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Diagnostic medical microbiology is concerned with the detection of infection.

Laboratory
procedures used in the diagnosis of infectious disease in humans include the following:
1. Morphologic identification of the agent in stains of specimens or sections of tissues (light and
electron microscopy).
2. Culture isolation and identification of the agent.
3. Detection of antigen from the agent by immunologic assay (e.g., latex agglutination or by
fluorescein-labeled antibody stains.
4. DNA-DNA or DNA-RNA hybridization to detect pathogen-specific genes in patients’
specimens.
5. Detection and amplification of organism nucleic acid in patients’ specimens.
6. Demonstration of meaningful antibody or cell-mediated immune responses to an infectious
agent.
DIAGNOSIS OF BACTERIAL & FUNGAL INFECTIONS
Specimens
 Laboratory examination usually includes microscopic study of fresh unstained and stained
materials and preparation of cultures with conditions suitable for growth of a wide variety of
microorganisms, including the type of organism most likely to be causative based on clinical
evidence. If a microorganism is isolated, complete identification may then be pursued.
Isolated microorganisms may be tested for susceptibility to antimicrobial drugs. When
significant pathogens are isolated before treatment, follow-up laboratory examinations during
and after treatment may be appropriate.
 A properly collected specimen is the single most important step in the diagnosis of an
infection, because the results of diagnostic tests for infectious diseases depend upon the
selection, timing, and method of collection of specimens. Bacteria and fungi grow and die,
are susceptible to many chemicals, and can be found at different anatomic sites and in
different body fluids and tissues during the course of infectious diseases. Because isolation of
the agent is so important in the formulation of a diagnosis, the specimen must be obtained
from the site most likely to yield the agent at that particular stage of illness and must be
handled in such a way as to favor the agent’s survival and growth.
 Recovery of bacteria and fungi is most significant if the agent is isolated from a site normally
devoid of microorganisms. Any type of microorganism cultured from blood, cerebrospinal
fluid, joint fluid, or the pleural cavity is a significant diagnostic finding. Conversely, many
parts of the body have a normal microbial flora that may be altered by endogenous or
exogenous influences. Recovery of potential pathogens from the respiratory, gastrointestinal,
or genitourinary tracts; from wounds; or from the skin must be considered in the context of
the normal flora of each particular site. Microbiologic data must be correlated with clinical
information in order to arrive at a meaningful interpretation of the results.
A few general rules apply to all specimens:
1. The quantity of material must be adequate.
2. The sample should be representative of the infectious process (e.g., sputum, not saliva; pus
from the underlying lesion, not from its sinus tract; a swab from the depth of the wound, not
from its surface).
3. Contamination of the specimen must be avoided by using only sterile equipment and aseptic
precautions.
4. The specimen must be taken to the laboratory and examined promptly. Special transport
media may be helpful.
5. Meaningful specimens to diagnose bacterial and fungal infections must be secured before
antimicrobial drugs are administered. If antimicrobial drugs are given before specimens are
taken for microbiologic study, drug therapy may have to be stopped and repeat specimens
obtained several days later.
The type of specimen to be examined is determined by the presenting clinical picture. If
symptoms or signs point to involvement of one organ system, specimens are obtained from that
source. In the absence of localizing signs or symptoms, repeated blood samples for culturing are
taken first, and specimens from other sites are then considered in sequence, depending in part
upon the likelihood of involvement of a given organ system in a given patient and in part upon
the ease of obtaining specimens.
Microscopy & Stains
 Microscopic examination of stained or unstained specimens is a relatively simple and
inexpensive but much less sensitive method than culture for detection of small numbers of
bacteria.
 Gram staining is a very useful procedure in diagnostic microbiology. Most specimens
submitted when bacterial infection is suspected should be smeared on glass slides, Gram-
stained, and examined microscopically.
 On microscopic examination, the Gram reaction (purple-blue indicates gram-positive
organisms; red, gram-negative) and morphology (shape: cocci, rods, fusiform, or other; of
bacteria should be noted. The appearance of bacteria on Gram-stained smears does not permit
identification of species. Reports of gram-positive cocci in chains are suggestive of, but not
definitive for, streptococcal species; gram-positive cocci in clusters suggest a staphylococcal
species. Gram-negative rods can be large, small, or even coccobacillary. Some non-viable
gram-positive bacteria can stain gram-negatively. Typically, bacterial morphology has been
defined using organisms grown on agar. However, bacteria in body fluids or tissue can have
highly variable morphology.
 Specimens submitted for examination for mycobacteria should be stained for acid-fast
organisms, using either Ziehl-Neelsen stain.
 Specimens to be examined for fungi can be examined unstained after treatment with a
solution of 10% potassium hydroxide, which breaks down the tissue surrounding the fungal
mycelia to allow a better view of the hyphal forms.
 Darkfield microscopy is used to detect Treponema pallidum in material from primary or
secondary syphilitic lesions.
SEROLOGY
This is study of antigen–antibody reactions and their use in tests to detect specific antigens and
antibodies.
Terminologies
Antibody - A glycoprotein synthesized in a plasma cell, which is derived from a B cell that has
interacted with a specific antigen. The antibody molecule can bind specifically to this antigen.
Antigen - A molecule that:
 triggers synthesis of antibody and/or a T cell response;
 binds specifically to an antibody or a lymphocyte receptor.
Seropositive - A person or an animal is said to be seropositive for an antigen, e.g. a virus, if their
blood contains antibodies specific for that antigen.
Introduction
The detection of antibodies as a measure of infection by a particular organism may be used for
diagnosis. One may measure the presence of antibody as:
 an indication of immune status, following natural infection or vaccination;
 seroconversion (i.e. appearance of antibody) or an increase in titer as an indicator of recent
infection.
 a particular class of immunoglobulin to determine the nature of the infection.
 responses to various antigens associated with the same virus to indicate disease progression.
 a combination of antibody and antigen levels also to monitor disease state or progression.
 levels in different body fluids, such as serum versus CSF, to determine involvement of
possible site of infection.
Agglutination
Agglutination is the visible aggregation of particles caused by combination with specific
antibody. Antibodies that produce such reactions are often called agglutinins. Because this
reaction takes place on the surface of the particle, antigen must be exposed and able to bind with
antibody. Agglutination is actually a two-step process, involving sensitization or initial binding
followed by lattice formation, or formation of large aggregates. Types of particles participating
in such reactions include erythrocytes, bacterial cells, and inert carriers such as latex particles.
Each particle must have multiple antigenic or determinant sites, which are cross-linked to sites
on other particles through the formation of antibody bridges.
Direct agglutination
 Direct agglutination occurs when antigens are found naturally on a particle. One of the best
examples of direct agglutination testing involves known bacterial antigens used to test for the
presence of unknown antibodies in the patient.
 Typically, patient serum is diluted into a series of tubes or wells on a slide and reacted with
bacterial antigens specific for the suspected disease. Detection of antibodies is primarily used
in diagnosis of diseases for which the bacterial agents are extremely difficult to cultivate.
One such example is the Widal test, a rapid screening test to help determine the possibility of
typhoid fever. The antigens used in this procedure include Salmonella O (somatic) and H
(flagellar) antigens. A significant finding is a fourfold increase in anti-body titer over time
when paired dilutions of serum samples are tested with any of these antigens. While more
specific tests are now available, this test is still considered useful in diagnosing typhoid fever
in developing countries, and it remains in use in many areas throughout the world.

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