Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. II. Measurement of intestinal antibodies by radioimmunoassay.

Antibodies to the lipopolysaccharide (LPS) and protein antigens of S. typhi in secretions of small intestine obtained from 12 typhoid patients, four typhoid carriers and 16 non-typhoid control subjects were measured by a solid-phase radioimmunoassay technique. Intestinal secretions obtained from typhoid patients as a group had significantly higher anti-LPS and anti-protein antibodies than those from the control group. These antibodies were both IgM and IgA classes. There was no correlation between the IgM or IgA antibody levels in serum and those in the intestinal secretions. In the intestinal secretions obtained from typhoid carriers, on the other hand, only IgA-class antibodies to the LPS and protein antigens of S. typhi were present at high levels.     

Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. I. Measurement of serum antibodies by radioimmunoassay.

Serum antibody responses to the lipopolysaccharide and protein antigens of S. typhi in typhoid patients were studied using a solid-phase radioimmunoassay technique. Sera from 24 adult typhoid patients and 20 non-typhoid adult controls were compared. As a group, sera from typhoid patients showed increased IgA, IgG and IgM immunoglobulin levels and gave significantly higher anti-LPS and anti-protein antibody titres in all three major immunoglobulin classes than did non-typhoid controls. Levels of antibodies against LPS or protein in sera of typhoid patients were highly variable with a skew distribution. A good correlation was found between antibody titres to the LPS antigen and those to a protein antigen. No correlation, however, was found between the anti-LPS antibody titres measured by radioimmunoassay and the anti-O antibody titres measured by the Widal agglutination test. Titration of anti-LPS or anti-protein antibodies by radioimmunoassay was found to be more sensitive and specific than Widal test for the serological diagnosis of typhoid fever. The advantages of measuring antibody response by radioimmunoassay over conventional Widal test are discussed.     

Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans.

To determine whether lipopolysaccharide (LPS) structures of Campylobacter fetus are related to the three known heat-stable serogroups, proteinase K-treated whole cell lysates obtained from strains of each serogroup were electrophoresed in polyacrylamide gels. All strains had smooth-type LPS with multiple high-molecular-weight repeating units. The profiles of serogroup A from C. fetus subsp. fetus and from C. fetus subsp. venerealis were identical, but they were different from those of C. fetus subsp. fetus serogroups B and AB. When we immunoblotted the LPS of these serogroups with normal or immune rabbit serum we found homologous recognition between serogroups A from C. fetus subsp. fetus and C. fetus subsp. venerealis. Similarly, serogroups AB and B from C. fetus subsp. fetus showed homologous recognition. However, antiserum against serogroup A did not recognize serogroups B and AB and vice versa. Absorption studies confirmed the identity of LPS from all serogroup A C. fetus strains and cross-reactivity of the serogroup B and AB strains with one another. Serogroup A strains were resistant to the bactericidal activity in normal human serum, whereas serogroup B and AB strains generally were susceptible; isolates from humans predominantly belonged to serogroup A. Results of these studies suggest that the LPS composition forms the basis for the heat-stable serotyping system for C. fetus and that the structural and antigenic variants are associated with differential serum susceptibility.     

Monoclonal antibodies delineate multiple epitopes on the O antigens of Salmonella typhi lipopolysaccharide.

Fifteen monoclonal antibodies (MAbs) directed against Salmonella typhi were produced and characterized. The specificities of the antibodies were determined by their binding patterns in an enzyme immunoassay, with a panel of lipopolysaccharides isolated from different bacteria. Seven MAbs reacted with S. typhi, Salmonella enteritidis, and Salmonella dublin (all belonging to serogroup D). One MAb also reacted with Salmonella paratyphi A and S. paratyphi B. Five MAbs reacted with S. typhi, S. enteritidis, S. dublin, and S. paratyphi B. Two MAbs did not bind to any lipopolysaccharide but showed reactivity with bacterial sonic extracts isolated from S. typhi, S. paratyphi A, S. paratyphi B, Escherichia coli, and Shigella sonnei. These antibodies would be helpful in studying the complexity of antigenic determinants expressed by S. typhi and the nature of the antibody response during typhoid and paratyphoid fevers and also in the diagnosis of the disease.     

Molecular analysis of isolates of Salmonella typhi obtained from patients with fatal and nonfatal typhoid fever.

Molecular characterization of a total of 52 human isolates of Salmonella typhi from Papua New Guinea was performed by using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases, XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Of the 52 isolates tested, 11 were obtained from patients with fatal typhoid fever and 41 were obtained from patients with nonfatal disease. The 52 isolates showed limited genetic diversity as evidenced by only three different PFGE patterns detected following digestion with XbaI (patterns X1 to X3; F [coefficient of similarity] = 0.86 to 1.0), four patterns detected following digestion with AvrII (patterns A1 to A4; F =0.78 to 1.0), and two patterns detected following digestion with SpeI (patterns S1 and S2; F = 0.97 to 1.0). Of the 52 isolates, 37 were phage typed, and all belonged to phage type D2. All 11 isolates obtained from patients with fatal typhoid fever were identical (F = 1.0) and possessed the PFGE pattern combination X1S1A1, whereas the 41 isolates from patients with nonfatal typhoid fever had various PFGE pattern combinations, the most common being X2S1A2 (39%), X1S1A1 (24%), and X1S1A2 (15%). Thus, all the isolates from patients with the fatal disease had the X1 and A1 patterns, whereas the majority of the isolates from patients with nonfatal typhoid fever possessed the X2 and A2 patterns. The data suggest that there is an association among strains of S. typhi between genotype, as assessed by PFGE patterns, and the capability to cause fatal illness. Analysis of blood and fecal isolates of S. typhi from the same patient also indicated that some genetic changes occur in vivo during the course of infection.     

Detection of Salmonella typhi D, Vi, and d antigens, by slide coagglutination, in urine from patients with typhoid fever.

Salmonella typhi antigens D, Vi, and d were detected in the urine of 59 out of 61 (97%) bacteriologically confirmed typhoid fever patients by slide coagglutination with monovalent antisera coupled to protein A-rich staphylococci. These antigens were also detected in the urine of an additional 22 patients, 16 of whom subsequently demonstrated seroconversion by S. typhi O antibody agglutination, but from whom the bacterium was not isolated. The remaining 13 patients had negative urine coagglutination results, no isolation of S. typhi from blood or stool specimens, and no demonstration of seroconversion. These results suggest that the method of slide coagglutination of urine can be used to screen patients with suspected typhoid fever with a high degree of reliability. The method may also have potential importance in the diagnosis of typhoid when the bacterium is not isolated.     

Enzyme-linked immunosorbent assay for detection of Salmonella typhi Vi antigen in urine from typhoid patients.

Because typhoid fever continues to be a major cause of illness in many developing countries, there is a clear need for a sensitive and specific test that will permit rapid laboratory diagnosis of the disease. An enzyme-linked immunosorbent assay (ELISA) has recently been developed and tested, both in the laboratory and in a clinical situation, for its ability to detect Vi antigen in urine. The ELISA was capable of detecting as little as 1 ng of purified Vi antigen per ml in urine, compared with 100 ng/ml detectable by a previously tested coagglutination method. It could also detect antigen in urine diluted as much as 1:1,024 in normal urine. In tests of urine specimens from six stool culture-positive persons in a small typhoid outbreak in the United States, the ELISA detected antigen in specimens from four of the six patients. The ELISA also proved to be specific, giving no false-positive results for specimens from 50 persons who did not have typhoid fever. The apparent high sensitivity and specificity of this ELISA make it a promising test for rapid diagnosis of typhoid fever.     

TLR2 and TLR4 signaling shapes specific antibody responses to Salmonella typhi antigens

TLR directly induce innate immune responses by sensing a variety of microbial components and are critical for the fine‐tuning of subsequent adaptive immune responses. However, their impact and mechanism of action on antibody responses against bacterial antigens are not yet fully understood. Salmonella enterica serovar Typhi (S. typhi) porins have been characterized as inducers of long‐lasting specific antibody responses in mice. In this report, we show that immunization of TLR4‐deficient (TLR4^?/?), myeloid differentiating gene 88‐deficient and Toll/IL‐R domain‐containing adaptor‐inducing IFN‐β‐deficient mice with S. typhi porins led to significantly reduced B‐cell responses. TLR2^?/? mice, as well, showed reduced IgG titers with a more pronounced impairment in the production of IgG3 anti‐porins antibodies. Adoptive transfer of TLR2^?/?‐ or TLR4^?/?‐B cells into B‐cell‐deficient mice revealed a direct effect of TLR4 on B cells for the primary IgM response, whereas stimulation of B cells via TLR2 was important for IgG production. Furthermore, S. typhi porins were found to efficiently elicit maturation of CD11c⁺ conventional DC. Taken together, S. typhi porins represent not only a suitable B‐cell antigen for vaccination, but exhibit potent TLR‐dependent stimulatory functions on B cells and DC, which help to further enhance and shape the antibody response.     

Detection with monoclonal antibody of Salmonella typhi antigen 9 in specimens from patients.

Monoclonal antibodies were raised against Barber antigen (Ba) of Salmonella typhi 0901. Antibodies produced to antigen 9 of group D salmonellae were used in double- and triple-sandwich antibody enzyme-linked immunosorbent assays (ELISAs) for detecting antigen 9 in urine and plasma specimens from three groups of patients and 49 controls. The triple-antibody ELISA detected the antigen in urine samples from 11 of 18 (65%) patients with hemoculture-proven typhoid (group 1) and 12 of 39 (31%) patients with clinical features compatible with typhoid but whose hemocultures were negative (group 2). This ELISA was negative in three patients from whom Salmonella paratyphi A, Escherichia coli, and Klebsiella pneumoniae (group 3) were isolated by hemoculture and in all healthy controls. The double-antibody sandwich ELISA was positive in 41 and 15% of urine samples from patients in groups 1 and 2, respectively, and was negative with samples from two patients from group 3 and all controls. The sensitivity and specificity compared with those for healthy controls were 65 and 100%, respectively, for the triple-antibody ELISA. Although as little as 7.8 ng of homologous lipopolysaccharide could be detected, background in clinical specimens prevented accurate interpretation of the detection of this antigen in serum. Results were best with urine specimens.     

Induction and characterization of heat shock proteins of Salmonella typhi and their reactivity with sera from patients with typhoid fever.

The heat shock protein (HSP) response of Salmonella typhi following exposure to elevated growth temperatures was studied. Three major proteins with molecular sizes of 58, 68, and 88 kDa were abundantly expressed when S. typhi cells were shifted from 37 to 45 degrees C and to 55 degrees C. These proteins were also constitutively expressed at 37 degrees C. Western blotting and immunoprecipitation studies with anti-HSP monoclonal antibodies revealed that the 58- and 68-kDa proteins were analogous to the GroEL and DnaK proteins, respectively, of Escherichia coli. These HSPs are also abundantly present in the outer membrane fraction of disrupted cells and, to a lesser extent, in the cytosol. Immunoblotting experiments with sera from patients with a culture-positive diagnosis of typhoid fever showed the presence of antibodies to these HSPs. Nine of twelve sera reacted with the 58-, 68-, and 88-kDa proteins, while three sera reacted only with the 68- and 88-kDa proteins. All 10 sera from healthy individuals showed no binding to these HSPs. In light of the well-documented roles of HSPs in the pathogenesis of microbial infections and as immunodominant antigens, these findings may be relevant for a better understanding of disease processes and for the future development of diagnostic and preventive strategies.     

Lactose-fermenting, multiple drug-resistant Salmonella typhi strains isolated from a patient with postoperative typhoid fever

Two lactose-fermenting Salmonella typhi strains were isolated from bile and blood specimens of a typhoid fever patient who underwent a cholecystectomy due to cholelithiasis. One lactose-fermenting S. typhi strain was also isolated from a pus specimen which was obtained at the tip of the T-shaped tube withdrawn from the operative wound of the common bile duct of the patient. These three lactose-fermenting isolates: GIFU 11924 from bile, GIFU 11926 from pus, and GIFU 11927 from blood, were phenotypically identical to the type strain (GIFU 11801 = ATCC 19430 = NCTC 8385) of S. typhi, except that the three strains fermented lactose and failed to blacken the butt of Kligler iron agar or triple sugar iron agar medium. All three lactose-fermenting strains were resistant to chloramphenicol, ampicillin, sulfomethoxazole, trimethoprim, gentamicin, cephaloridine, and four other antimicrobial agents. The type strain was uniformly susceptible to these 10 drugs. The strain GIFU 11925, a lactose-negative dissociant from strain GIFU 11926, was also susceptible to these drugs, with the sole exception of chloramphenicol (minimal inhibitory concentration, 100 micrograms/ml).     

Simultaneous detection of Salmonella typhi antigen and antibody in serum by counter-immunoelectrophoresis for an early and rapid diagnosis of typhoid fever

Serum samples were collected from 26 proved cases and 15 clinically suspected cases of typhoid fever and from 23 normal controls. The convalescent phase serum samples were obtained in 18 of the 26 proved cases of typhoid fever. Salmonella typhi antigen and antibody were detected simultaneously in the serum samples by counter-immunoelectrophoresis (CIE). The conventional Widal test was also performed to elicit S. typhi H and O agglutinins. Out of the 26 cases proved by culture examination, 25 were detected during early stages of the disease (24 positive for S. typhi antigen and 1 for antibody) by CIE. Out of 15 clinically suspected cases of typhoid fever, 4 were positive for S. typhi antigen and none positive for antibody. All the 18 convalescent phase serum samples were positive for antibody, but were negative for S. typhi antigen. None of the 23 normal controls gave any false positive reaction for either antigen or antibody. In contrast, the Widal test could detect only 1 out of 26 cases in the acute stage, and all the 18 cases where convalescent serum samples were obtained were positive for antibodies at diagnostic titres. None of the 15 clinically suspected cases and 23 normal controls were positive by the Widal test. The feasibility of using simultaneous S. typhi antigen and antibody detection by CIE in diagnosis of typhoid fever is discussed.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

Epidemic typhoid in vietnam: molecular typing of multiple-antibiotic-resistant Salmonella enterica serotype typhi from four outbreaks

Multidrug-resistant Salmonella enterica serotype Typhi isolates from four outbreaks of typhoid fever in southern Vietnam between 1993 and 1997 were compared. Pulsed-field gel electrophoresis, bacteriophage and plasmid typing, and antibiotic susceptibilities showed that independent outbreaks of multidrug-resistant typhoid fever in southern Vietnam are caused by single bacterial strains. However, different outbreaks do not derive from the clonal expansion of a single multidrug-resistant serotype Typhi strain.     

Monoclonal antibodies against flagellar antigen of Salmonella typhi

Two hybrid cell clones secreting monoclonal antibodies against flagellar antigen isolated from Salmonella typhi, were produced and characterized. The antibodies bound specifically to the flagellar strain of S. typhi and did not show any reactivity with a flagellar S. typhi or with flagellar strains of S. dublin, S. paratyphi A, S. paratyphi B, S. typhimurium, E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The antibodies recognized a determinant present on a group of proteins migrating between 45 Kd and 60 Kd. These monoclonal antibodies would be useful reagents for clinical and epidemiological studies.     

Diagnosis of typhoid fever by detection of Salmonella typhi antigen in urine.

A monoclonal antibody specific for group D Salmonella antigen 9 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting the antigen in urine specimens collected from patients with clinical typhoid fever in Jakarta, Indonesia. The ELISA had a sensitivity of 95% in identifying patients in whom Salmonella typhi was isolated from hemocultures, 73% in patients in whom S. typhi was isolated from stool specimens, and 40% in patients in whom the organism was isolated from bone marrow cultures. Among patients in whom S. typhi was isolated from blood cultures, the ELISA had a sensitivity of 65% when a single urine specimen was examined and 95% when serially collected urine specimens were examined. A dot blot immunoassay performed on a nitrocellulose filter in parallel had a sensitivity of 85%, versus 83% for the plate ELISA in which S. typhi was isolated from blood, bone marrow, and/or stool specimens. Since S. typhi antigen is intermittently excreted in the urine of patients with typhoid fever, serially collected urine from patients with typhoid should be tested for antigen 9.     

Salmonella typhi VI antigen co-agglutination test for the rapid diagnosis of typhoid fever

A slide Co-agglutination test for the detection of Salmonella typhi Vi antigen in blood was evaluated for its efficiency in rapid diagnosis of Typhoid fever. The results were compared with conventional methods like Blood culture and Widal test. The test showed a sensitivity of 86.67% and specificity of 88.83% when compared with blood culture positivity or Widal titre above 160. This is a useful rapid diagnostic test for the early diagnosis of Typhoid fever.     

Duodenal isolation of Salmonella typhi by string capsule in acute typhoid fever.

Three of seven volunteers with acute typhoid fever had Salmonella typhi isolated from the duodenum using a string capsule device. The string capsule device provides a simple method for culturing S. typhi from the duodenal contents. Its possible use in typhoid carriers is discussed.