Diagnostic microbiology-bacterial
and fungal infections
DR NAZIA KHAN
OBJECTIVES
1.
Outline the principles of diagnostic microbiology
2. Discuss conventional techniques and newer modalities
indiagnosis of bacterial and fungal diseases
3. Describe the knowledge of normal microbial flora
ininterpretation of conventional microbiological
diagnostictests
4. Discuss principle of various immunological techniques
usedin diagnostic microbiology
PRINCIPLES OF DIAGNOSTIC MICROBIOLOGY
• Diagnostic medical microbiology is concerned with the etiologic diagnosis 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 (latex agglutination, EIA, etc) or by
fluorescein-labeled (or peroxidase-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.
• Laboratory test results depend largely on the quality of the specimen, the timing and the care with which it
is collected, and the technical proficiency and experience of laboratory personnel.
Diagnosis of Bacterial & Fungal Infections
• A properly collected specimen is must
• 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.
• 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 (a normally
sterile area like CSF, Joint fluid, blood, pleural cavity)
• 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.
:
(
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
(eg, sputum, not saliva)
3.
Contamination of the specimen must be avoided.
4.
The specimen must be taken to the laboratory and examined
promptly.
5.
Specimens must be taken before antimicrobial drugs are
administered.
What specimens to be taken:
• CSF, blood,sputum,stool,urine,pleural fluid,peritoneal fluid etc
• For fungal infections: scrapings from the edges of the lesions
Microscopic examination
• simple and inexpensive but much less sensitive method than
culture
• A specimen must contain at least 105 organisms per milliliter
• For bacteria
1. Gram staining
2. Ziehl-Neelsen stain & Kinyoun stain for Acid fast bacteria
like Tb bacilli
3. Immunofluorescent antibody (IF) staining :specific than
other staining techniques but also more cumbersome to
perform
4. special structures e.g. Endospores, granule and capsule can
be used to give an initial presumptive identification
• For fungi
i.
calcofluor white: binds to cellulose and
chitin in the cell walls of fungi and
fluoresces under long-wave length
ultraviolet light
ii.
methenamine silver, useful in staining
carbohydrates
iii. periodic acid-Schiff (PAS):stain tissue
sections
iv. lactophenol cotton blue:distinguish fungal
growth and to identify organisms by their
morphology
v. 10% potassium hydroxide: it breaks
down the tissue surrounding the fungal
mycelia to allow a better view of the
hyphal forms.
CULTURE SYSTEMS
• Culture media may be classified as
1. Solid, liquid or semisolid
2. Simple, complex, special media(
enriched, enrichment, selective,
transport)
3. Aerobic or anerobic media
Fungal Cultures are done in paired
sets, one set incubated at 25–30 °C
and the other at 35–37 °C
Chocolate agar
Sabaroud’s dextrose agar
CULTURE TECHNIQUES-ADVANCED
MGIT for TB culture
Liquid medium
Specimen inoculated
A fluorescent compound is
embedded in silicone on the
bottom
The fluorescent compound is
sensitive to O2
Actively respiring microorganisms
consume the oxygen and allow the
fluorescence to be detected
specimen biochemical test (rapid test methods)
• These include enzymes
(Catalase, Oxidase,
Decarboxylase), fermentation of
sugars, capacity to digest or
metabolize complex polymers
and sensitivity to drugs can be
used in identification.
Antimicrobial susceptibility testing
SENSITIVITY TESTING
The isolated colonies are further
incubated on the sensitivity agar
Multiple antimicrobial discs are
applied
Incubated overnight
Note zones of inhibition
Predict antimicrobial sensitivities
of particular isolate
Immunological Methods(for Antigen detection)
• Culturing of certain viruses, bacteria, fungi, and parasites may not be possible
because the methodology remains undeveloped ( Treponema pallidum; Hepatitis
A, B, C; and Epstein-Barr virus ), is unsafe (rickettsias), or is impractical for all but
a few clinical microbiology laboratories ( Mycobacteria ).
• Cultures may be negative because of prior antimicrobial therapy.
• Under these circumstances, detection of antibodies or antigens may be quite
valuable diagnostically
• Immunological methods involve the interaction of a microbial antigen with an
antibody (produced by the host immune system).
• Testing for microbial antigen or the production of antibodies is often easier than
test for the microbe itself.
• Lab kits based on this technique is available for the identification of many
microorganisms.
Enzyme immunoassay(Enzyme linked immunosorbent assay-ELISA
 Known antigen/ antibody is coated onto
microtitre plate
 Patient specimen containing unknown
antigen/antibody is added,
 Enzyme labeled antibody is added
 Substrate for enzyme is added
 Enzyme activity occurs and is measured as color
change
 Color change is measured in a
spectrophotometer(stronger the color;stronger
would be the enzyme activity and thus
antigen/antibody level)
 Many versions of ELISA(direct, indirect, sandwich
etc.) ; increasing the steps increases the
specificity(correctness) of the test
 One of the most commonly used test for diagnosis
of microorganisms in lab
Western blot
• Confirmatory test in the
diagnosis of HIV
• In western blot the viral proteins
are separated by
electrophoresis transferred to
a filter paperreacted with
patients serum containing
antibodiesenzyme coated
antihuman antibody is
addedsubstrate is added and
colored bands are noted
Problems With Traditional Methods
• Cultivation-based methods insensitive for detecting some organisms.
• Cultivation-based methods limited to pathogens with known growth requirements.
• Failure to detect infections caused by uncultivated organisms
• Visual appearance of microorganisms is nonspecific.
• Examples of Failures With Traditional Approaches
1.
Detection and speciation of slow-growing organisms takes weeks (e.g., M.
tuberculosis ).
2.
A number of visible microorganisms cannot be cultivated (e.g., Whipple bacillus ).
3.
Diseases presumed to be infectious remain ill-defined with no detected
microorganism (e.g., abrupt fever after tick bite)
Molecular methods
• The initiation of new molecular technologies in genomics and
proteomics is shifting traditional techniques for bacterial
classification, identification, and characterization in the 21st century
towards methods based on the elucidation of specific gene sequences
or molecular components of a cell.
• Genotypic methods of microbe identification include the use of :
1.
2.
3.
4.
5.
6.
Nucleic acid probes
PCR (RT-PCR, RAPD-PCR)
Nucleic acid sequence analysis
16s rRNA analysis
RFLP
Plasmid fingerprinting.
INDIRECT TESTS
MOLECULAR TECHNIQUES (PCR)
Org DNA is obtained
Mixed in special tube containing the
primers, Taq polymerase enzyme,
nucleotides etc.
Temperature changes-DNA strand opens
Polymerase starts producing
transcription using primers
Duplicate copy of DNA is made
Temp lowered, repeat cycle
PCR
Multiple cycles repeated
Millions copies of original DNA is
obtained
DNA is then either
electrophoresed or tagged with
anti DNA antibodies
Visualize the bands
Compare with known control
Can detect DNA of bacteria,
fungus
Can also detection antimicrobial
sensitivity by resistant gene
PCR PRINCIPLE
The Importance of Normal Bacterial & Fungal
Flora
• Organisms such as Mycobacterium tuberculosis, Salmonella Typhi, and
Brucella species are considered pathogens whenever they are found in
patients.
• However, many infections are caused by organisms that are permanent
or transient members of the normal flora. For example, E coli is part of
the normal gastrointestinal flora and is also the most common cause of
urinary tract infection.
• Similarly, the vast majority of mixed bacterial infections with
anaerobes are caused by organisms that are members of the normal
flora.
NORMAL MICROBIAL FLORA INTERPRETATION
OF TESTS
How to differentiate between commensal,
colonizer and pathogen?
Pure colony
Heavy growth
Sensitive isolate
Elaboration of toxin e.g. by Cl. perferingens,
C. diphtheriae