Infectious Diseases - Projects
Longitudinal Study of the Vaginal Microbiome Prior to Incident STI
There are approximately 20 million new sexually transmitted infections (STIs) in the U.S. each year that add $16 billion in medical costs to the healthcare system. One approach to prevention of STIs in women that has not been fully explored is harnessing the protective features of the vaginal microbiome. The vaginal microbiota play an important role in preventing colonization by pathogenic organisms. Vaginal Lactobacillus spp. provide broad-spectrum antimicrobial activity in part through their production of lactic acid, which creates an acidic and hostile environment to pathogens. To date, only studies based on bacterial cultivation techniques or microscopic evaluation are available to describe the temporal association between the vaginal bacteria and risk of STI. However, a majority of microbial species (>90%) resist cultivation and microscopy only provides morphological information. Complete characterization of the vaginal microbiota requires molecular approaches as we propose. We have shown using metagenomic analysis that specific genotypes of Lactobacillus spp. are associated with both chlamydial infection and vaginal microbiota instability. We are putting forth a novel hypothesis that, in addition to a low-Lactobacillus state, specific Lactobacillus genotypes are associated with increased risk for STIs, and that the mixture of D- and L- lactate isomers produced by Lactobacillus spp. differentially affects STI risk. Inflammation may cause destruction of the vaginal epithelium, allowing pathogens to access deeper tissues, and therefore, we also hypothesize that local immune responses associated with specific vaginal microbiota may facilitate STIs. We seek to utilize archived cervicovaginal lavage (CVL) samples collected from the 1999 NIH Longitudinal Study of Vaginal Flora in which 3,620 women were followed for one year with quarterly assessments. A total of 681 women were observed to acquire an incident Chlamydia trachomatis (CT), Neisseria gonorrhea (GC), or Trichomonas vaginalis (TV) genital infection. The repository samples provide a unique opportunity to characterize the vaginal microbiome prior to acquisition of STIs in a well-powered study. We will conduct a nested case-control study with incidence density sampling. Cases will be defined as women who had an incident STI and we will analyze the sample at the visit prior to the first STI detection. Controls will be matched on age, ethnicity and visit number to cases and will be women who did not develop a STI by the time of the case visit. Specific aims are to (1) Evaluate the association between vaginal microbiota and incidence of CT, GC, and TV genital infection using 16S rRNA gene analysis; (2) Compare levels of D- and L-lactic acid isomers in cases and controls; (3) Investigate if specific strains of Lactobacillus (or other bacterium) are associated with STI risk using metagenomic analysis; (4) Determine if cervicovaginal immune responses independently predict incident STIs. All four aims will be modeled in the context of comprehensive behavioral data. This study will provide a functional understanding of the vaginal microbiome's role in STI protection and may reveal novel targets to prevent STIs and improve women's health.
Lubricant Use and the Vaginal Microbiome
Twenty-five percent of U.S. women report frequent use of vaginal lubricants to reduce discomfort and increase pleasure during sex. Clinically, lubricants are also used for digital exams and procedures such as trans-vaginal ultrasound (T-VUS). Among postmenopausal women, 50% use lubricants due to vaginal dryness and dyspareunia. The first line treatment for these women is non-hormonal vaginal lubricants. Recent studies have questioned the safety of current lubricants, yet there is little available data A healthy vagina is protected by Lactobacillus spp. that acidify the vagina with lactic acid, a potent bacteriocide and viricide. Disruption of this healthy microbiome markedly increases susceptibility to reproductive tract infections. We hypothesize that lubricants can disrupt the vaginal microbiome and increase risk to infections. Most vaginal lubricants contain hypertonic humectants such as glycerol or propylene glycol together with antibacterial preservatives, parabens and sometimes chlorhexidine. The active ingredients are "generally recognized as safe" (GRAS) by the FDA, hence are not subject to human clinical trials. However, at high concentrations or with frequent use, these ingredients may have toxic effects. It is unknown if hypertonic antibacterial lubricants affect the vaginal microbiota. We propose three aims to study the effect of lubricant on the vaginal microenvironment: 1) Evaluate the effects of lubricant on the temporal dynamics of vaginal microbiota with two methods: 1A) Perform a controlled observational study of one-time use of vaginal lubricant by recruiting women who are presenting for T-VUS, a procedure in which lubricant is applied to a T-VUS probe. This study will standardize the lubricant brand and dose by using the hypertonic lubricant currently in use by the Department of Radiology. Participants will self-collect daily samples prior to T-VUS for 1 week and twice-weekly for 9- weeks after TVUS. Changes in bacterial composition will be assessed using 16S rRNA gene analysis. 1B) Conduct a secondary analysis of existing data (behavioral diaries, clinical exams and 16S rRNA sequences) collected from a cohort of 130 women who were followed for 10 weeks in a prior study as part of the Human Microbiome Project (HMP). Participants self-collected mid-vaginal swabs daily and all samples are stored in a freezer repository. Lubricant use was reported by 37 women on 140 occasions. Samples collected every other day have already undergone DNA extraction and 16S rRNA sequencing (NIH RO1-GM103604). 2) Examine the effects of lubricant on the vaginal microenvironment in samples obtained from Aims 1A and 1B by assessing pH, D- and L-isomers of lactic acid and inflammatory cytokines. 3) Determine if lubricants have toxic effects on the vaginal epithelium using measurements of cell shedding rates, glycogen (necessary for lactobacilli survival), fibronectin (a marker of epithelial disruption), and lactate dehydrogenase (a marker of cell death) in samples collected in Aim 1A. This study will contribute to the development of safer formulations for personal and clinical lubricants.
Vaginal Microbiota, Immune Responses and the Vulvovaginal Symptoms During Menopause
Studies demonstrate that between 25-50% of menopausal women report vulvovaginal symptoms including vaginal dryness, irritation, burning, itching, and dyspareunia, all of which severely affect quality of life. As women approach menopause, decreased estrogen levels are accompanied by changes in the vagina including decreased glycogen accumulation in the epithelium, increased pH, and deterioration of vaginal tissues, which lead to symptoms of vaginal discomfort. Hormone therapy is the primary treatment for vulvovaginal symptoms in menopausal women, however it is contraindicated in some women. Non-hormonal therapeutic approaches for improving symptoms and quality of life in postmenopausal women have not been well studied. The vaginal microbial communities (termed the vaginal microbiota) play a critical role in protecting the female genital tract from disease; however, surprisingly little is known about the composition of vaginal bacteria across the different stages of menopausal transition, how they differ between individuals, the correlation with vaginal immunologic microenvironment, or the associations with gynecologic complaints and sexual functioning. Between 2009-2012, 885 women aged 35-60 years were enrolled in a prospective study of menopause transition and followed every six months for two years (R01-CA123467). Taking advantage of that resource, the proposed study will utilize 2500 longitudinal vaginal samples stored in a freezer archive to address the following three specific aims: (1) To describe the changes in the vaginal microbiota by age and stage of menopause over a 2-year period, (2) To evaluate the association between the vaginal microbiota and vulvovaginal complaints and (3) To assess the correlation between the vaginal immunologic microenvironment with the microbiota. Participants were categorized according to menopausal categories by the Stages of Reproductive Aging Workshop (STRAW) guidelines. The vaginal microbiota will be characterized using 16S rRNA gene analysis amplified from whole genomic DNA isolated from clinician-collected mid-vaginal swabs. Cervical secretions will be used to quantify concentrations of 27 different immune markers (cytokines, chemokines, and growth factors) using multiplexed bead-based immunoassays with total protein adjustment. By bringing together multidisciplinary expertise in bioinformatics, epidemiology, genomics, gynecology, microbiology and statistics, this study will be able to provide critical descriptive data on (1) differences in the composition and stability of the vaginal microbiome by menopausal stage, (2) association between microbial changes and presence of vulvovaginal complaints, and (3) the possible correlation between vulvovaginal microbial and immunologic microenvironmental changes during the menopausal transition. These data will be used to evaluate the potential for non-hormonal, anti-inflammatory, or probiotic interventions for the treatment of common vulvovaginal complaints in peri- and postmenopausal women.
Evolution of Eukaryotic Parasites
The phylum Apicomplexa comprises a diverse group of unicellular, eukaryotic parasites, many of which infect humans, or mammalian species on which human livelihood greatly depends. This phylum includes the causative agents of malaria, babesiosis, cryptosporidiosis, and toxoplasmosis in humans, as well as theileriosis and East Coast fever in cattle. The genome sequence for over a dozen apicomplexan species is available in either complete of draft form, including those of eight Plasmodium species, and three species of each in the Theileria, Babesia and Cryptosporidium genera.
Lyme disease and comparative genomics of the Lyme agent, Borrelia
Lyme disease is the fifth most common Nationally Notifiable disease and the most commonly reported vector-borne illness in the United States. It is caused by spirochetes of the bacterial species group Borrelia burgdorferi sensu lato that is obligatorily transmitted by hard-bodied ticks (e.g., Ixodes scapularis in the US). Whereas incidence of Lyme disease in the USA is currently concentrated in the Northeast, Upper Midwest, and West Coast, the disease prevalence continues to rise and the geographic range of pathogen and tick populations continue to expand, with climate changes and declining biodiversity across the global as likely driving factors. Three specific public-health risks have emerged as results of the ongoing geographic expansion of Lyme disease pathogens and vectors. The first risk is the growth of previously rare and isolated pathogen populations ("spill-over"). For example, a new, highly human-virulent species B. mayonii has recently been discovered in the Upper Midwest. The second risk is the colonization of new areas by well-established pathogen populations elsewhere (“invasion”). For example, at least four different types of B. burgdorferi sensu stricto, the main pathogenic species in the U.S., are found in both Europe and North America, likely due to recent cross-Atlantic migration. The third risk is the increasing hybridization between previously segregated species or strains due to recombination (“mixing”). For example, Borrelia populations consists of recombinant genomes in North America and cross-species recombinants in Europe.
Given these heightening Lyme disease risks, there is a pressing need to characterize the full extent of genetic variations of Lyme disease pathogens by whole-genome sequencing. IGS scientists have previously sequenced and published complete genome sequences of over twenty Borrelia bacteria and performed extensive comparative analysis of these genomes. High-quality genome sequences of additional Borrelia strains that span the world-wide range of genetic and epidemiological diversity, including newly discovered Borrelia species and emerging strains of this clinically important bacteria, are currently in the works. Whole-genome sequencing are essential for deducing mechanisms of pathogenesis through comparative analyses, as well as for identifying genetic targets for vaccines, diagnostics, and therapeutics. In addition, high-quality reference genomes representing national and global Borrelia diversity have to potential to provide unprecedented resolution for epidemiological tracking, prediction, and control of the spread of Lyme pathogens.
[PI's: Dr. Sherwood Casjens, U. Of Utah, Dr. Benjamin Luft, Stonybrook U., Dr. Weigang Qiu, Hunter College of U. Of New York, Dr. Steven Schutzer, Rutgers U.]
This project explores the dynamic interactions between pathogens, hosts, microbiota, the immune system, and the environment, with the goal to provide a comprehensive understanding of the determinants of infectious disease. The project includes work on bacteria (Escherichia coli), fungi (Candida, Aspergillus, and Mucormycosis species), and eukaryotic parasites (Plasmodium and Brugia).
NIAID-funded Genome Center for Infectious Diseases Integrated Genomics Research in Parasitic Tropical Diseases — Lymphatic Filariasis Subproject
Parasitic diseases impose a tremendous toll on the global public health. Malaria causes up to 1.24 million deaths every year, while human filariasis is a neglected tropical disease that remains a major cause of disability in the developing world. This subproject focuses on the filarial nematode Brugia malayi, which causes lymphatic filariasis. Population genomics, multi-species transcriptomics, and whole genome sequencing are used to improve our understanding of the organisms responsible for this important neglected tropical disease.
NIAID-funded Genome Center for Infectious Diseases Core Leader – Tech Core
Genomics has revolutionized research into infectious diseases and is poised to revolutionize the clinic. Through the activities in this technology core, we provide high-throughput genome sequencing and analysis focused on understanding host, pathogen, and microbiome interactions as determinants of disease outcome. We provide state-of-the-art, large-scale, high-throughput sequencing data for analysis of genomes, transcriptomes, metagenomes, metatranscriptomes, and microRNAs using the best methodologies and technologies available.
EPSTI: Eco-Pathogenomics of Sexually Transmitted Infections
The "Eco-Pathogenomics of Sexually Transmitted Infections" (EPSTI), project builds upon the research started under the "Eco-Pathogenomics of Chlamydial Reproductive Tract Infection"(EPCRTI) project and examines the triangular relationship between human genetic variation, sexually transmitted infections (STIs) and co-infections caused by Chlamydia trachomatis andNeisseria gonorrhoeae, and microbiota composition among partners with distinct infection outcomes. Sexually transmitted infections pose a major health challenge in the United States where Chlamydia trachomatis (CT) genital infections, with an estimated 2.8M cases yearly, are the most frequently reported bacterial infectious disease. Likewise there are an estimated 820,000 cases of Neisseria gonorrhoeae (GC) each year. The sequelae of infections and co-infections caused by these two pathogens are insidious and account for the majority of the 750,000 annual cases of pelvic inflammatory disease (PID) in the United States, a precursor to life-threatening ectopic pregnancy and tubal factor infertility (TFI) in women. Thus, research to prevent, control and treat these STIs will provide broad health and economic benefits.
In the battleground of an infection site, both the host cells and the microbes employ complex signaling mechanisms and weaponry to destabilize, neutralize or kill the other. Identifying and understanding these biomarkers of infection and disease are the main research goals of this Cooperative Research Center (CRC). The anticipated impact will be to reduce the incidence of sexually transmitted infections and diseases (STIs & STDs) in humans worldwide. We believe that: a) the genetic variance of the infected host, the genetic diversity of the infecting pathogen(s), and the composition and function of the resident microbiota directly impact the evolution of STIs and could be biomarkers of disease severity or protection from STIs; b) genes, RNAs and proteins that are expressed or produced by the host, the pathogens, and/or the genital microbiota in response to one another are biomarkers of a specific type of STI or STD. The aim of the CRC is therefore to identify host, pathogen and/or microbiota biomarkers of STIs that may reveal mechanisms of pathogenesis and therapeutic or diagnostic targets that can be exploited for the development of translational curative or prophylactic interventions that have a direct impact in public health.
To test these hypotheses and realize these objectives, this CRC, EPSTI, will build on data acquired by the parent CRC, EPCRTI, a Chlamydia-centric program that laid the methodological and conceptual foundations of EPSTI. Within EPSTI, STING (STI Network Groups), consisting of multiple networks of sexual partners, will be leveraged to examine the triangular relationship between human genetic variation, CT, GC infections and co-infections, and microbiota composition among partners with distinct infection outcomes. The experimental approach will adopt a systems biology strategy focused on the identification of biomarkers of genital/reproductive infection and disease and are eminently amenable to translational applications in clinical and public health. These are expected to include predictive diagnosis for individuals at greatest risk of STI and STD based on their biological and microbiome characteristics, development of sensitive and specific point-of-care diagnostic tests and highly specific targets for vaccine development.
This Sexually Transmitted Infections Cooperative Research Center (STI CRC) is funded through grant U19 AI08044 from the National Institutes of Health, National Institute for Allergies and Infectious Disease.
Genital Microbiome-Pathogen Interactions in a Sexual Transmission Network
Abundant lactobacilli in the human vagina are thought to protect against invasion by non-indigenous bacteria, including sexually transmitted infections caused by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). The means by which this happens are not well understood. It could be that these exclusionary mechanisms are properties of the vaginal microbiome, features of the host immune system and physiology, or some combination of both. The goal of this project is to employ a systems biology approach to identify biomarkers of the vaginal and penile microbiome, the host and the pathogens that are associated with increased or decreased risks of infection by CT, GC or both. Project 3 of this research program will rely on samples collected by the Clinical Core C from STING networks of sex partners who have been exposed to and possibly infected by CT, GC, or both. In these networks we expect that about 20-40% of the participants will have been exposed to, but not infected by these pathogens. This will give us the unique opportunity to assess the role of the microbiome in preventing or facilitating infections by CT and GC. Our overarching hypothesis is that when pathogen transmission does not occur the genetic traits of the infecting pathogen(s) may be insufficient to overcome the host response or the exclusionary mechanisms of the microbiome environment; or that features of the microbiome are protective or induce a protective mucosal environment. In this project, we will build on these findings and use modern ‘omic technologies to identify specific functional features of the vaginal and penile microbiota associated with susceptibility and resistance to infection and co-infection and the importance of host and pathogen genetic variation in this infection process, which will be done in collaboration with Projects 1 & 2. We will achieve these goals by addressing three integrated specific aims: Aim 1. Characterize the genomic variations in CT/GC in participants of the STING networks of sex partners; Aim 2. Use ‘omic approaches and system biology analysis characterize the molecular interactions between the host, the pathogens and the genital microbiota in discordant and concordant couples for CT/GC infections; Aim 3. Validate and explore mechanistic explanations for how the microbiota prevent or facilitate infection by CT/GC using an in vitro three-dimensional model of endocervical epithelial cells. Our long-term goal is to leverage the information generated in this project to develop improved diagnostic methods, identify novel targets for new drug development and develop targeted and effective curative or preventive therapies, and ultimately, promote health, reduce risk to unintended adverse sequelae of STI and improve the quality of life for men and women who are at risk of STIs.
The Interaction Between Vaginal Microbiota, Immunogenetics and Chlamydia trachomatis Susceptibility
The vaginal microbiota is thought to play a major role in preventing Sexually Transmitted Infections (STIs) through ecological competition, lactic acid and target-specific bacteriocins production by Lactobacillus species. A condition known as bacterial vaginosis (BV) characterized by the depletion of Lactobacillus spp. and an overabundance of anaerobic bacteria has been shown to be associated with acquisition of STIs including HSV-2 and HIV-1. Chlamydia trachomatis (Ct) is an obligate intracellular bacterial pathogen associated with cervicitis, pelvic inflammatory disease, and subsequent tubal factor infertility and ectopic pregnancy. It is the most common bacterial STI worldwide, including in the United States. Epidemiological data suggest that the incidence of this disease has increased recently despite efforts to prevent it, which motivates the search for vaccine candidates and updates to current recommendations for Ct screening. The long-term goal of this research is to leverage biological and epidemiological data to adapt strategies for Ct control by integrating these data types. Our hypothesis is that the composition of the vaginal microbiota, and potentially its disruption, could lead to an increased susceptibility to Ct infection, independent of the effect immunogenetics may have on this relationship. If such a role could be highlighted, a change to the treatment and prevention guidelines for Ct and STI in general would be warranted. However, modification to the current recommendations for Ct screening, prevention and treatment would need to be supported by information on the natural history of Ct infection. Therefore, the objective of this project is to study the role of the vaginal microbiota in Ct acquisition and clinical presentation in a unique and already established cohort of young women prospectively followed in France. A multidisciplinary international research network has been assembled to answer these questions by focusing on two specific aims: 1) Characterize the vaginal microbiota composition, abundance and dynamics over 2 years, and the frequency of a set of immunogenetic biomarkers in a cohort of 18-24 years old women; 2) Model the interactions between the vaginal microbiota, immunogenetic factors and C. trachomatis infection using a cohort of 18-24 years old women prospectively followed. In the first aim, we will characterize the vaginal microbiota (composition and abundance) of samples from 300 women prospectively followed at four time points. We will then evaluate under the second aim the independent effects of vaginal microbiota composition and immunogenetic biomarkers on susceptibility to Ct infection as well as presence of symptoms, in order to build host-pathogen-microbiota interaction models. This research is novel in that the role of the vaginal microbiota composition in the susceptibility to Ct infection has never been studied in a large-scale prospective cohort. Its innovative potential lies in the use of novel molecular methods that allow us to refine our approach to “health” and “disease” by integrating individual predisposition to STIs, thus leading the way for personalized medicine.
Diversity of enteric pathogens including E. coli/Shigella
Diarrheal disease is a problem on a global scale as recognized by United Nations Millennium Development Goal number 4, which states as a main goal, "to combat death due to diarrhea diseases in children less than 5 years of age". The most comprehensive diarrheal studies indicate that there are greater than 110 million cases of diarrhea in children under 5 each year and approximately 2 million people die each year as a direct result of diarrheal disease, and a large proportion of those are children. The major bacterial pathogens that contribute to diarrheal disease are Escherichia coli and Shigella species. While E.coli and Shigella are very similar from an organismal perspective they can exist as commensal organisms, causing no disease in humans and animals to infection causing significant disease that results in death. We still do not fully grasp the diversity of these pathogens and commensals. We are trying to capture this diversity on both the genomic and transcriptomic level using the latest-generation sequencing technologies. A current project will address the genomic content of greater than 100 isolates of E. coli and Shigella, leading to further functional characterization of pathogenic features that will result in additional therapeutic and vaccine targets.
Omics-based Identification of Novel Vaccine Targets Against Neisseria gonorrhoeae
The diplococcic bacterium Neisseria gonorrhoeae (Gc) is the causative agent of the sexually transmitted infection (STI) gonorrhea. The ability of major surface proteins to vary their expression significantly complicates the development of a vaccine against Gc. Our project focuses on Gc changes that occur under relevant physiological conditions that would be encountered in vivo. We hypothesize that specific differentially regulated Gc surface proteins are selected for during infection and these events enhance infectivity across multiple strains, thus providing targets for vaccine development. We will first characterize these profiles using Gc laboratory strains then extrapolate them to similarities in profiles observed between Gc clinical isolates that are readily transmitted and/or display enhanced infectivity. Targets identified from this innovative and integrative research strategy will constitute a novel foundation of Gc host interaction determinants relevant to enhanced infectivity that will be pursued in follow-up larger studies and clinical trials to determine their viability as vaccine candidates.
[PI: Hervé Tettelin; Vonetta Edwards, postdoctoral fellow]
Cardiac Micro-lesion Formation During Invasive Pneumococcal Disease
I have performed dual RNA-seq profiling of the host-pathogen interplay during disseminated Streptococcus pneumoniae infection in a mouse model. We were able to simultaneously profile bacterial and mouse gene responses using whole infected hearts, as well as infected blood. These studies led to the identification of novel anatomical site-specific expression of determinants of pneumococcal pathogenesis, gave insights into their mechanism of interaction with the host, and provided key information on currently considered and future protein candidates for vaccine development. We are now extending this project to study multiple anatomical sites in the mouse and combine transcriptomics with metabolomics studies. As a part of the GCID, we are also studying host-pneumococcus interactions and biofilms, with or without the influence of the influenza virus, using an ex-vivo primary cadaver lung cell culture system.
[PI: Carlos Orihuela, U. of Alabama at Birmingham]
Fitness Profiling of Streptococcus gordonii in Oral Microenvironments
Streptococcus gordonii is an initial colonizer of the biofilm that forms tooth surfaces. Identification of S. gordonii genes essential for species survival and proliferation under synergistic or antagonistic conditions will provide essential insights into community dynamics that favor or disfavor a state of oral health. We are using Tn-seq to identify and examine genes involved in S. gordonii fitness under selected in vitro model conditions relevant to those in the oral cavity.
[PI: Meg Virckerman, U. at Buffalo]
Regulation of Plasmodium vivax Parasites
Plasmodium vivax is a unicellular parasite responsible for the majority of the cases of human malaria outside sub-Saharan Africa. While on-going malaria elimination efforts are reducing the burden of falciparum malaria worldwide, the situation is much less promising for P. vivax, which displays very different features than P. falciparum. Unfortunately, our understanding of the biology of P. vivax parasites dramatically lags behind that of P. falciparum, primarily due to our inability to propagate these parasites in vitro. We are using a combination of genomic approaches, including single cell gene expression profiling, to better understand these parasites and how they are regulated throughout their life cycle in patients as well as during their development in mosquitoes.
Host/pathogen Interactions and the Acquisition of Immunity to Malaria Parasites
Plasmodium falciparum causes more than 200 million cases of clinical malaria and half a million deaths every year. Falciparum malaria disproportionally affects young children as individuals living in endemic areas gradually acquire immunity against the disease. We are interested in understanding the role of the host and parasite gene expression in this acquisition of immunity against falciparum malaria and are conducting a study of infected Malian children followed for four years to better characterize the molecular changes accompanying the gradual acquisition of resistance to malaria parasites.
Characterization of Eukaryotic Parasites Causing Diarrhea
Diarrheal diseases are responsible for more than two billion cases every year and constitute one of the main global health challenges. The disease is particularly common in developing countries, where it disproportionally affects young children who, on average, get diarrhea three times a year. Diarrhea results from an infection in the intestinal tract, which can be caused by a variety of bacteria, viruses and eukaryotic parasites. In most studies, less than half of the diarrhea cases can be attributed to a known pathogen. We have developed new tools to comprehensively characterize all eukaryotic parasites present in a sample and are screening 3,600 stool samples collected by the GEMS study in four countries from infants with diarrhea and matched controls to identify novel pathogens and assess their contributions to the disease etiology.