Abstract

Typhoid fever is a medical condition that needed to taken seriously since it can

lead to morbidity and mortality. This infection is caused by a microorganism called

Salmonella Typhi. The transmission of this disease occurs through ingestion of food or

water that contains the pathogenic microorganism. The Salmonella then affects the lower

part of the ileum and invade the mesenteric lymph nodes. Then they would enter the

blood and spread to various target organs such as the bone marrow, spleen, kidney, liver

and gallbladder. The S. Typhi is able to avoid the immune system of the human due to

their stealth characteristic. This trait allows them to replicate and colonize and after 8 to

14 days, these bacteria are then shed back into the bloodstream and cause the onset of

clinical illness. In diagnosing the typhoid fever, laboratory test like the Widal

agglutination test, is an essential component. The test can be done in two ways either

through slide agglutination or tube agglutination. Although many new techniques have

been discovered and a lot of debate has questions the ability of this test, but with proper

knowledge and skill the Widal test can be a reliable test in detecting Salmonella infection

until today. The Widal test is also cheap and easy to perform.

Key Words: Typhoid fever, Widal test, diagnosis

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Chapter I

Introduction

Typhoid fever is a systemic bacterial infection that may affect all ages and is

essentially a human infection. It is cause by Salmonella Typhi, (S. Typhi) the most serious

etiology of enteric fever. The pathogen can survive for days in groundwater, pond water,

or seawater, and for months in contaminated eggs and frozen oysters. The infection is

transmitted by ingestion of food or water contaminated with feces. Known risk factors

include contaminated water supply, raw fruit and vegetables grown in fields fertilized

with sewage, history of contact with other patients before illness, poor personal hygiene

and housing and also past history of infection with Helicobacter pylori. Most S. Typhi

have a polysaccharide capsule (Vi), which is associated with increased infectiousness and

virulence, but Vi-negative strains may also cause the disease.1,10

The Salmonella Typhi affects mainly the lower part of the ileum where it enters

the lymphoid follicles. The bacteria adhere to the intestinal mucosa in the terminal ileum

through interaction with an epithelial receptor, the cystic fibrosis trans-membrane

conductance regulator protein. These affected parts undergo necrosis and ulceration while

the mesenteric lymph nodes become infected. The Salmonella then invades the

bloodstream via the thoracic duct. Bacteremia begins during the first week and may

continue up to the fourth week. During this phase, the bone marrow, spleen, kidney, liver,

and gallbladder may become infected, where the bacteria survive and replicate in cells of

monocytic lineage. S. Typhi is able to avoid an early inflammatory response in the gut of

the human host because it has a stealth approach that allows colonization of deeper

tissues of the body. After an incubation period of 8-14 days, these bacteria are shed back

into the bloodstream, marking the onset of the clinical illness. The gallbladder may re-

infect the intestines causing further acute inflammation of the lymphoid follicles.1,10

Typhoid fever is estimated to have caused 21.6 million illnesses and 216 500

deaths globally in 2000.4 The incidence of typhoid was high (>100 cases per 100 000

population per year) in south-central Asia, southeast Asia, and possibly southern Africa,

medium (10-100 cases per 100 000) in the rest of Asia, Africa, Latin America, and

Oceania, except for Australia and New Zealand, and low in the other parts of the world

(<10 cases per 100 000).1,2 There are a lot of laboratory procedures that can be used to

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diagnose typhoid fever, but in this paper I am going to focus on the Widal agglutination

test. The Widal test identifies the agglutinating antibodies against the O (somatic) and H

(flagellar) S. Typhi antigens, which will appear a week to 10 days after disease onset.1,3

The sensitivity, specificity, and predictive values reported from different centre vary

because of sharing of O and H antigens and cross-reacting epitopes with cither

Enterobacteriaceae. The high number of false-positive and false-negative Widal test

results limit its clinical usefulness.1,4 To make a diagnosis, results from a single acute

sample should be interpreted against the appropriate local cut-off values or there should

be a four-fold rise in the antibody titers between convalescent and acute sera.

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Chapter II

Discussion

Widal test is an agglutination test used in the serological diagnosis of typhoid

fever. The test is named after its founder, Georges Fernand Isidore Widal, a French

physician and bacteriologist.5 Basically, Widal test is a demonstration of agglutinating

antibodies against antigens O-somatic and H-flagellar in the blood. Therefore this test is

performed with standardized Salmonella antigen; S. Typhi O (TO) and S. Typhi H (TH)

antigens and Salmonella enterica subsp. enterica serovar Paratyphi A (S. Paratyphi A) O

and H antigens.3,5,6 The Widal test can be performed through two methods, using the slide

agglutination or tube agglutination.

Slide agglutination Widal test

A slide Widal test is more popular as it gives rapid results.5,6 One drop each of

undiluted patients’ serum samples for the four antigens are placed on the circled card and

one drop of each of the four Salmonella antigens are added separately and gently rotated

for one minute. Appearance of agglutination gives qualitative results. Positive (+) result

is obtain when there are antigens reacting while negative (-) result is when there are no

reaction of the antigens. To know the titer for each of the antigens, the test is repeated

with dilutions of the serum. 80 μl, 40 μl, 20 μl, 10 μl and 5 μl of patient’s serum each for

the four antigens are placed on the slide or circled card. To each series of serum

specimen, one drop of specific antigen is added to each. Both were gently mixed and

rotated for one minute. Agglutination in each of these is noted. Reacting antigens were

classified as positive (+) while non-reactive antigens were classified as negative (-).

Reactive titers of 1:80 and above were classified as positive (+), while titers less than

1:80 were classified as negative (-).5,7 But this titers cut off points varies among countries

especially in countries where typhoid is an endemic such as 1:160 in Philippines and

Turkey or 1:80 in Nigeria.4,7

4

Tube agglutination Widal test

Fresh blood samples that had been taken from patient are then centrifuged to

obtain the sera for the screening test. The next step is make a series of serum dilutions for

each antigen to be tested including tubes with 0.5 ml saline for control of each antigen to

be used. Prepare the dilutions beginning with 1:10 and doubling through 1:640 or so in

three to four rows. Make sure to use perfectly clean and dry test tubes. Add 0.1 ml of

serum to 0.9 ml of physiological saline and then dilute serially by mixing 0.5 ml diluted

serum with 0.5 ml saline and discarding 0.5 ml from the last tube. All the tubes in a row

including control are mixed with 0.5 ml of antigen suspension. The first row is tested

with S. Typhi O antigen, the second row with S. Typhi H antigen, the third row with S.

Paratyphi AH antigen and the fourth row with S. Paratyphi BH antigen. But since

infections by S. Paratyphi B are rare, this antigen is usually omitted in the test. After all

the tubes have been mixed with antigens, they are incubated at 37°C overnight and

checked visually for agglutination. The titer was determined as the highest dilution that

showed visible agglutination. The titers for all the antigens are noted. Also, they should

be no agglutination in the control tubes.2,3,4,5,8

Figure 1. Tube agglutination Widal test (Taken from www.microrao.com)

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The Widal test had gone a long way since its discovery. It is also very easy to

perform, which makes it practical for use in the field.4 The Widal test in the present time,

it is still considered one of the important test in diagnosing salmonella infection

especially the typhoid fever although there are a lot of controversy regarding its low

sensitivity, specificity, and positive and negative predictive values, which changes with

geographical area. There are many false negative and false positive results produced by

the Widal test.1,4,6

Factors that should be taken into consideration to avoid false negativity include;

1. Timing of the test. Samples that have been obtained too early in the first week

may be negative while obtaining samples too late like in the after the fourth week

may also causes confusion as the antibodies would starting to decline.4 Therefore,

the timing of test is very important. The titers increases during second, third and

fourth week after which it gradually declines.5

2. Widal test is performed only once on patients suspected with infection of

Salmonella.4,8

3. The early administration of an antibiotic.2,4,8,9

4. Technical difficulty or errors in performing the test.4,5,9

Factors that should be taken into consideration to avoid false positivity include;

1. Patient had received vaccines against Salmonella. This can be differentiated from

true infection by repeating the test after a week.3

2. Patients from Salmonella endemic regions.3,8,9

In order to counter its low specificity and sensitivity, The Widal test is best

performed twice, on convalescent and acute-phase serum.1,8 A three to fourfold raise in

antibody titers can be detected. That way the test would produce a more reliable result

with higher specificity and sensitivity.1,2,8,10 It is also recommend that doctors always

consider and exclude more common causes of fever in the tropics before proceeding with

a Widal test in order to obtain a more accurate result.2,9

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In this era of modern technology, companies have discovered newer and easier

tool to diagnose typhoid fever. Examples such as the Tubex test, Typhidot tests and

Polymerase Chain Reaction tests have proven to shown high index of sensitivity and

specificity in detecting typhoid fever.6,10 As a result, the use of Widal test is losing its

relevance in Western and European nations but still it used in countries in the South East

Asia.5 This is due to the low cost of Widal test which is more affordable to the public

compared to the modern tests. But then it does not show that the Widal test is inferior to

the modern tests. According to a study by B. Dong et all, newer serological tests did not

provide substantial advantages over the established Widal test. Only the time advantages

in generating a result may be of an importance to clinicians.3,10

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Chapter III

Summary

Typhoid fever is a systemic bacterial infection that may affect all ages that is

caused by Salmonella Typhi which may be acquired by ingestion of food or water

contaminated by feces from typhoid patients or carriers. Typhoid is estimated to have

caused high cases of mortality and morbidity globally. There are a lot of laboratory

procedures that can be used to diagnose typhoid fever; one of it is Widal agglutination

test. The Widal test identifies the agglutinating antibodies against the O (somatic) and H

(flagellar) S. Typhi antigens, which will appear a week to 10 days after disease onset. The

sensitivity, specificity, and predictive values of this are often questions. The high number

of false-positive and false-negative test results that had been acquired limits its clinical

usefulness. From the articles and journal that I had reviewed, I come to a conclusion that

The Widal test can still be considered as an important tool in diagnosing typhoid fever.

Even though the Widal test is an old technique with a lot of controversies, I think with

proper knowledge on the test and disease, the Widal test will be able to provide us with

the correct value needed supported with other serological test.

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References

1. Bhan MK, Bahl R, Bhatnagar S. Typhoid and paratyphoid fever. The Lancet. 2005; 366.

2. Smith SI, Odunukwe NN, Niemogha MT, Ahmed AO. Diagnostic methods for typhoid fever in Nigeria. British Journal of Biomedical Sciences. 2004; 61.

3. Dong B, Galindo CM, Shin E, et al. Optimizing typhoid fever case definitions by combining serological tests in a large population study in Hechi City, China. Epidermiol. Infect. 2007; 135: 1014-1020.

4. Hosoglu S, Bosnak V, Akalin S, et al. Evaluation of false negativity of the Widal test among culture proven typhoid fever cases. Journal Infect Developing Countries. 2008; 2(6): 475-478.

5. Sridhar Rao PN. Widal test. www.microrao.com. 2009.

6. Devrim I, Ergunay K, Kara A, et al. The comparison of cultures, widal agglutination test and polymerase chain reaction as a diagnostic tool in typhoid fever. Cent. Eur. J. Med. 2008; 3(4): 470-474

7. Itah AY, Uweh EE. Bacteria isolated from blood, stool and urine of typhoid patients in a developing country. Southeast Asian Journal of Tropical Medicine and Public Health 2005; 36: 3.

8. Willke A, Ergonul O, Bayar B. Widal test in diagnosis of typhoid fever in Turkey. Clinical and Diagnosic Laboratory Immunology. 2002: 938-941

9. Adeleke SI, Nwokedi EE. Diagnostic value of Widal test in febrile children. African Scientist. 2008; 9: 5-8.

10. Ismail TF. Rapid diagnosis of typhoid fever. Indian Journal of Medical Research. 2006: 489-492

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