Diagnosis laboratorium tifoid IPD dr Maimun.pdf

tifoid

1. Maimun Z Arthamin
Clinical Pathology Dept.
FKUB - RSSA

2. Introduction
•Typhoid fever is acute enteric infectious
disease caused by Salmonella enterica
subsp. enterica serotype Typhi
•The signs and symptoms: prolonged
fever, relative bradycardia, apathetic
facial expressions, roseola,
splenomegaly, hepatomegaly,
leukopenia.
•Complications: intestinal perforation,
intestinal hemorrhage

3. •The diagnosis on clinical grounds is difficult, as
the presenting symptoms are diverse and
similar to other common febrile illnesses.
•The isolation of serotype Typhi from blood
remains the method of choice for the
laboratory diagnosis .
•However, the availability of microbiological
culturing facilities is often limited in endemic
area, and blood cultures can be negative when
patients have received prior antibiotic therapy.
•Bone marrow culturing has a higher sensitivity
than blood culturing but is a more invasive
procedure

4. Fig 1. A schematic diagram of a single Salmonella typhi cell
showing the locations of the H (flagellar), 0 (somatic), and Vi (K
envelope) antigens.
Etiology

5. Susceptibility and immunity
•All people equally susceptible to infection
•Most cases in school-age children and
young adults.
•Acquired immunity can keep longer,
reinfection are rare
•Immunity is not associated with antibody
level of “H”, “O”and “VI”.
•No cross immunity between typhoid and
paratyphoid.

6. S. Typhi
stomach
Lower
ileum
peyer's patches &
mesenteric lymph nodes
thoracic
duct
1st bacteremia
(Incubation stage)
10-14d
(monon
uclear
phagocy
tes )
2nd bacteremia
Liver, spleen, gall,
BM, etc
early stage & acme stage
(1-3w）
LN Proliferate,swell
necrosis
defervescence stage
（3-4w）
Bac. In gall
Bac. In
feces
S.Typhi eliminated
convalvescence stage
(4-5w)
Enterorrha
gia,
intestinal
perforation
Pathogenesis
Fig. 2. Pathogenesis typhoid fever

7. Fig. 3. Immunopathogenesis typhoid fever

8. Fig. 4. The course of infection

9. Fig. 5. The serological profile

10. Diagnosis

• Epidemiology data

•Typical symptoms and signs

•Laboratory findings

11. Identification of the causative pathogen
Three Main Categories
•Identification of microorganism by
isolation and culture
•Identification of specific microbial
products (cell wall antigen toxins)
•Detection of specific antibodies to a
pathogen (IgM, IgG antibodies)
•Other laboratory test

12. Isolation of bacilli
Specimens: Blood
Urine
Faeces
Aspirated duodenal fluid,
etc..

13. Blood culture
Positive in,
90%-1
st
week
75%-2
nd
week
60% -3
rd
week
25%-till the subsidence of pyrexia

14. Fig. 6. Bacterial cuture and DST

15. The Serological diagnostic tests
•Serology should not be relied upon for diagnosis
•Serodiagnosis of Typhoid:
–Detection of Antibodies in serum---1. Widal
test, 2. Typhidot assay 3. Tubex system, 4.
Dipstick assay.
–Detection of Antigens in serum.--- a. Tubex
system b. Counter current
Immunoelectrophoresis. c. Co-agglutination
test. d. ELISA
–Detection of Antigens in urine: 1. Tubex system
2. Counter Immuno Electrophoresis, 3. Latex
agglutination 4. and Co-agglutination

16. The Widal Test
•The Widal test, which detects agglutinating
antibodies to lipopolysaccharide (LPS) (TO
test) and flagella (TH test)
•Interpretation of results is difficult.
•The sensitivity and specificity of the test is
moderate and may be negative in up to 30%
of culture proven cases.
•Result reported as ‘titres’: Highest dilution
where agglutination is seen
•Titres will depend on the stage of disease:
Agglutinins will appear by the end of 1st wk;
Rise till 3rd or 4th wk, later decline

17. Fig. 7. Widal: Slide and tube agglutination

18. •It is difficult to determine an appropriate
cut-off titre in areas with endemic typhoid
fever, a number of patients may have
raised titres from previous infection, and a
raised titre may not indicate acute typhoid
fever.
•Paired sera should be done. One taken
acutely and then repeated at day 7-14. A
four fold rise in titre is then diagnostic.
•A single positive serology specimen is not
diagnostic but may support the diagnosis
with consistent clinical features and other
laboratory parameters e.g.: leukopenia.

19. Widal: Test results
Causes of negative Widal
agglutination tests
•absence of infection by S typhi
•the carrier state
•an inadequate inoculum of
bacterial antigen in the host to
induce antibody production
•technical difficulty or errors in
the performance of the test
•previous antibiotic treatment
•variability in the preparation of
commercial antigens

Causes of positive Widal
agglutination tests
•typhoid fever
•previous immunisation with
Salmonella antigen.
•cross-reaction with non-
typhoidal Salmonella.
•variability and poorly
standardised commercial
antigen preparation
•infection with malaria or
other enterobacteriaceae
•other diseases such as
dengue

20. Enzyme-linked immunosorbent assays
•Enzyme-linked immunosorbent assays
(ELISAs) have been considered an
alternative approach for the diagnosis of
typhoid fever.
•For the most part, these assays have been
based on the detection of anti-LPS
antibodies and have been reported to be
more sensitive than the Widal TO test.
•More recently, ELISAs for the detection of
antiflagellum antibodies have been
developed

21. Fig. 8. ELISA: the principle methods

22. TUBEX TF (IDL Biotech, Sweden)
•It is a 5 minute semi quantitative calorimetric
test.
•It detects antibodies to the Salmonella enterica
Serotype Typhi lipopolysaccharide (LPS)O9
antigen.
•Tubex system is suited for diagnosis of infections
as it allows IgM to be detected early & rapidly
from serum.
•The antibodies are detected by their ability to
inhibit the interaction between two types of
reagent particles.
•Interpretation: Scores of 0-2 considered negative,
3-10 positive.

23. Fig. 9. The TUBEX test. (a) Cartoon
illustrating how TUBEX works for
detecting anti-O9 antibodies or
for detecting O9 antigen. (b)
Photograph of four sets of
reaction wells (six wells each)
containing various reaction
mixtures, (c) Detection of S. Typhi
LPS by TUBEX using the original
protocol for antibody detection,
or the new protocol for antigen
detection.
Reasonable sensitivities (75–90%)
and specificities (70-97%) have
been observed.

24. Dipstick assay (Netherlands)
•It consists of a strip of nitrocellulose membrane
containing a 2 mm wide line of immobilized
antigen as a detection band & a separate line
immobilized anti human IgM antibody as reagent
control.
•It is based on binding of S. Typhi specific IgM
antibodies in samples to S. Typhi LPS antigen and
the staining of bound antibodies by an antihuman
IgM antibody conjugated to colloidal dye particles.
•Cross reaction: sepsis by another species of
Salmonella that share antigen O9 determinant
with Salmonella enteritidis (Salmonella group D1)

25. Typhidot assay
•Typhidot assay: (Malaysian Biodiagnostics
Research, Selangor, Malaysia): It is a rapid ELISA
in the dot test format which detects IgM & IgG
antibodies against OMP of Salmonella typhi
antigen in human whole blood, serum or
plasma.
•The advantages: • early and specific diagnosis of
typhoid fever • fast, simple and reliable • simple
to perform and no additional sample
preparation required • no special equipment is
needed • results are easy to interpret • minimal
sample volume used
•Typhidot-M®, was recently developed to detect
specific IgM antibodies only.

26. •Typhidot has a sensitivity of 92.3%,specificity of
98.8%,PPV-85.7%& NPV- 99.4%.
•The test becomes positive within 2-3 days of
infection.
•Limitation of the test:
1.This product is designed for use with human
whole blood, serum and plasma only.
2.The test is a qualitative assay & is not for
quantitative determination of antibodies.
3.The results obtained should only be
interpreted in conjunction with other
diagnostic results and clinical information.
4.Due to the limitations of the test, for cases
where interpretation of result seems difficult,
repeating the test, TYPHIDOT 1 hour or
TYPHIDOT 3 hours is strongly recommended.

27. Electro chemical Immunoassay
Recently, copper, silver, and
gold-enhanced colloidal gold
have been reported for
immunoglobin G (IgG)
determination, which is the
model of electrochemical
immunoassay with low
detection limits ranged from
1.0ng/ml to 0.25 pg/ml.

28. Latex Agglutination
Agglutination test in which
inert particles (latex beads
or heat-killed S aureus
Cowan 1 strain with
protein A) are coated with
antibody to any of a
variety of antigens and
then used to detect the
antigen in specimens or in
isolated bacteria

29. Co agglutination
•Co agglutination is similar to the latex
agglutination technique for detecting antigen.
•Protein A, a uniformly distributed cell wall
component of Staphylococcus aureus, is able to
bind to the Fc region of most IgG isotype
antibodies leaving the Fab region free to
interact with antigens present in the applied
specimens.
•The visible agglutination of the S. aureus
particles indicates the antigen-antibody
reactions

30. •Many known carriers of typhoid bacilli
possess antibody against the Vi
(virulence) antigen of S. typhi.
•This is a surface antigen easily lost during
cultivation.
•Vi tires seem to correlate better with the
carrier state than do O or H titres. For this
reason, Felix et al. suggested the use of Vi
agglutination for detection of carriers.

31. Conclusion
•Diagnosis of typhoid fever should not rely upon serology
test, but also is based on epidemiology, clinical, and other
laboratory data.
•Rapid diagnostic test for typhoid fever should not be used
as a screening tests for all febrile illness. Request should
be limited to those with high clinical suspicion of typhoid
fever
•Widal: Despite the limitations the test may be useful in
areas that cannot afford the more expensive diagnostic
methods. This is acceptable so long as the results are
interpreted in accordance with local cut-off values for the
positivity.
•Tubex® & Typhidot: the test performed better than the
Widal test in both sensitivity and specificity