WIDAL TEST ( simple steps)

         Widal test is a tube agglutination test employed in the serological diagnosis of enteric fever. The test is named after
Georges Fernand Isidore Widal, a French physician and bacteriologist, born March 9, 1862, Algeria; died January
14, 1929, Paris.
Principle: Patients’ suffering from enteric fever would possess antibodies in their sera which can react and
agglutinate serial doubling dilutions of killed, coloured Salmonella antigens in a tube agglutination test.
Requirements: Widal rack, round-bottomed Felix tubes, conical-bottomed Dreyer’s tubes, water bath, doubly
diluted patient serum in three-four rows, Killed coloured suspensions of S.typhi O antigen, S.typhi H antigen,
S.paratyphi AH antigen and optionally S.paratyphi BH antigen.
Preparation of antigens: Salmonella typhi 901 strain is used to prepare S.typhi O and S.typhi H antigens. O
antigens for S.paratyphi A and S.paratyphi B are not taken as they cross-react with S.typhi O antigen. H antigen
suspension is prepared by treating overnight broth culture or saline suspension of Salmonella with 0.1% formalin.
For preparing O antigen suspension, Salmonella are grown on phenol agar (1:800) to inhibit flagella. The growth is
then emulsified in small volume of saline, mixed with 20 times its volume of alcohol, heated at 40oC to 50oC for 30
minutes and centrifuged. The antigens are treated with chloroform (preservative) and appropriate dyes are added
for easy identification of antigens.
Procedure:
© Sridhar Rao P.N (www.microrao.com)
Patient serum is doubly diluted by mixing and transferring from 1:10 to 1:640 in three-four rows. First row usually
comprises of Felix tubes, where somatic S.typhi O antigen is added. For all the remaining rows, Dreyer’s tubes are
taken; where different flagellar H antigens are added. Each tube must contain 0.5ml of diluted serum. A test tube
with only saline is kept in each row as control. All the tubes (including control) in a row are mixed with 0.5ml of
antigen suspension. The first row is treated 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. Since infections by S.paratyphi B
are rare, this antigen is usually omitted in the test. After all the tubes have been treated with specific antigen
suspensions, the widal rack is placed in a thermostatically controlled water bath maintained at 37oC for overnight
incubation. Another approach is to incubate the tubes at 50-55oC.
Reading the results: The control tubes must be examined first, where they should give no agglutination. The
agglutination of O antigen appears as a “matt” or “carpet” at the bottom. Agglutination of H antigens appears loose,
wooly or cottony. The highest dilution of serum that produces a positive agglutination is taken as titre. The titres for
all the antigens are noted.
Slide widal test:
A slide widal test is more popular among diagnostic laboratories as it gives rapid results.
Qualitative test: 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. To know the titre for each of the antigens, the test is repeated
with dilutions of serum.
Quatitative test: 80 µl, 40 µl, 20 µl, 10 µl and 5 µl of patient’s serum each for the four antigens are placed on the
circled card. To each series of serum specimen, one drop of specific antigen is added to each, mixed and rotated
for one minute. Agglutination in each of these is noted. 80 µl corresponds to 1in 20 dilution, 40 µl to 1 in 40, 20 µl to
1 in 80, 10 µl to 1 in 160 and 5 µl corresponds to 1 in 320 titre.
Interpretation of widal test:
  Timing of test is important, as antibodies begin to arise during end of first week. The titres increase during
second, third and fourth week after which it gradually declines. The test may be negative in early part of first
week.
  Single test is usually of not much value. A rise in titre between two sera specimens is more meaningful than
a single test. If the first sample is taken late in the disease, a rise in titre may not be demonstrable. Instead,
there may be a fall in titre.
  Baseline titre of the population must be known before attaching significance to the titres. The antibody
levels of individuals in a population of a given area give the baseline titre. A titre of 100 or more for O
antigen is considered significant and a titre in excess of 200 for H antigens is considered significant.
  Patients already treated with antibiotics may not show any rise in titre, instead there may be fall in titre.
Patients treated with antibiotics in the early stages may not give positive results.
  Patients who have received vaccines against Salmonella may give false positive reactions. This can be
differentiated from true infection by repeating the test after a week. True untreated infection results in rise in
titre whereas vaccinated individuals don’t demonstrate any rise in titre.
  Those individuals, who had suffered from enteric fever in the past, sometimes develop anti-Salmonella
antibodies during an unrelated or closely related infection. This is termed anamnestic response and can be
differentiated from true infection by lack of any rise in titre on repetition after a week.
  Antigen suspensions with fimbrial antigens may sometimes give false positive reactions due to sharing of
fimbrial antigens by some Enterobacteriaceae members. Antigen suspension must be devoid of fimbrial
antigens.
* Widal test is losing its relevance in Western and European nations but continues to be used in India.       

Widal test

       The Widal test is a presumptive serological test for enteric fever or undulant fever. In case of Salmonella infections, it is a demonstration of agglutinating antibodies against antigens O-somatic and H-flagellar in the blood. For brucellosis, only O-somatic antigen is used.

The Widal test is not a very specific test, since patients are often exposed to other bacteria (e.g. Salmonella enteritidis, Salmonella typhimurium and some types of E. coli) in this species that induce cross-reactivity; many people have antibodies against these enteric pathogens, which also react with the antigens in the Widal test, causing a false-positive result. Test results need to be interpreted carefully in the light of past history of enteric fever, typhoid vaccination, and the general level of antibodies in the populations in endemic areas of the world. Typhidot is the other test used to ascertain the diagnosis of typhoid fever. As with all serological tests, the rise in antibody levels needed to make the diagnosis takes 7-14 days, which limits. Other means of diagnosing Salmonella typhi (and paratyphi) include cultures of blood, urine and faeces. The organism also produces H₂S from thiosulfate.

Often 2-mercaptoethanol is added. This agent more easily denatures the IgM class of antibodies, so if a decrease in the titer is seen after using this agent, it means that the contribution of IgM has been removed leaving the IgG component. This differentiation of antibody classes is important; as it allows for the distinction of a recent (IgM) from an old infection (IgG)

The Widal test is positive if TO antigen titer is more than 1:160 in an active infection, or if TH antigen titer is more than 1:160 in past infection or in immunized persons. A single Widal test is of little clinical relevance due to the number of cross reacting infections, including malaria. If no other tests (either bacteriologic culture or more specific serology) are available, a four fold increase in the titer (e.g., from 1:40 to 1:160) in the course of the infection, or a conversion from an IgM reaction to an IgG reaction of at least the same titer, would be consistent with a typhoid infection.

The Widal test is "a test involving agglutination of typhoid bacilli when they are mixed with serum containing typhoid antibodies from an individual having typhoid fever; which may be used to detect the presence of Salmonella typhi and S. paratyphi

Testing the Stool Sample

     In general, the results of stool tests are usually reported back within 3 to 4 days, although it often takes longer for parasite testing to be completed.

Examining the Stool for Blood

Your doctor will sometimes check the stool for blood, which can be caused by certain kinds of infectious diarrhea, bleeding within the gastrointestinal tract, and other conditions. However, most of the time, blood streaking in the stool of an infant or toddler is from a slight rectal tear, called a fissure, which is caused by straining against a hard stool (this is fairly common in infants and kids with ongoing constipation).

Testing for blood in the stool is often performed with a quick test in the office that can provide the results immediately. First, stool is smeared on a card, then a few drops of a developing solution are placed on the card. An instant color change shows that blood is present in the stool. Sometimes, stool is sent to a laboratory to test for blood, and the result will be reported within hours.

Culturing the Stool

Stool can be cultured for disease-causing bacteria. A stool sample is placed in an incubator for at least 48 to 72 hours and any disease-causing bacteria are identified and isolated. Remember that not all bacteria in the stool cause problems; in fact, about half of stool is bacteria, most of which live there normally and are necessary for digestion. In a stool culture, lab technicians are most concerned with identifying bacteria that cause disease.

For a stool culture, the lab will need a fresh or refrigerated sample of stool. The best samples are of loose, fresh stool; well-formed stool is rarely positive for disease-causing bacteria. Sometimes, more than one stool will be collected for a culture.

Swabs from a child's rectum also can be tested for viruses. Although this is not done routinely, it can sometimes give clues about certain illnesses, especially in newborns or very ill children. Viral cultures can take a week or longer to grow, depending on the virus.

Testing the Stool for Ova and Parasites

Stool may be tested for the presence of parasites and ova (the egg stage of a parasite) if a child has prolonged diarrhea or other intestinal symptoms. Sometimes, the doctor will collect two or more samples of stool to successfully identify parasites. If parasites — or their eggs — are seen when a smear of stool is examined under the microscope, the child will be treated for a parasitic infestation. The doctor may give you special collection containers that contain chemical preservatives for parasites.

Collecting a Stool Specimen

     Unlike most other lab tests, stool is sometimes collected by the child's family at home, not by a health care professional. Here are some tips for collecting a stool specimen:

• Collecting stool can be messy, so be sure to wear latex gloves and wash your hands and your child's hands well afterward.
• Many kids with diarrhea, especially young children, can't always let a parent know in advance when a bowel movement is coming. Sometimes a hat-shaped plastic lid is used to collect the stool specimen. This catching device can be quickly placed over the toilet bowl or your child's rear end to collect the specimen. Using a catching device can prevent contamination of the stool by water and dirt. If urine contaminates the stool sample, it will be necessary to take another sample. Also, if you're unable to catch the stool sample before it touches the inside of the toilet, the sample will need to be repeated. Fishing a bowel movement out of the toilet does not provide a clean specimen for the laboratory to analyze.
• Another way to collect a stool sample is to loosely place plastic wrap across the rim of the toilet, under the seat. Then place the stool sample in a clean, sealable container before taking to the laboratory. Plastic wrap can also be used to line the diaper of an infant or toddler who is not yet using the toilet.

The stool should be collected into clean, dry plastic jars with screw-cap lids. You can get these from your doctor or through hospital laboratories or pharmacies, although any clean, sealable container could do the job. For best results, the stool should then be brought to the laboratory immediately.

If it's impossible to get the sample to the laboratory right away, the stool should be refrigerated, then taken to the laboratory to be cultured as soon as possible after collection. When the sample arrives at the lab, it is either examined and cultured immediately or placed in a special liquid medium that attempts to preserve potential bacteria or parasites.

The doctor or the hospital laboratory will usually provide written instructions on how to successfully collect a stool sample; if written instructions are not provided, take notes on how to collect the sample and what to do once you've collected it.

If you have any questions about how to collect the specimen, be sure to ask. The doctor or the lab will also let you know if a fresh stool sample is needed for a particular test, and if it will need to be brought to the laboratory right away.

Most of the time, disease-causing bacteria or parasites can be identified from a single stool specimen. Sometimes, however, up to three samples from different bowel movements must be taken. The doctor will let you know if this is the case.

Stool Tests

     Stool (or feces) is usually thought of as nothing but waste — something to quickly flush away. But bowel movements can provide doctors with valuable information as to what's wrong when a child has a problem in the stomach, intestines, or another part of the gastrointestinal system.
A doctor may order a stool collection to test for a variety of possible conditions, including:

• allergy or inflammation in the body, such as part of the evaluation of milk protein allergy in infants
• infection, as caused by some types of bacteria, viruses, or parasites that invade the gastrointestinal system
• digestive problems, such as the malabsorption of certain sugars, fats, or nutrients
• bleeding inside of the gastrointestinal tract
  

The most common reason to test stool is to determine whether a type of bacteria or parasite may be infecting the intestines. Many microscopic organisms living in the intestines are necessary for normal digestion. If the intestines become infected with harmful bacteria or parasites, though, it can cause problems like certain types of bloody diarrhea, and testing stool can help find the cause.
Stool samples are also sometimes analyzed for what they contain; for instance, examining the fat content. Normally, fat is completely absorbed from the intestine, and the stool contains virtually no fat. In certain types of digestive disorders, however, fat is incompletely absorbed and remains in the stool.