Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype.

Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.

OMIP 082: A 25-color phenotyping to define human innate lymphoid cells, natural killer cells, mucosal-associated invariant T cells, and γδ T cells from freshly isolated human intestinal tissue.

We developed a 25-color flow cytometry panel to comprehensively interrogate innate lymphoid cells (ILC), mucosal-associated invariant T (MAIT) cells, natural killer (NK) cells and γδ T cells in human tissues. The ability to isolate and interrogate these cells from fresh human tissue is crucial in understanding the role these cells play at immune-privileged mucosal surfaces like the intestine in health and disease settings. However, liberating these cells from tissue is extremely challenging as many key surface identification markers are susceptible to enzymatic cleavage. Choosing the correct enzyme-antibody clone combination within a high-parameter panel is, therefore, a critical consideration. Here, we present a comprehensive, in-depth analysis of the effect different common digestive enzyme blends have on key surface markers used to identify these cell types. In addition, we compared multiple antibody clones for surface markers that are highly susceptible to enzymatic cleavage, such as CD127 and NKp44, to achieve the most consistent and superior staining patterns among donors.

Attitudes towards Enhanced Recovery after Surgery (ERAS) interventions in colorectal surgery: nationwide survey of Australia and New Zealand colorectal surgeons.

BACKGROUND: Whilst Enhanced Recovery after Surgery (ERAS) has been widely accepted in the international colorectal surgery community, there remains significant variations in ERAS programme implementations, compliance rates and best practice recommendations in international guidelines. METHODS: A questionnaire was distributed to colorectal surgeons from Australia and New Zealand after ethics approval. It evaluated specialist attitudes towards the effectiveness of specific ERAS interventions in improving short term outcomes after colorectal surgery. The data were analysed using a rating scale and graded response model in item response theory (IRT) on Stata MP, version 15 (StataCorp LP, College Station, TX). RESULTS: Of 300 colorectal surgeons, 95 (31.7%) participated in the survey. Of eighteen ERAS interventions, this study identified eight strategies as most effective in improving ERAS programmes alongside early oral feeding and mobilisation. These included pre-operative iron infusion for anaemic patients (IRT score = 7.82 [95% CI: 6.01-9.16]), minimally invasive surgery (IRT score = 7.77 [95% CI: 5.96-9.07]), early in-dwelling catheter removal (IRT score = 7.69 [95% CI: 5.83-9.01]), pre-operative smoking cessation (IRT score = 7.68 [95% CI: 5.49-9.18]), pre-operative counselling (IRT score = 7.44 [95% CI: 5.58-8.88]), avoiding drains in colon surgery (IRT score = 7.37 [95% CI: 5.17-8.95]), avoiding nasogastric tubes (IRT score = 7.29 [95% CI: 5.32-8.8]) and early drain removal in rectal surgery (IRT score = 5.64 [95% CI: 3.49-7.66]). CONCLUSIONS: This survey has demonstrated the current attitudes of colorectal surgeons from Australia and New Zealand regarding ERAS interventions. Eight of the interventions assessed in this study including pre-operative iron infusion for anaemic patients, minimally invasive surgery, early in-dwelling catheter removal, pre-operative smoking cessation, pre-operative counselling, avoidance of drains in colon surgery, avoiding nasogastric tubes and early drain removal in rectal surgery should be considered an important part of colorectal ERAS programmes.

Antibiotics Do Not Reduce Length of Hospital Stay for Uncomplicated Diverticulitis in a Pragmatic Double-Blind Randomized Trial.

BACKGROUND & AIMS: Antibiotic treatment is the standard care for patients with uncomplicated acute diverticulitis. However, this practice is based on low-level evidence and has been challenged by findings from 2 randomized trials, which did not include a placebo group. We investigated the non-inferiority of placebo vs antibiotic treatment for the management of uncomplicated acute diverticulitis. METHODS: In the selective treatment with antibiotics for non-complicated diverticulitis study, 180 patients hospitalized for uncomplicated acute diverticulitis (determined by computed tomography, Hinchey 1a grade) from New Zealand and Australia were randomly assigned to groups given antibiotics (n = 85) or placebo (n = 95) for 7 days. We collected demographic, clinical, and laboratory data and answers to questionnaires completed every 12 hrs for the first 48 hrs and then daily until hospital discharge. The primary endpoint was length of hospital stay; secondary endpoints included occurrence of adverse events, readmission to the hospital, procedural intervention, change in serum markers of inflammation, and patient-reported pain scores at 12 and 24 hrs. RESULTS: There was no significant difference in median time of hospital stay between the antibiotic group (40.0 hrs; 95% CI, 24.4-57.6 hrs) and the placebo group (45.8 hrs; 95% CI, 26.5-60.2 hrs) (P = .2). There were no significant differences between groups in adverse events (12% for both groups; P = 1.0), readmission to the hospital within 1 week (1% for the placebo group vs 6% for the antibiotic group; P = .1), and readmission to the hospital within 30 days (11% for the placebo group vs 6% for the antibiotic group; P = .3). CONCLUSIONS: Foregoing antibiotic treatment did not prolong length of hospital admission. This result provides strong evidence for omission of antibiotics for selected patients with uncomplicated acute diverticulitis. ACTRN: 12615000249550.

Chyme reinfusion nutritional management for enterocutaneous fistula: first international application of a novel pump technique.

AIM: High-output enterocutaneous fistulas (ECFs) are an established cause of intestinal failure. Parenteral nutrition (PN) remains the gold standard for nutritional management but is complex, expensive and associated with significant complications. Chyme reinfusion (CR) has been reported by multiple centres as a viable option for nutritional management that improves nutritional status, provides the capacity to cease PN and is cost-effective. The aim of this paper is to describe the first use of a novel pump device (The Insides System™) by an independent centre in Australia for the nutritional management of a patient with high-output ECF. METHOD: CR was performed on a 66-year-old woman with a high-output ECF. The device consists of two main components: a centrifugal pump that sits inside the stoma appliance and a battery-powered driver that is magnetically coupled externally onto the pump. The device allows for bolus CR at a rate of infusion that is manually controlled by the patient based on comfort, volume and effluent viscosity. RESULTS: CR provided adequate nutritional support, with successful cessation of PN. Effective use of the device was learnt easily by the patient with minimal demands on nursing assistance. Side effects of CR (diarrhoea, abdominal cramping) were overcome by the patient's ability to manually adjust the reinfusion rate. CONCLUSION: Our experience with the novel Insides System™ device showed promising results in maintaining nutritional status as well as providing a minimally invasive, easy to use and low-cost system for CR. CR should be considered as a viable alternative for the nutritional management of patients with a high-output ECF.

The role of mechanical bowel preparation and oral antibiotics for left-sided laparoscopic and open elective restorative colorectal surgery with and without faecal diversion.

BACKGROUND: There is significant variation in the use of mechanical bowel preparation and oral antibiotics prior to left-sided elective colorectal surgery. There has been no consensus internationally. METHODS: This was a retrospective analysis of the 2015 American College of Surgeons National Surgical Quality Improvement Program database. Patients were divided into four groups: those who had mechanical bowel preparation with oral antibiotics, mechanical bowel preparation alone, oral antibiotics alone and no preparation. The main outcome measures included overall, superficial, deep and organ/space surgical site infections. Secondary outcomes included anastomotic leak, ileus and rate of Clostridium difficile. RESULTS: A total of 5729 patients were included for analysis. The overall surgical site infection rate (any superficial, deep or organ/space infection) was significantly lower in the mechanical bowel preparation and oral antibiotics approach when compared to no preparation (OR = 0.46, 95% CI 0.36-0.59, P < 0.0001). On multivariable logistic regression analysis, mechanical bowel preparation with oral antibiotics maintained a lower risk of overall surgical site infections. MBP and OAB also had a protective effect on anastomotic leak in both the laparoscopic and open cohorts (laparoscopic multivariable adjusted OR = 0.42 (0.19-0.94), P = 0.035; open multivariable adjusted OR = 0.3 (0.12-0.77), P = 0.012). Mechanical bowel preparation alone and oral antibiotics alone was not associated with a significant decrease in surgical site infections. There was no increase in C. difficile occurrences with the use of oral antibiotics. CONCLUSION: Mechanical bowel preparation with oral antibiotics significantly minimised surgical site infections and anastomotic leak following both laparoscopic and open left-sided restorative colorectal surgery. Mechanical bowel preparation alone did not reduce surgical site infections. There was a trend to reduction in surgical site infections with oral antibiotics alone.

The impact of variations in care and complications within a colorectal Enhanced Recovery After Surgery program on length of stay.

PURPOSE: Enhanced Recovery After Surgery (ERAS) has become standard of care in colorectal surgery. However, there is not a universally accepted colorectal ERAS protocol and significant variations in care exist between institutions. The aim of this study was to examine the impact of variations in ERAS interventions and complications on length of stay (LOS). METHODS: This study was a single-center review of the first 200 consecutive patients recruited into our prospectively collected ERAS database. The primary outcome of this study was to examine the rate of compliance to ERAS interventions and the impact of these interventions on LOS. The secondary outcome was to assess the impact of complications (anastomotic leak, ileus, and surgical site infections) on LOS. ERAS interventions, rate of adherence, LOS, readmissions, morbidity, and mortality were recorded, and statistical analysis was performed. RESULTS: ERAS variations and complications significantly influenced patient LOS on both univariate and multivariate analysis. ERAS interventions identified as the most important strategies in reducing LOS included laparoscopic surgery, mobilization twice daily postoperative day (POD) 0 to 1, discontinuation of intravenous fluids on POD 0 to 1, upgrading to solid diet by POD 0 to 2, removal of indwelling catheter by POD 0 to 2, avoiding nasogastric tube reinsertion and removing drains early. Both major and minor complications increased LOS. Anastomotic leak and ileus were associated with the greatest increase in LOS. CONCLUSION: Seven high-yield ERAS interventions reduced LOS. Major and minor complications increased LOS. Reducing variations in care and complications can improve outcomes following colorectal surgery.

Neutrophil Extracellular Trap Density Increases With Increasing Histopathological Severity of Crohn's Disease.

BACKGROUND: Intestinal neutrophil recruitment is a characteristic feature of the earliest stages of inflammatory bowel disease (IBD). Neutrophil elastase (NE) and myeloperoxidase (MPO) mediate the formation of neutrophil extracellular traps (NETs); NETs produce the bactericidal oxidant hypochlorous acid (HOCl), causing host tissue damage when unregulated. The project aim was to investigate the relationship between NET formation and clinical IBD in humans. METHODS: Human intestinal biopsies were collected from Crohn's disease (CD) patients, endoscopically categorized as unaffected, transitional, or diseased, and assigned a histopathological score. RESULTS: A significant linear correlation was identified between pathological score and cell viability (TUNEL+). Immunohistochemical analysis revealed the presence of NET markers NE, MPO, and citrullinated histone (CitH3) that increased significantly with increasing histopathological score. Diseased specimens showed greater MPO+-immunostaining than control (P < .0001) and unaffected CD (P < .0001), with transitional CD specimens also showing greater staining than controls (P < .05) and unaffected CD (P < .05). Similarly, NE+-immunostaining was elevated significantly in diseased CD than controls (P < .0001) and unaffected CD (P < .0001) and was significantly higher in transitional CD than in controls (P < .0001) and unaffected CD (P < .0001). The CitH3+-immunostaining of diseased CD was significantly higher than controls (P < .05), unaffected CD (P < .0001) and transitional CD (P < .05), with transitional CD specimens showing greater staining than unaffected CD (P < .01). Multiplex immunohistochemistry with z-stacking revealed colocalization of NE, MPO, CitH3, and DAPI (cell nuclei), confirming the NET assignment. CONCLUSION: These data indicate an association between increased NET formation and CD severity, potentially due to excessive MPO-mediated HOCl production in the extracellular domain, causing host tissue damage that exacerbates CD.

Our data show for the first time that the density of neutrophil extracellular trap formed in the bowel of Crohn's disease patients increases with increasing disease severity, suggesting that myeloperoxidase-mediated host-tissue damage may play a role in disease pathogenesis.

An in situ analysis pipeline for initial host-pathogen interactions reveals signatures of human colorectal HIV transmission.

The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4+ T cells, the primary targets of downstream infection. HIV+ DCs accumulate near and within lymphoid aggregates, which act as early sanctuaries of high viral titers while facilitating HIV passage to the submucosa. Finally, HIV entry induces recruitment and clustering of target cells, facilitating DC- and macrophage-mediated HIV transfer and enhanced infection of CD4+ T cells. These data demonstrate a rapid response to HIV structured to maximize the likelihood of mucosal infection and provide a framework for in situ studies of host-pathogen interactions and immune-mediated pathologies.

Acute Gastroenteritis in the Etiology of Inflammatory Bowel Disease: Systematic Review and Meta-analysis.

Background: Inflammatory bowel disease (IBD) consists of a spectrum of disorders including ulcerative colitis and Crohn's disease, with a rising incidence worldwide. However, despite this prevalence the etiology of IBD remains uncertain. It has been suggested that an episode of gastroenteritis may precipitate IBD. Methods: Studies were identified using a literature search of Pubmed/Medline and Embase/Ovid. This review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The primary outcome was incidence of new-onset IBD after gastroenteritis. Secondary outcomes included incidence of IBD after bacterial, viral, and parasitic gastrointestinal infections. Results: Eleven studies (n = 923 608 patients) were included. Four studies assessed patients with gastroenteritis, subsequently developing IBD as the primary outcome. Patients with gastroenteritis had a higher incidence of subsequent IBD but this did not reach statistical significance (odds ratio [OR] 3.81, 95% CI 0.52-27.85, P = .19). Seven studies examined the incidence of antecedent gastroenteritis (primary outcome) in patients with a confirmed diagnosis of IBD, compared to the controlled population. There was no difference between incidence of antecedent gastroenteritis across the 2 population groups (OR 1.07, 95% CI 0.55-2.08, P = .85). There was no association between IBD and bacterial, viral, or parasitic infections. Conclusions: In summary, our meta-analysis has shown that there is considerable heterogeneity in the literature regarding the role of gastroenteritis in the development of IBD. Further higher quality studies need to be performed to ascertain the true nature of this.

Outcomes of young patients diagnosed with locally advanced rectal cancer.

BACKGROUND: The incidence of rectal cancer is higher in the older population. In developed nations, there has been a rise in incidence in young onset colorectal cancer (CRC). We examined the outcomes of locally advanced rectal cancer (LARC) in younger patients (yRC) compared with older patients, using a retrospective audit. METHODS: All cases of LARC referred to two tertiary referral cancer centres in Western Sydney were examined. Patient demographics, presenting symptoms, treatment, relapse free survival (RFS), overall survival (OS) and progression free survival (PFS) were obtained. Under 50 years old was used as the cut-off age for defining yRC. RESULTS: All 145 consecutive patients were treated for LARC, including 28 in the yRC and 117 in the older patient group. Median follow-up was 54 months. yRC were more likely to complete neoadjuvant therapy (100% vs. 86%; P=0.032) and to undergo more extensive surgical procedures (24% vs. 2%, P<0.0001). yRC were more likely to have microsatellite high (MSI) tumours (30% vs. 4.7%; P=0.003). yRC demonstrated significantly poorer RFS compared with the standard group (HR 2.79; median RFS 4.67 vs. 16.02 months; P=0.023). In the relapsed setting, yRC had poorer PFS compared with the standard group (median PFS 2.66 vs. 9.70, P=0.006, HR 3.04). A difference in OS was also seen between the two groups, with yRC demonstrating poorer OS (median OS 40.46 vs. 58.26 months, HR 3.48, P=0.036). CONCLUSIONS: Patients under 50 years with LARC are more likely to have MSI tumours with a more aggressive disease course and poorer RFS, PFS and OS. Initiatives to improve early detection of these patients may improve outcomes. Further research is necessary to understand this disease and optimise its treatment.

Bowel Preparation and Oral Antibiotic Agents for Selective Decontamination in Colorectal Surgery: Current Practice, Perspectives, and Trends in Australia and New Zealand, 2019-2020.

Background: There has been much debate as to the importance of mechanical bowel preparation (MBP) and oral antibiotic agents (OAB) prior to elective colorectal surgery over the past two decades. There is no consensus between international guidelines. Methods: The Australia and New Zealand Mechanical Bowel Preparation and Oral Antibiotics (ANZ-MBP-OAB) questionnaire was distributed to colorectal surgeons after institutional board approval assessing specialist attitudes toward 18 enhanced recovery after surgery (ERAS) interventions. Data were analyzed using a rating scale and graded response model in item response theory (IRT) on Stata MP, version 15 (StataCorp LP, College Station, TX). Specialist attitudes toward the effectiveness of MBP and OAB strategies in providing better short-term outcomes was ranked alongside other ERAS interventions. This was followed by specific questions examining current practice, perspectives, and trends. Results: Ninety-five of 300 (31.7%) colorectal surgeons in Australia and New Zealand participated in the survey. Statistical modeling was achieved in 13 ERAS interventions. Compared with other ERAS interventions, the use of MBP with OAB and MBP alone ranked nine of 13 and 10 of 13, respectively, in order of effectiveness in providing better short-term outcomes after colorectal surgery. Oral antibiotic agents alone was not considered effective. Mechanical bowel preparation with OAB was considered to be the best strategy in both colon (37%) and rectal surgery (48%) but current practice varied substantially from perspective. Mechanical bowel preparation alone was strongly favored in rectal surgery (81%) with only 14% using MBP with OAB. In colon surgery, only 10% used MBP with OAB, with MBP alone (45%) and no preparation (45%) being equally the most commonly used strategies. Conclusions: Among Australian and New Zealand colorectal surgeons, MBP with OAB was considered the best bowel preparation strategy. However, despite an awareness of its benefits, MBP with OAB has yet to be widely adopted into clinical practice or guidelines in Australia and New Zealand.

Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.

Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

Iatrogenic rectal injury associated with fleet enema administration masquerading as Fournier's Gangrene.

Rectal injuries secondary to fleet enemas are the result of a chemical irritation as well as mechanical trauma causing localized perforation and mucosal necrosis. These injuries can be managed expectantly. Fournier's Gangrene is a life-threatening, fulminant soft tissue infection of the genito-perineal region commonly secondary to a perianal infection, requiring extensive debridement of all affected tissue and judicious antibiotic therapy. This distinction is critical in the management of the current case, allowing for limited debridement and judicious observation without a resection of the injured rectum. A diverting colostomy allows for effective faecal management and minimizes contamination of the perineal wound.

Dangers of delayed diagnosis of perianal abscess and undrained perianal sepsis in Fournier's gangrene: a case series.

Fournier's gangrene (FG) is a rapidly progressing infective necrotising fasciitis of the perianal, perineal and genital region. It is characterised by its aggressive nature and high mortality rates of between 15% and 50%. While it has been commonly found to primarily develop from urological sources, there have been increasing reports of the role of colorectal sources as the underlying aetiology of FG. Presented is a case series of four FG presentations at a single institution during a 12-month period as a result of underlying untreated perianal disease highlighting its dangers in progressing to a deadly infection, advocating for early and aggressive surgical debridement, and the role of adjunct scoring systems, such as Laboratory Risk Indicator for Necrotising Fasciitis, in guiding clinical diagnosis.

Metronidazole in the Management of Post-Open Haemorrhoidectomy Pain: Systematic Review.

PURPOSE: Open haemorrhoidectomy is associated with significant postoperative pain. Metronidazole is commonly prescribed in the postoperative period as an adjunct to analgesia in pain management. METHODS: In our systematic review, studies were identified using PubMed/MEDLINE, Embase/Ovid and Cochrane Register of Controlled Trials databases. Studies were included if they were randomised controlled trials (RCTs) involving interventions with oral metronidazole at any dose over any time period. The primary outcome was pain score (visual analogue scale, VAS) after open haemorrhoidectomy. Secondary outcomes included time to return to normal daily activities, additional analgesia usage, and postoperative complications. RESULTS: Of 14 RCTs reviewed, 4 met inclusion criteria and were selected. The studies comprised 336 study subjects and 169 subjects were randomised to metronidazole while 167 were in the control group. There was a significant reduction in VAS across all time points, with maximal reduction seen on day 5 posthaemorrhoidectomy (mean difference, -2.28; 95% confidence interval, -2.49 to -2.08; P < 0.001). There was no difference in incidence of complications (P = 0.13). The Cochrane Risk of Bias Tool showed 3 of 4 of the studies had a risk of bias. CONCLUSION: Metronidazole may be associated with decreased pain but there is insufficient evidence from RCTs to provide a strong grade of recommendation. Further RCTs are required.