Introduction
Diagnostic tests help in identifying the presence of disease in a suspected patient. Some microorganisms take time to grow in culture, so faster methods like antigen–antibody reactions and molecular techniques are commonly used. Serological tests may be direct (detecting antigen) or indirect (detecting antibody). Other common diagnostic tests include skin tests, allergy tests and hypersensitivity tests.
Schick’s Test
Schick’s test is used to check a person’s susceptibility to diphtheria. A small amount of diphtheria toxin (0.1 ml) is injected into the skin of one forearm, and heat-inactivated toxoid is injected into the other arm as control.
- Positive test: Redness and swelling appear only at the toxin site – indicates lack of immunity.
- Negative test: No reaction on both arms – person is immune.
- Pseudo-positive: Early redness on both arms disappearing in 4 days.
- Combined reaction: Control arm inflames early but toxin arm reacts later – indicates hypersensitivity.
This test is rarely used today as children receive diphtheria vaccines.
ELISA (Enzyme Linked Immunosorbent Assay)
ELISA detects antigen or antibody in a sample using enzyme-linked antibodies. When substrate is added, a colour reaction appears and is measured using a microplate reader.
It is widely used for diagnosing HIV, hepatitis and in food testing.
1. Competitive ELISA
The microplate is coated with antibody. Labelled and unlabelled antigens compete for binding. More sample antigen gives a weaker colour reaction.
2. Direct ELISA
Antigen is coated on the plate. Enzyme-linked antibody binds directly, followed by substrate addition to give colour.
3. Sandwich ELISA
Antibody is coated first, antigen binds to it, and then another enzyme-linked antibody binds on the other side, forming a “sandwich”. Colour appears after substrate addition.
4. Indirect ELISA
Antigen is coated, then primary antibody binds. A secondary enzyme-linked antibody detects the primary antibody.
Molecular Diagnostic Techniques
Molecular tests detect specific DNA or RNA sequences of pathogens. They are important for genetic disorders and infectious diseases.
PCR (Polymerase Chain Reaction)
PCR amplifies DNA or RNA segments into millions of copies. The steps include:
- Denaturation: Heating at 90°C separates DNA strands.
- Annealing: Primers bind to the single strands.
- Extension: Taq polymerase builds new complementary strands at 72°C.
PCR is used to detect HIV, viruses, fungi, genetic mutations and in forensic investigations.
Blotting Techniques
- Southern Blot: Detects DNA fragments.
- Northern Blot: Detects RNA sequences.
- Western Blot: Detects proteins using primary and enzyme-linked secondary antibodies.
Widal Test
Widal test is a serological agglutination test used to diagnose typhoid fever caused by Salmonella typhi and S. paratyphi. The test detects antibodies against O and H antigens.
Principle
Patient’s serum is mixed with killed bacterial antigen suspensions (O, H, AH, BH). Visible clumping indicates presence of specific antibodies.
Rapid Slide Method
- Place serum drops on labelled circles.
- Add O, H, AH, BH antigens.
- Mix and look for agglutination within 1 minute.
Quantitative Tests
The highest dilution showing visible agglutination is recorded as the antibody titre.
An antibody titre above 1:80 is generally considered significant, but rising titres are more reliable.
Limitations
- False positives may occur due to prior vaccination or infection.
- Cross-reactivity with other Salmonella species.
- False negatives in early antibiotic use or immunodeficiency.
- Results vary between regions and labs.
QBC (Quantitative Buffy Coat) Test for Malaria
QBC is a fluorescent microscopy method for detecting malaria parasites.
- Blood is taken in an acridine-orange coated capillary tube.
- A plastic float helps separate blood layers after centrifugation.
- Malarial parasites fluoresce under UV light.
- Different stages appear in different blood layers (e.g., rings near RBC layer, gametocytes near platelet interface).
Mantoux Test (Delayed Hypersensitivity Test)
The Mantoux test screens for tuberculosis. Purified protein derivative (PPD) is injected intradermally.
- Induration 5–15 mm = positive.
- No induration = negative.
- False positives occur after BCG vaccination or infection with non-TB mycobacteria.
- False negatives in immunodeficiency.
Life Cycle of Malarial Parasite (Short Overview)
Malaria is transmitted by the female Anopheles mosquito. The parasite Plasmodium has two hosts:
- Mosquito (Primary host): Sporogony cycle producing sporozoites.
- Human (Secondary host): Schizogony cycle occurring in liver and RBCs.
After a mosquito bite, sporozoites enter the liver, multiply, and then infect RBCs. RBCs rupture, releasing merozoites that cause fever and chills. Some parasites form gametocytes, which continue the cycle when taken up by another mosquito.
Symptoms of Malaria
- Shivering, chills and fever spikes
- Weakness and sweating
- Recurring fever every 2–3 days depending on species
Detailed Notes:
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