Strains of Salmonella typhi implicated in two separate cases of laboratory acquired infection from patients and the medical laboratory technologists who processed the patients' samples were analysed by pulsed-field gel electrophoresis. Although all four isolates were of bacteriophage type E1, PFGE was able to demonstrate that the strains responsible for the two laboratory acquired cases were not genetically related. The PFGE patterns of the isolates from the MLTs were found to be identical to those of the corresponding patients after digestion with restriction enzyme AvrII. This provided genetic as well as epidemiological evidence for the source of the laboratory acquired infections.
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).
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.
A retrospective study of 137 patients with blood culture-positive typhoid fever admitted to the paediatric unit of the Hospital Universiti Sains Malaysia was carried out to study epidemiological, clinical, laboratory and treatment aspects of typhoid fever in Kelantanese children in hospital. The male:female ratio was 1:1.1. School-children were the most affected. Cases were seen throughout the year. The five most frequently presenting features were fever, hepatomegaly, diarrhoea, vomiting and cough. Rose spots were seen in only two patients. Complications included gastritis, bronchitis, ileus, psychosis, encephalopathy, gastro-intestinal bleeding and myocarditis. Relative bradycardia was not seen. Blood and stool cultures were positive in the 1st, 2nd and 3rd weeks of illness. There was no significant difference between percentages of elevated O and H titres, whether done during or after the 1st week of illness. A four-fold rise in (O) titres occurred in 50% of cases tested. We would miss 50% of typhoid fever cases if a titre (O) equal to more than 1/160 were relied upon for diagnosis. Altogether, 46% of patients had leucopenia. Chloramphenicol was the most commonly used antibiotic. There were two deaths.
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.
Titres of melioidosis haemagglutinating antibodies of 1/40 or more were found in 18 of 905 British, Australian, and New Zealand soldiers serving in West Malaysia. Previous mild unsuspected melioidosis seemed to be responsible for these positive titres, which were more common in men exposed to surface water at work and during recreation. This accords with the current view that soil and surface water is the normal habitat of Pseudomonas pseudomallei, the causal organism. Pyrexia of unknown origin after arriving in Malaysia was significantly more common in men with titres of 1/40 or more than in the remainder. It is suggested that mild melioidosis may present as pyrexia of unknown origin. Pyrexias of unknown origin should be investigated vigorously in patients who are in or who have visited endemic areas.
Salmonella Typhi (S. Typhi) causes typhoid fever which is a disease characterised by high mortality and morbidity worldwide. In order to curtail the transmission of this highly infectious disease, identification of new markers that can detect the pathogen is needed for development of sensitive and specific diagnostic tests. In this study, genomic comparison of S. Typhi with other enteric pathogens was performed, and 6 S. Typhi genes, that is, STY0201, STY0307, STY0322, STY0326, STY2020, and STY2021, were found to be specific in silico. Six PCR assays each targeting a unique gene were developed to test the specificity of these genes in vitro. The diagnostic sensitivities and specificities of each assay were determined using 39 S. Typhi, 62 non-Typhi Salmonella, and 10 non-Salmonella clinical isolates. The results showed that 5 of these genes, that is, STY0307, STY0322, STY0326, STY2020, and STY2021, demonstrated 100% sensitivity (39/39) and 100% specificity (0/72). The detection limit of the 5 PCR assays was 32 pg for STY0322, 6.4 pg for STY0326, STY2020, and STY2021, and 1.28 pg for STY0307. In conclusion, 5 PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution for diagnosis of typhoid fever.
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.
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.
The hemolysin (HlyE) protein of Salmonella enterica serovar Typhi was reported to be antigenic. This work describes the cloning, expression, and purification of a hexahistidine-tagged HlyE protein under native conditions. Immunoblot analysis and a competitive enzyme-linked immunosorbent assay using sera from typhoid patients showed the presence of HlyE-specific antibodies in circulation.
Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.
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.
Molecular surveillance of Chikungunya virus (CHIKV) is important as it provides data on the circulating CHIKV genotypes in endemic countries and enabling activation of measures to be taken in the event of a pending outbreak. Molecular surveillance is carried out by first detecting CHIKV in susceptible humans or among field-caught mosquitoes. This is followed by sequencing a selected region of the virus which will provide evidence on the source of the virus and possible association of the virus to increased cases of Chikungunya infections.
The Typhidot test, which detects IgM and IgG antibodies to a Salmonella typhi-specific outer membrane protein, is as sensitive as, and more specific than, the Widal test in the diagnosis of enteric fever in Malaysian children. It is easier and quicker to perform. In order to increase diagnostic accuracy in an area of high endemicity, the Typhidot-M test has been developed in which IgG is first removed. This theoretically allows improved detection of IgM, and thus would differentiate new from recent infections. We evaluated both tests in 134 unselected febrile children admitted to the General Hospital Kota Bharu, Malaysia. The children were divided into two groups: (i) those who were blood and/or stool culture positive for S. typhi and/or who had clinical features strongly suggestive of enteric fever (n = 62); and (ii) those who were both culture-negative and had clinical evidence of another diagnosis (n = 72). The sensitivity and specificity of the Typhidot and Typhidot-M tests were identical at 90.3 and 93.1%, respectively. Both tests had comparable sensitivity but greater specificity than those of the Widal test (91.9 and 80.6%, respectively). When used together, a positive result for Typhidot and/or Typhidot-M was more specific than either test alone (95.2%) but specificity was lower (87.5%). We conclude that the Typhidot and Typhidot-M tests have comparatively high diagnostic accuracy, suggesting that IgM can be detected in children who may have a predominant IgG response to S. typhi. Using these tests in combination increases the negative predictive value but at the cost of a lower positive predictive value.
We evaluated the diagnostic sensitivity and specificity of two dot-enzyme-linked immunoassays (Typhidot and Typhidot-M; Malaysian Biodiagnostic Research SDN BHD, Kuala Lumpur, Malaysia), assessing IgG and IgM antibodies against the outer membrane protein (OMP) of Salmonella typhi, and the Widal test in comparison with blood culture in a consecutive group of children with suspected typhoid fever. Of 97 children with suspected typhoid fever, the disease was confirmed bacteriologically in 46 (47%), whereas 25 (26%) were considered to have typhoid fever on clinical grounds. An alternative diagnosis was made in 26 (27%). The Typhidot and Typhidot-M were superior to the Widal test in their diagnostic sensitivity and specificity, although values (sensitivity = 85-94% and specificity = 77-89%) were significantly lower than in other regional reports. The lower specificity of the Typhidot in our series may represent regional differences in the genomic structure and plasticity of the OMP of S. typhi and merits further evaluation of these tests in diverse geographic locations.
Data are presented for 2382 children investigated for fever in a Malaysian hospital between 1984 and 1987 when Widal tests and blood cultures were a routine part of every fever screen. There were 145 children who were culture positive (TYP-CP) for Salmonella typhi, while 166 were culture negative but were diagnosed as having typhoid (TYP-CN). Analyses of the sensitivity and specificity of combinations of initial Widal titres in predicting a positive S. typhi culture in a febrile child (culture positive vs the rest) showed the best model to be an O- and/or H-titre of > or = 1 in 40 (sensitivity 89%; specificity 89%). While the negative predictive value of the model was high (99.2%) the positive predictive value remained below 50% even for very high titres of O and H (> 1 in 640), at which point the specificity was 98.5%, supporting the clinical view that a high proportion of the TYP-CN patients really were typhoid but were missed by culture. The TYP-CN patients showed a very similar clinical and age profile to TYP-CP patients. The length of history of fever did not affect the initial Widal titre in culture positive cases. The Widal test in children remains a sensitive and specific 'fever screen' for typhoid although it will not identify all cases. In children, lower cut-off points for O- and H-titres should be used than are generally recommended.