Jhansi L. Leslie

Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era

IMPORTANCE Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision making and public health reporting. Molecular tests are increasingly used to diagnose C difficile infection (CDI), but many molecular test-positive patients lack toxins that historically defined disease, making it unclear if they need treatment. OBJECTIVE To determine the natural history and need for treatment of patients who are toxin immunoassay negative and polymerase chain reaction (PCR) positive (Tox-/PCR+) for CDI. DESIGN, SETTING, AND PARTICIPANTS Prospective observational cohort study at a single academic medical center among 1416 hospitalized adults tested for C difficile toxins 72 hours or longer after admission between December 1, 2010, and October 20, 2012. The analysis was conducted in stages with revisions from April 27, 2013, to January 13, 2015. MAIN OUTCOMES AND MEASURES Patients undergoing C difficile testing were grouped by US Food and Drug Administration-approved toxin and PCR tests as Tox+/PCR+, Tox-/PCR+, or Tox-/PCR-. Toxin results were reported clinically. Polymerase chain reaction results were not reported. The main study outcomes were duration of diarrhea during up to 14 days of treatment, rate of CDI-related complications (ie, colectomy, megacolon, or intensive care unit care) and CDI-related death within 30 days. RESULTS Twenty-one percent (293 of 1416) of hospitalized adults tested for C difficile were positive by PCR, but 44.7% (131 of 293) had toxins detected by the clinical toxin test. At baseline, Tox-/PCR+ patients had lower C difficile bacterial load and less antibiotic exposure, fecal inflammation, and diarrhea than Tox+/PCR+ patients (P < .001 for all). The median duration of diarrhea was shorter in Tox-/PCR+ patients (2 days; interquartile range, 1-4 days) than in Tox+/PCR+ patients (3 days; interquartile range, 1-6 days) (P = .003) and was similar to that in Tox-/PCR- patients (2 days; interquartile range, 1-3 days), despite minimal empirical treatment of Tox-/PCR+ patients. No CDI-related complications occurred in Tox-/PCR+ patients vs 10 complications in Tox+/PCR+ patients (0% vs 7.6%, P < .001). One Tox-/PCR+ patient had recurrent CDI as a contributing factor to death within 30 days vs 11 CDI-related deaths in Tox+/PCR+ patients (0.6% vs 8.4%, P = .001). CONCLUSIONS AND RELEVANCE Among hospitalized adults with suspected CDI, virtually all CDI-related complications and deaths occurred in patients with positive toxin immunoassay test results. Patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without C difficile by either method. Exclusive reliance on molecular tests for CDI diagnosis without tests for toxins or host response is likely to result in overdiagnosis, overtreatment, and increased health care costs.

Persistence and Toxin Production by Clostridium difficile within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function

ABSTRACT Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.

Outcomes in patients tested for Clostridium difficile toxins

Clostridium difficile testing is shifting from toxin detection to C. difficile detection. Yet, up to 60% of patients with C. difficile by culture test negative for toxins and it is unclear whether they are infected or carriers. We reviewed medical records for 7046 inpatients with a C. difficile toxin test from 2005 to 2009 to determine the duration of diarrhea and rate of complications and mortality among toxin-positive (toxin+) and toxin- patients. Overall, toxin- patients had less severe diarrhea, fewer diarrhea days, and lower mortality (P < 0.001, all comparisons) than toxin+ patients. One toxin- patient (n = 1/6121; 0.02%) was diagnosed with pseudomembranous colitis, but there were no complications such as megacolon or colectomy for fulminant CDI among toxin- patients. These data suggest that C. difficile-attributable complications are rare among patients testing negative for C. difficile toxins. More studies are needed to evaluate the clinical significance of C. difficile detection in toxin- patients.

Evaluation of Clostridium difficile Fecal Load and Limit of Detection during a Prospective Comparison of Two Molecular Tests, the illumigene C. difficile and Xpert C. difficile/Epi Tests

ABSTRACT In a large prospective comparison, the illumigene test detected Clostridium difficile in 98% of toxin-positive and 58% of toxin-negative samples confirmed positive by other methods. The Xpert was uniformly sensitive. Most samples with discrepant results had C. difficile concentrations below the illumigene limit of detection. The significance of low-level C. difficile detection needs investigation.

Clostridium difficile Colonizes Alternative Nutrient Niches during Infection across Distinct Murine Gut Microbiomes

Infection by the bacterium Clostridium difficile causes an inflammatory diarrheal disease which can become life threatening and has grown to be the most prevalent nosocomial infection. Susceptibility to C. difficile infection is strongly associated with previous antibiotic treatment, which disrupts the gut microbiota and reduces its ability to prevent colonization. In this study, we demonstrated that C. difficile altered pathogenesis between hosts pretreated with antibiotics from separate classes and exploited different nutrient sources across these environments. Our metabolite score calculation also provides a platform to study nutrient requirements of pathogens during an infection. Our results suggest that C. difficile colonization resistance is mediated by multiple groups of bacteria competing for several subsets of nutrients and could explain why total reintroduction of competitors through fecal microbial transplant currently is the most effective treatment for recurrent CDI. This work could ultimately contribute to the identification of targeted, context-dependent measures that prevent or reduce C. difficile colonization, including pre- and probiotic therapies. ABSTRACT Clostridium difficile is the largest single cause of hospital-acquired infection in the United States. A major risk factor for Clostridium difficile infection (CDI) is prior exposure to antibiotics, as they disrupt the gut bacterial community which protects from C. difficile colonization. Multiple antibiotic classes have been associated with CDI susceptibility, many leading to distinct community structures stemming from variation in bacterial targets of action. These community structures present separate metabolic challenges to C. difficile. Therefore, we hypothesized that the pathogen adapts its physiology to the nutrients within different gut environments. Utilizing an in vivo CDI model, we demonstrated that C. difficile highly colonized ceca of mice pretreated with any of three antibiotics from distinct classes. Levels of C. difficile spore formation and toxin activity varied between animals based on the antibiotic pretreatment. These physiologic processes in C. difficile are partially regulated by environmental nutrient concentrations. To investigate metabolic responses of the bacterium in vivo, we performed transcriptomic analysis of C. difficile from ceca of infected mice across pretreatments. This revealed heterogeneous expression in numerous catabolic pathways for diverse growth substrates. To assess which resources C. difficile exploited, we developed a genome-scale metabolic model with a transcriptome-enabled metabolite scoring algorithm integrating network architecture. This platform identified nutrients that C. difficile used preferentially between pretreatments, which were validated through untargeted mass spectrometry of each microbiome. Our results supported the hypothesis that C. difficile inhabits alternative nutrient niches across cecal microbiomes with increased preference for nitrogen-containing carbon sources, particularly Stickland fermentation substrates and host-derived glycans. IMPORTANCE Infection by the bacterium Clostridium difficile causes an inflammatory diarrheal disease which can become life threatening and has grown to be the most prevalent nosocomial infection. Susceptibility to C. difficile infection is strongly associated with previous antibiotic treatment, which disrupts the gut microbiota and reduces its ability to prevent colonization. In this study, we demonstrated that C. difficile altered pathogenesis between hosts pretreated with antibiotics from separate classes and exploited different nutrient sources across these environments. Our metabolite score calculation also provides a platform to study nutrient requirements of pathogens during an infection. Our results suggest that C. difficile colonization resistance is mediated by multiple groups of bacteria competing for several subsets of nutrients and could explain why total reintroduction of competitors through fecal microbial transplant currently is the most effective treatment for recurrent CDI. This work could ultimately contribute to the identification of targeted, context-dependent measures that prevent or reduce C. difficile colonization, including pre- and probiotic therapies.

The rest of the story: the microbiome and gastrointestinal infections

Bacterial infectious diseases are studied primarily as a host-pathogen dyad. However it is increasingly apparent that the gut microbial community is an important participant in these interactions. The gut microbiota influences bacterial infections in a number of ways, including via bacterial metabolism, stimulation of host immunity and direct bacterial antagonism. This review focuses on recent findings highlighting the interplay between the gastrointestinal microbiota, its host and bacterial pathogens; and emphasizes how these interactions ultimately impact our understanding of infectious diseases.

Erratum: Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: Is quantitation the next step in C. Difficile testing? (European Journal of Clinical Microbiology and Infectious Diseases DOI: 10.1007/s10096-012-1695-6)

All Log C. difficile DNA concentrations reported in the manuscript are incorrectly low by a factor of 10 and, thus, represent the number of C. difficile DNA copies/100uL feces, not DNA copies/mL, as labeled. This includes the Y-axis labels for Figure 1 and the 95 % sensitivity cutoff for prediction of toxin status. The appropriate quantitative cutoff to detect 95 % of toxin positive samples is ≥5.1 Log C. difficile tcdB DNA copies/mL. All other proportions, relationships and conclusions remain accurate and valid. The authors regret any confusion this error may cause.

Clostridium difficile differentially alters the structure and metabolism of distinct cecal microbiomes to promote persistent colonization during infection

Clostridium difficile has become the most common single cause of hospital-acquired infection over the last decade in the United States. Susceptibility is primarily associated with previous exposure to antibiotics, which compromise the structure and function of the gut bacterial community. Furthermore, specific antibiotic classes correlate more strongly with recurrent or persistent C. difficile infection. We used a murine model of infection to explore the effect of distinct antibiotic classes on sustained C. difficile colonization, as well as the impact of infection on community-level gene expression and metabolism 18 hours post-infection. Utilizing untargeted metabolomic analysis, we found that C. difficile infection has larger impacts on the metabolic activity of the microbiota across cefoperazone and streptomycin-pretreated mice, which become persistently colonized compared to clindamycin-pretreated mice that cleared the infection within 8 days. Through metagenome-enabled metatranscriptomics, we observed that the infected microbial communities were enriched in pathways associated with amino acid metabolism and particularly in non-dominant species relative to mock-infected controls. Conversely, in mice pretreated with clindamycin, the effect of infection on the microbiota was only detectable in changes to the community structure but not in metabolic activity or gene expression. Our results suggest that C. difficile is able to restructure the nutrient-niche landscape in certain gut environments in order to promote persistent infection.Susceptibility to Clostridium difficile infection is primarily associated with previous exposure to antibiotics, which compromise the structure and function of the gut bacterial community. Specific antibiotic classes correlate more strongly with recurrent or persistent C. difficile infection. As such, we utilized a mouse model of infection to explore the effect of distinct antibiotic classes on the impact that infection has on community-level transcription and metabolic signatures shortly following pathogen colonization and how those changes may associate with persistence of C. difficile. Untargeted metabolomic analysis revealed that C. difficile infection had significantly larger impacts on the metabolic environment across cefoperazone and streptomycin-pretreated mice, which become persistently colonized compared to clindamycin-pretreated mice where infection quickly became undetectable. Through metagenome-enabled metatranscriptomics we observed that transcripts for genes associated with carbon and energy acquisition were greatly reduced in infected animals, suggesting those niches were instead occupied by C. difficile. Furthermore, the largest changes in transcription were seen in the least abundant species indicating that C. difficile may “attack the loser” in gut environments where sustained infection occurs more readily. Overall, our results suggest that C. difficile is able to restructure the nutrient-niche landscape in the gut to promote persistent infection.