2. Introduction
Microbial diagnostics are essential tools in the field of microbiology and infectious
disease medicine, enabling the detection and identification of microbial pathogens
that cause diseases in humans, animals, and plants.
Their primary goal is to accurately identify the causative agents of infections,
facilitating appropriate treatment and management strategies
advances in technology, molecular diagnostics, such as Polymerase Chain Reaction
(PCR) and Next-Generation Sequencing (NGS), have revolutionized the field,
offering rapid and highly specific identification of pathogens at the genetic level.
microbial diagnostics are at the forefront of combating antimicrobial resistance
(AMR). Overuse and misuse of antibiotics have led to the emergence of resistant
strains of bacteria, posing a significant public health threat. By precisely
identifying pathogens and their susceptibility patterns, microbial diagnostics guide
the use of antibiotics, promoting their judicious use and contributing to the
stewardship of these critical drugs
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Laboratory diagnosis of infections involves direct detection of the pathogen or its
components and indirect detection through antibodies. The accuracy of these tests is
determined by their sensitivity and specificity, with their predictive value highly
influenced by the prevalence of the disease. Proper sample collection and transport are
crucial for direct diagnosis, which traditionally includes microscopy and culturing based on
the pathogen's physical, physiological, and chemical properties.
4. Types of Microbial Diagnostic Tests
Culture Techniques: Traditional methods where microbes are grown on specific media for
identification based on growth patterns and biochemical tests. Example: Diagnosing strep
throat with a throat culture.
Molecular Diagnostics: Techniques like PCR and RT-PCR detect genetic material of
pathogens, offering rapid and specific identification. Example: RT-PCR for COVID-19
detection.
Immunodiagnostic Tests: These tests detect antigens or antibodies using the immune
response, suitable for both qualitative and quantitative analysis. Example: ELISA for HIV
antibody detection.
Biosensors: Advanced devices that combine biological components with physicochemical
detectors for rapid pathogen detection. Example: Influenza virus detection in respiratory
samples.
5. LABORATORY DIAGNOSIS OF BACTERIA
1.Microscopy
Microscopy is a vital tool in microbiology, allowing the observation of bacteria and other microorganisms.
Bacteria are so small that a magnification of 1000X is required to view them properly, which is at the limit of
light microscope capability. At this magnification, bacteria can only be discerned in a preparation in which their
density is at least 10^4 – 10^5 bacteria per ml.
Types of Microscopy:
Native Preparations: These are slides where bacteria are observed in their natural state, with or without vital
staining. The poor contrast of such preparations makes it necessary to amplify this aspect (dark field and phase
contrast microscopy). Native preparations include the coverslip and suspended drop types.
Stained Preparations: These are slides where bacteria are stained to enhance contrast. The staining procedure
kills the bacteria. The material is first applied to a slide in a thin layer, dried in the air, and fixed with heat or
methyl alcohol. Simple and differential staining techniques are used. The best-known simple staining technique
employs methylene blue.
Fluorescence Microscopy: This is a special technique where a fluorochrome absorbs shortwave light and emits
light with a longer wavelength. Preparations stained with fluorochromes are exposed to light at the required
wavelength. The stained particles appear clearly against a dark background in the color of the emitted light.
This technique requires special equipment. Its practical application is in the observation of mycobacteria. In
immunofluorescence detection, a fluorochrome (e.g., fluorescein isothiocyanate) is coupled to an antibody to
reveal the presence of antigens on particle surfaces.
6. Culturing Methods
Culturing methods are fundamental in microbiology for detecting and identifying bacteria. Nutrient
mediums, such as nutrient broth or nutrient agar, are used for this purpose. Enrichment mediums,
like blood agar plates, encourage the growth of various bacterial species.
Types of Nutrient Mediums:
Selective Mediums: Allow only certain bacteria to grow while suppressing others.
Indicator Mediums: Register metabolic processes.
Bacteria exhibit various proliferation forms in mediums, such as:
Diffuse proliferation in liquid mediums.
"Crumbs" or grainy bottom sediment.
Biofilm skin at the surface (pseudomonads).
Isolated colonies on nutrient agar, indicating pure cultures.
7. Ct …. Identification of Bacteria:
Morphological Characteristics: Form, size, staining (Gram-positive/negative),
presence of flagella, capsule, and spores.
Physiological Characteristics: Respiratory chain enzymes, carbohydrate
breakdown enzymes, protein and amino acid metabolism enzymes, and
resistance/sensitivity to chemicals.
Chemical Characteristics: DNA structure, cell wall murein, antigen structure,
fatty acids in membranes and cell wall.
8. Molecular Methods
Molecular methods of bacterial identification aim to directly recognize pathogen-specific nucleotide sequences
in the test material, particularly for bacteria that are not culturable, are very difficult to culture, or
proliferate slowly. These methods can also be used for pure bacterial cultures. Species-specific sequences,
especially those coding for 16S rRNA and 23S rRNA, are useful for identification.
Methods Used:
DNA Probes: Detect single-strand sequences using hybridization techniques with complementary marking of
single strands. Probes can be marked with radioactivity or nonradioactive reporter molecules. Techniques
include solid phase hybridization (colony blot, dot blot), liquid phase hybridization, and in-situ hybridization.
Amplification: Increase sensitivity to find the "needle in a haystack." Techniques include amplification of the
target sequence (e.g., PCR, real-time PCR), probe amplification, and signal amplification.
Direct Detection of Bacterial Antigens:
Antigens specific to certain species or genera can be detected directly using polyclonal or monoclonal
antibodies in the test material, enabling rapid diagnosis. For example, bacterial antigens in cerebrospinal fluid
can be detected in cases of acute purulent meningitis.
Diagnostic Animal Tests:
Animal testing in diagnostic bacteriology is now largely replaced by molecular genetics methods. Previously,
bacterial toxins (e.g., diphtheria toxin, tetanus toxin, botulinum toxin) were confirmed through animal tests.
Now, toxin gene presence is detected using molecular genetics methods, often involving an amplification step.
9. LABORATORY DIAGNOSIS OF FUNGI
Laboratory diagnosis of fungi involves identifying the pathogen through various methods:
Microscopy: Native preparation involves briefly heating the material under a coverslip with 10% KOH.
Stained preparations use dyes like methylene blue, lactophenol blue, periodic acid-Schiff (PAS), or
ink.
Culturing: Fungi can be cultured on universal and selective mediums like Sabouraud dextrose agar,
which may contain selective agents. Morphological structures, especially reproductive structures, are
key for identification.
Serology: Identifying antibodies to specific fungal antigens in patient serum. Interpretation of
serological findings can be challenging.
Antigen Detection: Finding specific antigens in diagnostic material using known antibodies, possible
in some fungal infections like cryptococcosis.
Cutaneous Test: Allergy tests with specific fungal antigens can help diagnose fungal infections.
Nucleic Acid Detection: Combined with amplification, these tests are useful for rapidly detecting
mycotic diseases, especially in immunocompromised patients.
10. Laboratory diagnosis of virus infections
Diagnosing virus infections involves identifying the virus or parts of it in patient material or demonstrating
a host immune response to the virus, often antibodies. Several techniques are used for virus detection:
Virus Isolation in Tissue Culture: Viruses require living cells for growth. Clinical specimens are inoculated
into cell cultures, and the development of cytopathic effects (CPE) indicates the presence of a virus.
Electron Microscopy: Allows direct visualization of viral particles, useful for high-titer samples like feces
or vesicle fluid.
Antigen Detection: Demonstrates the presence of viral antigens or genomes in clinical samples, often
using fluorescently labeled monoclonal antibodies.
Nucleic Acid Detection: Involves detecting viral DNA or RNA sequences. Techniques like polymerase chain
reaction (PCR) amplify specific sequences for sensitive detection.
Diagnosis by serological methods involves:
Measuring Virus-Specific IgG: Rise in IgG titers between acute and convalescent sera indicates recent
infection.
Measuring Virus-Specific IgM: Presence of specific IgM in acute serum suggests recent infection.
Detection of Viral Antigens: Demonstrating viral antigens, like HBsAg in hepatitis B, in serum indicates
infection.
11. Diagnostic Tests in Microbiological Specimen
Analysis
Objectives and Learning Outcomes
Acquire basic skills in the identification and isolation of microbial pathogens.
Demonstrate understanding of the principles used in diagnostic medical
microbiology and the clinical correlations.
Make observations, understand the fundamental elements of experimental
design, generate and analyse data on pathogenesis and control of diseases.
12. Outline
Normal Bacterial & Fungal Flora
Laboratory Aids in the Selection of Antimicrobial Therapy
Diagnosis of Chlamydial Infections
Diagnosis of Viral Infections
13. Normal Bacterial & Fungal Flora
Some organisms are considered pathogens whenever they are found in
patients. Eg. Many infections are caused by organisms that are permanent
members of normal flora.
Gram-negative rods are strongly suspect as the cause of pneumonia eg.
Klebsiella pneumonia
In some cases, identification of normal flora is more warranted, eg. in
abdominal abscesses.
Yeast in small numbers are common flora, not others.
14. Laboratory Aids in the Selection of
Antimicrobial Therapy
Drug testing is essential in those bacteria commonly showing resistance,
primarily Staphylococcus sp., Neisseria gonorrhoea etc.
Testing on fungal and protozoan infections is difficult and often unnecessary.
Identification of infectious agents should be attempted as soon as possible,
secured before the antimicrobic drug is given.
Choice of drug is based on “informed best guess”.Disk diffusion susceptibility
(or Kirby-Bauer) test – common test, that measures the ability of drugs to
inhibit the growth of bacteria.
The size of zones of inhibition vary with each different drug.
Tube dilution tests give more sensitive and quantitative results.
15. Laboratory Aids in the Selection of
Antimicrobial Therapy
Minimum inhibitory concentration (MIC) = measures the concentration of an
antibiotic necessary to inhibit growth of a standardised inoculum under
defined conditions. The end point, or minimum inhibitory concentration is the
last lowest concentration of drug that gives clear broth, ie. free from
microbial growth. This is very useful in gauging the dosage regimen necessary
for patients.
In addition, bactericidal effects can be estimated by subculturing the clear
broth onto antibiotic-free solid media. The result, which is a reduction of
colony-forming units by 99.9% below that of the control, is called the minimal
bactericidal concentration (MBC).
The results of antimicrobic sensitivity tests guide the physician’s choice of a
suitable drug. Continuous observation of patient’s clinical response is
imperative once antimicrobial therapy has begun
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Drug toxicity – choose one with high/low selective toxicity for the infectious agent and
high/low human toxicity.
Therapeutic index (TI) = the ratio of the dose of the drug that is toxic to humans as
compared to its minimum effective (therapeutic) dose:
TI = toxic dose (µg/ml)
effective dose (µg/ml)
When antimicrobic treatment fails, this may be due to:i) the inability of the drug to diffuse
into body compartment (brain, joints)ii) a few resistant cells that did not appear in the
sensitivity test in-vitro) infection caused by more than one pathogen
Other considerations before administrating drugs: allergy, pregnancy, underlying liver or
kidney diseases, age, any genetic or metabolic abnormalities, drugs incompatibility, cost.
17. Diagnosis of Chlamydia
Chlamydia: Structure
Outer cell wall resembles Gram-negative; with relatively high lipid content.
Cell wall is rigid but no peptidoglycan, hence no effect on lysozyme.
Perhaps it contains tetrapeptide-linked matrix.Penicillin-binding proteins
occur in chlamydiae.
DNA and RNA are present in elementary and reticulate bodies.
Although Chlamydia sp. are bacteria, they are obligate intracellular parasites.
Hence, procedures are much likely resembled those of diagnosing viruses.
Specimens are collective from respective sites of infections, and carefully
placed in transport medium
18. Species Infected sites
C. trachomatis Oculogenital epithelial, conjunctival scraping,
endometrium scraping
C. pneumoniae Nasopharyngeal swab
C. psittaci Sputum, blood, biopsy material.
19. Chlamydiae Staining properties:
Giemsa stains: Elementary bodies – purple; larger, noninfective reticulate bodies stain – blue and host
cytoplasm - blue.
Fully formed, mature intracellular inclusions of C. trachomatis are compact masses near nucleus (dark
purple) in Giemsa stain.
Gram staining – negative
Chlamydia Microscopy:
Micro-organisms can sometimes be found in the cytoplasm of squamous cells from conjunctival
scrapings due to Chlamydia psittaci infection.
This organism forms compact, oval-shaped grape-like cluster in the cells’ cytoplasm (yellow arrow).
Sometimes dispersed elementary bodies can be seen.
20. Diagnosis of Chlamydia
Cell culture techniques for isolation of chlamydia species involves inoculation of the C trachomatis
and C psittaci onto cycloheximide-treated McCoy cells (antimetabolites that inhibit host cell
replication but allows chlamydiae to use available cell nutrient for growth), and C pneumoniae
requires pretreated HEp-2 cells.
Immunoassay –EIAs are used to detect chlamydial antigens from genital swab.
Nucleic acid Hybridization – commercial kits with nonradioisotopic probes are available for C
trachomatis 16S RNA sequences. PCR and ligase chain reaction (LCR) are much more sensitive than
culture.
Serology: Complement fixation test is widely used to diagnose psittacosis. The
microimmunofluorescence method is more sensitive than CF for measuring antichlamydial antibodies.
Detection of IgM against C trachomatis is helpful in infant pneumonitis
21. Detection of Viral Growth
Lytic or cytopathic viruses replicate in cells and produce alterations in cellular morphology (or cell
death) and the effect can be seen directly by light microscopy. For example, enteroviruses often
produce cell rounding, pleomorphism and eventual cell death; and measles and RSV cause fusion of
cells to produce ____________.
Other viruses are detectable by their production of haemagglutinins; and by a method called
interference whereby the virus which produces no cytopathic effect in susceptible cell culture, but can
be detected by “challenging” the cell culture with a different virus that normally produces a
characteristic cytopathic cell. The second virus fails to infect the cell culture because of the
interference by the first virus, which is thus detected. Eg. Rubella virus
22. In vivo Isolation Methods
Embryonated hen’s egg is still used for the initial isolation and propagation of influenza A virus. The egg is
incubated to permit viral replication and recognition.
Animal host, commonly the mouse; suckling mice in the first 48h of life are especially susceptible to
many viruses. Viral replication is based on development of illness, with signs such as paralysis,
convulsions, poor feeding or death.
Further test to elucidate the nature of infecting virus – by histologic and immunofluorescent examinations
of tissues or by antibody detection.
Virus Identification
On isolation, virus can usually be identified by its cultural characteristics. Further identification may
require adequate quantities for testing.
Several diagnostic methods for viral identification are:
1. Antigen detection
2. nucleic acid amplification and detection
3. nucleic acid hybridization
4. measuring immune response to virus infection
5. immune electron microscopy
23. Diagnostic Virology
Antigen Detection: EIA, immunofluorescence, latex agglutination.
Nucleic Acid Amplification & Detection: PCR, reverse-transcriptase PCR.
Nucleic Acid Hybridisation: Highly sensitive and specific.
Measuring Immune Response: Assessing cellular and humoral immunity.
Immune Electron Microscopy (IEM): For detecting viruses causing enteritis
and diarrhoea
24. Assays for Anti-HIV Antibodies
A. ELISA Enzyme-Linked Immunosorbent Assay:
Primary screening test for HIV-1 infection.HIV-1 antigens immobilised on a solid surface (plastic wells, or beads). Add patient’s serum. HIV-1
antibodies bound to immobilised antigens are then detected with an enzyme labeled anti-human IgG and a colorimetric reaction.Results
interpretation: the amount of colour is proportionately higher with higher HIV-1 antibodies.ELISA for HIV-1 is extremely sensitive and specific.
Follow up question: What about infants born with HIV-infected mothers?
Read about the principle of immunoassay and howto perfoem ELISA test
Western Blot:To measure specific HIV-1 antibodies to confirm a positive ELISA result.
Western Blot 1. HIV-1 proteins are separated by electrophoresis.
HIV-1 proteins are transferred onto nitrocellulose strip.
The strip is incubated with patient’s serum.
The specific HIV-1 antibodies are subsequently detected using an enzyme-linked antihuman IgG.
Positive colorimetric reaction forms bands on the nitrocellulose paper
Western Blot: Results
i) Positive test : any 2 bands corresponding to the proteins.
ii) no bands : negative result
iii) bands that do not meet the criterion for a positive test is an indeterminate results.
False-positive and false-negative results are relatively uncommon.
Repeat positive ELISAs and indeterminate Western blots.
