Serological diagnosis in medical microbiology

Question 1

Laboratory Investigation of Microbial
infections: Examining specimens to detect isolate and identify pathogens:

Answer 1

1- Microscopy
2- Culture techniques
3- Biochemical reactions
4- Serological identification:
5- Molecular biology techniques
6- Bacteriophage typing

Question 2

What is the serological diagnosis of
infections?

Answer 2

It is the diagnosis of infectious diseases based upon the Antigen-Antibody (Ag-Ab) reactions. - Ag - Ab reaction is the union of an Ag with its specific Ab, best detected when each is in optimal concentration. -The type of such reaction depends upon the nature of both Ag and Ab used. - These reactions are mostly used in the laboratory (In Vitro) to detect either the Ag (Direct diagnosis) or the Ab (indirect diagnosis).

Question 3

Types of Ag-Ab Reactions (In Vitro)

Answer 3

1. Agglutination.
2. Precipitation.
3. Complement fixation Test (CFT).
4. Neutralization.
5. Immunofluorescence Test (IF).
6. Enzyme linked immunosorbent assay (ELISA).
7. Radio immunoassay (RIA).
8. ImmunochromatographY (ICT) .
   9- Immuno-blotting assays.

Question 4

Application of Ag-Ab Reactions (Serology):

Answer 4

1. Diagnosis of many infectious diseases (direct antigen detection or measuring Immunity to infection) especially for non-culturable or
   delayed growing microorganisms.
2. Estimation of the Severity or stage of diseases.
3. Determination of the Response to treatment.
4. Epidemiological studies.
5. Diagnosis of congenital infections.
6. Screening donation of blood and tissues. 7. Non-infectious diagnostic applications (Tumors,
   Autoimmune diseases, endocrinology, …etc.

Question 5

Agglutination

Answer 5

• Is the visible clumping of particulate (insoluble) Ag with its specific Ab forming visible lattice. The Ab is the divalent agglutinating one either IgG or IgM
type.
• The reaction can be either on slide or tube agglutination.
• It can be direct or via the use of carrier (Passive or indirect) e.g. Latex, RBCs (hemagglutination), or Staph protein A (Coagglutination). Indirect more
easily visible.
• The reaction can be qualitatively or quantitatively expressed

Question 6

Examples of agglutination reactions

Answer 6

1. Direct slide detection of a bacterial or viral antigens in a lesion or culture.
2. Direct tube agglutination (Classical Widal test) for diagnosis of typhoid fever (replaced now by Latex)
3. Indirect Latex tests commonly used in most microbiology laboratories nowadays.
4. Indirect passive hemagglutination tests e.g. Treponema Pallidum Hemagglutination (TPHA) for diagnosis of syphilis which is more specific. 5. Brucella slide or tube agglutination test (Prozone phenomenon).

5- Widal latex slide agglutination tests for diagnosis
of typhoid fever. 6- Weil-Felix Test used for the diagnosis of rickettsial disease e.g., epidemic typhus which is non- specific test using the Heterophil Ag of proteus
species.

Question 7

Why are latex tests more common?

Answer 7

• Easy to manufacture, to use, cheap, clearly visible.
• No instruments
• Can be coated with any Ag (Soluble) to detected Ab
• Can be coated with the Ab to detect the Ag.
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Question 8

Prozone Phenomenon

Answer 8

• It is the absence of agglutination in the first tubes containing high concentration of Abs (appearing as being false-negative) and its occurrence in the following tubes containing lower concentrations
(higher dilution i.e. > 1/40 or in 1/80).

• May be caused by the blocking effect of IgA. • Overcome by the use of higher dilution and use of the human antiglobulin (Coombs Reagent).

Question 9

Advantages of Agglutination

Answer 9

•The most widely used tests for diagnosis and
screening.
• Easy and simple.
• Very cheap
• No instrument is required
•somewhat sensitive

Question 10

Disadvantages of agglutination

Answer 10

• low specificity (have false positive results) need
confirmatory tests
• low sensitivity (have false negative results).
• Results affected by vaccine-induced Ab.
•Subjective errors in reading.
• Agglutinating Ab appears late in the infections.

Question 11

Precipitation Tests

Answer 11

• It the reaction between soluble Ag and its specific Ab.
• It can be Precipitation in solution or in gel. • Example of Precipitation in solution include the Ring test for detection of infectious Ags in CSF.
• Another example of precipitation in solution is the slide test for detection of pneumococcal capsule.
• Precipitation in gel is used for the quantification of Immunoglobulins in patient serum (less common) in form of single, double radial immuno-diffusion or
immuno-electrophoresis.

Question 12

Flocculation tests

Answer 12

• Flocculation is an antigen-antibody reaction that occurs if the antigen is neither cellular nor soluble, but is an insoluble particulate.

• The flocculation reaction is a special type of precipitation reaction.

• Example of the flocculation reaction in current use are the Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) used for the diagnosis of syphilis.

• These tests are non-specific (non-treponemal) or called standard test of syphilis (STS).

• The antigen used is cardiolipin, a hapten from normal beef heart those cross-reacts with a heterophile (Heterogenetic) antigen of the spirochete of syphilis so to detects antibodies to Treponema pallidum.

•Cholesterol particles with water-insoluble
cardiolipin on their surface are used in the test.

• Visible aggregates form in the presence of an antibody (reagin) in the serum of patients with
syphilis.

•VDRL has many limitation and not in current use
nowadays and the in current use is the RPR.

Question 13

Complement Fixation Test (CFT)

Answer 13

• It is the detection of complement fixing Abs (IgM or IgG) against the causative organism in the patient serum.

• The (Guinea pig) complement and the Ag used in the test are added in the lab to pt. serum after serum heating to destroy the internal complement
proteins and incubated in Wassermann tubes.

•So, no hemolysis mean positive test.
•The titre is last tube showing no hemolysis.

•The result are affected by anti-complementary substances in the patient serum e.g. in SLE or
heparin therapy.

• Then, an RBCs and Anti-RBCs are added as indicator system to be destroyed by the
complement if there is no Ab in the serum.

• If there is Ab the complement will be fixed in the first reaction so, no hemolysis will occur to certain dilution after which it will occur.

•So, no hemolysis mean positive test.

•The titre is last tube showing no hemolysis.

•The result are affected by anti-complementary substances in the patient serum e.g. in SLE or heparin therapy.

Question 14

Application of CFTs

Answer 14

• Bacterial: Syphilis and chronic gonorrhea.
•Viral: Influenza.
•Systemic fungal infections: Systemic candidiasis.
• The CFTs are laborious and are rarely used nowadays as being replaced by other tests.

Question 15

Advantages of CFT

Answer 15

• The CF Ab do not persist long (recent infections).
• Stable antigen used in the test for years (>10 ys).
• High specificity
• Quantitative results.

Question 16

Disadvantages of CFT

Answer 16

• Low sensitivity (false negative)
• Time consuming.
• Not standardized
• Needs high technical skills
•Sample contamination giving anticomplentary
• Still false positive results occur.
• Still in little use for some viral and protozoal infections

Question 17

Neutralization Tests

Answer 17

• It is an Ag-Ab reaction in which the Ab neutralizes the effect of the Ag (bacterial toxin or virus).
• Example of neutralization test in vitro is the Anti- Streptolysin O titre (ASOT) for the diagnosis of post- Streptococal Complications e.g. Rheumatic fever.

• Alkaline phosphatase is a commonly used enzyme as it can be covalently couple to mAbs without affecting either the Ag binding capacity of the Ab or
inhibiting the activity of the enzyme. • Many formats of ELISA are present and used in
clinical laboratories.

Question 18

Enzyme Linked Immunosorbant
Assay [ELISA]

Answer 18

• Monoclonal antibody (mAb) or recombinant Ag is fixed to a surface of a microtiter plate wells.
•The test sample (with unknown Ag or Ab) is applied and incubated to react then washed.
•The bound material is detected by a secondary, enzymatically labeled mAb (Conjugate) which is incubated and then washed to remove unbound.
•Then a substrate for the enzyme is added later on and the colored reaction is stopped by acid.
• The color reaction is directly proportionate to the amount of the unknown (Ag or Ab) present and is
measured bv a spectronhotometer.

Question 19

Advantages of ELISA

Answer 19

1. Simplicity:
   (a) Reagents added in small volumes. (b) Separation of bound and free reactants is
   made by simple washing procedures. (c) Passive adsorption of proteins to plastic is
   easy.
   (d) Specialized equipment readily available.
2. Reading: (a) Colored end-product can be read by naked eye to
   see test is working before measurement.
   (b) Multichannel spectrophotometers is used.

•3. Rapidity:
(a) Tests can be performed in a few hours.
(b) Spectrophotometric reading of results is rapid (96 wells read in 5 s).

•4. Sensitivity: • Detection levels of 0.01 to 1 ug/mL ideal for most diagnostic purposes. •5. Reagents Commercially available: with great flexibility and open to work in different Equipment.
•6. Cost:
a) Startup costs are low.
b) Reagent costs are low.

7. Acceptability: Fully standardized ELISAs in many fields are now
   accepted as “gold standard” assays.
8. Safety:
   Safe non-mutagenic reagents are available.
   Disposal of waste has no problem (unlike radioactive).
9. Availability: ELISAs can be performed anywhere, even in laboratories where facilities are less than state of the
   art.
10. Kits ELISA kits are widespread and
    successful.

•11. Standardization: • Quantification of data allows easier
standardization.

Question 20

Main drawbacks of ELISA

Answer 20

is lacking the absolute specificity, so, still there are some false-positive
results to be confirmed by molecular testing.

Question 21

Radioimmunoassay (RIA)

Answer 21

• RIA uses radioisotopes as an Ab or Ag conjugates, unlike the enzymes used in ELISA.

• The isotope iodine-125 (21- Gamma-emitting isotope) is commonly used as the conjugate because Ab or Ag can be readily iodinated without disrupting their immune specificity. C14 and H3
(Beta-emitting isotopes) can also be used.

• RIA was used clinically (Endocrinology) to measure serum proteins such as human growth hormone, testosterone and insulin present in humans in extremely small amounts.

Question 22

Immunofluorescence Assay (IFA)

Answer 22

• It is Ag-Ab reaction where the Ab is labeled with fluorescent dye.

• The fluorescent dye is either Fluorescin or
Rhodamine linked to isothiocyanate (ITC).

• The reaction is seen by the mean of a special fluorescent microscope.

•The IFA reaction can be direct to detect specific Ag by the mean of labeling specific Ab.

•Or indirect by using unlabeled Ab and fluorescently labeled Anti-Ab which is better technique to only label one Anti-Ab and not all specific Abs to plenty of Antigens needed to be detected.

•IFA has the advantages of detecting Ags in tissues.

The most common example for IFA is Treponema Pallidum Immunofluorescent Antibody test (TP-IFA) and infections caused by Chlamydia species.

Question 23

Advantages of IFT

Answer 23

• Fixed slides can persist for long time
• High sensitivity.

Question 24

Disadvantages of IFT

Answer 24

• Subjective errors in reading.
• Expensive equipment.

Question 25

Immunochromatography (ICT):

Answer 25

• Immunochromatographic assays, termed also (Rapid Test), (Point-of-care test, POCT) or (lateral-flow dipstick) immunoassays.

•Based upon Sandwich ELISA assay and run on chromatographic papers or Nitrocellulose membrane by the capillary action.
•The test strips or cartridge contains two type of fixed monoclonal antibodies one at the test (T) zone which is specific to the Ag or Ab of interest (to be tested for), and the other Ab at the control (C) zone which is anti-antibody to the human IgG for validation of reaction components of the test strip or cartridge.

The 3rd type of mAb is the colloidal gold-labeled (Conjugate) free moving and infiltrating in the strip or cartridge pad and specific to the Ag or Ab of interest (Tested for) and ready to react with the Ag or Ab of interest (if present in sample) and move across
the other side of the strip.
• If the test is positive the Ag or Ab will be trapped giving colored band at test (T) zone and the human (e.g.) IgG will react with the Anti-antibody in the control (C) areas giving colored band.
•On the other hand, if the sample is negative for the Ag or Ab of interest only colored band will appear at the C zone due to the presence of human immuno-globulins indicating the validity of the test.

Question 26

Applications of Rapid tests in clinical diagnosis

Answer 26

:
• 1- plenty and increasing range of usage of the rapid ICT tests including HIV, Hepatitis, malaria, influenza, Pregnancy, filaria, Leishmannia, Trichomoniasis, Chagas disease, Entamoeba histolytica/ dispar,
Cryptosporidium and Giardia, ..etc.

Question 27

Rapid tests Advantages:

Answer 27

• Easy to use immediate and fast results (10-20
minutes) so used in screening.
• Can be used at home or clinic.
• Inexpensive
•Some tests are FDA approved
• Done upon whole blood or saliva or urine. • No equipment, refrigeration, No electricity, No multiple timing steps required.
• Built in controls

Question 28

Disadvantages of rapid tests.

Answer 28

• Positive late after infection; after seroconversion.
• Need confirmation for reactive tests in some cases.
•Subjective variability in result reading.
•Qualitative and not quantitative.
• Less sensitive than ELISA.

Question 29

Immuno-blotting assays

Answer 29

• Western blotting (WB) is analysis and detection of proteins was first developed in 1979.

• The method is based on building an antibody: protein complex via specific binding of antibodies to immobilized proteins on a membrane and detecting the
bound antibody with one of several detection methods.

• WB is based on the principles of immunochromato- graphy where denaturated proteins are separated into poly acrylamide gel according to the isoelectric point and molecular weight by electrophoresis.

• Then these separated proteins are transferred (blotted) on a nitrocellulose or Polyvinylidene fluoride (PVDF) membrane.

Specific labeled antibodies are allowed to react to these proteins fixed on the nitrocellulose or PVDF membrane (Ag-Ab reaction).

• This Ag-Ab reaction then visualized according to the method of labeling of the antibody mainly
staining and photography.

• The photographed films can be kept for long time.

• Several procedures and reagents are needed for sample preparation, gel electrophoresis, transter,
antibody probing, detection, imaging and analysis.

• The WB technique (replaced by or /with PCR) are the current confirmatory diagnosis of HIV in our
laboratories

Question 30

What are the most common serological tests used in our hospital and reference laboratories?

Answer 30

1. Latex tests in the lab are the most common (Widal, Brucella, ASOT, CRP, bacterial and viral identification in
   the lab…etc.
2. Rapid tests are commonly used in clinics as POCT (HIV,
   Pregnancy, hepatitis, etc). 3. ELISA are commonly used for viral infections (HIV,
   HBV, HCV, HSV, ..etc) 4. Flocculation tests (RPR) are used for screening
   syphilis.
3. Syphilis is confirmed by TPHA or ELISA or FTA
4. WB is used as a confirmatory test for HIV.
5. Rarely to non used tests include: RIA, IFA, CFT,