strep
throat.
 Specific cultures may be necessary when
Neisseria
gonorrhoeae
or
Corynebacterium
diphtheriae
are
suspected.
Section VI. Microbiology Within Healthcare Facilities
CHAPTER 13: DIAGNOSING INFECTIOUS DISEASES
WOUND AND ABSCESS SPECIMENS
 Whenever possible, wound and abscess
specimens should be an aspirate (i.e., pus
that has been collected using a small
needle and syringe assembly), rather than
a swab specimen.
 Specimens collected by swab are
frequently contaminated with indigenous
skin microbes and do not provide useful
information concerning the cause of the
infection.
 The laboratory test requisition that
accompanies a wound specimen must
indicate the type of wound and its
anatomical location.
GENITAL/STD SPECIMEN
 N. gonorrhoeae is a fastidious (fussy"),
microaerophilic, and capnophilic organism.
 Dacron, calcium alginate, or nontoxic
cotton swabs should be used for GC
specimen collection to avoid toxicity to the
bacterium.
 Swabs should be inoculated immediately
onto Thayer Martin or Martin-Lewis
medium or into a tube/bottle containing an
appropriate culture medium and 5-10%
CO₂ atmosphere.
 Incubate cultures at 37°C overnight, then
ship to a microbiology laboratory for
positive identification of N. gonorrhoeae.
 Transport swabs in a transport medium;
never refrigerate GC swabs as low
temperatures may kill N. gonorrhoeae.
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Diagnosis involves direct microscopic
examination, culture, biochemical tests, and
immunologic tests to identify various bacteria (e.g.,
E. coli, Salmonella, Shigella, Clostridium), fungi
(Candida),
intestinal
protozoa
(Giardia,
Entamoeba), and intestinal helminths.
THE PATHOLOGY DEPARTMENT (“THE LAB")
The clinical specimen just described are submitted
to the CML. Within hospital setting, the CML, is an
integral part of Pathology department (which is
frequently referred to simply as “the lab”).
The pathology department is under the direction of
a pathologist (a physician who has had extensive,
specialized training in Pathology – the study of the
structural and functional manifestation of disease).

2 major divisions of pathology department:
- Anatomical Pathology
- Clinical Pathology
FECAL SPECIMENS




Ideally collected at the laboratory and
processed
immediately
to
prevent
temperature decrease, which can lead to
pH drop and death of Shigella and
Salmonella species.
Fecal bacteria are obligate aerobes,
aerotolerant, and facultative anaerobes
due to the anaerobic nature of the colon.
Anaerobic culture performed only when
Clostridium difficile-associated disease or
clostridial food poisoning is suspected.
In gastrointestinal infections, pathogens
often predominate over indigenous flora.
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ANATOMICAL PATHOLOGY
 Most pathologists work in Anatomical
Pathology, where they perform autopsies in
the morgue and examine diseased organs,
stained tissue sections, and cytology
specimens.
 Other healthcare professionals employed
in
Anatomical
Pathology
include
cytogenetic
technologists,
cytotechnologists, histologic technicians,
histotechnologists,
and
pathologist’s
assistants.
Section VI. Microbiology Within Healthcare Facilities
CHAPTER 13: DIAGNOSING INFECTIOUS DISEASES
CLINICAL PATHOLOGY
 Personnel working in Clinical Pathology
include pathologists; specialized scientists
such as chemists and microbiologists, who
have graduate degrees in their specialty
areas; clinical laboratory scientists (also
known as medical technologists or MTs).
THE CLINICAL MICROBIOLOGY LABORATORY
ORGANIZATION

Depending on the size of the hospital, the
CML may be under the direction of a
pathologist, a microbiologist (having either
a master or doctor of clinical microbiology
degree), or, in smaller hospitals, a medical
technologist who has had many years of
experience working in microbiology. Most
of the actual bench work that is performed
in the CML is performed by CLSs and CLTs.

The primary mission of the CML is to assist
clinicians in the diagnosis and treatment of
infectious diseases.
To accomplish this mission, the 4 major,
day-to-day responsibilities of the CML are
to:
1. Process the various clinical
specimen that are submitted to the
CML
1. Isolate pathogens from specimen
2. Identify (speciate)the pathogens
3. Perform antimicrobial susceptibility
testing when appropriate to do so
ISOLATION
AND
PATHOGENS
In the Bacteriology Section, various types of
clinical specimens are processed, bacterial
pathogens are isolated from the specimens, tests
are performed to identify the bacterial pathogens,
and antimicrobial susceptibility testing is
performed whenever it is appropriate to do so.
Once they are isolated from clinical specimens,
bacterial pathogens are identified by gathering
clues (phenotypic characteristics).
The various phenotypic characteristics (clues)
useful in identifying bacteria include the following:
- Gram reaction (i., Gram-positive or Gramnegative)
-Cell shape (e., cocci, bacilli, curved, spiralshaped, filamentous, branching)
- Morphologic arrangement of cells (e.,
pairs, tetrads, chains, clusters)
- Growth or no growth on various types of
plated media; etc.
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The overall responsibility of the Bacteriology
Section of the CML is to assist clinicians in the
diagnosis of bacterial diseases.
IDENTIFICATION
OF
MYCOLOGY SECTION



To isolate bacteria and fungi from clinical
specimens, specimens are inoculated into liquid
culture media or onto solid culture media.
The overall responsibility of the Mycology
Section of the CML is to assist clinicians in
the diagnosis of fungal infections
(mycoses). In the Mycology Section,
various types of clinical specimens are
processed, fungal pathogens are isolated,
and tests are performed to identify the
fungal pathogens.
In general, the specimens processed in the
Mycology Section are the same types of
specimens that are processed in the
Bacteriology Section.
3 types are specimens commonly
submitted to the Mycology Section than to
the Bacteriology Section: hair clippings,
nail clippings, and skin scrapings.
PARASITOLOGY SECTION
BACTERIOLOGY SECTION
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
The Parasitology Section of a CML is
responsible for aiding clinicians in
Section VI. Microbiology Within Healthcare Facilities
CHAPTER 13: DIAGNOSING INFECTIOUS DISEASES

diagnosing parasitic diseases caused by
endoparasites like protozoa and helminths.
Parasitic infections are typically diagnosed
by observing and identifying different life
cycle stages of parasites in clinical
specimens, such as trophozoites and cysts
of protozoa, or eggs and larvae of
helminths. Identification is based on the
characteristic appearance, including size,
shape, and internal details. In some cases,
whole worms or worm segments may be
observed in fecal specimens.
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characteristics and various biochemical
tests.
- Mycobacterium tuberculosis, the primary
cause of tuberculosis, is slow-growing, but the
acid-fast stain allows for a rapid presumptive
diagnosis.
MOLECULAR SECTION

VIROLOGY SECTION


The overall responsibility of the Virology
Section of the CML is to assist clinicians in
the diagnosis of viral diseases. Many viral
diseases
are
diagnosed
using
immunodiagnostic procedures.
Other techniques used to identify viral
pathogens are:
- Observation of intracytoplasmic or
intranuclear viral inclusion bodies in specimens by
cytologic or histologic examination
- Observation of viruses in specimens
using electron microscopy
Nonmolecular Methods for Direct Detection of
Infectious Agents.


MYCOBACTERIOLOGY SECTION


TB Lab aid clinicians in diagnosing
tuberculosis - achieved by processing
various types of specimens, primarily
sputum specimens, and performing acidfast staining, isolation and identification of
mycobacteria, and susceptibility testing.
Identification of Mycobacterium spp. is
done using a combination of growth
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Via fluorescent antibody stains that target
antigens on the organism surface or
immunodiagnostic methods called enzyme
immunoassays (EIAs) that are based on an
antigen/antibody reaction.
Thes are rapid, relatively inexpensive, and
easy to perform, but lack the sensitivity of
molecular-based assays.
Organism Identification from Culture

- Molecular techniques such as nucleic acid
probes and polymerase chain reaction assays
(described on the CD-ROM)
- Virus isolation by use of cell cultures;
viruses are identified primarily by the type(s) of cell
lines that they are able to infect and the physical
changes (called cytopathic effect or CPE) that they
cause in the infected cells.
Specializes in molecular diagnostic
techniques for identifying pathogens, either
in direct patient specimens or following
growth in culture.
Problem organisms that cannot be
identified by phenotypic characteristics
often can be identified either by nucleic
acid sequencing or by a newer technique
utilizing
mass
spectrometry
instrumentation.
16S Ribosomal RNA Gene Sequencing

Has highly conserved and highly variable
regions that can be used for microbial
identification.
- The DNA is first extracted from the organism; the
target segment of about 500 base pairs is amplified
through PCR technology and then sequenced on
an automated sequencer.
- The resulting nucleotide sequence is compared
to known sequences in public databases such as
GenBank, a repository of sequences maintained
by the National Institutes of Health.
- The MicroSeq Microbial Identification System
(Applied Biosystems, Foster City, CA) has kits and
Section VI. Microbiology Within Healthcare Facilities
CHAPTER 13: DIAGNOSING INFECTIOUS DISEASES
an electronic database that can be used for
bacterial or fungal identification. The results can be
obtained in approximately 5 hours.
Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF
MS)



Measures the mass-to-charge ratio of
ionized particles such as proteins from an
organism.
Produces a proteomic fingerprint that can
be compared to known reference strains.
Results can be obtained in minutes and
has the potential of rapid identification of
bacteria. fungi. and mycobacteria.
Antimicrobial Susceptibility Testing



Once a pathogen is identified, the organism
is tested against a battery of antimicrobial
agents to determine which agents might be
useful for therapy.
Viruses can be tested by molecular
sequencing assays to determine if
resistance genes are present.
3 main methods are used for susceptibility
testing:
1. Disk diffusion - requires a
standardized inoculum of the organism to be
tested.
2. Broth microdilution - requires a
dilution series of the antibiotic and can provide
a minimum inhibitory concentration (or MIC}
that is defined as the lowest concentration of
the antibiotic that results in no visible growth of
the organism after incubation.
3. Gradient diffusion - combines some
of the properties of the disk diffusion and broth
dilution.
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