Xanthogranulomatous inflammation requiring small bowel anastomosis revision: A case report.

BACKGROUND: Xanthogranulomatous inflammation (XGI) is an uncommon process involving an accumulation of inflammatory cells, commonly lipid-laden macrophages. XGI has been described to occur throughout the body but only rarely in the lower gastrointestinal tract. We describe a case of XGI contributing to chronic obstructive symptoms in the terminal ileum, in which the patient had an initial diagnostic laparoscopy, continued to have symptoms, then proceeded to have the definitive treatment. To our knowledge, this is the first report of XGI associated with a prior small bowel anastomosis. CASE SUMMARY: We report the case of a 42-year-old female who presented with intermittent epigastric pain and subjective fevers. She had undergone a laparoscopic small bowel resection for Meckel's diverticulum five years prior. Her workup was notable for computed tomography scan demonstrating mild inflammation and surrounding stranding at the level of the prior anastomosis. She underwent a laparotomy, resection of the prior anastomosis and re-anastomosis, with final histopathological examination findings consistent with mural XGI. CONCLUSION: XGI can occur at the site of a prior bowel anastomosis and cause chronic obstructive symptoms.

Surgical Infection Society Guidelines: 2022 Updated Guidelines for Antibiotic Use in Open Extremity Fractures.

Background: Open fractures, defined as fractures communicating with the environment through a skin wound, cause substantial morbidity after traumatic injury. Current evidence supports administration of prophylactic systemic antibiotic agents to patients with open extremity fractures to decrease infectious complications. Methods: The Therapeutic and Guidelines Committee of The Surgical Infection Society convened to revise guidelines for antibiotic use in open fractures. PubMed was queried for pertinent studies. Evaluation of the published evidence was performed using the GRADE framework. All committee members voted to accept or reject each recommendation. Results: In type I or II open extremity fractures, we recommend against administration of extended-spectrum antibiotic coverage compared with gram-positive coverage alone to decrease infections complications, hospital length of stay or mortality. In type III open extremity fractures, we recommend antibiotic therapy for no more than 24 hrs after injury, in the absence of clinical signs of active infection, to decrease infectious complications, hospital length of stay or mortality, and we recommend against extended antimicrobial coverage beyond gram-positive organisms to decrease infectious complications, hospital length of stay or mortality. In type III open extremity fractures with associated bone loss, we recommend antibiotic therapy in addition to systemic therapy to decrease infectious complications. Conclusions: Although antibiotic agents remain a standard of care for infection prevention after open extremity fractures, our findings and surveys of clinical practice patterns clearly show that additional robust clinical trials are needed to provide stronger corroborating evidence.

Evidence-based surgery for laparoscopic cholecystectomy.

Background: Laparoscopic cholecystectomy is frequently performed for acute cholecystitis and symptomatic cholelithiasis. Considerable variation in the execution of key steps of the operation remains. We conducted a systematic review of evidence regarding best practices for critical intraoperative steps for laparoscopic cholecystectomy. Methods: We identified 5 main intraoperative decision points in laparoscopic cholecystectomy: (1) number and position of laparoscopic ports; (2) identification of cystic artery and duct; (3) division of cystic artery and duct; (4) indications for subtotal cholecystectomy; and (5) retrieval of the gallbladder. PubMed, EMBASE, and Web of Science were queried for relevant studies. Randomized controlled trials and systematic reviews were included for analysis, and evidence quality was assessed using the Grading of Recommendations, Assessment, Development, and Evaluation framework. Results: Fifty-two articles were included. Although all port configurations were comparable from a safety standpoint, fewer ports sometimes resulted in improved cosmesis or decreased pain but longer operative times. The critical view of safety should be obtained for identification of the cystic duct and artery but may be obtained through fundus-first dissection and augmented with cholangiography or ultrasound. Insufficient evidence exists to compare harmonic-shear, clipless ligation against clip ligation of the cystic duct and artery. Stump closure during subtotal cholecystectomy may reduce rates of bile leak and reoperation. Use of retrieval bag for gallbladder extraction results in minimal benefit. Most studies were underpowered to detect differences in incidence of rare complications. Conclusion: Key operative steps of laparoscopic cholecystectomy should be informed by both compiled data and surgeon preference/patient considerations.

Developing and leading a sustainable organization for early career acute care surgeons: Lessons from the inaugural American Association for the Surgery of Trauma Associate Member Council.

ABSTRACT: The associate membership of the American Association for the Surgery of Trauma (AAST) was established in 2019 to create a defined but incorporated entity within the larger AAST for the next generation of acute care surgeons. The Associate Member Council (AMC) was subsequently established in 2020 to provide the new AM with an elected group of leaders who would represent them within the AAST. In its inaugural year, this cohort of junior faculty and surgical trainees had developed for the AM a set of bylaws, a mission statement, a strategic vision, and a succession plan. The experience of the AAST AMC is exemplary of what can be accomplished with collaboration, mentorship, innovation, and tenacity. It has the potential to serve as a template for the creation and vitalization of future professional groups. In this piece, the AMC proposes a blueprint for the successful conception of a new organization.

Fatal Case of Perforated Cytomegalovirus Colitis: Case Report and Systematic Review.

Objective: We describe a patient with history of heart transplant on maintenance immunosuppression who presented with sigmoid colon perforation from cytomegalovirus (CMV) colitis and performed a systematic review of outcomes after perforated CMV colitis. Background: Cytomegalovirus enterocolitis is uncommon among solid organ transplant patients and can result in small or large bowel perforation. Methods: We systematically reviewed articles describing patients with CMV enterocolitis with small or large bowel perforations from PubMed, Embase, and Web of Science from database inception to February 2021. Results: Seventy-seven articles were identified containing 84 patients with perforated CMV enterocolitis. The most prevalent comorbid diagnosis was human immunodeficiency virus (HIV; 27 patients, 32%), and 37 patients (44%) were taking corticosteroids at time of presentation. The ileum was the most common location for a perforation (26 patients, 31%). Odds of survival were lower for patients with small bowel perforation (odds ratio [OR], 0.38; 95% confidence interval [CI], 0.14-0.98) and HIV/acquired immunodeficiency syndrome (AIDS; OR, 0.32; 95% CI, 0.11-0.88). Odds of survival were higher for patients with large bowel perforation (OR, 2.64; 95% CI, 1.03-7.09), radiographically diagnosed perforation (OR, 3.45; 95% CI, 1.12-11.60) and those who received a CMV antiviral (OR, 9.19; 95% CI, 3.26-28.48). Conclusions: Perforated CMV enterocolitis is uncommon even in immunocompromised hosts. Clinicians should maintain a high level of suspicion for CMV-induced bowel perforation in this population because antiviral treatment is associated with increased odds of survival.

Evidence-based surgery for laparoscopic appendectomy: A stepwise systematic review.

INTRODUCTION: Appendectomy is a common emergency surgery performed globally. Despite the frequency of laparoscopic appendectomy, consensus does not exist on the best way to perform each procedural step. We identified literature on key intraoperative steps to inform best technical practice during laparoscopic appendectomy. METHODS: Research questions were framed using the population, indication, comparison, outcome (PICO) format for 6 key operative steps of laparoscopic appendectomy: abdominal entry, placement of laparoscopic ports, division of mesoappendix, division of appendix, removal of appendix, and fascial closure. These questions were used to build literature queries in PubMed, EMBASE, and the Cochrane Library databases. Evidence quality and certainty was assessed using Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) definitions. RESULTS: Recommendations were rendered for 6 PICO questions based on 28 full length articles. Low quality evidence favors direct trocar insertion for abdominal entry and establishment of pneumoperitoneum. Single port appendectomy results in improved cosmesis with unclear clinical implications. There was insufficient data to determine the optimal method of appendiceal stump closure, but use of a specimen extraction bag reduces rates of superficial surgical site infection and intra-abdominal abscess. Port sites made with radially dilating trocars are less likely to necessitate closure and are less likely to result in port site hernia. When port sites are closed, a closure device should be used. CONCLUSION: Key operative steps of laparoscopic appendectomy have sufficient data to encourage standardized practice.

Impact of COVID-19 on presentation, management, and outcomes of acute care surgery for gallbladder disease and acute appendicitis.

BACKGROUND: The ongoing coronavirus disease 2019 (COVID-19) pandemic has significantly disrupted both elective and acute medical care. Data from the early months suggest that acute care patient populations deferred presenting to the emergency department (ED), portending more severe disease at the time of presentation. Additionally, care for this patient population trended towards initial non-operative management. AIM: To examine the presentation, management, and outcomes of patients who developed gallbladder disease or appendicitis during the pandemic. METHODS: A retrospective chart review of patients diagnosed with acute cholecystitis, symptomatic cholelithiasis, or appendicitis in two EDs affiliated with a single tertiary academic medical center in Northern California between March and June, 2020 and in the same months of 2019. Patients were selected through a research repository using international classification of diseases (ICD)-9 and ICD-10 codes. Across both years, 313 patients were identified with either type of gallbladder disease, while 361 patients were identified with acute appendicitis. The primary outcome was overall incidence of disease. Secondary outcomes included presentation, management, complications, and 30-d re-presentation rates. Relationships between different variables were explored using Pearson's r correlation coefficient. Variables were compared using the Welch's t-Test, Chi-squared tests, and Fisher's exact test as appropriate. RESULTS: Patients with gallbladder disease and appendicitis both had more severe presentations in 2020. With respect to gallbladder disease, more patients in the COVID-19 cohort presented with acute cholecystitis compared to the control cohort [50% (80) vs 35% (53); P = 0.01]. Patients also presented with more severe cholecystitis in 2020 as indicated by higher mean Tokyo Criteria Scores [mean (SD) 1.39 (0.56) vs 1.16 (0.44); P = 0.02]. With respect to appendicitis, more patients were diagnosed with a perforated appendix at presentation in 2020 [20% (36) vs 16% (29); P = 0.02] and a greater percentage were classified as emergent cases using the emergency severity index [63% (112) vs 13% (23); P < 0.001]. While a greater percentage of patients were admitted to the hospital for gallbladder disease in 2020 [65% (104) vs 50% (76); P = 0.02], no significant differences were observed in hospital admissions for patients with appendicitis. No significant differences were observed in length of hospital stay or operative rate for either group. However, for patients with appendicitis, 30-d re-presentation rates were significantly higher in 2020 [13% (23) vs 4% (8); P = 0.01]. CONCLUSION: During the COVID-19 pandemic, patients presented with more severe gallbladder disease and appendicitis. These findings suggest that the pandemic has affected patients with acute surgical conditions.

Intercostal nerve cryoablation during surgical stabilization of rib fractures.

BACKGROUND: Intercostal nerve cryoablation (IC) offers potential for targeted and durable analgesia for patients with traumatic rib fractures. Our pilot study aimed to investigate thoracoscopic IC's safety, feasibility, and preliminary efficacy for patients undergoing surgical stabilization of rib fractures (SSRF). We hypothesized that concurrent surgical stabilization of rib fractures and intercostal nerve cryoablation (SSRF-IC) is a safe and feasible procedure without immediate or long-term complications. METHODS: We retrospectively evaluated patients 18 years or older who underwent SSRF (with or without IC) for acute rib fractures at our level I trauma center between September 1, 2019, and September 30, 2020. We performed IC under thoracoscopic visualization (-70°C for 2 minutes per intercostal nerve bundle). Among patients whose only operative procedure during hospitalization was SSRF, we evaluated post-SSRF length of stay, operative times, opioid requirements (oral morphine equivalents), and pain scores (Numerical Rating Scale). Generalized estimating equations compared SSRF and SSRF-IC group outcomes (population mean [robust standard error]). We assessed long-term outcomes of patients who underwent SSRF-IC. RESULTS: Thirty-four patients (144 ribs) underwent SSRF; of these, 20 patients (135 ribs) underwent SSRF-IC. Patients who did and did not undergo concurrent IC had no significant difference demographic, injury, or hospitalization characteristics. Among 20 patients who did not undergo other operations, 12 underwent SSRF-IC. We did not find significant difference between SSRF and SSRF-IC groups' median operative times or post-SSRF length of stay. Compared with SSRF group, SSRF-IC group did not have statistically significant change in pain score (0.2 [1.5] lower) or opioid use (43.9 [86.1] mg/d greater) between 12 hours before SSRF and last 24 admission hours. Among 17 SSRF-IC patients who followed-up postdischarge (median [range], 160 [9-357] days), one reported mild chest wall paresthesia; no other complications were reported. CONCLUSION: This pilot study performing 135 intercostal nerve cryoablations on 20 patients suggests that IC is safe and feasible for patients undergoing SSRF. Evaluating IC's analgesic efficacy for rib fractures requires further study. LEVEL OF EVIDENCE: Therapeutic, Level V.

Epidemiology of pediatric trauma during the COVID-19 pandemic shelter in place.

INTRODUCTION: The first COVID-19 cases occurred in the US in January of 2020, leading to the implementation of shelter in place. This study seeks to define the impact of shelter in place on the epidemiology of pediatric trauma. METHODS: We examined pediatric trauma admissions at 5 Level 1 and 1 Level 2 US pediatric trauma centers between January 1 and June 30, 2017-2020. Demographic and injury data were compared between pre- and post-shelter in place patient cohorts. RESULTS: A total of 8772 pediatric trauma activations were reviewed. There was a 13% decrease in trauma volume in 2020, with a nadir at 16 days following implementation of shelter in place. Injury severity scores were higher in the post-shelter in place cohort. The incidence of nonmotorized vehicle accidents and gunshot wounds increased in the post-shelter in place cohort. CONCLUSION: We found an overall decrease in pediatric trauma volume following shelter in place. However, injuries tended to be more severe. Our findings help inform targeted injury prevention campaigns during future pandemics.

Functional and Taxonomic Dysbiosis of the Gut, Urine, and Semen Microbiomes in Male Infertility.

BACKGROUND: Little is known about the role of the genitourinary and gastrointestinal microbiota in the pathogenesis of male infertility. OBJECTIVE: To compare the taxonomic and functional profiles of the gut, semen, and urine microbiomes of infertile and fertile men. DESIGN, SETTING, AND PARTICIPANTS: We prospectively enrolled 25 men with primary idiopathic infertility and 12 healthy men with proven paternity, and we collected rectal swabs, semen samples, midstream urine specimens, and experimental controls. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We performed comprehensive semen analysis, 16S rRNA sequencing for quantitative high-resolution taxonomy, and shotgun metagenomics with a median of 140 million reads per sample for functional metabolic pathway profiling. RESULTS AND LIMITATIONS: We identified a diverse semen microbiome with modest similarity to the urinary microbiome. Infertile men harbored increased seminal α-diversity and distinct ß-diversity, increased seminal Aerococcus, and decreased rectal Anaerococcus. Prevotella abundance was inversely associated with sperm concentration, and Pseudomonas was directly associated with total motile sperm count. Vasectomy appeared to alter the seminal microbiome, suggesting a testicular or epididymal contribution. Anaerobes were highly over-represented in the semen of infertile men with a varicocele, but oxidative stress and leukocytospermia were associated with only subtle differences. Metagenomics data identified significant alterations in the S-adenosyl-L-methionine cycle, which may play a multifaceted role in the pathogenesis of infertility via DNA methylation, oxidative stress, and/or polyamine synthesis. CONCLUSIONS: This pilot study represents the first comprehensive investigation into the microbiome in male infertility. These findings provide the foundation for future investigations to explore causality and identify novel microbiome-based diagnostics and therapeutics for men with this complex and emotionally devastating disease. PATIENT SUMMARY: We explored the resident populations of bacteria living in the gut, semen, and urine of infertile and fertile men. We found several important bacterial and metabolic pathway differences with the potential to aid in diagnosing and treating male infertility in the future.

Prospectively Assigned AAST Grade versus Modified Hinchey Class and Acute Diverticulitis Outcomes.

BACKGROUND: The American Association for the Surgery of Trauma (AAST) recently developed a classification system to standardize outcomes analyses for several emergency general surgery conditions. To highlight this system's full potential, we conducted a study integrating prospective AAST grade assignment within the electronic medical record. METHODS: Our institution integrated AAST grade assignment into our clinical workflow in July 2018. Patients with acute diverticulitis were prospectively assigned AAST grades and modified Hinchey classes at the time of surgical consultation. Support vector machine-a machine learning algorithm attuned for small sample sizes-was used to compare the associations between the two classification systems and decision to operate and incidence of complications. RESULTS: 67 patients were included (median age of 62 y, 40% male) for analysis. The decision for operative management, hospital length of stay, intensive care unit admission, and intensive care unit length of stay were associated with both increasing AAST grade and increasing modified Hinchey class (all P < 0.001). AAST grade additionally showed a correlation with complication severity (P = 0.02). Compared with modified Hinchey class, AAST grade better predicted decision to operate (88.2% versus 82.4%). CONCLUSIONS: This study showed the feasibility of electronic medical record integration to support the full potential of AAST classification system's utility as a clinical decision-making tool. Prospectively assigned AAST grade may be an accurate and pragmatic method to find associations with outcomes, yet validation requires further study.

Creation and implementation of a novel clinical workflow based on the AAST uniform anatomic severity grading system for emergency general surgery conditions.

OBJECTIVE: Emergency general surgery (EGS) conditions encompass a variety of diseases treated by acute care surgeons. The heterogeneity of these diseases limits infrastructure to facilitate EGS-specific quality improvement (QI) and research. A uniform anatomic severity grading system for EGS conditions was recently developed to fill this need. We integrated this system into our clinical workflow and examined its impact on research, surgical training, communication, and patient care. METHODS: The grading system was integrated into our clinical workflow in a phased fashion through formal education and a written handbook. A documentation template was also deployed in our electronic medical record to prospectively assign severity scores at the time of patient evaluation. Mixed methods including a quantitative survey and qualitative interviews of trainees and attending surgeons were used to evaluate the impact of the new workflow and to identify obstacles to its adoption. RESULTS: We identified 2291 patients presenting with EGS conditions during our study period. The most common diagnoses were small bowel obstruction (n=470, 20.5%), acute cholecystitis (n=384, 16.8%), and appendicitis (n=370, 16.1%). A total of 21 qualitative interviews were conducted. Twenty interviewees (95.2%) had a positive impression of the clinical workflow, citing enhanced patient care and research opportunities. Fifteen interviewees (75.0%) reported the severity grading system was a useful framework for clinical management, with five participants (25.0%) indicating the system was useful to facilitate clinical communication. Participants identified solutions to overcome barriers to adoption of the clinical workflow. CONCLUSIONS: The uniform anatomic severity grading system can be readily integrated into a clinical workflow to facilitate prospective data collection for QI and research. The system is perceived as valuable by users. Educational initiatives that focus on increasing familiarity with the system and its benefits will likely improve adoption of the classification system and the clinical workflow that uses it. LEVEL OF EVIDENCE: Level III.

Appendicitis in Low-Resource Settings.

Background: Acute appendicitis is one of the most common surgical emergencies globally. Its incidence is increasing in low- and middle-Human Development Index countries (LMHDICs). Although a proportion of patients can be treated successfully with non-operative management consisting of antibiotics, supportive therapy, and close observation, current diagnostic algorithms lack the granularity to identify these patients accurately. Methods: We reviewed published literature describing practice patterns and clinical outcomes for appendicitis in LMHDICs and compared them with studies from high-Human Development Index countries, as well as guidelines published by international surgical societies. Results: We identified shortcomings in current diagnostic and therapeutic strategies used in LMHDICs. Delays in obtaining surgical care inherent in many LMHDIC healthcare systems make prompt surgical care the mainstay for the treatment of acute appendicitis. Laparoscopic appendectomy leads to better outcomes than open appendectomy in resource-constrained settings and when available should be the surgical technique of choice. Conclusions: Acute appendicitis is common in LMHDICs, and if possible, laparoscopic appendectomy should be the procedure of choice.

A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.

Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria box.

The apicomplexan parasites Cryptosporidium parvum and Cryptosporidium hominis are major etiologic agents of human cryptosporidiosis. The infection is typically self-limited in immunocompetent adults, but it can cause chronic fulminant diarrhea in immunocompromised patients and malnutrition and stunting in children. Nitazoxanide, the current standard of care for cryptosporidiosis, is only partially efficacious for children and is no more effective than a placebo for AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidium biology impede the development of better drugs for treating cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture (MMV) Open Access Malaria Box for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited submicromolar potency against C. parvum. Potency was conserved in a subset of compounds from each scaffold with varied physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvum growth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of the compounds in the allopurinol-based series against T. gondii and C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvum and other apicomplexan parasites.

Drug repurposing: mining protozoan proteomes for targets of known bioactive compounds.

OBJECTIVE: To identify potential opportunities for drug repurposing by developing an automated approach to pre-screen the predicted proteomes of any organism against databases of known drug targets using only freely available resources. MATERIALS AND METHODS: We employed a combination of Ruby scripts that leverage data from the DrugBank and ChEMBL databases, MySQL, and BLAST to predict potential drugs and their targets from 13 published genomes. Results from a previous cell-based screen to identify inhibitors of Cryptosporidium parvum growth were used to validate our in-silico prediction method. RESULTS: In-vitro validation of these results, using a cell-based C parvum growth assay, showed that the predicted inhibitors were significantly more likely than expected by chance to have confirmed activity, with 8.9-15.6% of predicted inhibitors confirmed depending on the drug target database used. This method was then used to predict inhibitors for the following 13 disease-causing protozoan parasites, including: C parvum, Entamoeba histolytica, Giardia intestinalis, Leishmania braziliensis, Leishmania donovani, Leishmania major, Naegleria gruberi (in proxy of Naegleria fowleri), Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei and Trypanosoma cruzi. CONCLUSIONS: Although proteome-wide screens for drug targets have disadvantages, in-silico methods can be developed that are fast, broad, inexpensive, and effective. In-vitro validation of our results for C parvum indicate that the method presented here can be used to construct a library for more directed small molecule screening, or pipelined into structural modeling and docking programs to facilitate target-based drug development.

Drug repurposing screen reveals FDA-approved inhibitors of human HMG-CoA reductase and isoprenoid synthesis that block Cryptosporidium parvum growth.

Cryptosporidiosis, a diarrheal disease usually caused by Cryptosporidium parvum or Cryptosporidium hominis in humans, can result in fulminant diarrhea and death in AIDS patients and chronic infection and stunting in children. Nitazoxanide, the current standard of care, has limited efficacy in children and is no more effective than placebo in patients with advanced AIDS. Unfortunately, the lack of financial incentives and the technical difficulties associated with working with Cryptosporidium parasites have crippled efforts to develop effective treatments. In order to address these obstacles, we developed and validated (Z' score = 0.21 to 0.47) a cell-based high-throughput assay and screened a library of drug repurposing candidates (the NIH Clinical Collections), with the hopes of identifying safe, FDA-approved drugs to treat cryptosporidiosis. Our screen yielded 21 compounds with confirmed activity against C. parvum growth at concentrations of <10 µM, many of which had well-defined mechanisms of action, making them useful tools to study basic biology in addition to being potential therapeutics. Additional work, including structure-activity relationship studies, identified the human 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor itavastatin as a potent inhibitor of C. parvum growth (50% inhibitory concentration [IC(50)] = 0.62 µM). Bioinformatic analysis of the Cryptosporidium genomes indicated that the parasites lack all known enzymes required for the synthesis of isoprenoid precursors. Additionally, itavastatin-induced growth inhibition of C. parvum was partially reversed by the addition of exogenous isopentenyl pyrophosphate, suggesting that itavastatin reduces Cryptosporidium growth via on-target inhibition of host HMG-CoA reductase and that the parasite is dependent on the host cell for synthesis of isoprenoid precursors.

Control of Entamoeba histolytica adherence involves metallosurface protease 1, an M8 family surface metalloprotease with homology to leishmanolysin.

Invasive amebiasis due to Entamoeba histolytica infection is an important cause of morbidity in developing countries. The E. histolytica genome contains two homologues to the metalloprotease leishmanolysin gene, Entamoeba histolytica MSP-1 (EhMSP-1) and EhMSP-2, while the commensal ameba Entamoeba dispar has lost EhMSP-1. In this study, we sought to characterize E. histolytica metallosurface protease 1 (EhMSP-1). Using immunoprecipitation and a model substrate, we found that EhMSP-1 was a functional metalloprotease. Confocal microscopy and flow cytometry revealed that EhMSP-1 localized to the cell surface and revealed the existence of distinct, nonclonal trophozoite populations with high and low EhMSP-1 surface abundance that became synchronized following serum starvation. Phenotypic assays were performed after silencing EhMSP-1. Adherence of EhMSP-1-deficient trophozoites to tissue culture cell monolayers was more than five times greater than that of control amebas, but surface staining of several antigens, including the galactose adherence lectin, was unchanged. EhMSP-1 silencing similarly increased adherence to both viable and apoptotic Jurkat lymphocytes. Tissue culture cell monolayer destruction was reduced by EhMSP-1 silencing, although it was blocked almost completely by inhibiting cysteine proteases. Consistent with a primary defect in regulation of amebic adherence, EhMSP-1 silencing also resulted in reduced mobility on tissue culture cell monolayers and in increased phagocytosis. In conclusion, EhMSP-1 was shown to be a surface metalloprotease involved in regulation of amebic adherence, with additional effects on cell motility, cell monolayer destruction, and phagocytosis.