Evaluation of an Electricity-free,Culture-based Approach for Detecting Typhoidal Salmonella Bacteremia during Enteric Fever in a High Burden,Resource-limited Setting

Background

In many rural areas at risk for enteric fever, there are few data on Salmonella enterica serotypes Typhi (S. Typhi) and Paratyphi (S. Paratyphi) incidence, due to limited laboratory capacity for microbiologic culture. Here, we describe an approach that permits recovery of the causative agents of enteric fever in such settings. This approach involves the use of an electricity-free incubator based upon use of phase-change materials. We compared this against conventional blood culture for detection of typhoidal Salmonella.

Methodology/Principal Findings

Three hundred and four patients with undifferentiated fever attending the outpatient and emergency departments of a public hospital in the Kathmandu Valley of Nepal were recruited. Conventional blood culture was compared against an electricity-free culture approach. Blood from 66 (21.7%) patients tested positive for a Gram-negative bacterium by at least one of the two methods. Sixty-five (21.4%) patients tested blood culture positive for S. Typhi (30; 9.9%) or S. Paratyphi A (35; 11.5%). From the 65 individuals with culture-confirmed enteric fever, 55 (84.6%) were identified by the conventional blood culture and 60 (92.3%) were identified by the experimental method. Median time-to-positivity was 2 days for both procedures. The experimental approach was falsely positive due to probable skin contaminants in 2 of 239 individuals (0.8%). The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% (95% CI: 80.0%–97.0%) and 96.0% (92.7%–98.1%), respectively. After initial incubation, Salmonella isolates could be readily recovered from blood culture bottles maintained at room temperature for six months.

Conclusions/Significance

A simple culture approach based upon a phase-change incubator can be used to isolate agents of enteric fever. This approach could be used as a surveillance tool to assess incidence and drug resistance of the etiologic agents of enteric fever in settings without reliable local access to electricity or local diagnostic microbiology laboratories.     

Genetic diversity of Salmonella Paratyphi A isolated from enteric fever patients in Bangladesh from 2008 to 2018

BackgroundThe proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh.Principal findingsWe conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms.SignificanceThis study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies.     

Non-Protein Coding RNA Genes as the Novel Diagnostic Markers for the Discrimination of Salmonella Species Using PCR

Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we formulated a strategy of detection and differentiation of salmonellosis by a multiplex polymerase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecular target for infectious disease.     

Antimicrobial Resistance,Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata,India during 2009-2013

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (bla _TEM-1, catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.     

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

Background

Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.

Methods

Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.

Results

An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.

Conclusions

The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.     

Differential Epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu,Nepal: A Matched Case Control Investigation in a Highly Endemic Enteric Fever Setting

Background

Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.

Methodology and principal findings

A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.

Conclusions and significance

Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.     

Construction of an attenuated Salmonella enterica serovar Paratyphi A vaccine strain harboring defined mutations in htrA and yncD

The global epidemic features of enteric fever have changed greatly in recent years. The incidence of enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased. In some areas of Asia, infections with S. Paratyphi A have exceeded those with S. Typhi, resulting in S. Paratyphi A becoming the main causative agent of enteric fever. However, two currently licensed typhoid vaccines do not confer adequate cross‐protection against S. Paratyphi A infection. Therefore, development of specific vaccines against enteric fever caused by S. Paratyphi A is urgently needed. In the present study, an attenuated strain was constructed by double deletion of the htrA and yncD genes in a wild‐type strain of S. Paratyphi A and its safety and immunogenicity assessed. In a mouse model, the 50% lethal dose of the double deletion mutant and the wild‐type strain were 3.0 × 10⁸ CFU and 1.9 × 10³ CFU, respectively, suggesting that the double deletion resulted in remarkably decreased bacterial virulence. Bacterial colonization of the double deletion mutant in the livers and spleens of infected mice was strikingly less than that of the wild‐type strain. A single nasal administration of the attenuated vaccine candidate elicited high concentrations of anti‐LPS and anti‐flagellin IgG in a mouse model and protected immunized mice against lethal challenge with the wild‐type strain. Thus, our findings suggest that the attenuated vaccine strain is a promising candidate worthy of further evaluation both as a human enteric fever vaccine and as a vaccine delivery vector for heterologous antigens.     

CRISPR Is an Optimal Target for the Design of Specific PCR Assays for Salmonella enterica Serotypes Typhi and Paratyphi A

Background

Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms.

Methodology

Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats), as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers.

Principal findings

We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species.

Conclusions

The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.     

Differential Killing of Salmonella enterica Serovar Typhi by Antibodies Targeting Vi and Lipopolysaccharide O:9 Antigen

Salmonella enterica serovar Typhi expresses a capsule of Vi polysaccharide, while most Salmonella serovars, including S. Enteritidis and S. Typhimurium, do not. Both S. Typhi and S. Enteritidis express the lipopolysaccharide O:9 antigen, yet there is little evidence of cross-protection from anti-O:9 antibodies. Vaccines based on Vi polysaccharide have efficacy against typhoid fever, indicating that antibodies against Vi confer protection. Here we investigate the role of Vi capsule and antibodies against Vi and O:9 in antibody-dependent complement- and phagocyte-mediated killing of Salmonella. Using isogenic Vi-expressing and non-Vi-expressing derivatives of S. Typhi and S. Typhimurium, we show that S. Typhi is inherently more sensitive to serum and blood than S. Typhimurium. Vi expression confers increased resistance to both complement- and phagocyte-mediated modalities of antibody-dependent killing in human blood. The Vi capsule is associated with reduced C3 and C5b-9 deposition, and decreased overall antibody binding to S. Typhi. However, purified human anti-Vi antibodies in the presence of complement are able to kill Vi-expressing Salmonella, while killing by anti-O:9 antibodies is inversely related to Vi expression. Human serum depleted of antibodies to antigens other than Vi retains the ability to kill Vi-expressing bacteria. Our findings support a protective role for Vi capsule in preventing complement and phagocyte killing of Salmonella that can be overcome by specific anti-Vi antibodies, but only to a limited extent by anti-O:9 antibodies.     

Typhoidal Salmonella: Distinctive virulence factors and pathogenesis

Although nontyphoidal Salmonella (NTS; including Salmonella Typhimurium) mainly cause gastroenteritis, typhoidal serovars (Salmonella Typhi and Salmonella Paratyphi A) cause typhoid fever, the treatment of which is threatened by increasing drug resistance. Our understanding of S. Typhi infection in human remains poorly understood, likely due to the host restriction of typhoidal strains and the subsequent popularity of the S. Typhimurium mouse typhoid model. However, translating findings with S. Typhimurium across to S. Typhi has some limitations. Notably, S. Typhi has specific virulence factors, including typhoid toxin and Vi antigen, involved in symptom development and immune evasion, respectively. In addition to unique virulence factors, both typhoidal and NTS rely on two pathogenicity‐island encoded type III secretion systems (T3SS), the SPI‐1 and SPI‐2 T3SS, for invasion and intracellular replication. Marked differences have been observed in terms of T3SS regulation in response to bile, oxygen, and fever‐like temperatures. Moreover, approximately half of effectors found in S. Typhimurium are either absent or pseudogenes in S. Typhi, with most of the remaining exhibiting sequence variation. Typhoidal‐specific T3SS effectors have also been described. This review discusses what is known about the pathogenesis of typhoidal Salmonella with emphasis on unique behaviours and key differences when compared with S. Typhimurium.     

Oral Wild-Type Salmonella Typhi Challenge Induces Activation of Circulating Monocytes and Dendritic Cells in Individuals Who Develop Typhoid Disease

A new human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently developed. In this model, ingestion of 104 CFU of Salmonella resulted in 65% of subjects developing typhoid fever (referred here as typhoid diagnosis -TD-) 5–10 days post-challenge. TD criteria included meeting clinical (oral temperature ≥38°C for ≥12h) and/or microbiological (S. Typhi bacteremia) endpoints. One of the first lines of defense against pathogens are the cells of the innate immune system (e.g., monocytes, dendritic cells -DCs-). Various changes in circulating monocytes and DCs have been described in the murine S. Typhimurium model; however, whether similar changes are present in humans remains to be explored. To address these questions, a subset of volunteers (5 TD and 3 who did not develop typhoid despite oral challenge -NoTD-) were evaluated for changes in circulating monocytes and DCs. Expression of CD38 and CD40 were upregulated in monocytes and DCs in TD volunteers during the disease days (TD-0h to TD-96h). Moreover, integrin α4β7, a gut homing molecule, was upregulated on monocytes but not DCs. CD21 upregulation was only identified in DCs. These changes were not observed among NoTD volunteers despite the same oral challenge. Moreover, monocytes and DCs from NoTD volunteers showed increased binding to S. Typhi one day after challenge. These monocytes showed phosphorylation of p38MAPK, NFkB and Erk1/2 upon stimulation with S. Typhi-LPS-QDot micelles. In contrast, monocytes from TD volunteers showed only a moderate increase in S. Typhi binding 48h and 96h post-TD, and only Erk1/2 phosphorylation. This is the first study to describe different activation and migration profiles, as well as differential signaling patterns, in monocytes and DCs which relate directly to the clinical outcome following oral challenge with wild type S. Typhi.     

Comparison of Culture Methods for Isolation of Salmonella in Yak Fecal Samples

To compare the effectiveness of culture methods for identifying yak Salmonella, three selective enrichment broths (SC, TTB, MSRV) and three media (SS, XLD, CAS) for detecting Salmonella were evaluated in this study. The results showed that TTB broth was better than SC broths and MSRV broths, and SS medium has the highest isolation rate, significantly higher than those of CAS and XLD media (P < 0.05). It is worth noticing that there was no overlapping of the positive results given by TTB, SC and MSRV broths. In addition, all of the yak Salmonella isolates were detected positive by the five reported PCR assays, targeting the invA, srfC, invE, stn and 16S–23S rRNA genes. The combination of TTB and MSRV broths and SS and CAS media (or XLD) recommended in this study was relatively efficient in recovering Salmonella from yak feces, and the five PCR assays can be successfully used to identify yak Salmonella.     

Estimating the Burden of Paratyphoid A in Asia and Africa

Despite the increasing availability of typhoid vaccine in many regions, global estimates of mortality attributable to enteric fever appear stable. While both Salmonella enterica serovar Typhi (S. Typhi) and serovar Paratyphi (S. Paratyphi) cause enteric fever, limited data exist estimating the burden of S. Paratyphi, particularly in Asia and Africa.We performed a systematic review of both English and Chinese-language databases to estimate the regional burden of paratyphoid within Africa and Asia. Distinct from previous reviews of the topic, we have presented two separate measures of burden; both incidence and proportion of enteric fever attributable to paratyphoid. Included articles reported laboratory-confirmed Salmonella serovar classification, provided clear methods on sampling strategy, defined the age range of participants, and specified the time period of the study.A total of 64 full-text articles satisfied inclusion criteria and were included in the qualitative synthesis. Paratyphoid A was commonly identified as a cause of enteric fever throughout Asia. The highest incidence estimates in Asia came from China; four studies estimated incidence rates of over 150 cases/100,000 person-years. Paratyphoid A burden estimates from Africa were extremely limited and with the exception of Nigeria, few population or hospital-based studies from Africa reported significant Paratyphoid A burden.While significant gaps exist in the existing population-level estimates of paratyphoid burden in Asia and Africa, available data suggest that paratyphoid A is a significant cause of enteric fever in Asia. The high variability in documented incidence and proportion estimates of paratyphoid suggest considerable geospatial variability in the burden of paratyphoid fever. Additional efforts to monitor enteric fever at the population level will be necessary in order to accurately quantify the public health threat posed by S. Paratyphi A, and to improve the prevention and treatment of enteric fever.     

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26’s role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity.     

Proteomic comparison between Salmonella Typhimurium and Salmonella Typhi

The genus Salmonella contains more than 2500 serovars. While most cause the self-limiting gastroenteritis, a few serovars can elicit typhoid fever, a severe systemic infection. S. enterica subsp. enterica serovar Typhimurium and S. Typhi are the representatives of the gastroenteritis and typhoid fever types of Salmonella. In this study, we adopted Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technology to quantitatively compare the proteomes of the two serovars. We found several proteins with serovar-specific expression, which could be developed as new biomarkers for clinical serotype diagnosis. We found that flagella and chemotaxis genes were down-regulated in S. Typhi in comparison with S. Typhimurium. We attributed this observation to the fact that the smooth cellular structure of S. Typhi may better fit its systemic lifestyle. Instead of known virulence factors that were located within Salmonella Pathogenecity Islands, a number of core genes, which were involved in metabolism and transport of carbohydrates and amino acids, showed differential expression between the two serovars. Further studies on the roles of these differentially-expressed genes in the pathogenesis should be undertaken.     

Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. Typhi Challenge Model

Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, causes significant morbidity and mortality worldwide. Currently available vaccines are moderately efficacious, and identification of immunological responses associated with protection or disease will facilitate the development of improved vaccines. We investigated S. Typhi-specific modulation of activation and homing potential of circulating regulatory T cells (T_reg) by flow and mass cytometry using specimens obtained from a human challenge study. Peripheral blood mononuclear cells were obtained from volunteers pre- and at multiple time-points post-challenge with wild-type S. Typhi. We identified differing patterns of S. Typhi-specific modulation of the homing potential of circulating T_reg between volunteers diagnosed with typhoid (TD) and those who were not (No TD). TD volunteers demonstrated up-regulation of the gut homing molecule integrin α4ß7 pre-challenge, followed by a significant down-regulation post-challenge consistent with T_reg homing to the gut. Additionally, S. Typhi-specific T_reg from TD volunteers exhibited up-regulation of activation molecules post-challenge (e.g., HLA-DR, LFA-1). We further demonstrate that depletion of T_reg results in increased S. Typhi-specific cytokine production by CD8+ T_EM in vitro. These results suggest that the tissue distribution of activated T_reg, their characteristics and activation status may play a pivotal role in typhoid fever, possibly through suppression of S. Typhi-specific effector T cell responses. These studies provide important novel insights into the regulation of immune responses that are likely to be critical in protection against typhoid and other enteric infectious diseases.     

Bloodstream Infection among Children Presenting to a General Hospital Outpatient Clinic in Urban Nepal

Background

There are limited data on the etiology and characteristics of bloodstream infections in children presenting in hospital outpatient settings in South Asia. Previous studies in Nepal have highlighted the importance of murine typhus as a cause of febrile illness in adults and enteric fever as a leading bacterial cause of fever among children admitted to hospital.

Methods

We prospectively studied a total of 1084 febrile children aged between 2 months and 14 years presenting to a general hospital outpatient department in Kathmandu Valley, Nepal, over two study periods (summer and winter). Blood from all patients was tested by conventional culture and by real-time PCR for Rickettsia typhi.

Results

Putative etiological agents for fever were identified in 164 (15%) patients. Salmonella enterica serovar Typhi (S. Typhi) was identified in 107 (10%), S. enterica serovar Paratyphi A (S. Paratyphi) in 30 (3%), Streptococcus pneumoniae in 6 (0.6%), S. enterica serovar Typhimurium in 2 (0.2%), Haemophilus influenzae type b in 1 (0.1%), and Escherichia coli in 1 (0.1%) patient. S. Typhi was the most common organism isolated from blood during both summer and winter. Twenty-two (2%) patients were PCR positive for R. typhi. No significant demographic, clinical and laboratory features distinguished culture positive enteric fever and murine typhus.

Conclusions

Salmonella infections are the leading cause of bloodstream infection among pediatric outpatients with fever in Kathmandu Valley. Extension of immunization programs against invasive bacterial disease to include the agents of enteric fever and pneumococcus could improve the health of children in Nepal.     

The Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Salmonella enterica serovar Typhi

Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol.     

O:2-CRM197 Conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM₁₉₇, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM₁₉₇ as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.