INTRODUCTION

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Salmonella enterica subsp. enterica serotype Typhi is the etiological agent of typhoid fever. Typhoid is an important cause of morbidity in many regions of the world (19), with an estimated 13 million cases occurring annually in Asia alone (B. Ivanhof, Abstr. Third Asia-Pacific Symp. Typhoid Fever Other Salmonelloses, abstr. S1-1, 1997). The diagnosis of typhoid fever on clinical grounds is difficult, as the presenting symptoms are diverse (30) and similar to those observed with other common febrile illnesses, such as malaria and nonsevere dengue fever. The isolation of serotype Typhi from blood remains the method of choice for the laboratory diagnosis (33). However, the availability of microbiological culturing facilities is often limited in regions in which typhoid is endemic, and blood cultures can be negative when patients have received prior antibiotic therapy. Bone marrow culturing has a higher sensitivity than blood culturing (6, 31) but is a more invasive procedure.

The Widal test, which detects agglutinating antibodies to lipopolysaccharide (LPS) (TO test) and flagella (TH test), was introduced over a century ago and is widely used for the serological diagnosis of typhoid fever (24). In the original format, the Widal test required acute- and convalescent-phase serum samples taken approximately 10 days apart. More recently, the test has been adapted for use with a single, acute-phase serum sample (2, 3, 13, 20, 21, 23, 25, 26). Enzyme-linked immunosorbent assays (ELISAs) have been considered an alternative approach for the diagnosis of typhoid fever. For the most part, these assays have been based on the detection of anti-LPS antibodies and have been reported to be more sensitive than the Widal TO test (7, 17, 27-29). More recently, ELISAs for the detection of antiflagellum antibodies have been developed (11, 14).

Typhoid fever is the major cause of community-acquired septicemia in southern Vietnam and many other areas in the developing world (8). In the southern provinces of Vietnam, microbiological culturing facilities are limited to a few centers, and the Widal test is widely used for the laboratory diagnosis of typhoid fever (21). In this study, we used antibody-class-specific ELISAs to describe the antibody responses to the LPS and flagellum antigens of serotype Typhi in typhoid patients and community members living in this area. We also report on the potential use of serotype Typhi anti-LPS and antiflagellum levels in serum for the diagnosis of typhoid fever.

	MATERIALS AND METHODS

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Patients and samples. The serological studies were performed at the Centre for Tropical Diseases (CTD), Ho Chi Minh City, and the Dong Thap Provincial Hospital, Cao Lanh, Dong Thap Province, in the Mekong Delta region of southern Vietnam. All studies were approved by the scientific and ethical committees of the participating institutions, and informed consent was obtained from all participants or, in the case of children, from a parent or guardian. Serum samples were collected from children (<15 years) and adults with typhoid fever and also from hospital and community control subjects. These subjects were being recruited into either treatment or epidemiology studies of typhoid fever which have been reported elsewhere (16, 18, 32).

Antigens. The LPS and flagellum antigens were purified from serotype Typhi vaccine strains BRD 985 (Vi negative) (22) and BRD 691 (4), respectively. LPS was purified using a modified aqueous phenol method (12). The preparation showed a typical ladder pattern on a silver-stained sodium dodecyl sulfate gel (data not shown) and contained <2% (wt/wt) protein, as determined by the Bradford assay (Bio-Rad, Hemel Hempstead, United Kingdom). Flagella were purified using the method of Ibrahim et al. (9). A large polypeptide band of 50 kDa and corresponding to the flagella was visible on a Coomassie blue-stained 10% sodium dodecyl sulfate gel (data not shown), and the LPS content was determined to be 1% (wt/wt) by the Limulus amebocyte lysate assay (Sigma, Poole, Dorset, United Kingdom).

ELISAs. In-house indirect sandwich ELISAs were established to detect anti-LPS IgA, IgM, and IgG and antiflagellum IgG. All reagents used were purchased from Sigma unless stated otherwise. Immulon 1b flat-bottom 96-well microtiter plates (Dynex Technologies, Billinghurst, United Kingdom) were coated overnight at 4°C with 100 µl of either 1 µg of antigen/ml in coating buffer (0.1 M carbonate buffer [pH 9.4], antigen positive) or coating buffer alone (antigen negative). The plates were blocked for 1 h at 37°C with 200 µl of phosphate-buffered saline containing 1% bovine serum albumin (BSA). Sera were either assayed at a single dilution (1/1,000 for anti-LPS IgG, 1/500 for anti-LPS IgA, or 1/250 for anti-LPS IgM and antiflagellum IgG) or serially diluted (starting at a dilution of 1/50). Sera were diluted in phosphate-buffered saline containing 0.1% BSA and 0.05% Tween 20, 100 µl was applied to the appropriate wells, and the plates were incubated for 4 h at room temperature. Bound antibodies (IgA, IgG, or IgM) were detected using heavy-chain-specific goat antibodies directly conjugated to alkaline phosphatase. The latter were diluted (anti-IgG, 1/5,000; anti-IgA, 1/500; and anti-IgM, 1/2,500) in Tris-buffered saline containing 0.1% BSA and 0.05% Tween 20. One hundred microliters was added to each well, and the plates were incubated overnight at 4°C. One hundred microliters of p-nitrophenyl phosphate (1 mg/ml) was added to each well, and the plates were incubated at ambient temperature in the dark for 30 to 40 min. The absorbance at 405 nm (reference filter, 450 nm) was determined using an automated ELISA reader (Bio-Rad).

Widal test. The Widal tube agglutination test (Sanofi Diagnostics Pasteur, Marnes la Coquette, France) was performed according to the manufacturer's instructions by a single operator blind to the patient's diagnosis. Briefly, serial tube dilutions of sera were made in 0.85% saline, starting at a dilution of 1/100. Standard preparations of Salmonella O and H antigens were added, and the tubes were incubated at 37°C for 1 h. The tubes were centrifuged for 5 min, and agglutination was determined by eye. The Widal TO or TH titer was taken as the highest dilution of serum with visible agglutination.

IDeaL TUBEX. The IDeaL TUBEX (IDL Biotech AB, Borlange, Sweden) was performed according to the manufacturer's instructions. The test is a semiquantitative competitive agglutination test for the detection of anti-O9 antibodies. The test was performed by a single operator, and the results were interpreted by three members of the laboratory staff who had no knowledge of the samples being tested. Samples were graded as 0 to 10 according to the color of the reaction mixture at the end of the procedure. Those with a grade of >2 were considered positive.

Serotype Typhi IgM dipstick assay. The serotype Typhi IgM dipstick assay was provided by H. L. Smits, Department of Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands. Briefly, serum samples were diluted (1/50) in the detecting reagent (containing dye-labeled, anti-human IgM antibodies). Nitrocellulose dipsticks coated with heat-inactivated serotype Typhi were immersed in the diluted serum and incubated at room temperature for 4 h. The strips were then washed and dried at room temperature. The sera were graded (0 to 4) according to the staining intensity of the colored band corresponding to the antigen. The test was performed by a single operator, and the results were interpreted by three members of the laboratory staff who had no knowledge of the samples being tested.

Statistics and calculations. Statistical analyses were performed using the computer package SPSS for Windows (SPSS Benelux Inc., Gorinchem, The Netherlands). The Mann-Whitney U test was used for comparison of nonpaired samples, and the Wilcoxon sign rank test was used for comparison of paired samples. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the diagnostic tests were calculated using the following formulae: sensitivity is a/(a + c), specificity is d/(d + b), PPV is a/(a + b), and NPV is d/(d + c); in these formulae, a is test positive and true positive, b is test positive and true negative, c is test negative and true positive, and d is test negative and true negative, where true positive and true negative are culture (blood or bone marrow) positive or negative, respectively.

	RESULTS

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Serum anti-LPS and anti-flagellum antigen antibody titers in culture-confirmed typhoid patients admitted to the CTD. To initially assess the serum antibody responses to serotype Typhi LPS and flagellum antigens in patients infected with serotype Typhi, serum samples were collected from 160 patients with culture-confirmed typhoid fever and admitted to the CTD. Of these individuals, 84 were male and 76 were female. The median (interquartile range [IQR]) age of the group was 18 (11 to 26) years. The median (IQR) duration of illness (time from onset of symptoms to time of sample collection) was 12 (9 to 17) days. Clinical details for these patients have been reported elsewhere (18, 32).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Serum anti-LPS IgM (a), anti-LPS IgG (b), anti-LPS IgA (c), and antiflagellum IgG ELISA OD values for children (14 years) and adults (>14 years) with typhoid fever. Solid line, median; box, quartile; bar, range; circles, outliers; asterisks, extremes.

Evaluation of serological tests for the diagnosis of typhoid fever. Based on the results of the above study, an evaluation of a panel of serological tests for the diagnosis of typhoid fever was undertaken. Sera were collected from patients with culture-confirmed typhoid fever and hospital control subjects admitted to Dong Thap Provincial Hospital. The hospital control was the next febrile patient of the same age group (within 5 years) and sex and admitted to the hospital with a diagnosis other than typhoid fever. Sera also were collected from healthy community control subjects. These were persons who resided in the household adjacent to the homes of the typhoid patients and who were of the same age group (within 5 years) and sex as the typhoid patients.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Serum anti-LPS IgM (a), anti-LPS IgG (b), anti-LPS IgA (c), and antiflagellum IgG (d) titers for patients with typhoid fever (TF) and age- and sex-matched hospital control (HC) and community control (CC) subjects.

View this table: [in this window] [in a new window]   TABLE 2.   Number (percentage) of control subjects with raised serum IgG titers (1/50) by age

	DISCUSSION

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This report describes serum antibody titers to the flagellum and LPS antigens of serotype Typhi in patients with typhoid fever and control subjects and evaluates the potential of serological approaches in the diagnosis of typhoid fever. In southern Vietnam, blood culture facilities are available in very few centers, and there is a need for a rapid, affordable diagnostic test for typhoid fever. There have been numerous reports on the single Widal test but no consensus as to its diagnostic value in regions in which typhoid is endemic (1, 3, 15, 20, 23, 25, 26). The results reported here show that the Widal TO test lacks either sensitivity or specificity in southern Vietnam, supporting the findings of a previous study from this region (21). Although the anti-LPS IgM ELISA (titers, 1/400) was more sensitive than the Widal TO test (titers, 1/400) in detecting culture-confirmed typhoid cases, none of the assays for the detection of anti-LPS antibodies (ELISAs and IDeaL TUBEX) had both high sensitivity and specificity (i.e., 0.95). The IgM dipstick assay (1+), which detects IgM antibodies against whole-cell serotype Typhi, performed as well as the anti-LPS IgM ELISA and was more sensitive than the Widal TO test (titers, 1/400). In agreement with previous studies (11, 14), the antiflagellum IgG ELISA and the Widal TH test were found to be highly specific but lacked sensitivity. In the present study, this result was probably due to the young age and/or the relatively short length of illness (52 of 65 ill for <14 days) of the typhoid patients. The sensitivity of an antiflagellum IgG diagnostic test might be increased if the test were used only for adults, particularly those with a long history of fever. However, this strategy would clearly limit the usefulness of such a test.

It could be argued that the raised serum anti-LPS and antiflagellum antibody titers seen in the sera of some of the hospital controls was due to current or recent infection with serotype Typhi. However, this possibility is unlikely, as all the hospital control patients had an alternative diagnosis (16). Furthermore, elevated anti-LPS and antiflagellum antibody titers were found in similar proportions of sera from healthy control subjects, indicating that background immunity to the two antigens can be high in this region of typhoid endemicity. The frequency of raised IgG titers (i.e., >1/50) increased with age, in agreement with previous studies showing that the prevalence of raised Widal TO and TH titers increases with age in regions of typhoid endemicity (5, 15). Titers of antibodies to the LPS and flagellum antigens can be raised following exposure to either serotype Typhi or another microorganism sharing common antigens (15), and this fact presumably contributes to the lack of specificity seen with serological diagnostic tests in regions of typhoid endemicity.

The results presented here show that ELISAs or dipstick tests for the detection of IgM antibodies to serotype Typhi LPS or whole bacteria perform better than the Widal TO test, as has been reported previously (7, 17, 28). Thus, a simple, inexpensive, rapid solid-phase immunoassay based on the detection of anti-LPS IgM or the prototype anti-serotype Typhi IgM dipstick test should be of greater diagnostic use than the Widal TO test for patients with suspected typhoid fever but who are blood culture negative or in areas where culturing facilities are not available. Similarly, Bhutta and Mansurali (1) recently reported that the Typhidot and Typhidot-M tests, which detect antibodies against a 50-kDa serotype Typhi outer membrane protein first described by Ismail et al. (10), had higher sensitivity (0.85 to 0.94) and specificity (0.77 to 0.89) than the Widal test (0.63 and 0.81, respectively). However, none of the serological diagnostic tests described in either this study or earlier studies have been shown to have both high sensitivity and high specificity (>0.95) for the diagnosis of typhoid fever in regions of typhoid endemicity when used with a single, acute-phase serum sample. Further studies to evaluate the sensitivity and specificity of these tests with paired serum samples taken 3 to 4 days apart are warranted.