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.
We used Bayes' theorem to calculate the probability of enteric fever in 260 patients presenting with undiagnosed fever, without recourse to blood or stool culture results. These individuals were divided into 110 patients with enteric fever (63 culture positive, 47 culture negative) and 150 patients with other causes of fever. Comparison of the frequencies of occurrence of 19 clinical and laboratory events, said to be helpful in the diagnosis of enteric fever, in the two groups revealed that only 8 events were significantly more frequent in enteric fever. These were: a positive Widal test at a screening dilution of 1:40; a peak temperature greater than = 39 degrees C; previous treatment for the fever; a white blood cell count less than 9 X 10(6)/litre; a polymorphonuclear leucocyte count less than 3.5 X 10(6)/litre; splenomegaly; fever duration greater than 7 d; and hepatomegaly. When the probability of enteric fever was determined prospectively in 110 patients, using only 6 of these discriminating events, the probability of patients with a positive prediction having enteric fever (diagnostic specificity) was 0.80 (95% confidence interval: 0.68 to 0.91) and the probability of those with a negative prediction not having enteric fever (diagnostic sensitivity) was 0.92 (0.85 to 0.99). Using all 19 events did not alter the diagnostic specificity or diagnostic sensitivity. This study shows that a small number of clinical and laboratory features can objectively discriminate enteric fever from other causes of fever in the majority of patients. Calculating the probability of enteric fever can aid in diagnosis, when culturing for salmonella is either unavailable or is negative.
To investigate the role of serum C-reactive protein (CRP) in the diagnosis of typhoid fever, we studied 227 febrile Malaysian children hospitalized during a 12-month period. The children were: culture-positive for Salmonella typhi (Group 1; n = 108); culture-negative but with typical clinical features of typhoid fever (Group 2; n = 60); or had non-typhoidal illness (Group 3; n = 59). Group 1 children had the highest serum CRP concentrations (geometric mean [SD range]; 43 [12-150] mg/l vs. 26 [8-85] mg/l in Group 2 and 21 [4-110] mg/l in Group 3; p < 0.001). In regression analysis, age, patient group and fever duration were independently associated with serum CRP (p < 0.05) but gender was not. In Group 1 patients, there was a significant positive association between serum CRP and Widal O and H agglutinin titres. In receiver-operator characteristic (ROC) analysis of serum CRP for Groups 1 and 2 combined, compared with Group 3, the area under the curve (AUC) was 0.65. These data show that the serum CRP is highest in culture-positive children with enteric fever and reflects the immune response to the infection in this group. Nevertheless, serum CRP had relatively low sensitivity and specificity for confirmed or clinically diagnosed typhoid fever (68 and 58 per cent, respectively at 'cut-off' concentration 30.0 mg/l), and an AUC value only moderately above that associated with no predictive power (0.5). Although of limited use as a primary diagnostic test, a raised serum CRP may still have a place as one of a range of features that facilitate assessment of a febrile child in a typhoid-endemic area.
ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.
Typhoid fever being a systemic infection can present in a multitude of ways, involving various systems. Here we describe a case of typhoid fever presenting with acute cerebellar ataxia and marked thrombocytopenia. This atypical presentation is not common in typhoid fever and can lead to misdiagnosis as well as a delay in the initiation of appropriate therapy. Prompt clinical improvement and the return of platelet counts to normal were noted after the patient was started on IV Ceftriaxone.
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.
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.
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.
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.
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.
We studied 1,629 febrile patients from a rural area of Malaysia, and made a laboratory diagnosis in 1,025 (62.9%) cases. Scrub typhus was the most frequent diagnosis (19.3% of all illnesses) followed by typhoid and paratyphoid (7.4%); flavivirus infection (7.0%); leptospirosis (6.8%); and malaria (6.2%). The hospital mortality was very low (0.5% of all febrile patients). The high prevalence of scrub typhus in oil palm laborers (46.8% of all febrile illnesses in that group) was confirmed. In rural Malaysia, therapy with chloramphenicol or a tetracycline would be appropriate for undiagnosed patients in whom malaria has been excluded. Failure to respond to tetracycline within 48 hours would usually suggest a diagnosis of typhoid, and indicate the need for a change in therapy.
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.
This report deals with a young man who developed features of haemophogocytosis during the course of typhoid fever. The pertinent clinical and laboratory features of typhoid-associated haemophagocytosis are discussed. The need for blood component replacement therapy in addition to specific anti-microbials to treat haemophagocytosis complicating typhoid fever is stressed.
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.
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.
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.