Introduction

Adverse outcomes of Clostridioides difficile infection (CDI) are derived from toxin presence, thus the gold standard has focused on cell cytotoxin assays (1,2). However, quantitative polymerase chain reaction’s (qPCR) cycle threshold values linked to genomic load may be significantly associated with disease outcome, aiding in the distinction between causative pathogens, coinfections and asymptomatic carriage (3). Genes encoding toxins A/B (tcdA/tcdB) are usually targeted by PCR diagnostics (3). While most virulent strains produce them simultaneously, both alone can also cause CDI (2,4,5). These genes are located in the genome within the Pathogenicity loci (PaLoc), which can be horizontally transferred (4,6). Recent studies have also discovered extrachromosomal molecules carrying tcdB (7). This suggests that the number of cells does not necessarily correlate to the number of toxin genes.

Aims & Methods

The aim was to develop a reliable qPCR method to quantify C. difficile toxin A/B genes to compare them to bacterial load by targeting a house-keeping gene (adK). Standard curves were validated according to official guidelines and reference publications (efficiency E=90-110%; slope S=(-3.1)-(-3.6); linearity R²≥0.98) (8–11), testing 10 replicates by 9 consecutive 10-fold dilutions starting from copies/mL of 3 quantified dsDNA gBlock stocks. The linear range was validated running 5 dilution points ( to copies/mL) in triplicate. The last detected dilution inside the linear range with a coefficient of variation ≤25% was considered limit of quantification. Precision, repeatability and reproducibility were evaluated. Commercially supplied C. difficile isolates and retrospective clinical samples confirmed to be C. difficile positive were quantified.

Results

All standard curve assays developed showed acceptable qPCR efficiency and qPCR linearity (E=96-103%; S=(-3.2)-(-3.4); R²≥0.99). Four commercially supplied C. difficile isolates and 14 clinical samples were tested with all 3 validated methods. For each sample, concentrations resulted in the same order of magnitude but different total numbers for all assays (adK to copies/mL; tcdA to copies/mL; tcdB to copies/mL), except for three samples that presented one order of magnitude less tcdA than adK. In general, adK gene copies resulted in higher numbers than the toxins, with tcdB more concentrated than tcdA.

Conclusion

Three reliable qPCR standard curve quantification methods were successfully developed and validated to allow for the detection of toxigenic C. difficile and quantification of bacterial load. Results indicate that the number of copies for each gene is not the same within one single sample. This raises doubts regarding CDI, such as the possibility of harbouring both toxigenic and nontoxigenic strains (PaLoc negative) within one single patient, which would explain the higher concentration of adK gene copies in samples. Since multiple copies of the PaLoc have not been described, a higher concentration of tcdB compared to tcdA could suggest either the existence of some tcdA-tcdB+ organisms or of multiple copies of tcdB+ extrachromosomal molecules, the percentage of which might have been underestimated. Further studies would be required to elucidate the relevance and implications that these findings might have to CDI diagnosis and treatment.

References

1. Garvey MI, et al. Antimicrob Resist Infect Control. 2017 Dec;6(1):127. doi: 10.1186/s13756-017-0283-z
2. Seugendo M, et al. Front Microbiol. 2018 Aug 7;9:1843. doi: 10.3389/fmicb.2018.01843
3. Bonacorsi S, et al. Front Med. 2021 Sep 23;8:711809. doi: 10.3389/fmed.2021.711809
4. Orrell KE, Melnyk RA. Microbiol Mol Biol Rev. 2021 Aug 18;85(3):e00064-21. doi: 10.1128/MMBR.00064-21
5. Marvaud JC, et al. New Microbes New Infect. 2019 Nov;32:100590. doi: 10.1016/j.nmni.2019.100590
6. Di Bella S, et al. Toxins. 2016 May 3;8(5):134. doi: 10.3390/toxins8050134
7. Smits WK, et al. Curr Opin Microbiol. 2022 Feb;65:87–94. doi: 10.1016/j.mib.2021.10.016
8. National Committee for Clinical Laboratory Standards. Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline, EP6-A. 2nd ed. Vol. 23 No. 16. 2003.
9. Broeders S, et al. Trends Food Sci Technol. 2014 Jun;37(2):115–26. doi: 10.1016/j.tifs.2014.03.008
10. Bustin SA, et al. Clin Chem. 2009 Apr 1;55(4):611–22. doi: 10.1373/clinchem.2008.112797
11. Forootan A, et al. Biomol Detect Quantif. 2017 Jun;12:1–6. doi: 10.1016/j.bdq.2017.04.001

Disclosure

QIAGEN provided support in the form of salaries for some authors: RSA, NC, LR, SH, KV, DL, FVD, CCF and LP.

Introduction

Clostridium difficile infection (CDI) presents a complex challenge in healthcare, particularly within hospitals and long-term care facilities where the use of antibiotics is usually unavoidable. Understanding the factors that contribute to CDI recurrence is crucial for effective treatment and management.

Aims & Methods

This study aimed to assess possible risk factors contributing to the recurrence of CDI. We performed a retrospective analysis of 107 adults diagnosed with CDI who were managed in the tertiary care center during 2018-2020, focusing on variables including demographics, comorbidities, clinical features, history of prior antibiotic and glucocorticoid treatments.

Results

Overall, this study included 63 (58.9%) females, with a median age for all patients of 77 years (IQR 68–82). The cohort comprised a diverse array of comorbid conditions: inflammatory bowel disease (3.7%), diabetes mellitus (10.3%), chronic kidney disease (CKD) (15.9%), with 4 (23.5%) patients requiring haemodialysis, and oncological diseases (21.5%). Obesity/overweight was noted in 28 (26.2%) patients, and a history of stroke was present in 14 (13.1%). Nursing care was required for 41 (38.3%) individuals, while dementia symptoms were reported in 31 (29 %) cases. There was a common history of prior hospitalization (65.4%), glucocorticoids were used by 14 (13.1%) patients, proton pump inhibitors (PPI) by 23 (21.5%) subjects, and 18 (16.8%) patients had surgeries within 6 months before hospitalization. Antibiotics were administered to 48.6% of patients before the onset of CDI.
During hospitalization, 4 (3.7%) patients died: 3 of whom had oncological history, 2 of them worsened after chemotherapy, and other 2 patients experienced progressive heart failure. Comparative analysis between patients with a first-time CDI diagnosis (n = 39), who all remained recurrence-free over a 3-year follow-up, and those with recurrences (n = 64), revealed that recurrence was more common in females (73.3% vs. 46.5%, p=0.006), while no significant age differences were observed. Neither surgical history (p = 0.974) nor PPI usage (p = 0.232) demonstrated a significant influence on CDI recurrence. Moreover, diabetes, oncological history, and CKD did not significantly impact CDI recurrence rates (p = 0.405, p = 0.078, and p = 0.974) either. In contrast, a significant correlation was found between dementia and increased CDI recurrence: 83.3% of patients with dementia experienced recurrence versus 53.4% without dementia (p = 0.004). While patients with a history of stroke and those requiring nursing care had higher rates of recurrence, these trends were not statistically significant (p = 0.533 and p = 0.065, respectively).

Conclusion

While many clinical and demographic factors were assessed, only female sex and dementia showed a significant influence on CDI recurrence. These findings suggest that among the array of potential risk factors, cognitive impairment presents a unique challenge in the management and prevention of CDI recurrence.

Introduction

The CRISPR/Cas9 system has emerged as a revolutionary tool for genome editing, enabling researchers to precisely modify genes of interest. However, achieving high knockout efficiency remains a challenge, especially in patient-derived organoid models. These models resemble in vivo conditions more accurately but are not well amenable to lipofection. In this study, we propose an innovative strategy to enhance the knockout efficiency of CRISPR/Cas9 ribonucleoproteins (RNPs) by co-targeting hypoxanthine-guanine phosphoribosyltransferase (HPRT), a key enzyme in the purine salvage pathway and subsequently treating with 6-thioguanine. Here, we demonstrate that this co-knockout strategy can significantly increase knockout efficiency of a gene of interest in patient-derived colorectal cancer (CRC) organoids.

Aims & Methods

HPRT is an enzyme involved in the salvage pathway of purine nucleotide synthesis. The anticancer agent, 6-thioguanine, relies on HPRT to be metabolized into cytotoxic 6-thioguanine nucleotides. Subsequent incorporation of these nucleotides into DNA and RNA mediates its cytotoxic effect. It has been previously shown in 2D cell lines that the functional significance of HPRT in the purine salvage pathway can be exploited to enhance the knockout efficiency of a gene of interest using CRISPR/Cas9: by co-targeting HPRT alongside the gene of interest, HPRT-deficient cells are unable to metabolize 6-thioguanine and thus become resistant to its cytotoxic effects. Consequently, 6-thioguanine can be used as a selective agent, enabling the enrichment of cells with HPRT KO together with the desired knockout. We have successfully redesigned and optimized this method for use in CRC organoids.

Results

We have established an easy-to-scale 384-well CRISPR/Cas9 RNP transfection platform to generate high-efficiency gene knockouts (KO) in adherent cell lines. Depending on the individual sgRNA used, we were able to achieve a genome editing efficiency of 40-60 percent 48 hours after transfection. We show that this editing efficiency can be further increased by co-targeting HPRT with CRISPR RNP transfection and subsequently treating with 6-thioguanine. In the effort to extend this approach to CRC organoids, we optimized electroporation conditions with CRISPR RNPs and sgRNA produced via in vitro transcription. We demonstrate high knockout efficiencies of more than 40 percent in FACS analysis after knockout of surface markers CD81, CD46 & CD151 with two sgRNAs, respectively. In Sanger sequencing genotyping (analysis with Synthego ICE CRISPR analysis tool), we were able to achieve knockout efficiencies of 20 percent at individual guide target sites.

Conclusion

Here, we report an innovative method for generating high-efficiency CRISPR/Cas9 RNP knockout pools in patient-derived CRC organoids. We intend to apply this method to verify and further investigate the knockout effects of specific genes of interest identified in arrayed CRISPR RNP screens conducted using our 384-well RNP transfection platform in adherent CRC cell lines. Furthermore, our method is particularly valuable for generating knockout organoids without the need for lentiviral transduction. Of note, CRISPR RNPs offer the advantage of quicker editing and lower off-target rates when compared with DNA-based CRISPR delivery methods. High-efficiency knockouts in patient-derived organoids without the need for lentiviral transduction are essential for developing new targeted therapies for colorectal cancer.

Introduction

Clostridioides difficile infection (CDI) is among of the most common causes of nosocomial diarrhea with high morbidity and mortality. Recurrent and refractory disease courses in particular represent a special challenge in the management of these patients. The identification of patients at risk for recurrent courses could improve management, e.g. through the use of new and effective therapies in patients with CDI.

Aims & Methods

The aim of this study was to investigate the relationship between the presence of different comorbidities and the course of CDI.
In a retrospective, monocentric cohort study of a tertiary centre over a period of 10 years, patients with CDI-related hospitalizations were examined. The study included 336 patients with available information on comorbidities (diverticulosis, biliary diseases (cholecysto-, docholithiasis, cholecystitis, cholangitis or cholangiectasis) inflammatory bowel diseases (IBD), liver cirrhosis and malignancies) and the diagnosis of the recurrent CDI (rCDI) vs. non-recurrent course of CDI (nrCDI). Patients on dialysis or immunosuppressed patients were also considered as comorbid.

Results

Among 336 patients included in the study, 227 (67.6%) presented with at least one comorbidity. The most common were malignancy (30.4%), biliary diseases (19.4%), diverticulosis (15.8%), liver cirrhosis (14.3%), and inflammatory bowel disease (IBD, 8.9%). rCDI occurred more frequently in patients with diverticulosis and biliary disease. Diverticulosis was present in 24.4% of rCDI cases vs. 12.6% in nrCDI cases (OR 2.24, 95% CI 1.22–4.17). Biliary diseases were observed in 26.7% of rCDI vs. 16.7% of nrCDI patients (OR 1.82, 95% CI 1.01–3.26). No significant associations with rCDI were found for IBD (OR 0.66, 95% CI 0.27–1.65), malignancy (OR 1.13, 95% CI 0.67–1.88), or liver cirrhosis (OR 0.79, 95% CI 0.38–1.61).

Conclusion

These findings suggest diverticulosis and biliary disease may be independent risk factors for rCDI, whereas other common comorbidities are not significantly associated with recurrence. Structured assessment of risk conditions may help to select patients for tailored management to prevent rCDI disease course.

Introduction

Faecal microbiota transplantation (FMT) has emerged as a promising therapy for patients with Clostridioides difficile infection (CDI) unresponsive to standard medical therapy. However, data on long-term efficacy and safety are scarce, particularly regarding to real-world data.

Aims & Methods

A single-centre retrospective study was performed on patients treated with FMT for recurrent or refractory CDI. FMT was done according to the institutional protocol and stool material was provided by the same donor, after a careful clinical and analytical workup. The clinical registries of patients with at least six months of follow-up were retrieved to document sustained efficacy, potential adverse events, and antibiotic exposure.

Results

Since June 2014, 47 patients (63.0% female; median age 78.5, interquartile range 69-84) received 54 FMT infusions for CDI (92.6% delivered through esophagogastroscopy). Twenty-three patients had recurrent CDI while 24 had refractory infection. Eight patients were immunosuppressed with either corticosteroids or chemotherapy and 43 had prior antibiotic exposure. The median follow-up duration was 27.5 months (6-105). Short-term efficacy, defined as resolution of diarrhoea without recurrence of CDI within 2 months, was 91,4% (in three cases more than one TMF administration was required), with CDI symptoms resuming after 1.5±0.7 days. Only one late recurrence was registered 7 months after FMT, in a patient that received, once again, broad-spectrum antibiotics. No adverse events were reported during the follow-up.

Conclusion

As far as the authors know, this is the largest cohort of FMT for CDI in a Portuguese hospital. This real-world study demonstrated the high effectiveness of FMT for recurrent and refractory CDI, both in the short- and long-term, with a good safety profile. The 16S rRNA sequencing of our “super donor” microbiome is underway.