The rapid diagnosis of salmonellosis by a determination of the antigen in the biosubstrates of patients using the coagglutination reaction]

The diagnostic potential of the coagglutination test was checked with the aim of improving the laboratory diagnosis of Salmonella infections by the detection of Salmonella specific antigen in different biological materials (feces, urine, saliva and immune complexes in blood sera). The study of all specimens resulted in the confirmation of the diagnosis in 78% of patients, often during the first days of the disease. The proportion of nonspecific reactions, as shown in the control groups of healthy donors and patients with dysentery and other acute enteric infections, did not exceed 5%.     

Latent persistence of specific antigens of the causative agent in the composition of circulating immune complexes in typhoid fever

This study revealed the presence of O-antigen of group D salmonellae and Vi-antigen in circulating immune complexes in patients with typhoid fever, bacteriologically confirmed (56 +/- 5.6% and 65 +/- 5.4% of cases, respectively) and not confirmed (15.5 +/- 5% and 39 +/- 7% of cases, respectively), in patients with diarrhea of nontyphoid etiology in the presence of negative results of the coagglutination test and in healthy persons. The level and dynamics of circulating immune complexes were established with respect to the antigens contained in these complexes. Altogether O- and Vi-antigens were detected in circulating immune complexes, respectively, in 92%, 96% and 94% of cases on weeks 1, 2 and 3 from the beginning of the disease, and in all cases on weeks 4 and 5, the antigens being determined together and separately. Thus, the latent persistence of S. typhi antigens as part of circulating immune complexes in the blood serum was established. The determination of such persistence is of great pathogenetic and diagnostic importance, which also applies to the early period of the disease. The use of such specific and sensitive method as the coagglutination test for this purpose accelerates and facilitates the diagnosis of typhoid fever.     

Development of specific cellular immunoreactivity in typhoid fever.

Development of cellular immunoreactivity to Salmonella typhi and Salmonella paratyphi-A was studied by the leukocyte migration inhibition test in 9 patients with typhoid fever and in 2 patients with paratyphoid fever. Cellular reactivity could be demonstrated from the first days of the disease in all the subjects. The most pronounced migration inhibition was observed during the febrile period. It is suggested that specific cellular reactivity may play a pathogenetic role in typhoid fever.     

Diagnostic significance of the coagglutination reaction in patients with the diarrhoea syndrome

Specific soluble Shigella, Salmonella and Yersinia enterocolitica antigens were determined in biological fluids (saliva, urine, coprofiltrate) from 268 patients with the diarrhoea syndrome using the coagglutination reaction. The findings suggest that the coagglutination reaction (COA) is a simple and efficient method suitable for the fast diagnosing of acute intestinal infection (AII) in the early days from the onset of the disease. COA enables the identification of specific antigens associated with the causative agents of intestinal infections in 79-54% of patients with shigellosis and salmonellosis. COA was shown to possess a high diagnostic potential in AII of unknown etiology. Shigella, Salmonella and Yersinia antigens were determined in 47.7, 23.4 and 10.8% of cases respectively where no bacterial excretion could be confirmed. Two and less frequently three antigens being identified simultaneously in 10.8% cases. The identification of opportunistic microorganisms in AII using the COA does not appear to be sufficient to confirm their etiological significance as Shigella and Salmonella antigens were simultaneously determined in most patients.     

Detection of typhoid fever by Widal and indirect fluorescent antibody (IFA) tests. A comparative study

Widal test is a conventional method for the detection of typhoid fever. However, it takes 18-24 hours to complete the test. In the present study indirect fluorescent antibody test has been compared with the Widal test using single serum specimens and was found to be rapid, sensitive and specific. Serum specimens from 41 culture proven cases of typhoid fever, 14 clinically suspected cases and 22 normal individuals were collected. Whereas Widal test detected 63.41% positive cases, IFA test detected 87.80% from among culturally proven typhoid cases. Among the clinically suspected cases of typhoid fever, IFA test detected 85.71% (28.57 + 57.14%) while Widal test detected only 57.13% (35.71 + 21.42%) positive cases out of above 14 cases.     

Coagglutination test and its application to the rapid diagnosis of meningococcal meningitis

Modified technique of slide coagglutination test for detecting meningococcal group-specific antigens in the spinal fluid of patients with meningococcal meningitis has been developed. Precipitating meningococcal sera, groups A, C, X, Y, Z, were conjugated with formalin-treated staphylococcal cells, strain Cowan-I. To prevent nonspecific reactions, 5-minute boiling of the spinal fluid specimens is suggested. 111 specimens of spinal fluid were taken from 75 patients at different periods of the disease. All patients were administered antibiotics, and therefore the etiology of the disease was bacteriologically confirmed only in 31% of patients. Coagglutination test was positive in 56.7% of patients, the frequency of positive results reaching 71% during the first 4 days of the disease. The specimens of spinal fluid taken from the control group of patients yielded not more than 2% of the positive results. Coagglutination test is recommended as a rapid test for diagnosing meningococcal meningitis.     

An immunoblotting as a method for the diagnosis of typhoid fever

The patients' sera had been referred to the National Salmonella Centre for routine Widal serology. Sera were predominately from patients suspected of having been infected with Salmonella Typhi, but also included one serum from patient with typhoid fever who was culture positive for Salmonella Typhi. The immunoblotting procedure using Salmonella Typhi somatic (O=9,12 LPS) and flagellar (H=d) antigens was used for preliminary testing of selected patients sera previously evaluated by Widal agglutination assay as containing different levels of antibodies against O and/or H antigens of Salmonella Typhi. Following Chart et al., immunoblotting reactions were graded between 0 and 3, with 0 indicating an absence of antibody binding, and 3 where antibody binding was readily observed. Sera giving reaction of 2 or 3 were considered to be antibody positive for this study. Positive immunoblotting reaction to O=9,12 LPS antigen was obtained only with the serum of patient with typhoid fever. Presence of specific anti-LPS antibodies was also observed in two other patients' sera diluted 1:50, and in case of one of them also in dilution 1:200, but intensity of antigen-antibody reaction was under positive result criterion. The most other sera positive to O=9,12 antigen in law dilutions (1:50, 1:100) by Widal assay, showed the traces of non-specific reaction by immunoblotting. Presence of positive antigen-antibody reaction was indicated for five sera in dilution 1:50 when tested with the >55 kDa H=d flagellar protein subunit, including the serum of patient with typhoid fever. Only in this serum the high level of specific antibodies was detected also in dilution 1:200, what was not observed in case of the other four, which appeared negative. All the other sera were shown not to contain antibodies to flagella antigen. Although the presented results are preliminary and additional study of more sera of people infected with Salmonella Typhi is needed, it can be concluded after Chart et al., that an immunoblotting procedure incorporating O=9,12 LPS and flagellar H=d antigens is a useful method for providing serological evidence of infection with Salmonella Typhi. In our opinion it can serve as a rapid test for the diagnosis of typhoid fever.     

Demonstration of an antigenic protein specific for Salmonella typhi

Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.     

Transient inhibition of glucose utilization by erythrocytes during the acute stage of typhoid fever

The exact reason for hemolysis of glucose-6-phosphate dehydrogenase-deficient (G6PD) erythrocytes in patients with typhoid fever is unknown. Therefore, glucose utilization by normal and G6PD-deficient erythrocytes was measured during incubation with plasma of healthy controls as well as from patients in acute or recovery stages of typhoid fever. Glucose utilization in normal and G6PD-deficient erythrocytes significantly decreased compared to the controls when incubated with plasma of patients with acute typhoid fever, which normalized to the baseline after recovery from typhoid fever, suggesting an acquired alteration in G6PD enzyme properties by Salmonella typhi or its endotoxins.     

Confirmatory identification of Neisseria gonorrhoeae by slide coagglutination

Neisseria gonorrhoeae was identified by the Phadebact gonococcus test, a rapid slide coagglutination technique, and the results obtained were compared with those obtained by conventional methods (Gram stain morphology, oxidase reaction, and carbohydrate utilization tests) for the confirmatory identification of gonococci. Of 308 clinical isolates examined, the coagglutination procedure correctly identified 97.8% of the isolates tested as N. gonorrhoeae and 93.9% of other bacteria as not N. gonorrhoeae. The coagglutination procedure also identified 29 laboratory strains correctly as not N. gonorrhoeae. The slide coagglutination test is easy to perform and offers a valuable alternative to other techniques for the confirmatory identification of N. gonorrhoeae.     

Immunofluorescent Identification of Salmonella typhi During a Typhoid Outbreak

Immunofluorescence and conventional bacteriological methods were compared for their ability to detect Salmonella typhi in 134 fecal specimens from 105 individuals associated with an outbreak of typhoid fever. Smears prepared from untreated fecal material (direct method) and after a preliminary incubation in selenite F broth (delayed method) were tested with an anti-Vi serum conjugated with fluorescein isothiocyanate. The delayed method was more sensitive than the direct method in detecting S. typhi. The delayed method was positive in 40 of 41 patients positive by culture methods, but gave positive or questionable reactions in 11 presumably uninfected individuals. The fluorescent-antibody test employing a Vi conjugate is a satisfactory screening procedure for detecting S. typhi, but all positives must be confirmed bacteriologically.     

Combined rapid (TUBEX) test for typhoid-paratyphoid A fever based on strong anti-O12 response: design and critical assessment of sensitivity

Rapid diagnostics can be accurate but, often, those based on antibody detection for infectious diseases are unwittingly underrated for various reasons. Herein, we described the development of a combined rapid test for two clinically-indistinguishable bacterial diseases, typhoid and paratyphoid A fever, the latter fast emerging as a global threat. By using monoclonal antibodies (mAbs) to bacterial antigens of known chemical structures as probes, we were able to dissect the antibody response in patients at the level of monosaccharides. Thus, a mAb specific for a common lipopolysaccharide antigen (O12) found in both the causative organisms was employed to semi-quantify the amounts of anti-O12 antibodies present in both types of patients in an epitope-inhibition particle-based (TUBEX) immunoassay. This colorimetric assay detected not only anti-O12 antibodies that were abundantly produced, but also, by steric hindrance, antibodies to an adjoining epitope (O9 or O2 in the typhoid or paratyphoid bacillus, respectively). Sensitivity and, particularly, reaction intensities, were significantly better than those obtained using an anti-O9 or anti-O2 mAb-probe in the examination of paired sera from 22 culture-confirmed typhoid patients (sensitivity, 81.8% vs 75.0%) or single sera from 36 culture-confirmed paratyphoid patients (52.8% vs 28.6), respectively. Importantly, sensitivity was better (97.1% for typhoid, 75.0% for paratyphoid) if allowance was made for the absence of relevant antibodies in certain specimens as determined by an independent, objective assay (ELISA)--such specimens might have been storage-denatured (especially the older paratyphoid samples) or procured from non-responders. Benchmarking against ELISA, which revealed high concordance between the two tests, was useful and more appropriate than comparing with culture methods as traditionally done, since antibody tests and culture target slightly different stages of these diseases. Paired sera analysis was insightful, revealing 64% of typhoid patients who had no change in antibody titer over 4-16 days, and 14% with no IgM-IgG class-switching.     

Surveillance for typhoid fever in Matsuyama city during 1974-1981 and detection of Salmonella typhi in sewage and river waters

Eighty-two cases of typhoid fever were found in Matsuyama city in the period from 1974 to 1981. Seventy-six cases were found to be infected with Salmonella typhi other three with Salmonella paratyphi A, and the remaining three were diagnosed only clinically. The strains of S. typhi isolated from these patients showed such a variety of Vi-phage types as D1, D2, E1, M1, 53 and degraded Vi-positive strain (DVS). The concurrent survey of the city sewage and river waters for typhoid bacilli was conducted with total 578 samples taken therefrom. S. typhi was isolated from 120 of those samples. The Vi-phage types of the isolates were closely related with those of the isolates from the patients. The periodical examinations of the city sewage and the draining river may serve as a useful means for the controlling typhoid fever epidemics.     

The complex serologic diagnosis of typhoid fever

A parallel serological study of the blood sera of typhoid patients has been made by the methods of countercurrent immunoelectrophoresis and the indirect hemagglutination test with a view to establish the presence of soluble typhoid antigens and their corresponding antibodies. As shown in this study, the occurrence of Salmonella typhi O- and Vi-antigens is essentially higher than the content of specific antibodies in diagnostically significant titers.     

Leukocyte migration inhibition by a specific antigen in human typhoid fever.

Cellular reactivity to heat-killed Salmonella typhi antigen was investigated by the leukocyte migration inhibition (LMI) method in 33 S. typhi infected patients and in 32 control persons. In the typhoid group a statistically significant LMI was observed as compared to the members of the control group. A correlation was found between the level of the cellular sensitivity and the time elapsed between onset of the disease and performance of the test. Previous typhoid vaccination had no influence on the LMI. No correlation was found between the agglutinin titres and the sensitivity demonstrated by the LMI test. The value of the method in studies of cellular immunity in typhoid fever is discussed.     

Isolation of a highly purified capsular polysaccharide from Salmonella enterica serovar Typhi (Salmonella typhi)--Vi-antigen and its use in serological diagnosis of typhoid fever

For use in differential diagnostics of typhoid fever, samples of the capsular polysaccharide from Salmonella enterica serovar Typhi (usually named Vi-antigen) were isolated and characterized by physicochemical and serological methods. It was shown that only the sample of Vi-antigen with the minimal (0.57%) admixture of the corresponding lipopolysaccharide (LPS) from S. typhi retained a high serological activity in the tests with monoreceptor anti-Vi sera. However, it exhibited a substantially weaker reaction with sera from normal donors and patients with acute nontyphoid salmonelloses, than Vi-antigen preparations with a higher (0.8-1.2%) LPS content. The chromatographically pure Vi-antigen was purified by triple reprecipitation with hexadecyltrimethylammonium bromide. The content of the LPS admixture in the resulting Vi-antigen samples was quantitatively determined by GC. A high purification level of the Vi-antigen from the LPS admixture allows us to hope that this preparation could serve as a basic component of the test system for the diagnostics of typhoid fever. The English version of the paper.     

Clinical and bacteriological profiles of patients with typhoid fever treated during 1975-1998 in the Tokyo Metropolitan Komagome Hospital

Patients with typhoid fever presenting to the Tokyo Metropolitan Komagome Hospital during the period 1975-1998 were retrospectively investigated. All cases were diagnosed by a positive culture for Salmonella typhi in either of their clinical specimens. Of the total number of 130 patients, 57% contracted the disease abroad; this population increased in later years as the total numbers of cases decreased. The period from disease onset to diagnosis averaged 14 days with 20% of the cases requiring over three weeks to establish a diagnosis. As for symptomatology relative bradycardia was seen in less than half of the cases, and rose spots or splenomegaly in less than one third. A positive blood culture was the most frequent test establishing the diagnosis followed by a positive stool culture. Intestinal bleeding was recognized in as many as 35 cases (27%) and even intestinal perforation occurred in two cases (1.5%). Chloramphenicol was most commonly employed during the early study period, however, during the late period it was replaced by fluoroquinolones. The clinical cure rate was 98% with regimens that include fluoroquinolones/quinolone; however it was 87% with the other antimicrobial regimens. Bacteriological relapse occurred in 25% of the non-fluoroquinolone group while only in 2.0% in the fluoroquinolone/quinolone group. Four strains of Salmonella typhi that were multi-resistant to chloramphenicol, ampicillin and cotrimoxazole were isolated in travelers from Asia. Early diagnosis by appropriate bacteriological examination regardless of classical symptomatology should be stressed and the use of fluoroquinolones is warranted in the treatment of typhoid fever.     

Salmonella vaccines for use in humans: present and future perspectives

In recent years there has been significant progress in the development of attenuated Salmonella enterica serovar Typhi strains as candidate typhoid fever vaccines. In clinical trials these vaccines have been shown to be well tolerated and immunogenic. For example, the attenuated S. enterica var. Typhi strains CVD 908-htrA (aroC aroD htrA), Ty800 (phoP phoQ) and chi4073 (cya crp cdt) are all promising candidate typhoid vaccines. In addition, clinical trials have demonstrated that S. enterica var. Typhi vaccines expressing heterologous antigens, such as the tetanus toxin fragment C, can induce immunity to the expressed antigens in human volunteers. In many cases, the problems associated with expression of antigens in Salmonella have been successfully addressed and the future of Salmonella vaccine development is very promising.     

Serotyping of Pseudomonas aeruginosa strains by slide coagglutination

Serological typing of Pseudomonas aeruginosa strains (228 strains) by slide coagglutination, using our own reagents (5 polyvalent and 22 monovalent ones, corresponding to the 22 serotypes in Meitert-Meitert scheme), led to identical results obtained by conventional slide agglutination. Utilization of live Ps. aeruginosa cells suspensions, killed by boiling or autoclaving, showed a 100% concordance of results, when using the second and the third suspension types and a 97.37% one between them and the live cells suspension. We noticed that reactions intensity was higher when using bacterial suspensions, boiled for 2.5 hours, in comparison with autoclaved cells suspensions, 30 minutes at 120 C. Compared to conventional slide agglutination, the slide coagglutination presents more advantages, being simple, rapid, specific and economical.     

Determination of Salmonella typhimurium antigens in the excreta of children with salmonellosis using the passive hemagglutination reaction

1,390 samples of different excreta obtained from salmonellosis patients have been tested for the presence of S. typhimurium O- and H-antigens. S. typhimurium antigens, detected with the use of antibody diagnostica, have been found to occur more frequently than S. typhimurium cells. Particulate O- and H-antigens capable of agglutinating antibody diagnostica are excreted differently with saliva and urine. Salmonella antigens are best detected in feces in the passive hemagglutination test with the use of antibody diagnostica, but not in the antibody neutralization test. The combination of the passive hemagglutination test, carried out with the use of antibody diagnostica, and bacteriological study considerably enhances the efficiency of diagnosing salmonellosis in children in comparison with bacteriological study alone.