The Humoral Response against <i>Salmonella</i> Typhi Protein Antigens During Acute, Convalescent, and Chronic Typhoid Fever

Enteric (typhoid) fever is a life-threatening disease caused by the Salmonella enterica subspecies enterica serovars Typhi (S. Typhi) and Paratyphi A, B, and C (S. Paratyphi A, B, and C). The disease still causes major public health problems in low- and middle-income countries, principally in Asia and Africa. The increasing frequency of multi-drug resistant (MDR) and extended-drug resistant isolates (XDR) of S. Typhi and an increasing incidence of S. Paratyphi A mean that the international dynamics of enteric fever are changing. These changes add urgency to the demand for more efficient enteric fever control campaigns. The aim of this thesis was to assess control measures for enteric fever in Vietnam and to develop techniques that can be used as further control methods. I firstly systemically reviewed retrospective information regarding enteric fever in Vietnam and combined these data with data on economic development. This investigation revealed that national economic growth, the provision of improved quality drinking water, and better sanitation were likely the greatest contributors to the decline and ultimate elimination of enteric fever in Vietnam. My work then evaluated the serodiagnostic potential of a panel of novel S. Typhi protein antigens and the Vi capsular polysaccharide (Vi) in a group of patients with febrile diseases in Bangladesh. These data demonstrated the utility of serology for typhoid diagnostics when exploiting a combination of Vi and at least one protein antigen. I then assessed the acquisition of antibody against typhoid toxin during natural S. Typhi and S. Paratyphi A infections and measured the capability of these antibodies to neutralise the toxin. The data provided supporting evidence for generating an antitoxin treatment for enteric fever (caused by both S. Typhi and S. Paratyphi A), and potentially encourages the use of typhoid toxin in vaccine formulations. Within the scope of searching for vaccine novel candidates, my work further identified a panel of immunogenic antigens shared between S. Typhi and S. Paratyphi A that can stimulate an antibody response which can instigate bactericidal killing during natural infection. Finally, by exploiting the unique immunological profiles of S. Typhi carriers (cytokines and antibody), I developed a method of identifying S. Typhi carriers and estimating the prevalence of S. Typhi carriage in a typhoid endemic population. My findings will potentially lead to the development of novel enteric fever control strategies. I conclude that improved case detection and widespread vaccination campaigns using polyvalent Salmonella vaccines should be initiated for reducing the burden of enteric fever in endemic areas

Temporal fluctuation of multidrug resistant salmonella typhi haplotypes in the mekong river delta region of Vietnam.

BACKGROUND: typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005. PRINCIPAL FINDINGS: the population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene. SIGNIFICANCE: the H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam

The Control of Typhoid Fever in Vietnam.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a diminishing public health problem in Vietnam, and this process may represent a prototype for typhoid elimination in Asia. Here, we review typhoid epidemiology in Vietnam over 20 years and assess the potential drivers associated with typhoid reduction. In the 1990s, multidrug resistant S. Typhi were highly prevalent in a sentinel hospital in southern Vietnam. A national typhoid incidence rate of 14.7/100,000 population per year was estimated around the new millennium. The Vietnamese government recognized the public health issue of typhoid in the 1990s and initiated vaccine campaigns to protect the most vulnerable members of the population. At their peak, these campaigns immunized approximately 1,200,000 children in 35 provinces. Concurrently, Vietnam experienced unprecedented economic development from 1998 to 2014, with the gross national income per capita increasing from $360 to$1,890 over this period. More recent typhoid incidence data are not available, but surveillance suggests that the current disease burden is negligible. This trajectory can be considered a major public health success. However, a paucity of systematic data makes it difficult to disaggregate the roles of immunization and water, sanitation, and hygiene (WASH) interventions in typhoid reduction in Vietnam. Given the limitations of typhoid vaccines, we surmise the practical elimination of typhoid was largely driven by economic development and improvement in general population living standards. Better designed WASH intervention studies with clinical endpoints and systematic incidence data are essential to glean a greater understanding of contextual factors that impact typhoid incidence reduction

The burden and characteristics of enteric fever at a healthcare facility in a densely populated area of Kathmandu

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.This work was supported by The Wellcome Trust, Euston Road, London, United Kingdom. MFB is supported by the Medical Research Council (grant G0600718). SB is supported by an OAK foundation fellowship through Oxford University

Gallbladder carriage generates genetic variation and genome degradation in Salmonella Typhi

Despite recent advances in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains poorly understood. Aiming to understand if S. Typhi becomes genetically adapted for long-term colonisation in the gallbladder, we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneously sampled sequences from organisms isolated from the blood of acute patients within the same population. We found that S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes, but was largely reflective of S. Typhi circulating in the general population. However, gallbladder isolates showed a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p = 2.8x10-9), respectively. Within gallbladder isolates of the predominant H58 genotype, variation was associated with a higher prevalence of nonsense mutations. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Specifically, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to the 134Kb deletion of SPI-7. S. Typhi is under strong selective pressure in the human gallbladder, which may be reflected phylogenetically by long terminal branches that may distinguish organisms from chronic and acute infections. Our work shows that selective pressures asserted by the hostile environment of the human gallbladder generate new antigenic variants and raises questions regarding the role of carriage in the epidemiology of typhoid fever

Two cases of bacteriemia caused by nontoxigenic, non-O1, non-O139 Vibrio cholerae isolates in Ho Chi Minh City, Vietnam.

The toxigenic bacterium Vibrio cholerae belonging to the O1 and O139 serogroups is commonly associated with epidemic diarrhea in tropical settings; other diseases caused by this environmental pathogen are seldom identified. Here we report two unassociated cases of nonfatal, nontoxigenic V. cholerae non-O1, non-O139 bacteremia in patients with comorbidities in Ho Chi Minh City, Vietnam, that occurred within a 4-week period

The ecological dynamics of fecal contamination and Salmonella Typhi and Salmonella Paratyphi A in municipal Kathmandu drinking water

One of the UN sustainable development goals is to achieve universal access to safe and affordable drinking water by 2030. It is locations like Kathmandu, Nepal, a densely populated city in South Asia with endemic typhoid fever, where this goal is most pertinent. Aiming to understand the public health implications of water quality in Kathmandu we subjected weekly water samples from 10 sources for one year to a range of chemical and bacteriological analyses. We additionally aimed to detect the etiological agents of typhoid fever and longitudinally assess microbial diversity by 16S rRNA gene surveying. We found that the majority of water sources exhibited chemical and bacterial contamination exceeding WHO guidelines. Further analysis of the chemical and bacterial data indicated site-specific pollution, symptomatic of highly localized fecal contamination. Rainfall was found to be a key driver of this fecal contamination, correlating with nitrates and evidence of S. Typhi and S. Paratyphi A, for which DNA was detectable in 333 (77%) and 303 (70%) of 432 water samples, respectively. 16S rRNA gene surveying outlined a spectrum of fecal bacteria in the contaminated water, forming complex communities again displaying location-specific temporal signatures. Our data signify that the municipal water in Kathmandu is a predominant vehicle for the transmission of S. Typhi and S. Paratyphi A. This study represents the first extensive spatiotemporal investigation of water pollution in an endemic typhoid fever setting and implicates highly localized human waste as the major contributor to poor water quality in the Kathmandu Valley

Role of Environmental Factors in Shaping Spatial Distribution of Salmonella enterica Serovar Typhi, Fij

Fiji recently experienced a sharp increase in reported typhoid fever cases. To investigate geographic distribution and environmental risk factors associated with Salmonella enterica serovar Typhi infection, we conducted a cross-sectional cluster survey with associated serologic testing for Vi capsular antigen–specific antibodies (a marker for exposure to Salmonella Typhi in Fiji in 2013. Hotspots with high seroprevalence of Vi-specific antibodies were identified in northeastern mainland Fiji. Risk for Vi seropositivity increased with increased annual rainfall (odds ratio [OR] 1.26/quintile increase, 95% CI 1.12–1.42), and decreased with increased distance from major rivers and creeks (OR 0.89/km increase, 95% CI 0.80–0.99) and distance to modeled flood-risk areas (OR 0.80/quintile increase, 95% CI 0.69–0.92) after being adjusted for age, typhoid fever vaccination, and home toilet type. Risk for exposure to Salmonella Typhi and its spatial distribution in Fiji are driven by environmental factors. Our findings can directly affect typhoid fever control efforts in Fiji.This study was supported by the World Health Organization, Division of Pacific Technical Support (grant 2013/334890-0); the Chadwick Trust; the Bill and Melinda Gates Foundation (grant OPP1033751); and the Wellcome Trust of Great Britain (grant 100087/Z/12/Z

Diagnostic metabolite biomarkers of chronic typhoid carriage.

BACKGROUND: Salmonella Typhi and Salmonella Paratyphi A are the agents of enteric (typhoid) fever; both can establish chronic carriage in the gallbladder. Chronic Salmonella carriers are typically asymptomatic, intermittently shedding bacteria in the feces, and contributing to disease transmission. Detecting chronic carriers is of public health relevance in areas where enteric fever is endemic, but there are no routinely used methods for prospectively identifying those carrying Salmonella in their gallbladder. METHODOLOGY/PRINCIPAL FINDINGS: Here we aimed to identify biomarkers of Salmonella carriage using metabolite profiling. We performed metabolite profiling on plasma from Nepali patients undergoing cholecystectomy with confirmed S. Typhi or S. Paratyphi A gallbladder carriage (and non-carriage controls) using two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOFMS) and supervised pattern recognition modeling. We were able to significantly discriminate Salmonella carriage samples from non-carriage control samples. We were also able to detect differential signatures between S. Typhi and S. Paratyphi A carriers. We additionally compared carriage metabolite profiles with profiles generated during acute infection; these data revealed substantial heterogeneity between metabolites associated with acute enteric fever and chronic carriage. Lastly, we found that Salmonella carriers could be significantly distinguished from non-carriage controls using only five metabolites, indicating the potential of these metabolites as diagnostic markers for detecting chronic Salmonella carriers. CONCLUSIONS/SIGNIFICANCE: Our novel approach has highlighted the potential of using metabolomics to search for diagnostic markers of chronic Salmonella carriage. We suggest further epidemiological investigations of these potential biomarkers in alternative endemic enteric fever settings

Role of Environmental Factors in Shaping Spatial Distribution of Salmonella enterica Serovar Typhi, Fiji.

Fiji recently experienced a sharp increase in reported typhoid fever cases. To investigate geographic distribution and environmental risk factors associated with Salmonella enterica serovar Typhi infection, we conducted a cross-sectional cluster survey with associated serologic testing for Vi capsular antigen-specific antibodies (a marker for exposure to Salmonella Typhi in Fiji in 2013. Hotspots with high seroprevalence of Vi-specific antibodies were identified in northeastern mainland Fiji. Risk for Vi seropositivity increased with increased annual rainfall (odds ratio [OR] 1.26/quintile increase, 95% CI 1.12-1.42), and decreased with increased distance from major rivers and creeks (OR 0.89/km increase, 95% CI 0.80-0.99) and distance to modeled flood-risk areas (OR 0.80/quintile increase, 95% CI 0.69-0.92) after being adjusted for age, typhoid fever vaccination, and home toilet type. Risk for exposure to Salmonella Typhi and its spatial distribution in Fiji are driven by environmental factors. Our findings can directly affect typhoid fever control efforts in Fiji.This study was supported by the World Health Organization, Division of Pacific Technical Support (grant 2013/334890-0); the Chadwick Trust; the Bill and Melinda Gates Foundation (grant OPP1033751); and the Wellcome Trust of Great Britain (grant 100087/Z/12/Z)