Chronic carriers of Salmonella Typhi act as reservoirs for the organism and become the agents of typhoid outbreaks in a community. In this study, chronic carriers in Kelantan, Malaysia were first identified using the culture and polymerase chain reaction method. Then, a novel serological tool, designated Typhidot-C, was evaluated in retrospect using the detected individuals as control positives. Chronic carriage positive by the culture and polymerase chain reaction method was recorded at 3.6% (4 out of 110) among individuals who previously had acute typhoid fever and a 9.4% (10 out of 106) carriage rate was observed among food handlers screened during outbreaks. The Typhidot-C assay was able to detect all these positive carriers showing its potential as a viable carrier screening tool and can be used for efficient detection of typhoid carriers in an endemic area. These findings were used to establish the first carrier registry for S Typhi carriers in Malaysia.
Signal transduction through protein-protein interactions and protein modifications are the main mechanisms controlling many biological processes. Here we described the implementation of MedScan information extraction technology and Pathway Studio software (Ariadne Genomics Inc.) to create a Salmonella specific molecular interaction database. Using the database, we have constructed several signal transduction pathways in Salmonella enterica serovar Typhi which causes Typhoid Fever, a major health threat especially in developing countries. S. Typhi has several pathogenicity islands that control rapid switching between different phenotypes including adhesion and colonization, invasion, intracellular survival, proliferation, and biofilm formation in response to environmental changes. Understanding of the detailed mechanism for S. Typhi survival in host cells is necessary for development of efficient detection and treatment of this pathogen. The constructed pathways were validated using publically available gene expression microarray data for Salmonella.
Salmonella osteomyelitis of the rib is a rare clinical entity. In our case, a muhidrug resistant Salmonella enterica serotype Typhi was isolated from an immuno-competent patient with osteomyclitis of the ribs, who was treated earlier with ciprotloxacin for typhoid fever. The patient was successfully treated for osteomyclitis with intravenous ceftriaxone.
The objective of this study was to develop an optimized assay for Salmonella Typhi biofilm that mimics the environment of the gallbladder as an experimental model for chronic typhoid fever. Multi-factorial assays are difficult to optimize using traditional one-factor-at-a-time optimization methods. Response surface methodology (RSM) was used to optimize six key variables involved in S. Typhi biofilm formation on cholesterol-coated polypropylene 96-well microtiter plates. The results showed that bile (1.22%), glucose (2%), cholesterol (0.05%) and potassium chloride (0.25%) were critical factors affecting the amount of biofilm produced, but agitation (275 rpm) and sodium chloride (0.5%) had antagonistic effects on each other. Under these optimum conditions the maximum OD reading for biofilm formation was 3.4 (λ600 nm), and the coefficients of variation for intra-plate and inter-plate assays were 3% (n = 20) and 5% (n = 8), respectively. These results showed that RSM is an effective approach for biofilm assay optimization.
Salmonella enterica serovar Typhi (S. Typhi) is a foodborne pathogen that causes typhoid fever and infects only humans. The ability of S. Typhi to survive outside the human host remains unclear, particularly in human carrier strains. In this study, we have investigated the catabolic activity of a human carrier S. Typhi strain in both planktonic and biofilm cells using the high-throughput Biolog Phenotype MicroArray, Minimum Biofilm Eradication Concentration (MBEC) biofilm inoculator (96-well peg lid) and whole genome sequence data. Additional strains of S. Typhi were tested to further validate the variation of catabolism in selected carbon substrates in the different bacterial growth phases. The analyzes of the carbon utilization data indicated that planktonic cells of the carrier strain, S. Typhi CR0044 could utilize a broader range of carbon substrates compared to biofilm cells. Pyruvic acid and succinic acid which are related to energy metabolism were actively catabolised in the planktonic stage compared to biofilm stage. On the other hand, glycerol, L-fucose, L-rhamnose (carbohydrates) and D-threonine (amino acid) were more actively catabolised by biofilm cells compared to planktonic cells. Notably, dextrin and pectin could induce strong biofilm formation in the human carrier strain of S. Typhi. However, pectin could not induce formation of biofilm in the other S. Typhi strains. Phenome data showed the utilization of certain carbon substrates which was supported by the presence of the catabolism-associated genes in S. Typhi CR0044. In conclusion, the findings showed the differential carbon utilization between planktonic and biofilm cells of a S. Typhi human carrier strain. The differences found in the carbon utilization profiles suggested that S. Typhi uses substrates mainly found in the human biliary mucus glycoprotein, gallbladder, liver and cortex of the kidney of the human host. The observed diversity in the carbon catabolism profiles among different S. Typhi strains has suggested the possible involvement of various metabolic pathways that might be related to the virulence and pathogenesis of this host-restricted human pathogen. The data serve as a caveat for future in-vivo studies to investigate the carbon metabolic activity to the pathogenesis of S. Typhi.
Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.
A dot enzyme immunoassay (EIA) using 50-kD outer-membrane proteins (OMPs) of Salmonella typhi was compared with the Widal test for the serodiagnosis of typhoid fever in 109 febrile children admitted to a hospital in an endemic area. In the culture-positive typhoid group, the initial dot EIA was positive in 40 of 42 cases and the initial Widal test was positive in 41. In the culture-negative clinical typhoid group, both the dot EIA and the Widal test were positive in 17 of 18 cases. In the nontyphoidal fever group, the dot EIA was negative in all of 49 cases and the Widal test was negative in 44. With culture used as the gold standard, the dot EIA is as sensitive as the Widal test (95% vs. 98%), has a similar high negative predictive value (96% vs. 98%), and is more specific (75% vs. 67%). In addition, the dot EIA offers the advantages of simplicity, speed, early diagnosis, economy, and flexibility (i.e., other diagnostic tests can be conducted simultaneously).
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.
A nitrocellulose membrane strip dotted with a specific 50 kDa outer membrane protein of Salmonella typhi was applied for the serodiagnosis of typhoid fever. Using horseradish peroxidase conjugated IgM and IgG antibodies with 4-chloronaphthol as substrate, antibodies in typhoid patients were clearly visualised as bluish purple dots while sera from patients with non-typhoid fevers gave negative results. The detection of specific IgM and IgG antibodies in typhoid patients suggest either recent or current infection. Combined with the high specificity, reliability and rapidity of the test, the dot EIA technique provides a simple and useful method for the serodiagnosis of typhoid using a single serum specimen.
Analysis of diarrhoeal disease patterns in Malaysia from 1981-1986 suggested that infectious hepatitis ranked as the most predominant diarrhoeal disease followed by typhoid, food poisoning, dysentery and cholera. Although these five major food and water-borne diseases are still endemic in this country, diarrhoeal diseases per se no longer become an important public health problem in Malaysia. Enforcement of the cholera control program brought the incidence of the disease to a minimal. Unfortunately, this fatal form of diarrhoeal disease caused the greatest mortality compared to the others. Seasonal influence also played a part in controlling the occurrence of the disease. There was a preponderance of diarrhoeal diseases during the rainy season implicating contaminated water as a source of transmission. Although greater than half of the population has been supplied with piped water and sanitary latrines, a lot more has to be done before diarrhoeal diseases could be eliminated from this country.
The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.
A simple adherence test to detect IgM antibodies in patients with typhoid is described. The test utilises the IgM-"capture" approach, in which the test serum is applied to microtitration plate wells previously coated with anti-human IgM, followed by application of a stained Salmonella typhi antigen suspension which shows adherence in positive cases. By this test, 58 (95%) of 61 sera from confirmed cases of typhoid possessed IgM antibodies to the H or O or both antigens of S. typhi. In patients for whom a diagnosis of typhoid was based only on a significant Widal-test titre, 31 (41%) of 76 sera had IgM antibodies to the H or O or both antigens of S. typhi. Some cross-reactivity of the IgM antibodies was detected, especially with the O antigens of S. paratyphi A and B. A total of 82 sera from non-typhoidal fevers (leptospirosis, typhus, dengue fever) showed no reactivity in this test. In normal sera there was no detectable IgM to the O antigen of S. typhi and only a small number (3.9%) had low levels of IgM to the H antigen. The significance and potential importance of this simple, sensitive, specific and economical test is discussed.
With the development of de novo binders for protein targets from non-related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single-chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking "disembodied" amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein-antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen-based detection agents for typhoid diagnostics.
This paper deals with two patients with typhoid fever in whom hepatic manifestations were the dominant and presenting features of the illness. The ability of typhoid hepatitis to simulate other common infectious diseases in this region is highlighted. It is recommended that typhoid hepatitis should be included in the differential diagnosis of patients presenting with fever and jaundice particularly in the tropics.
Salmonella enterica serovar Typhi is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths globally. Herein, we describe the whole-genome sequence of the Salmonella Typhi strain ST0208, isolated from a sporadic case of typhoid fever in Kuala Lumpur, Malaysia. The whole-genome sequence and comparative genomics allow an in-depth understanding of the genetic diversity, and its link to pathogenicity and evolutionary dynamics, of this highly clonal pathogen that is endemic to Malaysia.
Salmonella enterica subsp. enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Even though it is a human-restricted pathogen, the bacterium is also isolated from environments such as groundwater and pond water. Here, we describe the genome sequence of the Salmonella enterica subsp. enterica serovar Typhi PM016/13 which was isolated from well water during a typhoid outbreak in Kelantan, Malaysia, in 2013.
To find the incidence, markers and nature of complications of typhoid fever, we studied 102 children with cultures positive for Salmonella typhi in a cross-sectional study, prospectively, over a period of almost 5 years. All isolates were sensitive to commonly used antibiotics. One third of these children developed complications which included: anicteric hepatitis, bone marrow suppression, paralytic ileus, myocarditis, psychosis, cholecystitis, osteomyelitis, peritonitis, pneumonia, haemolysis, and syndrome of inappropriate release of antidiuretic hormone (SIADH). Twelve children developed multiple complications. If hepatitis is excluded from the complications, the rate of complications in bacteriologically confirmed cases of typhoid fever drops to 11 per cent. These complications were not related to: the age or sex of patients, duration of illness before admission, use of antibiotics before admission, nutritional status, level of 'O' or 'H' titre, presence of IgM or IgG antibodies, or treatment with chloramphenicol or ampicillin. Children with splenomegaly, thrombocytopenia or leukopenia were more likely to develop complications.
Typhoid fever remains a common problem in Malaysia, but for its diagnosis both blood culture and the Widal test have drawbacks. A dot enzyme immunoassay (EIA) has been developed which detects IgM and IgG antibodies to a specific 50 kDa outer membrane protein on Salmonella typhi. This study was performed among outpatients attending the university hospital in Kelantan, a state on the east coast of Peninsular Malaysia where typhoid is endemic. The dot EIA was done on 149 outpatients of all ages in whom typhoid was suspected. Of these, 60 were not analysable due to insufficient data. The other 89 were retrospectively classed as typhoid (total = 21), or not typhoid (total = 68). The criteria for diagnosis of typhoid was either, blood culture was positive, or with blood culture negative, temperature was at least 38 degrees C and Widal O and/or H titer greater than or equal to 1/160. We then compared the diagnosis with the EIA result. For the result where either IgM or IgG was positive, sensitivity was 90%, specificity 91% and negative predictive value 97%. For IgM positive, specificity was 100%. But the specificity of IgG positive alone was reduced by six false positives, which were probably due to persistence of IgG after acute infection. Other cases were found where IgG positive alone appeared in the first week of typhoid fever, probably due to rapid response in a second or subsequent infection. We also found that IgM-producing patients were significantly younger than those showing IgG alone positive.
Study site: Community Medicine clinic, Accident & emergency department, Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia