Laboratory Diagnosis of Enteric Fever

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The diagnosis of typhoid fever on clinical presentations alone is difficult, as the presenting symptoms are diverse and similar to those observed with other febrile illnesses, especially during the first weeks of the infection. Therefore, laboratory-based investigations are essential for supporting the diagnosis of the disease. The “gold standard” for diagnosis of typhoid fever is the isolation of Salmonella typhi from appropriate samples including blood, bone marrow aspirates, stool, urine and rose spots.


Serodiagnosis depends upon the 100-year-old Widal test, and other serological diagnostic tools have limitations because of their low sensitivity and/or specificity. The development of molecular methods for diagnosis of infectious diseases, including typhoid fever has improved the sensitivity and specificity of diagnosis. One of the molecular methods, Polymerase chain reaction (PCR) is the most sensitive and rapid method to detect microbial pathogens in clinical specimens. Antigen detection has not been investigated for well over three decades and detecting an immune response specific for typhoid fever has been done only with antibody detection.

Isolation of the organism

Culture isolation of the S. typhi remains the most effective diagnostic procedure in suspected typhoid fever. Where culture is available, typhoid fever may account for two thirds of cases of community acquired septicaemia admitted to hospital. Blood has been the mainstay of culture for S. typhi since 1900. S. typhi maximally isolated from blood in the first week of disease; from faeces in the second and subsequent weeks and urine in the third and fourth weeks. The various culture methods available are blood culture, clot cultures, faeces culture, bone marrow culture, urine culture, bile culture and duodenal aspirate culture.

Blood Culture

Blood culture is the gold standard diagnostic method for diagnosis of typhoid fever. The sensitivity of blood culture is highest in the first week of the illness and reduces with advancing illnesses. The organisms may be recovered from bloodstream at any stage of the illness, but are most commonly found during the first 7 to 10 days and during relapses. 

 

Blood culture is the method of choice and has the great advantage over culture from the faeces, urine or bile. It is showing not only that patient is infected with the bacillus but that the infection is active. Though it is gold standard, the yield of blood culture is quite variable. In the untreated patient, blood culture is usually positive in about 80.0% during first week and declining 20.0 to 30.0% later in the course of the disease.

Sensitivity of cultures can be affected by antibiotic treatment of the patient, inadequate sampling, type of culture medium, lengths of incubation, and variations of bacteraemia in the patients. In addition, Salmonella cultures take 4 to 7 days for isolation and identification of the organisms. Adequate volumes of medium should be used in blood culture system to avoid negative results. A study finding suggested that 50 ml of medium was adequate for 8 ml of blood, presumably because of very low degrees of bacteraemia in some patients.

If the whole blood is to be cultured it is essential to prevent bactericidal effects of serum either by adequate dilution of the sample in an adequate medium volume or by inhibition of serum bactericidal factors. Sodium polyanethol sulfonate (SPS) and bile salt inhibit this bactericidal effect. The SPS in concentration of 0.025% to 0.03% is the best anticoagulant for blood. It is also ant complementary and ant phagocytic, and interferes with the activity of some anti-microbial agents, notably amino glycosides.

A study was reported that SPS aids in early recovery of S. typhi and S. Paratyphi A from blood cultures. Taking samples of blood on several occasions may improve the results of culture. Three types of blood cultures have been in use such as traditional or conventional blood culture, lysis centrifugation and automated blood culture.

Automated Technique

Modern blood culture techniques (automated) permit the bacteriological confirmation of typhoid fever in a higher proportion of cases. These systems employ equipment that automatically detects an early sign of bacterial growth in a special blood culture bottle. An isolation rate of 92% of blood culture with the Bactec 460 Radiometric system using a blood: broth ratio of 1:6 was found in a study.

Lysis Centrifugation

Lysis centrifugation system consists of a tube containing anticoagulant (SPS), EDTA, and saponin. After the tube is filled with blood during phlebotomy, the contents are mixed and centrifuged and the resulting pellet is inoculated onto agar media taking all aseptic measures. The system effectively recovers S. typhi from blood specimen. The benefit of this system include; the more rapid and greater recovery of the organism; the presence of actual colonies for direct identification and susceptibility testing after initial incubation; the ability to quantify the colony forming units present in the blood; rapid detection of
polymicrobial bacteraemia; and possible recovery of intracellular microorganisms caused by lysis of host cells. Limitation of the method is high rate of possible contamination

Clot Culture

The blood clot culture was found to be much more sensitive for S. typhi than whole blood culture. Bacterial growth was significantly faster in cultures of blood clot compared to whole blood. The rapid confirmation of the aetiological agent would facilitate an early institution of appropriate antimicrobial therapy, thereby reducing clinical morbidity, especially in an endemic population.

Blood clot from which serum has been removed often gives a positive result when a similar volume of whole blood yields no growth. A method of clot culture with streptokinase has been recommended. An amount of 8 ml quantities of venous blood is taken from patient and allowed to clot in sterile screw capped universal containers. The separated serum is removed. The medium used consists of a Wilson and Blair agar slope in a 120 ml bottle to which is added 15 ml of streptokinase bile salt broth.

The streptokinase causes rapid clot lysis with release of bacteria trapped in the clot. The cultures are then incubated and positive results may be obtained in less than 24 hours. Clot culture is more sensitive than blood cultures with isolation rate of 92% and the clot technique has many advantages over conventional whole blood culture, both in reliability and in cost.

Bone Marrow Culture

Salmonella typhi is an intracellular pathogen in the reticuloendothelial cells of the body including the bone marrow. The overall sensitivity of bone marrow cultures ranges from 80.0 to 95.0% and is good even in the late phase of the disease and despite prior antibiotic therapy. Bone marrow aspirates are known to yield a higher rate of positive cultures than peripheral blood in typhoid fever cases20-21. Bone marrow culture may give a positive result when blood culture fails, particularly in patients admitted to hospital while on antibiotic treatment. As a result unlike blood culture bone marrow culture is highly (90%) sensitive.

Another study reported that the concentration of S. typhi

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in the bone marrow was found considerably higher than in peripheral blood23. In the bone marrow there were over 10 times more bacteria than in peripheral blood. it seems likely to the positively rate of a 1 ml bone marrow culture  is equivalent to the result of 10 ml of peripheral blood23. The invasive nature of bone marrow aspiration discourage from its use as a first line investigation for diagnosis of typhoid fever.

Stool Culture

In typhoid fever, stool cultures are usually positive from the second week of the infection. Stool is usually plated on desoxycholate- citrate agar and also inoculated into fluid enrichment media such as tetrathionate or selenite broth. The limitation of liquid of medium is that the growth of fluid enrichment medium is sub cultured appropriate medium for proper identification. Suspicious colonies from culture plates are tested directly for the presence of salmonella O antigens by slide agglutination and subcultured to peptone water for determination of H antigen structure and for further biochemical analysis

Urine Culture

Urine cultures are not recommended for diagnosis in view of poor sensitivity. Bacteria are not excreted continuously and therefore, several specimens may need to be cultured before organisms can be isolated. In typhoid fever, urine cultures are usually positive from the third week of the infection. The centrifuged urine deposit is plated on desoxycholate-citrate agar and is also inoculated into fluid enrichment media such as tetrathionate or selenite broth. The growth of fluid enrichment medium is sub cultured appropriate medium for proper identification.

Duodenal String: Capsule Culture

Duodenal string test was found to be a simple, noninvasive and a reliable test which when used in combination with blood culture could identify almost all cases of typhoid fever irrespective of duration of fever and prior use of antibiotics. Duodenal content cultures have been proved to be more sensitive (86%) in diagnosis than bone marrow (75%) and more effective than blood (42%) and stool (26%) cultures in recovery of S. typhi. The sensitivity of duodenal content cultures was found not modified by the duration of illness at admission or by previous antibacterial therapy. Culture of duodenal aspirate is important in the detection of typhoid carriage. Individuals can excrete S. typhi in the bile and yet be undetected by stool culture. Because of patient’s discomfort and the time required for tube placement, duodenal aspiration has not been widely used.

Widal test

The information regarding Widal test has been noted in Britannica encyclopedia. The Widal agglutination test was introduced as a serologic technique to aid in diagnosis of typhoid fever. The test was named after Georges Fernand Isidore Widal, a French physician and bacteriologist. In 1896, Widal developed a procedure for diagnosing typhoid fever based on the fact that antibodies in the blood of an infected individual cause the bacteria to bind together into clumps (the Widal reaction).

The test was based on demonstrating the presence of agglutinin (antibody) in the serum of an infected individual, against the H (flagellar) and O (somatic) antigens of Salmonella typhi. The “O” antigen is the somatic antigen of S. typhi and is shared by S. paratyphi A, S. paratyphi B, other Salmonella species and other members of the Enterobacteriaceae family. Antibodies against the O antigen are predominantly IgM, rise early (appear on day 6 to 8) in the illness and disappear early. The H antigens are flagellar antigens of S. typhi, paratyphi A and paratyphi B. Antibodies to H antigens are both IgM and IgG, rise late (on days 10 to 12) in the illness and persist for a longer time.

Haemagglutination (HA) Tests

Many researchers have evaluated the usefulness of HA tests in different countries. In a study from India, the anti-LPS HA test showed a sensitivity of 60% and specificity of 98.2%. The positive predictive value and negative predictive value were 66.7% and 96.7% respectively. In the same study, the haemagglutination inhibition test targeted Salmonella antigens and was found useful for helping the early detection of S. typhi in culture. In another study, a Reverse Passive Haemagglutination Test (RPHA) was designed for the detection of S. typhi antigen. The test was found to be 70% sensitive and 92% specific for acute typhoid fever diagnosis.

Immunochromatographic method

ICT has been studied in many countries and they found significantly higher sensitivity and specificity. An evaluation of ICT (Typhidot) in India was found to be 100% sensitive and 80% specific compared to a blood culture as gold standard.

Countercurrent Immunoelectophoresis (CIE)

This test is based on electrophoresis and the visualization of the precipitin band of antigenantibody complexes that form. The sensitivity is similar to that of the Widal test and the procedure may be quicker if tests are batched (about one hour for a gel), but bands are often difficult to see, the cost is higher than that of the Widal, and some studies conclude that CIE has a low sensitivity with Vi antigen. A panel of antigens (somatic (O), flagellar (H) and capsular polysaccharide (Vi) antigens of S. typhi is recommended for rapid diagnosis of typhoid fever.

Dot Enzyme Immunoassay (EIA) Test

A dot enzyme immunoassay that detects IgG and IgM antibodies against a 50 KD outer membrane protein, distinct from the somatic (O), flagellar (H) or capsular (Vi) antigen of Salmonella typhi is commercially available as Typhidot. Commercially it is available in two different properties like the Typhidot M that detects only IgM antibodies of S.typhi has been reported to be slightly more specific in a couple of studies. Typhidot® test detects specific IgM and IgG antibodies to S. typhi. It has undergone full-scale multinational clinical evaluation of its diagnostic value50. In areas of high endemicity, where the rate of S. typhi transmission is high, the detection of specific IgG increases. The IgG can persist for more than two years after typhoid fever Infection. The detection of specific IgG cannot differentiate between acute and convalescent cases

IgM Dipstick Test

A rapid dipstick assay for the detection of S. typhi specific IgM antibodies in serum and whole blood samples was previously reported and the sensitivity and specificity was evaluated. The dipstick assay may thus also be useful for the serodiagnosis of culture-negative patients with clinical signs and symptoms consistent with typhoid fever. The advantages of the dipstick assay are that the result can be obtained on the same day allowing a prompt treatment, that only a small volume of serum is needed, and that no special laboratory equipment is needed to perform the assay. The stability of the reagents of the dipstick and the simplicity of the assay allows its use in places that lack
laboratory facilities

PCR 

The PCR has been used to enable diagnosis of typhoid fever within few hours and more specific and sensitive than blood cultures. In addition nested PCR has been shown promising results. These results show that the nested PCR has good potential to be a rapid tool for the definitive, differential diagnosis of typhoid and is superior to conventional method

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