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Overexpression, purification and validation of antigenic Salmonella enterica serovar Typhi proteins identified from LC-MS/MS

Chin CF, Teh BA, Anthony AA, Aziah I, Ismail A, Ong EB, et al.

Appl Biochem Biotechnol, 2014 Nov;174(5):1897-906.
PMID: 25149461 DOI: 10.1007/s12010-014-1173-y

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.

Matched MeSH terms: Typhoid Fever/microbiology
Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method

Abdullah J, Saffie N, Sjasri FA, Husin A, Abdul-Rahman Z, Ismail A, et al.

Braz J Microbiol, 2014;45(4):1385-91.
PMID: 25763045

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.

Matched MeSH terms: Typhoid Fever/microbiology
Fever/clinical signs and association with neutropenia in solid cancer patients--bacterial infection as the main cause

Rasool Hassan BA, Yusoff ZB, Othman SB

Asian Pac J Cancer Prev, 2010;11(5):1273-7.
PMID: 21198276

INTRODUCTION: Neutropenia remains one of the serious side effects of chemotherapeutics drugs making cancer patients face serious risk of infections. Fever and clinical signs are considered as important indicators. The objectives of this study were to assess fever and clinical signs with neutropenia onset and/ or severity in solid cancer cases, using culture tests to determine the type of bacteria predominating, whether gram positive or gram negative.
METHODS: This observational retrospective study was conducted on files of all solid cancer patients who admitted to a general hospital between 1 January 2003 and 31 December 2006. All data were categorical and analyzed for association with neutropenia.
RESULTS: 117 neutropenic patients were studied, 83 (70.9%) of them suffering from fever ranging between 38.5-39 °C, with hypotension (53; 27.3%) and headache 51 (26.3%) as the most common clinical signs. Only 34 (29.1%) neutropenic patients underwent culture testing and only 14 (41.2%) showed positive growth, gram negative types predominating (9; 64.2%), mainly Escherichia coli (5; 35.7%), with gram positive only in 5 (35.7%). Significant associations were found for fever and clinical signs with neutropenia severity (P<0.05), but not neutropenia onset (P>0.05). Logistic regression results showed strong significant association between presence of fever (P=0.02, OR=1.3) (95% confidence interval (CI)) hypotension and headache (P=0.001, OR=1.148) (95% CI) with neutropenia severity.
CONCLUSION: Fever and clinical signs specifically headache and hypotension are symptoms associated with severe neutropenia in solid cancer patients. Both may primarily result from bacterial infection, particularly gram negative forms.

Matched MeSH terms: Fever/microbiology*
Amplification of ST50 gene using dry-reagent-based polymerase chain reaction for the detection of Salmonella typhi

Aziah I, Ravichandran M, Ismail A

Diagn Microbiol Infect Dis, 2007 Dec;59(4):373-7.
PMID: 17964105

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.

Matched MeSH terms: Typhoid Fever/microbiology
Risk factors of leptospirosis among febrile hospital admissions in northeastern Malaysia

Rafizah AA, Aziah BD, Azwany YN, Imran MK, Rusli AM, Nazri SM, et al.

Prev Med, 2013;57 Suppl:S11-3.
PMID: 23295174 DOI: 10.1016/j.ypmed.2012.12.017

Leptospirosis is a worldwide zoonotic disease. Risk factors for the disease may vary among countries.

Matched MeSH terms: Fever/microbiology
Risk assessment and microbiological profile of infections in paediatric cancer patients with febrile neutropenia

Latiff Z, Zulkifli SZ, Jamal R

Malays J Pathol, 2002 Dec;24(2):83-9.
PMID: 12887165

Febrile neutropenia is a common and potentially fatal problem encountered in cancer patients undergoing chemotherapy. We carried out an observational study to evaluate the possible risk factors of developing fever amongst neutropenic children with an underlying malignancy. We also looked at the microbiological profile of causative pathogens in patients with febrile neutropenia. During a study period of 1 year, a total of 90 neutropenic episodes were recorded amongst 57 patients who were on treatment and follow-up during the study period. Multivariate analysis showed that factors such as chemotherapy status, underlying disease, existing central venous catheters, presenting white blood cell counts at chemotherapy, use of steroid therapy or hospitalisation at the onset of neutropenia, were not significant risk factors for developing fever during neutropenic episodes. Although the presence of a central venous catheter was associated with a higher risk of developing fever, it did not reach statistical significance (p=0.11). Of the 90 neutropenic episodes, 59 (65.6%) developed fever and 25 of these had positive blood cultures. The causative organisms include gram-negative bacteria (64%), gram positive bacteria (16%) and fungus (20%). Of the gram-negative organisms, Klebsiella spp. predominated (28%) with the extended spectrum beta-lactamase producing strain forming the majority (16%). Amongst those with fungaemia, Candida spp. and Candida tropicalis formed the majority (8% each) of the isolates.

Matched MeSH terms: Fever/microbiology
Update: Outbreak of acute febrile illness among athletes participating in Eco-Challenge-Sabah 2000--Borneo, Malaysia, 2000

Stone SC, McNutt E

Ann Emerg Med, 2001 Jul;38(1):83-4.
PMID: 11423818
Matched MeSH terms: Fever/microbiology*
From the Centers for Disease Control and Prevention. Update: outbreak of acute febrile illness among athletes participating in Eco-Challenge-Sabah 2000--Borneo, Malaysia, 2000

JAMA, 2001 Feb 14;285(6):728-30.
PMID: 11236770
Matched MeSH terms: Fever/microbiology
Group A streptococcal infections in hospitalized patients

Jamal F, Pit S, Kasni S, Yasin MS, Aton SB, Singh K

Adv Exp Med Biol, 1997;418:35-7.
PMID: 9331592
Matched MeSH terms: Rheumatic Fever/microbiology
Septicaemia in paediatric cancer patients: a 5-year surveillance study in university hospital, Kuala Lumpur, Malaysia

Ariffin H, Ariffin W, Peng LH, Parasakthi N

J Trop Pediatr, 1997 10;43(5):279-81.
PMID: 9364125 DOI: 10.1093/tropej/43.5.279

Infectious complications are the major cause of morbidity and mortality in children with malignancy. Empirical antimicrobial therapy in the management of fever of unknown origin should be tailored to local bacteriological data and antibiotic sensitivity patterns. Five-hundred-and-fifty-nine cases of culture-proven septicaemia occurring in pediatric cancer patients between 1990 and 1994 were retrospectively analysed and compared with a similar study done in our centre between 1976 and 1979. A wide spectrum of organisms was isolated. Staphylococcus epidermidis, Staphylococcus aureus, and Klebsiella pneumoniae were the most common and consistent bacteria isolated during the 5 year period. More than 70 per cent of the staphylococci were sensitive to methicillin and universally sensitive to vancomycin. However, a worrying trend of ceftazidime-resistance amongst gram-negative organisms was found. In these situations, the use of imipenem is recommended as resistance to this antimicrobial agent was exceedingly rare.

Matched MeSH terms: Fever/microbiology
Usefulness of the Widal test in diagnosing childhood typhoid fever in endemic areas

Choo KE, Razif AR, Oppenheimer SJ, Ariffin WA, Lau J, Abraham T

J Paediatr Child Health, 1993 Feb;29(1):36-9.
PMID: 8461177

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.

Matched MeSH terms: Typhoid Fever/microbiology
DNA fingerprinting of human isolates of Salmonella enterica serotype Paratyphi B in Malaysia

Goh YL, Yasin R, Puthucheary SD, Koh YT, Lim VK, Taib Z, et al.

J Appl Microbiol, 2003;95(5):1134-42.
PMID: 14633043

DNA fingerprinting of Salmonella enterica serotype Paratyphi B isolated in Malaysia during 1982-83, 1992 and 1996-2002 was carried out by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility tests and D-tartrate utilization tests to assess the extent of genetic diversity of these isolates in Malaysia.

Matched MeSH terms: Paratyphoid Fever/microbiology*
Fulltext Penicilliosis in lupus patients presenting with unresolved fever: a report of 2 cases and literature review

Chong YB, Tan LP, Robinson S, Lim SK, Ng KP, Keng TC, et al.

Trop Biomed, 2012 Jun;29(2):270-6.
PMID: 22735849 MyJurnal

Penicilliosis is a rare occurrence among non human immunodeficiency virus (HIV) infected patients. We report here two cases of Penicillium marneffei infection in patients with systemic lupus erythematosus (SLE). Both patients had a recent flare of lupus and were on immunosuppressive drugs when they presented with prolonged fever without an obvious foci of infection, unresponsive to broad-spectrum antibiotics. They were leucopaenic upon admission, with rapid deterioration during the course of the illness. Diagnosis of penicilliosis via fungal isolation from blood culture was delayed resulting in the late initiation of antifungal agents. While both patients ultimately recovered, the delay in diagnosis led to a prolonged hospital stay with increased morbidity. Clinicians should be aware of this uncommon but emerging fungal pathogen in SLE patients and maintain a high index of suspicion in diagnosing this potentially fatal but treatable disease.

Matched MeSH terms: Fever/microbiology*
Typhoid fever--important issues still remain

Pang T, Levine MM, Ivanoff B, Wain J, Finlay BB

Trends Microbiol, 1998 Apr;6(4):131-3.
PMID: 9587187
Matched MeSH terms: Typhoid Fever/microbiology
Increasing genetic diversity of Salmonella enterica serovar typhi isolates from papua new guinea over the period from 1992 to 1999

Thong KL, Goh YL, Yasin RM, Lau MG, Passey M, Winston G, et al.

J Clin Microbiol, 2002 Nov;40(11):4156-60.
PMID: 12409390

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA was performed on 133 strains of Salmonella enterica serovar Typhi obtained from Papua New Guinea, with the objective of assessing the temporal variation of these strains. Fifty-two strains that were isolated in 1992 and 1994 were of one phage type, D2, and only two predominant PFGE profiles, X1 and X2, were present. Another 81 strains isolated between 1997 and 1999 have shown divergence, with four new phage types, UVS I (n = 63), UVS (n = 5), VNS (n = 4), and D1 (n = 9), and more genetic variability as evidenced by the multiple and new PFGE XbaI profiles (21 profiles; Dice coefficient, F = 0.71 to 0.97). The two profiles X1 and X2 have remained the stable, dominant subtypes since 1992. Cluster analysis based on the unweighted pair group method using arithmetic averages algorithm identifies two main clusters (at 87% similarity), indicating that the divergence of the PFGE subtypes was probably derived from some genomic mutations of the X1 and X2 subtypes. The majority of isolates were from patients with mild and moderate typhoid fever and had various XbaI profiles. A single isolate from a patient with fatal typhoid fever had a unique X11 profile, while four of six isolates from patients with severe typhoid fever had the X1 pattern. In addition, 12 paired serovar Typhi isolates recovered from the blood and fecal swabs of individual patients exhibited similar PFGE patterns, while in another 11 individuals paired isolates exhibited different PFGE patterns. Three pairs of isolates recovered from three individuals had different phage types and PFGE patterns, indicating infection with multiple strains. The study reiterates the usefulness of PFGE in assessing the genetic diversity of S. enterica serovar Typhi for both long-term epidemiology and in vivo stability and instability within an individual patient.

Matched MeSH terms: Typhoid Fever/microbiology
Molecular analysis of isolates of Salmonella typhi obtained from patients with fatal and nonfatal typhoid fever

Thong KL, Passey M, Clegg A, Combs BG, Yassin RM, Pang T

J Clin Microbiol, 1996 Apr;34(4):1029-33.
PMID: 8815078

Molecular characterization of a total of 52 human isolates of Salmonella typhi from Papua New Guinea was performed by using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases, XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Of the 52 isolates tested, 11 were obtained from patients with fatal typhoid fever and 41 were obtained from patients with nonfatal disease. The 52 isolates showed limited genetic diversity as evidenced by only three different PFGE patterns detected following digestion with XbaI (patterns X1 to X3; F [coefficient of similarity] = 0.86 to 1.0), four patterns detected following digestion with AvrII (patterns A1 to A4; F =0.78 to 1.0), and two patterns detected following digestion with SpeI (patterns S1 and S2; F = 0.97 to 1.0). Of the 52 isolates, 37 were phage typed, and all belonged to phage type D2. All 11 isolates obtained from patients with fatal typhoid fever were identical (F = 1.0) and possessed the PFGE pattern combination X1S1A1, whereas the 41 isolates from patients with nonfatal typhoid fever had various PFGE pattern combinations, the most common being X2S1A2 (39%), X1S1A1 (24%), and X1S1A2 (15%). Thus, all the isolates from patients with the fatal disease had the X1 and A1 patterns, whereas the majority of the isolates from patients with nonfatal typhoid fever possessed the X2 and A2 patterns. The data suggest that there is an association among strains of S. typhi between genotype, as assessed by PFGE patterns, and the capability to cause fatal illness. Analysis of blood and fecal isolates of S. typhi from the same patient also indicated that some genetic changes occur in vivo during the course of infection.

Matched MeSH terms: Typhoid Fever/microbiology*
Analysis of Salmonella typhi isolates from Southeast Asia by pulsed-field gel electrophoresis

Thong KL, Puthucheary S, Yassin RM, Sudarmono P, Padmidewi M, Soewandojo E, et al.

J Clin Microbiol, 1995 Jul;33(7):1938-41.
PMID: 7665677

Pulsed-field gel electrophoresis (PFGE) revealed that multiple genetic variants of Salmonella typhi are simultaneously present in Southeast Asia and are associated with sporadic cases of typhoid fever and occasional outbreaks. Comparative analysis of PFGE patterns also suggested that considerable genetic diversity exists among S. typhi strains and that some PFGE patterns are shared between isolates obtained from Malaysia, Indonesia, and Thailand, implying movement of these strains within these regions of Southeast Asia, where they are endemic.

Matched MeSH terms: Typhoid Fever/microbiology
Epidemiologic analysis of sporadic Salmonella typhi isolates and those from outbreaks by pulsed-field gel electrophoresis

Thong KL, Cheong YM, Puthucheary S, Koh CL, Pang T

J Clin Microbiol, 1994 May;32(5):1135-41.
PMID: 7914202

Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.

Matched MeSH terms: Typhoid Fever/microbiology*
Fulltext Genome sequence and comparative pathogenomics analysis of a Salmonella enterica Serovar Typhi strain associated with a typhoid carrier in Malaysia

Yap KP, Gan HM, Teh CS, Baddam R, Chai LC, Kumar N, et al.

J Bacteriol, 2012 Nov;194(21):5970-1.
PMID: 23045488 DOI: 10.1128/JB.01416-12

Salmonella enterica serovar Typhi is a human pathogen that causes typhoid fever predominantly in developing countries. In this article, we describe the whole genome sequence of the S. Typhi strain CR0044 isolated from a typhoid fever carrier in Kelantan, Malaysia. These data will further enhance the understanding of its host persistence and adaptive mechanism.

Matched MeSH terms: Typhoid Fever/microbiology
Molecular analysis of environmental and human isolates of Salmonella typhi

Thong KL, Cordano AM, Yassin RM, Pang T

Appl Environ Microbiol, 1996 Jan;62(1):271-4.
PMID: 8572705

Molecular characterization of a total of 54 isolates of Salmonella typhi from Santiago, Chile, was performed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases: XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Thirteen of the 54 isolates were obtained from environmental sources (sewage and river water), and the rest were isolates from clinical cases of typhoid fever. Considerable genetic diversity was detected among the human isolates obtained in 1994, as evidenced by the presence of 14 to 19 different PFGE patterns among 20 human isolates, with F (coefficient of similarity) values ranging from 0.69 to 1.0 (XbaI), 0.61 to 1.0 (AvrII), and 0.70 to 1.0 (SpeI). A total of eight phage types were detected among these 20 isolates, with 50% possessing the E1 or 46 phage type. There was no correlation between PFGE pattern and phage types. Similar diversity was seen among 21 isolates obtained in 1983, with 17 to 19 PFGE patterns detected and F values of 0.56 to 1.0 (XbaI), 0.55 to 1.0 (AvrII), and 0.67 to 1.0 (SpeI). Comparison of these two groups of human isolates obtained 11 years apart indicated that certain molecular types of S. typhi are shared and are able to persist for considerable periods. A similar degree of genetic diversity was also detected among the environmental isolates of S. typhi, for which 10 to 12 different PFGE patterns were detected among the 13 isolates analyzed, with F values ranging from 0.56 to 1.0 (XbaI), 0.52 to 1.0 (AvrII), and 0.69 to 1.0 (SpeI). Certain molecular types present among the environmental isolates of S. typhi were also found among the human isolates from the same time period, providing evidence for the epidemiological link between environmental reservoirs and human infection.

Matched MeSH terms: Typhoid Fever/microbiology

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