The Efficacy of Bezlotoxumab in the Prevention of Recurrent Clostridium difficile: A Systematic Review

Clostridium difficile infection (CDI) is the most common nosocomial infection in hospitals. Despite the fact that CDI has treatment options, recurrence is common after the treatment, recurrence will occur in approximately 20%-35% of people initially affected, with 40%-60% of these having a second recurrence. Patients are more likely to have several recurrences after the second, which can lead to antibiotic overuse, and as a result, CDI-related health care expenses, hospitalizations, and mortality are on the rise. The first treatment to receive Food and Drug Administration (FDA) approval for the prevention of C. difficile recurrence is bezlotoxumab, a novel human monoclonal antibody against C. difficile toxin B. In the present systematic review, we assessed various studies from PubMed, PubMed Central (PMC), Google Scholar, and Science direct that evaluated the efficacy of bezlotoxumab in the prevention of recurrent C. difficile (rCDI), and we also briefly discussed the pathophysiology of C. difficile and the risk factors for recurrence of C. difficile. The major MODIFY trial has proven the efficacy, pooled analysis of MODIFY 1 AND 2 trials demonstrated the following results as compared to placebo (bezlotoxumab: 129/781 [16.5] placebo:206/773 [26.6] -10.0% [95% CI -14.0 to -6.0], p<0.0001) with number needed to treat (NNT) of 10. All other observational studies also showed a positive response with bezlotoxumab in the prevention of C. difficile. In conclusion, bezlotoxumab is a great option adjunctive with standard of care CDI antibiotics for the prevention of rCDI in high-risk adults.


Introduction And Background
Despite strong national and international concerns, the incidence of primary and recurrent C. difficile infection (PCDI and rCDI), respectively, has increased dramatically. Between 2000 and 2009, the prevalence of C. difficile in the United States more than doubled, and current estimates imply that C. difficile infects >500,000 patients yearly, resulting in over 14,000 deaths [1]. In 2011, C. difficile was responsible for about half a million infections and was linked to nearly 29,000 deaths. From 2012 to 2017, nucleic acid amplification test (NAAT) use was adjusted to 55% there was a decline in health-care-associated infections, and the anticipated total national burden of C. difficile infection (CDI) in the United States was reduced by an adjusted 24%. In 2017, community-associated infections did not decline and accounted for nearly half of all CDIs [2]. C. difficile has become the most common cause of health-care-associated infections in U.S. hospitals, with excess health-care expenses associated with CDI estimated at $4.8 billion for acute-care facilities alone in 2008 [3]. C. difficile, now Clostridioides difficile, is strongly linked to antibiotic use [4]. C. difficile is a gram-positive, toxin-producing anaerobic bacillus bacterium. The bacterium was difficult to culture when it was first described in newborns in 1935, subsequently named Bacillus difficilis. C difficile is ubiquitous and can be found in river water, soil, and meats. C difficile is also a spore-forming bacteria that can survive in harsh environments [5]. CDI is more common in the elderly and individuals with chronic medical conditions [6]. Following a primary bout of CDI, approximately 25% of individuals will develop a recurrent infection. After a first recurrence, the likelihood of a future recurrence rises to 40% [7]. The majority of recurrences are due to relapses of CDI with the original strain, rather than re-infection with a different strain [8]. Recurrent CDI infections put patients at risk for consequences such as toxic dilatation of the colon and septicemia, both of which have a high death rate [6]. C. difficile virulence factors include toxin A (TcdA) and toxin B (TcdB). TcdA and TcdB bind to and enter the colonic epithelium, resulting in the generation of proinflammatory chemokines and cytokines, neutrophil influx, tight junction rupture, fluid secretion, and epithelial cell death [4]. Toxin B is 10 times more potent than toxin A, thus strains that do not produce toxin A can be just as virulent as ones that produce both. NAP1/BI/027, a "hypervirulent" strain, produces a third toxin termed C. difficile binary toxin (CDT). The CDT toxin causes the gut wall to break down, facilitating adhesion [9].
CDI occurs most frequently during antibiotic usage and within the first month after treatment, but the risk can last up to 90 days. Proton pump inhibitor exposure has been detected in roughly 31% of communityacquired CDI patients, with no antibiotic exposure [8]. Metronidazole, vancomycin, and fidaxomicin are among the drugs used to treat primary illness, although they do little to prevent CDI recurrence. Fidaxomicin may lower the occurrence of rCDI when given early in patients with a non-NAP1/BI/027 strain; nevertheless, it did not appear to be more effective than oral vancomycin for the treatment of mild to moderate CDI. Fecal microbiota transplantation (FMT) has shown significant cure rates for rCDI, although there are concerns about regulatory issues and long-term safety. FMT's, widespread use, has been hindered by the lack of standardized ways of delivering fecal bacteria. In light of this, a human monoclonal antibody against toxin B (bezlotoxumab) was developed as an adjunct to antibiotic therapy to prevent rCDI [9]. The primary aim of this systematic review is to determine the efficacy of bezlotoxumab in the prevention of recurrent C. difficile.

Review Methods
Relevant studies were found by searching PubMed, PubMed Central(PMC), Google Scholar, and Science Direct. This review was carried out using the preferred reporting items for systematic review and metaanalysis (PRISMA) guidelines [10]. The following keywords were used for the search C. difficile, recurrent C. difficile, pseudomembranous colitis bezlotoxumab , Zinplava, and broadly neutralizing antibody. Additionally, a combination of the above regular keywords and the Medical Subject Headings (MeSH) strategy was used to identify relevant records from the PubMed databases.

Study Selection
Inclusion criteria were: published as randomized clinical trials (RCT) and observational cohort studies, Systematic Reviews, and Narrative reviews in the last 10 years (2012-2022), full free text. For Google Scholar, only the first 300 articles were reviewed because of the vast number of articles. There were no restrictions regarding age, sex, and duration of the study. We imposed no geographic or language restrictions. In observational studies, we included studies using bezlotoxumab to prevent recurrent CDI, with or without a comparator, in an adult population (18+ years) and a follow-up period of at least 90 days. Studies that failed to report clinical outcomes were excluded. All references were imported and managed in Zotero (Reference Manager). As shown in Table 1, the field search employed in the procedure was chosen based on keywords used in prior literature and Medical Subject Headings (Mesh), based on the database used.

Study Results and Bias Assessment
The initial search identified 3,259 publications. Of these, 2,162 articles were excluded after applying inclusion criteria. 1,097 articles were screened and an additional 922 were removed after screening titles and abstracts. Consequently, 175 papers were retrieved in full text. Of these, 30 articles were reviewed and 13 articles (One RCT, one systematic review, seven observational studies, and four reviews) were included in the review. Figure 1 shows a flow diagram depicting the screening process and study selection.

Risk of Bias
Randomized controlled trials were assessed with the Cochrane Collaboration's risk of bias tool (CCRBT) [11]. For observational studies, the Newcastle Ottawa tool (NOS) [12] was used. For systematic review, Assessment of Multiple Systematic Reviews 2 (AMSTAR 2) checklist [13] was used and for Narrative reviews, Scale for the Assessment of Narrative Review Articles 2 (SANRA 2) checklist [14] was used. Each assessment tool required a score of at least 70% to be accepted ( al. [15] Herroro et al. [16], NOS [12] Non-Randomised

Control Trials and
Observational studies Eight items: (1) Representativeness of the exposed cohort (2) Selection of the non-exposed cohort (3) Ascertainment of exposure (4) Demonstration that an outcome of interest was not present at the start of study (5) Comparability of cohorts on the basis of the design or analysis* (6). Assessment of outcome (7)

Discussion
The following sections discuss the pathophysiology of C. difficile, risk factors for recurrent CDI and the role of bezlotoxumab in the prevention of rCDI.

Pathophysiology of C. difficile
C. difficile's potential to cause enteritis is determined by two host characteristics: colonization resistance and immunological response to C. difficile. The indigenous flora of the large intestine, which consists of around 4,000 bacterial species and is collectively known as the fecal microbiome, protects it from invasive diseases. By competing for vital nutrients and attachment sites to the gut wall, these microorganisms give colonization resistance against pathogenic species. Antibiotics disrupt the barrier microbiota and reduce colonization resistance, creating an environment for gut infections to colonize. Antibiotic reduction of the Bacteroides and Firmicutes phyla appears to be particularly relevant in the pathogenesis of C. difficile [26]. C. difficile pathophysiology is mostly based on the effects of toxin A and toxin B. The toxins are encoded by the tcdA and tcdB genes, which are located within a chromosomally integrated DNA sequence known as the pathogenicity locus or PaLoc. Three more genes are found in the PaLoc: (1) tcdR, which codes for an alternative RNA polymerase sigma factor that regulates the expression of tcdA and tcdB. (2) tcdE, which encodes a putative holin-like protein required for both toxins' extracellular release; and (3) tcdC, which inhibits TcdA and TcdB production. PaLoc can be horizontally transferred from non-pathogenic strains lacking tcdA and tcdB, transforming them into pathogenic strains to producers. In theory, while a "healthy" gut microbiota converts primary bile acids into secondary bile acids (which inhibit C. difficile germination), a disrupted microbiota, deficient in primary bile acid converters, may enable C. difficile germination and overgrowth following broad-spectrum antibiotic therapy [24]. and cell proliferation is hindered as a result of the suppression of both cell cycle progression and actindependent cytokinesis. Type I (apoptosis) and type III (necrosis) programmed cell death can result from the cytotoxic effects of TcdA and TcdB. TcdA can induce apoptosis predominantly via activating caspase-8 and cytochrome c/caspase-9, which has drawn attention to the role of TcdA glucosyltransferase activity in this process And is dependent on Rho protein monoglucosylation.TcdB has the ability to activate a variety of apoptotic pathways in host cells. In addition to glucosylation, TcdB activities also result in cytotoxicity, mostly through caspase-dependent (activation of caspase-3) and caspase-independent mechanisms (i.e., Bcl-2-dependent mechanism) apoptotic mechanisms. TcdB has also been shown to cause apoptosis through the involvement of mitochondrial ATP-dependent potassium channels. This process is linked to an increase in cytosolic calcium concentration and hyperpolarization of the mitochondrial membrane, a state that is likely to influence commitment to cell death. The cytotoxic effects are also linked to the activation of the inflammasome by glycosylated RhoA, which is thought to be the cause of C. difficile-induced inflammation and colitis [27].

Risk Factors for Recurrent C. difficile
CDI was defined as diarrhea (≥Three unformed bowel movements [types 5 to 7 on the Bristol stool scale] in 24 hours) with a stool test result that was positive for toxigenic C. difficile [15]. Recurrences are linked to a weakened immune response to C. difficile toxins and/or changes in the microbiota in the colon [28]. Advanced Age: The most frequently reported risk factor for rCDI is advanced age. The reason for the recurrence in elderly people is unclear, decreased immune response to CDI and increased comorbidity may play a role [29]. Antibiotic use: The alteration of the gut microbiota by antibiotics impacts nutrient sources in at least two ways. Antibiotics reduce competition for limited resources and open up previously unoccupied ecological niches by lowering the diversity of the intestinal microbiota. Second, bacterial cell lysis provides carbon sources that can be consumed by the remaining community. The altered intestinal microbiota by antibiotics also influences bile acid composition in the colon, thereby promoting the growth of C. difficile [29,30]. Gastric acid suppression: These agents may reduce stomach acidity, weakening defenses against C. difficile and increasing the risk of CDI. The rate of rCDI in patients with stomach acid suppression was higher than in patients without gastric acid suppression, according to a recent metaanalysis that included 16 [29,32]. Other important risk factors: Host genetics, compromised immune system, Chronic renal failure, Prior CDI episode Severity of CDI episode and Severe Primary CDI, and Prolonged Hospital stay [24,29].

Role of Bezlotoxumab in the Prevention of Recurrent C. difficile
Bezlotoxumab is a fully humanized ImmunoglobulinG1 (IgG1) monoclonal antibody that only binds to C. difficile toxin B. Binding to toxin B neutralizes the toxin and prevents damage to mammalian colonic cells. Currently, bezlotoxumab is approved for the prevention of rCDI in adult patients at high risk for rCDI. The product must be administered during the active CDI antibacterial treatment and is available as 1000 mg/40 mL single-dose vials. Reconstituted vials should be diluted in 0.9% sodium chloride or 5% dextrose to a final concentration between 1 and 10 mg/mL. The recommended dosage is based on the patient's body weight, with 10 mg/kg intravenously over 60 min in a single administration, up to treatment day 14 [2,3].
MODIFY I and MODIFY II were two separate but largely identical trials by Wilcox et al. that was designed to see if bezlotoxumab alone or in conjunction with actoxumab (Monoclonal antibody against C. difficile toxin A) could prevent rCDI. MODIFY I also included an actoxumab alone arm, which was dropped early after being linked to significantly higher rates of sepsis-related death and a lack of efficacy when compared to the bezlotoxumab and actoxumab arm. Randomization was stratified based on oral standard-of-care antibiotics and hospitalization status (inpatient or outpatient). The primary endpoint was the proportion of participants with rCDI (defined as a new episode of CDI after the initial clinical cure of the baseline episode) during 12 weeks of follow-up in the modified intention-to-treat population. The initial clinical cure was defined as no loose stools for two consecutive days after completing standard-of-care (SoC) antibiotic therapy for ≤16 days. A secondary endpoint, also called global cure or sustained clinical response, was the sustained cure rate, which meant initial clinical cure as defined above and no recurrence of CDI through 12 weeks [15].
In the trials, 2,580 (97%) of the 2,655 participants were treated, and 2,559 (96%) were included in the modified intention-to-treat population. In the modified intention-to-treat population, 2,174 people (85%) completed the 12-week trial. In both trials, the percentage of participants with recurrent infection in the modified intention-to-treat population was significantly lower in the bezlotoxumab group than in the placebo group ( adjusted difference, −10.7 percentage points; 95% CI, −16.4 to −5.1; both p < 0.001). The majority of recurrences (71%) occurred within four weeks of the study infusion. Differences in the risk of recurrent infection between the bezlotoxumab and placebo regimens were visible as early as two weeks after infusion and lasted until week 12. The number needed to treat (NNT) to prevent one episode of rCDI was 10. In subgroups of 65 years of age or older and those with previous CDI the NNT was 6. The outcomes of MODIFY I and MODIFY II, taken separately and together, reveal that bezlotoxumab was associated with a significantly reduced rate of recurrent infection than placebo among participants receiving SoC antibiotic therapy for primary or rCDI [4,15]. The most common adverse events reported during the first four weeks after infusion were nausea, vomiting, abdominal pain, diarrhea, fatigue, pyrexia, urinary tract infection, and headache [15].
In the observational study by Valerio et al., bezlotoxumab was only financed for patients with three or more risk factors. Recurrent CDI (r-CDI) was defined as CDI symptoms and positive stool samples that occurred in the first 10 to 90 days after recovery from a previous CDI episode. Out of 16 patients in the study, two patients received fidaxomicin (because of vancomycin allergy) and 14 patients received vancomycin. As two patients died, out of 14 patients there were only three recurrences with a recurrence rate of 21.4%. Limitations of this study include there was no comparison group, and it was done in a small group of patients in an institution [18].
In the observational study by Johnson et al., the effectiveness of bezlotoxumab was assessed only in the transplant patients as they're at high risk for rCDIn and its sequela of graft loss and mortality. 39 in the bezlotoxumab (BEZ) group and 56 in the SoC group. During BEZ administration, one patient suffered nausea and vomiting, prompting her to stop taking medication. In unadjusted analysis, there was no difference between BEZ and SoC participants in the primary outcome of 90-day rCDI (16% vs 29%, p = 0.13). In a multivariable study of 90-day rCDI incidence in the general population, BEZ was linked to a 72% decreased risk of rCDI when compared to those who did not take BEZ (odds ratio, 0.28 [95% CI, .08-0.91]; p = 0.03). The number of previous CDI episodes was likewise linked to a higher risk of rCDI. The most common CDI treatment (86%) was PO Vancomycin (VAN), which was consistent across cohorts (p = 0.99;). BEZ patients were more likely to receive Fidaxomicin (FDX) (34% vs 11%, p = 0.01), while SoC recipients were more likely to use combination treatments (mainly PO VAN + IV Metronidazole (MTZ), 13% vs 41%, p = 0.01). Less than 50% of patients in this study have more than four risk factors and Immunocompromised patient populations have been underrepresented in the pivotal trials, accounting for about 20% of the MODIFY I/II. Patients in the MODIFY studies were given BEZ a median of three days after starting CDI-directed drugs. Due to the average hospital stay being quite long, reimbursement issues with inpatient administration, insurance prior permission restrictions, and scheduling issues for outpatient infusion, patients in this study received BEZ at a median of 25 days after CDI treatment initiation. Limitations of the study include: the study's primary and secondary outcomes were under powered, and these findings should be taken as exploratory, Patients who received prophylaxis during courses of broad-spectrum antibiotics and tapering regimens with recurrent CDI had longer durations of CDI therapy when receiving BEZ [19].
In the observational study by Oksi et al., out of 46 patients, seventeen patients were outpatients (eight of whom were awaiting FMT), while 29/46 patients were in the hospital at the time of BEZ infusion. Due to background comorbidity or immunosuppressive treatment, 28 (61%)/46 patients were immunocompromised. After treatment with BEZ, 20 (71%) / 28 immunocompromised patients did not have a rCDI. 19 (42%) of the 45 patients (one patient was unknown) were treated with at least one antibacterial antibiotic (other than anti-C. difficile). In 37 of the 46 patients with SoC, vancomycin was used partly or entirely, metronidazole in nine, fidaxomicin in seven, and tigecycline in two. In 73% of patients, adjunctive treatment to SoC was effective in preventing rCDI, and the performance remained at 71% effective even among immunocompromised individuals. In cases with severe CDI, 63% of cases remained rCDI-free over the next three months. BEZ had no significant side effects, but one patient reported shocking feelings after the infusion, and another patient developed a fever the next day after the infusion. Within a three-month follow-up period, 42% of participants in this study received concurrent antibiotics. The best results for preventing rCDI were achieved when BEZ was given in a stable setting, such as right before discharge from the hospital or after all antibiotic treatments, including SoC antibiotics, had been stopped (as was the case with those receiving BEZ when waiting for fecal microbiota transplantation [FMT]). In this condition, the intestinal flora has more time to recuperate, which helps with healing. Limitations include use of solely toxin gene PCR to detect CDI. C. difficile is likely to colonize patients for some time following the SOC. However, in the hospitals where the study was conducted, there is a high bar for using the test-it is not permitted to be used unless there is a genuine clinical suspicion of CDI or its return. Furthermore, the use of BEZ was always supervised by a specialist in infectious disease [20].
In the observational study by Hengel et  . Limitations of the review include: The RCT's included are of variable quality, as more than 50% of the studies did not report blinding of the participants. Furthermore, the RCTs investigating FMT differed in terms of design, donor selection, FMT preparation, follow-up time, lag time between feces collection and infusion, and lag time between antibiotic discontinuation and FMT infusion; whereas mABs were infused either during or immediately after antibiotic discontinuation. The number of previous recurrences was not reported in any of the included RCTs. Furthermore, due to the early termination of the majority of the included RCTs and inconsistent reporting of adverse events, safety outcomes were limited [23].
In the narrative reviews by Giacobbe et al. and Alonso and Mahoney, MODIFY trials and Post hoc analysis of MODIFY trials were reviewed. In one of the post hoc analysis, Participants in the MODIFY trials who had sustained clinical cure at 12 weeks had no rCDI after another nine months of follow-up (0/69, 0%) versus 2/65 (3%) and 1/34 (3%) in the bezlotoxumab plus actoxumab and placebo groups, respectively [2]. Zeng and colleagues used whole-genome sequencing to distinguish recurrences from new infections. Recurrences due to relapse of infection by the same ribotype of the index CDI episode (198/259 evaluable patients, 76%) were distinguished from recurrences due to a different ribotype (50/259 evaluable patients, 19%) [33]. The researchers noted that patients receiving bezlotoxumab had a lower cumulative incidence of relapses (as measured by a competing risk model) than patients who did not receive bezlotoxumab (actoxumab or placebo) [33]. Decreased CDI-related hospital readmissions in patients who are at risk of rCDI [5.1% (27/530) vs. 11.2% (58/520), with a difference of 6.1%, 95%] CI -9.5 to -2.8 was observed in post hoc analysis by prabhu et al. [34]. Bezlotoxumab resulted in a gain of 0.12 QALYs (Quality Adjusted Life Years) compared to placebo, and appeared cost-effective in terms of the prevention of rCDI in the entire study population, with an incremental cost-effectiveness ratio of US$19 824/QALY gained in the post hoc analysis by prabhu et al. [35]. Endogenous antibodies against toxin B were protective compared to antibodies against toxin A, which is consistent with the results of bezlotoxumab as compared to actoxumab [36]. In terms of bezlotoxumab administration timing, efficacy in preventing rCDI was unaffected by the time of administration in relation to the onset of antibiotic treatment (i.e. 0-2, 3-4, and greater than five days after onset) [2,3,37]. The proportion of patients developing rCDI in 382 MODIFY I/II participants with cancer was lower in the bezlotoxumab arms (26/146, 17.8%) than in the placebo arms (42/138, 30.4%), with an absolute difference of -12.6%, 95% CI -22.5 to -2.7% [38]. An exploratory study looked into whether human genetic variants can affect how bezlotoxumab works in patients included in the MODIFY trials. In individuals treated with bezlotoxumab, the single nucleotide polymorphism rs2516513 and the human leukocyte antigen alleles HLA-DRB1*07:01 and HLADQA1*02:01, which are found in the extended major histocompatibility complex on chromosome 6, were linked to a lower incidence of rCDI. The same was not observed in patients who were given a placebo [2,3,39].

Limitations
There are some limitations to our analysis, the minimal number of publications from only four databases were included and grey literature and other databases are excluded, the evaluation of solely free full-text papers, and the small sample sizes and lack of comparison group in the majority of the observational studies. The criteria for patient selection, the definition of recurrence, the timing of antibiotic administration, and the method of testing are varied between studies. Because of the significant level of variability among the studies included in our analysis, our results should be interpreted with caution.

Conclusions
In summary, this systematic review shows that bezlotoxumab, a recently approved novel agent by FDA, has proven to be effective in the prevention of rCDI in high-risk individuals. It has also shown to be effective in decreasing hospital readmission and improved quality of life from Post hoc studies. Its negligible impact on the fecal microbiome, and ability to use in outpatient infusion centers, make it a promising supplementary therapy for rCDI prophylaxis. To assess the efficacy of FMT versus bezlotoxumab in preventing recurrence, head-to-head randomized clinical trials will be needed. In light of few therapeutic options available for the prevention of rCDI, bezlotoxumab is a great option with a good safety profile.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.