CHAPTER 4
Principles of Laboratory Diagnosis of Infectious
Diseases
The diagnosis of a microbial infection begins with an assessment of clinical and
epidemiologic features, leading to the formulation of a diagnostic hypothesis. Anatomic
localization of the infection with the aid of physical and radiologic findings (for example,
right lower lobe pneumonia, subphrenic abscess) is usually included. This clinical
diagnosis suggests a number of possible etiologic agents based on knowledge of
infectious syndromes and their courses. The general approaches to laboratory diagnosis
vary with different microorganisms and infectious diseases. However, the types of
methods are usually some combination of direct microscopic examinations, culture,
antigen detection, and antibody detection (serology). Nucleic acid amplification assays
which allow direct detection of genomic components of pathogens are now numerous but
few are practical for routine use. All diagnostic approaches begin with some kind of
specimen collected from the patient.
THE SPECIMEN
I. Direct Tissue or Fluid Samples
1. Direct specimens are collected from normally sterile tissues (lung, liver) and body
fluids (cerebrospinal fluid, blood).
2. Direct samples give highest quality but some carry risk
II. Indirect Samples
1. Indirect samples are specimens of inflammatory exudates (expectorated
sputum, voided urine) that have passed through sites known to be colonized with
normal flora.
2. Bypassing the normal flora requires extra effort
3. Results require interpretive evaluation of contamination
III. Samples from Normal Flora Sites
1. Frequently the primary site of infection is in an area known to be colonized
with many organisms (pharynx and large intestine)
2. Strict pathogens must be specifically sought
3. Lack of normal viral flora simplifies interpretation
IV. Specimen Collection and Transport
1. Swabs are convenient but limit volume and survival
2. Viability may be lost if specimen is delayed
3. Transport media stabilize conditions and prevent drying
DIRECT EXAMINATION - MICROSCOPY
I. Light Microscopy
1. Bacteria are visible if optics are maximized
2. Bacteria must be stained
A. The Gram Stain
1. Stain applies an iodine solution to cells previously stained with crystal violet
2. The difference is a decolorizing step in which mixtures of acetone and alcohol
may or may not extract the purple iodine–dye complexes
3. Gram-positive bacteria retain purple iodine-dye complexes
4. Gram-negative bacteria do not retain the complexes and are demonstrated with a
red counterstain
5. Properly decolorized background should be red
6. Gram reaction plus morphology guide clinical decisions
B. The Acid-Fast Stain
1. Acid fastness is a property of the mycobacteria (eg, Mycobacterium
tuberculosis) and related organisms.
2. Acid-fast bacteria take stains poorly due to high lipid content in cell wall
3. Only stained by prolonged application of concentrated dyes, penetrating agents
or heat
4. Once in the stain is retained strongly
5. In classic stain acid-fast organisms appear red against a blue background
6. A variant uses a fluorescent dye which allows visualization by fluorescence
microscopy
C. Fungal and Parasitic Stains
1. The smallest fungi are the size of large bacteria and all parasitic forms are larger
2. Fungi can be seen in a potassium hydroxide (to dissolve debris) or with the
fluorescent dye calcofluor
3. Detection of the cysts and eggs of parasites can be visualized with a simple iodine
stain but stool specimens require a concentration
D. Darkfield and Fluorescence Microscopy
1. Some bacteria, such as Treponema pallidum, the cause of syphilis, are too thin
to be visualized with the usual bright-field illumination.
2. Darkfield microscopy creates a halo around organisms too thin to see by brightfield
3. Fluorescent stains convert darkfield to fluorescence microscopy
E. Electron Microscopy
1. Electron microscopy demonstrates structures by transmission of an electron beam
and has 10 to 1000 times the resolving power of light microscopy
2. Individual viruses are visible only by electron microscopy
CULTURE
I.
Isolation and Identification of Bacteria and Fungi
1. Bacteria and fungi grow in soup-like media prepared from digests of animal or
vegetable protein supplemented with nutrients such as glucose
2. Large numbers of bacteria in broth produce turbidity
3. Agar is a convenient gelling agent for solid media
4. Bacteria and fungi may be separated in isolated colonies on agar plates
5. Colonies have consistent and characteristic features
6. Optical, chemical, and electrical methods can detect growth
A. Culture Media
1. The nutrient component of a medium is designed to satisfy the growth
requirements of the organism to permit isolation and propagation.
2. Media are prepared with enzymatic or acid digests of animal or plant products
such as muscle, milk, or beans
3. To this nutrient base, salts, vitamins, or body fluids such as serum may be
added to provide pathogens with the conditions needed for optimum growth
4. Selective media are used when specific pathogenic organisms are sought in
sites with an extensive normal flora
5. Unwanted organisms are inhibited with chemicals or antimicrobics
6. Indicator media contain substances designed to demonstrate biochemical or
other features characteristic of specific pathogens or organism groups.
7. Metabolic properties of bacteria are demonstrated by substrate and indicator
systems like a pH indicator
B. Atmospheric Conditions
1. Aerobic - Most pathogenic bacteria and fungi that are not obligate anaerobes
will grow in air at 35 to 37°C
2. CO2 is required by some and enhances the growth of others
3. Anaerobic - Anaerobes require reducing conditions and protection from oxygen
4. A convenient commercial system produces anaerobiosis chemically in a packet
to which water is added before the jar is sealed.
C. Clinical Microbiology Systems
1. Combinations of media and incubation must be matched to the organisms
expected at any particular clinical circumstance
2. Routine systems are designed to detect the most common organisms
a. Identification
1. Identification involves the use of methods to obtain pure cultures followed by
tests to identify the isolate
2. Extent of identification is linked to medical relevance
i. Features Used to Classify Bacteria and Fungi
1. Cultural characteristics include unique nutritional requirements,
pigment production, and hemolysis on blood agar plates
2. For fungi growth as a yeast colony or a mold is the primary separator
3. The ability to attack various substrates or to produce particular
metabolic products has broad application to the identification of bacteria
and yeast
4. Biochemical reactions analyzed by tables and computers give
identification probability
5. In some diseases a specific toxin may be detected in cell cultures or by
immunologic methods
6. Antigenic structure - Antibodies of known specificity can detect
antigens present on whole organisms or free in extracts
7. Genomic Structure- Nucleic acid sequence relatedness have become a
primary determinant of taxonomic decisions
II. Isolation and Identification of Viruses
A. Cell and Organ Culture
1. Cell cultures derived from human or animal tissues are used to isolate viruses
2. Monkey kidney is used in primary and secondary culture
3. Primary cultures either die out or transform
4. Cell strains regrow a limited number of times
5. Cells from cancerous tissue may grow continuously
B. Detection of Viral Growth
1. Viral cytopathic effect (CPE) is due to morphologic changes or cell death in the
cell cultures
2. CPE is characteristic for some viruses
3. Hemadsorption (erythrocyte adherence to infected cells) or interference marks
cells that may not show CPE
4. Epstein–Barr virus (EBV) and HIV antigens are expressed on lymphocytes
5. Immunologic or genomic probes detect incomplete viruses
C. In Vivo Isolation Methods
1. Embryonated eggs and animals are used for isolation of some viruses
2. Specimen preparation is required
3. Time to detection varies from days to weeks
D. Viral Identification
1. Nature of CPE and cell cultures affected may suggest virus
2. Neutralization of biologic effect with specific antisera confirms identification
3. Inclusions and giant cells suggest some viruses
4. Immune electron microscopy shows agglutinated viral particles
5. Not all viruses grow in culture
IMMUNOLOGIC SYSTEMS
I. Methods for Detecting an Antigen–Antibody Reaction
A. Precipitation
1. When antigen and antibody combine in the proper proportions, a visible
precipitate is formed
2. Optimum antigen–antibody ratios can be produced by allowing one to diffuse into
the other, most commonly through an agar matrix (immunodiffusion)
3. Both the speed and the sensitivity of immunodiffusion are improved by
counterimmunoelectrophoresis (CIE)
4. RBCs and latex particles coated with antigen or antibody enhance demonstration
5. Simple mixing on slide may cause agglutination
B. Neutralization
1. Some observable function of the agent, such as cytopathic effect of viruses or
the action of a bacterial toxin, is neutralized
2. Usually done by reacting the agent with antibody before placing the antigen–
antibody mixture into a test system.
C. Complement Fixation
1. Complement fixation assays depend on the fixation of complement to antigen–
antibody complexes and the ability of bound complement to cause hemolysis of
antibody sensitized RBCs
2. Action of complement on RBCs is used as indicator system
D. Labeling Methods
1. Labeling antibody allows detection by fluorescence, radioactivity, or enzyme
immunoassay
2. In immunofluorescence antibody labeled with a fluorescent dye is applied to a
slide of material that may contain the antigen sought
3. Light halo enhances microscopic visualization
4. Indirect methods use a second antibody
5. Radioimmunoassay (RIA) and Enzyme Immunoassay (EIA) are liquid phase
particularly used in virology
6. Liquid phase RIA and EIA methods have many variants
II. Serologic Classification
1. Antigenic systems classify below the species level as serotype, serogroup
2. Serologic classification is primarily of epidemiologic value
3. Proof of etiologic relationship is enhanced on antigen detection
III. Antibody Detection (Serology)
1.
2.
3.
4.
5.
Antibodies formed in response to infection may be diagnostic
Antibodies may indicate current, recent, or past infection
Paired acute and convalescent specimens are compared
In quantitation titer is the highest serum dilution demonstrating activity
Seroconversion or fourfold rise in titer is most conclusive for infection in that
time period
6.
7.
8.
9.
Single titers may be useful in some circumstances
IgM responses indicate acute infection
Experience with systems and temporal relationships aids interpretation
Western blot confirms specificity of antibodies for protein components of the
agent (e.g., HIV)
IV. Antigen Detection
1. Theoretically, any of the methods described for detecting antigen–antibody
interactions can be applied directly to clinical specimens
2. An antibody of known specificity is used to detect its complementary antigen
3. Immunofluorescence detects agents in respiratory secretions
4. Soluble antigens may be detected in body fluids by agglutination, CIE, RIA,
and EIA
5. Rapid detection can replace culture and be used at point of care
NUCLEIC ACID ANALYSIS
I. Methods of Nucleic Acid Analysis
A. DNA Hybridization and Probes
1. If the DNA double helix is opened complementary sequences of a second DNA
molecule will hybridize to it
2. A probe is a cloned DNA fragment which has been labeled so it can be detected
if it hybridizes to complementary sequences in such a test system
3. A probe derived from the gene for a known protein detects that gene
4. A variant in which the DNA is separated by agarose gel electrophoresis before
binding to the membrane is called Southern hybridization.
B. Agarose Gel Electrophoresis
1. Agarose gel electrophoresis separates DNA fragments or plasmids based on size
2. Restriction endonucleases digest at specific nucleotide sequences and refine the
analysis
3. Probes detect sequences in fragments
C. Nucleic Acid Amplification
1. Nucleic acid amplification (NAA) methods like the polymerase chain reaction
(PCR) allow the detection and selective replication of a targeted portion of the
genome
2. PCR instruments use temperature to manipulate primers and polymerases
3. The targeted DNA can be amplified one million to one billion times
II. Application of Nucleic Acid Methods to Infectious Diseases
A. Bacterial and Viral Genomes
1. Bacterial plasmids and viral genomes are small enough to be directly detected
and sized by agarose gel electrophoresis
2. Number and size of plasmids differentiates strains
3. Endonuclease digestion of plasmids refines their comparison
4. Bacterial chromosomes must be digested prior to electrophoresis
B. DNA Probes
1. Probes may be cloned or synthesized from known sequences
2. Probes can detect DNA of pathogen directly in clinical specimens
3. Sensitively is enhanced by prior NAA in the specimen
C. Applications of Polymerase Chain Reaction (PCR)
1. PCR combined with probes gives the greatest sensitivity
2. PCR from tissue allows study of organisms that cannot be cultured
3. Ribotyping makes use of the conserved nature of bacterial ribosomal RNA and
of the ability of RNA to hybridize to DNA under certain conditions
4. Labeled ribosomal RNA of one organism can be hybridized with restriction
endonuclease–digested chromosomal DNA of another