PRINCIPES OF

DIAGNOSIS

BY DR BUHENDWA CIKURU RODRIGUE

DR MAMADI OWERA FRANCIS

DR NU’MAN OMAR

SUPERVISOR DR ISMAIL
 Outline
General concept
Introduction
MANIFESTATIONS OF INFECTION
MICROBIAL CAUSES OF INFECTION
SPECIMEN SELECTION, COLLECTION AND PROCESSING
MICROBIOLOGIC EXAMINATION
Microbial Identification
Interpretation of Culture Results
Serodiagnosis
Antimicrobial Susceptibility
GENERAL CONCEPTS
• Manifestations of Infection:
reflects the interaction between the host and the microorganism.
This interaction is affected by the host immune status and microbial
virulence factors.
• Microbial Causes of Infection:
may be caused by bacteria, viruses, fungi, and parasites. The
pathogen may be exogenous (acquired from environmental or
animal sources or from other persons) or endogenous (from the
normal flora).
Con’t….
• Specimen Selection, Collection, and Processing
Specimens are selected on the basis of signs and symptoms, should
be representative of the disease process, and should be collected
before administration of antimicrobial agents. The specimen amount
and the rapidity of transport to the laboratory influence the test results.
• Microbiologic Examination
Direct Examination and Techniques:
Direct examination of specimens reveals gross pathology.
Microscopy may identify microorganisms.
Immunofluorescence, immuno-peroxidase staining, and other
immunoassays may detect specific microbial antigens. Genetic probes
identify genus- or species-specific DNA or RNA sequences.
Cont
Culture:
Isolation of infectious agents frequently requires specialized media.
Nonselective (noninhibitory) media permit the growth of many
microorganisms.
Selective media contain inhibitory substances that
permit the isolation of specific types of microorganisms.
Microbial Identification:
Colony and cellular morphology may permit preliminary
identification. Growth characteristics under various conditions,
utilization of carbohydrates and other substrates, enzymatic activity,
immunoassays, and genetic probes are also used.
General concept
• Serodiagnosis:
A high or rising titre of specific IgG antibodies or the presence of
specific IgM antibodies may suggest or confirm a diagnosis.
• Antimicrobial Susceptibility:
Microorganisms, particularly bacteria, are tested in vitro to
determine whether they are susceptible to antimicrobial agents.
INTRODUCTION
• Some infectious diseases are distinctive enough to be identified clinically. Most
pathogens, however, can cause a wide spectrum of clinical syndromes in
humans.
• Conversely, a single clinical syndrome may result from infection with any one of
many pathogens(Influenza virus infection, for example).
• Therefore, it is necessary to use microbiologic laboratory methods to identify a
specific etiologic agent.
• Diagnostic medical microbiology is the discipline that identifies etiologic agents
of disease.
• The job of the clinical microbiology laboratory is to test specimens from patients
for microorganisms that are, or may be, a cause of the illness and to provide
information (when appropriate) about the in vitro activity of antimicrobial drugs
against the microorganisms identified
• The staff of a clinical microbiology laboratory should be qualified to
advise the physician as well as process specimens.
• The physician should supply salient information about the patient,
such as age and sex, tentative diagnosis or details of the clinical
syndrome, date of onset, significant exposures, prior antibiotic
therapy, immunologic status, and underlying conditions.
• The clinical microbiologist participates in decisions regarding the
microbiologic diagnostic studies to be performed, the type and timing
of specimens to be collected, and the conditions for their
transportation and storage.
• Above all, the clinical microbiology laboratory, whenever appropriate,
should provide an interpretation of laboratory results.
MANIFESTATIONS OF INFECTION

• The manifestations of an infection depend on many factors, including:

the site of acquisition or entry of the microorganism; organ or system
tropisms of the microorganism; microbial virulence; the age, sex, and
immunologic status of the patient; underlying diseases or conditions;
and the presence of implanted prosthetic devices or materials.
• The signs and symptoms of infection may be localized, or systemic
with fever, chills, and hypotension.
• In some instances the manifestations of an infection are sufficiently
characteristic to suggest the diagnosis; however, they are often
nonspecific.
MICROBIAL CAUSES OF INFECTION
• Infections may be caused by bacteria (eg, mycobacteria, chlamydiae,
mycoplasmas, and rickettsiae), viruses, fungi, or parasites.
• Infection may be endogenous or exogenous.
• It is important to establish the cause of an infection, the differential
diagnosis is based on a careful history, physical examination, and
appropriate radiographic and laboratory studies, including the
selection of appropriate specimens for microbiologic examination.

• Results of the history, physical examination, and radiographic and

laboratory studies allow the physician to request tests for the
microorganisms most likely to be the cause of the infection.
 SPECIMEN SELECTION, COLLECTION AND
PROCESSING

• Specimens selected for microbiologic examination should reflect the disease process
and be collected in sufficient quantity to allow complete microbiologic examination.
• The number of microorganisms per millilitre of a body fluid or per gram of tissue is
highly variable, ranging from less than 1 to 10 8 or 1010 colony-forming units (CFU).
• Swabs, although popular for specimen collection, frequently yield too small a
specimen for accurate microbiologic examination and should be used only to collect
material from the skin and mucous membranes.
• Skin and mucous membranes have a large and diverse indigenous flora, every effort
must be made to minimize specimen contamination during collection
• It is often impossible to collect an uncontaminated specimen, and decontamination
procedures, cultures on selective media, or quantitative cultures must be used.
• Specimens collected by invasive techniques, particularly those obtained
intraoperatively, require special attention.
• Enough tissue must be obtained for both histopathologic and
microbiologic examination.
• Histopathologic examination is used to distinguish neoplastic from
inflammatory lesions and acute from chronic inflammations.
• The type of inflammation present can guide the type of microbiologic
examination performed. If, for example, a caseous granuloma is observed
histopathologically, microbiologic examination should include cultures for
mycobacteria and fungi.
• The surgeon should obtain several samples for examination from a
single large lesion or from each of several smaller lesions.
• If an abscess is found, the surgeon should collect several millilitres of
pus, as well as a portion of the wall of the abscess, for microbiologic
examination. Swabs should be kept out of the operating room.
• If possible, specimens should be collected before the administration
of antibiotics.
• Above all, close communication between the clinician and the
microbiologist is essential to ensure that appropriate specimens are
selected and collected and that they are appropriately examined.
MICROBIOLOGIC EXAMINATION

• Direct Examination
• Direct examination of specimens frequently provides the most rapid
indication of microbial infection.
• A variety of microscopic, immunologic, and hybridization techniques
have been developed for rapid diagnosis
• Sensitivity and Specificity
The sensitivity of a technique usually depends on the number of microorganisms in
the specimen.
Its specificity depends on how morphologically unique a specific microorganism
appears microscopically or how specific the antibody or genetic probe is for that
genus or species.
An increase in the sensitivity of a test is often accompanied by a decrease in
specificity.
 In some instances, the sensitivity of direct examination tests can be improved by
collecting a better specimen.
The sensitivity may also be affected by the stage of the disease at which the
specimen is collected.
 Finally, sensitivity may be improved through the use of an enrichment or
enhancement step in which microbial or genetic replication occurs to the point at
which a detection method can be applied.
• Techniques
For microscopic examination it is sufficient to have a compound binocular
microscope equipped with low-power (1OX), high-power (40X), and oil
immersion (1OOX) achromatic objectives, 10X wide-field oculars, a
mechanical stage, a substage condenser, and a good light source.
For examination of wet-mount preparations, a darkfield condenser or
condenser and objectives for phase contrast increases image contrast. An
exciter barrier filter, darkfield condenser, and ultraviolet light source are
required for fluorescence microscopy.
For immunologic detection of microbial antigens, latex particle
agglutination, coagglutination, and enzyme-linked immunosorbent assay
(ELISA) are the most frequently used techniques in the clinical laboratory.
 Antibody to a specific antigen is bound to latex particles or to a heat-killed and
treated protein A-rich strain of Staphylococcus aureus to produce agglutination.
 There are several approaches to ELISA; the one most frequently used for the
detection of microbial antigens uses an antigen-specific antibody that is fixed to
a solid phase, which may be a latex or metal bead or the inside surface of a well
in a plastic tray. Antigen present in the specimen binds to the antibody.
The test is then completed by adding a second antigen-specific antibody bound
to an enzyme that can react with a substrate to produce a coloured product.
The initial antigen antibody complex forms in a manner similar to that shown.
When the enzyme-conjugated antibody is added, it binds to previously unbound
antigenic sites, and the antigen is, in effect, sandwiched between the solid phase
and the enzyme-conjugated antibody. The reaction is completed by adding the
enzyme substrate.
Genetic probes are based on the detection of unique nucleotide
sequences with the DNA or RNA of a microorganism.
Once such a unique nucleotide sequence, which may represent a portion
of a virulence gene or of chromosomal DNA, is found, it is isolated and
inserted into a cloning vector (plasmid), which is then transformed into
Escherichia coli to produce multiple copies of the probe.
The sequence is then reisolated from plasmids and labeled with an
isotope or substrate for diagnostic use.
 Hybridization of the sequence with a complementary sequence of DNA
or RNA follows cleavage of the double-stranded DNA of the
microorganism in the specimen.
The use of molecular technology in the diagnoses of infectious diseases
has been further enhanced by the introduction of gene amplication
techniques, such as the polymerase chain reaction (PCR) in which DNA
polymerase is able to copy a strand of DNA by elongating
complementary strands of DNA that have been initiated from a pair of
closely spaced oligonucleotide primers.
This approach has had major applications in the detection of infections
due to microorganisms that are difficult to culture (e.g. the human
immunodeficiency virus) or that have not as yet been successfully
cultured (e.g. the Whipple's disease bacillus).
• Culture
In many instances, the cause of an infection is confirmed by isolating and
culturing microorganism either in artificial media or in a living host. Bacteria
(including mycobacteria and mycoplasmas) and fungi are cultured in either
liquid (broth) or on solid (agar) artificial media.
Liquid media provide greater sensitivity for the isolation of small numbers of
microorganisms; however, identification of mixed cultures growing in liquid
media requires subculture onto solid media so that isolated colonies can be
processed separately for identification. Growth in liquid media also cannot
ordinarily be quantitated.
Solid media, although somewhat less sensitive than liquid media, provide
isolated colonies that can be quantified if necessary and identified.
Some genera and species can be recognized on the basis of their colony
morphologies.
In some instances one can take advantage of differential carbohydrate
fermentation capabilities of microorganisms by incorporating one or
more carbohydrates in the medium along with a suitable pH indicator.
Such media are called differential media (e.g., eosin methylene blue or
MacConkey agar) and are commonly used to isolate enteric bacilli.
Culture media can also be made selective by incorporating compounds
such as antimicrobial agents that inhibit the indigenous flora while
permitting growth of specific microorganisms resistant to these
inhibitors
The number of bacteria in specimens may be used to define the presence of
infection.
For this reason, quantitative cultures of urine must always be performed.
For most other specimens a semiquantitative streak method over the agar
surface is sufficient.
For quantitative cultures, a specific volume of specimen is spread over the agar
surface and the number of colonies per milliliter is estimated.
For semiquantitative cultures, an unquantitated amount of specimen is applied
to the agar and diluted by being streaked out from the inoculation site with a
sterile bacteriologic loop.
The amount of growth on the agar is then reported semi quantitatively as
many, moderate, or few (or 3+, 2+, or 1+ ), depending on how far out from the
inoculum site colonies appear.
An organism that grows in all streaked areas would be reported as 3+.
Chlamydiae and viruses are cultured in cell culture systems, but virus
isolation occasionally requires inoculation into animals, such as
suckling mice, rabbits, guinea pigs, hamsters, or primates.
Rickettsiae may be isolated with some difficulty and at some hazard
to laboratory workers in animals or embryonated eggs.
For this reason, rickettsial infection is usually diagnosed serologically.
Some viruses, such as the hepatitis viruses, cannot be isolated in cell
culture systems, so that diagnosis of hepatitis virus infection is based
on the detection of hepatitis virus antigens or antibodies.
Cultures are generally incubated at 35 to 37°C in an atmosphere
consisting of air, air supplemented with carbon dioxide (3 to 10
percent), reduced oxygen (microaerophilic conditions), or no oxygen
(anaerobic conditions), depending upon requirements of the
microorganism.
 Since clinical specimens from bacterial infections often contain
aerobic, facultative anaerobic, and anaerobic bacteria, such
specimens are usually inoculated into a variety of general purpose,
differential, and selective media, which are then incubated under
aerobic and anaerobic conditions
The duration of incubation of cultures also varies with the growth
characteristics of the microorganism.
Most aerobic and anaerobic bacteria will grow overnight, whereas
some mycobacteria require as many as 6 to 8 weeks.
• Microbial Identification
Microbial growth in cultures is demonstrated by the appearance of
turbidity, gas formation, or discrete colonies in broth; colonies on agar;
cytopathic effects or inclusions in cell cultures; or detection of genus- or
species-specific antigens or nucleotide sequences in the specimen,
culture medium, or cell culture system.
Identification of bacteria (including mycobacteria) is based on growth
characteristics (such as the time required for growth to appear or the
atmosphere in which growth occurs), colony and microscopic
morphology, and biochemical, physiologic, and, in some instances,
antigenic or nucleotide sequence characteristics.
The selection and number of tests for bacterial identification depend upon the
category of bacteria present (aerobic versus anaerobic, Gram-positive versus
Gram-negative, cocci versus bacilli) and the expertise of the microbiologist
examining the culture.
Gram-positive cocci that grow in air with or without added CO2 may be
identified by a relatively small number of tests.
The identification of most Gram-negative bacilli is far more complex and often
requires panels of 20 tests for determining biochemical and physiologic
characteristics.
The identification of filamentous fungi is based almost entirely on growth
characteristics and colony and microscopic morphology.
Identification of viruses is usually based on characteristic cytopathic effects in
different cell cultures or on the detection of virus- or species-specific antigens
or nucleotide sequences.
• Interpretation of Culture Results
Some microorganisms, such as Shigella dysenteriae, Mycobacterium
tuberculosis, Coccidioides immitis, and influenza virus, are always
considered clinically significant.
Others that ordinarily are harmless components of the indigenous flora of
the skin and mucous membranes or that are common in the environment
may or may not be clinically significant, depending on the specimen
source from which they are isolated.
Therefore, their isolation from superficial ulcers, wounds, and sputum
cannot usually be interpreted as clinically significant.
Physicians must also consider that the composition of microbial species
on the skin and mucous membranes may be altered by disease,
administration of antibiotics, endotracheal or gastric intubation, and the
hospital environment.
• Serodiagnosis
Infection may be diagnosed by an antibody response to the infecting
microorganism.
This approach is especially useful when the suspected microbial agent
either cannot be isolated in culture by any known method or can be
isolated in culture only with great difficulty.
The diagnosis of hepatitis virus and Epstein-Barr virus infections can be
made only serologically, since neither can be isolated in any known cell
culture system.
Although human immunodeficiency virus type 1 (HIV-1) can be isolated in
cell cultures, the technique is demanding and requires special
containment facilities.
HIV-1 infection is usually diagnosed by detection of antibodies to the virus.
The disadvantage of serology as a diagnostic tool is that there is
usually a lag between the onset of infection and the development of
antibodies to the infecting microorganism.
Although IgM antibodies may appear relatively rapidly, it is usually
necessary to obtain acute- and convalescent-phase serum samples to
look for a rising titer of IgG antibodies to the suspected pathogen.
In some instances the presence of a high antibody titer when the
patient is initially seen is diagnostic; often, however, the high titer
may reflect a past infection, and the current infection may have an
entirely different cause.
Another limitation on the use of serology as a diagnostic tool is that
immunosuppressed patients may be unable to mount an antibody
response.
• Antimicrobial Susceptibility
The responsibility of the microbiology laboratory includes not only
microbial detection and isolation but also the determination of microbial
susceptibility to antimicrobial agents.
Many bacteria, in particular, have unpredictable susceptibilities to
antimicrobial agents, and their susceptibilities can be measured in vitro
to help guide the selection of the most appropriate antimicrobial agent.
Antimicrobial susceptibility tests are performed by either disk diffusion
or a dilution method.
 In the former, a standardized suspension of a particular microorganism
is inoculated onto an agar surface to which paper disks containing
various antimicrobial agents are applied.
Following overnight incubation, any zone diameters of inhibition
about the disks are measured and the results are reported as
indicating susceptibility or resistance of the microorganism to each
antimicrobial agent tested.
An alternative method is to dilute on a log2 scale each antimicrobial
agent in broth to provide a range of concentrations and to inoculate
each tube or, if a microplate is used, each well containing the
antimicrobial agent in broth with a standardized suspension of the
microorganism to be tested.
The lowest concentration of antimicrobial agent that inhibits the
growth of the microorganism is the minimal inhibitory concentration
(MIC).
The MIC and the zone diameter of inhibition are inversely correlated .
In other words, the more susceptible the microorganism is to the
antimicrobial agent, the lower the MIC and the larger the zone of
inhibition.
Conversely, the more resistant the microorganism, the higher the MIC
and the smaller the zone of inhibition.
The term susceptible means that the microorganism is inhibited by a
concentration of antimicrobial agent that can be attained in blood
with the normally recommended dose of the antimicrobial agent and
implies that an infection caused by this microorganism may be
appropriately treated with the antimicrobial agent.
The term resistant indicates that the microorganism is resistant to
concentrations of the antimicrobial agent that can be attained with
normal doses and implies that an infection caused by this
microorganism could not be successfully treated with this
antimicrobial agent.
References
• Medical Microbiology. 4th edition