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DIAGNOSING INFECTIOUS
DISEASES
Dr. Mohammad Shakeeb ,MD
Specialist in clinical
pathology/microbiology and
immunology
INTRODUCTION
• The proper diagnosis of an infectious disease
requires
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taking a complete patient history
physical examination of the patient
Carefully evaluating the patient’s signs and symptoms
implementing the proper selection, collection, transport,and
processing of appropriate clinical specimens
CLINICAL SPECIMENS
CLINICAL SPECIMENS
Role of Healthcare Professionals in the Submission
of Clinical Specimens
• 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.
• 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 personnel who collect clinical specimens must
strictly adhere to the safety policies known as Standard
Precautions.
• All specimens should be collected or transferred into a leakproof primary container with a secure closure.
Importance of High-Quality Clinical Specimens
• High-quality clinical specimens are required to achieve
accurate, clinically relevant laboratory results.
• The three components of specimen quality are:
o Proper specimen selection
o proper specimen collection
o Proper transport of the specimen to the laboratory
• The laboratory must provide written guidelines regarding
specimen selection, collection, and transport in the form of a
manual.
Proper Selection, Collection, and Transport of
Clinical Specimens
• Appropriate type of specimen for diagnosis of the suspected
infectious disease.
• Specimens must be collected in a manner that will eliminate
or minimize contamination of the specimen with indigenous
microflora.
• Specimens should be obtained before antimicrobial therapy
has begun.
• The acute stage of the disease is the appropriate time to
collect most specimens.
• If the patient is to collect the specimen, such as sputum or
urine, the patient must be given clear and detailed collection
instructions.
• 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.
• Specimens should be protected from heat and cold and
promptly delivered to the laboratory.
• Specimens must be placed in a sealed plastic bag for
immediate and careful transport to the laboratory.
• The specimen container must be properly labeled and
accompanied by an appropriate laboratory test requisition
containing adequate instructions.
• Labels should contain :
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The patient’s name.
Unique hospital identification number.
Hospital room number.
Requesting clinician’s name.
Culture site.
Date and time of collection.
• Laboratory test requisitions should contain:
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The patient’s name, age, sex.
Unique hospital identification number.
Name of the requesting clinician.
Specific information about the type of specimen .
The site from which it was collected.
Date and time of collection.
Initials of the person who collected the specimen.
Information about any antimicrobial agent(s) that the patient is receiving.
Contamination of Clinical
Indigenous Microflora.
Specimens
with
• when present in specimens, these organisms might merely be
contaminants, but it is also possible that they are causing an
infection. (Opportunistic pathogens).
Types of Clinical Specimens Usually Required to
Diagnose Infectious Diseases.
 Blood
 Urine
 Cerebrospinal Fluid
 Sputum
 Throat Swabs
 Wound Specimens
 Fecal Specimens
Blood
• Blood is usually sterile.
• The presence of bacteria in the bloodstream (bacteremia)
may indicate a disease.
• temporary or transient bacteremias may occur after oral
surgery, tooth extraction, or even aggressive tooth brushing
that causes bleeding.
• Bacteremia may occur during certain stages of many
infectious diseases.
• Septicemia is a serious disease characterized by chills, fever,
prostration, and the presence of bacteria or their toxins in the
bloodstream.
• The most severe types of septicemia are those caused by
Gram-negative bacilli, owing to the endotoxin that is released
from their cell walls.
• Endotoxin can induce fever and septic shock, which can be
fatal.
• To diagnose either bacteremia or septicemia, it is
recommended that at least three blood cultures be collected
during a 24-hour period.
 The person drawing the blood must wear sterile gloves, and
gloves must be changed between patients.
 Blood for culture is usually obtained from a vein located at the
antecubital fossa.
 After locating a suitable vein, the skin at the site is disinfected
with 70% isopropyl alcohol and then with an iodophor.
 When disinfecting the site, a concentric swabbing motion is
used.
 The iodophor is then allowed to dry.
 A tourniquet is applied and the appropriate amount of blood
is withdrawn.
 It is important not to touch the site after it has been
disinfected.
 there are many different types of blood culture systems
currently available.
 The rubber tops of blood culture bottles must be disinfected
prior to insertion of the needle.
 Then an appropriate volume of blood is injected.
 The blood culture bottle(s) should be transported promptly to
the laboratory for incubation at 37°C.
Urine
• Urine is ordinarily sterile while it is in the urinary bladder.
• during urination, it becomes contaminated by indigenous
microflora of the distal urethra.
• Contamination can be reduced by collecting a clean-catch,
midstream urine.
• “Clean-catch”: area around the external opening of the
urethra is cleansed by washing with soap and rinsing with
water before urinating.
• removes the indigenous microflora.
• “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.
• the microorganisms that live in the distal urethra are flushed
out of the urethra by the initial portion of the urine stream,
into the toilet or bedpan, rather than into the specimen
container.
• the clinician may prefer to collect a catheterized specimen or
use the suprapubic needle aspiration technique to obtain a
sterile sample of urine.
• all urine specimens must be processed within 30 minutes of
collection, or refrigerated at 4°C until they can be analyzed.
• Refrigerated urine specimens should be cultured within 24
hours.
• Failure to refrigerate a urine specimen will cause an inflated
colony count------incorrect diagnosis of UTI.
• Complete urine culture consists of a colony count, isolation
and identification of the pathogen, and antimicrobial
susceptibility testing.
• A calibrated loop is used to perform the colony count.
• contains a precise volume of urine.
• two types:
 calibrated to contain 0.01 ml.
 calibrated to contain 0.001 ml.
 The calibrated loop is dipped into the CCMS urine specimen.
 volume of urine within the calibrated loop is inoculated over
the entire surface of a blood agar plate, which is then
incubated overnight at 37°C
• After incubation, 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.
• The dilution factor is 100 if a 0.01-mL calibrated loop was
used, or 1,000 if a 0.001-mL calibrated loop was used.
• A CFU count that is 100,000 (1 × 10*5) CFU/mL or higher is
indicative of a UTI.
• Cerebrospinal Fluid
• 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 the meninges.
• To diagnose these diseases, CSF must be collected into a
sterile tube by a lumbar puncture (spinal tap) under surgically
aseptic conditions.
• difficult procedure is performed by a physician.
• CSF specimens must be rushed to the laboratory and must not
be refrigerated.
• Refrigeration might kill any fragile pathogens present in the
specimen.
• Emergency specimen.
• workup of the specimen will be initiated immediately.
• Gram stain of the spinal fluid sediment will be reported by
telephone to the clinician immediately.
• enable clinicians to make diagnoses and initiate therapy, and
often save patients’ lives.
Sputum
• Sputum is pus that accumulates deep within the lungs of a
patient with pneumonia, tuberculosis, or other lower
respiratory infection.
• Many of the sputum specimens that are submitted to the CML
are actually saliva.
• If tuberculosis is suspected, extreme care in collecting and
handling the specimen should be exercised because one could
easily be infected with the pathogens.
• Sputum specimens may be refrigerated for several hours
without loss of the pathogens.
Throat Swabs
• Routine throat swabs are collected to determine whether a
patient has strep throat (Streptococcus pyogenes
pharyngitis).
• If a clinician suspects a pathogen other than S. pyogenes to be
causing a patient’s pharyngitis, that information must be
included on the laboratory test requisition.
Wound Specimens
• Wound specimen should be an aspirate (using a small needle
and syringe) rather than a swab specimen.
• swab are frequently contaminated with indigenous skin
microflora and often dry out
• The laboratory test requisition that accompanies a wound
specimen must indicate the type of wound and its anatomical
location.
Fecal Specimens
• Ideally, fecal specimens (stool specimens) should be collected
at the laboratory and processed immediately.
• Alternatively, the specimen may be placed in a container with
a preservative that maintains a pH of 7.0.
• In gastrointestinal infections, the pathogens frequently
overwhelm the indigenous intestinal microflora, so that they
are the predominant organisms seen in smears and cultures.
• Enteropathogenic Escherichia coli, Salmonella spp., Shigella
spp., Clostridium perfringens, C. difficile, Vibrio cholerae,
Campylobacter spp.
THE PATHOLOGY DEPARTMENT
(“THE LAB”)
THE PATHOLOGY DEPARTMENT
(“THE LAB”)
THE CLINICAL MICROBIOLOGY
LABORATORY
THE CLINICAL MICROBIOLOGY
LABORATORY
• The four major responsibilities of the CML are:
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Processing clinical specimens.
Isolate pathogens.
Identifying pathogens
Perform antimicrobial susceptibility testing when appropriate
to do so.
• In general, the processing of clinical specimens in the
CML includes :
 Examining the specimen macroscopically.
 Examining the specimen microscopically.
 Inoculating the specimen to appropriate culture media.
• A less frequent responsibility of the CML is to process
environmental samples whenever there is an outbreak or
epidemic within the hospital.
• Environmental samples include those collected from
appropriate hospital sites (floors, sink drains, showerheads,
respiratory therapy equipment.) , and employees (e.g., nasal
swabs, material from open wounds).
• To isolate bacteria and fungi from clinical specimens,
specimens are inoculated into liquid culture media or onto
solid culture media.
Bacteriology Section
• CML professionals gather “clues” (phenotypic
characteristics) about a pathogen until they have
sufficient information to identify (speciate) it.
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Gram reaction.
Cell shape.
Morphologic arrangement of cells.
Growth or no growth on various types of plated 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 (e.g., catalase, coagulase, oxidase, urease)
Ability to catabolize various carbohydrates and amino acids
Ability to reduce nitrate
Ability to produce indole from tryptophan
Atmospheric requirements
Type of hemolysis produced
Mycology Section
• Three types are specimens are much more commonly
submitted to the Mycology Section than to the Bacteriology
Section:
 hair clippings.
 nail clippings.
 skin scrapings.
• A potassium hydroxide preparation (KOH prep) is performed
on hair clippings, nail clippings, and skin scrapings.
• The KOH acts as a clearing agent by dissolving keratin in the
specimens.
• This enables the technologist to see into the specimens when
they are examined microscopically.
• determine whether any fungal elements (e.g., yeasts or
hyphae) are present in the specimen.
• Specimens will also be inoculated onto Sabouraud dextrose
agar, a selective medium for fungi.
• When isolated from clinical specimens, yeasts are identified
using various biochemical tests , primarily based on their
ability to catabolize various carbohydrates.
• When isolated from clinical specimens, moulds are identified
using a combination of rate of growth and macroscopic and
microscopic observations.
• Susceptibility testing of fungi is not currently performed in
most CMLs.
Parasitology Section
• Parasitic infections are diagnosed by observing and
recognizing various parasite life cycle stages (e.g.,
trophozoites and cysts of protozoa; microfilariae, eggs, and
larvae of helminths) in clinical specimens.
Virology Section
• 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 cytological or histological
examination.
 Observation of viruses in specimens using electron
microscopy.
 Molecular techniques
 Virus isolation by use of cell cultures.
Mycobacteriology Section
• Various types of specimens (primarily sputum specimens) are
processed.
• Acid-fast staining is performed.
• Mycobacteria are isolated and identified.
• Susceptibility testing is performed.
• Mycobacterium spp. are identified using a combination of
growth characteristics and various biochemical tests.