Enterobacteriaceae II Salmonella
Question 1. Describe laboratory diagnosis of typhoid fever.
Or
Write a note on laboratory diagnosis of enteric fever.
Answer:
Laboratory diagnosis of typhoid fever is done as:
- Identification of bacteria
- Demonstration of antibodies
- Demonstration of antigens
Identification of bacteria:
- Microscopy: In samples such as pus, urine, and CSF, prepare the smear and stain it with Gram’s stain to demonstrate the gram-negative bacilli.
- Culture: Blood and bone marrow: After completion of the overnight incubation of bile broth, it should be subcultured over blood agar and MacConkey agar and selective media.
Stool: Add stool sample to an enrichment media, i.e. selenite F and tetrathionate F broth to increase the rate of isolation. Inoculate part of the sample on blood agar, MacConkey’s agar, and Wilson and Blair’s agar. - Urine: Inoculate the sediment into enrichment media and plating media.
- Bile, sputum, pus, or CSF: Samples of bile, sputum, pus, or CSF should be inoculated directly over the plating media.
Diagnosis Of Enteric Fever Colony Characteristics
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Incubation should be done at 37 °C:
- Blood agar demonstrates translucent colonies which are large, 2 to 3 mm in diameter, circular, convex and is grayish-white in color.
- MacConkey agar demonstrates colorless, non-lactose fermenting colonies.
- The colony present on the Wilson and Blair medium helps in the identification of species based on their color.
- If growth is not seen on the first subculture of blood or bone marrow, then repeat subculturing every day for 10 days and then declare it as negative.
- Biochemical reactions:
- All the species lead to the fermentation of glucose and mannitol with acid and gas, while S. typhi forms acid but no gas.
- All species are citrate and MR positive, while S. typhi is citrate negative.
- S. typhi is urease-negative.
- Serology:
- From nutrient agar, a loopful growth should be added to a few drops of normal saline on the slide to prepare the emulsion.
- This emulsion acts as a control to show that strain is autoagglutinable.
- If there is suspicion of S. typhi, a loopful of typhoid antisera factor 9 should be added and observed for agglutination after rotating the slide manually.
- Prompt agglutination is indicative of an isolated that belongs to Salmonella group D. Its identification as S. typhi is done by agglutination with flagella antisera.
- Fresh isolates of S. Typhi are in the V form so these strains do not agglutinate along with O antisera.
- These strains are either tested with Vi antisera or growth emulsifier in normal saline and are boiled for 20 minutes.
- These are tested for agglutination with O antisera.
Diagnosis Of Enteric Fever Demonstration of Antibodies Demonstration of antibodies in the serum of the patient should be done by:
- Widal test
- Indirect hemagglutination test
- Counter immunoelectrophoresis
- ELISA
- Radioimmunoassay
Diagnosis Of Enteric Fever WIDAL test This test is the most widely used.
Diagnosis Of Enteric Fever Principle: This is an agglutination test that detects antibodies that are produced against S. typhi, S. paratyphi A, and B.
Diagnosis Of Enteric Fever Antigens used:
- In this test, four antigens are used:
- H and O of S. Typhi
- H of S. paratyphi A
- H of S. paratyphi B
- Usually, antigens of paratyphoid O are not used in this test, as they cross-react with typhoid O antigens by sharing factor 12.
Diagnosis Of Enteric Fever Procedure:
- The widal rack has four rows of test tubes.
- Add an equal volume of serial dilution of serum to all test tubes.
- Add antigen to the respective rows.
- Incubate the rack in a water bath and set it at 37°C overnight.
- Control tubes consist of normal saline and antigens are used to check auto-agglutination.
Diagnosis Of Enteric Fever Interpretation:
- H agglutination: Appear as loose cotton woolly clumps.
- O agglutination: It appears as a granular disc-like pattern at the bottom of the test tube.
- Significant liters depend on titers found in local healthy persons.
- Titers of 1:200 for H and 1:100 for O are considered to be significant.
- The following points shall be considered while interpreting the results of the Widal test:
- A single positive test result cannot diagnose enteric fever, and a negative test result should not rule out the presence of enteric fever.
- Demonstrating a rising titer in the test made in the first and third week is of higher significance than a single test. If the collection of the first sample is late in the disease, the rise is not demonstrated.
- Antibodies appear during the 7th to 10th day of enteric fever so that negative test results may be obtained from samples that are collected directly. Titer increases steadily till 3rd or 4th week of enteric fever.
- Serum taken from the individuals who are immunized by the TAB vaccine demonstrate high titer antibodies against all antigens but in patients having typhoid fever rise is only against one serotype.
- The persistence of H agglutinins is for many months after vaccination but O agglutinins disappear soon. The rise in O is indicative of recent infection.
- For a demonstration of stereotype on an infecting organism, H agglutinin is more reliable as compared to O because serotypes have shared O antigen.
- Persons having past history of typhoid fever or immunization can develop an anamnesis response during unrelated fever. This difference is demonstrated by repeating the test after a week.
- Anamnesis response demonstrates transient rise, while in typhoid fever rise can sustain.
- Poor agglutinin response is shown by the patient treated with chloramphenicol.
- In non-specific conditions such as rheumatoid arthritis, multiple myeloma, etc. due to fibrial antigen false positive results can be produced.
Diagnosis Of Enteric Fever Demonstration of Antigens
- During the early phase of the disease, antigens are present in the serum as well as the urine of a patient.
- Tests used to detect antigens are agglutination and ELISA.
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