Typhoid fever

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• Faculty of Allied Medical Sciences
• Clinical Immunology Serology Practice
• (MLIS 201)

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Typhoid fever
Prof. Dr. Ezzat M Hassan Prof. of Immunology Med
Res Inst, Alex Univ E-mail elgreatlyem_at_hotmail.co
m
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Teaching Objectives

• To define Typhoid Fever
• To know the causes and symptoms of the disease
• To understand how the bacteria cause the disease
• To know different methods to diagnose Typhoid
  fever
• To define Widal test and its components
• To describe the procedure of tube Widal test
• To know how to interpret the results
• To describe the procedure of slide Widal test
• To know the causes of false ve and flase ve
  results
• To know the limitations of the test

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Countries endemic for typhoid?(U.S. CDC 2006)
Alex LaPointe, Wikimedia Commons
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Definition

• An infectious feverish disease caused by the
  bacterium Salmonella typhi and less commonly by
  Salmonella paratyphi.
• The infection always comes from another human,
  either an ill person or a healthy carrier of the
  bacterium.
• Persons with typhoid fever carry the bacteria in
  their bloodstream and intestinal tract
• Transmitted through the ingestion of food or
  drink contaminated by the feces or urine of
  infected people
• The bacterium can withstand both drying and
  refrigeration.

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Causes

• 1. Caused by the bacterium Salmonella
  Typhi .
• 2. Ingestion of contaminated food or
  water.
• 3. Contact with an acute case of typhoid
  fever.
• 4. Contact with a chronic asymptomatic
  carrier.

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Salmonella typhi

• Rod shaped, flagellated, aerobic, Gram -ve
  bacilli.
• Refrigeration and freezing could slow their
  growth.
• Pasteurizing and food irradiation kill Salmonella
  for commercially-produced foodstuffs
• Foods prepared in the home from raw eggs can
  spread salmonella if not properly cooked before
  consumption.

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How does the bacteria cause disease ?

• Ingestion of contaminated food or water
• Salmonella typhi
• Carried by white blood cells into the liver,
  spleen, and bone marrow
• Multiply and reenter the bloodstream (Clinical
  illness)
• Bacteria invade the gallbladder, biliary system,
  and the lymphatic tissue of the bowel and
  multiply in high numbers
• Then pass into the intestinal tract (can be
  identified for diagnosis in cultures from the
  stool)
• Typhoid ulcers can cause perforation and
  hemorrhage

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How does the bacteria cause disease ?
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Symptoms

• No symptoms - if only a mild exposure some
  people
• become "carriers" of typhoid.
• Poor appetite, Headaches and generalized pains,
• Fever, Lethargy
• Rose spots on chest wall
• Diarrhea / constipation and abdominal pain
• Chest congestion develops in many patients,
• slow heartbeat.
• Enlarged spleen and liver

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Symptoms

• Rose spots

Aches and pains
High fever
Diarrhea
Chest congestion
Typhoid Meningitis
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Diagnosis

• Diagnosis of typhoid fever is made by
• Clinical examination
• Blood, bone marrow, or stool cultures for S.
  typhi
• Serological Tests

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Serodiagnosis of Typhoid

• 1.Detection of Antibodies in serum
• 1.Widal test (Tube or Slide),
  2.Typhidot assay
• 3.Tubex system,
  4. Dipstick assay.
• 2. Detection of Antigens in serum
• 1. Tubex system 2. Countercurrent
  Immunoelectrophoresis (CIE).
• 3. Co-agglutination test. 4. ELISA
• 3. Detection of Antigens in urine
• 1.Tubex system 2. CIE,
• 3. Latex agglutination 4.
  Co-agglutination

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Widal test
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• Antigenic structure of Salmonella

H( flagella ) antigens O (somatic) antigens Vi
(Virulence) capsular polysaccharide antigens
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O (somatic) antigens H (flagella) antigens

• LPS in the cell wall
• Heat stable
• Less immunogenic
• Agglutination with antisera
• Fine, compact, granular chalky clumps

• Present in flagella
• Heat labile
• Strongly immunogenic
• Induce rapid High Ab titres
• Agglutination with antisera
• Large, loose, cotton wool clumps

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Vi (virulence) antigen

• Capsular polysaccharide expressed on certain
  serotypes
• Heat labile
• Poorly immunogenic, BUT antibodies are
  protective
• Detection of Vi antibody not helpful in diagnosis
• Absence in a case of typhoid ? poor prognosis
• Persistence of Vi antibody carrier state

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WIDAL Test

• Tube agglutination test.
• Detects anti O and H antibodies in serum
• Diagnosis of Typhoid and Paratyphoid cases
• Carriers of typhoid bacilli possess antibody
  against the Vi antigen of S. typhi. (Vi tires
  seem to correlate better with the carrier state
  than do O or H titres).
• For this reason, the use of Vi agglutination for
  detection of carriers was suggested .

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Widal test

• Significance
• I st week negative.
• Titers raise in 2nd week
• Raise of titers is diagnostic

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Materials

• Antigens
• Suspension of S. typhi "O" antigen, O
• Suspension of S. typhi "H" antigen H
• Suspension of S. paratyphi A "H" antigen, PA
• Suspension of S. schottmuelleri "H" antigen, PB
• Antibody serum of suspected patient
• Normal saline
• Test tubes and pipettes

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PROCEDURE
1 2 3 4 5 6 7
O
H
PA
PB

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PROCEDURE

• Make the mark of tubes
• Dilute patients serum 110 (0.1 ml serum 0.9
  ml saline).
• Add reagents as the following
• Reagent (ml) 1 2
  3 4 5 6 7

Final serum dil. 140 180 1160
1320 1640 11280 -ve
Bacteria suspension O H PA PB
Shake several times, put it in 370C water bath
for 16-18 hours. Then let it stand at room
temperature over night.
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Observation

• Do not shake tubes before reading the results
• 1. Control tube (Tube No. 7) no
  agglutination (-)
• 2. Lowest titer tube absolutely
  agglutination ()
• 3. Other tubes
• ¾ agglutination() ½ agglutination
  ()
• ¼ agglutination () no
  agglutination (-)
• Interpretation
• Agglutination titer the highest dilution
  of serum
• which appears () bacteria agglutination.

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Agglutination how it appears after reactivity

• O

Dreyers tube Conical bottom H agglutination
Felix tube Round bottom O agglutination
Compact granular agglutination
Loose Cotton woolly clumps

• Observed for agglutination
• H Loose , cotton woolly clumps
• O Compact Fine granular agglutination
• Supernatant should be clear

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 How do you read Widal test results for typhoid
fever?

• The highest dilution of the patients serum in
• which agglutinations occurs is noted, ex. if
  the
• dilution is 1 in 160 then the titer is 160.
• Agglutination in dilution up to lt160 is seen
• in normal individuals . Agglutination in
  dilution
• 1160 is suggestive of Salmonella infection.
• Agglutination in dilution of gt1320 is
• confirmatory of Enteric fever .

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Interpretation of results
Olt180, Hlt1160, PHlt180 Normal value
O 180 H 1160 or O 180 PH 180 Typhoid fever Paratyphoid fever
O 180 H lt1160 or O 180 PH lt180 Early infection or other salmonella infections
O lt180 H 1160 or O lt180 PH 180 Vaccination or nonspecific memory reaction
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Conclusion
1 2 3 4
5 6 7 O
- - -
H
- - - PA
- - - -
- PB - -
- - - - -
140 180 1160 1320
1640 1280
Negative control

• N.B.
• Single test not diagnostic (Except at high titers
  gt 320).
• Paired samples tests with rising titer is
  diagnostic
• Diagnostic.
• O gt 1 80
• H gt 1 160
• H agglutinins appear first

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Prozone phenomenon in Agglutination tests

• Prozone effect - Occasionally, it is observed
  that when the concentration of antibody is high
  (i.e. lower dilutions), there is no agglutination
  and then, as the sample is diluted, agglutination
  occurs.
• Lack of agglutination in the prozone is due to
  antibody excess resulting in very small complexes
  that do not clump to form visible agglutination

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Limitation of Widal Test

• The Widal test is time consuming and often times
  when diagnosis is reached it is too late to start
  an antibiotic regimen.
• In spite of several limitation many Physicians
  depend on Widal Test

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Interpretation of Widal test

• Test results need to be interpreted carefully in
  the light of
• Past history of enteric fever,
• Typhoid vaccination,
• general level of antibodies in the healthy
  populations in endemic areas of the world.

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False Positive Reactions with WIDAL Test

• patients who have had previous vaccination or
  infection with S typhi.
• Cross-reaction with non typhoidal Salmonella.
• in association with some autoimmune diseases.
• Infection with malaria

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False Negative Reactions with WIDAL Test

1. Early treatment,
2. Relapses of typhoid fever.
3. Occasionally the infecting strains are poorly
   immunogenic.

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Typical Serological Profile After Acute Infection

• Note that during Reinfections, IgM may be absent
  or present at a low level transiently

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Slide Widal test

• Slide Widal test is more popular as it gives
  rapid results.
• Qualitative test
• 1 drop of undiluted
  patients serum sample for
• the 2 antigens is placed
  on the circled card.
• 1 drop of each of 2 salmonella antigens
  are added separately
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  rotated gently for 1 min.
• Appearance of agglutination gives
  qualitative results.
• (semiquantitative test is repeated
  with dilutions of serum)

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Slide Widal test (Cont.)

• Semi-quantitative test
• 80µl, 40µl, 20µl, 10µl, 5µl, of patients
  serum each for 2 salmonella antigens are placed
  on the circled card.
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• one drop of specific antigen is added to each
  series of serum.
• Agglutination of each of these is
  noted.
• Interpretation
• 80µl corresponds to 1 in 20 dilution.
• 40µl corresponds to 1 in 40 dilution.
• 20µl corresponds to 1 in 80 dilution.
• 10µl corresponds to 1 in 160 dilution.
• 5µl corresponds to 1 in 320 dilution.

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Prevention And Treatment
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Prevention

• Two main typhoid fever prevention
  strategies 
• 1. Vaccination
• . 
• 2. Avoid risky food and drinks

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• Medication
• Antibiotics
• Antibiotics, such as ampicillin, chloramphenicol,
  fluoroquinolone
• trimethoprim-sulfamethoxazole, Amoxicillin
  and ciprofloxacin etc used to treat typhoid
  fever.
• Prompt treatment of the disease with antibiotics
  reduces the case-fatality rate to approximately
  1.

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Study Questions

• Write short note
• Symptoms Prevention of typhoid fever

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Assigment

• Write shortly on Widal test
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• THANK YOU