The pattern of phage types of 2553 strains of Salmonella typhi isolated over the 10-year period 1970-9 was studied. During the period 29 different phage types were encountered, not including the categories of 'untypable strains', 'degraded Vi-strains' and Vi negative strains. For the period as a whole, the commonest phage types encountered were A (20.9%), E1 (14.8%), D1 (10.3%), degraded Vi positive strains (10.3%), untypable Vi strains (7.3%), C4 (7.1%), D2 (4.4%), E2 (3.9%) and type 25 (2.6%). There were phage types which appeared in the early years of the period and then disappeared (types B2, D9 and D1-N). Others only made their appearance in recent years (K1 and 53). Notable differences were also seen in the predilection of some phage types for certain geographical areas.
The diagnostic value of the Widal test was assessed in an endemic area. The test was done on 300 normal individuals, 297 non-typhoidal fevers and 275 bacteriologically proven cases of typhoid. Of 300 normal individuals, 2% had an H agglutinin titre of 1/160 and 5% had an O agglutinin titre of 1/160. On the basis of these criteria a significant H and/or O agglutinin titre of 1/320 or more was observed in 93-97% of typhoid cases and in only 3% of patients with non-typhoidal fever. Of the sera from typhoid cases which gave a significant Widal reaction, the majority (79.9%) showed increases in both H and O agglutinins and 51 of 234 (21.8%) of these sera were collected in the first week of illness. The significance and implications of these findings are discussed.
Salmonella enterica serovar Typhi (S. Typhi) is strictly a human intracellular pathogen. It causes acute systemic (typhoid fever) and chronic infections that result in long-term asymptomatic human carriage. S. Typhi displays diverse disease manifestations in human infection and exhibits high clonality. The principal factors underlying the unique lifestyle of S. Typhi in its human host during acute and chronic infections remain largely unknown and are therefore the main objective of this study.
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.
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.
We performed genome size estimation of 17 recent human isolates of Salmonella typhi from geographically diverse regions using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with restriction endonucleases XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3') and SpeI (5'-ACTAGT-3'), and summation of the sizes of restriction fragments obtained. All 17 isolates had circular chromosomes, and genome sizes differed by as much as 959 kb, ranging from 3,964 to 4,923 kb (mean genome size = 4,528 kb). The data obtained confirm the usefulness of PFGE in studies of bacterial genome size and are in agreement with recent results indicating considerable genetic diversity and genomic plasticity of S. typhi. The variation in genome sizes noted may be relevant to the observed biological properties of this important human pathogen, including its virulence.
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.
The results of serotyping of 10 953 salmonella isolates from humans over a 10-year period, 1973-82 at the Bacteriology Division, Institute for Medical Research, Malaysia are presented. A total of 104 serotypes from 22 'O' groups were encountered; 95 isolates were considered untypable. The three most predominant serotypes, namely Salmonella typhi, S. typhimurium and S. weltevreden together accounted for 54.1% of all isolates whilst the 25 most frequent serotypes accounted for 93.6% of the total. Whilst the commoner serotypes occurred regularly throughout the study period, the rarer ones tended to appear only in one year, when they might be associated with an outbreak, and never again. The pattern of serotypes, though quite similar to the one seen in neighbouring Singapore, is different from those experienced in other places such as Hong Kong, Jakarta, Bangladesh and Manchester.
Four strains of S. typhi isolated in Malaysia were found to show resistance to chloramphenicol and other antibiotics. In two of these strains it was possible to show that this resistance was transferable.
This problem which is widespread in neighbouring countries and undetected in Malaysia till recently has now been shown to exist in this country. Fears that the incidence of such strains will increase in the future are expressed and the need for vigilance is emphasised.
A clinical isolate of Salmonella typhi (Vi phage type 25), resistant to chloramphenicol, streptomycin and tetracycline, was examined for the presence of R plasmids. Results from conjugation, agarose gel electrophoresis and transformation experiments indicated that it harboured a single large self-transmissible R plasmid which coded for both the chloramphenicol and tetracycline resistance traits.
Two tsunami survivors from Banda Acheh, Sumatra, presented with pyrexia of unknown origin and a nonresolving left-sided empyema, respectively. Both children had mixed infections of tuberculosis and melioidosis; Salmonella typhi was also present in the second patient. Mixed infections are common late sequela complications in post-tsunami victims.
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.
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.
We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.
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.
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.