We designed an open, per-protocol, two-arm pilot trial using oral vancomycin (250mg every 6 h for 10–14 days) or FMT-FURM as an alternative treatment for the first CDI episode among hospitalized patients. Patients were included in the study from February 2015 to October 2015 in Hospital Universitario “Dr. Jose Eleuterio Gonzalez” in Monterrey, Mexico; a 450-bed teaching hospital with an average of 22,000 annual discharges. Patients’ cases were reviewed and evaluated by the study investigators and research coordinators. Adult patients, i.e., over 18 years old, who had been hospitalized for any cause and diagnosed with a first CDI episode >48 h after admission were included. None of the patients had a history of CDI or had received prior treatment for the current CDI episode. CDI cases were defined as follows: > 3 bowel movements during the prior 24 h, a Bristol scale > 5, positive test results for C. difficile toxins A/B detected by either immunoassay (Meridian immunocard C. difficile toxins A/B) or real-time PCR (Cepheid Xpert C. difficile/Epi, Cepheid, Sunnyvale CA) in accordance with the manufacturers´ instructions. Patients with a toxic megacolon, suspected or documented intestinal perforation, pregnancy, or the concomitant presence of colon neoplasms were excluded.
Xpert c difficile epi
Manufactured by Cepheid
Sourced in United States
The Xpert C. difficile/Epi is a molecular diagnostic test that detects the presence of Clostridioides difficile (C. diff) and the binary toxin gene associated with increased disease severity in a single test. The test is designed to provide rapid and accurate results to assist healthcare professionals in the diagnosis and management of C. diff infections.
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9 protocols using xpert c difficile epi
1
Pilot Trial of Vancomycin vs. FMT-FURM for First CDI
2
Clostridium difficile Recurrence and FMT
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Recurrence of CDI was defined as patients with a primary diagnosis ≥3 loose stools and positive test results for toxins using the ImmunoCard toxins A&B assay (Meridian Bioscience, Cincinnati, OH, USA) or real-time PCR (Cepheid Xpert C. difficile/Epi, Cepheid, Sunnyvale, CA) followed by an adequate response to treatment (absence of diarrhea, leukocytosis, and abdominal pain at the end of treatment) and developed new diarrhea (≤3 loose stools) within 8 weeks after treatment.
CDI resolution was considered by the absence of diarrhea, leukocytosis, and abdominal pain at the end of treatment. A new FMT was administrated if after 72 h from the first dose, the patient had an inadequate clinical response deemed as a reduction of less than 50% bowel movements and failure to improve consistency of stool.
CDI resolution was considered by the absence of diarrhea, leukocytosis, and abdominal pain at the end of treatment. A new FMT was administrated if after 72 h from the first dose, the patient had an inadequate clinical response deemed as a reduction of less than 50% bowel movements and failure to improve consistency of stool.
3
Perirectal Swabbing for Clostridium difficile Detection
4
Rapid Detection of Toxigenic Clostridium difficile
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Clinical stool samples were collected from patients with diarrhea at Zhejiang Provincial People’s Hospital between August 1 and 30 December 2020. Liquid, soft, or semi-solid stool samples of sufficient volume were stored at −80°C and transported to Hangzhou Medical College within 48 h for further testing. Each stool sample was divided into two aliquots (1 mL per each), one was analyzed using the CQ100 system, and the other was analyzed by Xpert C. difficile/Epi (Cepheid, Sunnyvale, CA, United States). Toxigenic culture (TC) was used as the reference method for evaluation of these two assays as previously described (Neuendorf et al., 2016 (link)).
Stool samples were thawed to room temperature (20°C) and genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN), according to the manufacturer’s instructions. The toxin A (tcdA) and toxin B gene (tcdB) of C. difficile were selected as target genes, and sequences were obtained from GenBank. The primers and probes were designed using DNASTAR V5 (DNASTAR, Madison, WI, United States), and the specificity of the primers and probes was verified using the NCBI Primer BLAST database. The primer and probe sequences are listed inSupplementary Table S1 . All sequences were synthesized by General Biosystems (Anhui) Co., Ltd.
Stool samples were thawed to room temperature (20°C) and genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN), according to the manufacturer’s instructions. The toxin A (tcdA) and toxin B gene (tcdB) of C. difficile were selected as target genes, and sequences were obtained from GenBank. The primers and probes were designed using DNASTAR V5 (DNASTAR, Madison, WI, United States), and the specificity of the primers and probes was verified using the NCBI Primer BLAST database. The primer and probe sequences are listed in
5
Hospital-Acquired Clostridium difficile Infection
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Patients included in the study were adults with a suspected diagnosis of CDI who reported ≥3 bowel movements with loose stools (Bristol 5–7) in the preceding 24 h. For CDI confirmation at least one of the following tests had to be positive: detection of toxins using the ImmunoCard toxins A&B assay (Meridian Bioscience, Cincinnati, OH, USA) in stools, real-time PCR (Cepheid Xpert C. difficile/Epi, Cepheid, Sunnyvale CA) in stools, or an endoscopic image consistent with pseudomembranous colitis. All hospitals used the same diagnostic methods with the exception of GEA and INCAN where no PCR-testing was performed. At the time of the study, none of the hospitals used glutamate deshidrogenase test (GDH).
CDI was classified as hospital-onset healthcare facility-associated CDI (HO-HCFA) when patients had been in-hospital for at least 48 h and were CDI free at admission, or as community-onset health care facility-associated (CO-HCFA) when patients were hospitalized for at least 48 h during the previous 12 weeks at CDI onset.11 (link) Data on community-acquired CDI was very limited, with no cases included. Treatment failure was defined as persistence of diarrhea after five days of treatment.12
CDI was classified as hospital-onset healthcare facility-associated CDI (HO-HCFA) when patients had been in-hospital for at least 48 h and were CDI free at admission, or as community-onset health care facility-associated (CO-HCFA) when patients were hospitalized for at least 48 h during the previous 12 weeks at CDI onset.11 (link) Data on community-acquired CDI was very limited, with no cases included. Treatment failure was defined as persistence of diarrhea after five days of treatment.12
6
Retrospective Study of CDI Outcomes
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We performed a retrospective review of hospitalized patients with a diagnosis of CDI between January 1, 2011 to September 30, 2015, who underwent abdominal CT within 72 h of diagnosis. The study was performed at the University Hospital “Dr. José Eleuterio González”, a 450-bed tertiary care hospital in Monterrey, Mexico.
Patients included were 18 years or older, and the diagnosis was determined by the Immunocard toxins A&B assay (Meridian Bioscience, Cincinnati, OH, USA), positive PCR (Cepheid XpertC. difficile/Epi) or presence of pseudomembranous colitis on colonoscopy.
Clinical data was collected from the time of diagnosis and throughout hospitalization. The primary outcome was defined as fulminant colitis with colectomy, and the secondary outcome was all-cause mortality within 30 days of diagnosis.
Patients included were 18 years or older, and the diagnosis was determined by the Immunocard toxins A&B assay (Meridian Bioscience, Cincinnati, OH, USA), positive PCR (Cepheid XpertC. difficile/Epi) or presence of pseudomembranous colitis on colonoscopy.
Clinical data was collected from the time of diagnosis and throughout hospitalization. The primary outcome was defined as fulminant colitis with colectomy, and the secondary outcome was all-cause mortality within 30 days of diagnosis.
7
Retrospective C. difficile Diagnostic Testing
8
Detecting C. difficile Toxins via PCR
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Starting in April 2014, all stool samples were tested for C. difficile toxins using real-time polymerase chain reaction (PCR) (Cepheid Xpert C. difficile/Epi, Cepheid, Sunnyvale CA) to identify toxin-producing C. difficile strains, including strain NAP1/B1/027. Prior to the availability of PCR-based diagnostic approaches all diarrhea specimens were tested by enzyme immunoassay (Meridian Bioscience, Cincinnati, OH, USA). All positive specimens were saved for future testing. All stool specimens were stored at 4° C for five days, and then frozen at −70° C. After PCR retesting, only positive samples were included in the final analysis.
9
Whole Genome Sequencing of C. difficile
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Patients’ stools were tested for CDI using a two-step testing algorithm which includes GDH screening by ELISA (Diasorin LIAISON® C. difficile GDH) followed by toxin testing (Cepheid Xpert® C. difficile/Epi) from outpatient physician offices. C. difficile isolates were selected on cycloserine cefoxitin fructose agar (CCFA) and species confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Genomic DNA was extracted for whole genome sequencing using an in-house modified protocol from the QIAamp DNA Mini Extraction Kit (Qiagen, USA). Colonies were re-suspended in 0.9% saline to a McFarland standard of 2.4–3.0 and subsequently centrifuged at 13,000 rpm (rpm) on a tabletop centrifuge for one minute with supernatant discarded. The remaining pellet was re-suspended in 180 μL of enzyme solution (20 mg/mL lysozyme, 20 mM Tris-HCl, 2 mM EDTA and 1.2% Triton X). The solution was then incubated for 30 min at 37 °C until clearing of the solution was noted. Twenty μL of reconstituted Proteinase K solution (Sigma-Aldrich, USA) was added with a further incubation at 56 °C overnight. The remainder of the protocol followed instructions as denoted in the manufacturer’s protocol. The final pellet was re-suspended in 50 μl DNase/RNase free water.
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