Category Archives: C. diff. Research & Development

Immuron Announced First Patients Enrolled In Phase 1/2 (first-in-human) Clinical Trials For Immuron’s IMM-529 For Treatment of C.difficile Infections

The Australian biopharmaceutical company Immuron announced that the first patients have enrolled in phase 1/2 (first-in-human) clinical trials for Immuron’s IMM-529, an oral immunotherapeutic medication for treatment of Clostridium difficile infections (CDI).

As published in MD Mag February 16, 2018

To review this publication in its entirety please click on the following link to be redirected:

http://www.mdmag.com/medical-news/a-powerful-new-weapon-in-the-fight-against-clostridium-difficile-infection

According to Dan Peres, MD, senior vice president and head of medical development at Immuron, IMM-529 “has shown promise in successfully treating Clostridium-difficile” through its “unique delivery of antibodies.”

If the trials are successful, IMM-529 may be a powerful new weapon in the global fight against CDI. Peres reports that IMM-529 that has been effective in preclinical studies for prophylactic use, treatment of disease, and the prevention of recurrence in relation to CDI, and that the company is excited to enroll the first patients.

The placebo-controlled study to test the safety, tolerability and efficacy of IMM-529 will take place at Hadassah Medical Center in Jerusalem and include 60 CDI diagnosed patients in the 28 day study.

Patients enrolled in the study, led by Yoseph Caraco, MD, head of the clinical pharmacology unit at Hadassah Medical Center, will receive IMM-529 or a placebo 3 times a day during the 28 -day trial period, and be monitored for 2 additional months, determining any recurrence of the disease.

In a statement, Caraco said that he was optimistic about IMM-529 based on pre-clinical trial results and that IMM-529 could “be the answer we’re all looking for” when it comes to treatment of CDI.

IMM-529 targets CDI in 2 ways: by neutralizing toxin B (TcdB), a cytotoxin responsible for inflammation and diarrhea that characterizes CDI, and by binding Clostridium difficile spores and vegetative cells preventing further colonization. Caraco reported that IMM-529 approaches CDI by “targeting the main virulence factors of the disease with only minor disturbance to the natural biome” which could be extremely valuable in treating CDI.

In the earlier pre-clinical proof-of-concept study by led by Dena Lyras, MD, PhD with Monash University in Melbourne, Australia, IMM-529 was shown to be 80% effective in both the treatment of and prevention of CDI without the use of antibiotics.

In a December 2015 statement from Immuron, Lyras stated that she was “excited by the potential of these therapeutics in treating patients with both the acute and the relapse phase, of the disease.”

According to data supplied by the American Gastroenterological Association, approximately 500,000 people in the US are diagnosed with CDI each year, and CDI-associated deaths range from 14,000 to 30,000 per year.

In the European Union, according to a 2016 study led by Alessandro Cassini, MD, with the European Centre for Disease Prevention and Control in Stockholm, Sweden, more than 150,000 cases of hospital-acquired CDI infections (134,053–173,089; 95% CI) occur each year.

According to Immuron, the cost of CDI globally (calculated by CIDRAP, the Center for Infectious Disease and Policy at the University of Minnesota) is an estimated annual economic burden of more than $10 billion and increases in hypervirulent and antibiotic-resistant strains have led to CDI becoming a major medical concern.

Caraco stated that CDI poses “a growing risk amongst a greater population of patients, including those recently treated with antibiotics, the elderly, institutionalized and hospitalized.”

If IMM-529 is found to be safe and effective in clinical trials, it could prove a significant boon to the global fight against CDI at all 3 stages of the disease.

Researchers Utilize Deep Metagenomic Sequencing to Profile FMT ‘s Retracting the Gut Microbiome Features That Coincided With Successful Fecal Transplant Engraftment

A team led by investigators at the Broad Institute have started untangling the bacterial strains that influence successful fecal microbiota transplantation (FMT) engraftment in individuals treated for recurrent Clostridium difficile infection.

As they reported in Cell Host & Microbe today, researchers from the Broad Institute, Massachusetts Institute of Technology, Massachusetts General Hospital, and elsewhere used deep metagenomic sequencing to profile FMT in four FMT donors and 19 recipients with C. difficile infections, retracing the gut microbiome features that coincided with successful fecal transplant engraftment.

The initial gut microbial communities in both the donors and the recipients seemed to influence this process, the team noted, particularly bacterial abundance and strain phylogeny. The final gut microbe composition differed between donors and post-FMT recipients, though, with specific strains that originated in the host either taking hold or falling by the wayside in recipients in an “all-or-nothing” manner.

“This paper provides a context for understanding how to make these live biological therapeutics as an alternative to transferring raw fecal matter,” co-senior author Eric Alm, co-director of MIT’s Center for Microbiome Informatics and Therapeutics, said in a statement.

“We describe a model focused on three elements, including bacterial engraftment, growth, and mechanism of action, that need to be considered when developing these live therapies targeting the gut microorganisms, or microbiome,” added Alm, who is also affiliated with the Broad Institute and Finch Therapeutics.

Along with its use for treating recurrent C. difficile infection, the team noted that FMT has been proposed in other conditions such as inflammatory bowel disease and metabolic syndrome. Even so, there is a ways to go in understanding the factors influencing bacterial engraftment and effectiveness in the recipient gut — information needed to move the approach from a shotgun approach using fecal donor material to microbe-based treatments based on purified collections of specific bacteria.

“Although the success of FMT requires donor bacteria to engraft in the patient’s gut, the forces governing engraftment in humans are unknown,” the authors wrote.

To follow this process, the researchers used the Illumina GAIIx instrument to do deep metagenomic sequencing on seven stool samples from four healthy donors and 67 samples collected over time from 19 individuals treated for C. difficile infection with FMT.

With the help of statistical modeling and a new computational method dubbed Strain Finder, the team looked at the bacterial species that successfully engrafted in FMT recipients and followed strain genotypes over time. It also mapped the metagenomes to Human Microbiome Project reference genomes to take a look at bacterial taxa abundance.

Prior to treatment, for example, FMT recipients had lower-than-usual gut microbiome diversity. And while gut microbial community patterns shifted in recipients after FMT, the resulting gut microbiomes continued to differ from the original donor microbiomes, the researchers reported.

Even so, their analytical methods made it possible predict post-FMR metagenomic operational taxa unit abundance and incidence.

With nearly 1,100 bacterial strains in the 79 samples considered, the team traced transmission of certain strains from FMT donors to recipients, noting that bacterial strains tended to engraft in an “all-or-nothing” manner, “whereby no strains or complete sets of strains colonize the patients.”

“We find that engraftment can be predicted largely from the abundance and phylogeny of bacteria in the donor and the pre-FMT patient,” Alm and co-authors wrote. “Furthermore, donor strains within a species engraft in an all-or-nothing manner and previously undetected strains frequently colonize patients receiving FMT.”

Such patterns were supported by the researchers’ follow-up analyses on 16S ribosomal RNA sequence data for stool samples from 10 more FMT donors and 18 recipients, as well as an analysis of metagenomic sequence data for samples from five individuals treated with FMT for metabolic syndrome.

“Together,” they authors said, “these findings suggest that the principles of engraftment we discovered for recurrent C. difficile infection may generalize to other disease indications, including metabolic syndrome.”

 

To read article in its entirety please click on the following link to be redirected:

https://www.genomeweb.com/sequencing/donor-recipient-strain-analyses-offer-fecal-transplant-engraftment-clues

Da Volterra Shared Results From Phase I Clinical Trial of DAV132 to Prevent Gut Microbiome Disruption Caused by Antibiotics and Prevent C. diff. Infections

DAV132 is a first-in-class product to protect the microbiome during antibiotic treatments and prevent Clostridium difficile infections.

French biotech Da Volterra is developing products to fight the rapidly rising rates of antibiotic resistance. During a Phase I study, the company’s medical device, DAV132, was used with the antibiotic, moxifloxacin, and successfully protected the intestinal microbiota from antibiotic residues. Overall, the product managed to reduce exposure of the microbiota to the antibiotic by 99% and maintain 97.8% of the microbiome’s genetic richness without affecting the drugs therapeutic efficacy.

In a randomized, controlled clinical trial performed in 44 healthy human volunteers, DAV132 was used in association with moxifloxacin, a widely used fluoroquinolone antibiotic. It was demonstrated that DAV132 is able to effectively capture residual antibiotics in the colon and reduce their concentration to very low levels. DAV132 reduced exposure of the intestinal microbiota to moxifloxacin by 99%. Meanwhile the plasma concentration of the antibiotic was essentially unaffected by the co-administration DAV132, meaning that its therapeutic efficacy will be maintained.

The ability of DAV132 to protect the intestinal microbiome was explored by identifying changes in bacterial gene richness as well as a detailed statistical analysis of the evolution of bacterial species throughout the study. In volunteers who received moxifloxacin alone, gene richness was drastically diminished to 54.6% of baseline after antibiotic treatment and failed to return to baseline even one month after treatment; 39% of bacterial species identified in the intestinal microbiota were affected. The co-administration of DAV132 with moxifloxacin largely protected the intestinal microbiome from disruption (97.8% of baseline for bacterial gene richness, and 93% of bacterial species protected).

The primary endpoints for the study were fully achieved and DAV132 showed an excellent tolerability profile.

Annie Ducher MD, Chief Medical Officer of Da Volterra, declared: “This clinical study is indicative of the potential of DAV132 to become one of the first preventative solutions to protect the intestinal microbiome and further avoid the detrimental consequences of antibiotic treatments, such as Clostridium difficile infections, for patients. We look forward to advancing the development of DAV132 in a pivotal patient study in 2018.”

Jean de Gunzburg PhD, Chief Scientific Officer of Da Volterra, added: “This study constitutes the first scientific demonstration of the protection of the intestinal microbiome from dysbiosis caused by a fluoroquinolone antibiotic treatment; our data suggests that this effect should be extendable to many different antibiotics from several therapeutic classes. The metagenomics analysis is outstanding and thoroughly convincing that DAV132 is highly effective at protecting the commensal bacteria in the intestines.”

The results are available under the reference: Gunzburg et al. Protection of the human gut microbiome from antibiotics. The Journal of Infectious Diseases, jix604, https://doi.org/10.1093/infdis/jix604.

***

About DAV132:
With a novel and unique mechanism of action, DAV132 is a product candidate aiming to protect the intestinal microbiome from the side effects of antibiotics, hence preventing the onset of C. difficile infections. DAV132 is an adsorbent with a proprietary coating with colon targeted delivery. DAV132 has been tested in four Phase 1 clinical studies with no adverse safety events. DAV132 is currently entering a Phase 2 clinical trial.

About Da Volterra:
Da Volterra is a biopharmaceutical company based in France that develops new strategies aimed at protecting the intestinal microbiome from the deleterious effects of antibiotics, and preventing multi-resistant and life-threatening infections. Da Volterra’s innovative approaches promise a substantial medical progress to combat deadly pathogens. http://www.davolterra.com

(January 2018)

To read the article in its entirety, please click on the following link to be redirected:

https://www.businesswire.com/news/home/20180116005398/en/Da-Volterra-Announces-Publication-Full-Results-Clinical

Gut Microbiome Research High-resolution Profiling Reveals the Extent of Clostridium difficile (C.diff.) Burden

Microbiome profiling through 16S rRNA gene sequencing has proven to be a valuable tool to characterize the diversity and composition of gut microbial communities, including in studies of CDI development and recurrence.8

 

Authors:

  • Ninalynn Daquigan,
  • Anna Maria Seekatz,
  • K. Leigh Greathouse,
  • Vincent B. Young &
  • James Robert White
Published online:

Abstract

Microbiome profiling through 16S rRNA gene sequence analysis has proven to be a useful research tool in the study of C. difficile infection (CDI); however, CDI microbiome studies typically report results at the genus level or higher, thus precluding identification of this pathogen relative to other members of the gut microbiota.

Accurate identification of C. difficile relative to the overall gut microbiome may be useful in assessments of colonization in research studies or as a prognostic indicator for patients with CDI.

To investigate the burden of C. difficile at the species level relative to the overall gut microbiome, we applied a high-resolution method for 16S rRNA sequence assignment to previously published gut microbiome studies of CDI and other patient populations. We identified C. difficile in 131 of 156 index cases of CDI (average abundance 1.78%), and 18 of 211 healthy controls (average abundance 0.008%).

We further detected substantial levels of C. difficile in a subset of infants that persisted over the first two to 12 months of life. Correlation analysis of C. difficile burden compared to other detected species demonstrated consistent negative associations with C. scindens and multiple Blautia species.

These analyses contribute insight into the relative burden of C. difficile in the gut microbiome for multiple patient populations, and indicate that high-resolution 16S rRNA gene sequence analysis may prove useful in the development and evaluation of new therapies for CDI.

Introduction

Clostridium difficile infection (CDI) poses a major healthcare burden to the global population, with an estimated 450,000 cases and 29,000 deaths in the United States annually.1,2 CDI is often associated with antibiotic treatment and is frequently acquired by patients during hospitalization.

Multiple diagnostic tests for CDI are available and hospitals commonly use a combination of enzyme immunoassay (EIA) and glutamate dehydrogenase (GDH) testing in tandem with real-time polymerase chain reaction (PCR) for increased sensitivity and shorter turnaround time.3

After diagnosis, patients with CDI are typically treated with metronidazole and/or vancomycin depending on symptom severity.3 Treatment failure is estimated to occur in 20% of patients, resulting in a recurrent CDI population that may require other treatment strategies.4,5 The development of microbial-based therapeutics, such as fecal microbiota transplantation (FMT) and combinations of selected microbes for the treatment of recurrent CDI suggests that mixtures of commensal microbes may be routinely utilized in the future as an alternative to powerful antibiotics.6,7

Microbiome profiling through 16S rRNA gene sequencing has proven to be a valuable tool to characterize the diversity and composition of gut microbial communities, including in studies of CDI development and recurrence.8

Given the intricate relationship between the gut microbiota and CDI, accurate                                   identification of C. difficile directly from 16S rRNA profiles in patient populations could be a valuable measure in future studies. However, a fundamental challenge to studying C. difficile through these approaches has been the level of taxonomic resolution provided through short 16S rRNA sequences.

As a result, most microbiome sequencing studies of CDI utilize higher aggregate taxonomic categories (e.g., the Clostridium XI cluster, which encompasses many other organisms related to C. difficile) as a proxy for the organism itself or simply avoid quantification altogether.9,10,11,12,13,14,15,16,17

Here we utilize a high-resolution method (Resphera Insight) for assigning species-level context to 16S rRNA gene sequence data to estimate C. difficile burden in different patient populations. This method was recently validated for detection of Listeria monocytogenes18 and Salmonella enterica,19,20 and was applied in this study to determine the relative abundance of C. difficile in several clinically relevant patient groups. Re-examining published 16S rRNA gene sequence datasets has confirmed previous associations of C. difficile with C. scindens, and identified new positive and negative correlations of C. difficile with other species, both of which may help provide insight into community aspects of C. difficile colonization and resistance against CDI.

Results

Evaluation of sensitivity and specificity for C. difficile identification

One of the challenges of 16S rRNA gene sequencing is the limited information available in these short DNA fragments to distinguish related microbial members below the genus-level. To accurately assess C. difficile at the species level from 16S rRNA gene sequence data, we used a method developed specifically for species level characterization (Resphera Insight, see Methods). We first validated this approach by obtaining full-length 16S rRNA gene sequences from 804 novel C. difficile isolates derived from multiple sources, and subsequently simulated noisy 16S rRNA gene sequence reads for taxonomic assignment (see Methods). Performance was measured using the Diagnostic True Positive Rate (DTP), defined as the percentage of sequences with an unambiguous assignment to C. difficile. The method achieved an average DTP of 99.9% (ranging from 98.92 to 100% per isolate, Table S1), indicating sufficient sensitivity to detect C. difficile from short 16S rRNA gene sequence reads.

In addition to establishing sufficient sensitivity to detect C. difficile, we also sought to evaluate false positive rates in which the method incorrectly assigns a sequence to C. difficile. As this species is a member of the Clostridium XI cluster, a false positive assessment was performed based on in silico simulations of 22 other members of this group, including the very similar Clostridium irregulare. Simulating 10,000 16S rRNA gene sequence reads per species with a 0.5% error rate, 20 of 22 species resulted in zero false positive assignments to C. difficile, with the highest false positive rate (0.07%) attributed to Clostridium irregulare (Table 1).

Table 1: False positive rates for 22 related species

Representation of C. difficile relative to the microbiota in adult cases of CDI and healthy individuals

To examine the presence of C. difficile in different human populations, we re-examined existing published 16S rRNA gene sequencing datasets with our validated method. We first compared the relative abundance of C. difficile across a cohort of healthy individuals to two cohorts of patients diagnosed with CDI (symptomatic index cases) from Seekatz et al.10 and Khanna et al.21 (Table S2). The Seekatz protocol for CDI diagnosis followed a two-stage algorithm employing enzyme immunoassay for GDH antigen and toxins A and B, with confirmation of tcdB gene presence via PCR if toxin and GDH results were discordant; the Khanna et al. protocol for CDI diagnosis was not reported in the original publication. The healthy patient cohort and Seekatz CDI datasets were generated using equivalent processing and sequencing methods.10 Average analyzed sequencing depths per sample for CDI and healthy groups were 16,114 and 14,937, respectively.

Overall, C. difficile was detected in 58 of 70 CDI index patients (82.9%) in the Seekatz study with an average abundance of 3.04% (Fig. 1a). In the Khanna dataset, C. difficile was detected in 73 of 86 CDI index patients (84.9%) with an average abundance of 0.76% (Fig. 1b). Among healthy controls, only 18 of 211 (8.5%) harbored detectable levels of C. difficile, with an average abundance of 0.008%, significantly less than both Seekatz and Khanna index cases (P < 2e-16; Mann–Whitney test).

Fig. 1
Fig. 1

Relative burden of C. difficile in the gut microbiome of two cohorts of CDI index patients and healthy controls. a Index cases of recurrent CDI (Seekatz et al.) and b CDI index patients (Khanna et al.) frequently harbored moderate to high levels of C. difficile. c Healthy controls. Overall 91.5% of controls had no detectable C. difficile and 0.9% maintained C. difficile levels higher than 0.1%

We were further interested in determining whether the ability to detect C. difficile or varying levels of C. difficile relative abundance from 16S rRNA gene sequences was related to disease outcome. The Seekatz dataset included samples collected from patients that went on to develop recurrent CDI, a serious outcome following primary diagnosis, or from patients who were later reinfected with CDI beyond the standard time recurrence window.10 Additionally, a severity score22 was available for some of the patients. Across the full Seekatz CDI positive sample set, our method detected C. difficile above 0.1% abundance in 59.2% of samples (Table 2). On average, patients with CDI for index (at primary diagnosis), recurrence or reinfection events had C. difficile abundances greater than 1% regardless of the calculated severity status using Infectious Diseases Society of America (IDSA) standards. We found no significant associations of C. difficile abundance with IDSA severity status among index samples or at the time of recurrence or reinfection (P > 0.05, Mann–Whitney test).

Table 2: C. difficile relative abundances in cases of CDI from Seekatz et al.10 compared to healthy controls

Representation of C. difficile relative to the microbiota in infants

To assess the levels of C. difficile carriage among infants relative to the total gut microbiome, we re-examined 16S rRNA gene sequence datasets describing longitudinal studies of pre-term infants in the neonatal intensive care unit (NICU) by Zhou et al.23 and a single infant profiled during the first 18 months of life by Davis et al.16 In the Zhao dataset, 12 necrotizing enterocolitis (NEC) cases and 26 age-matched controls (all treated at Brigham and Women’s Hospital NICU, Boston, MA) were sequenced with an average of seven samples per subject. The Davis asymptomatic case study consisted of profiling 50 fecal samples over time, during which researchers noted colonization switching between toxigenic and non-toxigenic strains and observed 100,000-fold fluctuations of C. difficile spore counts.16

In these two 16S rRNA gene sequence datasets, moderate levels of C. difficile (>1.0% abundance) appeared consistently within infants over time. In the Zhao dataset, C. difficile was detected in 25 of 38 (66%) infants, including 6 of 12 (50%) infants with NEC, and 19 of 26 (73%) normal infants. There was no significant difference in overall C. difficile presence between NEC and normal infants (P = 0.27, Fisher’s exact test), and both groups maintained statistically similar C. difficile abundance distributions relative to their total gut microbial communities under multivariate regression after adjustment for patient source (Fig. 2a). As the original Davis case study determined C. difficile carriage using spore counts and GDH concentration, we detected substantial representation of C. difficile (up to 7.1% abundance) until the time of weaning and transition to cow’s milk (Fig. 2b). We further found a statistically significant correlation between our C. difficile relative abundance estimates and GDH concentration measurements from the Davis study (Spearman correlation = 0.817; P = 5e-13).

Fig. 2
Fig. 2

Distribution of C. difficile during longitudinal gut microbiome sampling of infants. a Pre-term infants in a NICU, including those developing necrotizing enterocolitis (purple) and normal (grey). Each boxplot reflects a single patient with multiple time points (total samples per patient shown along the x-axis). b A longitudinal case study of an infant before (red) and after (blue) weaning during the first 18 months of life. During the transition to cow’s milk, C. difficile relative abundance fell to undetectable levels

Correlations of C. difficile with other bacterial species

Recent studies in animal models have indicated that certain species may generate metabolites that inhibit C. difficile, such as the production of secondary bile acids by C. scindens.15 However, previous studies correlating the abundance of C. difficile with other taxa did not utilize the microbiome-based abundances directly, but rather quantified C. difficile abundance through other means such as real-time PCR, colony forming units through culture, measuring GDH concentration or spore counts.15,16,17

We sought to determine whether high-resolution analysis of the 16S rRNA gene sequence data itself could reveal the same associations, and perhaps other relevant species. Computing correlations using Compositionality Corrected by REnormalization and PErmutation (CCREPE)24 across our re-analyzed cohorts, we found a significant negative association between C. difficile and C. scindens for the Khanna CDI patient cohort and the Davis infant longitudinal study (P < 0.02 for both datasets), with a supporting trend in the other studies (Fig. 3, Table S3). Additionally, multiple members of Blautia spp. displayed a consistent negative correlation like that of C. scindens (Fig. 3, Table S3). In contrast, other Clostridia such as C. neonatale and C. paraputrificum and members of Veillonella showed strong positive associations with C. difficile abundance. In silico simulations of noisy 16S rRNA gene sequence reads from these species confirmed a low mis-assignment rate (average 0.08%; see Table S4).

Fig. 3
Fig. 3

Correlation analysis identifies species positively or negatively associated with C. difficile. The CCREPE N-dimensional checkerboard score (y-axis) incorporates the ratio of co-variation to co-exclusion patterns normalized to a range of (−1, +1). In addition to C. scindens, we identify significant negative correlations with C. difficile for members of Blautia and positive correlations with other Clostridia and Veillonella spp. (*P ≤ 0.05). Ambiguous species level assignments are denoted by slashes. Key for re-analyzed datasets from the following studies: Recurrent CDI=10, Index CDI=21, FMT=9, Infant longitudinal=16, NICU=23 (Table S2)

Discussion

In this study, we sought to identify species-level abundances of C. difficile in 16S rRNA gene sequence datasets from different patient populations using a validated algorithm (Resphera Insight). Similar to previous studies of Listeria monocytogenes18 and Salmonella enterica,19,20 validation using a high-resolution taxonomic assignment method from 804 novel C. difficile isolates established an overall sensitivity of 99.9% with a marginal false positive rate less than 0.1%, suggesting that C. difficile could be distinguished from other related microbiota members.

Compared to the microbiota of healthy individuals, we observed a higher presence and relative abundance of C. difficile in microbiota data collected from two CDI patient cohorts. 8.5% of healthy individuals were positive for C. difficile using our approach, supporting previous epidemiological assessments of asymptomatic carriage rates.25,26,27,28 Although analysis of CDI datasets revealed a wide distribution of C. difficile relative abundances (ranging from virtually undetectable to above 50% of total sequences), the relative abundance of detected C. difficile in relation to other members of the microbiota was significantly lower in healthy individuals than that of CDI patients. The ability to assess C. difficile levels as part of the microbiota community is potentially more important within population surveys compared to diagnosis using traditional PCR or GDH/EIA tests that merely account for the presence of C. difficile using toxin B or GDH as a proxy.

While detection of C. difficile from 16S rRNA gene sequence data is limited by sequencing depth, our results suggest that C. difficile does not generally reside in healthy adults. In contrast, we did not detect C. difficile in all patients with CDI. The relative presence of C. difficile in these patients is likely below the detection limit given the available sequencing depth, however some of the samples collected from patients in the Seekatz dataset were collected during antibiotic treatment, thus potentially limiting growth of C. difficile during those time points. Indeed, Seekatz et al. report that they were unable to retrieve C. difficile strains from all patient time points via anaerobic cultivation, generally the gold standard for C. difficile detection and diagnosis.

In a third cohort of 14 recurrent CDI patients receiving fecal microbiota transplantation from nine healthy donors (FMT; Table S2, Fig. 3), C. difficile was less frequently detected than the Seekatz and Khanna index CDI patient groups. Only 4 of 14 FMT patients had any detectable levels of C. difficile before treatment, and 3 of 14 had observations of C. difficile post-FMT. Notably, Resphera Insight detected C. difficile presence in both patients who went on to develop symptomatic CDI post-FMT (recipient IDs 005 and 006).9 Prior to FMT, all patients were treated with vancomycin (125 mg 4× per day) for at least 4 days before and the day of transplantation. Thus, we attribute the reduced detection of C. difficile in this cohort to differences in patient treatment before sampling.

Applying our approach to a longitudinal dataset of 38 premature infants in a single NICU, we identified C. difficile in two-thirds of this patient cohort. Asymptomatic carriage of C. difficile among infants has been observed to be higher than for adults, and it remains unknown whether infant cases of CDI represent true disease.29,30 While CDI testing of infants is not recommended,30 recent epidemiological studies indicate 26% of children hospitalized with CDI are infants under 12 months of age, and 5% are neonates.31 In one study of 753 pediatric patients 0 to 12 years of age, 2.9% of CDI outpatients, 4.6% of CDI inpatients, and 6.6% of healthy controls were positive for C. difficile toxin B.32 Another recent study of C. difficile in 338 healthy infants (<2 yrs) in the United Kingdom found 10% were colonized at enrollment with a toxigenic strain, and 49% became colonized with a toxigenic strain post-enrollment.33 Symptomatic Clostridium difficile infections are believed not to occur in infants due to the expected lack of specific toxin receptors and under-developed signaling pathways in the gut; however, these proposed mechanisms have not been rigorously evaluated in studies of humans.34,35,36 Multiple case studies have argued that CDI can occur in this patient population,36 and there is ongoing debate about the appropriate policy for treatment of symptomatic children who test positive for C. difficile.37,38

Our analysis of an infant case study of asymptomatic colonization during the first 18 months of life identified a reduction in C. difficile relative abundance after abrupt transition from human milk to cow’s milk. Yet in a large longitudinal study by Stoesser and colleagues, multivariate analysis demonstrated that breastfeeding (mixed with formula or exclusively) was protective against asymptomatic C. difficile colonization.33 As noted by Davis and colleagues,16C. difficile does not carry the functional capacity for cleaving monosaccharides from oligosaccharide side chains and thus depends on the generation of monomeric glucose by other commensal members of the gut microbiome.39 Additionally, C. difficile relies on sialic acid as a carbon source for expansion made available by other commensals such as Bifidobacterium species.40 Therefore, the reduction of C. difficile after transition to cow’s milk is potentially the result not of milk source alone, but shifting microbial community composition and the presence of substrates by which C. difficile may thrive.

We were also able to identify a significant negative correlation between the abundance of C. difficile and C. scindens in one of the CDI cohorts, confirming similar trends reported by Buffie et al.15C. scindens, a secondary bile acid producer of deoxycholic acid which has been shown to protect against CDI, may have important translational implications.13,41 New and consistent negative correlations were also identified between C. difficile and multiple species within the Blautia genus including B. faecis, B. luti, B. schinkii, and B. wexlerae. Notably, some members of the Blautia genus are known for 7α-dehydroxylating activity of primary bile acids,42,43,44 however this remains to be evaluated for the species we identified in this study. These data suggest that species other than C. scindens may provide relevant functional capabilities in the context of CDI and prove to be informative in the development of future microbial-based therapeutics. One exception to these findings was the lack of negative correlations identified within the NICU infant cohort, which can be attributed to the very limited observations of these Blautia species and C. scindens in the overall dataset (Table S3). Indeed, among the 322 NICU infant samples analyzed, only B. luti and B. wexlerae were observed at all, and only in 5 (1.6%) and 2 (0.6%) samples, respectively, which precluded their evaluation with the CCREPE method.

While microbiome profiling through 16S rRNA gene sequencing is unlikely to replace existing methods for routine diagnosis of CDI, sequence-based assessment of C. difficile levels in the context of microbiota profiling rather than presence alone may prove valuable in surveillance of C. difficile in patient populations, prediction of disease outcome, or the development of new therapies for CDI. Although our study is limited to 16S rRNA gene-based identification of C. difficile and cannot predict whether a strain produces toxin or carries a functional pathogenicity locus,45 consideration for accurate identification of C. difficile and related members may be useful in assessing clinical outcomes of new microbial therapies that rely on 16S rRNA gene sequencing to validate recovery of the microbiota.

Methods

Validation of Resphera Insight for identification of C. difficile

Whole-genome shotgun sequence datasets available from (i) The Wellcome Trust Sanger Institute and (ii) The University of Maryland Institute for Genome Sciences designated as novel C. difficile isolates were downloaded from the NCBI Sequence Read Archive (see Table S1 for accessions), trimmed for quality using Trimmomatic46 and assembled into contigs using Minia.47 Contigs containing portions of 16S rRNA genes were identified using BLASTN48 and extracted for amplicon simulations. For each isolate, we subsequently simulated 16S rRNA amplicon sequence reads (10,000 per isolate) from the V4 region (the primary amplicon region selected in the real datasets) with a random nucleotide error rate of 0.5%. The Diagnostic True Positive Rate was computed as the percentage of sequences unambiguously assigned by Resphera Insight to C. difficile.

For false positive assessment, simulated V4 sequences were generated from reference 16S rRNA genes for 22 unique species within the Clostridium XI cluster (10,000 per species, 0.5% nucleotide error rate). False positives were defined as unambiguous assignments to C. difficile.


Processing of 16S rRNA gene sequence datasets

Raw 16S rRNA gene sequence datasets were processed as follows: Raw overlapping paired-end reads were merged into consensus fragments by FLASH49 requiring a minimum 20 bp overlap with 5% maximum mismatch density, and subsequently filtered for quality (targeting error rates < 1%) and length (minimum 200 bp) using Trimmomatic46 and QIIME.50 Spurious hits to the PhiX control genome were identified using BLASTN and removed. Sequences were then trimmed of their associated primers, evaluated for chimeras with UCLUST (de novo mode),51 and screened for human-associated contaminants using Bowtie252 searches of NCBI Homo sapiens Annotation Release 106. Mitochondrial contaminants were detected and filtered using the RDP classifier53 with a confidence threshold of 50%, and passing high-quality 16S rRNA gene sequences were subsequently assigned to a high-resolution taxonomic lineage using Resphera Insight (Baltimore, MD).18,19,20,54,55 Briefly, the method relies on (i) a manually curated 16S rRNA gene database including 11,000 unique species and (ii) a hybrid global-local alignment strategy to assign sequences a species-level taxonomic lineage. While the method attempts to achieve species-level resolution, if the internal statistical model indicates uncertainty in final species membership, the tool minimizes false positives by providing “ambiguous assignments” i.e., a list of species reflecting all relevant candidates. For example, if a 16S rRNA gene fragment is ambiguous between Veillonella atypica and Veillonella dispar, the algorithm will provide the ambiguous assignment: “Veillonella_atypica:Veillonella_dispar.”


Statistical analyses

Correlations between C. difficile and other species were computed using CCREPE (v.1.10.0)24 (http://huttenhower.sph.harvard.edu/ccrepe). CCREPE (Compositionality Corrected by REnormalization and PErmutation) utilizes an N-dimensional extension of the checkerboard score particularly suited to similarity score calculations between compositions derived from ecological relative abundance measurements of co-occurrence or co-exclusion. Two sample statistical comparisons utilized the Mann-Whitney U test unless otherwise noted.

In silico evaluation for species identified in CCREPE analysis

For single species reported in CCREPE correlation analysis, we simulated noisy 16S rRNA gene sequences (V4 region; 0.5% error rate; 1000 seqs per species), and calculated the frequency of (1) assignments that included the correct species (allowing for ambiguous assignments), (2) unambiguous assignments to the correct species, and (iii) mis-assignments that did not include the correct species (Table S4).


Ethics approvals and consent to participate

IRB approval and patient consent statements from each study: Recurrent CDI (Seekatz et al.10)—All subjects signed written consent to participate in this study. This study was approved by the University of Michigan Institutional Review Board (Study HUM33286; originally approved 8/26/2009).

Index CDI (Khanna et al.21)—We prospectively recruited 88 patients (median age 52.7 years, interquartile range 36.9–65.1; 60.2% female) with their first CDI episode (from 3/2012–9/2013) as identified from the Clinical Microbiology Laboratory at Mayo Clinic, Rochester, Minnesota and collected an aliquot from the stool samples that led to the diagnosis. Clinical data including demographics, hospitalization status, concomitant medications, CDI severity, laboratory parameters, prior and concomitant antibiotic use, initial CDI treatment, treatment response and recurrent CDI were obtained by a review of the electronic medical record.

NICU Infants (Zhou et al.23)—Samples were collected following a protocol that was approved by the Partner’ s Human Research Committee (IRB) for Brigham and Women’ s Hospital. All study procedures were approved by the IRBs at both Brigham and Women’ s Hospital in Boston, MA and at The Genome Institute in St. Louis, MO. The IRB deemed this study to be of minimal risk with no interaction and no intervention with human subjects and thus, was exempt from consent.

Infant Longitudinal (Davis et al.16)—The study was approved by the TechLab Institutional Review Board and included informed consent obtained from the mother.

FMT (Seekatz et al.9)—Informed consent was received from all participants under an approved Institutional Review Board (IRB) protocol at Essentia Health Duluth Clinic (IRB no. SMDC-09068; principal investigator, Timothy Rubin, FDA Investigational New Drug [IND] no. 15460).

Healthy Controls (Seekatz et al. submitted)—All subjects signed written consent to participate in this study. This study was approved by the University of Michigan Institutional Review Board (Study HUM33286; originally approved 8/26/2009).


Data availability

NCBI BioProject accessions of publicly available 16S rRNA gene sequence datasets used in this study: PRJNA307992, PRJNA342347, PRJNA264177, PRJNA331150, PRJNA238042, and PRJNA386260 (Table S2).

Additional Information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank Cynthia Sears, Karen Carroll, and David Cook for helpful suggestions on this work. This work was supported in part by the ERIN CRC (Enteric Research Investigative Network Cooperative Research Center), (U19AI09087, NIAID), awarded to V.B.Y. A.M.S. supported by the National Center for Advancing Translational Sciences (UL1TR000433).

To review the article in its entirety please click on the following link:

https://www.nature.com/articles/s41522-017-0043-0

Researchers Find Health Care Costs Associated With a Clostridium difficile Infection (CDI) and Recurrent CDI Shows a Significant Increase

 

“This study is consistent with previous literature that has demonstrated a significant and substantial increase in health care resource utilization for CDI over and above similar patients without CDI,” researcher Dongmu Zhang, PhD, of Merck’s Center for Observational and Real-World Evidence, and colleagues wrote. “It has also shown that having rCDI is associated with substantial health care resource use as compared to similar CDI patients who do not have a recurrence.”

To estimate costs and time of hospitalization associated with CDI and rCDI, the researchers conducted a retrospective observational study. They assessed patient records using databases of commercial and Medicare health care claims. Both databases included information on demographics, diagnoses and prescriptions, among other data.

The researchers matched patients without CDI to those with the infection in a 1:1 ratio to estimate costs and lengths of hospital stay due to primary CDI. They then matched patients with primary CDI 1:1 to those with rCDI in a similar comparison. Each patient was followed for 6 months.

The study included records for 55,504 patients diagnosed with CDI between

July 2010 and July 2014.

The mean patient age was 61.3 years,

62% of patients were women.

Nearly a quarter of patients — 24.8% — had rCDI.

The estimated cumulative hospital stays due to CDI and rCDI were 5.2 days and 1.95 days, respectively.

The estimated health care costs due to CDI and rCDI were $24,205 and $10,580, respectively.

Zhang and colleagues said the data show that clinicians must act to control CDI.

“The health care resource utilization and economic burden associated with primary and rCDI are quite substantial,” they wrote. “Better prevention and treatment of CDI, especially rCDI, are needed.” – by Joe Green

 

To read the article in full entirety please click on the following link:

https://www.healio.com/infectious-disease/nosocomial-infections/news/in-the-journals/%7Bce566ea4-98f0-41d3-a8a3-6e0f2125e3dc%7D/cdi-costs-approach-25000-per-patient

Fecal Microbiota Transplantation – Regulatory Harmonization Is Lacking

 

 

 

Abstract

During faecal microbiota transplantation, stool from a healthy donor is transplanted to treat a variety of dysbiosis-associated gut diseases.

Competent authorities are faced with the challenge to provide adequate regulation. Currently, regulatory harmonization is completely lacking and authorities apply non-existing to most stringent requirements.

A regulatory approach for faecal microbiota transplantation could be inserting faecal microbiota transplantation in the gene-, cell- and tissue regulations, including the hospital exemption system in the European Advanced Therapy Medicinal Products regulation, providing a pragmatic and efficacy-risk balanced approach and granting all patients as a matter of principle access to this therapy.

https://www.ncbi.nlm.nih.gov/pubmed/29179687?dopt=Abstract&utm_source=dlvr.it&utm_medium=twitter

C.difficile Study Using C. difficile Conditioned Medium of Six Different C. difficile Strains

 

 

 

 

Abstract

Clostridium difficile infection (CDI) is typically associated with disturbed gut microbiota and changes related to decreased colonization resistance against C. difficile are well described.

However, nothing is known about possible effects of C. difficile on gut microbiota restoration during or after CDI.

In this study, we have mimicked such a situation by using C. difficile conditioned medium of six different C. difficile strains belonging to PCR ribotypes 027 and 014/020 for cultivation of fecal microbiota.

A marked decrease of microbial diversity was observed in conditioned medium of both tested ribotypes. The majority of differences occurred within the phylum Firmicutes, with a general decrease of gut commensals with putative protective functions (i.e. Lactobacillus, Clostridium_XIVa) and an increase in opportunistic pathogens (i.e. Enterococcus). Bacterial populations in conditioned medium differed between the two C. difficile ribotypes, 027 and 014/020 and are likely associated with nutrient availability. Fecal microbiota cultivated in medium conditioned by E. coli, Salmonella Enteritidis or Staphylococcus epidermidis grouped together and was clearly different from microbiota cultivated in C. difficile conditioned medium suggesting that C. difficile effects are specific.

Our results show that the changes observed in microbiota of CDI patients are partially directly influenced by C. difficile.

https://www.ncbi.nlm.nih.gov/pubmed/29180685?dopt=Abstract&utm_source=dlvr.it&utm_medium=twitter