A double-strain TM (gp45) polypeptide antigen and its application in the serodiagnosis of equine infectious anemia.

Lentiviruses, including equine infectious anemia virus (EIAV), are considered viral quasispecies because of their intrinsic genetic, structural and phenotypic variability. Immunoenzymatic tests (ELISA) for EIAV reported in the literature were obtained mainly by using the capsid protein p26, which is derived almost exclusively from a single strain (Wyoming), and do not reflect the great potential epitopic variability of the EIAV quasispecies. In this investigation, the GenBank database was exploited in a systematic approach to design a set of representative protein antigens useful for EIAV serodiagnosis. The main bioinformatic tools used were clustering, molecular modelling, epitope predictions and aggregative/ solubility predictions. This approach led to the design of two antigenic proteins, i.e. a full sequence p26 capsid protein and a doublestrain polypeptide derived from the gp45 transmembrane protein fused to Maltose Binding Protein (MBP) that were expressed by recombinant DNA technology starting from synthetic genes, and analyzed by circular dichroism (CD) spectroscopy. Both proteins were used in an indirect ELISA test that can address some of the high variability of EIAV. The novel addition of the gp45 double-strain antigen contributed to enhance the diagnostic sensitivity and could be also useful for immunoblotting application.

Minimally invasive distal pancreatectomy: mapping surgical maneuvers towards operative standardization.

AIM: To analyze the minimally invasive surgical maneuvers currently performed to remove pancreatic tail, with or without preservation of the spleen, for benign and borderline malignant neoplasms. MATERIAL AND METHODS: We described operative steps and technical pitfalls encountered during laparoscopic and robotic distal pancreatectomy. The methodology of research focused on recruitment of evidence-based surgical strategies and critical analysis of modern minimally invasive techniques. RESULTS: Laparoscopic and robotic distal pancreatectomy have gradually accepted by pancreatic surgeons and clinical evidences document its growing interest. The choice of patient positioning, port placement, surgical dissection and operative techniques used for pancreatic parenchymal transection is not codified and changes according to personal preference. The technical variability in minimally invasive approach to pancreatic surgery strongly depends depends on surgeon's training and to limited application of these procedures in single institutions. CONCLUSIONS: Pancreatic surgeons worldwide accept laparoscopic and robotic distal pancreatectomy but the best intraoperative praxis is not defined in clinical routine. To date, the pancreatic resection adopts hybrid techniques and the conduction of minimally invasive resection depends to surgeon's experience, patient body habitus and location of pancreatic lesion. Although several technical variations have described, no standardization of the operative minimally invasive surgical method is convincingly built. KEY WORDS: Laparoscopy, Minimally invasive surgery, Pancreas, Robotic pancreatectomy.

Design and structural bioinformatic analysis of polypeptide antigens useful for the SRLV serodiagnosis.

Due to their intrinsic genetic, structural and phenotypic variability the Lentiviruses, and specifically small ruminant lentiviruses (SRLV), are considered viral quasispecies with a population structure that consists of extremely large numbers of variant genomes, termed mutant spectra or mutant cloud. Immunoenzymatic tests for SRLVs are available but the dynamic heterogeneity of the virus makes the development of a diagnostic "golden standard" extremely difficult. The ELISA reported in the literature have been obtained using proteins derived from a single strain or they are multi-strain based assay that may increase the sensitivity of the serological diagnosis. Hundreds of SRLV protein sequences derived from different viral strains are deposited in GenBank. The aim of this study is to verify if the database can be exploited with the help of bioinformatics in order to have a more systematic approach in the design of a set of representative protein antigens useful in the SRLV serodiagnosis. Clustering, molecular modelling, molecular dynamics, epitope predictions and aggregative/solubility predictions were the main bioinformatic tools used. This approach led to the design of SRLV antigenic proteins that were expressed by recombinant DNA technology using synthetic genes, analyzed by CD spectroscopy, tested by ELISA and preliminarily compared to currently commercially available detection kits.

The Potential of Colonic Tumor Tissue Fusobacterium nucleatum to Predict Staging and Its Interplay with Oral Abundance in Colon Cancer Patients.

BACKGROUND: Intestinal microbiota dysbiosis may enhance the carcinogenicity of colon cancer (CC) by the proliferation and differentiation of epithelial cells. Oral Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) have the ability to invade the gut epithelium, promoting tumor progression. The aim of the study was to assess whether the abundance of these odontopathogenic bacteria was associated with colon cancer. We also investigated how lifestyle factors could influence the oral Fn and Pg abundance and CC. METHODS: Thirty-six CC patients were included in the study to assess the Pg and Fn oral and colon tissue abundance by qPCR. Oral health data, food habits and lifestyles were also recorded. RESULTS: Patients had a greater quantity of Fn in the oral cavity than matched CC and adjacent non-neoplastic mucosa (adj t) tissues (p = 0.004 and p < 0.001). Instead, Pg was not significantly detected in colonic tissues. There was an association between the Fn quantity in the oral and CC tissue and a statistically significant relation between the Fn abundance in adenocarcinoma (ADK) and staging (p = 0.016). The statistical analysis revealed a tendency towards a greater Fn quantity in CC (p = 0.073, η2p = 0.12) for high-meat consumers. CONCLUSION: In our study, Pg was absent in colon tissues but was correlated with the oral inflammation gingival and plaque indices. For the first time, there was evidence that the Fn oral concentration can influence colon tissue concentrations and predict CC prognosis.

Extracorporeal versus intracorporeal anastomosis after laparoscopic right hemicolectomy: cost-effectiveness analysis.

AIM: The cost effectiveness of the laparoscopic right hemicolectomy is still debated, and the current literature does not allow to be drawn certain conclusion. Our study compared direct clinical costs and outcomes for laparoscopic right hemicolectomy with the two most used type of anastomosis, such as ExtraCorporeal Anastomosis (ECA) and IntraCorporeal Anastomosis (ICA). MATERIAL AND METHODS: In this retrospective study, all patients who underwent laparoscopic right hemicolectomy with intracorporeal and extracorporeal anastomosis between January 2016 and April 2018 were evaluated. Patients were divided into two groups according to the type of anastomosis: ECA or ICA. RESULTS: Thirty ECA and twenty-nine ICA patients were included in the study. Operative time was significantly longer in ICA group than ECA group (p < 0.001). No significant differences between the groups were seen in terms of timeto- first flatus, postoperative complications and re-admission rate. ICA group showed a shorter hospitalization (5 vs 6; p < 0.022). In the ICA group, considering only the surgical tools were more expensive than in ECA (1435.6 € vs 72 €). Nevertheless, the total cost of the two procedures in similar (14451.36 € in ECA group vs 14631.04 € in ICA group). CONCLUSION: ECA and ICA are comparable in terms of postoperative outcomes. ICA requires much more expensive charges, compared to a minor hospitalization. The ECA seems to be less expensive in terms of surgical supplies but the longer recovery determines an increase in the total cost resulting in a non-inferiority of one compared to the other technique. KEY WORDS: Cost-analysis, ExtraCorporeal Anastomosis, IntraCorporeal Anastomosis, Laparoscopy, Right Hemicolectomy.

Meropenem and ciprofloxacin in complicated gastric surgery for cancer patients: A simple SPE-UHPLC-PDA method for their determination in human plasma.

A simple and rapid ultra-high-performance liquid chromatographic (UHPLC) for the simultaneous determination of meropenem and ciprofloxacin in human plasma was developed and validated. All of the analytes were separated in <5 min. A solid-phase extraction method was applied from sample preparation. Analytical separation was performed on a Poroshell SB C18 column (50 × 2.1 mm, 2.7 µm particle size) with photodiode array (PDA) detection. Meropenem and ciprofloxacin were determined at wavelengths of 300 and 277 nm, respectively. The mobile phase was a mixture of acetonitrile-10 mm ammonium acetate-methanol in gradient elution. The method has been validated for both drugs in gastric surgery for cancer patients. The method showed good linearity with correlation coefficients, r2  = 0.994 for the two drugs, as well as high precision (RSD < 10.5% in each case); accuracy ranged from -5.8 to +6.0%. The limit of quantitation of the two drugs was established at 0.02 and 0.01 µg/mL, respectively. Meropenem, ciprofloxacin and the internal standard were extracted from human plasma with a mean recovery ranging from 92.5 to 98.6%. The method was applied to quantify the drugs dosage in complicated gastric surgery patients.

Impact of one-to-one tutoring on fundamentals of laparoscopic surgery (FLS) passing rate in a single center experience outside the United States: a randomized controlled trial.

BACKGROUND: Outside the US, FLS certification is not required and its teaching methods are not well standardized. Even if the FLS was designed as "stand alone" training system, most of Academic Institution offer support to residents during training. We present the first systematic application of FLS in Italy. Our aim was to evaluate the role of mentoring/coaching on FLS training in terms of the passing rate and global performance in the search for resource optimization. METHODS: Sixty residents in general surgery, obstetrics & gynecology, and urology were selected to be enrolled in a randomized controlled trial, practicing FLS with the goal of passing a simulated final exam. The control group practiced exclusively with video material from SAGES, whereas the interventional group was supported by a mentor. RESULTS: Forty-six subjects met the requirements and completed the trial. For the other 14 subjects no results are available for comparison. One subject for each group failed the exam, resulting in a passing rate of 95.7%, with no obvious differences between groups. Subgroup analysis did not reveal any difference between the groups for FLS tasks. CONCLUSION: We confirm that methods other than video instruction and deliberate FLS practice are not essential to pass the final exam. Based on these results, we suggest the introduction of the FLS system even where a trained tutor is not available. This trial is the first single institution application of the FLS in Italy and one of the few experiences outside the US. Trial Number: NCT02486575 ( https://www.clinicaltrials.gov ).

Is right colectomy a complete learning procedure for a robotic surgical program?

This study analyses the utility of right colectomy as a learning procedure at the beginning of a robotic surgical program. The hypothesis is that right colectomy contains all the technical steps necessary to acquire basic abilities in robotics surgery. The first 23 consecutive robotic right colectomy performed at the beginning of a robotic program were analysed. All surgical times were recorded in the operating room and second checked on a dedicated video-database. Specific robotic times were analysed using CUSUM method to evaluate the learning curve. CUSUM-derived learning phases were compared. Fourteen males and nine females with a mean age of 68.7 (46-84) underwent robotic right colectomy. The mean overall time was 265.3 min (180-320 min), docking time was 7 min (5-12 min), console time was 205.9 min (145-260 min), and anastomotic time was 43.6 (25-60 min). CUSUM analyses identified two learning phases: "starting phase" and "consolidation phase". Interphase comparison confirmed the significant (p < 0.05) differences between the two phases. Robotic technology facilitates the training process in minimally invasive colorectal surgery. At the beginning of the learning curve, right colectomy could represent a complete procedure to be proficient in robotic colorectal surgery.

Meropenem, levofloxacin and linezolid in human plasma of critical care patients: A fast semi-automated micro-extraction by packed sorbent UHPLC-PDA method for their simultaneous determination.

An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was developed and validated for the simultaneous quantification of meropenem, linezolid, and levofloxacin in human plasma and applied in human plasma of critical care patients. A semi-automated microextraction by packed sorbent (MEPS) for sample preparation was used. All parameters in the extraction step (pH, sample volume, sample dilution and number of aspiration - ejection cycles) and in the desorption step (percentage of acetonitrile in the solvent of elution and number of aspirations of elution solvent through the device) were statistically significant when the recovery was used as response. The method showed good linearity with correlation coefficients, r2>0.9991 for the three drugs, as well as high precision (RSD%<10.83% in each case). Accuracy ranged from -7.8% to +6.7%. The limit of quantification of the three drugs was established at 0.01µg/mL for linezolid and levofloxacin and 0.02µg/mL for meropenem. Linezolid, meropenem, levofloxacin and the internal standard were extracted from human plasma with a mean recovery ranged from 92.4% to 97.4%. During validation, the concentration of meropenem, linezolid and levofloxacin was found to be stable after 3 freeze-thaw cycles and for at least 24h after extraction. This method will be subsequently used to quantify the drugs in patients to establish if the dosage regimen given is sufficient to eradicate the infection at the target site.

Development of a dried blood spot HPLC-PDA method for the analysis of linezolid and ciprofloxacin in hospital-acquired pneumonia patients.

This study developed a high performance liquid chromatography (HPLC) method involving dried blood spotting (DBS) as a sampling method for the therapeutic drug monitoring of antimicrobial combination therapy with linezolid and ciprofloxacin. DBS for standards, quality control samples, and patient samples was excised and then extracted using a mixture of methanol/water/formic acid 80:20:0.1 (v/v/v), respectively. Linezolid (LZD) and ciprofloxacin (CPR) were measured by HPLC using a Kinetex EVO C18 (100 × 4.6 mm I.D. 2.6 µm particle size). Mobile phase consisted of 10 mM ammonium acetate (A) and a mixture of acetonitrile and methanol (B), both containing 0.1% triethylamine, in gradient elution. Detection was carried out at 251 nm for linezolid (LZD) and 277 for ciprofloxacin (CPR). Ulifloxacin was used as an internal standard. The internal standard, LZD, and CPR were eluted in 7.90, 7.18, and 8.89 min, respectively. Total run-time was 20 min. Calibration curves were constructed in the range of 0.05-30 µg/mL for LZD and 0.02-10 µg/mL for CPR, respectively. The intra- and inter-day precision (RSD values) did not exceed 9.43%, the intra- and inter-day accuracy (accuracy %) ranged between 96.2 and 106.2%. Haematocrit (Hct) effects were investigated to obtain a linear correlation between haematocrit values and volume of blood. Copyright © 2017 John Wiley & Sons, Ltd.

Development and validation of a fast micro-extraction by packed sorbent UHPLC-PDA method for the simultaneous determination of linezolid and ciprofloxacin in human plasma from patients with hospital-acquired pneumonia.

An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was developed and validated for the simultaneous quantification of linezolid and ciprofloxacin in human plasma and applied in hospital acquired pneumonia patients (HAP). The method uses a semi-automated microextraction by packed sorbent for sample preparation. All parameters in the extraction step (pH, sample volume, sample dilution and number of aspiration - ejection cycles) and in the desorption step (percentage of acetonitrile in the solvent of elution and number of aspirations of elution solvent through the device) were statistically significant when the recovery was used as response. The method showed good linearity with correlation coefficients, r2>0.9995 for the two drugs, as well as high precision (RSD%<9.77% in each case), accuracy ranged from -6.2% to +8.2. The limit of quantification of the two drugs was established at 0.01 and 0.02µg/mL for ciprofloxacin and linezolid, respectively. Linezolid, ciprofloxacin and internal standard were extracted from human plasma with a mean recovery ranging from 92.4% to 97.4%. During validation, the concentrations of linezolid and ciprofloxacin were found to be stable after 3 freeze-thaw cycles and for at least 24h after extraction. This method will subsequently be used to quantify the drugs dosage in patients with HAP to establish if the dosage regimen given is sufficient to eradicate the infection at the target site.

Left colon obstruction due to non-reducible Spigelian hernia of the right side. Report of a case and literature review.

AIM: Describe a rare case of intestinal obstruction due to sigmoid colon incarceration into a right Spigelian hernia and provide a literature review about its surgical management. MATERIAL OF STUDY: An 88 year-old man presented with a 3-day history of right lower quadrant pain and abdominal distension. Clinical examination revealed a not reducible palpable mass on right side of the anterior abdominal wall. Computed tomography shown signs of intestinal obstruction and a Spigelian hernia of about 3 cm. RESULTS: Sigmoid colon was incarcerated in the defect but no signs of intestinal ischemia was found during the surgical exploration. The hernia content was reduced and the defect was repaired with a polypropylene mesh placed in the pre-peritoneal space. Postoperative period was uneventful and the patient was discharged on the fourth postoperative day. DISCUSSION: Spigelian hernia occurs through congenital or acquired defects in the Spigelian fascia at the level of the semicircular line. Clinical presentation depends on the size and the pattern of the hernia defect. In some cases, symptoms are no specific and uncommon findings have been reported so far. Preoperative establishment of the hernia content and the location of the sac is mandatory to plan a correct surgical strategy. Intestinal incarceration often occurs and suggests a prompt surgical exploration. CONCLUSIONS: Colonic obstruction secondary to an incarcerated Spigelian hernia is a rare but dangerous occurrence. Surgical approach is selected based on patient's conditions and surgeon's expertise. In a non-contaminated surgical field, the use of prosthesis is recommended. KEY WORDS: Intestinal obstruction, Mesh repair, Spigelian hernia.

Vertebral rotation in adolescent idiopathic scoliosis calculated by radiograph and back surface analysis-based methods: correlation between the Raimondi method and rasterstereography.

PURPOSE: The aim of the present research is to evaluate the relationship between an X-ray-based method (i.e. the Raimondi method) and rasterstereography in the evaluation of vertebral rotation (VR) in a sample of adolescent idiopathic scoliosis (AIS) patients. METHODS: A total of 25 patients (9 males; mean age 14 ± 3 years; mean height 160.7 ± 11.9 cm; mean weight 52.4 ± 10.7 kg) were considered for the present analysis. The mean Cobb angle was 30° ± 9°. The evaluation of VR on radiographs was made using the Raimondi method regolo (Marrapese Editore--Demi S.r.1., Rome). Rasterstereography was performed by means of Formetric 4D(®) (Diers International GmbH, Schlangenbad, Germany). Correlations between rasterstereographic and radiographic measurement of VR were calculated, both for the whole sample and for thoracic and lumbar spinal segments considered separately, as well as for subgroups of patients with a Cobb angle <30° and ≥ 30° using Spearman's correlation coefficient by rank (r (s)). RESULTS: When applied to the entire spine, measurement of VR by means of the two methods highlighted a significant correlation in the whole group (r = 0.52; p < 0.0001), as well as in the <30° Cobb (r = 0.47; p = 0.0001) and ≥ 30° Cobb (r = 0.42; p < 0.0001) subgroups. A significant correlation was found also when lumbar and thoracic VR were considered as separated groups (r = 0.30, p = 0.024 and r = 0.47, p = 0.002, respectively). CONCLUSIONS: Rasterstereographic evaluation of VR shows a good correlation with the Raimondi method, thereby confirming the possibility to use this non-invasive method for deformity assessment in AIS patients.

Robot-assisted subtotal pancreas-preserving duodenectomy.

BACKGROUND: Angiodysplasia of the duodenum is a rare disorder, often requiring surgical resection. Technical difficulties have made the use of the minimally invasive approach uncommon. Herein, we present a subtotal pancreas-preserving duodenectomy using robotic assistance. METHODS: The patient is a 60-y-old female with a long medical history including chronic gastrointestinal bleeding due to angiodysplasia with intermittent melena, and requiring multiples blood transfusions. A capsule endoscopy and double-balloon upper endoscopy showed angiectasis, which appeared to be limited to the third and fourth portion of the duodenum and the proximal loops of the jejunum. Despite multiple endoscopic cauterizations, the patient continued to require blood transfusion for several years. The patient underwent a robot-assisted subtotal pancreas-preserving duodenectomy. RESULTS: The operation lasted 420 min with minimal blood loss. The postoperative course was uneventful. The pathology report showed multiple small bowel mucosal and submucosal distorted and dilated vasculature, consistent with angiodysplasia. At 2-mo follow-up, the patient was totally asymptomatic. A barium swallow study showed contrast passed antegrade through the duodenojejunostomy with no evidence of obstruction, stricture, or leakage. CONCLUSION: The use of robotic assistance to perform a subtotal pancreas-preserving duodenectomy for the treatment of benign duodenal disease, such as angiodysplasia, is feasible and safe. The technical advantages include a high degree of freedom offered by the robotic instruments, as well as enhanced visualization, which allows for precise microdissection and microsuture, thereby preserving the benefits of minimally invasive surgery. The use of robotic technology allows for a wider range of indications for minimally invasive surgery.