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    Lab Diagnosis of Enteric Fever

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    saket
    This document summarizes the laboratory diagnosis of enteric fever, which is caused by Salmonella Typhi and Paratyphi bacteria. For patients presenting in the first week of fever, blood culture is the primary diagnostic test to detect bacteria in the bloodstream. For patients presenting after the first week, the Widal test is used to detect antibodies against Salmonella antigens, as bacteria are no longer present in high levels in the blood. Faecal and urine cultures can also be used for diagnosis after the first week as bacteria may be present in these samples even after antibiotics are administered. The document outlines the procedures for blood culture, Widal test, and biochemical and serological identification of Salmonella isolates.

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    Lab Diagnosis of Enteric Fever

    Uploaded by

    saket
    112 views7 pages
    This document summarizes the laboratory diagnosis of enteric fever, which is caused by Salmonella Typhi and Paratyphi bacteria. For patients presenting in the first week of fever, blood culture is the primary diagnostic test to detect bacteria in the bloodstream. For patients presenting after the first week, the Widal test is used to detect antibodies against Salmonella antigens, as bacteria are no longer present in high levels in the blood. Faecal and urine cultures can also be used for diagnosis after the first week as bacteria may be present in these samples even after antibiotics are administered. The document outlines the procedures for blood culture, Widal test, and biochemical and serological identification of Salmonella isolates.

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    Lab Diagnosis of Enteric Fever

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    10. Lab Diagnosis of Enteric Fever

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    This document summarizes the laboratory diagnosis of enteric fever, which is caused by Salmonella Typhi and Paratyphi bacteria. For patients presenting in the first week of fever, blood culture is the primary diagnostic test to detect bacteria in the bloodstream. For patients presenting after the first week, the Widal test is used to detect antibodies against Salmonella antigens, as bacteria are no longer present in high levels in the blood. Faecal and urine cultures can also be used for diagnosis after the first week as bacteria may be present in these samples even after antibiotics are administered. The document outlines the procedures for blood culture, Widal test, and biochemical and serological identification of Salmonella isolates.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    112 views7 pages

    Lab Diagnosis of Enteric Fever

    Uploaded by

    saket
    This document summarizes the laboratory diagnosis of enteric fever, which is caused by Salmonella Typhi and Paratyphi bacteria. For patients presenting in the first week of fever, blood culture is the primary diagnostic test to detect bacteria in the bloodstream. For patients presenting after the first week, the Widal test is used to detect antibodies against Salmonella antigens, as bacteria are no longer present in high levels in the blood. Faecal and urine cultures can also be used for diagnosis after the first week as bacteria may be present in these samples even after antibiotics are administered. The document outlines the procedures for blood culture, Widal test, and biochemical and serological identification of Salmonella isolates.

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    of 7

    LABORATORY DIAGNOSIS OF ENTERIC FEVER

    A. Patient presenting in the 1st wk of fever


    I

    BLOOD CULTURE

    a. Indications:
    For the detection of septicemic conditions. Apart from the clinical setting of
    Enteric fever in the 1st wk, such conditions are also encountered in:
    1. Brucellosis
    2. Infective endocarditis
    3. Puerperal sepsis
    4. Sepsis in a surgical wound
    5. Meningitis
    6. Osteomyelitis
    7. Pneumonia
    NOTE: In conditions 2 to 7, bacteria spills over into the bloodstream from the
    primary site of infection.
    As part of diagnostic work up in a case of pyrexia of unknown origin (PUO)
    Septicemia- The presence of bacteria in the blood of a patient with clinical signs &
    symptoms of infection. Signs & symptoms of septicemia may include fever or
    hypothermia, chills, hyperventilation & subsequent respiratory alkalosis,
    hypotension or shock, skin lesions, change in mental status & diarrhea.
    PUO- Any febrile illness lasting more than a few days, the cause of which is
    obscure due to the absence of obvious specific or localizing signs & symptoms.
    b. Principle of Blood Culture Examination
    In this technique, detection of bacteria in the bloodstream is undertaken by
    inoculating a sample of venous blood into an appropriate broth. To neutralize the
    antibacterial substances present in the blood ratio of 1:10 is maintained between
    volume of sample & medium. This broth is incubated at 37C & monitored at
    specific time intervals for the detection of turbidity. On the appearance of turbidity,
    subculture is done on an appropriate solid medium for the identification & further
    processing of the organism growing in the broth. In the absence of turbidity, the
    broth is incubated for 7-10 days before declaring culture results as negative.
    Page 1 of 7

    c. Procedure
    1) Care should be taken while sample collection, it should be taken before
    antibiotic therapy.
    2) Skin preparation Use 70% ethanol/ betadine soaked cotton pad to scrub skin
    vigorously for 60 secs.( Dont touch the sterilized part again, or contamination
    may give false positivity). Allow to dry.
    3) Preparation of Blood culture vials- Wipe tops of vials with single alcohol swab
    & allow to dry (Dont touch the sterilized part again, or contamination may give
    false positivity).
    4) Phlebotomy- Insert the needle into the prepared vein & collect 5-10ml blood (
    in adults) & 2-5 ml blood in children. Add blood into the blood culture bottle
    through the needle.
    5) Transport to laboratory at room temperature with minimum delay. NEVER
    REFRIGERATE.
    NOTE: The addition of SPS (Sodium Polyanethol Sulphonate) in culture media is
    currently being done by some manufacturers to counteract the
    bactericidal action of blood.
    To reduce the risk of contamination during repeated subcultures, Castanedas
    method of culture or biphasic medium is used.
    In this biphasic medium, broth as well as agar slant is there.
    After inoculation of blood, the bottle is incubated in the upright position. For
    subculture, the bottle is tilted so that the broth runs over the surface of the
    agar.
    It is reincubated in the upright position.
    If organisms are present, colonies will appear on the slant.
    Media & Inoculation
    Medium

    Amount of blood to be
    inoculated
    Adults- 10ml
    Children- 5 ml
    Neonates- 2ml

    Aerobic bacteria
    Brain Heart Infusion broth
    Bile broth
    Glucose broth
    Anaerobic bacteria
    Thioglycollate both
    Robertson Cooked Meat Broth (RCM)
    d. Observation
    Time of

    Broth observed

    If positive
    Page 2 of 7

    If negative

    Reading
    After
    Overnight
    incubation
    On 3rd day

    for
    Turbidity

    On 5th day

    Turbidity

    On 7th day

    Turbidity

    Turbidity

    Subculture on
    Blood agar &
    MacConkey agar
    Subculture on
    Blood agar &
    MacConkey agar
    Subculture on
    Blood agar &
    MacConkey agar
    Subculture on
    Blood agar &
    MacConkey agar

    Reincubate
    till 3rd day
    Reincubate
    till 5th day
    Reincubate
    till 7th day
    Declare as
    Culture
    negative
    sample

    Colony morphology
    Culture media
    1. Blood agar
    2. MacConkey agar

    Colony
    characterstics
    after
    Subculture at 37 C
    Grayish color & Non-Haemolytic
    Pale color

    e. Further tests for speciation


    1. Biochemical tests
    Tests

    Result

    1. Oxidase

    Negative

    2. Nitrate reduction
    test
    3. Catalase

    Positive

    4. Sugar
    fermentation test

    Glucose fermented : S.Typhi (with acid no gas),


    S.Paratyphi A (with acid & gas), S.Paratyphi B (with acid
    & gas).
    Lactose not fermented
    Mannitol fermented : S.Typhi (with acid no gas),
    S.Paratyphi A (with acid & gas), S.Paratyphi B (with acid
    & gas).
    Maltose fermented : S.Typhi (with acid no gas),
    S.Paratyphi A (with acid & gas), S.Paratyphi B (with acid
    & gas).
    Sucrose not fermented
    Xylose fermented S.Typhi (with acid no gas),
    S.Paratyphi A (not fermented), S.Paratyphi B (with acid &
    gas).

    Positive

    Page 3 of 7

    5. Motility
    6. Indole
    production
    7. Methyl red
    8. Voges-Proskauer
    9. Citrate
    Utilization test
    10. Urease
    11. Triple Sugar
    Iron agar

    12. Phenylalanine
    deaminase

    Motile
    Indole not produced
    Positive
    Negative
    Not utilized: S.Typhi & S.Paratyphi A
    Utilized: S.Paratyphi B
    Negative
    Alkaline slant / Acidic butt ( K/A, no gas, H2S ): S.Typhi
    Alkaline slant / Acidic butt ( K/A, gas, no H2S ):
    S.Paratyphi A
    Alkaline slant / Acidic butt ( K/A, gas, H2S ): S.Paratyphi
    B
    Not produced

    2. Slide agglutination test


    Choice of antisera to be guided by biochemical reactions given by the
    recovered isolate.
    Serotyping

    O antisera

    H antisera

    For S.Typhi
    For S.Paratyphi A
    For S.Paratyphi B

    D9
    A2
    B4

    D
    A
    B

    B. Patient presenting in the second or subsequent week of Enteric Fever:


    Note
    1. Following the first week of enteric fever, the sensitivity of blood culture gradually
    decreases to about 75%of cases in second week of fever, 60% in the third week and
    25% thereafter till the subsidence of pyrexia.
    2. From the second week of enteric fever, the sensitivity of Widal test starts rising
    owing to the appearance of specific antibodies in the peripheral circulation.
    3. Since Salmonella is excreted in the faeces throughout the course of the disease,
    faecal culture can also be done for diagnosis of enteric fever. Importance of faecal
    culture rises in the later stages of the disease and convalescence,since the bacteria
    is no larger present in the blood stream during that period. Faecal culture is
    valuable in patients on antibiotics as drug does not eliminate the bacilli from the

    Page 4 of 7

    gut as rapidly as it does from the blood. However faecal culture can also be positive
    in carriers.
    4. The role of urine culture in enteric fever is relatively less owing to irregular and
    infrequent excretion of bacteria in urine.
    WIDAL TEST
    Indication: For the diagnosis of enteric fever, beyond the first week of disease,
    through the detection of specific antibodies to enteric fever pathogens.
    Principle: It is a tube agglutination test employed for the serological diagnosis of
    enteric fever. Patient serum, in doubling dilutions, is reacted with 4 antigens of
    Salmonella taken in separate tubes, viz. (i) common O antigen shared between S.
    typhi/ S.paratyphi A/ S. Paratyphi B (ii) Salmonella Typhi H(TH) (iii) Salmonella
    Paratyphi AH (AH), (iv) Salmonella Paratyphi B H (BH)antigens. Presence of specific
    antibodies is demonstrated by the formation of antigen- antibody complexes, in the
    form of clumps, at the bottom of the tubes.
    Procedure:
    1. Test tubes are arranged in four rows, with 6 tubes in each row.
    2. Patient serum is diluted (in doubling dilution) with normal saline through a series
    of tubes (Felixs Tube, a short rounded bottom tube for O antigen and Dreyers
    Tube, a narrow tube with a conical bottom for H antigen) from left to right,
    ranging from 1:10 to 1:160.
    3. In each row, a control tube is set up that contains normal saline & antigen.
    4. Final dilution is done by adding a fixed amount of standard suspension of O
    antigen and H antigen in each tube.
    5. Tubes are incubated overnight at 37 C.
    6. Tubes are examined for agglutination comparing with the respective antigen
    control tubes.
    Serum dilutions:
    Felixs Tube for S.Typhi Oantigen

    1:10

    1:20

    1:40

    Control Tubes

    1:80
    Page 5 of 7

    1:160

    Normal Saline

    Dreyers Tube for S.TyphiH antigen

    1:10

    1:20

    1:40

    1:80

    1:160

    Normal Saline

    1:160

    Normal Saline

    1:160

    Normal Saline

    Dreyers Tube for S.Paratyphi A AH antigen

    1:10

    1:20

    1:40

    1:80

    Dreyers Tube for S.Paratyphi B BH antigen

    1:10

    1:20

    1:40

    1:80

    Final Dilution (After adding equal volume of respective antigen)

    1:10

    1:20

    1:40

    1:80

    1:160 Normal Saline + Antigen

    Observation:
    The control tubes are examined first, for the absence of agglutination.
    The agglutination of O antigen appears as a matt or carpet at the bottom.
    Agglutination of H antigens appears as large, loose, fluffy clumps.
    The highest dilution of serum that produces a visible agglutination is taken as endtitre. The titres against all four antigens are recorded.
    Interpretation:
    Generally antibody titers above 1:80 for O & 1: 160 for H antigens are considered
    clinically and significant. However these cut- off titres show geographical variation,
    depending on the endemicity of Enteric fever.
    Positive Widal test (antibody titers above cut-off level) is indicative of the presence
    of antibodies to Enteric fever pathogens.
    False negative Widal test is observed in early stage (<1 week) of Enteric fever or
    with the early administration of antibiotics.

    Page 6 of 7

    False positive Widal test may be observed as an anamnestic response in several


    febrile illnesses.
    Repeat performance of Widal test after a gap of 7- 10 days demonstrates a drop in
    antibody titer, in case of an anamnestic response. In case of Enteric fever, a repeat
    test shows rise in antibody titer.
    TAB vaccinated patients may show a high titre of antibodies to each of the

    antigens.

    Page 7 of 7

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