Ramiro L. Gutierrez

An Evidenced-Based Scale of Disease Severity following Human Challenge with Enteroxigenic Escherichia coli

Background Experimental human challenge models have played a major role in enhancing our understanding of infectious diseases. Primary outcomes have typically utilized overly simplistic outcomes that fail to entirely account for complex illness syndromes. We sought to characterize clinical outcomes associated with experimental infection with enterotoxigenic Escherichia coli (ETEC) and to develop a disease score. Methods Data were obtained from prior controlled human ETEC infection studies. Correlation and univariate regression across sign and symptom severity was performed. A multiple correspondence analysis was conducted. A 3-parameter disease score with construct validity was developed in an iterative fashion, compared to standard outcome definitions and applied to prior vaccine challenge trials. Results Data on 264 subjects receiving seven ETEC strains at doses from 1x105 to 1x1010 cfu were used to construct a standardized dataset. The strongest observed correlation was between vomiting and nausea (r = 0.65); however, stool output was poorly correlated with subjective activity-impacting outcomes. Multiple correspondence analyses showed covariability in multiple signs and symptoms, with severity being the strongest factor corresponding across outcomes. The developed disease score performed well compared to standard outcome definitions and differentiated disease in vaccinated and unvaccinated subjects. Conclusion Frequency and volumetric definitions of diarrhea severity poorly characterize ETEC disease. These data support a disease severity score accounting for stool output and other clinical signs and symptoms. Such a score could serve as the basis for better field trial outcomes and gives an additional outcome measure to help select future vaccines that warrant expanded testing in pivotal pre-licensure trials.

Campylobacter jejuni transcriptional and genetic adaptation during human infection

Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.A human challenge trial with Campylobacter jejuni uncovers transcriptional and genomic changes during infection, highlighting pathogen factors associated with acute and persistent infection.

Review: chronic and persistent diarrhea with a focus in the returning traveler

BackgroundTravelers’ diarrhea is a common malady afflicting up to 50% of travelers after a 2-week travel period. An appreciable percentage of these cases will become persistent or chronic. We summarized the published literature reporting persistent/chronic diarrhea in travelers elucidating current understanding of disease incidence, etiology and regional variability.MethodsWe searched electronic databases (Medline, Embase, and Cochrane database of clinical trials) from 1990 to 2015 using the following terms: “chronic or persistent diarrh* and (returning) travel* or enteropathogen, GeoSentinel, and travel-associated infection. Included studies published in the English language on adult returning travelers (duration < 3-months) reporting denominator data. Point estimates and standard 95% confidence intervals were calculated for incidence using a random-effects model. Study incidence heterogeneity rates were assessed using x2 heterogeneity statistics, graphically represented with Forest plots.ResultsWe identified 19 studies meeting the inclusion criteria (all published after 1999). 18 studies reported upon the incidence of persistent/chronic diarrhea as a syndromic diagnosis in returning travelers; one study reported adequate denominator data from which to assess pathogen specific etiology. Giardiasis comprise an appreicaible percentage of infectious mediated persistent/chronic diarrhea in returning travelers. The overall estimate of persistent/chronic diarrhea incidence was 6% (0.05–0.07) in 321,454, travelers; with significant heterogeniety observed across regions. The total number of regional travelers, and point estimates for incidence (95% CI) for Latin American, African, and Asian travelers were [15816 (0.09 [0.07–0.11]), 42290 (0.06 [0.05–0.07]), and 27433 (0.07 [0.06–0.09])] respectively. We identified lower published rates of chronic diarrhea from Sub-Saharan Africa relative to [North Africa, South Central Asia, and Central America]. Persistent/chronic diarrhea ranked fourth as a syndromic diagnosis in all regions.ConclusionsPersistent/Chronic diarrhea is a leading syndromic diagnosis in returning travelers across all regions. The 6% incidence [proportionate morbidity (PM) of 60] observed in over >300,000 global travelers is comparable to prior estimates. We identified lower published rates of chronic diarrhea from Sub-Saharan Africa relative to [North Africa, South Central Asia, and Central America]. Giardiasis comprises an appreciabile percentatge of travel-associated infectious mediated persistent/chronic diarrhea. There’s a dearth of published data characterizing the incidence of specific enteropathogenic etiologies for persistent/chronic diarrhea in returning travelers.

Enterotoxigenic E. coli virulence gene regulation in human infections

Significance Bacterial pathogens must sense cues in the host environment and adapt with appropriate transcriptional responses to cause infections. Although identifying global transcriptional changes in a pathogen during infections would reveal these mechanisms of success, such work in human infections is difficult and rare. Here we use samples from a controlled human model of enterotoxigenic Escherichia coli (ETEC) infection to identify ETEC’s global transcriptional response to the human host. We found ETEC senses environmental oxygen to coordinate virulence gene expression during human infections via the transcriptional regulator FNR, and that FNR regulates biofilm formation in ETEC. This work identified a global regulator of virulence genes in ETEC where toxin and adhesin expression is coordinated with pathogen proximity to the epithelium. Enterotoxigenic Escherichia coli (ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC’s response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion of fnr in ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

Enterotoxigenic Escherichia coli blood group A interactions intensify diarrheal severity

Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A–specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.

Giardia lamblia infection increases risk of chronic gastrointestinal disorders

BackgroundGiardia lamblia is a common parasitic cause of infectious gastroenteritis in the United States and the world and may be linked to an increased risk of chronic gastrointestinal (GI) disorders. We sought to assess the risk of several chronic GI disorders following Giardia infection among active duty US military personnel.MethodsThis study was designed as a retrospective cohort study in which active duty military personnel with documented G. lamblia infection were assessed for the subsequent risk of developing a chronic GI disorder including irritable bowel syndrome (IBS), dyspepsia and gastroesophageal reflux disease (GERD). Post-giardia chronic GI disorder risk was compared to risk in uninfected personnel matched on several demographic characteristics and medical encounter information. Data were obtained from the Defense Medical Surveillance System and exposures (1998–2009) with outcomes identified based on documented medical encounters with specific medical billing codes. Modified Poisson regression was used to evaluate the relationship between G. lamblia infection and chronic GI disorders.ResultsA total of 80 Giardia cases were identified for an estimated incidence of 0.55 cases per 100,000 person-years. Cases were matched to 294 unexposed subjects. After adjusting for important covariates, there was an increased risk of IBS (relative risk: 2.1, p = 0.03) associated with antecedent Giardia infection.ConclusionThese data add to a growing body of literature and demonstrate an increased risk of IBS after infection with G. lamblia.

Clinical endpoints in the controlled human challenge model for Shigella: A call for standardization and the development of a disease severity score

Background Since 1946 the controlled human infection model (CHIM) for Shigella has been used to improve understanding of disease pathogenesis, describe clinical and immunologic responses to infection and as a tool for vaccine development. As the frequency and intent for use in vaccine comparisons increases, standardization of the primary endpoint definition is necessary. Methods Subject-level data were obtained from previously conducted experimental Shigella CHIM studies. Signs and symptoms severity were categorized consistently across all studies. Sign and symptom correlations were estimated and univariate models were utilized to describe the association between stool output and other Shigella-attributable signs and symptoms. Multiple correspondence and hierarchical clustering analyses were performed to describe the co-occurrence of signs and symptoms. A disease score is proposed based on the co-occurrence of these events. Results Data were obtained on 54 subjects receiving 800 to 2000 colony forming units (cfu) of S. flexneri. The median maximum 24 hour stool output was 514 ml (IQR: 300, 998 ml) with a median frequency of 6 (IQR: 4, 9). Subjects reported abdominal pain or cramps (81.5%), headache (66.7%) and anorexia (64.8%), 50.0% had a fever and 27.8% had gross blood in multiple loose stools. Multiple correspondence analyses highlighted co-occurrence of symptoms based on severity. A 3-parameter disease severity score predicted shigellosis endpoints and better differentiated disease spectrum. Conclusion Dichotomous endpoints for Shigella CHIM fail to fully account for disease variability. An ordinal disease score characterizing the breadth of disease severity may enable a better characterization of shigellosis and can decrease sample size requirements. Furthermore, the disease severity score may be a useful tool for portfolio management by enabling prioritization across vaccine candidates with comparable efficacy estimates using dichotomous endpoints.