Fecal Indole as a Biomarker of Susceptibility to Cryptosporidium Infection.

Cryptosporidium causes significant diarrhea worldwide, especially among children and immunocompromised individuals, and no effective drug treatment is currently available for those who need it most. In this report, previous volunteer infectivity studies have been extended to examine the association between fecal indole and indole-producing (IP) gut microbiota on the outcome of a Cryptosporidium infection. Fecal indole concentrations (FICs) of 50 subjects and 19 taxa of common gut microbiota, including six IP taxa (11 subjects) were determined in stool samples collected before and after a challenge with Cryptosporidium oocysts. At the baseline, the mean FIC (± the standard deviation) was 1.66 ± 0.80 mM in those who became infected after a challenge versus 3.20 ± 1.23 mM in those who remained uninfected (P = 0.0001). Only 11.1% of the subjects with a FIC of >2.5 mM became infected after a challenge versus 65.2% of the subjects with a FIC of <2.5 mM. In contrast, the FICs of infected subjects at the baseline or during diarrhea were not correlated with infection intensity or disease severity. The relative abundances (percent) of Escherichia coli, Bacillus spp., and Clostridium spp. were greater ≥2.5-fold in volunteers with a baseline FIC of >2.5 mM, while those of Bacteroides pyogenes, B. fragilis, and Akkermansia muciniphila were greater in those with a baseline FIC of <2.5 mM. These data indicate that some IP bacteria, or perhaps indole alone, can influence the ability of Cryptosporidium to establish an infection. Thus, preexisting indole levels in the gut join the oocyst dose and immune status as important factors that determine the outcome of Cryptosporidium exposure.

A rapid and specific method for the detection of indole in complex biological samples.

Indole, a bacterial product of tryptophan degradation, has a variety of important applications in the pharmaceutical industry and is a biomarker in biological and clinical specimens. Yet, specific assays to quantitate indole are complex and require expensive equipment and a high level of training. Thus, indole in biological samples is often estimated using the simple and rapid Kovács assay, which nonspecifically detects a variety of commonly occurring indole analogs. We demonstrate here a sensitive, specific, and rapid method for measuring indole in complex biological samples using a specific reaction between unsubstituted indole and hydroxylamine. We compared the hydroxylamine-based indole assay (HIA) to the Kovács assay and confirmed that the two assays are capable of detecting microgram amounts of indole. However, the HIA is specific to indole and does not detect other naturally occurring indole analogs. We further demonstrated the utility of the HIA in measuring indole levels in clinically relevant biological materials, such as fecal samples and bacterial cultures. Mean and median fecal indole concentrations from 53 healthy adults were 2.59 mM and 2.73 mM, respectively, but varied widely (0.30 mM to 6.64 mM) among individuals. We also determined that enterotoxigenic Escherichia coli strain H10407 produces 3.3 ± 0.22 mM indole during a 24-h period in the presence of 5 mM tryptophan. The sensitive and specific HIA should be of value in a variety of settings, such as the evaluation of various clinical samples and the study of indole-producing bacterial species in the gut microbiota.

Tryptophan Metabolites And Their Predicted Microbial Sources In Fecal Samples From Healthy Individuals.

Gut microbiota produce tryptophan metabolites (TMs) important to homeostasis. However, measuring TM levels in stool and determining their microbial sources can be difficult. Here, we measured TMs from the indole pathway in fecal samples from 21 healthy adults with the goal to: 1) determine fecal TM concentrations in healthy individuals; 2) link TM levels to bacterial abundance using 16S and whole genome shotgun (WGS) sequencing data; and 3) predict likely bacterial sources of TM production. Within our samples, we identified 151 genera (16S) and 592 bacterial species (WGS). Eight TMs were found in ≥17 fecal samples, including four in all persons. To our knowledge, we are the first to report fecal levels for indole-3-lactate, indole-3-propionate, and 3-indoleacrylate levels in healthy persons. Overall, indole, indole-3-acetate (IAA), and skatole accounted for 86% of the eight TMs measured. Significant correlations were found between seven TMs and 29 bacterial species.  Predicted multiple TM sources support the notion of a complex network of TM production and regulation. Further, the data suggest key roles for Collinsella aerofaciens and IAA, a metabolite reported to maintain intestinal homeostasis through enhanced barrier integrity and anti-inflammatory/antioxidant activities. These findings extend our understanding of TMs and their relationship to the microbial species that act as effectors and/or regulators in the healthy intestine and may lead to novel strategies designed to manipulate tryptophan metabolism to prevent disease and/or restore health to the dysbiotic gut.

Fully resolved assembly of Cryptosporidium parvum.

BACKGROUND: Cryptosporidium parvum is an apicomplexan parasite commonly found across many host species with a global infection prevalence in human populations of 7.6%. Understanding its diversity and genomic makeup can help in fighting established infections and prohibiting further transmission. The basis of every genomic study is a high-quality reference genome that has continuity and completeness, thus enabling comprehensive comparative studies. FINDINGS: Here, we provide a highly accurate and complete reference genome of Cryptosporidium parvum. The assembly is based on Oxford Nanopore reads and was improved using Illumina reads for error correction. We also outline how to evaluate and choose from different assembly methods based on 2 main approaches that can be applied to other Cryptosporidium species. The assembly encompasses 8 chromosomes and includes 13 telomeres that were resolved. Overall, the assembly shows a high completion rate with 98.4% single-copy BUSCO genes. CONCLUSIONS: This high-quality reference genome of a zoonotic IIaA17G2R1 C. parvum subtype isolate provides the basis for subsequent comparative genomic studies across the Cryptosporidium clade. This will enable improved understanding of diversity, functional, and association studies.

Opportunities and Challenges in Developing a Cryptosporidium Controlled Human Infection Model for Testing Antiparasitic Agents.

Cryptosporidiosis is a leading cause of moderate-to-severe diarrhea in low- and middle-income countries, responsible for high mortality in children younger than two years of age, and it is also strongly associated with childhood malnutrition and growth stunting. There is no vaccine for cryptosporidiosis and existing therapeutic options are suboptimal to prevent morbidity and mortality in young children. Recently, novel therapeutic agents have been discovered through high-throughput phenotypic and target-based screening strategies, repurposing malaria hits, etc., and these agents have a promising preclinical in vitro and in vivo anti-Cryptosporidium efficacy. One key step in bringing safe and effective new therapies to young vulnerable children is the establishment of some prospect of direct benefit before initiating pediatric clinical studies. A Cryptosporidium controlled human infection model (CHIM) in healthy adult volunteers can be a robust clinical proof of concept model for evaluating novel therapeutics. CHIM could potentially accelerate the development path to pediatric studies by establishing the safety of a proposed pediatric dosing regimen and documenting preliminary efficacy in adults. We present, here, perspectives regarding the opportunities and perceived challenges with the Cryptosporidium human challenge model.

Prevalence of Clostridium difficile infections among Kenyan children with diarrhea.

BACKGROUND: Diarrhea causes significant morbidity and mortality among children worldwide. Regions most affected by diarrhea include Sub-Saharan Africa and Southeast Asia, where antibiotics are in common use and can make children more vulnerable to Clostridium difficile and pathogens that are not affected by these drugs. Indeed, C. difficile is a major diarrhea-associated pathogen and poses a significant threat to vulnerable and immunocompromised populations. Yet, little is known about the role and epidemiology of C. difficile in diarrhea-associated illness among young children. As a result, C. difficile is often neglected in regions such as Sub-Saharan Africa that are most impacted by childhood diarrhea. The purpose of this study was to establish the frequency of C. difficile in young children (<5 years) with diarrhea. METHODS: Children presenting with diarrhea at a national hospital in Kenya from 2015 to 2018 were enrolled consecutively. Following informed consent by a parent or legal guardian, stool samples were obtained from the children and demographic data were collected. The stools were examined for the presence of four common pathogens known to cause diarrhea: C. difficile, rotavirus, Cryptosporidium parvum, and Giardia lamblia. C. difficile was verified by toxigenic culture and PCR. The presence of C. parvum and/or G. lamblia was determined using the ImmunoCard STAT! Crypto/Giardia Rapid assay. Rotavirus was detected by ELISA. RESULTS: The study population comprised 157 children; 62.4% were male and 37.6% were female and their average age was 12.4 months. Of the 157 stool specimens investigated, 37.6% were positive for C. difficile, 33.8% for rotavirus, 5.1% for Cryptosporidium, and 5.1% for Giardia. PCR analysis identified at least one of the C. difficile-specific - genes (tcdA, tcdB, or tcdC). Further, 57.6% of the stools had C. difficile colonies bearing a frame-shift deletion in the tcdC gene, a mutation associated with increased toxin production. The frequency of C. difficile was 32.6% in children ≤12 months old and increased to 46.6% in children 12-24 months old. CONCLUSIONS: In Kenyan children presenting with diarrhea, C. difficile is more prevalent than rotavirus or Cryptosporidium, two leading causes of childhood diarrhea. These findings underscore the need to better understand the role of C. difficile in children with diarrhea, especially in areas with antibiotic overuse. Understanding C. difficile epidemiology and its relationship to co-infecting pathogens among African children with diarrhea will help in devising ways of reducing diarrhea-associated illness.

Adenovirus 36 antibody detection: Improving the standard serum neutralization assay.

Adenovirus 36 (AdV36) causes weight gain in animal models, including non-human primates. In humans, AdV36-neutralizing antibodies are associated with adiposity; however, longitudinal studies in large populations are needed to clarify AdV36's contribution. The current gold standard for detection of AdV36-specific antibody is the serum neutralization assay (SNA), which requires long incubation times and highly trained personnel. The standard SNA was modified using an immunocytochemical (ICC) approach, which allows for a more rapid and objective assessment of AdV36 antibodies. Using the ICC assay, virus-infected cells were detected as early as day 1 (D1) and by D5 were detected in 100% of microtiter wells versus 20.3% of wells detected by observing the cytopathic effect. Further, human sera tested with the ICC assay at D5 had a sensitivity and specificity of 80.0% and 95.7%, respectively, when compared to the standard SNA read at D11. Thus, the ICC assay decreased assay incubation time, provided a more objective and easily interpreted assessment, and had a high degree of sensitivity and specificity in determining serological status. The more rapid and objective ICC method will make large population studies feasible, improve comparability among laboratories, and contribute to understanding the role of AdV36 in obesity.

Cryptosporidium hominis: experimental challenge of healthy adults.

Cryptosporidium hominis causes diarrhea in humans and has been associated with community outbreaks. This study describes the infectivity, illness, and serologic response after experimental challenge of 21 healthy adult volunteers with 10-500 C. hominis (TU502) oocysts. Sixteen subjects (76.2%) had evidence of infection; the 50% infectious dose (ID(50)) was estimated to be 10-83 oocysts using clinical and microbiologic definitions of infection, respectively. Diarrhea occurred in 40% of subjects receiving 10 oocysts with a stepwise increase to 75% in those receiving 500 oocysts. A serum IgG response was seen in those receiving more than 30 oocysts. Greatest responses were seen in volunteers with diarrhea and oocyst shedding. Volunteers with no evidence of infection had indeterminant or negative IgG responses. Cryptosporidium hominis 10 oocysts) and is clinically is infectious for healthy adults (ID(50) = similar to C. parvum-induced illness. In contrast to C. parvum, C. hominis elicted a serum IgG response in most infected persons.

Cryptosporidium muris: infectivity and illness in healthy adult volunteers.

Although Cryptosporidium parvum and C. hominis cause the majority of human cryptosporidiosis cases, other Cryptosporidium species are also capable of infecting humans, particularly when individuals are immunocompromised. Ten C. muris cases have been reported, primarily in human immunodeficiency virus (HIV) -positive patients with diarrhea. However, asymptomatic cases were reported in two HIV-negative children, and in another case, age and immune status were not described. This study examines the infectivity of C. muris in six healthy adults. Volunteers were challenged with 10(5) C. muris oocysts and monitored for 6 weeks for infection and/or illness. All six patients became infected. Two patients experienced a self-limited diarrheal illness. Total oocysts shed during the study ranged from 6.7 × 10(6) to 4.1 × 10(8), and the number was slightly higher in volunteers with diarrhea (2.8 × 10(8)) than asymptomatic shedders (4.4 × 10(7)). C. muris-infected subjects shed oocysts longer than occurred with other species studied in healthy volunteers. Three volunteers shed oocysts for 7 months. Physical examinations were normal, with no reported recurrence of diarrhea or other gastrointestinal complaints. Two persistent shedders were treated with nitazoxanide, and the infection was resolved. Thus, healthy adults are susceptible to C. muris, which can cause mild diarrhea and result in persistent, asymptomatic infection.

Cryptosporidium virulence determinants--are we there yet?

Exposure to Cryptosporidium parvum in healthy individuals results in transient infection that may be asymptomatic or can result in self-limited diarrhoea. In contrast, acquired immune deficiency syndrome patients with cryptosporidiosis can experience severe manifestations of disease. Volunteer studies have demonstrated that as few as 10 oocysts can cause infection in otherwise healthy adults and that isolates from geographically diverse regions differ in infectivity and, perhaps, virulence. Variability in isolate pathogenicity and infectivity has also been seen in bovine and murine models, respectively. Furthermore, isolate specific differences in protein composition and in host immunoreactivity have been observed. The molecular basis for differences in pathogenicity is not understood. Determining which factors are responsible for host selectivity and for the initiation, establishment, and perpetuation of infection with Cryptosporidium is key to rational drug design and vaccine development. To date, no specific virulence factors have been unequivocally shown to individually cause direct or indirect damage to host tissues nor have mutant strains been produced that could prove that particular deletions result in less virulent strains. Nevertheless, a number of candidate molecules have been identified by immunological and molecular methods. Here, we review the salient characteristics of some of these putative virulence determinants, including molecules that are involved in adhesion, protein degradation and the modulation of the host responses.

Molecular cloning and analysis of the Cryptosporidium parvum aminopeptidase N gene.

Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules found on the surface of sporozoites and merozoites. In this study, we cloned and expressed the C. parvum aminopeptidase N gene by screening a large insert, P1 artificial chromosome library with a probe identified from a Cryptosporidium genome survey-sequencing project. Analysis of the predicted protein encoded by the 2.3 kb gene demonstrated a high degree of homology with prokaryotic and eukaryotic aminopeptidases. The 783 amino acid sequence predicted a M(r) of approximately 89,000. The active site sequence was found to be highly conserved when compared with other Apicomplexan aminopeptidases. Motifs commonly found in aminopeptidases of this class and a unique single Arg-Gly-Asp (RGD) tripeptide motif predictive of cell adhesion were identified. The aminopeptidase N mRNA was expressed in infective sporozoites and during the infection of human HCT-8 enterocytes as revealed by reverse transcription PCR.

Interleukin-8, tumor necrosis factor-alpha, and lactoferrin in immunocompetent hosts with experimental and Brazilian children with acquired cryptosporidiosis.

To examine the importance of intestinal inflammation in the diagnosis and pathogenesis of human cryptosporidiosis, stools of healthy adult volunteers before and after experimental infection were tested for fecal lactoferrin, interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha). Stool samples of Brazilian children with well-defined Cryptosporidium infection, with or without diarrhea, were also tested for IL-8 and TNF-alpha. Only one of the 14 volunteers challenged with Cryptosporidium had increased fecal lactoferrin. However, of 17 stool specimens from children with only Cryptosporidium infection from a previous study, 12 had mild to moderately elevated lactoferrin despite negative work-up for inflammatory enteritides. One of 10 adult volunteers who developed diarrhea with experimental cryptosporidiosis and three of 11 children with cryptosporidiosis and diarrhea had detectable fecal IL-8. The level of TNF-alpha was increased only in one of 14 volunteers and in none of the children. Although considered relatively non-inflammatory. cryptosporidiosis is often associated with mild inflammation, especially in children in an endemic area.

Cryptosporidiosis in children.

Cryptosporidiosis is an important enteric parasitic infection that is associated with significant morbidity and mortality, especially among individuals who are immunosuppressed and infants and children in the developing world. The seroprevalence of this pathogen is high worldwide, suggesting that exposure occurs commonly. The routes of Cryptosporidium spp. transmission are waterborne, food-borne, and occasionally person-to-person. Infected patients can be asymptomatic or develop watery diarrhea and associated enteric symptoms, which are self-limited in immunocompetent persons. In contrast, immunodeficient individuals develop severe, chronic diarrhea that rarely can lead to extra intestinal cryptosporidiosis. Although the diagnosis of Cryptosporidium infection can be established by examining a modified acid-fast stain of stool for the presence of oocysts, enzyme-linked immunoassays are now the diagnostic modalities of choice. Recent clinical trials in pediatric cryptosporidiosis have shown nitazoxanide to be effective therapy.

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi, Encephalitozoon hellem, and Vittaforma corneae.

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.

Cryptosporidium meleagridis: infectivity in healthy adult volunteers.

Most Cryptosporidium infections in humans are caused by C. parvum or C. hominis. However, genotyping techniques have identified infections caused by unusual Cryptosporidium species. Cryptosporidium meleagridis has been identified in ≤ 1% of persons with diarrhea, although prevalence is higher in developing nations. We examined the infectivity of C. meleagridis in healthy adults. Five volunteers were challenged with 10(5) C. meleagridis oocysts and monitored six weeks for fecal oocysts and clinical manifestations. Four volunteers had diarrhea; three had detectable fecal oocysts; and one infected volunteer remained asymptomatic. Fecal DNA from two volunteers was amplified by using a polymerase chain reaction specific for the Cryptosporidium small subunit ribosomal RNA gene. Nucleotide sequence of these amplicons was diagnostic for C. meleagridis. All infections were self-limited; oocysts were cleared within ≤ 12 days of challenge. These studies establish that healthy adults can be infected and become ill from ingestion of C. meleagridis oocysts.

Texas needs physicians trained in public health: a new 4-year integrated MD/MPH degree program.

Traditionally, medicine and public health have not worked as synergistic disciplines because they are based on fundamentally different models. However, a number of very recent imperatives emphasize the need for dual training in these fields to address major public health problems facing society as well as the documented and forecasted workforce shortages. In response to this need, two University of Texas institutions based in San Antonio, Texas, partnered in 2007 to offer a dual 4-year Doctor of Medicine/Master of Public health (MD/MPH) degree program, one of a handful in the nation. Approximately 65 students (or 10% of three consecutive medical school classes) are currently enrolled. The dual-degree program meets the requirements of both degree programs while giving shared MPH credit for relevant courses taken in the medical curriculum and medical school credit for some courses in the public health curriculum. However, 75% of the MPH coursework originates at the School of Public Health. Initial results from focus groups conducted after the first year showed a high degree of student satisfaction, with frequent comments that the program was broadening their perspective on medicine and influencing their career and life goals. A dual MD/MPH degree is an important option for all medical students as a means of addressing pressing health issues in our society through combined training in medicine and the broader areas of prevention and population health. The four-year MD/MPH program, while posing challenges for faculty and students, attracts community- and prevention-minded medical students, reduces training costs (housing/living costs and lost time and wages before entering residency), and allows students to progress with the rest of their class.

Cryptosporidium muris in a Texas canine population.

Molecular technology has led to the discovery of previously unrecognized Cryptosporidium species in new hosts, such as C. canis in humans. The notion that dogs may transmit Cryptosporidium species to humans has significant public health implications, and additional studies are merited. The purpose of this study was to examine a group of kenneled dogs to determine the prevalence of Cryptosporidium species infection and to identify parasite species. Prevalence of active infection was 71%. Six positive samples were analyzed by polymerase chain reaction amplification of the 18S ribosomal RNA gene followed by restriction fragment length polymorphism analysis to identify the Cryptosporidium species. Restriction digest patterns identified C. muris as the infecting species in all six dogs; species identity was confirmed by genetic sequencing. To our knowledge, this is the first report of a naturally occurring C. muris infection in a canine host. The finding of C. muris in asymptomatic canines supports the notion of dogs as potential sources of human infection.

High levels of CXCL10 are produced by intestinal epithelial cells in AIDS patients with active cryptosporidiosis but not after reconstitution of immunity.

Chemokines play key roles in attracting immune cells to sites of infections. However, few data on chemokine expression in the gut during human infections are available. We examined expression of chemokines in intestinal tissues of AIDS patients during active Cryptosporidium infection and during resolution of such an infection. The chemokines and cytokines in cell lysates from jejunal biopsy tissues were assayed by a 22-multiplex bead immunoassay. CXCL10 (IP-10) and its receptor, CXCR3, in sections were studied by immunohistochemistry. In biopsies from AIDS patients with active cryptosporidiosis, four chemokines (CXCL10, CCL11 [eotaxin], CCL5 [RANTES], and CCL2 [monocyte chemoattractant protein 1]) and three cytokines (interleukin-1alpha [IL-1alpha], IL-10, and granulocyte colony-stimulating factor) were detected. The level of CXCL10 was significantly increased in AIDS patients with cryptosporidiosis compared to the level in AIDS patients without cryptosporidiosis or in normal volunteers (median in AIDS patients with cryptosporidiosis, 508 pg/mg protein, compared to 111 pg/mg and 72 pg/mg protein in AIDS patients without cryptosporidiosis and in normal volunteers, respectively [P < 0.05 and P < 0.005, respectively, as determined by a Mann-Whitney test]). The level of CXCL10 correlated with the parasite burden (as measured by the number of Cryptosporidium oocysts in the stools) and also with the IL-1alpha concentration (Pearson correlation values, 0.961 [P < 0.01] and 0.737 [P < 0.05]). As determined by immunohistochemistry, CXCL10 localized to epithelial cells at the site of infection. Following effective antiparasite and antiretroviral therapy, Cryptosporidium infections resolved, and the levels of CXCL10 decreased to normal levels. We hypothesized that CXCL10 plays an important role in the resolution of cryptosporidiosis by attracting immune effector cells to the site of infection. By contrast, in AIDS patients lacking effector cells, CXCL10 may contribute to the immunopathogenesis by recruiting inflammatory cells.