Perioperative Considerations for the Surgical Treatment of Crohn's Disease with Discussion on Surgical Antibiotics Practices and Impact on the Gut Microbiome.

Crohn's disease, a chronic inflammatory process of the gastrointestinal tract defined by flares and periods of remission, is increasing in incidence. Despite advances in multimodal medical therapy, disease progression often necessitates multiple operations with high morbidity. The inability to treat Crohn's disease successfully is likely in part because the etiopathogenesis is not completely understood; however, recent research suggests the gut microbiome plays a critical role. How traditional perioperative management, including bowel preparation and preoperative antibiotics, further changes the microbiome and affects outcomes is not well described, especially in Crohn's patients, who are unique given their immunosuppression and baseline dysbiosis. This paper aims to outline current knowledge regarding perioperative management of Crohn's disease, the evolving role of gut dysbiosis, and how the microbiome can guide perioperative considerations with special attention to perioperative antibiotics as well as treatment of Mycobacterium avium subspecies paratuberculosis. In conclusion, dysbiosis is common in Crohn's patients and may be exacerbated by malnutrition, steroids, narcotic use, diarrhea, and perioperative antibiotics. Dysbiosis is also a major risk factor for anastomotic leak, and special consideration should be given to limiting factors that further perturb the gut microbiota in the perioperative period.

Hepatic lipid-associated macrophages mediate the beneficial effects of bariatric surgery against MASH.

For patients with obesity and metabolic syndrome, bariatric procedures such as vertical sleeve gastrectomy (VSG) have a clear benefit in ameliorating metabolic dysfunction-associated steatohepatitis (MASH). While the effects of bariatric surgeries have been mainly attributed to nutrient restriction and malabsorption, whether immuno-modulatory mechanisms are involved remains unclear. Here we report that VSG ameliorates MASH progression in a weight loss-independent manner. Single-cell RNA sequencing revealed that hepatic lipid-associated macrophages (LAMs) expressing the triggering receptor expressed on myeloid cells 2 (TREM2) increase their lysosomal activity and repress inflammation in response to VSG. Remarkably, TREM2 deficiency in mice ablates the reparative effects of VSG, suggesting that TREM2 is required for MASH resolution. Mechanistically, TREM2 prevents the inflammatory activation of macrophages and is required for their efferocytotic function. Overall, our findings indicate that bariatric surgery improves MASH through a reparative process driven by hepatic LAMs, providing insights into the mechanisms of disease reversal that may result in new therapies and improved surgical interventions.

Diet-induced shifts in the gut microbiota influence anastomotic healing in a murine model of colonic surgery.

Host diet and gut microbiota interact to contribute to perioperative complications, including anastomotic leak (AL). Using a murine surgical model of colonic anastomosis, we investigated how diet and fecal microbial transplantation (FMT) impacted the intestinal microbiota and if a predictive signature for AL could be determined. We hypothesized that a Western diet (WD) would impact gut microbial composition and that the resulting dysbiosis would correlate with increased rates of AL, while FMT from healthy, lean diet (LD) donors would reduce the risk of AL. Furthermore, we predicted that surgical outcomes would allow for the development of a microbial preclinical translational tool to identify AL. Here, we show that AL is associated with a dysbiotic microbial community characterized by increased levels of Bacteroides and Akkermansia. We identified several key taxa that were associated with leak formation, and developed an index based on the ratio of bacteria associated with the absence and presence of leak. We also highlight a modifiable connection between diet, microbiota, and anastomotic healing, potentially paving the way for perioperative modulation by microbiota-targeted therapeutics to reduce AL.

Fecal Microbiota Transplantation for Fulminant Clostridioides Difficile Infection: A Combined Medical and Surgical Case Series.

INTRODUCTION: Urgent abdominal colectomy is indicated for patients with fulminant Clostridioides difficile infection (CDI) when other medical therapies fail, yet mortality remains high. Fecal microbiota transplant is a less invasive alternative approach for patients with fulminant CDI. We report the 30-day complications of patients with fulminant CDI who underwent either abdominal colectomy, fecal microbiota transplantation (FMT), or FMT followed by abdominal colectomy (FMT-CO). Methods: We performed a single-center, retrospective case review of combined medical and surgical patients with CDI at a large academic medical center between 2008 and 2016. Cohorts were identified as patients with fulminant CDI who underwent total abdominal colectomy alone (CO), FMT alone (FMT), or FMT-CO. We analyzed patient demographics, history, comorbidities, clinical and laboratory variables, CDI severity scores, and mortality outcomes at 30 days. Results: We identified 5,150 patients with CDI at our center during the review period; 16 patients met the criteria for fulminant CDI and were included in this study, with four patients in the CO cohort, eight patients in the FMT cohort, and four patients in the FMT-CO cohort. Demographics and CDI severity scores were similar for all three groups, although the selected comorbidity profiles differed significantly among the three cohorts. The 30-day mortality rates for patients in the CO, FMT, and FMT-CO groups were 25%, 12.5%, and 25%, respectively. Conclusions: FMT is an alternative or adjunctive therapy to colectomy for patients with fulminant CDI that is not associated with increased mortality. Implementation of FMT protocols in clinical practice would be dependent on the availability of qualified transplant material and successful early identification of patients likely to benefit from FMT.

The Influence of the Microbiome on Anastomotic Leak.

Anastomotic leak, defined by the International Study Group of Rectal Cancer as "a communication between the intra- and extraluminal compartments owing to a defect of the integrity of the intestinal wall at the anastomosis," is one of the most devastating complications in colorectal surgery. Much work has been done to identify causes of leak; however, despite advances in surgical technique, the prevalence of anastomotic leak has remained at around 11%. The potential causative role of bacteria in the etiopathology of anastomotic leak was established in the 1950s. More recently, alterations in the colonic microbiome have been shown to affect rates of anastomotic leak. Multiple perioperative factors that alter the homeostasis of the gut microbiota community structure and function have been linked to anastomotic leak after colorectal surgery. Here, we discuss the role of diet, radiation, bowel preparation, medications including nonsteroidal anti-inflammatory drugs, morphine, and antibiotics, and specific microbial pathways that have been implicated in anastomotic leak via their effects on the microbiome.

Implementation and Analysis of an Institution-Wide EHR-Integrated Handoff Note.

EHR-Integrated Handoff Notes are becoming increasingly prevalent, especially among inpatient clinical service teams composed of physicians in training (resident physicians). We describe the implementation of such a tool at our institution, and then describe the usage of the tool, as well as changes made to its content, structure, and format, years after original implementation. We also describe frequency and temporal distribution of updates made to free text elements of the Handoff Note. At the initial implementation there were three versions available; medical/surgical, pediatric, and ICU. Years after implementation, 57% of clinical service teams continue to utilize the note, including all medical, pediatric, behavioral health, obstetrics/gynecology, and neurology services, as well as most surgical services, that serve as "primary" teams. Several interesting changes were noted to the content and structure of the Handoff Note, namely that more complicated versions were abandoned in favor of simpler versions. The Patient Summary and To Do free text boxes are updated an average of 1.0 and 1.6 times per day. Around 60% of updates to both free text boxes occur between 12 pm - 5:59 pm, likely in preparation for the daytime-nightime team handoff.

A pilot study demonstrating the impact of surgical bowel preparation on intestinal microbiota composition following colon and rectal surgery.

The intestinal microbiota has been implicated in the pathogenesis of complications following colorectal surgery, yet perioperative changes in gut microbiome composition are poorly understood. The objective of this study was to characterize the perioperative gut microbiome in patients undergoing colonoscopy and colorectal surgery and determine factors influencing its composition. Using Illumina amplicon sequencing coupled with targeted metabolomics, we characterized the fecal microbiota in: (A) patients (n = 15) undergoing colonoscopy who received mechanical bowel preparation, and (B) patients (n = 15) undergoing colorectal surgery who received surgical bowel preparation, composed of mechanical bowel preparation with oral antibiotics, and perioperative intravenous antibiotics. Microbiome composition was characterized before and up to six months following each intervention. Colonoscopy patients had minor shifts in bacterial community composition that recovered to baseline at a mean of 3 (1-13) days. Surgery patients demonstrated substantial shifts in bacterial composition with greater abundances of Enterococcus, Lactobacillus, and Streptococcus. Compositional changes persisted in the early postoperative period with recovery to baseline beginning at a mean of 31 (16-43) days. Our results support surgical bowel preparation as a factor significantly influencing gut microbial composition following colorectal surgery, while mechanical bowel preparation has little impact.

Network-based approaches for the investigation of microbial community structure and function using metagenomics-based data.

Network-based approaches offer a powerful framework to evaluate microbial community organization and function as it relates to a variety of environmental processes. Emerging studies are exploring network theory as a method for data integration that is likely to be critical for the integration of 'omics' data using systems biology approaches. Intricacies of network theory and methodological and computational complexities in network construction, however, impede the use of these tools for translational science. We provide a perspective on the methods of network construction, interpretation and emerging uses for these techniques in understanding host-microbiota interactions.

The study of the contribution of the microbiome to health and disease offers a promising avenue to better understand disease processes and offer more personalized therapies. These communities comprise thousands of species and strains that interact among each other and with the host in a variety of complex ways to influence patient health. Studying these communities as a network of interactions, rather than looking at specific species or functions individually, will provide a more comprehensive understanding of how the microbiome contributes to disease processes. Here, we describe the current methods for studying microbiome networks and interpreting how network features may be related to health outcomes.

Human Oral Mucosal Stem Cells Reduce Anastomotic Leak in an Animal Model of Colonic Surgery.

BACKGROUND: Anastomotic leak is regarded as one of the most feared complications of bowel surgery; avoiding leaks is a major priority. Attempts to reduce or eliminate leaks have included alternate anastomotic techniques. Human oral mucosa stem cells (hOMSC) are self-renewing and expandable cells derived from buccal mucosa. Studies have shown that hOMSC can accelerate tissue regeneration and wound healing. The objective of this study was to evaluate whether hOMSC can decrease anastomotic leak rates in a murine model of colon surgery. METHODS: Two experiments were performed. In the first study, mice underwent colonic anastomosis using five interrupted sutures. hOMSC (n = 7) or normal saline (NS; n = 17) was injected into the colon wall at the site of the anastomosis. To evaluate whether hOMSC can impact anastomotic healing, the model was stressed by repeating the first experiment, reducing the number of sutures used for the construction of the anastomosis from five to four. Either hOMSC (n = 8) or NS (n = 20) was injected at the anastomosis. All mice that survived were sacrificed on postoperative day 7. Anastomotic leak rate, mortality, daily weight, and daily wellness scores were compared. RESULTS: In the five-suture anastomosis, there were no differences in anastomotic leak rate, mortality, or daily weight. Mice that received hOMSC had significantly higher wellness scores on postoperative day 2 (p < 0.05). In the four-suture anastomosis, there was a significant decrease in leak rate (70% [NS] vs. 25% [hOMSC], p = 0.029) and higher wellness scores in mice that received hOMSC (p < 0.05). CONCLUSION: Our study suggests that injecting hOMSC at the colonic anastomosis can potentially reduce anastomotic leak and improve postoperative wellness in a murine model of colon surgery.

Peri-operative antibiotics acutely and significantly impact intestinal microbiota following bariatric surgery.

Bariatric surgery is the most effective treatment for weight loss. Vertical sleeve gastrectomy (VSG) involves the resection of ~ 80% of the stomach and was conceived to purely restrict oral intake. However, evidence suggests more complex mechanisms, particularly postoperative changes in gut microbiota, in facilitating weight loss and resolving associated comorbidities. VSG in humans is a complex procedure and includes peri-operative antibiotics and caloric restriction in addition to the altered anatomy. The impact of each of these factors on the intestinal microbiota have not been evaluated. The aim of this study was to determine the relative contributions of each of these factors on intestinal microbiota composition following VSG prior to substantial weight loss. Thirty-two obese patients underwent one of three treatments: (1) VSG plus routine intravenous peri-operative antibiotics (n = 12), (2) VSG with intravenous vancomycin chosen for its low intestinal penetrance (n = 12), and (3) caloric restriction (n = 8). Fecal samples were evaluated for bacterial composition prior to and 7 days following each intervention. Only patients undergoing VSG with routine peri-operative antibiotics showed a significant shift in community composition. Our data support the single dose of routine peri-operative antibiotics as the most influential factor of intestinal microbial composition acutely following VSG.

Antibiotic-induced Disruption of Intestinal Microbiota Contributes to Failure of Vertical Sleeve Gastrectomy.

OBJECTIVE: The aim of this study was to test whether the perioperative composition of intestinal microbiota can contribute to variable outcomes following vertical sleeve gastrectomy (VSG). SUMMARY OF BACKGROUND DATA: Although bariatric surgery is the most effective treatment for obesity, metabolic outcomes are variable. METHODS: Diet-induced obese mice were randomized to VSG or sham surgery, with or without exposure to antibiotics that selectively suppress mainly gram-positive (fidaxomicin, streptomycin) or gram-negative (ceftriaxone) bacteria on postoperative days (POD) 1-4. Fecal microbiota was characterized before surgery and on POD 7 and 28. Mice were metabolically characterized on POD 30-32 and euthanized on POD 35. RESULTS: VSG resulted in weight loss and shifts in the intestinal microbiota composition relative to sham-operated mice. Antibiotic exposure resulted in sustained reductions in alpha (within-sample) diversity of microbiota and shifts in its composition. All antibiotic treatments proved to be detrimental to metabolic VSG outcomes, regardless of antimicrobial specificity of antibiotics. These effects involved functionally distinct pathways. Specifically, fidaxomicin and streptomycin markedly altered hepatic bile acid signaling and lipid metabolism, while ceftriaxone resulted in greater reduction of key antimicrobial peptides. However, VSG mice exposed to antibiotics, regardless of their specificity, had significantly increased subcutaneous adiposity and impaired glucose homeostasis without changes in food intake relative to control VSG mice. CONCLUSION: Dysbiosis induced by brief perioperative antibiotic exposure attenuates weight loss and metabolic improvement following VSG. Potential mechanisms include disruption of bile acid homeostasis and reduction in the production of gut antimicrobial peptides. Results of this study implicate the intestinal microbiota as an important contributor to metabolic homeostasis and a potentially modifiable target influencing clinical outcomes following VSG.

Serum FABP4 concentrations decrease after Roux-en-Y gastric bypass but not after intensive medical management.

BACKGROUND: Serum concentrations of fatty acid binding protein 4, an adipose tissue fatty acid chaperone, have been correlated with insulin resistance and cardiovascular risk factors. The objective of this study were to assess relationships among Roux-en-Y gastric bypass, intensive lifestyle modification and medical management protocol, fatty acid binding protein 4, and metabolic parameters in obese patients with severe type 2 diabetes mellitus; and to evaluate the relative contribution of abdominal subcutaneous adipose and visceral adipose to the secretion of fatty acid binding protein 4. METHODS: Participants were randomly assigned to intensive lifestyle modification and medical management protocol (n = 29) or to intensive lifestyle modification and medical management protocol augmented with Roux-en-Y gastric bypass (n = 34). Relationships among fatty acid binding protein 4 and demographic characteristics, metabolic parameters, and 12-month changes in these values were examined. Visceral and subcutaneous adipose tissue explants from obese nondiabetic patients (n = 5) were obtained and treated with forskolin to evaluate relative secretion of fatty acid binding protein 4 in the different adipose tissue depots. RESULTS: The intensive lifestyle modification and medical management protocol and Roux-en-Y gastric bypass cohorts had similar fasting serum fatty acid binding protein 4 concentrations at baseline. At 1 year, mean serum fatty acid binding protein 4 decreased by 42% in Roux-en-Y gastric bypass participants (P = .002) but did not change significantly in the intensive lifestyle modification and medical management protocol cohort. Percentage of weight change was not a significant predictor of 12-month fatty acid binding protein 4 within treatment arm or in multivariate models adjusted for treatment arm. In adipose tissue explants, fatty acid binding protein 4 was secreted similarly between visceral and subcutaneous adipose tissue. CONCLUSION: After Roux-en-Y gastric bypass, fatty acid binding protein 4 is reduced 12 months after surgery but not after intensive lifestyle modification and medical management protocol in patients with type 2 diabetes mellitus. Fatty acid binding protein 4 was secreted similarly between subcutaneous and visceral adipose tissue explants.

Supplementing Resident Research Funding Through a Partnership With Local Industry.

OBJECTIVE: To develop a model for the supplementation of resident research funding through a resident-hosted clinical immersion with local industry. DESIGN: Designated research residents hosted multiple groups of engineers and business professionals from local industry in general surgery-focused clinical immersion weeks. The participants in these week-long programs are educated about general surgery and brought to the operating room to observe a variety of surgeries. SETTING: This study was performed at the University of Minnesota, in Minneapolis, Minnesota, at a tertiary medical center. PARTICIPANTS: Ten designated research residents hosted general surgery immersion programs. Fifty-seven engineers and business professionals from 5 different local biomedical firms have participated in this program. RESULTS: General surgery research residents (in collaboration with the University of Minnesota's Institute for Engineering in Medicine) have hosted 9 clinical immersion programs since starting the collaborative in 2015. Immersion participant response to the experiences was very positive. Two full-time resident research positions can be funded annually through participation in this program. CONCLUSIONS: With decreasing funding available for surgical research, particularly resident research, innovative ways to fund resident research are needed. The general surgery clinical immersion program at the University of Minnesota has proven its value as a supplement for resident research funding and may be a sustainable model for the future.

Bariatric Surgery: A Perspective for Primary Care.

IN BRIEF Bariatric surgery is the most efficacious treatment for obesity, type 2 diabetes, and other obesity-related comorbidities. In this article, the authors review the current indications for bariatric surgery and discuss the most commonly performed procedures. They analyze medical outcomes of bariatric procedures by reviewing key prospective trials and discuss changes in physiology after these procedures. They conclude by discussing long-term management of bariatric patients by reviewing current guidelines for nutritional support and listing common complications related to these procedures.

Outcomes of Endoscopic Retrograde Cholangiopancreatography (ERCP) and Sphincterotomy for Suspected Sphincter of Oddi Dysfunction (SOD) Post Roux-En-Y Gastric Bypass.

BACKGROUND: Sphincter of Oddi dysfunction (SOD) is thought to be a cause of chronic abdominal pain post Roux-en-Y gastric bypass, and current practice of performing endoscopic retrograde cholangiopancreatography (ERCP) with or without sphincterotomy is not supported by evidence. In addition to the complexity and risks of the procedure in patients with Roux-en-Y anatomy, the outcomes are uncertain and debatable. We performed a retrospective review and analysis of post-gastric bypass patients who had undergone ERCP with sphincterotomy to determine the effectiveness in patients with suspected SOD. METHODS: Over a period of 5 years at the University of Minnesota, we retrospectively reviewed a prospectively collected database of a cohort of patients whom had a previous Roux-en-Y gastric bypass and whom had a subsequent ERCP for suspected SOD. Patients were categorized by modified Milwaukee classification, and outcomes were evaluated by patients' self-reporting of symptoms at follow-up. RESULTS: We identified 50 patients who underwent laparoscopic-assisted gastrostomy for endoscopic retrograde cholangiopancreatography post Roux-en-Y gastric bypass over the study period. Within this group, 35 patients (70%) were suspected to have SOD. Nine patients (25.7%) were classified as type I, 19 patients (54.3%) type II, and seven patients (20%) type III. Thirty-four patients (97.1%) had biliary sphincterotomy, and 17 patients (48.6%) had both biliary and pancreatic sphincterotomy. Fourteen (40%) had repeated ERCP. At median follow-up of 11.5 months, type I SOD had two responders (25%), type II had nine responders (52.9%), and type III had one responder (14.3%). A subgroup analysis did not show significant differences in improvement of symptoms between patients whom had single versus repeated ERCP or biliary sphincterotomy alone versus both biliary and pancreatic sphincterotomy. Three patients (9%) had post-ERCP pancreatitis. CONCLUSIONS: SOD in patients post Roux-en-Y gastric bypass is complex due to multiple confounding factors. Rome III and Milwaukee classification systems assist us in the diagnosis and treatment of sphincter dysfunction until we have a better way to predict treatment response post sphincterotomy. Current treatment is based on the type of disorder and anatomy of biliary ducts. Types I and II sphincter dysfunction particularly associated with dilated biliary duct on imaging have the best response to endoscopic sphincterotomy and therefore should be considered taking into account the risks and benefit. Repeated sphincterotomy and concurrent pancreatic sphincterotomy is generally not useful.

National Trends in Bariatric Surgery 2012-2015: Demographics, Procedure Selection, Readmissions, and Cost.

BACKGROUND: Bariatric surgery is widely accepted as the best treatment for obesity and type 2 diabetes mellitus (T2DM). The Roux-en-Y gastric bypass (RYGB) and the sleeve gastrectomy (SG) have become the predominant bariatric procedures in the USA over the last several years, although the most recent trends in selection are unknown. OBJECTIVE: The objective of this study is to assess selection trends, readmission rates, and cost of bariatric procedures in the USA from 2012 to 2015. METHODS: We used the Premier database from 2012 to 2015 to examine trends in incidence of RYGB, adjustable gastric banding (LAGB), and SG; readmissions; and cost. Multivariate regression was performed to identify predictors of readmission. RESULTS: The proportion of SG went up from 38 to 63% while the RYGB decreased from 44 to 30% over this time period. LAGB has decreased in use from 13 to 2%. In comparison to RYGB, readmission was less likely for SG (OR 0.64), males (OR 0.91), and more likely for black race (OR 1.27). The overall proportion of patients seeking RYGB with type 2 diabetes was higher than with SG (36 versus 25%), but SG has now overtaken RYGB as the most common procedure among diabetics. The SG is less costly than RYGB ($11,183 versus $13,485). CONCLUSIONS: There is a continued overall trend in the increased popularity of the SG and decreased utilization of the RYGB and LAGB, although growth of the SG appears to be slowing. This is also true among patients with type 2 diabetes mellitus. Regardless of surgery type, underinsured and African-American race were more likely to be readmitted.

Sleeve gastrectomy drives persistent shifts in the gut microbiome.

BACKGROUND: Changes in the gut microbiome following bariatric surgery have been causally linked to metabolic benefits. OBJECTIVES: We sought to characterize and assess the stability of gut microbiome shifts following sleeve gastrectomy (SG). SETTING: University laboratories. METHODS: Diet-induced obese mice were randomized to SG or sham surgery. Mice were housed individually or cohoused such that one SG mouse was housed with one weight-matched, sham-operated mouse. Fecal samples were collected before and on postoperative days 7 and 28. Bacterial composition in feces was characterized by using next-generation Illumina sequencing of 16 S rRNA. RESULTS: SG mice lost more weight and were more insulin sensitive than sham mice independent of housing status (P<.05). One week following surgery, fecal samples from all mice showed shifts in the microbiome that only persisted in SG-operated mice. Cohousing did not alter the microbial composition of SG-operated mice. Cohoused sham-operated mice showed a unique shift in microbial composition on postoperative day 28 that differed from individually housed, sham-operated mice (P<.001). Cohousing did not affect metabolic outcomes of either SG or sham surgeries. CONCLUSION: SG results in acute and sustained shifts in the gut microbiome. SG associated shifts are not altered by reexposure to obesity-associated gut microbiota.

Long-Term Body Composition Changes in Women Following Roux-en-Y Gastric Bypass Surgery.

BACKGROUND: Although most individuals experience successful weight loss following Roux-en-Y gastric bypass (RYGB), weight regain is a concern, the composition of which is not well documented. Our aim was to evaluate changes in body composition and handgrip strength as a measure of functional status in participants from a previous 1-year post-RYGB longitudinal study who had undergone RYGB approximately 9 years prior. METHODS: Five women from an original larger cohort were monitored pre-RYGB and 1.5 months, 6 months, 1 year, and 9 years post-RYGB. Body composition was assessed at all time points using dual energy x-ray absorptiometry and multiple dilution. Handgrip strength was measured using a digital isokinetic hand dynamometer (Takei Scientific Instruments, Ltd, Tokyo, Japan). RESULTS: Mean time to final follow-up was 8.7 years. Lean soft tissue (LST) loss over the ~9-year period was on average 11.9 ± 5.6 kg. Compared with 1-year post-RYGB, 9-year LST was 4.4 ± 3.0 kg lower ( P = .03). Fat-free mass decreased over the 9-year period by 12.6 ± 5.8 kg. Mean fat mass (FM) decreased from 75.4 ± 22.6 kg pre-RYGB to 35.5 ± 21.5 kg 1 year post-RYGB but then trended toward an increase of 8.6 ± 7.0 kg between 1 year and 9 years post-RYGB ( P = .053). Loss of LST was correlated with loss of handgrip strength ( r = 0.64, P = .0005). CONCLUSION: The continued loss of lean mass associated with decreased handgrip strength occurring with long-term trend toward FM regain post-RYGB is concerning. The loss of LST and functional strength carries particular implications for the aging bariatric population and should be investigated further.