Enteric fever – Part 2 – Typhoid / Enteric fever Diagnosis, Widal test, Its Procedure and Interpretation

Sample

1. It is done on the serum of the patient for the widal test.
   1. A random sample can be taken.
2. For blood, culture collects the blood in sterile test tubes or the specialized culture tubes.
3. For urine culture, clean the area and then collect the mid-stream sample.
4. Stool culture samples may be collected in sterile containers.
5. Bone marrow, if needed, will be done with all precautions and in a sterile manner.

Purpose of the test (Indication)

• This test is done to diagnose enteric fever (Typhoid and paratyphoid fever).

Pathophysiology

1. Diseases caused by salmonella are divided into two groups:
   1. Typhoidal infection by the S.typhi and S.paratyphi.
      1. Humans are the only host of these pathogens, and spread is person-to-person or via contaminated foodstuffs and water.
   2. Non-typhoidal infection from all other organisms.
      1. The most common non-typhoidal organisms in the USA are S.typhimurium, S.enteriditis.
      2. These infections 95% are food born.
2. History of widal test:
   1. In 1896 widal test was discovered by George-Fernand Widal for the diagnosis of enteric fever.
3. Enteric fever includes typhoid and paratyphoid fever.
   1. Paratyphoid fever is milder than typhoid fever.
   2. Enteric fever is also called typhoid fever.
4. Typhoid fever is caused by the bacterium Salmonella Typhi.
   1. Salmonella typhi has an incubation period of 14 to 21 days. Sometimes may even longer.
   2. 30% of the cases become chronic carriers due to persistent infection of the gallbladder.
5. This has flagellar antigen H antigen, and Lipopolysaccharides antigen is somatic or O antigen.
   

   Typhoid bacilli structure

3. The incubation period is 5 to 21 days.
4. Typhoid fever is common in the developing world, where it affects about 21.5 million persons each year.
5. Salmonella Typhi lives in humans and is also found in chickens and eggs.
6. It is present in the blood and intestinal tract.
7. The carrier can harbor bacilli in the gallbladder.
8. Both patients and carriers shed Salmonella Typhi in their feces (stool).

Mode of spread

1. Man is the only natural host for the S. Typhi, transmitted in contaminated drinks and foods.
2. Food: In the case of contaminated foods and drinks.
3. Spread from the carrier, who is shedding Salmonella Typhi in their feces.
4. Contaminated drinking water: If sewage water is contaminated with Salmonella Typhi.
5. Typhoid fever is more common in areas of the world where handwashing is less frequent.
6. Once Salmonella Typhi bacteria are eaten or drunk, they multiply and spread into the bloodstream.
7. After the bacteria’s ingestion, invade the small intestine and its Peyer’s patches and spread to regional lymph nodes.
   1. From it goes into the blood circulation and gives rise to bacteremia.

Microbiology of salmonella:

Salmonella Typhi

1. Salmonella infection facts are:
   1. 10⁵ to 10⁹ organisms are needed to cause infection.
   2. The above number is lower in:
      1. Infants.
      2. Patients with pernicious anemia.
      3. Patients taking antacids or H2 receptor blockers.
   3. The other factors (more prone) for infections are:
      1. Old age.
      2. Patients treated with antimicrobial therapy.
      3. HIV infection.
      4. Therapeutic immunosuppressive treatment.
      5. Patients with malarial infection.
      6. Sickle cell anemia.
      7. Patient with splenectomy.
      8. Patients with diabetes mellitus.
      9. Patients with malignant tumors.
      10. Autoimmune diseases like SLE
2. This can grow well on ordinary media.
3. From the feces, MacConkey agar and Desoxycholate agar culture will show pale, non-lactose fermenter colonies.
   1. Xylose lysine desoxycholate agar shows red colonies with a black center.
   2. Wilson and Blair’s bismuth sulfite agar shows black metallic colonies due to H2S production.
4. Non-lactose fermenter.
5. These are motile.
6. These bacteria produce acid and gas H2S from the carbohydrate, except for S.typhi, which does not produce gas.
7. Salmonella serotypes, clinically divided, are:
   1. Salmonella typhi.
   2. Salmonella cholerae-suis.
   3. Samonella eneteritidis.
8. Vi antigen is a polysaccharide capsule that surrounds the O antigen.
   1. This will protect the bacteria from the antibody attack on the O antigen.
   2. This antigen is also called Vi for virulence.
      

      Pathogenesis of enteric fever

Signs and symptoms

1. Salmonella can cause following diseases in the human:
   1. Typhoid fever.
   2. A carrier state.
   3. Sepsis.
   4. Gastroenteritis (Diarrhea).
2. The onset is insidious, and there are nonspecific symptoms with intermittent fever and headache.
   1. Initially, there is diarrhea followed by fever, and later on, develops anorexia, headaches, malaise, myalgia, arthralgia, cough, and sore throat.
   2. After one week later, there is nausea, vomiting, and chest pain. Systemic toxemia, neuropsychiatric manifestations, cough, coryza, sore throat, and chest pain.
   3. There may be abdominal pain.
   4. These symptoms resolve in 2 to 4 weeks.
3. The patient has a fever, and the pattern of the fever is typical.
   1. Fever is persistent and does not touch the normal level. This may be as high as 103 to 104 F.
      

      Enteric fever signs, symptoms, and the course of the disease in detail

4. The patient may feel weak.
5. May have stomach pains, abdominal pain, headaches, or loss of appetite.
6. In some cases, patients have a rash of flat type and rose-colored spots.
   

   The enteric fever rose spot rashes.

    

7. You may see abdominal tenderness, hepatosplenomegaly, lymphadenopathy, and a maculopapular rash called rose spots in the early stages.
8. After the recovery, 5 to 10% of the cases will continue to excrete organisms for several months; these are called convalescent carriers.
9. 1 to 4% of the cases will carry the bacteria for more than one year and are called chronic carriers.
   1. The common site of the organisms is the gall bladder. These carriers may have gall stones.
10. In 50% of the cases, there is splenomegaly and hepatomegaly.
11. In 30% of the cases, rose spot rashes are seen.
    1. These are 2 to 4 mm, slightly raised, discrete, irregular in shape, and blanching pink macules.
    2. These rose spots are seen on the anterior chest in crops of 5 to 15 in number.
    3. These spots last for 3 to 4 days and fade away without any scar formation.
       

       Salmonella typhi infection and its course of the disease in the patient

 

The complications of enteric fever:

1. Without treatment or even after the treatment, severe complications may occur.
2. These complications are usually seen in the third week (2 to 4 weeks) of the illness. These includes:
   1. Intestinal perforation was seen in 1% to 2% of the cases.
   2. Intestinal hemorrhage was seen in 3% to 20% of the cases.
   3. Meningitis.
   4. Lobar pneumonia is seen in 5% of the cases.
   5. Osteomyelitis.
   6. Acute cholecystitis was sen in 2% of the cases.
   7. Acute myocarditis was seen in 1% to 2% of the cases.
   8. Secondary suppurative lesions may be seen like pneumonia, parotitis, and furunculosis.
   9. Rarely seen complications are:
      1. Pericarditis.
      2. Endocarditis.
      3. Splenic or liver abscess.
      4. Orchitis.
3. The fourth week of illness is characterized by improvement.
4. The Carrier state:
   1. The carrier state is defined as when the organisms are found in the stool for more than one year.
   2. 3% of the S.typhi patients become a carrier.
   3. The site of the chronic infection is the biliary tree.
   4. The presence of the stones or chronic scarring makes it difficult to eradicate the infection.
5. In developing countries, the mortality is around 30%.
   1. 10% of untreated cases will have a relapse.
6. The mortality in the USA is 1 to 2%.

Diagnosis of Typhoid fever:

1. WBCs are decreased and maybe in the range of 4000 to 6000/cmm during the first two weeks.
   1. 3000 to 5000/cmm during the next 2 weeks.
   2. WBCs count around 10,000/cmm or more may suggest perforation of the intestine or suppuration.
2. The peripheral blood smear shows normocytic anemia.
   1. In case of bleeding, anemia may become hypochromic and microcytic.
3. Blood culture is positive during the first 10 days of fever in 90% of the cases.
   1. <30% positive after the third week of illness.
4. Stool cultures are positive after the 10th day of illness, increasing frequency up to 4th or 5 the week.
   1. Stool culture-positive after the 4 months indicates carrier state.
5. Urine culture is positive during the second to the third week in 25% of the cases, even if the blood culture is negative.
6. Widal test is positive after 7 to 10 days of infection. O and H antigens are found.
   1. There are an increase and the peak in the third to the fifth week of infection.
   2. After that, the titer starts to fall for several weeks.
   3. O antigen appears before the appearance of H antigen.
   4. The rising titer of O antigen is very important for the diagnosis.
   5. During the convalescent stage, the H antigen is higher than the O antigen.
   6. Positive widal test may be seen in:
      1. Typhoid fever.
      2. After the vaccination.
      3. In the case of a previous infection.
   7. The early treatment of the infection will not raise the titer, or it main remains negative or low.
   8. H antigen has little value in the diagnosis of typhoid fever.

The procedure of the Widal test:

1. Serial dilution of the patient serum is taken 1:40 to 1:320
2. Now add an equal volume of Salmonella antigen.
3. This can be done as a Slide method or as a Tube method.
4. When running in the tube, then incubate tubes for 12 hours or overnight.
5. Prepare the serial dilution, as shown in the diagram.
   

   Serial dilution in the widal test

Widal test reading:

It is done as follows and can read the result where there is the start of the agglutination, as shown in the following diagram.

Drawback: This test will be positive after 7 to 10 days of Enteric fever.

1. Serologically salmonella has 5 serogroups: A, B, C, D, and E, based on O somatic antigen.
   1. Based on H-antigen are 1200 serotypes.
      1. 7 to 10 days antibody to D-somatic antigen appears.
         1. These antibodies against O antigens reach their peak by 3 to 5 weeks.
      2. H-flagellar antibodies appear later on.
   2. Widal test will be positive after 7 to 10 days of infection.
   3. The titer of O-antibodies 1:80 is suspicious in unvaccinated patients.
   4. The titer of 1:160 is strongly suggestive of infection in unvaccinated individuals.
   5. The titer of 1:40 for Antibody to flagellar-antigen (H) are suspicious in unvaccinated individual.
      1. While 1:160 is strongly suggestive.
   6. Titers are much higher in the vaccinated individual.
   7. Vi antigen for S.typhi is used to screen the carrier. antibodies to Vi-antigen are positive in

Interpretation of the widal the test:

1. The highest dilution of the serum is noted where there is agglutination.
2. If it ends at 1:320, then that is the titer.
3. The Widal test is positive
   1. if “O” antigen titer is    >1:160 = active infection.
   2. If the “H” antigen titer is     >1:160, it indicates past infection or in immunized persons.
   3. A fourfold increase in the titer (e.g., from 1:40 to 1:160) is diagnostic.

O antigen

1. In the acute stage, Antigen O will be positive, and titer will be more than 1:160.
2. O antigen appears early and also disappear early.

H antigen

1. H antigen rises late and disappears late.
2. This will be positive in the recovery stage.

Vi antigen

1. This is an indicator of the carrier stage.

The false-positive test may be seen due to cross-reacting infections, including malaria.

Other diagnostic tests for the diagnosis of Typhoid (Enteric) fever are:

1. Blood culture, which will be positive in the first week when the Widal test is negative.
2. Stool culture.
   1. 60 to 70% of cases are negative during the first week, and more patients positive in the third week of infection.
   2. Stool 90% of the cases are cleared of the bacteria by the 8th week of infection.
3. Urine culture.
4. Bone marrow culture. This is a very sensitive test.
   1. Bone marrow 90% of the cases are positive despite antibiotic therapy if these are ≤5 days.
5. Another serological test is Typhidot.
   1. In this test, the serum of the patient is taken.
   2. This kit claims that enteric fever may be diagnosed in the first week of fever.
      
      

      Interpretation of the Typhidot test in enteric fever

   3. The gold standard is the blood culture, where 90% of cases are positive in the first week.
      1. 50% of cases are positive by the third week.
      2. For better yield, centrifuge the blood and take a buffy coat for culture.
6. Other samples are stool, urine, Rose spot, bone marrow, and gastric or intestinal secretions.
7. When culture is done simultaneously on blood, bone marrow, and intestinal secretions, positivity is >90%.
8. A small % become the carrier, and their stool culture remains positive for at least one year.
   

   Enteric fever diagnosis summary

Summary of the Culture in Enteric fever patients from  a different site:

Time period	Clinical S/S	Presence of Rose spot	Blood	Stool	Urine	Bone marrow
During I.P 0 to 1 week	Diarrhea (10% to 20%) Or constipation	Negative	negative	± (Transiently positive)	Negative	Negative
1 to 2 weeks Invasion of the intestine Invasion of the lymphoid tissue	Anorexia Fever Headache Myalgia Malaise Cough Sore throat	Negative	Positive in 80 to 90% of the cases	Negative	Negative	Negative
2 to 4 weeks Enteric fever confirmed	Systemic toxemia Nausea, vomiting Abdominal pain Neuropsychiatric manifestation Coryza and cough Sore throat Chest pain	Positive in 60% of the cases	Positive in 80 to 90% of the cases	Positive in 80% of the cases	Positive in 25% of the cases	Positive in 90% of the cases
4 to 5 weeks Convalescent stage	The patient is in recovering stage	Positive cases start decreasing.	Negative in ongoing disease Negative in relapse	Positive in 50% of the cases	Positive in 10% of the cases	Positive cases start decreasing
>5 weeks Late complications	Cholecystitis Osteomyelitis Soft tissue abscess formation	Negative	Negative in ongoing disease Negative in relapse	Decreasingly positive	Decreasingly positive	Negative

Treatment

1. The drug of choice is chloramphenicol, but it has many side-effects.
2. Ciprofloxacin is now a drug of choice.
3. Ampicillin, amoxicillin, 3rd generation cephalosporins, and fluoroquinolones can be used.
4. C0-trimoxazole is better than chloramphenicol with fewer side-effect.

Vaccination for the Enteric fever.

There are various types of vaccines, oral as well as I/M are available. The best one is the oral vaccine.

Type of the available vaccine	The dose of the vaccine	The success rate for protection	Relative information
Ty21a (Vivotif Berna)	One capsule one hour before the meal for 4 days One capsule every day for 4 days Give booster dose every 5 years.	Efficacy is 43% to 96% Don’t give antibiotics. Don’t give with antimalarial drugs.	This is a live attenuated vaccine. Not recommended in children under the age of 6 years Don’t give in immunosuppressed patients.
S.typhi (whole organism) This is a heat-killed vaccine.	0.5 mL I/M for 2 doses Give the second dose after >4 weeks apart Booster dose 0.1 mL, every 3 years The dose for the children 6 months to 10 years of age is 0.25 mL	Efficacy is 51% to 77%	The side-effects are: Severe headache in 10% of the cases Fever is seen in 17% to 29% of the cases Pain at the site of injection is seen in 35% to 60% of the cases Rarely severe  reaction is seen
Vi capsular polysaccharide [ViCP] OR Typhim Vi	0.5 mLI/M in 2 doses Give the second dose after >4 weeks apart Booster dose every 2 years Not recommended for children younger than 2 years	Efficacy is 55%	The side-effects are: local erythema or induration in 7% Headache in 1.5% to 3% Fever in 1%

Carriers:

1. 1. It is difficult to treat carriers because of gall bladder involvement.
   2. Ampicillin is the most useful drug, but it is to be given in larger doses and for an extended period of time.
   3. Chloramphenicol is less effective than ampicillin.
   4. Ciprofloxacin can also be tried.
   5. The ultimate end is cholecystectomy.

For Nonmedical (non-specialists) person explanation of Widal test (Enteric fever):

1. Please advise the Widal test if the fever persists for more than 5 days and is constant. It does not touch the normal level.
2. The titer of O antigen 1:40 is considered negative.
3. Just see the O antigen titer if it is 1:160, indicate Enteric fever.
4. If the O antigen is 1:80, repeat the test after 5 to 7 days, and now if the titer is 1:160, the rising titer again indicates acute infection.
5. While H antigen does not indicate acute infection or acute enteric fever.