Typhoid fever & vaccine development: a partially answered question.

Typhoid fever is a systemic disease caused by the human specific Gram-negative pathogen Salmonella enterica serovar Typhi (S. Typhi). The extra-intestinal infections caused by Salmonella are very fatal. The incidence of typhoid fever remains very high in impoverished areas and the emergence of multidrug resistance has made the situation worse. To combat and to reduce the morbidity and mortality caused by typhoid fever, many preventive measures and strategies have been employed, the most important being vaccination. In recent years, many Salmonella vaccines have been developed including live attenuated as well as DNA vaccines and their clinical trials have shown encouraging results. But with the increasing antibiotic resistance, the development of potent vaccine candidate for typhoid fever is a need of the hour. This review discusses the latest trends in the typhoid vaccine development and the clinical trials which are underway.

Immunoblot analysis of sera in uncomplicated typhoid fever & with typhoid ileal perforation.

Background & objectives: Ileal perforation is a serious complication of typhoid fever. The exact reasons for the development of perforation in only a few of those infected with Salmonella Typhi is unknown, and it is likely that immunological factors are involved. Therefore we undertook this study to compare the antibody profile in patients with uncomplicated typhoid fever with those having ileal perforation by immunoblotting. Methods: Two groups of patients were included in the study. Group II comprised patients with uncomplicated typhoid fever (n=47), and group I with typhoid ileal perforation (n=33). The flagellar (H), lipopolysaccharide (LPS) and outer membrane protein (OMP) antigens of Salmonella Typhi were extracted and used to test patient sera for antibodies by immunoblotting Results: Immunoblotting using S. Typhi antigens enabled the detection of S. Typhi antibodies in the two groups of patients. A significant difference was seen in the response of these two groups of patients with respect to antibodies to flagella, lipopolysaccharide and outer membrane proteins. Antibodies to flagella were more pronounced among patients with uncomplicated typhoid fever, while anti-OMP antibodies were significantly associated with typhoid ileal perforation. Interpretation & conclusions: A comparison of antibodies in patients with uncomplicated typhoid fever and with ileal perforation revealed the differences in the antibody profiles of the two groups. Our study suggests that the difference in antibody response may in some way play a role in the pathogenesis of typhoid ileal perforation which can also potentially be exploited to develop suitable diagnostic tests.

Baseline Widal titres in healthy children.

OBJECTIVE: To determine the baseline Widal titres in apparently healthy children in Davangere. METHODS: Cross-sectional study was done on 250 children. Widal titers were found using tube agglutination test. RESULTS: Out of 250,64.2% had a titre of less than 1:20,22.4% had a titre equal to 1:20,9.6% had a titre of 1:40 and 3.6% had a titre of 1:80 to 'O' antigen and 67.2% had a titre of less than 1:20, 21.2% had a titre equal to 1:20, 8% had a titre of 1:40 and 3.6% had a titre of 1:80 to 'H' antigen of S. enterica subsp. enterica ser. Typhi. No children in age group 6 months-2 years had a titre of 1:80 to either antigen. All children in this age group had a titre of less than 1:20 to AH antigen and older children had a titre upto 1:40 dilution. CONCLUSION: Baseline titres for either S. enterica subsp. enterica ser. Typhi antigen in 6 month-2 year was 1:40 and older children was 1:80. Baseline titres for H antigen of S. enterica subsp. enterica ser. Paratyphi A in 6 month-2 year was less than 1:20 and for older children was 1:40 dilution.

Immunological, cellular and molecular events in typhoid fever.

Salmonella, a facultative intracellular Gram-negative bacterium infects a wide range of hosts causing several gastrointestinal diseases and enteric fever in humans and certain animal species. Typhoid caused by Salmonella typhi remains a major health concern in India and worldwide. Also, with emergence of multidrug resistant strains, Salmonella has acquired increased virulence, communicability and survivability, resulting in increased morbidity and mortality. Though a number of vaccines for typhoid are available against S. typhi (or also against S. typhimurium), these have certain undesirable side effects and the search for new immunogens suitable for vaccine formulation is still continuing. The immune response to primary Salmonella infection involves both humoral and cell-mediated responses. The protective immunity against Salmonella depends on host- parasite interaction, however; the detailed mechanism of virulence, innate resistance and susceptibility of host remains unclear. This review focuses on the molecular, immunological and cellular mechanisms of pathogenesis of Salmonella infection to provide an insight to counteract bacterial infections and allow a better understanding of its clinical manifestations. It also reviews better technological possibilities combined with increased knowledge in related fields such as immunology and molecular biology and allow for new vaccination strategies. Some new approaches such as subunit and nucleic acid vaccines and recombinant antigen which are becoming increasingly important for the development of potential vaccines have also been discussed. A significant progress has been made in our understanding of Salmonella pathogenesis. Despite these efforts, however, many challenges exist, especially for investigators who aim to understand how the pathogenic mechanisms operating in vitro apply to in vivo model systems. However, unyielding work and collaborations between Salmonella researchers and clinicians worldwide have made significant contributions to understanding the interaction between virulence determinants and immunity required to stop the spread of this pathogen.

Prospective evaluation of a rapid diagnostic test Typhidot for typhoid fever.

BACKGROUND & OBJECTIVE: Typhoid fever still continues to be a major public health problem around the world. A simple, reliable and affordable rapid diagnostic test has been a long felt need of the clinicians. We therefore prospectively evaluated the sensitivity and specificity of Typhidot test. METHODS: The study was carried out between January 2002 and December 2003, on a total of 563 samples from patients clinically suspected to have typhoid fever; blood culture as well as serum for Typhidot test were received. RESULTS: Of the 563 samples, Typhidot test and blood culture were positive in 36 patients, both the tests were negative for 503 patients. Typhidot test was positive for 9 patients with S. Paratyphi A infection. The sensitivity and specificity of the test using blood culture as gold standard were 92.3 and 98.8 per cent respectively for the typhoid fever. INTERPRETATION & CONCLUSION: Typhidot test is rapid, easy to perform and reliable test for diagnosing typhoid fever, useful for small less equipped laboratories as well as for those with better facilities.

The role of epidemiology in the introduction of vi polysaccharide typhoid fever vaccines in Asia.

Despite the availability of at least two licensed typhoid fever vaccines--injectable sub-unit Vi polysaccharide vaccine and live, oral Ty21a vaccine--for the last decade, these vaccines have not been widely introduced in public-health programmes in countries endemic for typhoid fever. The goal of the multidisciplinary DOMI (Diseases of the Most Impoverished) typhoid fever programme is to generate policy-relevant data to support public decision-making regarding the introduction of Vi polysaccharide typhoid fever immunization programmes in China, Viet Nam, Pakistan, India, Bangladesh, and Indonesia. Through epidemiological studies, the DOMI Programme is generating these data and is offering a model for the accelerated, rational introduction of new vaccines into health programmes in low-income countries. Practical and specific examples of the role of epidemiology are described in this paper. These examples cover: (a) selection of available typhoid fever vaccines to be introduced in the programme, (b) generation of policy-relevant data, (c) providing the 'backbone' for the implementation of other multidisciplinary projects, and (d) generation of unexpected but useful information relevant for the introduction of vaccines. Epidemiological studies contribute to all stages of development of vaccine evaluation and introduction.

Comparison of the immunogenicity and safety of two different brands of Salmonella typhi Vi capsular polysaccharide vaccine.

BACKGROUND: The recent emergence of multi-drug-resistant Salmonella strains highlights the need for better preventive measures, including vaccination. Safe and immunologic vaccines have been developed based on purified Vi polysaccharide. OBJECTIVE: To compare the immune response elicited by two different brands of Salmonella Vi capsular polysaccharide vaccine (ViCPS). SETTING AND DESIGN: Double blind, randomized (3:1), controlled, parallel, phase III study was conducted at two centres in India to compare the safety and immunogenicity of Typbar, the investigational vaccine with an already marketed vaccine "X", in healthy subjects aged between 12 -25 years. MATERIAL AND METHODS: A sample size of 184 subjects was calculated. Subjects were randomly distributed in two groups, immunized with single dose of Typbar or Vaccine "X". Serum samples were taken before 7 days and 4 weeks after immunization for the determination of antibodies to Vi polysaccharide, by ELISA method. Safety was assessed by physical examination, laboratory parameters before and after vaccination and by monitoring adverse events. Statistics: The geometric mean antibody titre (GMT) 4 weeks after vaccination was compared from respective pre-vaccination values by Wilcoxon signed rank test. Geometric mean of antibody levels before and after immunization and the ratio between them (Mann-Whitney test), the Seroconversion rates (Z test of proportions) and the adverse events (Fisher's exact test and Chi square test), were compared between two groups. P value < 0.05 was considered statistically significant. P values and 95% confidence intervals were estimated in two-tailed fashion. RESULTS: 153 subjects (Typbar =116 and Vaccine "X" =37) were studied. 71.6% (95% CI=63.4%-79.8%) and 75.7% (95% CI=64.9% - 89.5%) were the seroconversion rates with Typbar and vaccine "X" respectively. The GMT values for Vi antibodies induced after Typbar and vaccine "X" were 10.23 Typbar and 13.46 mg/mL respectively and these values showed high significance when compared to their respective pre-immunization GMT values (P<0.0001) at 95% CI (-10.49 to -7.19 mg/mL for Typbar and -14.69 to -8.86 mg/mL for Vaccine "X"). The induction of antibody response appeared to be slightly stronger (P=0.032) with vaccine "X" when compared to that of Typbar. This is justifiable as the same group also had high pre-immunization GMT values (P=0.021). CONCLUSION: The immunogenicity and safety of the investigational vaccine Typbar was found to be similar to that of already marketed brand of Vi CPS, Vaccine "X". The availability of a single dose of vaccine that is safe and effective enhances the prospective for control of typhoid fever.

An unusually high occurrence of Salmonella enterica serotype paratyphi A in patients with enteric fever.

BACKGROUND & OBJECTIVES: Salmonella enterica serotype Paratyphi A has been reported less frequently as a causative agent of enteric fever. Reports on the antimicrobial susceptibility of this pathogen are few and varied. An unusually high occurrence of S. Paratyphi A was noted in a tertiary care hospital at Nagpur, Maharashtra during April 2001-September 2002. An effort was made to study the antimicrobial susceptibility pattern and phage types of the isolates. METHODS: Blood cultures of patients suspected to have enteric fever admitted to the Indira Gandhi Medical College and Hospital, Nagpur were processed by conventional methods. Antimicrobial susceptibility was tested by Kirby-Bauer disc diffusion method and the minimum inhibitory concentration (MIC) to chloramphenicol was determined. RESULTS: Eighteen (46.15%) of 39 Salmonella isolates were S. Paratyphi A and all were sensitive to ciprofloxacin and cephotaxime. Twelve (66.67%) strains were sensitive to ampicillin and 13 (72.22 %) to chloramphenicol. Two strains (11.11%) were resistant to three drugs (ampicillin, chloramphenicol and cotrimoxazole) simultaneously. The prevalent phage type in the local population was phage type I. INTERPRETATION & CONCLUSION: The high occurrence of S. Paratyphi A found in the present study indicated the emergence of this rare pathogen of enteric fever in the local population. Though some degree of resistance was encountered with ampicillin and chloramphenicol, all the isolates were sensitive to ciprofloxacin, currently a drug of choice for enteric fever. Multidrug resistance was rare.

Lipid profiles in typhoid fever patients

The serum Total Lipid [TL] concentrations in healthy adults and patients with typhoid fever were compared in order to detect the possible contribution of Lipid A component of Salmonella typhi endotoxins [Lipopolysaccharides] to the serum lipid concentration. A total of 177 hydrolysates of sera were evaluated. Eighty of the sera were obtained from apparently healthy adults, while 97 were from patients with symptoms of typhoid fever, who had S. typhi O antibody titre of equal or greater than 160. The results show that there was a significant difference [p<0.001] between the mean serum TL level of 253.1 +/- 84.7 mg/dl obtained in the healthy adults and the mean value of 949.5 +/- 542.5 mg/dl in the patients with typhoid fever. There was no significant difference [p>0.05] between the mean IL levels in males and females among both the healthy subjects and the patients. The findings suggest the diagnostic significance of lipid profiles in typhoid fever

Antibody response in typhoid fever in endemic Indonesia and the relevance of serology and culture to diagnosis.

Culture and serology were performed on blood and serum samples collected at or shortly after admission from 473 patients presented with suspected clinical typhoid. Clinical symptoms at first presentation including confusion, hepatomegaly, splenomegaly, abdominal pain, anemia, and gastrointestinal bleeding were non-specific as they were observed even more often in non-typhoid patients. Culture confirmed the diagnosis in 65.3% of the patients with typhoid fever as the final diagnosis. The sensitivity (58%) and specificity (98.1%) of a rapid dipstick assay for the detection of S. typhi-specific immunoglobulin M were somewhat lower than those of culture but higher than those of the Widal test. The dipstick assay thus may well be used in the serodiagnosis of typhoid in situation where culture facilities are not available. Combination of test results of dipstick and culture improved sensitivity to 82.5%. In laboratories that perform blood culture the dipstick assay may be used as a rapid screening tests to facilitate a rapid diagnosis. Sensitivity of the dipstick assay strongly increased with duration of illness and was higher for culture positive than for culture negative patients. Duration of illness, and different pathogen and host factors including dose of infection, pathogenicity and antigenicity, and prior antibiotic use are likely to influence the immune response, therefore the result of the dipstick assay. Duration of illness and presence of S. typhi in the blood are major factors that determine severity of disease.

Typhidot test to detect IgG & IgM antibodies in typhoid fever.

BACKGROUND & OBJECTIVES: As typhoid fever is endemic in India, there is a continuing search for a simple test which can be carried out in small laboratories for an early and rapid diagnosis. We have evaluated the Typhidot test for this purpose. METHODS: The Typhidot test was carried out on coded sera according to the manufacturer's instructions. The test was performed on 30 Widal positive sera, 30 sera from blood culture positive patients, 60 Widal negative sera and 30 samples from patients whose blood culture grew Gram negative bacilli (GNB) other than Salmonella Typhi. RESULTS: Typhidot test was positive for both IgG and IgM in 39 samples, IgM alone in 24 and IgG alone in 2. Of the 30 culture positive samples, 27 were positive by Typhidot. The Typhidot test gave a sensitivity of 100 per cent and specificity of 80 per cent when bacteraemic patients were analysed. INTERPRETATION & CONCLUSION: The Typhidot is easy to perform, and requires no special equipment or training of staff for interpretation of results. It will be a useful complementary test to blood culture and the Widal test in the diagnosis of typhoid fever.

Electrophoretic analysis of Salmonella typhi and other bacteria.

A comparative study involving SDS-PAGE of Salmonella typhi and other Bacteria was conducted. Protoplasmic antigens of Salmonella typhi. Salmonella paratyphi A, Salmonella typhimurium, Proteus sp, Klebsiellas sp. Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus were separated and compared on SDS-PAGE followed by checking of their cross reactivity by gel diffusion using antisera raised against whole cell and lysates of Salmonella typhi. Lines of identity between Salmonella typhi, Salmonella paratyphi A and Salmonella typhimurium were observed. No lines of identity were seen among Salmonella typhi, Pseudomonas aeruginosa and Sfaphylococcus aureus.

Molecular modelling of epitope presentation using membrane protein OmpC.

Three-dimensional models of the chimeric S. typhi OmpC protein carrying an epitope from rotavirus VP4 capsid protein on either of two exposed loops (fourth and sixth) were constructed separately, using computer-aided homology modelling. The theoretical model of S. typhi OmpC was used as a template. The monomers were initially energy minimized. The trimers were generated for both the chimeric S. typhi OmpC proteins and the structures were optimized after several cycles of minimization. The surface accessibility calculations for the resulting models show that epitope recognition should be more effective in the fourth loop than in the sixth loop, in accordance with the experimental results on the immunogenic nature of the rotaviral epitope inserted into the two putative loops of S. typhi OmpC.

Diagnostic value of dot-enzyme-immunoassay test to detect outer membrane protein antigen in sera of patients with typhoid fever.

Typhoid fever is still an important public health problem in many developing countries especially in tropical parts of the world, as in Indonesia. This problem opens the way for a further study with the aim of finding an alternative serological test with a high degree of reliability for the detection of typhoid fever. Given the above mentioned purpose, a study on the reliability of a laboratory test, the dot-enzyme-immunoassay outer membrane protein (DOT-EIA-OMP) was conducted comprising sera from 207 subjects (44 adult typhoid patients, 43 adult nontyphoid patients and sera from 120 adult healthy individuals serving as controls. The result of the study revealed that the diagnostic sensitivity of the DOT-EIA-OMP test for the detection of typhoid fever can be classified as high (93.16%), the specificity as moderate (76.74%), the efficiency (accuracy), positive predictive value and negative predictive value as high (85.06%, 80.39% and 91.66% respectively). The within run and between days reproducibility of this test was very high (CV=0%). Analysis of data obtained indicated that the DOT-EIA-OMP test was a reliable screening test for the establishment of the diagnosis of typhoid fever in health centers with simple laboratory facilities. The application of this test has to be more contemplated in countries where the cost of laboratory test is a problem.

Salmonella. typhi OMPs induced immunomodulation in peritoneal macrophages.

The immunomodulatory properties of outer membrane proteins (OMPs) from S. typhi Ty2 were studied in mouse model at 72 hr and 20 days post-infection. Inspite of reduction in the number of macrophages and their protein content observed in the immunized group vis-à-vis infected group, OMPs activated macrophages showed significant upregulation of NO. At 20 days post infection, the level remained almost the same suggesting the prolonged cytotoxic and cytostatic activity due to the long lasting effects of OMPs activated macrophages. Higher activity of SOD in these aged cells pointed out towards the protective efficacy of OMPs to keep the macrophages themselves away from the noxious effects of O2-. Lower level of acid phosphatase in the macrophages from immunized mice group indicated the involvement of oxygen dependent rather than oxygen independent killing process. The enhanced uptake of organisms and their killing could be related to the production of oxygen and nitrogen radicals in the OMPs immunized group.