typhoidserology

7/29/2019 typhoidserology

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Typhoid fever, also known as typhoid, is a common worldwideillness, transmitted by the ingestion of food or water contaminated

with the feces of an infected person, which contain the bacterium

Salmonella enterica entericaserovar Typhi. The bacteria then perforate through the intestinal wall and are

phagocytized by macrophages.

The organism is a Gram negative short bacillus that is motile due

to its peritrichous flagella. The term "enteric fever" is a collective term that refers to typhoid

and paratyphoid


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The Genus Salmonella

belongs to Enterobacteriaceae-Facultative anaerobe-Gram negative bacilli-Distinguished from other bacteria by

Biochemical and antigen structure.

BacteriologyTyphoid

fever


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Antigenic structure of Salmonella

H( flagellar ) antigensO (somatic) antigensVi (Virulence) capsular

polysaccharide antigens


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LPS in the cell wall; Extracted from the cell wall by treatment with

tetrachloroacetic acid (Boivin : Boivin antigen);

Less immunogenic than H antigen;

Agglutination with antisera:Compact, chalky granular clumps

Serogroupingof Salmonellae is based on characteristicO antigen;


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Present in flagella;

Heat labile;

2 phases : phase 1 and phase 2;

Strongly immunogenic; Induce rapid antibody formation in high titres;

Agglutination with antisera:

Large, loose, fluffy clumps


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Surface polysaccharide expressed on certain serotypes;Heat labile;

Interferes with agglutination by O antisera;

Lost on serial subcultures; Poorly immunogenic, BUT antibodies are protective:

Detection of Vi antibody not helpful in diagnosis

but their absence in a case of typhoidpoorprognosis;

Persistance of Vi antibody : carrier state


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Serodiagnosis of Typhoid :

1.Detection of Antibodies in serum---1.Widal test ,2.Typhidot assay

3.Tubex system,4. Dipstick assay.

2. Detection of Antigens in serum.---

a.Tubexsystem

b.CountercurrentImmunoelectrophoresis.

c. Co-agglutination test.

d. ELISA

3. Detection of Antigens in urine: 1.Tubex system 2.Counter

Immuno Electrophoresis, 3. Latex agglutination 4. and Co-agglu-tination


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Georges-Fernand-Isidor Widal

Widal & Sicard in 1896

described the Widal reaction.

In 1896 Widal A professor of

pathology and internal

medicine at the University of

Paris (191129), he

developed a procedure fordiagnosing typhoid fever

based on the fact that

antibodies in the blood of

an infected individual causethe bacteria to bind together

into clumps (the Widal

reaction).


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Widal Test

Serum agglutinins raise abruptly during the 2ndor 3rd week.

The Widal test detects antibodies against O and H antigens.

Two serum specimens obtained at intervals of 710 days to readthe raise of antibodies.

Serial dilutions on unknown sera are tested against the antigens forrespective Salmonella.

False positives and False negative limits the utility of the test.

The interpretative criteria when single serum specimens are testedvary.

Cross reactions limits the specificity.


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Widal testA standard tube agglutination test

Test can be performed by the tube dilution technique

In this, a constant amount of the antigen is added to a series of tubes containingserum dilutions.

After mixing, the tubes are incubated at a temperature of37c in athermostatic water bath and the highest dilution of serum showing

visible agglutination is determined.


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OFelix tube

Round bottomO agglutination

Compactgranularagglutination

HDreyers tubeConical bottomH agglutination

LooseCotton woollyclumps


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Equal volumes of serial dilutions of serum (1/10 to 1/640)mixed with H and O antigens in respective tubes;

Incubated in water bath at 370C overnight;

Observed for agglutination H : Loose , cotton woolly clumps;

O : compact granular agglutination;

Supernatant should be clear;


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0.9ml of normalsaline

+ 0.1 ml of serum

0.5 ml of S.Typhi O antigen control

0.5 ml NS.

0.5ml 0.5ml discarded.

0.5 ml of S.Typhi H antigen

0.5ml 0.5mldiscarded.


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Result reported as titres : Highest dilution whereagglutination 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; Demonstration in the rise of titre is significant;

Following Titers of antibodies against the antigens are

significant when single sample is tested.

Significant titre: O > 1 in 100

H > 1 in 200

Testing apaired sample for raise of antibodies carries a

greater significance.


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The Widal test (Widals agglutination reaction) is routinely practiced

for the Serodiagnosis of typhoid fever by most of the laboratories.

Several workers have expressed doubt regarding the reliability ofthe test.

Several factors have contributed to this uncertainty. These include

1.Poorly standardized antigens,

2.Sharingofantigenicdeterminants with other Salmonellae

3.Effects of immunization with TAB vaccine.

Another major problem relates to

the difficulty of interpretingWidal test results in areas whereSalmonella.Typhi is endemic and where the antibody titres of thenormal population are often not known.


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Classically, a four-fold rise of antibody in paired

seraWidal test is considered diagnostic of typhoid

fever.

However, paired sera are often difficult to obtain

and specific chemotherapy has to be instituted on the basisof a single Widal test.

Furthermore, in areas where fever due to infectious

causes is a common occurrence. So false positive reactions

may occur as a result of non-typhoid


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The Widal test is time consuming and most often it is too

late to start an antibiotic regimen when diagnosis isreached.


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Previous immunization with Salmonella antigen. Cross-reaction with non- typhoidal Salmonella. Variability and poorly standardized commercial antigen

preparation.

Infection with malaria

Brucellosis

other Enterobacteriaceae sharing the same s-LPS .

dysgammaglobulinaemia of chronic active hepatitis. Autoimmune diseases.


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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.

with "hidden organisms" in bone and joints.


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Prozone effect - Occasionally, it is observed thatwhen the concentration of antibody is high (i.e. lowerdilutions), there is no agglutination and then, as thesample is diluted, agglutination occurs.


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Slide Widal test is more popular as it gives rapid results.

Qualitative test: 1 drop of each undiluted patients serum sample for

the 2 antigens is placed on the circled card.

1 drop of each of 2 salmonella antigens are addedseparately

rotated gently for 1 min.

Appearance of agglutination gives qualitative results.(test is repeated is repeated with dilutions of serum)


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Quantitative test: 80l, 40l, 20l, 10l, 5l, of patients serum each for 2 salmonella

antigens are placed on the circled card.

one drop of specific antigen is added to each series of serum.

Agglutination of each of these is noted.

80l corresponds to 1 in 20 dilution.

40l corresponds to 1 in 40 dilution.

20l corresponds to 1 in 80 dilution.10 l corresponds to 1in 160 dilution.

5l corresponds to 1in 320 dilution.


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This test is performed with a loopful growth from nutrient agar

emulsified in 2 drops of 0.85% normal saline.For salmonella isolated for the first time, the following sera are tested

in order I to VI:

I. Salmonella polyvalent o serum(groups A-G,02-13)on one half of

slide.Test suspension alone on other half as a control to excludesaline auto agglutination.

II.Salmonella polyvalent H phases 1&2 serum and polyvalent H

phase 2 are placed on separated halves of the slide.

If polyvalent O & H reactions are negative, NO further slide testsare done.

If polyvalent O& H reactions are positive for salmonella, further

Slide tests are done.


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III. Test suspension is checked with individual Salmonella O group

sera 02-013 separately.IV. If the culture is in phase 1that is if it reacts positively with mixed

phase1 &2 serum, negatively with phase 2 H serum and if it reacts

with any single O group serum, then it is to be tested with single

H serum( a, b, d, i etc )---------salmonella.Enteritidis&salmonella.Typhimurium are the common serotypes in manycountries.

If salmonella belongs to O-group B (02) then it is tested with H-i

serum.If salmonella belongs to O-group D(09) then it is tested with H-d

serum.


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V. If the culture is in phase2, it is passed through Craigies tube or

Jameson strip containing phase 2 H serum and phase 2 phase 1.

VI. Salmonella. Typhi fails to agglutinate with group D(09) because

the bacilli are coated with vi antigen. If vi antiserum is added ,then it

agglutinates.If vi positive, saline suspension of salmonella is heated for 30 min,

cooled & retested with O antisera.

VII. If the isolate is nontyphiodal salmonella, producing gas fromsugars, it is tested for agglutination with O& H antisera for groups

A,B,C.


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TUBEX TF (IDL Biotech): It is a 5 minute semi quantitative calorimetric test for Typhoid

fever.

It detects antibodies to the salmonella entericaSerotype Typhilipopolysaccharide (LPS)o9 antigen.

Tubex system is ideally suited for use in diagnosis of infections as it

allows IgM antibodies to be detected early & rapidly from wholeserum.

The antibodies are detected by their ability to inhibit the interaction

between two types of reagent particles

a)colored indicator latex microspheres sensitized with an anti o9monoclonal antibody.

b)magnetic microspheres coated with S.Typhi LPS.


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Following rapid mixing of the serum with these reagents andsedimentation of the magnetic particles by the magnetic force, theconcentration of the particles left in suspension provides the measureof inhibition.

Tubex test for antibody detection:Test serum (25l) was mixed in a chamber of the reaction container

provided(which contains a set of six identical Vshaped chambers)with 25l of Brown reagent (antigen coated magnetic particles) for

2min.

Then 50 l of blue reagent (Ag coated indicator particles) is added &mixed for another 2 min.

The reaction mixture was placed on the magnet stand, and the resultant

color was read immediately and scored against the color chart(score 0-10).Scores of 0-2 considered negative.Scores of 3-10 considered positive.


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Tubex test for antigen detection: 25l of test serum is mixed with 50l of blue reagent in the reaction

well for 2 min.

then 25l of brown reagent is added& mixed for another 2 min.

The results are read after sedimentation of magnetic particles.

In conclusion,

Tubex kit can potentially be used in several ways to diagnose typhoidfever including:

1)Antibody &Antigen detection in serum.

2)Antigen detection from urine to complement serum detection.

3)Detection or identification of whole organisms from primary culture

plates or from blood culture (stool) broth.


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Results of Tubex TF:

results are scored against a color chart with a scale of 0 to10.

Score 0, negative and most red.

Score 10, most positive and most blue.

Reasonable sensitivities (7590%) and

specificities (70-97%) have been observed.

Di i k


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Dipstick assay:

It consists of a strip of nitrocellulose membrane containing a 2mmwide line of immobilized antigen as a detection band & a separateline immobilized anti human IgM antibody as reagent control.

It is developed in Netherlands.

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.

Antigen preparation:

culture of recent isolate of S.Typhi culture was grown in a Luria

Bertani broth & antigen was prepared by heating a washed & 30xconcentrated bacterial suspension of a 3 day old well grown culture

for 30min at 95c.

Cell debris was removed by centrifugation.


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The supernant containing the antigen was blotted in 2mm wide lanes

on the nitrocellulose membrane by incubation for 2hrs at 40c.

At the end of incubation, the lanes of blotting apparatus were rinsedwith phosphate buffered saline (PBS) to remove excess antigen.

Blotted strips rinsed with PBS, blocked with 3%skimmed milk,

rinsed & dried.

Detection reagent: It consists of a monoclonal antihuman IgMantibody conjugated to colloidal suspension of palanyl red.


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Procedure of Dipstick assay:

It is performed by incubating a wetted dipstick in a mixture of 5l ofsample & 250l of detection reagent for 3 hrs at room temperature.

Dipsticks are thoroughly rinsed with water & dried.Staining intensity of the antigen band was then graded by comparison

with a colored reference strip.

When no staining is observed ----- Test is scored negative.

Weak staining -----------------1+Moderate staining ---------------- 2+,3+

Strong staining -----------------4+


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Dipstick assay is applied to a single serum sample gives quick

result (3hours).

Testing of paired sera could increase the sensitivity of the assay.

It is a simple test & does not require any specific equipment for its

performance as the assay uses stabilized compounds.

Test is sufficiently sensitive(69.8%) and specific.


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Typhidot assay :(MalaysianBiodiagnosticsResearch,Selangor,Malaysia) :

It is an Enzyme linked Immunosorbent Assay in the dot test format

which detects IgM & IgG antibodies against outer membraneprotein of salmonella.Typhi antigen in human whole blood, serum orplasma.

It is a rapid serological procedure for the diagnosis of Typhoid fever.

The advantages of Typhidot includes: 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

Pr d r f th t t


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Procedure of the test:

The test is based on the presence of specific IgM & Ig G antibodies

to a specific 50kDa OMP antigen which is impregnated onnitrocellulose strips.

Reaction tray is divided into 2 columns as G and M.

250 l of sample diluent was dispensed in each well and 2.5l of test/control was added & then incubated for 20min.

Strips were washed with wash buffer thrice.

250l of antihuman IgG & IgM were dispensed in each well &

incubated for15 min.

wash& 250l of substrate is added for color development

,incubated for 15 min. Results were interpreted.


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Diagrammatic Representation of Assay ProcedureSerum/ Plasma sampleAdd 30l serum to the sample well. Makesure that there are no air bubbles. Add 1

drop of buffer after 15 seconds. Sample willstart wicking up. Read result within 15minutes.Whole Blood sampleAdd 40l of whole blood into the samplewell. Make sure that there are no air

bubbles. Add 1 drop of buffer after 15seconds. Sample will start wicking up.Read result within 15 minutes.[Note: If sample front stops wicking up aftera while, add an additional drop of buffer]


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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, serumand plasma only.

2. The test is a qualitative assay & is not for quantitativedetermination of antibodies concentration levels. The intensity of

the band does not have linear correlation with the antibody titer ofthe specimen.

3. The results obtained should only be interpreted in conjunctionwith other diagnostic results and clinical information.

4. Due to the limitations of the test, for cases where interpretationof result seems difficult, repeating the test

TYPHIDOT 1 hour or TYPHIDOT 3 hours isstronglyrecommended.


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This test, derived from Salmonella Typhi O and H antigens, was performed

in twoways:(i) as a semi quantitative slide agglutination test with visual

examination as per the package insert;

(ii) as a Widal test performed with a single tube, as described by Parry

et al.The presence or absence ofvisible agglutination indicates the

presence or absence of the corresponding antibody to the O and Hantigens of SalmonellaTyphi.

They defined the positivity cut-off point for the slide and tubeagglutination reactions for both O and H antigens as antibody titres

1:80.

Publication: Bulletin of the World Health Organization; June 2011,Type: Research Article ID: BLT.11.087627 .


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Electro chemical Immunoassay:

Recently, copper, silver, and gold-enhanced colloidal gold havebeen reported for immunoglobin G (IgG)determination, which

is the model of electrochemical immunoassay with low detectionlimits ranged from 1.0ng/ml to 0.25 pg/ml .


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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 IgGisotype 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


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Agglutination test in which inert particles(latex beads or heat-killed

S aureusCowan 1 strain with protein A)are coated with antibody to any of a

variety of antigensand then used to detect the antigenin specimens or in isolated bacteria.

P i f h l i S h l C 1


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Preparation of staphylococci: Staphylococcus aureus Cowan 1was grown on tryptic soy agar at 37C for about 18h.

The cells were harvested, washed in phosphate buffer, exposed to

0.5% formaldehyde, and heated to 80C and held for 5 min by theprocedure of Edwards and Hildebrand.

Attaching o antisera to staphylococci:One milliliter of the 10%suspension of stabilized, killed staphylococci was added to 0.2 ml ofnon- glycerolated, specific antiserum or to 0.4 ml of glycerolatedantiserum, mixed thoroughly, and allowed to react at roomtemperature for 3 h, with occasional gentle shaking .

The suspension was centrifuged at 800 x g for 30 min and washedtwice with 0.02 M phosphate (pH 7.3)-buffered 0.85% saline (PBS).

The cells were then suspended in PBS to a final volume of 5 ml, and1 drop of 5% sodium azide was added . The antibody-conjugatedstaphylococci made up a COAG. The COAGs were stored at 4C.

T i S l ll i b l i i S l ll i


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Testing Salmonella strains by co agglutination: Salmonella strainswere grown overnight on blood agar base (BBL MicrobiologySystems).

To test for agglutination,

1 drop of COAG is placed on a slide. Growth was taken from a slantwith a bacteriological loop and was mixed into the COAG drop.

Strong homologous reactions usually occurr immediately. If there wasno reaction, the slide was rocked gently for 1 min and then readunder fluorescent light against a black background.

The amount of agglutination was read as 4+, 3+, 2+, or 1+. A tracewas recorded as +/-.


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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.


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