FMT for Recurrent C. Difficile Infection Using Frozen Stool
Background Faecal microbial transplant (FMT) for recurrent Clostridium difficile infection (rCDI) is greatly facilitated by frozen stool banks. However, the effect of frozen storage of stool for greater than 2 months on the viability of stool bacteria is unknown and the efficacy of FMT is not clear.
Aim To evaluate the viability of bacteria in stool frozen for up to 6 months, and the clinical efficacy of FMT with stool frozen for 2–10 months, for the treatment of rCDI.
Methods Viability of six representative groups of faecal bacteria after 2 and 6 months of storage at −80 °C, in normal saline (NS) or 10% glycerol were assessed by culture on plate media. The clinical outcomes of 16 consecutive patients with rCDI treated with aliquots of stool frozen in 10% glycerol and stored for 2–10 months were also examined.
Results Viability at both 2 and 6 months was similar to baseline, in specimens stored in 10% glycerol and at 2 months in stool stored in NS, but was reduced by >1 log at 6 months for Aerobes (P < 0.01), total Coliforms (P < 0.01) and Lactobacilli (P < 0.01) in NS. Using stool frozen for 2–10 months in 10% glycerol, the cure rate for rCDI was 88% with one FMT and 100% after repeat FMT in those who relapsed.
Conclusion Stool for faecal microbial transplant to treat rCDI can be safely stored frozen in 10% glycerol for at least 6 months without loss of clinical efficacy or viability in the six bacterial groups tested.
Clostridium difficile is a spore forming gram-positive bacillus that is a common cause of health care associated diarrhoea, particularly in elderly or debilitated patients.Clostridium difficile infection (CDI) is thought to result from a diminished indigenous colonic flora, particularly after the use of broad spectrum antibiotics, that allows C. difficile to proliferate into the resultant ecological void. Recurrent Clostridium difficile infection (rCDI) is an increasing problem and hypervirulent strains have emerged resulting in increased morbidity and mortality. Faecal microbiota transplant (FMT) has become the standard of care for patients with rCDI as a result of randomised control trial evidence of its superiority to traditional antibiotic therapy alone. It is therefore now incumbent upon hospitals to establish FMT services so that rCDI can be effectively managed with this new therapy.
While many facilities use fresh stool from donors known to the recipients, there are a number of issues making this practice problematic. First, there are ethical concerns regarding coercion as well as confidentiality concerns in screening known donors in the event that pre-existing undeclared disease is found in a donor or transmitted to the recipient. There is also evidence from blood transfusion safety analyses that recipient-directed donors are more likely to test positive for infectious disease than unrelated volunteer donors. Stringent exclusion criteria can be more easily and dispassionately applied to volunteer donors from the community than recipient directed donors as there are a greater number of potential candidates and no perceived personal obligation between donors, recipients and healthcare workers. Last, donor recruitment and testing is labour intensive and costly and by using frozen stool bank with anonymous volunteers the process becomes more economical. Up to six treatments can be produced from each stool donation and suitable donors can give multiple samples over a short period of time.
Treating FMT donation in a similar way to blood banking with pre-screened, anonynmised donors, addresses many of the issues with fresh and/or recipient directed donors. The development of a frozen stool bank is the most efficient and reliable way to standardise the donor stool processing and screening and allows stool to be available for the clinician to use on demand. The precise elements of donor stool that determine the success of FMT are not known. A determinant of the success of a frozen stool bank may be the viability of the bacteria within the frozen stool specimens. There have also been suggestions that success may be attributable to nontoxigenic clostridial spores in the donor samples. There is evidence that FMT using stool frozen for less than 2 month is clinically as effective as fresh samples for rCDI and the successful use of stool frozen for up to 5 months has been reported. However, the concurrent clinical efficacy and bacterial viability of stool frozen for periods substantially greater than 2 months has not been reported to date.
Glycerol is commonly used as a cryoprotective agent for frozen faecal samples, however, it is not known for how long stool can be frozen and continue to deliver viable bacteria. We therefore examined the viability of six culturable bacterial populations within stool stored for 2 and 6 months in two different storage media; normal saline (0.9% sodium chloride) and a NS and 10% glycerol mix. In addition, we reviewed our prospectively maintained FMT for rCDI database for patients who received stool that had been in frozen storage in 10% glycerol for >2 months to assess its clinical effectiveness.
Abstract and Introduction
Abstract
Background Faecal microbial transplant (FMT) for recurrent Clostridium difficile infection (rCDI) is greatly facilitated by frozen stool banks. However, the effect of frozen storage of stool for greater than 2 months on the viability of stool bacteria is unknown and the efficacy of FMT is not clear.
Aim To evaluate the viability of bacteria in stool frozen for up to 6 months, and the clinical efficacy of FMT with stool frozen for 2–10 months, for the treatment of rCDI.
Methods Viability of six representative groups of faecal bacteria after 2 and 6 months of storage at −80 °C, in normal saline (NS) or 10% glycerol were assessed by culture on plate media. The clinical outcomes of 16 consecutive patients with rCDI treated with aliquots of stool frozen in 10% glycerol and stored for 2–10 months were also examined.
Results Viability at both 2 and 6 months was similar to baseline, in specimens stored in 10% glycerol and at 2 months in stool stored in NS, but was reduced by >1 log at 6 months for Aerobes (P < 0.01), total Coliforms (P < 0.01) and Lactobacilli (P < 0.01) in NS. Using stool frozen for 2–10 months in 10% glycerol, the cure rate for rCDI was 88% with one FMT and 100% after repeat FMT in those who relapsed.
Conclusion Stool for faecal microbial transplant to treat rCDI can be safely stored frozen in 10% glycerol for at least 6 months without loss of clinical efficacy or viability in the six bacterial groups tested.
Introduction
Clostridium difficile is a spore forming gram-positive bacillus that is a common cause of health care associated diarrhoea, particularly in elderly or debilitated patients.Clostridium difficile infection (CDI) is thought to result from a diminished indigenous colonic flora, particularly after the use of broad spectrum antibiotics, that allows C. difficile to proliferate into the resultant ecological void. Recurrent Clostridium difficile infection (rCDI) is an increasing problem and hypervirulent strains have emerged resulting in increased morbidity and mortality. Faecal microbiota transplant (FMT) has become the standard of care for patients with rCDI as a result of randomised control trial evidence of its superiority to traditional antibiotic therapy alone. It is therefore now incumbent upon hospitals to establish FMT services so that rCDI can be effectively managed with this new therapy.
While many facilities use fresh stool from donors known to the recipients, there are a number of issues making this practice problematic. First, there are ethical concerns regarding coercion as well as confidentiality concerns in screening known donors in the event that pre-existing undeclared disease is found in a donor or transmitted to the recipient. There is also evidence from blood transfusion safety analyses that recipient-directed donors are more likely to test positive for infectious disease than unrelated volunteer donors. Stringent exclusion criteria can be more easily and dispassionately applied to volunteer donors from the community than recipient directed donors as there are a greater number of potential candidates and no perceived personal obligation between donors, recipients and healthcare workers. Last, donor recruitment and testing is labour intensive and costly and by using frozen stool bank with anonymous volunteers the process becomes more economical. Up to six treatments can be produced from each stool donation and suitable donors can give multiple samples over a short period of time.
Treating FMT donation in a similar way to blood banking with pre-screened, anonynmised donors, addresses many of the issues with fresh and/or recipient directed donors. The development of a frozen stool bank is the most efficient and reliable way to standardise the donor stool processing and screening and allows stool to be available for the clinician to use on demand. The precise elements of donor stool that determine the success of FMT are not known. A determinant of the success of a frozen stool bank may be the viability of the bacteria within the frozen stool specimens. There have also been suggestions that success may be attributable to nontoxigenic clostridial spores in the donor samples. There is evidence that FMT using stool frozen for less than 2 month is clinically as effective as fresh samples for rCDI and the successful use of stool frozen for up to 5 months has been reported. However, the concurrent clinical efficacy and bacterial viability of stool frozen for periods substantially greater than 2 months has not been reported to date.
Glycerol is commonly used as a cryoprotective agent for frozen faecal samples, however, it is not known for how long stool can be frozen and continue to deliver viable bacteria. We therefore examined the viability of six culturable bacterial populations within stool stored for 2 and 6 months in two different storage media; normal saline (0.9% sodium chloride) and a NS and 10% glycerol mix. In addition, we reviewed our prospectively maintained FMT for rCDI database for patients who received stool that had been in frozen storage in 10% glycerol for >2 months to assess its clinical effectiveness.
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