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Find video protocols related to scientific articles indexed in Pubmed.
High genotypic diversity among rotavirus strains infecting Gambian children.
Pediatr. Infect. Dis. J.
PUBLISHED: 08-27-2014
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Rotavirus is the leading cause of diarrhea in children <5 years of age. In light of the implementation of rotavirus vaccines of limited valency, it is important to characterize the genotypic diversity of circulating rotavirus in sub-Saharan Africa.
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Gut-Homing Conventional Plasmablasts and CD27(-) Plasmablasts Elicited after a Short Time of Exposure to an Oral Live-Attenuated Shigella Vaccine Candidate in Humans.
Front Immunol
PUBLISHED: 08-20-2014
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Currently, there is no licensed Shigella vaccine; however, various promising live-attenuated vaccine candidates have emerged, including CVD1208S (?guaBA, ?set, ?sen S. flexneri 2a), which was shown to be safe and immunogenic in Phase 1 clinical trials. Here, we report the immune responses elicited in an outpatient Phase 2 clinical trial in which subjects were vaccinated with CVD 1208S. Oral immunization with CVD 1208S elicited high anti-S. flexneri 2a LPS and IpaB antibody responses as well as an acute plasmablast (PB) infiltration in peripheral blood 7?days after immunization. PB sorted based on their expression of homing molecules confirmed that cells expressing integrin ?4?7 alone or in combination with CD62L were responsible for antibody production (as measured by ELISpot). Furthermore, using high-color flow-cytometry, on day 7 after immunization, we observed the appearance of conventional PB (CPB, CD19(dim) CD20(-) CD27(+high) CD38(+high) CD3(-)), as well as a PB population that did not express CD27 (CD27(-) PB; pre-plasmablasts). The pattern of individual or simultaneous expression of homing markers (integrin ?4?7, CD62L, CXCR3, and CXCR4) suggested that CPB cells homed preferentially to the inflamed gut mucosa. In contrast, ~50% CD27(-) PB cells appear to home to yet to be identified peripheral lymphoid organs or were in a transition state preceding integrin ?4?7 upregulation. In sum, these observations demonstrate that strong immune responses, including distinct PB subsets with the potential to home to the gut and other secondary lymphoid organs, can be elicited after a short time of exposure to a shigella oral vaccine.
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Typhoid fever in Fiji: a reversible plague?
Trop. Med. Int. Health
PUBLISHED: 07-25-2014
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The country of Fiji, with a population of approximately 870 000 people, faces a growing burden of several communicable diseases including the bacterial infection typhoid fever. Surveillance data suggest that typhoid has become increasingly common in rural areas of Fiji and is more frequent amongst young adults. Transmission of the organisms that cause typhoid is facilitated by faecal contamination of food or water and may be influenced by local behavioural practices in Fiji. The Fijian Ministry of Health, with support from Australian Aid, hosted a meeting in August 2012 to develop comprehensive control and prevention strategies for typhoid fever in Fiji. International and local specialists were invited to share relevant data and discuss typhoid control options. The resultant recommendations focused on generating a clearer sense of the epidemiology of typhoid in Fiji and exploring the contribution of potential transmission pathways. Additionally, the panel suggested steps such as ensuring that recommended ciprofloxacin doses are appropriate to reduce the potential for relapse and reinfection in clinical cases, encouraging proper hand hygiene of food and drink handlers, working with water and sanitation agencies to review current sanitation practices and considering a vaccination policy targeting epidemiologically relevant populations.
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Shigella isolates from the global enteric multicenter study inform vaccine development.
Clin. Infect. Dis.
PUBLISHED: 06-23-2014
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Shigella, a major diarrheal disease pathogen worldwide, is the target of vaccine development. The Global Enteric Multicenter Study (GEMS) investigated burden and etiology of moderate-to-severe diarrheal disease in children aged <60 months and matched controls without diarrhea during 3 years at 4 sites in Africa and 3 in Asia. Shigella was 1 of the 4 most common pathogens across sites and age strata. GEMS Shigella serotypes are reviewed to guide vaccine development.
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A scalable method for biochemical purification of Salmonella flagellin.
Protein Expr. Purif.
PUBLISHED: 05-05-2014
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Flagellins are the main structural proteins of bacterial flagella and potent stimulators of innate and adaptive immunity in mammals. The flagellins of Salmonella are virulence factors and protective antigens, and form the basis of promising vaccines. Despite broad interest in flagellins as antigens and adjuvants in vaccine formulations, there have been few advances towards the development of scalable and economical purification methods for these proteins. We report here a simple and robust strategy to purify flagellin monomers from the supernatants of liquid growth culture. Phase 1 flagellins from Salmonella enterica serovars Typhimurium (i epitope) and Enteritidis (g,m epitopes) were purified directly from conditioned fermentation growth media using sequential cation- and anion-exchange chromatography coupled with a final tangential flow-filtration step. Conventional porous chromatography resin was markedly less efficient than membrane chromatography for flagellin purification. Recovery after each process step was robust, with endotoxin, nucleic acid and residual host-cell protein effectively removed. The final yield was 200-300 mg/L fermentation culture supernatant, with ?45-50% overall recovery. A final pH 2 treatment step was instituted to ensure uniformity of flagellin in the monomeric form. Flagellins purified by this method were recognized by monoclonal anti-flagellin antibodies and maintained capacity to activate Toll-like Receptor 5. The process described is simple, readily scalable, uses standard bioprocess methods, and requires only a few steps to obtain highly purified material.
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A rabbit model of non-typhoidal Salmonella bacteremia.
Comp. Immunol. Microbiol. Infect. Dis.
PUBLISHED: 04-30-2014
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Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.
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Association between moderate-to-severe diarrhea in young children in the global enteric multicenter study (GEMS) and types of handwashing materials used by caretakers in Mirzapur, Bangladesh.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-28-2014
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Handwashing practices among caretakers of case and control children < 5 years of age enrolled in the Global Enteric Multicenter Study in Mirzapur, Bangladesh were characterized and analyzed for association with moderate-to-severe diarrhea. Soap or detergent ownership was common, yet 48% of case and 47.7% of control caretakers also kept ashes for handwashing, including 36.8% of the wealthiest households. Soap, detergent, and ash were used for multiple hygiene purposes and were kept together at handwashing areas. Caretakers preferred soap for handwashing, but frequently relied on ash, or a detergent/ash mixture, as a low-cost alternative. Moderate-to-severe diarrhea was equally likely for children of caretakers who kept soap versus those who kept ash (matched OR = 0.91; 0.62-1.32). Contact with ash and water reduced concentrations of bacterial enteropathogens, without mechanical scrubbing. Thus, washing hands with ash is a prevalent behavior in Mirzapur and may help diminish transmission of diarrheal pathogens to children.
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Serum bactericidal assays to evaluate typhoidal and nontyphoidal Salmonella vaccines.
Clin. Vaccine Immunol.
PUBLISHED: 03-12-2014
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Invasive Salmonella infections for which improved or new vaccines are being developed include enteric fever caused by Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B and sepsis and meningitis in young children in sub-Saharan Africa caused by nontyphoidal Salmonella (NTS) serovars, particularly S. enterica serovars Typhimurium and Enteritidis. Assays are needed to measure functional antibodies elicited by the new vaccines to assess their immunogenicities and potential protective capacities. We developed in vitro assays to quantify serum bactericidal antibody (SBA) activity induced by S. Typhi, S. Paratyphi A, S. Typhimurium, and S. Enteritidis vaccines in preclinical studies. Complement from various sources was tested in assays designed to measure antibody-dependent complement-mediated killing. Serum from rabbits 3 to 4 weeks of age provided the best complement source compared to serum from pigs, goats, horses, bovine calves, or rabbits 8 to 12 weeks of age. For S. Enteritidis, S. Typhimurium, and S. Typhi SBA assays to be effective, bacteria had to be harvested at log phase. In contrast, S. Paratyphi A was equally susceptible to killing whether it was grown to the stationary or log phase. The typhoidal serovars were more susceptible to complement-mediated killing than were the nontyphoidal serovars. Lastly, the SBA endpoint titers correlated with serum IgG anti-lipopolysaccharide (LPS) titers in mice immunized with mucosally administered S. Typhimurium, S. Enteritidis, and S. Paratyphi A but not S. Typhi live attenuated vaccines. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccine candidates.
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An outpatient, ambulant-design, controlled human infection model using escalating doses of Salmonella Typhi challenge delivered in sodium bicarbonate solution.
Clin. Infect. Dis.
PUBLISHED: 02-10-2014
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Typhoid fever is a major global health problem, the control of which is hindered by lack of a suitable animal model in which to study Salmonella Typhi infection. Until 1974, a human challenge model advanced understanding of typhoid and was used in vaccine development. We set out to establish a new human challenge model and ascertain the S. Typhi (Quailes strain) inoculum required for an attack rate of 60%-75% in typhoid-naive volunteers when ingested with sodium bicarbonate solution.
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Advancing the management and control of typhoid fever: a review of the historical role of human challenge studies.
J. Infect.
PUBLISHED: 01-15-2014
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Typhoid infection causes considerable morbidity and mortality worldwide, particularly in settings where lack of clean water and inadequate sanitation facilitate disease spread through faecal-oral transmission. Improved understanding of the pathogenesis, immune control and microbiology of Salmonella Typhi infection can help accelerate the development of improved vaccines and diagnostic tests necessary for disease control. S. Typhi is a human-restricted pathogen; therefore animal models are limited in their relevance to human infection. During the latter half of the 20th century, induced human infection ("challenge") studies with S. Typhi were used effectively to assess quantitatively the human host response to challenge and to measure directly the efficacy of typhoid vaccines in preventing clinical illness. Here, the findings of these historic challenge studies are reviewed, highlighting the pivotal role that challenge studies have had in improving our understanding of the host-pathogen interaction, and illustrating issues relevant to modern typhoid challenge model design.
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Live oral Salmonella enterica serovar Typhi vaccines Ty21a and CVD 909 induce opsonophagocytic functional antibodies in humans that cross-react with S. Paratyphi A and S. Paratyphi B.
Clin. Vaccine Immunol.
PUBLISHED: 01-15-2014
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Live oral Salmonella enterica serovar Typhi vaccine Ty21a induces specific antibodies that cross-react against Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Paratyphi B, although their functional role in clearance remains unknown. We utilized an in vitro assay with THP-1 macrophages to compare the phagocytosis and survival of Salmonella opsonized with heat-inactivated human sera obtained before and after vaccination with Ty21a or a live oral S. Typhi vaccine, CVD 909. Opsonization with postvaccination sera predominantly increased the phagocytosis of S. Typhi relative to the corresponding prevaccination sera, and increases were also observed with S. Paratyphi A and S. Paratyphi B, albeit of lower magnitudes. Relative to prevaccination sera, opsonization with the postvaccination sera reduced the survival inside macrophages of S. Typhi but not of S. Paratyphi A or S. Paratyphi B. Higher anti-S. Typhi O antigen (lipopolysaccharide [LPS]) IgG, but not IgA, antibody titers correlated significantly with postvaccination increases in opsonophagocytosis. No differences were observed between immunization with four doses of Ty21a or one dose of CVD 909. Ty21a and CVD 909 induced cross-reactive functional antibodies, predominantly against S. Typhi. IgG anti-LPS antibodies may be important in phagocytic clearance of these organisms. Therefore, measurement of functional antibodies might be important in assessing the immunogenicity of a new generation of typhoid and paratyphoid A vaccines. (The CVD 909 study has been registered at ClinicalTrials.gov under registration no. NCT00326443.).
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Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition.
Genome Biol.
PUBLISHED: 01-14-2014
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Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.
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Community Based Case-Control Study of Rotavirus Gastroenteritis among Young Children during 2008-2010 Reveals Vast Genetic Diversity and Increased Prevalence of G9 Strains in Kolkata.
PLoS ONE
PUBLISHED: 01-01-2014
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Group A Rotaviruses are a major etiologic agent of gastroenteritis in infants and young children (<5 years) worldwide. Although rotavirus vaccines have been successfully administered in many countries, in India the introduction of rotavirus vaccine in national immunization program was approved in 2014. Since high disease burden and large number of genetic variants have been reported from low income countries including India, monitoring of rotavirus was initiated prior to implementation of the vaccine in the region.
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Age-dependent association among Helicobacter pylori infection, serum pepsinogen levels and immune response of children to live oral cholera vaccine CVD 103-HgR.
PLoS ONE
PUBLISHED: 01-01-2014
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Through its effects on gastric secretion, we hypothesized that Helicobacter pylori infection may influence oral immunization. Accordingly, we examined the association between H. pylori infection, serum pepsinogen (PG) (measures for H. pylori gastritis) and vibriocidal antibody (a correlate of protection) seroconversion following oral immunization with CVD 103-HgR live cholera vaccine among children of different ages.
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Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.
PLoS ONE
PUBLISHED: 01-01-2014
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Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response.
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Helicobacter pylori Infection Affects Immune Responses Following Vaccination of Typhoid-Naive US Adults With Attenuated Salmonella Typhi Oral Vaccine CVD 908-htrA.
J. Infect. Dis.
PUBLISHED: 11-23-2013
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Background.?We examined the association between Helicobacter pylori infection and the immune response following oral immunization of US adults with attenuated Salmonella Typhi vaccine CVD 908-htrA.Methods.?Baseline sera from 74 volunteers without a history of typhoid fever who were immunized orally with CVD 908-htrA were tested by enzyme-linked immunosorbent assay for immunoglobin G (IgG) antibodies to H. pylori, hepatitis A antibodies (a marker of low socioeconomic status and exposure to enteric infections), and pepsinogen (PG) I and II levels (measures of gastric inflammation). IgG against S. Typhi lipopolysaccharide (LPS) O and flagella was measured before and 28 days following immunization; a ?4-fold increase in titer from baseline constituted seroconversion.Results.?Seroconversion of S. Typhi IgG LPS antibodies was significantly higher among vaccinees infected with H. pylori versus uninfected subjects: adjusted odds ratio (OR) 3.8, 95% confidence interval (CI), 1.1-12.6 (P = .03). A low PG I:PG II ratio (<5), indicating more advanced corpus gastritis, increased the odds of seroconversion of IgG S. Typhi flagella antibody (adjusted OR 6.4, 95% CI, 1.3-31.4; P = .02). Hepatitis A infection did not influence the immune response to CVD 908-htrA.Conclusions.?H. pylori infection and gastric inflammation may enhance humoral immunity to oral attenuated S. Typhi vaccine.
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Safety and Immunogenicity of Single-Dose Live Oral Cholera Vaccine Strain CVD 103-HgR, Prepared from New Master and Working Cell Banks.
Clin. Vaccine Immunol.
PUBLISHED: 10-30-2013
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Currently, no cholera vaccine is available for persons traveling from the United States to areas of high cholera transmission and who for reasons of occupation or host factors are at increased risk for development of the disease. A single-dose oral cholera vaccine with a rapid onset of protection would be particularly useful for such travelers and might also be an adjunct control measure for cholera outbreaks. The attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR harbors a 94% deletion of the cholera toxin A subunit gene (ctxA) and has a mercury resistance gene inserted in the gene encoding hemolysin A. We undertook a phase I randomized placebo-controlled two-site trial to assess the safety and immunogenicity of a preliminary formulation of CVD 103-HgR prepared from new master and working cell banks. Healthy young adults were randomized (5:1 vaccinees to placebo recipients) to receive a single oral dose of ?4.4 × 10(8) CFU of vaccine or a placebo. Blood serum vibriocidal and cholera toxin-specific IgG antibodies were measured before and 10, 14, and 28 days following vaccination or placebo. Excretion of the vaccine strain in the stool was assessed during the first week postvaccination. A total of 66 subjects were enrolled, comprising 55 vaccinees and 11 placebo recipients. The vaccine was well tolerated. The overall vibriocidal and anti-cholera toxin seroconversion rates were 89% and 57%, respectively. CVD 103-HgR is undergoing renewed manufacture for licensure in the United States under the auspices of PaxVax. Our data mimic those from previous commercial formulations that elicited vibriocidal antibody seroconversion (a correlate of protection) in ?90% of vaccinees. (This study has been registered at ClinicalTrials.gov under registration no. NCT01585181.).
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Can Giardia lamblia infection lower the risk of acute diarrhea among preschool children?
J. Trop. Pediatr.
PUBLISHED: 10-23-2013
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There are inconsistent findings concerning the role of Giardia lamblia in pediatric diarrhea. A prospective cohort study of the incidence of acute diarrhea among Israeli Arab preschool children offered the opportunity to examine the association between G. lamblia infection (at baseline) and subsequent diarrhea. Following baseline screening by light microscopy for the presence of Giardia in their stools, a cohort was assembled of 142 children who were followed between October 2003 and August 2004 for the incidence of diarrhea. Surveillance was performed through maternal interviews. At baseline, 21 children tested Giardia-positive. During the prospective surveillance, acute diarrhea occurred less often among Giardia-positive children (9.5%) than among children who were not infected with Giardia (26.5%). G. lamblia infection was associated with lower risk of acute diarrhea; adjusted odds ratio of 0.18 (95% confidence interval 0.04-0.93) (p = 0.041). This prospective study provides additional evidence that Giardia may lower the risk of subsequent acute diarrhea among preschool children.
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Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.
J. Clin. Microbiol.
PUBLISHED: 07-24-2013
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Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.
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Quality of piped and stored water in households with children under five years of age enrolled in the Mali site of the Global Enteric Multi-Center Study (GEMS).
Am. J. Trop. Med. Hyg.
PUBLISHED: 07-08-2013
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Water, sanitation, and hygiene information was collected during a matched case-control study of moderate and severe diarrhea (MSD) among 4,096 children < 5 years of age in Bamako, Mali. Primary use of piped water (conditional odds ratio [cOR] = 0.45; 0.34-0.62), continuous water access (cOR = 0.30; 0.20-0.43), fetching water daily (cOR = 0.77; 0.63-0.96), and breastfeeding (cOR = 0.65; 0.49-0.88) significantly reduced the likelihood of MSD. Fetching water in > 30 minutes (cOR = 2.56; 1.55-4.23) was associated with MSD. Piped tap water and courier-delivered water contained high (> 2 mg/L) concentrations of free residual chlorine and no detectable Escherichia coli. However, many households stored water overnight, resulting in inadequate free residual chlorine (< 0.2 mg/L) for preventing microbial contamination. Coliforms and E. coli were detected in 48% and 8% of stored household water samples, respectively. Although most of Bamakos population enjoys access to an improved water source, water quality is often compromised during household storage.
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Housefly population density correlates with shigellosis among children in Mirzapur, Bangladesh: a time series analysis.
PLoS Negl Trop Dis
PUBLISHED: 06-01-2013
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Shigella infections are a public health problem in developing and transitional countries because of high transmissibility, severity of clinical disease, widespread antibiotic resistance and lack of a licensed vaccine. Whereas Shigellae are known to be transmitted primarily by direct fecal-oral contact and less commonly by contaminated food and water, the role of the housefly Musca domestica as a mechanical vector of transmission is less appreciated. We sought to assess the contribution of houseflies to Shigella-associated moderate-to-severe diarrhea (MSD) among children less than five years old in Mirzapur, Bangladesh, a site where shigellosis is hyperendemic, and to model the potential impact of a housefly control intervention.
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Health care seeking for childhood diarrhea in developing countries: evidence from seven sites in Africa and Asia.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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We performed serial Health Care Utilization and Attitudes Surveys (HUASs) among caretakers of children ages 0-59 months randomly selected from demographically defined populations participating in the Global Enteric Multicenter Study (GEMS), a case-control study of moderate-to-severe diarrhea (MSD) in seven developing countries. The surveys aimed to estimate the proportion of children with MSD who would present to sentinel health centers (SHCs) where GEMS case recruitment would occur and provide a basis for adjusting disease incidence rates to include cases not seen at the SHCs. The proportion of children at each site reported to have had an incident episode of MSD during the 7 days preceding the survey ranged from 0.7% to 4.4% for infants (0-11 months of age), from 0.4% to 4.7% for toddlers (12-23 months of age), and from 0.3% to 2.4% for preschoolers (24-59 months of age). The proportion of MSD episodes at each site taken to an SHC within 7 days of diarrhea onset was 15-56%, 17-64%, and 7-33% in the three age strata, respectively. High cost of care and insufficient knowledge about danger signs were associated with lack of any care-seeking outside the home. Most children were not offered recommended fluids and continuing feeds at home. We have shown the utility of serial HUASs as a tool for optimizing operational and methodological issues related to the performance of a large case-control study and deriving population-based incidence rates of MSD. Moreover, the surveys suggest key targets for educational interventions that might improve the outcome of diarrheal diseases in low-resource settings.
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Health care-seeking behavior for childhood diarrhea in Mirzapur, rural Bangladesh.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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We evaluated patterns of health care use for diarrhea among children 0-59 months of age residing in Mirzapur, Bangladesh, using a baseline survey conducted during May-June 2007 to inform the design of a planned diarrheal etiology case/control study. Caretakers of 7.4% of 1,128 children reported a diarrheal illness in the preceding 14 days; among 95 children with diarrhea, 24.2% had blood in the stool, 12.2% received oral rehydration solution, 27.6% received homemade fluids, and none received zinc at home. Caretakers of 87.9% sought care outside the home; 49.9% from a pharmacy, and 22.1% from a hospital or health center. The primary reasons for not seeking care were maternal perception that the illness was not serious enough (74.0%) and the high cost of treatment (21.9%). To improve management of childhood diarrhea in Mirzapur, Bangladesh, it will be important to address knowledge gaps in caretakers assessment of illness severity, appropriate home management, and when to seek care in the formal sector. In addition, consideration should be given to inclusion of the diverse care-giving settings in clinical training activities for diarrheal disease management.
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Determinants of health care seeking for diarrheal illness in young children in urban slums of Kolkata, India.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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Maternal practices regarding childrens health care have been recognized as an important factor associated with mortality rates among children < 5 years of age. We focused on health care-seeking practices of primary caretakers of children < 5 years of age with diarrheal disease in Kolkata. We interviewed caretakers of 1,058 children in a baseline survey and 6,077 children on six subsequent surveys. The prevalence of diarrhea during the preceding 2 weeks was 7.9% in the baseline survey and 5.7% (lowest 3.5% to highest 7.8%) in subsequent surveys. Multivariate logistic regression showed that formal education of primary caretakers was associated with seeking care outside the home (odds ratio [OR] = 15.5; 95% confidence interval [CI] [2.5-85.7]; P = 0.002). Multinomial logistic regression showed that formal education of the primary caretaker (OR = 21.4; 95% CI [3.2-139.0]; P = 0.002) and presence of dry mouth during diarrhea (OR = 17.3; 95% CI [2.7-110.9]; P = 0.003) were associated with seeking care from licensed providers compared with the children for whom care was not sought outside of the home. This health care utilization and attitudes survey (HUAS) can serve as a tool to identify the factors that influence a better health care-seeking pattern in urban slums of Kolkata.
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Seeking care for pediatric diarrheal illness from traditional healers in Bamako, Mali.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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Diarrhea is a leading cause of child mortality worldwide. Early recognition of symptoms and referral to medical treatment are essential. In 2007, we conducted a Healthcare Utilization and Attitudes Survey (HUAS) of 1,000 children randomly selected from a population census to define care-seeking patterns for diarrheal disease in Bamako, Mali, in preparation for the Global Enteric Multicenter Study (GEMS). We found that 57% of caretakers sought care for their childs diarrheal illness from traditional healers, and 27% of caretakers sought care from the government health center (GHC). Weighted logistic regression showed that seeking care from a traditional healer was associated with more severe reported diarrheal disease, like decreased urination (odds ratio [OR] = 3.35, 95% confidence interval [95% CI] = 1.19-9.41) and mucus or pus in stool (OR = 4.42, 95% CI = 1.35-14.51), along with other indicators of perceived susceptibility. A locally designed traditional healer referral system was, therefore, created that emphasized more severe disease. This system may serve as a model for health systems in West Africa.
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Health care-seeking behavior during childhood diarrheal illness: results of health care utilization and attitudes surveys of caretakers in western Kenya, 2007-2010.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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We interviewed caretakers of 1,043 children < 5 years old in a baseline cross-sectional survey (April to May 2007) and > 20,000 children on five separate subsequent occasions (May of 2009 to December 31, 2010) to assess healthcare seeking patterns for diarrhea. Diarrhea prevalence during the preceding 2 weeks ranged from 26% at baseline to 4-11% during 2009-2010. Caretakers were less likely to seek healthcare outside the home for infants (versus older children) with diarrhea (adjusted odds ratio [aOR] = 0.33, confidence interval [CI] = 0.12-0.87). Caretakers of children with reduced food intake (aOR = 3.42, CI = 1.37-8.53) and sunken eyes during their diarrheal episode were more likely to seek care outside home (aOR = 4.76, CI = 1.13-8.89). Caretakers with formal education were more likely to provide oral rehydration solution (aOR = 3.01, CI = 1.41-6.42) and visit a healthcare facility (aOR = 3.32, CI = 1.56-7.07). Studies calculating diarrheal incidence and healthcare seeking should account for seasonal trends. Improving caretakers knowledge of home management could prevent severe diarrhea.
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Health care use patterns for diarrhea in children in low-income periurban communities of Karachi, Pakistan.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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Diarrhea causes 16% of all child deaths in Pakistan. We assessed patterns of healthcare use among caretakers of a randomly selected sample of 959 children ages 0-59 months in low-income periurban settlements of Karachi through a cross-sectional survey. A diarrheal episode was reported to have occurred in the previous 2 weeks among 298 (31.1%) children. Overall, 280 (80.3%) children sought care. Oral rehydration solution and zinc were used by 40.8% and 2%, respectively; 11% were admitted or received intravenous rehydration, and 29% sought care at health centers identified as sentinel centers for recruiting cases of diarrhea for a planned multicenter diarrheal etiology case-control study. Odds ratios for independent predictors of care-seeking behavior were lethargy, 4.14 (95% confidence interval = 1.45-11.77); fever, 2.67 (1.27-5.59); and stool frequency more than six per day, 2.29 (1.03-5.09). Perception of high cost of care and use of home antibiotics were associated with reduced care seeking: odds ratio = 0.28 (0.1-0.78) and 0.29 (0.11-0.82), respectively. There is a need for standardized, affordable, and accessible treatment of diarrhea as well as community education regarding appropriate care in areas with high diarrheal burden.
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Health care utilization and attitudes survey in cases of moderate-to-severe diarrhea among children ages 0-59 months in the District of Manhica, southern Mozambique.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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In the predominantly rural Manhiça district, in southern Mozambique, diarrhea is one of the leading causes of death among children under 5 years. Caretakers randomly selected from the Demographic Surveillance Database were invited to participate in a community-based survey on use of healthcare services for gastroenteritis. Of those caretakers reporting an episode of diarrhea during the recall period, 65.2% in the first survey and 43.8% in the second survey reported seeking care at a health facility. Independent risk factors for seeking care in health facilities in the first survey included the presence of diarrhea with fever and not knowing any sign of dehydration; having a television at home was related with an independent decreased use of the health facilities. In the second survey, the use of health services was significantly associated with diarrhea with fever and vomiting. Establishment of continuous prospective monitoring allows accounting for changes in healthcare use that may occur because of seasonality or secular events.
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Health Care Utilization and Attitudes Survey: understanding diarrheal disease in rural Gambia.
Am. J. Trop. Med. Hyg.
PUBLISHED: 04-29-2013
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Diarrheal disease causes ?1.34 million deaths per year among children under 5 years of age globally. We conducted a Health Care Utilization and Attitudes Survey of 1,012 primary caregivers of children aged 0-11, 12-23, and 24-59 months randomly selected from a Demographic Surveillance population in rural Gambia. Point prevalence of diarrhea was 7.7% (95% confidence interval [CI] = 6.1-9.8); 23.3% had diarrhea within the previous 2 weeks. Caregivers of 81.5% of children with diarrhea sought healthcare outside their home, but only 48.4% of them visited a health center. Only 17.0% (95% CI = 12.1-23.2) of children with diarrhea received oral rehydration solution (ORS) at home. Abbreviated surveys conducted on six occasions over the subsequent 2 years showed no change in prevalence or treatment-seeking behavior. Diarrhea remains a significant problem in rural young Gambian children. Encouraging care-seeking behavior at health centers and promoting ORS use can reduce mortality and morbidity in this population.
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Safety and tolerability of a live oral Salmonella typhimurium vaccine candidate in SIV-infected nonhuman primates.
Vaccine
PUBLISHED: 04-17-2013
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Nontyphoidal Salmonella (NTS) serovars are a common cause of acute food-borne gastroenteritis worldwide and can cause invasive systemic disease in young infants, the elderly, and immunocompromised hosts, accompanied by high case fatality. Vaccination against invasive NTS disease is warranted where the disease incidence and mortality are high and multidrug resistance is prevalent, as in sub-Saharan Africa. Live-attenuated vaccines that mimic natural infection constitute one strategy to elicit protection. However, they must particularly be shown to be adequately attenuated for consideration of immunocompromised subjects. Accordingly, we examined the safety and tolerability of an oral live attenuated Salmonella typhimurium vaccine candidate, CVD 1921, in an established chronic simian immunodeficiency virus (SIV)-infected rhesus macaque model. We evaluated clinical parameters, histopathology, and measured differences in mucosal permeability to wild-type and vaccine strains. Compared to the wild-type S. typhimurium strain I77 in both SIV-infected and SIV-uninfected nonhuman primate hosts, this live-attenuated vaccine shows reduced shedding and systemic spread, exhibits limited pathological disease manifestations in the digestive tract, and induces low levels of cellular infiltration in tissues. Furthermore, wild-type S. typhimurium induces increased intestinal epithelial damage and permeability, with infiltration of neutrophils and macrophages in both SIV-infected and SIV-uninfected nonhuman primates compared to the vaccine strain. Based on shedding, systemic spread, and histopathology, the live-attenuated S. typhimurium strain CVD 1921 appears to be safe and well-tolerated in the nonhuman primate model, including chronically SIV-infected rhesus macaques.
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Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.
J. Clin. Microbiol.
PUBLISHED: 03-27-2013
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Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ? 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.
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Shigella antigen-specific B memory cells are associated with decreased disease severity in subjects challenged with wild-type Shigella flexneri 2a.
Clin. Immunol.
PUBLISHED: 03-11-2013
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The role of Shigella-specific B memory (BM) in protection has not been evaluated in human challenge studies. We utilized cryopreserved pre- and post-challenge peripheral blood mononuclear cells and sera from wild-type Shigella flexneri 2a (wt-2457T) challenges. Challenged volunteers were either naïve or subjects who had previously ingested wt-2457T or been immunized with hybrid Escherichia coli-Shigella live oral candidate vaccine (EcSf2a-2). BM and antibody titers were measured against lipopolysaccharide (LPS) and recombinant invasion plasmid antigen B (IpaB); results were correlated with disease severity following challenge. Pre-challenge IgA IpaB-BM and post-challenge IgA LPS-BM in the previously exposed subjects negatively correlated with disease severity upon challenge. Similar results were observed with pre-challenge IgG anti-LPS and anti-IpaB titers in vaccinated volunteers. Inverse correlations between magnitude of pre-challenge IgG antibodies to LPS and IpaB, as well as IgA IpaB-BM and post-challenge IgA LPS-BM with disease severity suggest a role for antigen-specific BM in protection.
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Progress and pitfalls in Shigella vaccine research.
Nat Rev Gastroenterol Hepatol
PUBLISHED: 02-19-2013
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Renewed awareness of the substantial morbidity and mortality that Shigella infection causes among young children in developing countries, combined with technological innovations in vaccinology, has led to the development of novel vaccine strategies in the past 5 years. Along with advancement of classic vaccines in clinical trials and new sophisticated measurements of immunological responses, much new data has been produced, lending promise to the potential for production of safe and effective Shigella vaccines. Herein, we review the latest progress in Shigella vaccine development within the framework of persistent obstacles.
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In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.
Front Immunol
PUBLISHED: 01-09-2013
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Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4?h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates.
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Sustained protection in mice immunized with fractional doses of Salmonella Enteritidis core and O polysaccharide-flagellin glycoconjugates.
PLoS ONE
PUBLISHED: 01-01-2013
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Non-typhoidal Salmonella (NTS) serovars S. Enteritidis and S. Typhimurium are a major cause of invasive bacterial disease (e.g., bacteremia, meningitis) in infants and young children in sub-Saharan Africa and also occasionally cause invasive disease in highly susceptible hosts (young infants, the elderly, and immunocompromised subjects) in industrialized countries. No licensed vaccines exist against human NTS infections. NTS core and O polysaccharide (COPS) and FliC (Phase 1 flagellin subunits) each constitute protective antigens in murine models. S. Enteritidis COPS conjugated to FliC represents a promising vaccine approach that elicits binding and opsonophagocytic antibodies and protects mice against lethal challenge with virulent S. Enteritidis. We examined the protective efficacy of fractional dosages of S. Enteritidis COPS:FliC conjugate vaccines in mice, and also established that protection can be passively transferred to naïve mice by administering sera from mice immunized with conjugate. Mice were immunized with three doses of either 10 µg, 2.5 µg (full dose), 0.25 µg, or 0.025 µg S. Enteritidis COPS:FliC conjugate at 28 day intervals. Antibody titers to COPS and FliC measured by ELISA fell consonant with progressively smaller vaccine dosage levels; anti-FliC IgG responses remained robust at fractional dosages for which anti-COPS serum IgG titers were decreased. Nevertheless, >90% protection against intraperitoneal challenge was observed in mice immunized with fractional dosages of conjugate that elicited diminished titers to both FliC and COPS. Passive transfer of immune sera from mice immunized with the highest dose of COPS:FliC to naïve mice was also protective, demonstrating the role of antibodies in mediating protection. These results provide important insights regarding the potency of Salmonella glycoconjugate vaccines that use flagellin as a carrier protein.
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Genomic characterization of enteroaggregative Escherichia coli from children in Mali.
J. Infect. Dis.
PUBLISHED: 12-19-2011
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Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood.
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Live attenuated Shigella dysenteriae type 1 vaccine strains overexpressing shiga toxin B subunit.
Infect. Immun.
PUBLISHED: 10-03-2011
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Shigella dysenteriae serotype 1 (S. dysenteriae 1) is unique among the Shigella species and serotypes in the expression of Shiga toxin which contributes to more severe disease sequelae and the ability to cause explosive outbreaks and pandemics. S. dysenteriae 1 shares characteristics with other Shigella species, including the capability of causing clinical illness with a very low inoculum (10 to 100 CFU) and resistance to multiple antibiotics, underscoring the need for efficacious vaccines and therapeutics. Following the demonstration of the successful attenuating capacity of deletion mutations in the guaBA operon in S. flexneri 2a vaccine strains in clinical studies, we developed a series of S. dysenteriae 1 vaccine candidates containing the fundamental attenuating mutation in guaBA. All strains are devoid of Shiga toxin activity by specific deletion of the gene encoding the StxA subunit, which encodes enzymatic activity. The StxB subunit was overexpressed in several derivatives by either plasmid-based constructs or chromosomal manipulation to include a strong promoter. All strains are attenuated for growth in vitro in the HeLa cell assay and for plaque formation and were safe in the Serény test and immunogenic in the guinea pigs. Each strain induced robust serum and mucosal anti-S. dysenteriae 1 lipopolysaccharide (LPS) responses and protected against wild-type challenge. Two strains engineered to overexpress StxB induced high titers of Shiga toxin neutralizing antibodies. These candidates demonstrate the potential for a live attenuated vaccine to protect against disease caused by S. dysenteriae 1 and potentially to protect against the toxic effects of other Shiga toxin 1-expressing pathogens.
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"IDEAL" vaccines for resource poor settings.
Vaccine
PUBLISHED: 08-19-2011
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In developing countries, immunization delivery would be more efficient, safer and economical if all vaccines could elicit long-term protection following needle-free administration of just a single dose and without need for a cold chain, and if immunization left an indelible (e.g., serologic) marker that would identify immunized persons. A few existing vaccines (e.g., yellow fever, measles) confer long-term protection following a single dose. To accomplish the same with less immunogenic live vaccines and with non-living antigens, potent (parenteral and mucosal) adjuvants are required. Emerging knowledge of how the innate immune system modulates adaptive immune responses is guiding development of modern adjuvants that can markedly enhance immune responses to vaccines by selective stimulation of components of the innate immune system. Needle-free immunization can be accomplished by administering vaccines via mucosal (oral or nasal) or transcutaneous routes or by parenteral injection using needle-free injection devices. Technologies such as vitrification (treatment with trehalose followed by drying) render vaccines resistant to temperature extremes. Ideally, immunization would lead to a biomarker such as a specific vaccine-derived antibody that allows differentiation of successfully immunized persons from susceptibles.
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Engineering and preclinical evaluation of attenuated nontyphoidal Salmonella strains serving as live oral vaccines and as reagent strains.
Infect. Immun.
PUBLISHED: 08-01-2011
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While nontyphoidal Salmonella (NTS) has long been recognized as a cause of self-limited gastroenteritis, it is becoming increasingly evident that multiple-antibiotic-resistant strains are also emerging as important causes of invasive bacteremia and focal infections, resulting in hospitalizations and deaths. We have constructed attenuated Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis strains that can serve as live oral vaccines and as "reagent strains" for subunit vaccine production in a safe and economical manner. Prototype attenuated vaccine strains CVD 1921 and CVD 1941, derived from the invasive wild-type strains S. Typhimurium I77 and S. Enteritidis R11, respectively, were constructed by deleting guaBA, encoding guanine biosynthesis, and clpP, encoding a master protease regulator. The clpP mutation resulted in a hyperflagellation phenotype. An additional deletion in fliD yielded reagent strains CVD 1923 and CVD 1943, respectively, which export flagellin monomers. Oral 50% lethal dose (LD??) analyses showed that the NTS vaccine strains were all highly attenuated in mice. Oral immunization with CVD 1921 or CVD 1923 protected mice against lethal challenge with wild-type S. Typhimurium I77. Immunization with CVD 1941 but not CVD 1943 protected mice against lethal infection with S. Enteritidis R11. Immune responses induced by these strains included high levels of serum IgG anti-lipopolysaccharide (LPS) and anti-flagellum antibodies, with titers increasing progressively during the immunization schedule. Since S. Typhimurium and S. Enteritidis are the most common NTS serovars associated with invasive disease, these findings can pave the way for development of a highly effective, broad-spectrum vaccine against invasive NTS.
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Salmonella enterica serovar enteritidis core O polysaccharide conjugated to H:g,m flagellin as a candidate vaccine for protection against invasive infection with S. enteritidis.
Infect. Immun.
PUBLISHED: 08-01-2011
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Nontyphoidal Salmonella enterica serovars Enteritidis and Typhimurium are a common cause of gastroenteritis but also cause invasive infections and enteric fever in certain hosts (young children in sub-Saharan Africa, the elderly, and immunocompromised individuals). Salmonella O polysaccharides (OPS) and flagellar proteins are virulence factors and protective antigens. The surface polysaccharides of Salmonella are poorly immunogenic and do not confer immunologic memory, limitations overcome by covalently attaching them to carrier proteins. We conjugated core polysaccharide-OPS (COPS) of Salmonella Enteritidis lipopolysaccharide (LPS) to flagellin protein from the homologous strain. COPS and flagellin were purified from a genetically attenuated (?guaBA) "reagent strain" (derived from an isolate from a patient with clinical bacteremia) engineered for increased flagellin production (?clpPX). Conjugates were constructed by linking flagellin monomers or polymers at random COPS hydroxyls with various polysaccharide/protein ratios by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or at the 3-deoxy-d-manno-octulosonic acid (KDO) terminus by thioether chemistry. Mice immunized on days 0, 28, and 56 with COPS-flagellin conjugates mounted higher anti-LPS IgG levels than mice receiving unconjugated COPS and exhibited high antiflagellin IgG; anti-LPS and antiflagellin IgG levels increased following booster doses. Antibodies generated by COPS-flagellin conjugates mediated opsonophagocytosis of S. Enteritidis cells into mouse macrophages. Mice immunized with flagellin alone, COPS-CRM???, or COPS-flagellin conjugates were significantly protected from lethal challenge with wild-type S. Enteritidis (80 to 100% vaccine efficacy).
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Cell-associated flagella enhance the protection conferred by mucosally-administered attenuated Salmonella Paratyphi A vaccines.
PLoS Negl Trop Dis
PUBLISHED: 07-28-2011
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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.
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Distribution of classical and nonclassical virulence genes in enterotoxigenic Escherichia coli isolates from Chilean children and tRNA gene screening for putative insertion sites for genomic islands.
J. Clin. Microbiol.
PUBLISHED: 07-20-2011
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Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNA-associated genetic insertions might identify new ETEC virulence determinants.
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Assessment of the epidemiology and burden of measles in Southern Mozambique.
Am. J. Trop. Med. Hyg.
PUBLISHED: 07-08-2011
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Measles has been a major killer among vaccine-preventable diseases in children < 5 years of age in developing countries. Despite progress in global efforts to reduce mortality, measles remains a public health problem. Hospital-based measles surveillance was conducted in Manhica, Mozambique (July 2001-September 2004). Suspected cases and community-based controls were enrolled, and blood was collected for immunoglobulin M (IgM) confirmation. Two hundred fifty-three suspected cases and 477 controls were enrolled, with 85% (216 of 253) cases reported during a measles outbreak. Measles-IgM confirmation was 30% among suspected cases and 5% in controls. Fifty-eight percent (14 of 24) of laboratory-confirmed cases had records indicating previous measles vaccination. Mortality was 3% (8 of 246) among cases and 1% among controls (6 of 426). Forty-five percent (33 of 74) of cases were < 24 months of age and 22% occurred in infants < 9 months of age and were associated with a high case-fatality rate (25%). Our data suggest that improved diagnostics, new tools to protect infants < 9 months of age, and a supplemental dose of measles vaccine could assist measles control.
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Safety, tolerability, and immunogenicity of inactivated trivalent seasonal influenza vaccine administered with a needle-free disposable-syringe jet injector.
Vaccine
PUBLISHED: 06-22-2011
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Jet injectors (JIs) avoid safety drawbacks of needle-syringe (N-S) while generating similar immune responses. A new generation of disposable-syringe jet injectors (DSJIs) overcomes the cross-contamination risk of multi-use-nozzle devices used in 20th-century campaigns. In the first study in humans, the newly-US-licensed LectraJet(®) model M3 RA DSJI was compared to N-S.
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Safety and immunogenicity of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) in Chilean children.
Hum Vaccin
PUBLISHED: 05-01-2011
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The safety and immunogenicity of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV, Synflorix™) were assessed in 240 healthy Chilean children randomized to receive 3 doses of PHiD-CV (PHiD-CV group) or hepatitis A vaccine (HAV control group) at 2-4-6 months of age. All were offered 1 HAV dose at 12 months (outside study). The PHiD-CV group received a second HAV dose at 18-21 months and PHiD-CV booster at 20-23 months. The HAV control group received 2 PHiD-CV catch-up doses at 18-21 and 20-23 months. Adverse events were recorded and pneumococcal antibody responses and opsonophagocytic activity (OPA) were measured. Both PHiD-CV vaccination schedules were well tolerated and immunogenic against the pneumococcal vaccine serotypes and protein D. The reactogenicity of PHiD-CV primary, booster and catch-up doses was in line with previous PHiD-CV studies, although generally higher than with HAV. For each vaccine serotype, the percentage of subjects with antibody concentrations ?0.2 µg/ml (GSKs 22F-inhibition ELISA) was at least 93.2% following 3 PHiD-CV primary doses and at least 97.4% post-booster; percentages with OPA titers ?8 were at least 91.7% post-booster. After 2-dose catch-up, at least 94.3% of children had antibody concentrations ?0.2 µg/ml against each serotype except 6B (84.3%); at least 95.2% had OPA titers ?8 except against serotypes 1, 5 and 6B. In conclusion, the safety profiles of 2 PHiD-CV vaccination schedules (3-dose primary plus booster and 2-dose catch-up) were in line with previous studies and PHiD-CV was immunogenic for all 10 vaccine serotypes and protein D.
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Mouse models to assess the efficacy of non-typhoidal Salmonella vaccines: revisiting the role of host innate susceptibility and routes of challenge.
Vaccine
PUBLISHED: 03-01-2011
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Non-typhoidal Salmonella enterica (NTS) serovars Typhimurium and Enteritidis are important causes of bacterial gastroenteritis in the USA and worldwide. In sub-Saharan Africa these two serovars are emerging as agents associated with lethal invasive disease (e.g., bacteremia, meningitis). The development of NTS vaccines, based on mucosally administered live attenuated strains and parenteral non-living antigens, could diminish the NTS disease burden globally. Mouse models of S. Typhimurium and S. Enteritidis invasive disease can accelerate the development of NTS vaccines. Live attenuated NTS vaccines elicit both cellular and humoral immunity in mice and their efficacy is well established. In contrast, non-living vaccines that primarily elicit humoral immunity have demonstrated variable efficacy. An analysis of the reported studies with non-living vaccines against S. Typhimurium and S. Enteritidis reveals that efficacy is influenced by two important independent variables: (1) the innate susceptibility to NTS infection that differs dramatically between commonly used mouse strains and (2) the virulence of the NTS strain used for challenge. Protection by non-living vaccines has generally been seen only in host-pathogen interactions where a sub-lethal infection results, such as challenging resistant mice with either highly virulent or weakly virulent strains or susceptible mice with weakly virulent strains. The immunologic basis of this discrepancy and the implications for human NTS vaccine development are reviewed herein.
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The role of research in viral disease eradication and elimination programs: lessons for malaria eradication.
PLoS Med.
PUBLISHED: 01-25-2011
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By examining the role research has played in eradication or regional elimination initiatives for three viral diseases--smallpox, poliomyelitis, and measles--we derive nine cross-cutting lessons applicable to malaria eradication. In these initiatives, some types of research commenced as the programs began and proceeded in parallel. Basic laboratory, clinical, and field research all contributed notably to progress made in the viral programs. For each program, vaccine was the lynchpin intervention, but as the programs progressed, research was required to improve vaccine formulations, delivery methods, and immunization schedules. Surveillance was fundamental to all three programs, whilst polio eradication also required improved diagnostic methods to identify asymptomatic infections. Molecular characterization of pathogen isolates strengthened surveillance and allowed insights into the geographic source of infections and their spread. Anthropologic, sociologic, and behavioural research were needed to address cultural and religious beliefs to expand community acceptance. The last phases of elimination and eradication became increasingly difficult, as a nil incidence was approached. Any eradication initiative for malaria must incorporate flexible research agendas that can adapt to changing epidemiologic contingencies and allow planning for posteradication scenarios.
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A research agenda to underpin malaria eradication.
PLoS Med.
PUBLISHED: 01-25-2011
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The interruption of malaria transmission worldwide is one of the greatest challenges for international health and development communities. The current expert view suggests that, by aggressively scaling up control with currently available tools and strategies, much greater gains could be achieved against malaria, including elimination from a number of countries and regions; however, even with maximal effort we will fall short of global eradication. The Malaria Eradication Research Agenda (malERA) complements the current research agenda--primarily directed towards reducing morbidity and mortality--with one that aims to identify key knowledge gaps and define the strategies and tools that will result in reducing the basic reproduction rate to less than 1, with the ultimate aim of eradication of the parasite from the human population. Sustained commitment from local communities, civil society, policy leaders, and the scientific community, together with a massive effort to build a strong base of researchers from the endemic areas will be critical factors in the success of this new agenda.
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Mucosal IgA responses in healthy adult volunteers following intranasal spray delivery of a live attenuated measles vaccine.
Clin. Vaccine Immunol.
PUBLISHED: 01-12-2011
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Measles remains an important cause of morbidity and mortality among children in the developing world. The goal of this study was to examine measles virus-specific mucosal immune responses in healthy immune (n = 24; plaque reduction neutralization [PRN] titers of ?200 mIU/ml) and nonimmune (n = 24) young adult volunteers who received the monovalent Moraten measles vaccine via intranasal (spray delivery) or subcutaneous immunization. Serum, oral fluid, and nasal wash samples were examined for measles virus-specific and total IgG and IgA on day 0 (prior to vaccination) and on days 14, 28, and 90 after vaccination. Nonimmune subjects vaccinated subcutaneously developed high levels of measles virus PRN, IgG, and IgA antibodies in serum, oral fluid, and nasal washes. Total IgG and secretory IgA (sIgA) titers were increased in nasal washes, and total IgG was increased in oral fluid specimens. There was a strong correlation between PRN and measles virus-specific IgG titers measured in serum, oral fluid, and nasal washes, whereas a weak correlation was found between PRN and measles virus-specific IgA titers. Notably, intranasal measles vaccination resulted in increased production of measles virus-specific sIgA in oral fluid and nasal washes in nonimmune individuals, without evidence of a systemic immune response. In contrast, no significant vaccine-induced responses were observed in immune subjects, regardless of the route of immunization. These results demonstrate that (i) intranasal measles immunization can elicit a mucosal response independent of the induction of serum antibodies and (ii) both mucosal and systemic antibody responses following nasal or subcutaneous immunization are blunted by preexisting measles immunity.
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Ultra-fast and sensitive detection of non-typhoidal Salmonella using microwave-accelerated metal-enhanced fluorescence ("MAMEF").
PLoS ONE
PUBLISHED: 01-10-2011
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Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1:1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids).
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Immunology of gut mucosal vaccines.
Immunol. Rev.
PUBLISHED: 01-05-2011
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Understanding the mechanisms underlying the induction of immunity in the gastrointestinal mucosa following oral immunization and the cross-talk between mucosal and systemic immunity should expedite the development of vaccines to diminish the global burden caused by enteric pathogens. Identifying an immunological correlate of protection in the course of field trials of efficacy, animal models (when available), or human challenge studies is also invaluable. In industrialized country populations, live attenuated vaccines (e.g. polio, typhoid, and rotavirus) mimic natural infection and generate robust protective immune responses. In contrast, a major challenge is to understand and overcome the barriers responsible for the diminished immunogenicity and efficacy of the same enteric vaccines in underprivileged populations in developing countries. Success in developing vaccines against some enteric pathogens has heretofore been elusive (e.g. Shigella). Different types of oral vaccines can selectively or inclusively elicit mucosal secretory immunoglobulin A and serum immunoglobulin G antibodies and a variety of cell-mediated immune responses. Areas of research that require acceleration include interaction between the gut innate immune system and the stimulation of adaptive immunity, development of safe yet effective mucosal adjuvants, better understanding of homing to the mucosa of immunologically relevant cells, and elicitation of mucosal immunologic memory. This review dissects the immune responses elicited in humans by enteric vaccines.
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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.
Clin. Immunol.
PUBLISHED: 09-09-2010
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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.
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Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised, double-blind, placebo-controlled trial.
Lancet
PUBLISHED: 08-06-2010
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Rotavirus gastroenteritis causes many deaths in infants in sub-Saharan Africa. Because rotavirus vaccines have proven effective in developed countries but had not been tested in developing countries, we assessed efficacy of a pentavalent rotavirus vaccine against severe disease in Ghana, Kenya, and Mali between April, 2007, and March, 2009.
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Immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine.
BMC Biol.
PUBLISHED: 07-12-2010
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Oral vaccines, whether living or non-living, viral or bacterial, elicit diminished immune responses or have lower efficacy in developing countries than in developed countries. Here I describe studies with a live oral cholera vaccine that include older children no longer deriving immune support from breast milk or maternal antibodies and that identify some of the factors accounting for the lower immunogenicity, as well as suggesting counter-measures that may enhance the effectiveness of oral immunization in developing countries. The fundamental breakthrough is likely to require reversing effects of the environmental enteropathy that is often present in children living in fecally contaminated, impoverished environments.
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A challenge model for Shigella dysenteriae 1 in cynomolgus monkeys (Macaca fascicularis).
Comp. Med.
PUBLISHED: 02-18-2010
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Shigella dysenteriae type 1 can cause devastating pandemics with high case fatality rates; a vaccine for Shigella is unavailable currently. Because of the risks associated with performing challenge studies with wild-type S. dysenteriae 1 in human clinical trials to advance vaccine development, an improved nonhuman primate model is needed urgently. In the present study, cynomolgus macaques (Macaca fascicularis) were challenged with various doses of S. dysenteriae 1 strain 1617 to establish a dose that would produce shigellosis. Further, different routes of delivery of S. dysenteriae 1 were compared to establish the most appropriate route for infection. Animals receiving 10(11) cfu S. dysenteriae 1 intragastrically consistently developed signs of shigellosis characterized by the onset of diarrhea and dysentery within 2 to 3 d. Administration of as many as 10(9) cfu S. dysenteriae 1 intraduodenally did not elicit signs characteristic of infection in macaques despite fecal shedding of bacteria for as long as 10 d. S. dysenteriae 1 administered intraduodenally at 10(9) cfu or intragastrically at 10(11) cfu elicited robust IgG and IgA antibody responses to LPS. We have developed a reliable challenge model of infection with wild-type S. dysenteriae 1 in cynomolgus macaques that reproducibly induces disease and elicits robust immune responses. We believe that this animal model may provide unique insights into the immunologic mechanisms of protection to S. dysenteriae 1 infection and in advancing development of a vaccine against shigellosis.
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Identification by PCR of non-typhoidal Salmonella enterica serovars associated with invasive infections among febrile patients in Mali.
PLoS Negl Trop Dis
PUBLISHED: 01-20-2010
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In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.
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A new generation of stable, nonantibiotic, low-copy-number plasmids improves immune responses to foreign antigens in Salmonella enterica serovar Typhi live vectors.
Infect. Immun.
PUBLISHED: 11-02-2009
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We hypothesized that adequately engineered attenuated Salmonella enterica serovar Typhi strains can serve as multivalent mucosal live vector vaccines to immunize against unrelated human pathogens. Toward this ultimate goal, we have developed a novel genetic stabilization system for antigen-expressing plasmids, engineered to encode the single-stranded binding protein (SSB), an essential protein involved in DNA metabolism which was deleted from the live vector chromosome. We utilized full-length protective antigen (PA83) of anthrax toxin from Bacillus anthracis as a foreign antigen and expressed PA83 as a fusion with the ClyA export protein, which allows export of ClyA-PA83 to the surface of S. Typhi live vectors. A series of SSB-encoding multicopy expression plasmids were introduced into reengineered S. Typhi strains previously tested in clinical trials, i.e., CVD 908-htrA and its less attenuated parent CVD 908. Immunogenicity was examined using a mouse model of intranasal immunization with live vector, followed by parenteral boosting with purified PA83. PA-specific antibody responses markedly improved as the copy number of the SSB-encoding plasmids decreased, and this effect was dramatically enhanced when the foreign antigen was delivered by the less attenuated live vector CVD 908ssb. These results suggest that antibody responses to antigens delivered by S. Typhi live vectors are inversely related to the metabolic burden imposed by expression of the foreign antigen and that these responses can be improved when antigens are expressed from low-copy-number plasmids and exported out of the cytoplasm of less attenuated live vectors.
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[Direct medical costs of invasive pneumococcal disease and radiologically-diagnosed pneumonia among Chilean children].
Rev. Panam. Salud Publica
PUBLISHED: 10-10-2009
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To determine the direct medical costs of health care services for cases of invasive pneumococcal disease (IPD) and pneumonia acquired in the community and confirmed by radiology (NAC-Rx) among Chilean children.
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Haemophilus influenzae Type B conjugate vaccine introduction in Mali: impact on disease burden and serologic correlate of protection.
Am. J. Trop. Med. Hyg.
PUBLISHED: 05-30-2009
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In Bamako, Mali, where surveillance revealed a high incidence of Haemophilus influenzae type b (Hib) invasive disease, Hib conjugate vaccine was introduced into the Expanded Program on Immunization and the impact assessed. Annual confirmed Hib hospitalizations for infants 0-11 months of age fell from 175/10(5) to 44/10(5) (P < 0.001); among infants 6-7 months of age Hib hospitalizations fell from 377/10(5) to 69/10(5), (82% decrease, P < 0.001). Invasive Streptococcus pneumoniae hospitalizations remained unchanged. In a baseline serosurvey, only 3/200 infants 6-7 months of age (1.5%) had protective anti-polyribosylribitol phosphate (PRP) titers > or = 0.15 microg/mL and 1(0.5%) had >or = 1.0 microg/mL. In serosurveys 18 and 30 months after vaccine introduction, 168/201 (84%) and 184/200 (92%) infants, respectively, had titers > or = 0.15 microg/mL and 141/201 (70%) and 163/200 (82%) had titers > or = 1.0 microg/mL. Introduction of Hib vaccine led to rises in anti-PRP seroprevalence, significant reductions in Hib disease, and all-cause hospitalizations, whereas S. pneumoniae disease remained unchanged.
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Salmonella enterica serovar Typhi live vector vaccines finally come of age.
Immunol. Cell Biol.
PUBLISHED: 05-05-2009
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Attenuated Salmonella Typhi vaccine strains hold great promise as live vectors for presentation of foreign antigens from unrelated bacterial, viral and parasitic pathogens to the immune system. Although this approach has proved quite successful in experimental animal models for eliciting antigen-specific mucosal, humoral and cellular responses, results have been disappointing for clinical trials carried out thus far. We hypothesize that the paucity of human responses to foreign antigens delivered by live vectors suggests that the strains and genetic approaches used to date have resulted in overattenuated vaccine strains with severely reduced immunogenicity. However, remarkable advances have now been made in the genetics of foreign antigen expression, understanding mechanisms of live vector immunity and refining immunization strategies. The time has now come for development of multivalent live vectors in which stable antigen expression and export is balanced with metabolic fitness to create highly immunogenic vaccines.
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Characterization of the most prevalent colonization factor antigens present in Chilean clinical enterotoxigenic Escherichia coli strains using a new multiplex polymerase chain reaction.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 04-01-2009
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Current methods to detect the colonization factor antigens (CFAs) associated with enterotoxigenic Escherichia coli (ETEC) strains are cumbersome, with some methods requiring antibodies that are not readily available. To achieve a gene-based method, we designed 2 multiplex polymerase chain reaction reactions to detect genes encoding the most common ETEC fimbrial colonization factors, including CFA/I and coli surface (CS) antigens CS1, CS2, CS3, CS4, CS5, and CS6. Analysis of 183 clinical ETEC strains shows that the most prevalent colonization factors were CFA/I only, CS1 and CS3, CS2 and CS3, and CS6 only. Interestingly, we identified 3 clinical isolates expressing CS1 only without its regulator rns. The method described here proved to be rapid and robust and correlates well with phenotypic expression of the CFAs, becoming a novel molecular diagnostic and research tool for future epidemiologic studies.
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Vaccines, global health and social equity.
Immunol. Cell Biol.
PUBLISHED: 03-24-2009
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Mortality rates of children less than 5 years old range from <10 per 1000 live births in industrialized countries to >100 in the worlds poorest countries. The fact that in New York City infant mortality fell from approximately 140 deaths per 1000 live births in 1900 to <60 per 1000 by 1930 indicates what can be achieved through improvements in public health, such as the provision of clean piped water, sewage disposal and fresh refrigeration. For children living in impoverished conditions today, excess mortality is largely due to infectious diseases for which there are effective vaccines. Thus, certain specific vaccines can reduce mortality and morbidity, improve quality of life and contribute to economic development. However, because many vaccines and the means to deliver them are beyond the financial resources of countries with the highest childhood mortality rates, strategies have been devised to provide vaccines to the most needy populations. These strategies include initiatives by the Global Alliance for Vaccines and Immunization, the International Finance Facility for Immunization and the Advanced Market Commitment, which together with various governments, international agencies and charitable foundations are providing funds to make life-saving vaccines available to the worlds most needy children.
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Mucosal immunization with attenuated Salmonella enterica serovar Typhi expressing protective antigen of anthrax toxin (PA83) primes monkeys for accelerated serum antibody responses to parenteral PA83 vaccine.
J. Infect. Dis.
PUBLISHED: 03-13-2009
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Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was genetically engineered for stable plasmid-based expression of protective antigen of anthrax toxin (PA83) fused with the export protein ClyA (ClyA-PA83). The priming potential of CVD 908-htrA expressing ClyA-PA83 was assessed in 12 rhesus and 20 cynomolgus macaques that were immunized mucosally (i.e., intranasally) on days 0 and 14. A parenteral booster with purified PA83 plus alum was given to rhesus macaques on days 42 and 225; cynomolgus monkeys received a booster with either PA or licensed anthrax vaccine (BioThrax; Emergent Biosolutions) only one time, 3 months after priming. Monkeys primed with S. Typhi expressing ClyA-PA83 developed high levels of serum toxin-neutralization activity (TNA) antibodies (50% effective dose [ED50], >1.3x10(3)), 7 days after receipt of the booster, whereas unprimed controls lacked serum TNA (ED50, 0). In nonhuman primates, the success of this anthrax vaccine strategy based on heterologous mucosal priming followed by a parenteral subunit vaccine booster paves the way for clinical trials.
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Immunology of combining CRM(197) conjugates for Streptococcus pneumoniae, Neisseria meningitis and Haemophilus influenzae in Chilean infants.
Vaccine
PUBLISHED: 02-03-2009
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We evaluated the immunogenicity and safety of an investigational combination of 9-valent pneumococcal conjugate vaccine (PCV9) and meningococcal group C conjugate (MnCC) vaccine (PCV9-MnCC) administered concomitantly with Haemophilus influenzae type b (Hib) conjugate vaccine, and of a combination of the three vaccines mixed together as a single injection (Hib-PCV9-MnCC), and compared them to separately administered PCV9 and MnCC dispensed to Chilean infants at 2, 4, and 6 months of age. The frequency of adverse events was similar among groups. Recipients of PCV9 alone or in combination with the other vaccines mounted significant antibody responses to the nine pneumococcal serotypes in PCV9, with >88% achieving protective levels of > or =0.35microg/mL. For serotypes 6B, 9V, and 5, recipients of PCV9 alone had significantly higher geometric mean concentrations (GMCs) than those of the other vaccine groups. Similarly, the GMC of anti-PRP antibodies was significantly lower among recipients of Hib-PCV9-MnCC than among those who received Hib vaccine separately from PCV9 or MnCC. In Chilean infants, PCV9, PCV9-MnCC, and Hib-PCV9-MnCC were highly immunogenic and safe. Overall, interactions of PCV9, MnCC and Hib affected the magnitude (GMC) of the primary antibody responses to some of the antigens, but not the percentage of subjects who achieved protective antibody thresholds.
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Characterization of rationally attenuated Francisella tularensis vaccine strains that harbor deletions in the guaA and guaB genes.
Vaccine
PUBLISHED: 02-02-2009
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Francisella tularensis, the etiologic agent of tularemia, can cause severe and fatal infection after inhalation of as few as 10 -- 100CFU. F. tularensis is a potential bioterrorism agent and, therefore, a priority for countermeasure development. Vaccination with the live vaccine strain (LVS), developed from a Type B strain, confers partial protection against aerosal exposure to the more virulent Type A strains and provides proof of principle that a live attenuated vaccine strain may be efficacious. However LVS suffers from several notable drawbacks that have prevented its licensure and widespread use. To address the specific deficiencies that render LVS a sub-optimal tularemia vaccine, we engineered F. tularensis LVS strains with targeted deletions in the guaA or guaB genes that encode critical enzymes in the guanine nucleotide biosynthetic pathway. F. tularensis LVSDeltaguaA and LVSDeltaguaB mutants were guanine auxotrophs and were highly attenuated in a mouse model of infection. While the mutants failed to replicate in macrophages, a robust proinflammatory cytokine response, equivalent to that of the parental LVS, was elicited. Mice vaccinated with a single dose of the F. tularensis LVSDeltaguaA or LVSDeltaguaB mutant were fully protected against subsequent lethal challenge with the LVS parental strain. These findings suggest the specific deletion of these target genes could generate a safe and efficacious live attenuated vaccine.
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Community-acquired bacteremia among children admitted to a rural hospital in Mozambique.
Pediatr. Infect. Dis. J.
PUBLISHED: 01-10-2009
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Although community-acquired bacteremia is an important cause of childhood mortality in Africa, recognition of disease burden and potential impact of bacterial vaccines is limited.
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Sindbis virus-based measles DNA vaccines protect cotton rats against respiratory measles: relevance of antibodies, mucosal and systemic antibody-secreting cells, memory B cells, and Th1-type cytokines as correlates of immunity.
J. Virol.
PUBLISHED: 01-07-2009
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Measles remains an important cause of pediatric morbidity and mortality in developing countries, especially among infants who are too young to receive the current licensed live attenuated measles vaccine. We developed two Sindbis virus DNA vaccines encoding the measles virus hemagglutinin (pMSIN-H) and fusion proteins (pMSINH-FdU) and examined their immunogenicities and protective efficacies when administered alone or followed by the live measles virus vaccine in cotton rats. Neutralizing antibodies, mucosal and systemic antibody-secreting cells, memory B cells, and gamma interferon-secreting T cells developed after priming and increased after boosting. pMSIN-H priming conferred 100% protection against pulmonary measles, whereas pMSINH-FdU protected only in conjunction with the live measles virus vaccine boost.
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