Laboratory Diagnosis of Salmonella Infections

Laboratory Diagnosis

Laboratory diagnosis of enteric fever is based on the following methods:

a)        Isolation of Salmonella spp. by culture,

b)       Serodiagnosis by demonstration Salmonella antibodies and antigens, and

c)        Molecular diagnosis by DNA probes and PCR.

◗ Specimens

Blood, blood clot, bone marrow, and stool are common speci-mens used for isolation of typhoidal bacilli for culture. Other specimens include the cerebrospinal fluid, peritoneal fluid, mesenteric lymph nodes, resected intestine, pharynx, tonsils, abscess, bone, and urine.

◗ Culture

Blood culture: Blood culture is a very useful procedure fordiagnosis of enteric fever. It is positive in approximately 90% of cases in the first week of fever, 75% of cases in the second week, 60% in the third week, and 25% thereafter till the subsidence of pyrexia. Blood cultures, however, rapidly become negative on treatment with antibiotics.

In this method, approximately 5–10 mL of blood is collected aseptically by venepuncture and inoculated into a culture bottle containing 50–100 mL of 0.5% bile broth. This 10-fold dilution of blood is achieved by adding 5–10 mL of blood to 50–100 mL of bile broth which is carried out to neutralize the bactericidal action of many substances that are present in the blood. The addition of liquid (sodium polyanethol sulfonate) further coun-teracts the bactericidal action of blood. Blood culture bottle is incubated at 37ºC for up to 7 days. After incubation overnight at 37ºC, the bile broth is subcultured on MacConkey agar.

Blood culture is positive in approximately 90% of cases in the first week of fever, 75% of cases in the 2nd week, 60% in the 3rd week, and 25% thereafter till the subsidence of pyrexia. Blood cultures, however, rapidly become negative on treatment with antibiotics.

Castañeda’s biphasic method of blood culture: It is a bettermethod of culture to reduce the risk of contamination during repeated subcultures. The method has additional advantage of being more safe and economical. In this method, the culture bottle has an agar slant in one side, which is flooded with bile broth. After inoculation of blood, the bottle is incubated in the upright position so that surface of the agar remains free without any broth covering the slant. Broth remains only in the lower part of the agar slope. For subculture, the bottle is simply tilted so that the broth flows over the surface of the agar slant and is reincubated in the upright position. If salmonellae are present, colonies appear on the agar slant (Color Photo 34).

Clot culture: Clot culture is a more sensitive method thanthe blood culture, because certain inhibitory substances that are found in the serum are absent in the clot proper. Another advantage of this method is that serum eluted from the blood during process of clotting can be used for demonstration of Salmonella antigens or antibodies.

In this method, 5 mL of blood is collected under strict aseptic conditions, from the patient, into a sterile test tube and allowed to clot. The serum is pipetted off and used for serological tests. The clot is broken up with a sterile glass rod and added to a bottle of bile broth containing streptokinase (100 units/mL). Streptokinase facilitates lysis of the clot with release of bacteria trapped inside the clot. The bile broth is incubated and subcul-tured on media in the same way as described for blood culture earlier.

Bone marrow culture: Bone marrow culture is a most sensitivemethod. It is positive in most cases even when blood cultures are negative. It is also positive even if patients have been taking antibiotics for several days, regardless of how long they have been suffering from enteric fever. This test is recommended for patients whose initial blood culture results are negative, pos-sibly due to prior antibiotic therapy.

Feces culture: Feces culture is also useful because salmonellaeare excreted in feces throughout the disease and even during convalescence with varying frequency. The feces is collected from the patient in a sterile container and sent immediately to the laboratory. If delay is anticipated, the stool specimens may be collected in a buffered glycerol saline transport medium. The fecal samples are inoculated directly on MacConkey, DCA, and Wilson–Blair media. Relatively, a heavy inoculation of stool is made on the Wilson–Blair media because it is highly selective. For enrichment, one tube each of selenite and tetrathionate broth is inoculated and incubated at for 12–18 hours before subculture onto selective media. The plates are incubated at 37ºC overnight. S. Typhi produces large black colonies, with a metallic sheen on the Wilson–Blair medium. On this medium, S. Paratyphi A produces green colonies due to the absence ofH2S production. Salmonellae form pale nonlactose fermenting colonies on MacConkey and DCA media. The colonies are iden-tified by tests as mentioned earlier. If no growth is observed after 7 days, then the culture is declared negative.

Other specimens for culture: Salmonellae may be isolatedfrom several specimens by culture, but they are not usually employed. These specimens include urine, bile, rose spots, pus from suppurative lesions, CSF, and sputum. Cultures may be obtained from the gallbladder, liver, spleen, and mesenteric lymph nodes at autopsy. Isolation of the organism from blood, blood clot, or bone marrow is definitive for diagnosis of enteric fever. Fecal cultures are useful, but a positive fecal culture may occur in carriers as well as in patients. Bile culture is useful for detection of carriers.

◗ Identification of bacteria

Colonies are identified by carrying out motility test, biochemical tests, and slide agglutination with specific Salmonella antisera.

Slide agglutination test: The test is performed with a loopfulof growth from nutrient agar plate or slope emulsified in two drops of saline on a clean slide. If S. Typhi is suspected, that is, when no gas is formed from glucose, a loopful of typhoid O antiserum (factor 9/group D) is added to one drop of bacterial emulsion on the slide. The slide is rocked gently. Development of immediate agglutination suggests that Salmonella strain tested belongs to Salmonella group D.

Identity of S. Typhi is established by agglutination with the flagellar antiserum (anti-d serum). Sometimes, fresh isolates of S. Typhi occur in the V form and do not agglutinate with the O antiserum. Such strains may be tested for agglutination against anti-Vi serum. Alternatively, the growth scraped off in a small amount of saline is tested for agglutination with the O antiserum after boiling for 20 minutes. If the isolate is a non-typhoidal Salmonella, producing gas from sugars, it is tested for agglutination with O and H antisera for groups A, B, C, etc.

◗ Serodiagnosis

Serodiagnosis of enteric fever is based on detection of specific Salmonella antibodies in the serum, or antigen in the serum andalso in urine by various serological tests.

Demonstration of serum antibodies

Widal test: Widal test is the traditional serologic test used forthe diagnosis of typhoid fever. The test measures agglutinating antibodies against flagellar (H) and somatic (O) antigens of S. Typhi for typhoid and paratyphoid bacilli in the patient’ssera. This is a tube agglutination test in which Dreyer’s narrow agglutination tube with a conical bottom is used for H agglutination, and Felix’s short round bottomed tube for O agglutination. The H and O antigens of S. Typhi and the H antigens of S. Paratyphi A and B are used in the test. The paratyphi O antigens are not used because they cross-react with the typhoid O antigen due to their sharing of antigenic factor 12. H antigens of S. Typhi and S. Paratyphi A and B are used individually because H antigens of these bacteria do not show any cross-reactivity with each other. S. Typhi 901, ‘O’ and ‘H’ strains are used for preparation of antigen.

The O antigen is prepared by culture of S. Typhi on phe-nol agar (1:800) and harvesting the growth in a small volume of saline. The saline bacterial suspension is then mixed with 20 times its volume of absolute alcohol, heated at 40–50ºC for 30 minutes, centrifuged, and the deposit is resuspended in saline to the appropriate density. Chloroform is used as a preservative. It is always essential to use standard smooth strains obtained from reference centers for antigen prepara-tion. After preparation of antigen, each batch of antigen is always compared with a standard. The H antigens are prepared by adding 0.1% formalin to a 24-hour broth culture or saline suspension of an agar culture.

The test is performed by taking equal volumes (0.4 mL) of serial dilutions of the serum (from 1/20 to 1/640) and H antigen of S.Typhi (TH), S. Paratyphi A (AH), S. Paratyphi B (BH) and O antigens of S. Typhi (TO) and mixing in Dreyer’s tubes and Felix’s tubes, respectively. The tubes are then incubated in a water bath at 37ºC overnight. Some recommend incuba-tion at 37ºC for 4 hours, followed by overnight incubation at 4ºC. Control tubes containing the antigen and normal saline are used to check for autoagglutination. The agglutination titers of the serum are read. H agglutination is characterized by the formation of loose, cotton woolly clumps, while O agglutination by a disc-like pattern at the bottom of the tube. The supernatant fluid remains clear in both types of agglutina-tions. The highest dilution of the serum showing agglutination with H or O antigens suggests the antibody titer of the patient’s serum. The results of the Widal test should be interpreted, tak-ing into account the following

a)       Antibodies against H and O antigens usually appear by 7th–10th day of the illness and increase steadily till the third or the fourth week, after which it declines gradually. Hence, the blood collected before 7–10 days will be nega-tive for antibodies.

b)       Demonstration of a fourfold or more rise in titer of antibod-ies in a paired sample, one sample collected in the first week and second sample is collected in the third week, is more useful than demonstration of antibodies in a single serum.

c)        A titer of 1/100 or more for O antibodies and 1/200 or more for H antibodies is usually suggestive of enteric fever. However, the results of a single test should be interpreted with caution. Moreover, it is necessary to obtain basal anti-body titer levels in “normal sera” in different areas, before arriving at a cut off diagnostic titer for H and O antibodies.

d)       An elevated level of antibodies may be present in sera of patients suffering from enteric fever in past and in sera of individuals with inapparent infection or vaccination against the enteric fever. Therefore, the mere demonstra-tion of antibodies in the Widal test should need not be considered to be suggestive of the enteric fever.

e)        Serum from an individual vaccinated with TAB vac-cine may show high titers of antibodies to S. Typhi and S. Paratyphi A and B. However, in case of infection, high titersof antibodies will be seen only against the infecting species. H antibodies persist for many months after vaccina-tion, but O antibodies disappear earlier within 6 months. However, in case of infection, high titres of antibodies will be seen only against the infecting species.

f)          Individuals who had suffered from enteric infections in past or who have been immunized may develop an anamnestic response during an unrelated fever, such as malaria, influenza, etc. The anamnestic response shows only a transient rise of antibodies, while the antibody rise is sustained in enteric fever.

g)        Patients treated early with antibiotics, such as chloram-phenicol, may show a poor antibody response.

The sensitivity, specificity, and predictive values of the Widal test have been shown to vary dramatically among laboratories. This wide variation is caused by differences in antigens, tech-niques, and patient population. The Widal test is positive in enteric fever in only 40–60% of patients at the time of admission.

Other serological tests: Indirect hemagglutination, counter-current immunoelectrophoresis, indirect fluorescent Vi antibody, and indirect enzyme-linked immunosorbent assay (ELISA) for immunoglobulin M (IgM) and IgG antibodies to S. Typhi polysaccharide are available for diagnosis of typhoidfever with varying sensitivity and specificity. Monoclonal antibodies against S. Typhi flagellin have been evaluated to increase specificity of the ELISA for antibodies.

Demonstration of serum antigens

In typhoid fever, circulating S. Typhi antigen is present in the serum as well as in the urine, but absent in serum of a cured case of typhoid. Hence, demonstration of serum antigen always indicates an active or recent typhoid fever. Counter-current immunoelectrophoresis, co-agglutination test, and ELISA are frequently employed for detection of circulating antigen in the serum and also in urine for diagnosis of typhoid fever with varying sensitivity and specificity.