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.     

Determination of antibody from typhoid patients against lipopolysaccharide and protein antigens of Salmonella typhi

Although the Widal test is simple, inexpensive and the most widely used for serodiagnosis of typhoid fever, the sensitivity and specificity of the test is sometimes doubtful. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum IgG and IgM antibodies to protein and lipopolysaccharide (LPS) antigens of Salmonella typhi which was compared with the Widal test in various groups of subjects. In typhoid patients with hemocultures positive for S. typhi (TP group), ELISA positivity was found on 100% for IgG antiprotein, 94.44% for IgG anti-LPS and 88.89% for IgM to both the protein and LPS antigens. In contrast, the Widal test was positive in only 61.11% for anti-O and 83.33% for anti-H antibodies. In healthy control subjects (HC group), only 5% of serum samples were positive for IgG anti-protein and none was positive for IgG anti-LPS or IgM to either the protein or LPS. In contrast, the Widal test was positive in 7.5% of HC group for anti-O and 17.5% for anti-H antibodies. In blood bank donors (BB group), both ELISA and Widal tests were positive in 23-40% of sera. Since the hospital records of BB group were incomplete. It might be possible that some of these subjects had recently been infected with S. typhi. Our data indicate that the standard Widal test was associated with false negative reactions in 16-39% of blood culture positive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serology of typhoid fever in an area of endemicity and its relevance to diagnosis

Currently, the laboratory diagnosis of typhoid fever is dependent upon either the isolation of Salmonella enterica subsp. enterica serotype Typhi from a clinical sample or the detection of raised titers of agglutinating serum antibodies against the lipopolysaccharide (LPS) (O) or flagellum (H) antigens of serotype Typhi (the Widal test). In this study, the serum antibody responses to the LPS and flagellum antigens of serotype Typhi were investigated with individuals from a region of Vietnam in which typhoid is endemic, and their usefulness for the diagnosis of typhoid fever was evaluated. The antibody responses to both antigens were highly variable among individuals infected with serotype Typhi, and elevated antibody titers were also detected in a high proportion of serum samples from healthy subjects from the community. In-house enzyme-linked immunosorbent assays (ELISAs) for the detection of specific classes of anti-LPS and antiflagellum antibodies were compared with other serologically based tests for the diagnosis of typhoid fever (Widal TO and TH, anti-serotype Typhi immunoglobulin M [IgM] dipstick, and IDeaL TUBEX). At a specificity of > or =0.93, the sensitivities of the different tests were 0.75, 0.55, and 0.52 for the anti-LPS IgM, IgG, and IgA ELISAs, respectively; 0.28 for the antiflagellum IgG ELISA; 0.47 and 0.32 for the Widal TO and TH tests, respectively; and 0.77 for the anti-serotype Typhi IgM dipstick assay. The specificity of the IDeaL TUBEX was below 0.90 (sensitivity, 0.87; specificity, 0.76). The serological assays based on the detection of IgM antibodies against either serotype Typhi LPS (ELISA) or whole bacteria (dipstick) had a significantly higher sensitivity than the Widal TO test when used with a single acute-phase serum sample (P < or = 0.007). These tests could be of use for the diagnosis of typhoid fever in patients who have clinical typhoid fever but are culture negative or in regions where bacterial culturing facilities are not available.     

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.     

A simple bactericidal antibody test for sero-diagnosis of typhoid fever

Serum samples were collected from 24 confirmed cases of typhoid fever, 15 clinically suspected cases and 23 normal healthy controls. The convalescent sera were obtained in 13 of the 24 confirmed typhoid cases. In all, 13 paired sera, 11 acute phase only, 15 clinically suspected and 23 normal serum samples were tested for eliciting bactericidal antibodies to Salmonella typhi. In addition, the Widal test was also performed for comparison. All the 24 acute phase sera as well as 13 convalescent sera were found to be positive by bactericidal antibody test (titre 1:80 or above). Of 15 clinically suspected cases, 5 were positive whereas one of the 23 normal controls sera gave a false positive reaction. In contrast, the Widal test could detect only one of the 24 cases in the acute stage, but all 13 cases showed antibodies at a diagnostic titre level during the convalescent stage. None of the 15 clinically suspected cases or 23 normal controls were positive by the Widal test. The feasibility of using a bactericidal antibody test in sero-diagnosis of typhoid fever is discussed.     

The serodiagnosis of infection with Salmonella typhi

BACKGROUND/AIMS: The serodiagnosis of infection with Salmonella typhi, using the Widal agglutination assay, relies on patients' antibodies to the O = 9,12 lipopolysaccharide (LPS) antigens, H = d flagellar antigens, and the Vi capsular antigens. A Vi agglutination titre of > 1/40 has traditionally been regarded as indicative of recent infection with S typhi. In this study, 91 sera were used to assess the reliability of the Widal agglutination assay based on antibodies to the Vi antigens. METHODS: The Widal agglutination assay was carried out using protocols established by the Central Public Health Laboratory, Colindale. Antibodies to the Vi capsular antigen were detected using a standard preparation of S typhi, ViI Bhatnagar variant strain (S typhi, ViI). Sera used in the study comprised 73 from patients who were culture positive for S typhi, 10 from patients who were culture positive for other species of Salmonella not expressing a Vi antigen (namely, S javiana, S enteritidis, S typhimurium, S stanley, S saint paul, S bareilly, or S mbandaka), and eight from healthy blood donors. RESULTS: Agglutination titres of > or = 1/40 were detected to S typhi ViI in 69 of 73 sera from patients with typhoid, although 27 of these also agglutinated an unrelated control antigen. The Widal assay also detected significant amounts of agglutinating antibodies to S. typhi ViI in all eight control sera and seven sera from patients infected with S bareilly, S enteritidis, S javiana, S mbandaka, S saint paul, and S stanley. CONCLUSIONS: Agglutinating antibodies to the Vi antigen can be detected by the Widal assay, but even with the appropriate control antigens the results were unreliable. The serodiagnosis of infections with S typhi should be based on the detection of antibodies to both the O = 9,12 LPS antigen and the H = d flagellar antigen by immunoblotting, and should not use the Vi antigen-based Widal assay. Conclusions should be made in the light of patients' clinical details and any knowledge of previous immunisation for typhoid.     

One-Step 2-Minute Test To Detect Typhoid-Specific Antibodies Based on Particle Separation in Tubes

Typhoid fever is caused by Salmonella typhi. Detection of anti-S. typhi antibodies in the patient is a useful diagnostic aid. Among the various methods developed over the years for this purpose, the Widal test, based on bacterial agglutination, has remained the most widely used, even though it is neither specific nor sensitive. Its popularity stems from the fact that it is simple to use and inexpensive. We describe a new test which also uses a simple one-step procedure but is more rapid and accurate than the Widal. The new test (TUBEX) detects anti-Salmonella O9 (both immunoglobulin M [IgM] and IgG) antibodies in patients by inhibiting the binding between an anti-O9 IgM monoclonal antibody (MAb) conjugated to colored latex particles and S. typhi lipopolysaccharide (LPS) conjugated to magnetic latex particles. The reactants are mixed in a specially designed microtube for 2 min, and the result is read based on the resultant color of the supernatant following forced sedimentation of the magnetic beads. In the absence of inhibitory antibodies, there is a color change (from blue to red) due to cosedimentation of the indicator particles with the magnetic particles, whereas if these antibodies are present, they prevent such a change to a degree dependent on their concentration. Preliminary examination of TUBEX using the anti-O9 MAb and irrelevant MAbs as inhibitors revealed the test to be specific and reproducible, with an analytical sensitivity of 16 μg per ml of antibody. The reagents remained stable for at least 9 months when kept at 4°C. In the examination of 16 stored sera obtained from 14 patients with proven cases of typhoid fever and 78 serum samples from 75 subjects without typhoid fever, TUBEX was found to be 100% sensitive and 100% specific. The nontyphoid group comprised 26 healthy blood donors, 30 antinuclear antibody (ANA)-negative patients, 9 ANA-positive patients, of whom 1 was positive for anti-DNA antibody, 4 typhus patients, and 6 septicemic patients. In addition, the sera obtained from 11 patients clinically diagnosed as having typhoid fever were all positive in the test. The TUBEX results correlated to some extent, albeit insignificantly (r = 0.38, P = 0.07), with those of an enzyme-linked immunoassay (ELISA) which used a similar detection format (inhibition) and reagents (S. typhi LPS and anti-O9 antibody). TUBEX correlated very well with ELISAs which detected anti-S. typhi LPS IgM (r = 0.58, P = 0.003) or IgG (r = 0.54, P = 0.006) antibodies from the typhoid patients. There was no correlation with the Widal test. The TUBEX test, if performed on slides (instead of tubes) or with soluble antigen (instead of antigen-conjugated magnetic beads), suffered significantly in sensitivity. Direct agglutination tests using LPS-conjugated indicator particles performed either on slides or in microwells also failed to detect antibodies from the majority of typhoid patients. Thus, TUBEX appears to be well designed and well suited for use in the laboratory or by the bedside as a simple, rapid aid to the routine diagnosis of typhoid fever.     

Comparison of passive haemagglutination test with Widal agglutination test for serological diagnosis of typhoid fever in an endemic area.

A passive haemagglutination test, using sheep red blood cells sensitised with Salmonella typhi lipopolysaccharide, was compared with the Widal test for the serological diagnosis of typhoid fever in an endemic area. The results obtained on sera from 152 patients with bacteriologically confirmed typhoid and 183 patients who did not have typhoid were analysed in terms of sensitivity, specificity, simplicity, and rapidity of the respective tests. The passive haemagglutination test was found to be more sensitive (80%) than the S typhi O antigen (71%) but marginally less sensitive than the H antigen (82%) of the Widal test. The false positive rate on control sera was 1.2% and 6.6%, respectively, for the Widal O and H antigens, and 1.6% for the passive haemagglutination test. Our findings indicate that the passive haemagglutination test is comparable with the Widal test for the serological diagnosis of typhoid fever in endemic areas, but is more simple, rapid, and economic. The passive haemagglutination test may be a useful alternative to the Widal test for the serological diagnosis of typhoid fever in busy microbiology laboratories in areas in which the disease is endemic.     

Serological investigations on Indian kala-azar.

Detailed serological investigations were carried out in forty-nine active kala-azar (KA) cases in North Bihar, India. Various classes of immunoglobulin (IgG, IgA, and IgM) and third component of complement (C3) levels were determined in these sera and results were compared with those obtained in normal controls. Antibody titres were determined by the indirect haemagglutination (IHA) method using soluble Leishmania antigen. Immunoglobulin G and M class-specific antibody titres were also determined separately by the enzyme-linked immunosorbent assay (ELISA) method. Polyclonal hypergammaglobulinaemia with marked increase in serum IgG (and to a lesser extent in IgM) level was a rather common feature in the majority of these sera. Much of this immunoglobulin increase, however, appeared to be non-specific in nature and no absolute correlation could be noted between serum IgG or IgM levels and corresponding IgG or IgM antibody titres. Significant decrease in serum C3 level was observed in KA and this decrease was found to be independent of immunoglobulin levels or specific antibody titres. A fairly good correlation between aldehyde test and serum IgG level was evident from this study. Aldehyde-positive KA sera usually gave higher antibody titres than aldehyde-negative ones. Anti-leishmanial antibodies belonged mostly to IgG class although some IgM antibodies were also demonstrable. The latter class of antibodies probably appeared early in KA infection although their serological specificity was poorer to IgG antibodies. Out of forty-nine KA sera examined in this study thirty-six (73.5%) gave positive IHA titres while forty-six (93.9%) were positive by IgG-ELISA which appeared to be a highly specific and sensitive serodiagnostic method particularly for the early detection of KA cases.     

Retrospective study to determine the diagnostic value of the Widal test in a non-endemic country

The purpose of this study was to determine the usefulness of the Widal test in the diagnosis of typhoid fever. Data were obtained by retrospective analysis of 311 Widal requests covering a six-year period. Nine cases of typhoid infection were diagnosed culturally. Of these, only three patients had samples for serological examination, all giving indicative titres. Of the 274 evaluated sera, 26 showed significant agglutinating titres; 23 of them were false positive. These results show that routine use of the Widal test is of limited value and should only be used for patients in whom repeated cultures remain negative.     

Development of immune response during typhoid fever in man.

The development of both humoral and cell-mediated immune responses (CMIR) to antigens prepared from Salmonella typhi was investigated in patients suffering from typhoid fever. The antibodies were determined by the standard Widal test while the leucocyte migration test was used for CMIR. These immunological parameters were correlated with the duration of illness, the duration of chloramphenicol therapy and the severity of the illness. It was found that CMIR appeared after the first week of illness in uncomplicated cases of typhoid fever, where as it remained negative in the patients who had complications. The antibody titres were similar in the two groups. On further follow up of complicated cases, the clinical recovery coincided with the development of CMIR. It may be concluded that for recovery in typhoid fever CMIR is more important than antibodies.     

Determination by Widal Agglutination of the Baseline Titre for the Diagnosis of Typhoid Fever in Two Nigerian States

This study was carried out in Borno and Plateau States of Nigeria to determine the baseline titre for the diagnosis of typhoid fever using a single Widal test. Of 172 patients with symptoms and signs of typhoid fever, 92.4% and 90.7% had reciprocal O and H antibody titres respectively of 160 or above. On the other hand, 95.3% and 66.3% of the 937 healthy control subjects had reciprocal O and H antibody titres respectively of 80 or less. The results of this study suggest that in Borno and Plateau States of Nigeria a reciprocal O antibody titre of 160 and above in persons with illness whose symptoms and signs are compatible with typhoid fever could be considered diagnostic using the single Widal test.     

Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Antibodies to Pseudomonas aeruginosa from 10 cystic fibrosis patients with chronic P. aeruginosa lung infections were quantitatively and qualitatively analyzed. The development of specific antibodies in patient serum was evaluated in a longitudinal study (1972 to 1987). The concentrations and specificities of immunoglobulin G (IgG) and IgM antibodies to purified lipopolysaccharides (LPS) from clinical isolates of P. aeruginosa and to a variety of other gram-negative bacteria were studied by immunoblotting and enzyme-linked immunosorbent assay techniques. Results were compared with the number of immunoprecipitates to P. aeruginosa whole-cell extracts detected by crossed immunoelectrophoresis. IgG, but not IgM, anti-Pseudomonas LPS concentrations increased significantly at the onset of chronic infection and continued to increase during the course of the infection. There was a good positive correlation between the concentration of IgG anti-Pseudomonas LPS antibodies and the number of crossed-immunoelectrophoresis precipitins. The increases in IgG anti-LPS antibody concentrations were much higher to Pseudomonas LPS than to other LPSs. Binding studies demonstrated an increase in binding of IgG anti-Pseudomonas LPS during infection, whereas the binding of other anti-LPS antibodies decreased. Immunoblotting studies confirmed that antibodies reacted strongly with Pseudomonas LPS and weakly with Escherichia coli core-lipid A. The specificity of the reaction with Pseudomonas LPS increased with the duration of infection. It is concluded that anti-LPS response in cystic fibrosis patients during chronic P. aeruginosa infection demonstrates a marked increase in IgG anti-Pseudomonas LPS antibody concentration, specificity, and affinity. The anti-LPS enzyme-linked immunosorbent assay is proposed as a routine test to diagnose and to follow the course of chronic P. aeruginosa lung infection in patients with cystic fibrosis.     

Studies of cellular and humoral immunity in typhoid fever and TAB vaccinated subjects.

An assessment of both humoral and cell-mediated immune responses to Salmonella typhi antigens in patients with acute typhoid infection, TAB inoculated subjects and in healthy controls is reported. Cell-mediated immunity as assessed by the leucocyte migration inhibition test (LMI), and developed in all cases with typhoid fever. Positive LMI was evident in the first week of the illness and was maintained during the evolution of disease and in some patients was still present after a year. It also developed at the end of 3 weeks in five out of nine TAB vaccinated subjects. Weakly positive LMI was noticed in only two of twenty asian and caucasian controls. Antibodies, determined by the standard Widal test, were significantly raised in both patients with typhoid fever and TAB inoculated subjects. The antibodies and cellular reactivity developed almost simultaneously but there was no correlation between the agglutination titres and LMI positivity, implying that they are independent of each other. Typhoid patients also showed significantly raised serum IgM and IgA levels and increased concentrations of secretory IgA in their saliva.     

Comparison of diffusion-in-gel enzyme-linked immunosorbent assay with conventional serological methods for detection of class-specific antibodies to Salmonella typhi O antigen

The diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) is a new and simple method for quantitation of antibodies, based on the ability of antibodies to diffuse from wells in gel and adsorb to antigen which is bound to a polystyrene surface. The antigen-antibody reaction is visualized with a color reaction caused by horseradish peroxidase-conjugated class-specific anti-immunoglobins. This method was used to study the immunoglobulin G, A, and M immune response to Salmonella typhi O antigen in individuals immunized with a monovalent heat-inactivated typhoid vaccine. The antibody values obtained by the DIG-ELISA method correlated with those evaluated by conventional direct agglutination (Widal) and indirect hemagglutination methods. The DIG-ELISA method was also found to be sensitive, specific, and economical, as well as suitable for handling large numbers of sera while requiring very simple equipment.     

Differentiation between active and inactive human brucellosis by measuring antiprotein humoral immune responses.

A preparation of Brucella abortus cytoplasmic proteins was depleted of lipopolysaccharide (LPS) by immunoadsorption with a monoclonal antibody (MAb), BC68, specific for the O antigen of B. abortus smooth LPS. Two enzyme-linked immunosorbent assay (ELISA) systems were developed and used in this study. The first system includes an LPS-free cytoplasmic protein preparation; the second one was based on antigen capture on MAb BC68. By using these systems, we have demonstrated that 94% (33 of 35) of the brucellosis patients studied showed immunoglobulin G antiprotein response and also that all of the patients showed a strong anti-LPS reactivity. Thirty-six serologically positive individuals with no active infection at the time of examination (SPI) were also included. No immunoglobulin G antibodies against proteins were detected in 34 of them (92%), whereas 31 SPI (86%) showed various degrees of anti-LPS reactivity. The use of the LPS-free protein extract in ELISAs made it possible to establish differential reactivity patterns between active and inactive brucellosis.     

Smooth muscle antibody in Burkitt''s lymphoma and in nasopharyngeal carcinoma.

Smooth muscle antibodies (SMA) with specificity for actin, were found with a higher frequency in sera from Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC) patients than in sera from matched controls. No correlation could be found between SMA and anti-Epstein-Barr virus (EBV) antibody titres. There was no parallelism, in individual sera, between the finding of SMA and the occurrence of cold lymphocytotoxins, aother antibody activity found with an abnormally high frequency among BL and NPC patients. The reason why actin, a weak antigen in experimental animals, may become immunogenic in humans remains unexplained.     

Early diagnosis of typhoid fever by an enzyme immunoassay usingSalmonella typhi outer membrane protein preparations

An enzyme immunoassay (EIA) for detection of serum antibodies in patients with typhoid fever was developed usingSalmonella typhi outer membrane protein (OMP) preparations as antigen. Acute phase (first week) sera from adult typhoid fever patients were tested as well as sera from the following control groups: adult travellers with diarrhea caused by enterotoxigenicEscherichia coli, children infected withCampylobacter jejuni, healthy Mexican adult blood donors, and adults with septicemia caused by other organisms. At a 1:3,125 serum dilution, the mean absorbance values were 1.41 in the typhoid fever patients, and 0.57, 0.55, 0.51 and 0.52 in the respective control groups. Inhibition EIA studies using OMP preparations or lipopolysaccharide (LPS) as free antigen indicated that proteins can play an important role in the detection of antibodies in early typhoid fever. This EIA may be useful for the diagnosis of typhoid fever since results were obtained within about five hours and in an endemic area antibodies againstSalmonella typhi OMP preparations appear early in the course of the disease.     

Monoclonal antibodies to 52-kilodalton protein of Salmonella typhi.

Ten monoclonal antibodies (6 immunoglobulin G1 kappa [IgG1 kappa] and 4 IgG2b kappa) from six hybrid clones specific for Salmonella typhi antigen were produced by immunizing BALB/cJ mice with affinity-purified S. typhi proteins (Bp). The latter were prepared by passing crude S. typhi Bp through an affinity column made from Sepharose conjugated to IgG antibodies against partially purified S. typhi Bp. The eluent was subsequently used as the immunogen for the production of monoclonal antibody. All 10 monoclonal antibodies reacted specifically with a 52-kilodalton (kDa) protein of S. typhi and were species specific. The presence of IgM antibody to the 52-kDa antigen in the sera of a majority of patients with acute typhoid fever suggested that this 52-kDa protein is also a good immunogen for humans. The potential usefulness of this antigen in the early diagnosis of typhoid fever is discussed.     

Typhoid is over-reported in Embu and Nairobi, Kenya

The paper looks at the usefulness of the Widal agglutination test in the context of variable normal antibody titres in two different populations in Kenya, and in comparison to the blood culture method of diagnosis. It presents a prospective case-control study. We examined 846 blood cultures and an equal number of serum samples, and 782 stools from adults who presented at two study sites; Kenyatta National Hospital and one hospital and 3 clinics in Embu District, with symptoms similar to typhoid. Examined also were 360 serum samples and stools from adults who were apparently healthy (controls) who sought routine medical examination at the study sites. From blood cultures, isolation rates for typhoid for Embu (3% ) and Nairobi (2.2%) were not significantly different (p>0.01). In addition the control population from the two study sites did not show any significant background O antibody titre levels characteristic of typhoid endemic areas. All the 7 commonly available Widal test kits including Murex, Europath, Biotech, Humatex, Biosystems, Microsystems and Typhex, that were evaluated for efficacy were equally specific in diagnosis of typhoid by Widal agglutination methods. However, there were minor differences in the sensitivities of the kits. The Widal test method gave a lower sensitivity (81.3%) than specificity (93%) when compared to the culture of blood for diagnosis of typhoid. Going by the reports of typhoid outbreaks in Embu and Nairobi (ca. 20-25% reported prevalence) we conclude that there has been over-reporting probably due to poor methodologies of performing the Widal test. We recommend adequate clinical examination in suspected cases of typhoid in addition to proper Widal in order to improve typhoid diagnosis. Newer improved methods that are more specific and sensitive than the Widal test need to be evaluated in improving laboratory diagnosis of typhoid.