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.     

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.     

Azithromycin and Ciprofloxacin Resistance in Salmonella Bloodstream Infections in Cambodian Adults

Background

Salmonella enterica is a frequent cause of bloodstream infection (BSI) in Asia but few data are available from Cambodia. We describe Salmonella BSI isolates recovered from patients presenting at Sihanouk Hospital Centre of Hope, Phnom Penh, Cambodia (July 2007–December 2010).

Methodology

Blood was cultured as part of a microbiological prospective surveillance study. Identification of Salmonella isolates was performed by conventional methods and serotyping. Antibiotic susceptibilities were assessed using disk diffusion, MicroScan and E-test macromethod. Clonal relationships were assessed by Pulsed Field Gel Electrophoresis; PCR and sequencing for detection of mutations in Gyrase and Topoisomerase IV and presence of qnr genes.

Principal Findings

Seventy-two Salmonella isolates grew from 58 patients (mean age 34.2 years, range 8–71). Twenty isolates were identified as Salmonella Typhi, 2 as Salmonella Paratyphi A, 37 as Salmonella Choleraesuis and 13 as other non-typhoid Salmonella spp. Infection with human immunodeficiency virus (HIV) was present in 21 of 24 (87.5%) patients with S. Choleraesuis BSI. Five patients (8.7%) had at least one recurrent infection, all with S. Choleraesuis; five patients died. Overall, multi drug resistance (i.e., co-resistance to ampicillin, sulphamethoxazole-trimethoprim and chloramphenicol) was high (42/59 isolates, 71.2%). S. Typhi displayed high rates of decreased ciprofloxacin susceptibility (18/20 isolates, 90.0%), while azithromycin resistance was very common in S. Choleraesuis (17/24 isolates, 70.8%). Two S. Choleraesuis isolates were extended spectrum beta-lactamase producer.

Conclusions and Significance

Resistance rates in Salmonella spp. in Cambodia are alarming, in particular for azithromycin and ciprofloxacin. This warrants nationwide surveillance and revision of treatment guidelines.     

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.     

Dynamic Modularity of Host Protein Interaction Networks in Salmonella Typhi Infection

Background

Salmonella Typhi is a human-restricted pathogen, which causes typhoid fever and remains a global health problem in the developing countries. Although previously reported host expression datasets had identified putative biomarkers and therapeutic targets of typhoid fever, the underlying molecular mechanism of pathogenesis remains incompletely understood.

Methods

We used five gene expression datasets of human peripheral blood from patients suffering from S. Typhi or other bacteremic infections or non-infectious disease like leukemia. The expression datasets were merged into human protein interaction network (PIN) and the expression correlation between the hubs and their interacting proteins was measured by calculating Pearson Correlation Coefficient (PCC) values. The differences in the average PCC for each hub between the disease states and their respective controls were calculated for studied datasets. The individual hubs and their interactors with expression, PCC and average PCC values were treated as dynamic subnetworks. The hubs that showed unique trends of alterations specific to S. Typhi infection were identified.

Results

We identified S. Typhi infection-specific dynamic subnetworks of the host, which involve 81 hubs and 1343 interactions. The major enriched GO biological process terms in the identified subnetworks were regulation of apoptosis and biological adhesions, while the enriched pathways include cytokine signalling in the immune system and downstream TCR signalling. The dynamic nature of the hubs CCR1, IRS2 and PRKCA with their interactors was studied in detail. The difference in the dynamics of the subnetworks specific to S. Typhi infection suggests a potential molecular model of typhoid fever.

Conclusions

Hubs and their interactors of the S. Typhi infection-specific dynamic subnetworks carrying distinct PCC values compared with the non-typhoid and other disease conditions reveal new insight into the pathogenesis of S. Typhi.     

East London Experience with Enteric Fever 2007-2012

Purpose

The clinical presentation and epidemiology for patients with enteric fever at two hospitals in East London during 2007–2012 is described with the aim to identify preventive opportunities and to reduce the cost of treatment.

Methods

A retrospective analysis of case notes from patients admitted with enteric fever during 2007 to 2012 with a microbiologically confirmed diagnosis was undertaken. Details on clinical presentation, travel history, demographic data, laboratory parameters, treatment, patient outcome and vaccination status were collected.

Results

Clinical case notes were available for 98/129 (76%) patients including 69 Salmonella enterica serovar Typhi (S. Typhi) and 29 Salmonella enterica serovar Paratyphi (S. Paratyphi). Thirty-four patients (35%) were discharged from emergency medicine without a diagnosis of enteric fever and then readmitted after positive blood cultures. Seventy-one of the 98 patients (72%) were UK residents who had travelled abroad, 23 (23%) were foreign visitors/new entrants to the UK and four (4%) had not travelled abroad. Enteric fever was not considered in the initial differential diagnosis for 48/98 (49%) cases. The median length of hospital stay was 7 days (range 0–57 days). The total cost of bed days for managing enteric fever was £454,000 in the two hospitals (mean £75,666/year). Median time to clinical resolution was five days (range 1–20). Seven of 98 (7%) patients were readmitted with relapsed or continued infection. Six of the 71 (8%) patients had received typhoid vaccination, 34 (48%) patients had not received vaccination, and for 31 cases (44%) vaccination status was unknown.

Conclusions

Further interventions regarding education and vaccination of travellers and recognition of the condition by emergency medicine clinicians in travellers to South Asia is required.     

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.     

High-resolution genotyping of the endemic Salmonella Typhi population during a Vi (typhoid) vaccination trial in Kolkata

Background

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem especially in developing countries. Vaccines against typhoid are commonly used by travelers but less so by residents of endemic areas.

Methodology

We used single nucleotide polymorphism (SNP) typing to investigate the population structure of 372 S. Typhi isolated during a typhoid disease burden study and Vi vaccine trial in Kolkata, India. Approximately sixty thousand people were enrolled for fever surveillance for 19 months prior to, and 24 months following, Vi vaccination of one third of the study population (May 2003–December 2006, vaccinations given December 2004).

Principal Findings

A diverse S. Typhi population was detected, including 21 haplotypes. The most common were of the H58 haplogroup (69%), which included all multidrug resistant isolates (defined as resistance to chloramphenicol, ampicillin and co-trimoxazole). Quinolone resistance was particularly high among H58-G isolates (97% Nalidixic acid resistant, 30% with reduced susceptibility to ciprofloxacin). Multiple typhoid fever episodes were detected in 22 households, however household clustering was not associated with specific S. Typhi haplotypes.

Conclusions

Typhoid fever in Kolkata is caused by a diverse population of S. Typhi, however H58 haplotypes dominate and are associated with multidrug and quinolone resistance. Vi vaccination did not obviously impact on the haplotype population structure of the S. Typhi circulating during the study period.     

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.     

A Large-Scale Community-Based Outbreak of Paratyphoid Fever Caused by Hospital-Derived Transmission in Southern China

Background

Since the 1990s, paratyphoid fever caused by Salmonella Paratyphi A has emerged in Southeast Asia and China. In 2010, a large-scale outbreak involving 601 cases of paratyphoid fever occurred in the whole of Yuanjiang county in China. Epidemiological and laboratory investigations were conducted to determine the etiology, source and transmission factors of the outbreak.

Methodology/Principal Findings

A case-control study was performed to identify the risk factors for this paratyphoid outbreak. Cases were identified as patients with blood culture–confirmed S. Paratyphi A infection. Controls were healthy persons without fever within the past month and matched to cases by age, gender and geography. Pulsed-field gel electrophoresis and whole-genome sequencing of the S. Paratyphi A strains isolated from patients and environmental sources were performed to facilitate transmission analysis and source tracking. We found that farmers and young adults were the populations mainly affected in this outbreak, and the consumption of raw vegetables was the main risk factor associated with paratyphoid fever. Molecular subtyping and genome sequencing of S. Paratyphi A isolates recovered from improperly disinfected hospital wastewater showed indistinguishable patterns matching most of the isolates from the cases. An investigation showed that hospital wastewater mixed with surface water was used for crop irrigation, promoting a cycle of contamination. After prohibition of the planting of vegetables in contaminated fields and the thorough disinfection of hospital wastewater, the outbreak subsided. Further analysis of the isolates indicated that the origin of the outbreak was most likely from patients outside Yuanjiang county.

Conclusions

This outbreak is an example of the combined effect of social behaviors, prevailing ecological conditions and improper disinfection of hospital wastewater on facilitating a sustained epidemic of paratyphoid fever. This study underscores the critical need for strict treatment measures of hospital wastewater and the maintenance of independent agricultural irrigation systems in rural areas.     

Expression and Function of S100A8/A9 (Calprotectin) in Human Typhoid Fever and the Murine Salmonella Model

Background

Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.

Methods and principal findings

S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9^-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9^-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.

Conclusion

S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.     

Cell-associated flagella enhance the protection conferred by mucosally-administered attenuated Salmonella Paratyphi A vaccines

Background

Antibiotic-resistant Salmonella enterica serovar Paratyphi A, the agent of paratyphoid A fever, poses an emerging public health dilemma in endemic areas of Asia and among travelers, as there is no licensed vaccine. Integral to our efforts to develop a S. Paratyphi A vaccine, we addressed the role of flagella as a potential protective antigen by comparing cell-associated flagella with exported flagellin subunits expressed by attenuated strains.

Methodology

S. Paratyphi A strain ATCC 9150 was first deleted for the chromosomal guaBA locus, creating CVD 1901. Further chromosomal deletions in fliD (CVD 1901D) or flgK (CVD 1901K) were then engineered, resulting in the export of unpolymerized FliC, without impairing its overall expression. The virulence of the resulting isogenic strains was examined using a novel mouse LD₅₀ model to accommodate the human-host restricted S. Paratyphi A. The immunogenicity of the attenuated strains was then tested using a mouse intranasal model, followed by intraperitoneal challenge with wildtype ATCC 9150.

Results

Mucosal (intranasal) immunization of mice with strain CVD 1901 expressing cell-associated flagella conferred superior protection (vaccine efficacy [VE], 90%) against a lethal intraperitoneal challenge, compared with the flagellin monomer-exporting mutants CVD 1901K (30% VE) or CVD 1901D (47% VE). The superior protection induced by CVD 1901 with its cell-attached flagella was associated with an increased IgG2a∶IgG1 ratio of FliC-specific antibodies with enhanced opsonophagocytic capacity.

Conclusions

Our results clearly suggest that enhanced anti-FliC antibody-mediated clearance of S. Paratyphi A by phagocytic cells, induced by vaccines expressing cell-associated rather than exported FliC, might be contributing to the vaccine-induced protection from S. Paratyphi A challenge in vivo. We speculate that an excess of IgG1 anti-FliC antibodies induced by the exported FliC may compete with the IgG2a subtype and block binding to specific phagocyte Fc receptors that are critical for clearing an S. Paratyphi A infection.     

Interferon-γ and proliferation responses to Salmonella enterica Serotype Typhi proteins in patients with S. Typhi Bacteremia in Dhaka, Bangladesh

Background

Salmonella enterica serotype Typhi is a human-restricted intracellular pathogen and the cause of typhoid fever. Cellular immune responses are required to control and clear Salmonella infection. Despite this, there are limited data on cellular immune responses in humans infected with wild type S. Typhi.

Methodology/Principal Findings

For this work, we used an automated approach to purify a subset of S. Typhi proteins identified in previous antibody-based immuno-affinity screens and antigens known to be expressed in vivo, including StaF-putative fimbrial protein-STY0202, StbB-fimbrial chaperone-STY0372, CsgF-involved in curli production-STY1177, CsgD- putative regulatory protein-STY1179, OppA-periplasmic oligopeptide binding protein precursor-STY1304, PagC-outer membrane invasion protein-STY1878, and conserved hypothetical protein-STY2195; we also generated and analyzed a crude membrane preparation of S. Typhi (MP). In comparison to samples collected from uninfected Bangladeshi and North American participants, we detected significant interferon-γ responses in PBMCs stimulated with MP, StaF, StbB, CsgF, CsgD, OppA, STY2195, and PagC in patients bacteremic with S. Typhi in Bangladesh. The majority of interferon-γ expressing T cells were CD4 cells, although CD8 responses also occurred. We also assessed cellular proliferation responses in bacteremic patients, and confirmed increased responses in infected individuals to MP, StaF, STY2195, and PagC in convalescent compared to acute phase samples and compared to controls. StaF is a fimbrial protein homologous to E. coli YadK, and contains a Pfam motif thought to be involved in cellular adhesion. PagC is expressed in vivo under the control of the virulence-associated PhoP-regulon required for intra-macrophage survival of Salmonella. STY2195 is a conserved hypothetical protein of unknown function.

Conclusion/Significance

This is the first analysis of cellular immune responses to purified S. Typhi antigens in patients with typhoid fever. These results indicate that patients generate significant CD4 and CD8 interferon-γ responses to specific S. Typhi antigens during typhoid fever, and that these responses are elevated at the time of clinical presentation. These observations suggest that an interferon-γ based detection system could be used to diagnose individuals with typhoid fever during the acute stage of illness.     

Salmonella Typhi in the Democratic Republic of the Congo: Fluoroquinolone Decreased Susceptibility on the Rise

Background

Drug resistance of Salmonella enterica serovar Typhi (Salmonella Typhi) to first-line antibiotics is emerging in Central Africa. Although increased use of fluoroquinolones is associated with spread of resistance, Salmonella Typhi with decreased ciprofloxacin susceptibility (DCS) has rarely been reported in Central Africa.

Methodology/Principal Findings

As part of a microbiological surveillance study in the Democratic Republic of the Congo (DR Congo), Salmonella Typhi isolates from bloodstream infections were collected prospectively between 2007 and 2011. The genetic relationship of the Salmonella Typhi isolates was assessed by pulsed-field gel electrophoresis (PFGE). The antimicrobial resistance profile of the isolates was determined and mutations associated with DCS were studied. In total, 201 Salmonella Typhi isolates were collected. More than half of the Salmonella Typhi isolates originated from children and young adults aged 5–19. Thirty different PFGE profiles were identified, with 72% of the isolates showing a single profile. Multidrug resistance, DCS and azithromycin resistance were 30.3%, 15.4% and 1.0%, respectively. DCS was associated with point mutations in the gyrA gene at codons 83 and 87.

Conclusions/Significance

Our study describes the first report of widespread multidrug resistance and DCS among Salmonella Typhi isolates from DR Congo. Our findings highlight the need for increased microbiological diagnosis and surveillance in DR Congo, being a prerequisite for rational use of antimicrobials and the development of standard treatment guidelines.     

Priming of Salmonella enterica Serovar Typhi-Specific CD8+ T Cells by Suicide Dendritic Cell Cross-Presentation in Humans

Background

The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8⁺ T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-γ secretion. However, how S. Typhi regulates the development of specific CD8⁺ responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8⁺ T cells.

Methodology/Principal Findings

We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-α, but low levels of IL-12 p70 and IFN-γ. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-γ and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3⁺CD8⁺CD45RA⁻CD62L⁻ effector/memory T cells.

Conclusions/Significance

This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8⁺ cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.     

The burden of invasive bacterial infections in Pemba, Zanzibar

Background

We conducted a surveillance study to determine the leading causes of bloodstream infection in febrile patients seeking treatment at three district hospitals in Pemba Island, Zanzibar, Tanzania, an area with low malaria transmission.

Methods

All patients above two months of age presenting to hospital with fever were screened, and blood was collected for microbiologic culture and malaria testing. Bacterial sepsis and malaria crude incidence rates were calculated for a one-year period and were adjusted for study participation and diagnostic sensitivity of blood culture.

Results

Blood culture was performed on 2,209 patients. Among them, 166 (8%) samples yielded bacterial growth; 87 (4%) were considered as likely contaminants; and 79 (4%) as pathogenic bacteria. The most frequent pathogenic bacteria isolated were Salmonella Typhi (n = 46; 58%), followed by Streptococcus pneumoniae (n = 12; 15%). The crude bacteremia rate was 6/100,000 but when adjusted for potentially missed cases the rate may be as high as 163/100,000. Crude and adjusted rates for S. Typhi infections and malaria were 4 and 110/100,000 and 4 and 47/100,000, respectively. Twenty three (51%), 22 (49%) and 22 (49%) of the S.Typhi isolates were found to be resistant toward ampicillin, chloramphenicol and cotrimoxazole, respectively. Multidrug resistance (MDR) against the three antimicrobials was detected in 42% of the isolates.

Conclusions

In the presence of very low malaria incidence we found high rates of S. Typhi and S. pneumoniae infections on Pemba Island, Zanzibar. Preventive measures such as vaccination could reduce the febrile disease burden.     

Head-to-Head Comparison of Humoral Immune Responses to Vi Capsular Polysaccharide and Salmonella Typhi Ty21a Typhoid Vaccines–A Randomized Trial

Background

The two typhoid vaccines, the parenteral Vi capsular polysaccharide and the oral live whole-cell Salmonella Typhi Ty21a vaccine, provide similar levels of protection in field trials. Sharing no antigens, they are thought to confer protection by different mechanisms. This is the first head-to-head study to compare the humoral immune responses to these two vaccines.

Methods

50 age- and gender-matched volunteers were immunized, 25 with the Vi and 25 with the Ty21a vaccine. Circulating plasmablasts reactive with whole-cell Salmonella Typhi or one of the typhoidal antigenic structures, Vi, O-9,12, and H-d antigens, were identified as antibody-secreting cells (ASC) with ELISPOT. Homing receptor (HR) expressions were determined. These results were compared with ASC in four patients with typhoid fever. Antibodies to S. Typhi lipopolysaccharides were assessed in cultures of ALS (antibodies in lymphocyte supernatants) and in serum with ELISA.

Results

In 49 out of 50 vaccinees, no typhoid-specific plasmablasts were seen before vaccination. On day 7, response to Vi antigen was mounted in 24/25 volunteers in the Vi, and none in the Ty21a group; response to S. Typhi and O-9,12 was mounted in 49/50 vaccinees; and to H-d in 3/50. The numbers of typhoid-specific plasmablasts (total of ASC to Vi, O-9,12 and H-d antigens) proved equal in the vaccination groups. The HR expressions indicated a mainly systemic homing in the Vi and intestinal in the Ty21a group, the latter resembling that in natural infection. Plasmablasts proved more sensitive than serum and ALS in assessing the immune response.

Conclusions

The typhoid-specific humoral responses to Vi and Ty21a vaccines are similar in magnitude, but differ in expected localization and antigen-specificity. The unforeseen O antigen-specific response in the Vi group is probably due to lipopolysaccharide contaminating the vaccine preparation. Only the response to Ty21a vaccine was found to imitate that in natural infection.

Trial Registration

Current Controlled Trials Ltd. c/o BioMed Central ISRCTN68125331 http://www.controlled-trials.com/ISRCTN68125331/     

The influence of reduced susceptibility to fluoroquinolones in Salmonella enterica serovar Typhi on the clinical response to ofloxacin therapy

Background

Infection with Salmonella enterica serovar Typhi (S. Typhi) with reduced susceptibility to fluoroquinolones has been associated with fluoroquinolone treatment failure. We studied the relationship between ofloxacin treatment response and the ofloxacin minimum inhibitory concentration (MIC) of the infecting isolate. Individual patient data from seven randomised controlled trials of antimicrobial treatment in enteric fever conducted in Vietnam in which ofloxacin was used in at least one of the treatment arms was studied. Data from 540 patients randomised to ofloxacin treatment was analysed to identify an MIC of the infecting organism associated with treatment failure.

Principal Findings

The proportion of patients failing ofloxacin treatment was significantly higher in patients infected with S. Typhi isolates with an MIC≥0.25 µg/mL compared with those infections with an MIC of ≤0.125 µg/mL (p<0.001). Treatment success was 96% when the ofloxacin MIC was ≤0.125 µg/mL, 73% when the MIC was between 0.25 and 0.50 µg/mL and 53% when the MIC was 1.00 µg/mL. This was despite a longer duration of treatment at a higher dosage in patients infected with isolates with an MIC≥0.25 µg/mL compared with those infections with an MIC of ≤0.125 µg/mL.

Significance

There is a clear relationship between ofloxacin susceptibility and clinical outcome in ofloxacin treated patients with enteric fever. An ofloxacin MIC of ≥0.25 µg/mL, or the presence of nalidixic acid resistance, can be used to define S. Typhi infections in which the response to ofloxacin may be impaired.