Vi antigen expression in Salmonella enterica serovar Typhi clinical isolates from Pakistan

The accurate identification of Salmonella enterica subsp. enterica serovar Typhi variants that fail to express the capsular polysaccharide, Vi, is an important and much discussed issue for medical microbiology. We have tested a multiplex PCR method which shows the presence or absence of the genetic locus required for Vi expression. Of 2,222 Salmonella serovar Typhi clinical isolates collected from patients' blood over a 4-year period in a region of Pakistan where typhoid is endemic, 12 tested negative for Vi expression by serological agglutination. However, only 1 of these 12 was Vi negative by the multiplex PCR method. This result was confirmed by immunofluorescence, the most sensitive method for Vi characterization in Salmonella serovar Typhi. The multiplex PCR described therefore represents a simple and accurate method for surveillance for Vi-negative variants of Salmonella serovar Typhi in Pakistan. Testing of clinical isolates of Salmonella serovar Typhi, before subculture, from other regions where Vi-negative Salmonella serovar Typhi has been described should be carried out so that the impact of vaccination with purified Vi antigen on the levels of Vi-negative Salmonella serovar Typhi in bacterial populations can be assessed.     

In vivo regulation of the Vi antigen in Salmonella and induction of immune responses with an in vivo-inducible promoter

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.     

Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR

We quantified the gene copies from Salmonella enterica serovar Typhi (S. Typhi) in the blood of patients suspected of having typhoid fever by using TaqMan-based real-time PCR (TaqMan assay) to target the S. Typhi flagellin gene in genomic DNAs isolated from blood samples. Of 55 blood samples taken from suspected typhoid fever patients, eight blood samples with a positive blood culture had S. Typhi loads ranging from 1.01 x 10(3) to 4.35 x 10(4) copies/ml blood, and from 47 blood samples with negative blood culture, there were 40 (85.1%) TaqMan assay-positive samples with loads ranging from 3.9 to 9.9 x 10(2) copies/ml blood. In the present study, the TaqMan assay detected more than 10(3) copies/ml blood of S. Typhi in all of the blood culture-positive samples, whereas less than 10(3) copies/ml blood of S. Typhi were detected in the blood culture-negative samples. Our findings suggest that a TaqMan assay may be useful for assessing S. Typhi loads, especially in cases of suspected typhoid fever with negative results from the standard blood culture test.     

Endemic, epidemic clone of Salmonella enterica serovar typhi harboring a single multidrug-resistant plasmid in Vietnam between 1995 and 2002

Salmonella enterica serovar Typhi strains resistant to ampicillin, chloramphenicol, tetracyclines, streptomycin, and cotrimoxazole, isolated from sporadic cases and minor outbreaks in Vietnam between 1995 and 2002, were typed and compared. Plasmid fingerprinting, Vi bacteriophage typing, XbaI pulsed-field gel electrophoresis, and PstI ribotyping showed that endemic, epidemic multidrug-resistant typhoid fever was due, for at least 74.1% of the isolates, to one or two clones of serovar Typhi harboring a single resistance plasmid. PstI ribotyping was used as a basic technique to ensure that a serovar Typhi expansion was clonal.     

Salmonella enterica serovar Dublin strains which are Vi antigen-positive use type IVB pili for bacterial self-association and human intestinal cell entry

Some strains of Salmonella enterica serovar Dublin are Vi antigen-positive. S. enterica serovar Typhi uses Type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association under conditions that favour DNA supercoiling. These pilus-mediated events may be important in typhoid fever pathogenesis. A survey of 17 isolates of S. enterica serovar Dublin showed that all strains which carried the viaB region also carried a serovar Typhi-like Type IVB pil operon, and all serovar Dublin Vi antigen-negative isolates lacked the pil operon. The pil operon was completely sequenced from one of the Vi(+) serovar Dublin strains, and was almost identical (4 nt changes; 3 aa changes, in over 10 kb) to that of serovar Typhi. A pilS mutant of one serovar Dublin strain was constructed, and shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. Purified prePilS protein inhibited INT407 cell entry by serovar Dublin. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggest that the serovar Dublin Type IVB pil operon may increase the human-invasiveness of serovar Dublin, compared to pil-free strains.     

Genetic determinants and polymorphisms specific for human-adapted serovars of Salmonella enterica that cause enteric fever

Salmonella enterica serovars Typhi, Paratyphi A, and Sendai are human-adapted pathogens that cause typhoid (enteric) fever. The acute prevalence in some global regions and the disease severity of typhoidal Salmonella have necessitated the development of rapid and specific detection tests. Most of the methodologies currently used to detect serovar Typhi do not identify serovars Paratyphi A or Sendai. To assist in this aim, comparative sequence analyses were performed at the loci of core bacterial genetic determinants and Salmonella pathogenicity island 2 genes encoded by clinically significant S. enterica serovars. Genetic polymorphisms specific for serovar Typhi (at trpS), as well as polymorphisms unique to human-adapted typhoidal serovars (at sseC and sseF), were observed. Furthermore, entire coding sequences unique to human-adapted typhoidal Salmonella strains (i.e., serovar-specific genetic loci rather than polymorphisms) were observed in publicly available comparative genomic DNA microarray data sets. These polymorphisms and loci were developed into real-time PCR, standard PCR, and liquid microsphere suspension array-based molecular protocols and tested for with a panel of clinical and reference subspecies I S. enterica strains. A proportion of the nontyphoidal Salmonella strains hybridized with the allele-specific oligonucleotide probes for sseC and sseF; but the trpS allele was unique to serovar Typhi (with a singular serovar Paratyphi B strain as an exception), and the coding sequences STY4220 and STY4221 were unique among serovars Typhi, Paratyphi A, and Sendai. These determinants provided phylogenetic data on the genetic relatedness of serovars Typhi, Paratyphi A, and Sendai; and the protocols developed might allow the rapid identification of these Salmonella serovars that cause enteric fever.     

Molecular Typing of Multiple-Antibiotic-Resistant Salmonella enterica Serovar Typhi from Vietnam: Application to Acute and Relapse Cases of Typhoid Fever

The rate of multiple-antibiotic resistance is increasing among Salmonella enterica serovar Typhi strains in Southeast Asia. Pulsed-field gel electrophoresis (PFGE) and other typing methods were used to analyze drug-resistant and -susceptible organisms isolated from patients with typhoid fever in several districts in southern Vietnam. Multiple PFGE and phage typing patterns were detected, although individual patients were infected with strains of a single type. The PFGE patterns were stable when the S. enterica serovar Typhi strains were passaged many times in vitro on laboratory medium. Paired S. enterica serovar Typhi isolates recovered from the blood and bone marrow of individual patients exhibited similar PFGE patterns. Typing of S. enterica serovar Typhi isolates from patients with relapses of typhoid indicated that the majority of relapses were caused by the same S. enterica serovar Typhi strain that was isolated during the initial infection. However, some individuals were infected with distinct and presumably newly acquired S. enterica serovar Typhi isolates.     

Differential bacterial survival, replication, and apoptosis-inducing ability of Salmonella serovars within human and murine macrophages

Salmonella serovars are associated with human diseases that range from mild gastroenteritis to host-disseminated enteric fever. Human infections by Salmonella enterica serovar Typhi can lead to typhoid fever, but this serovar does not typically cause disease in mice or other animals. In contrast, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, which are usually linked to localized gastroenteritis in humans and some animal species, elicit a systemic infection in mice. To better understand these observations, multiple strains of each of several chosen serovars of Salmonella were tested for the ability in the nonopsonized state to enter, survive, and replicate within human macrophage cells (U937 and elutriated primary cells) compared with murine macrophage cells (J774A.1 and primary peritoneal cells); in addition, death of the infected macrophages was monitored. The serovar Typhimurium strains all demonstrated enhanced survival within J774A.1 cells and murine peritoneal macrophages, compared with the significant, almost 100-fold declines in viable counts noted for serovar Typhi strains. Viable counts for serovar Enteritidis either matched the level of serovar Typhi (J774A. 1 macrophages) or were comparable to counts for serovar Typhimurium (murine peritoneal macrophages). Apoptosis was significantly higher in J774A.1 cells infected with serovar Typhimurium strain LT2 compared to serovar Typhi strain Ty2. On the other hand, serovar Typhi survived at a level up to 100-fold higher in elutriated human macrophages and 2- to 3-fold higher in U937 cells compared to the serovar Typhimurium and Enteritidis strains tested. Despite the differential multiplication of serovar Typhi during infection of U937 cells, serovar Typhi caused significantly less apoptosis than infections with serovar Typhimurium. These observations indicate variability in intramacrophage survival and host cytotoxicity among the various serovars and are the first to show differences in the apoptotic response of distinct Salmonella serovars residing in human macrophage cells. These studies suggest that nonopsonized serovar Typhimurium enters, multiplies within, and causes considerable, acute death of macrophages, leading to a highly virulent infection in mice (resulting in death within 14 days). In striking contrast, nonopsonized serovar Typhi survives silently and chronically within human macrophages, causing little cell death, which allows for intrahost dissemination and typhoid fever (low host mortality). The type of disease associated with any particular serovar of Salmonella is linked to the ability of that serovar both to persist within and to elicit damage in a specific host's macrophage cells.     

Evaluation of nested PCR in diagnosis of typhoid fever

In this study, nested PCR using H1-d primers, which is specific for Salmonella enterica serovar Typhi, was compared to blood culture and the single-tube Widal test. Results indicate that nested PCR can be used as a gold standard to determine the cutoff titer of the Widal test for diagnosis of typhoid fever.     

Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.Salmonella enterica is an important enteric pathogen and is involved in causing both systemic and intestinal diseases in humans and a wide range of other hosts (7, 21). Serotypes within subspecies I (Salmonella enterica subsp. enterica) are responsible for the vast majority of salmonellosis in warm-blooded animals. S. enterica subsp. enterica serovar Typhi and S. enterica subsp. enterica serovar Paratyphi A cause typhoid fever strictly in humans mostly in developing countries, with no age exemption, but it is less common in children younger than 2 years old. According to one estimate, the worldwide incidence of typhoid fever is 16 million cases annually and the mortality rate is 600,000 individuals per year (23). According to a press release from the Press Information Bureau, Government of India, dated 22 February 2006, the morbidity due to typhoid fever varies from 102 to 2,219 per 100,000 population in different parts of India, and in some areas, typhoid fever is responsible for 2 to 5% of all deaths. The problem of typhoid fever has been exacerbated by the appearance of multiple-drug-resistant strains (25), the treatment of which would depend on newer and advanced antibiotics and early and precise diagnosis.The existing modes of diagnosis are through the detection of antibodies against Salmonella bacteria by the Widal test and other serological tests like DOT enzyme immunoassay, dip stick assays, and semiquantitative tube agglutination test (22). Apart from this, the bacteremia observed in typhoid fever around day 6 to 9 enables it to be detected through the blood culture test (29) and PCR amplification of the bacterial DNA from blood. Of the commonly available diagnostic tests, Widal test and other serological diagnostic methods are limited because of the low specificity of the test. There are reports of a large number of false-positive cases especially in areas where typhoid fever is endemic and in patients exposed to typhoid fever earlier (6). The blood culture test has the major disadvantage of being a time-consuming test, which takes 2 to 3 days.PCR-based diagnoses are superior to the classical serological method, Widal test, and blood culture test in terms of their specificity and sensitivity. The modification by Sanchez-Jimenez and Cardona-Castro (26) where the initial DNA purification step is omitted and the whole blood is used directly as the template for PCR has been used in our assay system with minor modifications.The PCR-based systems currently use primers against flagellin genes (2, 4, 5, 12, 16, 17, 27), hilA (26), and invA and spvC genes (5). The different distributions of invA and spvC genes among Salmonella isolates from animals highlights the unsuitability of these two genes as PCR probes for Salmonella detection (20). Many genes carried on Salmonella pathogenicity islands have evolved differentially in typhoidal and nontyphoidal Salmonella serovars giving rise to different allelic variants of these genes (9). These genes are present in different Salmonella serovars, and their orthologs in other species of bacteria share various degrees of identity at the nucleotide levels (9). These differences, if minor, at certain PCR conditions can lead to promiscuous amplification, thereby leading to false-positive results. This problem can be overcome by choosing those regions that are unique to S. Typhi and S. Paratyphi A. Though certain pathogenicity islands are unique to S. Typhi and S. Paratyphi A, like Salmonella pathogenicity island 7 (SPI-7) and SPI-8, these islands are known to be unstable. These islands can be excised by the activation of certain recombinases as exemplified by isolation of the clinical variants lacking SPI-7 (19). Also, the presence of a gene encoding integrase on SPI-8 suggests that it is a mobile island (13). For the same reason, insertion sequences and bacteriophage genes are not good candidates for diagnostic purposes. However, a thorough examination of the whole-genome sequences of S. enterica serovar Typhi, S. Paratyphi A, and S. Typhimurium highlights the existence of genomic regions of unknown function with no homologous genes in related serovars and without the features of mobile DNA sequences. Using these criteria for the identification of a good diagnostic marker gene in S. Typhi CT18, we identified the genomic loci spanning the STY0312 gene, which is unique to S. Typhi CT18 and S. Paratyphi, the causative agents of typhoid fever. The adjoining locus spanning STY0313 to STY0316 was different in S. Typhi Ty2 and S. Paratyphi but otherwise conserved in most Salmonella strains. This region was found to be part of SPI-6, which is present in many Salmonella enterica subspecies I strains (10).We hypothesized that these novel primers could differentiate between typhoidal and nontyphoidal serovars of Salmonella enterica. Hence, the aim of the present work was to amplify the genomic region using the unique set of primers and demonstrate whether this method can be useful for the early diagnosis of typhoid fever.     

Variable number of tandem repeats in Salmonella enterica subsp. enterica for typing purposes

The genomic sequences of Salmonella enterica subsp. enterica strains CT18, Ty2 (serovar Typhi), and LT2 (serovar Typhimurium) were analyzed for potential variable number tandem repeats (VNTRs). A multiple-locus VNTR analysis (MLVA) of 99 strains of S. enterica supsp. enterica based on 10 VNTRs distinguished 52 genotypes and placed them into four groups. All strains tested were independent human isolates from France and did not reflect isolates from outbreak episodes. Of these 10 VNTRs, 7 showed variability within serovar Typhi, whereas 1 showed variability within serovar Typhimurium. Four VNTRs showed high Nei's diversity indices (DIs) of 0.81 to 0.87 within serovar Typhi (n = 27). Additionally, three of these more variable VNTRs showed DIs of 0.18 to 0.58 within serovar Paratyphi A (n = 10). The VNTR polymorphic site within multidrug-resistant (MDR) serovar Typhimurium isolates (n = 39; resistance to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline) showed a DI of 0.81. Cluster analysis not only identified three genetically distinct groups consistent with the present serovar classification of salmonellae (serovars Typhi, Paratyphi A, and Typhimurium) but also discriminated 25 subtypes (93%) within serovar Typhi isolates. The analysis discriminated only eight subtypes within serovar Typhimurium isolates resistant to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline, possibly reflecting the emergence in the mid-1990s of the DT104 phage type, which often displays such an MDR spectrum. Coupled with the ongoing improvements in automated procedures offered by capillary electrophoresis, use of these markers is proposed in further investigations of the potential of MLVA in outbreaks of salmonellosis, especially outbreaks of typhoid fever.     

Molecular basis of resistance displayed by highly ciprofloxacin-resistant Salmonella enterica serovar Typhi in Bangladesh

Highly ciprofloxacin-resistant (MIC, 512 microg/ml) strains of Salmonella enterica serovar Typhi were isolated from the blood of typhoid patients in Dhaka, Bangladesh. The strains were indistinguishable by their antibiograms, biotypes, and variable-number tandem repeat types and had matching point mutations at positions 83 and 87 of the gyrA gene. The isolation of these strains in an area of high endemicity indicates the need for continuous surveillance of antibiotic resistance of S. enterica serovar Typhi and for the rationalized use of ciprofloxacin.     

Contribution of phage typing and ribotyping in investigating a typhoid fever outbreak in Tunisia

A study was carried out to investigate an outbreak of typhoid fever that occurred in Sousse city and in the vicinity of Sousse (Tunisia) during summer 1999. Twenty four isolates of Salmonella enterica serotype Typhi were isolated in hospitalized patients with a typhoid fever in two hospitals (Farhat Hached Sousse and M'saken) and were studied with the help of two molecular typing methods: phage typing and automated ribotyping. Twenty one isolates with the Vi antigen had profile DVS (Degraded Vi Strain), one isolate with the Vi antigen belonged to phage type A and two isolates were non phage typable (no Vi antigen). The same ribotype was found in 22 out of 24 isolates. The results suggested that ribotyping is more discriminative than phage typing in this case in distinguishing strains and the strains shared the same source of the contamination. Unfortunately the precise source of the contamination could not be determined.     

Increasing genetic diversity of Salmonella enterica serovar typhi isolates from papua new guinea over the period from 1992 to 1999

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.     

Clinical application of a dot blot test for diagnosis of enteric fever due to Salmonella enterica serovar typhi in patients with typhoid fever from Colombia and Peru

Clinical application of a dot blot test to detect immunoglobulin G (IgG) (88% sensitivity and specificity) and IgM (12.1% sensitivity and 97% specificity) against flagellar antigen from Salmonella enterica serovar Typhi was performed in Peruvian and Colombian patients with typhoid fever. This test can be used as a good predictor of serovar Typhi infection in regions lacking laboratory facilities and in field studies.     

Oral priming with Salmonella Typhi vaccine strain CVD 909 followed by parenteral boost with the S. Typhi Vi capsular polysaccharide vaccine induces CD27+IgD-S. Typhi-specific IgA and IgG B memory cells in humans

Attenuated live oral typhoid vaccine candidate CVD 909 constitutively expresses Salmonella Typhi capsular polysaccharide antigen (Vi). A randomized, double-blind, heterologous prime-boost clinical study was conducted to determine whether immunity to licensed parenteral Vi vaccine could be enhanced by priming with CVD 909. Priming with CVD 909 elicited higher and persistent, albeit not significant, anti-Vi IgG and IgA following immunization with Vi, than placebo-primed recipients. Vi-specific IgA B memory (B(M)) cells were significantly increased in CVD 909-primed subjects. S. Typhi-specific LPS and flagella IgA B(M) cells were observed in subjects immunized with CVD 909 or with the licensed Vi-negative oral typhoid vaccine Ty21a. CVD 909-induced B(M) cells exhibited a classical B(M) phenotype (i.e., CD3(-)CD19(+)IgD(-)CD27(+)). This is the first demonstration of classical B(M) cells specific for bacterial polysaccharide or protein antigens following typhoid immunization. The persistent IgA B(M) responses demonstrate the capacity of oral typhoid vaccines to prime mucosally relevant immune memory.     

Salmonella enterica Serovar Typhi-Specific Immunoglobulin A Antibody Responses in Plasma and Antibody in Lymphocyte Supernatant Specimens in Bangladeshi Patients with Suspected Typhoid Fever

Many currently available diagnostic tests for typhoid fever lack sensitivity and/or specificity, especially in areas of the world where the disease is endemic. In order to identify a diagnostic test that better correlates with typhoid fever, we evaluated immune responses to Salmonella enterica serovar Typhi (serovar Typhi) in individuals with suspected typhoid fever in Dhaka, Bangladesh. We enrolled 112 individuals with suspected typhoid fever, cultured day 0 blood for serovar Typhi organisms, and performed Widal assays on days 0, 5, and 20. We harvested peripheral blood lymphocytes and analyzed antibody levels in supernatants collected on days 0, 5, and 20 (using an antibody-in-lymphocyte-supernatant [ALS] assay), as well as in plasma on these days. We measured ALS reactivity to a serovar Typhi membrane preparation (MP), a formalin-inactivated whole-cell preparation, and serovar Typhi lipopolysaccharide. We measured responses in healthy Bangladeshi, as well as in Bangladeshi febrile patients with confirmed dengue fever or leptospirosis. We categorized suspected typhoid fever individuals into different groups (groups I to V) based on blood culture results, Widal titer, and clinical features. Responses to MP antigen in the immunoglobulin A isotype were detectable at the time of presentation in the plasma of 81% of patients. The ALS assay, however, tested positive in all patients with documented or highly suspicious typhoid, suggesting that such a response could be the basis of improved diagnostic point-of-care-assay for serovar Typhi infection. It can be important for use in epidemiological studies, as well as in difficult cases involving fevers of unknown origin.Salmonella enterica serovar Typhi (serovar Typhi) is the cause of typhoid fever, an illness that affects over 20,000,000 individuals worldwide each year, killing over 200,000 (5, 8, 16). The largest burden of typhoid fever is borne by impoverished individuals in resource-poor areas of the world. Serovar Typhi is a human-restricted invasive enteric pathogen which, after ingestion, crosses the intestinal mucosa, is taken up by gut-associated lymphoreticular tissues, and enters the systemic circulation. Both mucosal and systemic host immune responses are stimulated after infection. Serovar Typhi is an intracellular pathogen, and antibody and cell-mediated immune responses occur after infection or immunization with live oral attenuated typhoid vaccines (10, 25, 34).Diagnostic tests for typhoid fever often lack sensitivity and/or specificity, especially in areas of the world that are endemic for typhoid fever, where clinically distinguishing typhoid fever from other febrile illnesses is difficult (5, 17, 39). Microbiologic culturing of blood is approximately 30 to 70% sensitive, with the highest sensitivity being associated with an absence of prior use of antibiotics and the culturing of larger volumes of blood, features that complicate this mode of diagnosis in young children (5, 6, 8, 36). Microbiologic culturing of bone marrow aspirates is more sensitive than blood but often clinically impractical (1, 11, 12). Serum Widal assay titers are often nonspecific in endemic settings and are of limited value unless titers are markedly elevated or are analyzed for changes from acute to convalescent phases of illness (18, 33, 38). Molecular diagnostic assays including PCR are promising, but issues of practicality, contamination, and quality control have limited their use in many resource-poor areas of the world (14).Since serovar Typhi interacts with both the mucosal and the systemic immune systems, we were interested to determine whether analyses of mucosal immune responses would give improved insight into this human-restricted infection. Activated mucosal lymphocytes migrate from intestinal tissue and circulate within peripheral blood before rehoming to mucosal tissues (20, 31). This migration peaks 1 to 2 weeks after intestinal infection and may be measured by using peripheral blood mononuclear cells (PBMC) in an antibody-secreting cell (ASC) assay (19, 26) or in supernatants recovered from harvested PBMC (the “antibody in lymphocyte supernatant” [ALS] assay) (7, 31). Although ALS and ASC responses have previously been measured after immunization with oral live attenuated typhoid vaccines, detailed analyses of ALS or ASC responses in individuals with wild-type typhoid fever are lacking (21, 24). In order to gain further insight into mucosal immune responses during wild-type serovar Typhi infection, we undertook a study to characterize the serum and ALS responses to serovar Typhi among individuals with suspected typhoid fever in Bangladesh.     

Vi capsular polysaccharide: Synthesis,virulence, and application

Vi capsular polysaccharide, a linear homopolymer of α-1,4-linked N-acetylgalactosaminuronate, is characteristically produced by Salmonella enterica serovar Typhi. The Vi capsule covers the surface of the producing bacteria and serves as an virulence factor via inhibition of complement-mediated killing and promoting resistance against phagocytosis. Furthermore, Vi also represents a predominant protective antigen and plays a key role in the development of vaccines against typhoid fever. Herein, we reviewed the latest advances associated with the Vi polysaccharide, from its synthesis and transport within bacterial cells, mechanisms involved in virulence, immunological characteristics, and applications in vaccine, as well as its purification and detection methods.