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1 Running Head: REVIEW Collegescribe.com 1 Duodenal Infusion of Donor Feces for Recurrent C. difficile Name University

2 REVIEW Collegescribe.com 2 Clostridium difficile is a colon infection that is acquired after extensive antimicrobial use and the disruption of normal intestinal flora (Harrisons, 2008, p. 818). It is an anaerobic, gram positive, spore-forming bacillus. Infection occurs after ingesting spores that colonize the gut and cause diarrhea and pseudomembranous colitis. Infection rates are >20% among adults hospitalized for more than one week. Traditionally, therapy has revolved around the use of vancomycin, metronidazole, nitazoxanide, or rifaximin as monotherapy or in combination to eliminate the disease, however, in a substantial number of patients, treatment fails and there is disease recurrence (Brandt, Borody, Campbell, 2011). In a study performed by van Nood, et al. (2013), a randomized, controlled clinical trial, the researchers assessed the efficacy of fecal transplantation compared to traditional antibiotic therapy as a treatment protocol for the disease. Theory and Hypothesis Formulation In the brief introduction to the study, the authors are effective at detailing the relevance and importance of their research. Namely, that antibiotic treatment fails to produce a successful response in nearly twenty-five percent of patients who undergo therapy. Furthermore, they outline that treatment has diminishing returns for recurrences and that the efficacy of treatment is sixty percent following the first recurrence with less success in each additional recurrence. Several mechanisms of the infection have been proposed which include persistence of the spores and reductions in commensal gut flora with each round of antibiosis. The authors point out that there were reports of fecal transplantation being effective in more than three hundred patients but that the experience of the treatment protocol is hindered by the lack of randomized clinical trials and the unappealing

3 REVIEW Collegescribe.com 3 nature of the therapy, however, there was no literature review that was explicitly highlighted in the article. The claim to three hundred patients being treated with this method was based on six articles describing case series of patients treated with fecal transplant. The sources cited had a wide range of publication years, the earliest two were from 2000 and 2003, and the remaining four were from after Bakken, J. in his review of the literature associated with fecal therapy found 100 cases reported between 1958 and 2008 (2009). Considering the growth of the problem due to increased antibiotic resistance and the increase in academic literature, it is surprising that the authors were only able to identify approximately three hundred cases some five years after Bakken s review. However, this does not substantially affect their research because of the valid assertion of a lack of a controlled clinical trial that assessed the suitability of this method compared with standard therapies. Furthermore, it would seem that the authors underutilized their own sources, as the 1958 report cited in the Bakken article was reference number 31 in this article. In total, there were 32 articles referenced by the authors in the article. Given the publication year of 2013, references published after were unlikely to have been available to the authors at the time of writing. A summary of the references, by year of publication, is presented in Table 1. Reference publication date Number of studies cited and earlier 5 Table 1: Summary of references in used by van Nood, et al. The concepts were very well defined, to the point that a layperson could likely pick up the article and understand the purpose of the experiment and the importance of

4 REVIEW Collegescribe.com 4 the problem. C. difficile infection was defined as diarrhea ( 3 loose or watery stools per day for at least 2 consecutive days or 8 loose stools in 48 hours) as well as a stool test showing c. difficile toxin with the isolates characterized by PCR ribotyping. The hypothesis was not explicitly stated, however it was rather obvious and flowed logically from the discussion of the problem that they sought to address. Effectively, the hypothesis was that fecal transplantation is more effective than antibiotics and employable in the treatment of clostridium difficile infections. It was assumed, based on their available literature that fecal transplant therapy would result in a cure rate of 90% and that antibiotic therapy would result in a cure rate of approximately 60%. It is clear that a controlled trial of the effectiveness of fecal transplant versus antibiotic therapy will contribute greatly to the advancement of clinical medicine. Firstly, antibiotic therapy for the problem is hit and miss to begin with. Some patients respond, however, a large number of patients do not respond to this therapy and therefore if there is another possibility for treatment of the problem, it would be of great benefit to the substantial numbers of patients who develop the infections while they are in hospital. Secondly, the use of antibiotics, in and of itself, is part of the problem. The increasing use of more powerful antibiotics is directly linked to the increasing incidence of c. difficile infections in hospitals in the western world. If a therapeutic methodology that restricted their use was developed, tested, and proven efficacious, a great leap forward would be had in the battle to limit the spread of anti-bacterial resistant diseases. Methods The methods section of the article by van Nood, et al. (2013), was divided into seven easily digestible headings. Their scheme is replicated in this critique.

5 REVIEW Collegescribe.com 5 Measurement The independent variables of the experiment were the treatments received; either 500 mg of oral vancomycin with bowel lavage followed by an infusion of donor feces, a course of standard vancomycin therapy over a fourteen-day period, or a standard vancomycin regimen with a bowel lavage on day four or five. The dependent variable was the c. difficile infective status of the patients. Another dependent variable was the fecal microbiota of the patient. The controlled variables of the experiment were the frequency and size of the dosing of vancomycin that the patients received, as well as the size of the lavage that they underwent when appropriate. The dependent variable of cure status was well operationalized as follows: cure without relapse within 10 weeks of initiation of therapy. Cure was defined as the absence of diarrhea or if diarrhea was present that it could be explained by other factors and three consecutive negative tests for c. difficile toxins. The validity of the measurements is beyond dispute. The definition of disease is a clinical one based on the absence of diarrhea and the absence of c. difficile toxin recovered from the stool samples. The authors failed to mention which method they used in the detection of the c. difficile toxin however, in the vast majority of the patients (39 of 43), the toxin test was confirmed by growing the anaerobe in culture. Furthermore, PCR analysis was done on 34 of the patients. It is safe to assume that the toxin was detected using ELISA testing which has a sensitivity of 90% and a specificity of 100% (Deshpande, et al., 2011). Using the adjudication committee and stool analysis for determination of the c. difficile status of the patient has face validity. Content validity is achieved by the very simple and elegant study design. Patients either have c. difficile

6 REVIEW Collegescribe.com 6 infections or they don t and the testing was clearly able to determine the narrow range of possibilities. The measures have criterion validity insofar as the negative toxin test is also backed up by the patients self-report of symptoms (the lack of diarrhea). Finally, construct validity is realized because searching for the c. difficile toxin in feces is a direct indication of infection as it is 100% specific. The feces of patients was also analyzed for microbiotia diversity. The method used was the HITChip (human intestinal tract chip) phylogenetic microarray. The HITChip uses over 4800 oligonucleotide probes and is able to detect 1140 phylotypes and quantify species as low as 0.1% of the total bacterial load (Rajilic-Stojanovic, et al., 2009). Like the presumed ELISA analysis for c. difficile toxin, this method has a high degree of validity. The method employs detection of the small subunit of rrna and its reproducibility is 99.9% and is thus very reliable (Rajilic-Stojanovic, et al., 2009). No direct evidence of validity and reliability of the variable measurements are given. However, the evidence is self-evident and easily quantifiable using the methods that were described. C. difficile infection causes pseudomembranous colitis, which in turn leads to severe diarrhea. The absence of diarrhea is easy to gauge using the provided definition of disease. Furthermore, the use of the HITChip method for determining the presence of normal gut flora is also valid and reliable given the well documented functions of the system (Rajilic-Stojanovic, et al., 2009; Paliy & Agans, 2012). Sample The study was performed at the Academic Medical Center of Amsterdam. Physicians conducting the study who performed the randomization visited patients admitted to referring hospitals. Participants provided written informed consent and the

7 REVIEW Collegescribe.com 7 research was approved by the ethics committee of the hospital. Patients admitted to the study were at least eighteen years old, had a life expectancy of at least three months, and had a relapsed c. difficile infection after at least one appropriate course of antibiotic therapy. Patients were excluded if they were immunocompromised for a number of reasons, prolonged use of steroids, pregnancy, use of antibiotics for treatment of anything except the c. difficle infection, admission to an ICU, or the need for vasopressor medication. In all, a total of 43 patients were randomly assigned into the three groups. 17 received the infusion of donor feces, 13 participants received a vancomycin regimen, and 13 received the vancomycin regimen plus a bowel lavage. Most patients in the control groups had relapses of the disease and therefore the monitoring board, during an interim efficacy analysis, advised termination of the trial. One of the patients from the original 43 was subsequently excluded from analysis due to his nephrologist objecting to the treatment protocol due to a deteriorating renal transplant. Another patient from the vancyomycin only group was discharged home and discontinued all medication at his own initiative because of heart failure and COPD and he died soon thereafter without providing data for the analysis. The base line demographics of the patient group are provided in table 2.

8 REVIEW Collegescribe.com 8 Table 2: Baseline demographic and clinical characteristics of the patients The sample was very appropriate for the research question at hand. The nature of c. difficile is that it is very hard to eradicate and highly likely to relapse (Kelly, de Leon & Jasutkar, 2011). Furthermore, the exclusion criteria made it possible to limit the confounding effects of other diseases and treatment protocols on the insult to gut flora. The absence of normal gut flora is what allows for the colonization of c. difficile, and with other treatments it is possible that any benefits to the gut flora derived from the fecal transplant would be compromised by continuing treatment for other pathologies. Design The study was described as an open-label, randomized, controlled trial and a summary was provided in the report and is shown in figure 1. The design was appropriate to the goals of the study. The population of interest was patients suffering from c. difficile infections in the hospital setting. The authors

9 REVIEW Collegescribe.com 9 believed that at least 38 people per group would be needed to reach a power of 80% to detect a difference between treatment groups with a one-sided level of significance of They had thus planned to recruit 40 patients per group due to the potential dropouts from the study. The study was ended early according to the Haybittle-Peto rule due to the obvious benefit the fecal donor group had in their therapy (P<0.001 for the cure end point), and the rate ratios were calculated to 99.9% confidence intervals. Two control groups were used in the study, which were the vancomycin treatment course groups. Until now, Vancomycin was considered the gold standard of therapeutic approaches to the treatment of c. difficile infections. The efficacy of fecal transplant above and beyond the use of vancomycin was what was being tested. 77 candidates for donor feces were reviewed and of them, 25 were selected. Feces from 15 donors was used for a total of 43 infusions conducted on the experimental group and for patients who relapsed with the vancomycin treatment. On average, 141 grams of feces was infused and the mean time from defecation to infusion was 3.1 ± 1.9 hours. Donors were volunteers younger than 60 years old and were screened for a variety of transmissible diseases including viral, bacterial, and patristic infections. Screening was repeated every four months and prior to donation another questionnaire was used to screen for recent illnesses. The independent variable, treatment protocol, consisted of three levels: donorfeces infusion, vancomycin only, and vancomycin with bowel lavage. The dependent variables were presence of c. difficile infection, and population of microbiota. The presence of c. difficile infection was measured daily by the patient in a diary, and clinically by physicians on days 7,14,21,35 and 70. The other dependent variable,

10 REVIEW Collegescribe.com 10 population of fecal microbiota, was not consistently measured in the study. Only 8 patients that underwent the fecal-transplant procedure completed before and after microbiota measurements. The nature of the study, using patients already diagnosed with refractory c.difficile and given the dynamism of the clinical medicine, it is difficult to conceptualize. Interventions occurred during the post-test phase of the exam. An attempt to simplify it is provided in table 3. Figure 1: Graphic summary of study design The method of randomization used in the experiment was ambiguous and the report just states that the participants underwent randomization. Comparability of the groups was easily established insofar as the experiment s primary endpoint was freedom from disease for 10 weeks in the control and variable groups.

11 REVIEW Collegescribe.com 11 Table 3: Alternate summary of design. There are no manifestly obvious threats to the internal validity of the study. There is no ambiguity in the temporal order of treatment of the patients, no substantial attrition was noted in any group and instrumentality was consistent throughout assessment. The baseline demographic information provided in table 1 shows that the three groups were very similar and that there were no statistically significant differences among them. In fact, the age of the fecal donor recipients was slightly older, and the particularly virulent strain o27 was slightly more common in the group, and in theory, harder to treat. Similarly, there were no obvious threats to the external validity of the study. The co-morbidities of the patients were well accounted for in the exclusion criteria and the results are well transferable and the study easily replicated. No attempt was made to explain other potential explanations of the findings as this was a very well designed experiment testing a very narrow hypothesis. Data Analysis and Deduction The primary endpoint was cure without relapse within 10 weeks of therapy. The small number of patients in the infusion group that underwent a second infusion were given an additional ten weeks following that infusion. Secondary endpoint was cure without relapse after five weeks. As stated before, cure was defined as an absence of diarrhea, or if there was diarrhea, it was explainable by some other cause and there were three consecutive stool tests negative for the c. difficile toxin. The adjudication

12 REVIEW Collegescribe.com 12 committee that decided if an individual was cured of disease were not aware of the group status of the patient and thus the experiment evaluators were blind and unlikely to be biased. The patients themselves kept a stool diary and were questioned about the frequency and consistency of their bowel habits, additional medications, and adverse effects on days 7, 14, 21, 35, and 70 after the initiation of the respective protocols. Tests for the c. difficile toxin occurred on days 14, 21, 35, 70, and whenever there was an occurrence of diarrhea. Also noted above was the use of the HITChip phylogenetic array that was used to estimate the diversity of intestinal flora before and after the infusion of donor feces. The experimenters required 38 patients per group to achieve a power of 80% to detect a difference between the groups with a one-sided level of significance of The statistical analysis was performed with a modified intention-to-treat basis with the exception of one of the patients who was excluded due to a required high-dose of prednisolone after randomization but prior to initiation. The differences in the cure rates were evaluated using Fisher s exact probability test. The significance of the change in patient gut flora was assessed with paired-samples Student t-test on the basis of Simpson s Reciprocal Index. Finally, Wilcoxon signed-rank tests were conducted with the use of the Benjamin-Hochberg approach to determine which microbial groups were significantly different in matched pairs of fecal samples obtained before and after infusion of feces. The statistical methodologies performed in the study were appropriate to the subject matter. Power represents the probability of identifying a difference between groups when one genuinely exists and therefore an avoidance of a type II error and

13 REVIEW Collegescribe.com 13 incorrect rejection of the null hypothesis (Whitley & Ball, 2002). The results using a level of significance of are admirable and ultimately shows an accurate rejection of the null hypothesis based on the data collected. The use of intention to treat paradigm is consistent with clinical experimental practice. It requires that patients final results are compared within the groups that they were randomized to regardless of the having received the assigned treatment, dropped out, or having violated the initial protocol. It analyzes the patient based on the treatment arm he was assigned to and nothing else. This method allows for pragmatic evaluation of the benefits of a new treatment (Soares & Vaz Carneiro, 2002). van Nood, et al. (2013), wisely state in their methods that they would be using a modified intention to treat basis, as many researchers violate the methodology with modifications that aren t explicitly disclosed (Soares & Vaz Carneiro, 2002). The use of Fisher s exact test is also appropriate since the data is of a categorical type. It is the commonly used analysis in smaller data sets and shows how much deviation from the null hypothesis there is in the data. The gut flora was appropriately measured with the matched pair student t-test as the patients gut flora was tested before and after the infusion of donor feces and the results of the comparison are shown in figure 2. The use of the Wilcoxon signed- rank test was a little bizarre in the present study due to the fact that the researchers had already employed a student t- test which essentially looks for the same thing, that is, the differences in the populations being examined in a matched pair analysis. However, its use in combination with the Benjamin- Hochberg approach lends statistical power to the analysis because of the

14 REVIEW Collegescribe.com 14 fact that multiple comparisons were being made (i.e. fecal infusion vis a vis vancomycin and vancomycin plus bowel lavage.) Figure 2: Patient gut flora The study hypothesis was very well tested and accurately reported in the article and the results were both practically and statistically very significant insofar as the Haybittle-Peto rule was implemented and that p<0.001 and confidence interval were calculated to 99.9%. The graph of the results is reproduced below as figure 3. As previously stated, extraneous variables were very well controlled through the use of the exclusion criteria that limited the confounding effects of treatments for co-morbidities. The treatments were delivered as intended by the study design and ultimately showed that compared to vancomycin alone, donor-feces infusion had a cure rate ratio of Compared to vancomycin and bowel lavage, donor-feces infusion had a cure rate ration of 4.05 with 99.9% confidence interval in both groups. The median time for relapse of disease was 23 days and 25 days respectively for the two vancomycin groups. Five weeks

15 REVIEW Collegescribe.com 15 after therapy there was a relapse in one patient in the donor-feces group, versus 8/13 in the vancomycin group and 7/13 in the combined vancomycin bowel lavage group. The average level of diversity of gut flora in patients prior to donor-feces infusion was 57, and within two weeks of infusion increased to 179 which was indistinguishable from the microbiota diversity of the donors (mean = 172). Principal component analysis was performed on the phylogenetic microarray profiles of each sample and this showed that nearly half the variation in gut flora could be explained by the first two principal components, which showed a major shift in the patients gut flora. The authors noted that they also thought that c. difficile infection is associated with low level of gut bacteria and that the phyla Firmicutes and Bacteroidetes were particularly important and highly associated with c. difficile infection. Figure 3 Percentage cured without relapse of disease Inferences

16 REVIEW Collegescribe.com 16 The results clearly provided support for the hypothesis that recipients of donorfeces treatment would have a cure rate of approximately 90%. After two infusions with donor feces, 15 of 16 patients were successfully treated and did not have relapse c. difficile infection 10 weeks after the therapy. This amounted to a cure rate of 93.8%. On the other hand, the authors assumed that treatment with vancomycin would cure approximately 60% of the relapsed c. difficile cases. In fact, vancomycin was only able to successfully cure 30.8% of patients, and those with bowel lavage fared even worse with a cure rate of 23.1%. The results were clearly stated and displayed as is shown in figure 3, which was reproduced from the study. In research undertaken to validate the therapy modality, Brandt, et al. (2012), discovered that outcomes appear to be very good and resolution of symptoms often happens within 3 days of therapy and the primary cure rate was 91%. The conclusions were fully consistent with the empirical findings namely that the treatment protocol involving fecal-donor transplantation was much more effective than a treatment protocol for c. difficile relying exclusively on methods of antibiosis. The authors noted several limitations to their study including the lack of patients from the intensive care unit, where c. difficile infections are particularly frequent and hazardous, and they excluded patients with extensive histories of immunodeficiency. An error that the authors note is that the power calculation they relied on was based on vancomycin therapy for a first occurrence of the disease. In fact, most patients had several relapses of infection before they were included in the study protocol and thus the efficacy of vancomycin therapy was lower than what they had originally predicted. The final limitation of the study that was noted was that another protocol for c. difficile treatment

17 REVIEW Collegescribe.com 17 includes a long tapering schedule of antibiotics. The authors did not incorporate this protocol because they felt it was too time consuming, however, they note that several participants had previously been on this tapered therapy to no avail. After the study was ended due to the clear efficacy of donor-fecal transplant over vancomycin treatment, which was visible very early on, the protocol was extended offlabel to an additional 18 patients who had originally been assigned to the antibiotic arms of the study. These results are very generalizable to other populations and the authors note that there is anecdotal evidence of this method being used to treat ICU patients as well. There is no reason to think that these findings would not be applicable to other populations. This research may also be transferable to other treatments or theories. For example, Shen, Obin, & Zhao have linked microbiota to obesity and insulin resistance (2013). This treatment method perhaps could be replicated in the treatment of obesity and perhaps obesity with all it s concomitant problems can also be solved with the eradication of gut flora and the implantation of a healthier nosocomial bacteria profile. Finally, in their discussion the authors seem to understand clearly the importance of their research and do not confuse the statistical significance with the practical significance of their work. These results are well integrated in the stream of scientific knowledge. They conform to a wide number of case-series on the subject that already showed the efficacy of fecal-donor transplantation for patients suffering from c. difficile infections. The authors seem to be aware of the major impact that their experiments will have on clinical practice in the coming years, but seem a little coy with the details. However, their final paragraph sums up the impact of their research succinctly when they state that infusion

18 REVIEW Collegescribe.com 18 of donor feces, as compared with vancomycin therapy, resulted in better treatment outcomes. Perhaps this was a little understated, but the impact is clear on clinical practice. There remains to be seen several practical elements to the use of the donor-feces in the treatment of c. difficile infections. Firstly, it would be useful to learn if the only method of administration of the donor-feces is through nasao-gastric tube or if it can be delivered through other means such as enema or colonoscopy. Furthermore, it would be interesting to see if the development of bacterial colonies separated from the fecal medium would improve the willingness of patients to undertake this therapy since the idea of ingesting feces seems to be a limiting factor in the uptake of this therapeutic regimen (Ettinger, Burton & Reid, 2013). In sum, this was a fascinating article that established an evidenced based foundation for the use of transplanted donor feces in the treatment of c. difficile infections. Sometimes, the writing was not explicitly clear and the structure of the article appeared to be falling apart as things seemed to be repeated and poorly placed under their headings. Furthermore there is an occasional lack of complete information such as not explicitly stating what method for stool toxin detection was used or explicitly outlining the randomization process that was used. However, the writer s style was ultimately simple enough that the message was easily understood, and the project interesting enough to maintain interest and readership through the end of the article. The researchers took a concept that had been floating around in case studies for some sixty years and finally applied a controlled clinical trial to measure the efficacy of the treatment concept. The experiment performed was simple, elegant, and very relevant to modern clinical practice

19 REVIEW Collegescribe.com 19 given the fact that c. difficile infections are increasing in number and severity throughout the western world. By finally running an experiment on this simple treatment method, they have revolutionized critical care and medicine.

20 REVIEW Collegescribe.com 20 Works Cited: Bakken, J.S. (2009). Fecal bacteriotherapy for recurrent Clostridium difficile infection. Anaerobe, 15, doi: /j.anaerobe Brandt, L.J., et al. (2012). Long-Term Follow-Up of Colonscopic Fecal Microbiota Transplant for Recurrent Clostridium difficile Infection. The American Journal of Gastroenterology, 107, doi: /ajg Deshpande, A., et al. (2011). Repeat Stool Testing to Diagnose Clostridium difficile Infection Using Enzyme Immunoassay Does Not Increase Diagnostic Yield. Clinical Gastroenterology and Hepatology, 9, doi: /j.cgh Ettinger, G., Burton, J.P., Reid, G. (2013) If microbial ecosystem therapy can change your life, what s the problem? Bioessays. DOI /bies Gerdine, D.N., Johnson, S. (2008). Clostridium difficile-associated Disease, Including Pseduomembranous Colitis. In S. Fauci, D. Kasper, D. Longo, E. Braunwald, S. Hauser, J.L. Jameson, & J. Loscalzo (Eds.), Harrison s Principles of Internal Medicine (17 th ed.) (pp ). New York: McGraw Hill Medical Kelly, C.R., de Leon, L., Jasutkar, N. (2011). Fecal Microbiota Transplantation for Relapsing Clostridium difficile Infection in 26 Patients. Journal of Clinical Gastrenterology, 46(2), van Nood, E., et al. (2013). Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile. The New England Journal of Medicine. DOI: /NEJMoa Paliy, O., Agans, R. (2011). Application of phylogenetic microarrays to interrogation of human microbiota. Microbiology Ecology, 79, DOI: /j x Rajilic-Stojanovic, M., et al. (2009). Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults. Environmental Microbiology, 11(7), doi: /j x. Shen, J., Obin, M.S., Zhao, L. (2013). The gut microbiota, obesity and insulin resistance. Molecular Aspects of Medicine, 34, Soares, I., Vaz-Carneiro, A. (2002). Intention-to-Treat Analysis in Clinical Trials: Principles and Practical Importance. Rev Port Cardiol, 21(10),

21 REVIEW Collegescribe.com 21 Whitley, E., Ball, J. (2002). Statistics review 4: Sample size calculations. Critical Care, 6(4),