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Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.

A multiplex rapid detection system, based on a PCR-lateral flow biosensor (mPCR-LFB) was developed to identify Salmonella Typhi and Salmonella Paratyphi A from suspected carriers. The lower detection limit for S. Typhi and S. Paratyphi A was 0.16 and 0.08 ng DNA equivalent to 10 and 102 CFU/mL, respectively. Lateral flow biosensor was used for visual detection of mPCR amplicons (stgA, SPAint, ompC, internal amplification control) by labeling forward primers with fluorescein-isothiocyanate (FITC), Texas Red, dinitrophenol (DNP) and digoxigenin (DIG) and reverse primers with biotin. Binding of streptavidin-colloidal gold conjugate with the amplicons resulted in formation of a red color dots on the strip after 15–20 min of sample exposure. The nucleic acid lateral flow analysis of the mPCR-LFB was better in sensitivity and more rapid than the conventional agarose gel electrophoresis. Moreover, the mPCR-LFB showed 100% sensitivity and specificity when evaluated with stools spiked with 100 isolates of Salmonella genus and other bacteria. A prospective cohort study on stool samples of 1176 food handlers in outbreak areas (suspected carriers) resulted in 23 (2%) positive for S. Typhi. The developed assay has potential to be used for rapid detection of typhoid carriers in surveillance program.

The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.

Conventional polymerase chain reaction (PCR) testing requires many pipetting steps and has to be transported and stored in cold chain. To overcome these limitations, we designed a ready-to-use PCR test for Salmonella typhi using PCR reagents, primers against the ST50 gene of S. typhi, a built-in internal amplification control (IAC), and gel loading dye mixed and freeze-dried in a single tube. The 2-step dry-reagent-based assay was used to amplify a 1238-bp target gene and an 810-bp IAC gene from 73 BACTEC blood culture broths (33 true positives for S. typhi and 40 true negatives for non-S. typhi). The sensitivity, specificity, positive predictive value, and negative predictive value of the PCR assay were 87.9%, 100%, 100%, and 90.9%, respectively. We suggest that this rapid 2-step PCR test could be used for the rapid diagnosis of typhoid fever.

An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

In this work we assessed the suitability of two different lipid membranes for the simulation of a TolC protein from Salmonella enterica serovar Typhi. The TolC protein family is found in many pathogenic Gram-negative bacteria including Vibrio cholera and Pseudomonas aeruginosa and acts as an outer membrane channel for expulsion of drug and toxin from the cell. In S. typhi, the causative agent for typhoid fever, the TolC outer membrane protein is an antigen for the pathogen. The lipid environment is an important modulator of membrane protein structure and function. We evaluated the conformation of the TolC protein in the presence of DMPE and POPE bilayers using molecular dynamics simulation. The S. typhi TolC protein exhibited similar conformational dynamics to TolC and its homologues. Conformational flexibility of the protein is seen in the C-terminal, extracellular loops, and α-helical region. Despite differences in the two lipids, significant similarities in the motion of the protein in POPE and DMPE were observed, including the rotational motion of the C-terminal residues and the partially open extracellular loops. However, analysis of the trajectories demonstrated effects of hydrophobic matching of the TolC protein in the membrane, particularly in the lengthening of the lipids and subtle movements of the protein’s β-barrel towards the lower leaflet in DMPE. The study exhibited the use of molecular dynamics simulation in revealing the differential effect of membrane proteins and lipids on each other. In this study, POPE is potentially a more suitable model for future simulation of the S. typhi TolC protein.

The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.

In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.

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.

Introduction Kelantan remains as the highest endemic area in Malaysia for typhoid cases with the incidence rate of 3.29 and 2.8 per 100,000 populations in 2008 and 2010. Salmonella Typhi is the causative pathogen of this illness which transmitted through fecal oral route, causing human is the only known reservoir for this organism. However, some typhoid cases related to contaminated water had also been reported. Objective To investigate the genetic diversity and relatedness of Salmonella Typhi isolated from water sample, and stool samples from food handlers and contacts in typhoid outbreak locality, Pasir Mas, Kelantan. Methods Nine Salmonella Typhi isolated from food handlers and contact while another one isolated from water sample were obtained from Makmal Kesihatan Awam Kota Bharu Kelantan between March to April 2013 and confirmed by biochemical test and serotyping. Pulsed field gel electrophoresis (PFGE) was performed according to CDC PulseNet Protocol and Bio-Rad CHEF MAPPER XA System. Dice Coefficient of Similarity (F value) was calculated for all isolates and a dendogram were constructed using Fingerprinting QuestTM software. The result was compared with previous data collected and interpreted according to the guidelines of Tenover et. al. (1995). Results & Discussion All nine isolates from food handlers and contacts showed similar PFGE pattern with the isolate from water sample, suggesting the disease was transmitted through water samples from well which is located in the typhoid outbreak area. Moreover, the PFGE pattern was unique and had never been found in S. Typhi previously isolated in Kelantan according to our PFGE database. Conclusion There are two possibilities of the source of the outbreak. The carriers among food handlers and contact might contaminated the water and causing the outbreak or vice versa.

Introduction Salmonella Typhi (S.Typhi) is a causative agent for typhoid fever, a major health problem especially in developing countries. In Malaysia, the disease is still an endemic with the occurrence of 1–4 cases per 100,000 populations reported from year 1996–2006. Early detection of the agent is important since immediate treatment given to the patients will save many lives. However, current methods for the diagnosis of S.Typhi did not satisfy the requirements for a rapid, simple, and cost-effective detection mode. Objective Our study is aimed to develop an in-house LAMP method for a rapid, sensitive, specific and cost-effective detection of S.Typhi. Methods An In-house method of LAMP was developed and optimized using genomic DNA of Salmonella Typhi ATCC7251. The test was further evaluated for its specificity, sensitivity and application on field samples. The result was compared with those obtained by PCR assay and culture method. Results & Discussion In-house LAMP method has been successfully developed and optimized. This LAMP method was shown to be more sensitive than PCR assay and highly specific where no cross-reactivity was observed with other tested bacteria. Results of LAMP on clinical samples were in accordance to the results obtained by PCR assay and culture method. Conclusion In-house LAMP method developed in this study can potentially be used as a rapid, sensitive, specific and cost-effective detection of Salmonella Typhi especially at low-resource settings. However, this LAMP method need to be further validated using larger number of clinical samples.

Introduction Salmonella Typhi is the causative agent for typhoid fever that infects people especially in underdeveloped and developing countries. Even though culture method is the gold standard for detection of this pathogen, there are some limitations such as time consuming and less sensitive that leads to the development of a more sensitive and efficient method such as polymerase chain reaction (PCR). Objective To evaluate the sensitivity of a multiplex PCR-DNA dipstick assay incomparison with culture method and EZTyphi Carrier DNA assay for the detection of S.Typhi in well water samples. Methods A total of 580 well water samples were collected and filtered using filter membrane. Bacteria trapped on the membrane was enriched in a selective Selenite F and evaluated using these three methods; (i) Culture method: Bacteria culture was plated on a selective media agar plate and further tested by biochemical tests and serotyping (test was performed within 3 days); (ii) EZTyphi Carrier DNA assay: Extracted DNA from bacteria culture was amplified using two sets of primers targeted for S. Typhi (1238 bp) and internal amplification control (IAC) (810 bp) (3 hours); (iii) Multiplex PCR-DNA dipstick assay: Extracted DNA was amplified using four sets of primers targeted for S. Typhi (70 bp), S. Paratyphi A (93 bp), pan-Salmonella (146 bp) and IAC (123 bp) (2 hours). Results & Discussion Fifteen out of 580 water samples were positive S.Typhi by multiplex PCR-DNA dipstick assay while one sample was positiveS. Typhi by EZTyphi Carrier DNA and culture method. This finding proved that the multiplex PCR-DNA dipstick was superior to culture method and PCR-agarose gel electrophoresis since it has higher positivity compared to the other two methods. Conclusion A rapid and sensitive multiplex PCR-DNA dipstick assay can be used to detect S. Typhi in well water especilally in typhoid endemic area.