In vitro cell culture of amniotic fluid keratinocytes on amniotic membrane: the ideal tissue for repairing skin ulcers.

OBJECTIVE: The amniotic fluid contains a large population of stem keratinocytes demonstrating minimal immunological rejection. Recent evidence suggests that stem cells from the amniotic fluid can be employed in the field of tissue engineering. In this work we identified precursors of the epithelial cells and expanded them in vitro. MATERIALS AND METHODS: After collecting samples of amniotic fluid and separating the cells via centrifugation, we seeded a portion of these cells in selection media to analyze the proliferation of epithelial cells. The stem cells precursors of keratinocytes were identified through specific markers. The expression of these markers was evaluated by immunofluorescence and reverse transcription polymerase chain reaction (PCR). RESULTS: The stem cells demonstrated 90% confluence, after undergoing proliferation in the selection medium for 15 days. Most of these cells tested positive for the keratinocyte-specific markers, but negative for stem cell specific markers. Of note, the identity of the keratinocytes was well established even after several subcultures. CONCLUSIONS: These results suggested that it is feasible to isolate and expand differentiated cell populations in the amniotic fluid from precursor cells. Furthermore, amniotic membranes can be utilized as scaffolds to grow keratinocytes, which can be potentially exploited in areas of skin ulcer transplantation and tissue engineering interventions.

SVEP1 is important for morphogenesis of lymphatic system: Possible implications in lymphedema.

SVEP1, also known as Polydom, is a large extracellular mosaic protein with functions in protein interactions and adhesion. Since Svep1 knockout animals show severe edema and lymphatic system malformations, the aim of this study is to evaluate the presence of SVEP1 variants in patients with lymphedema. We analyzed DNA from 246 lymphedema patients for variants in known lymphedema genes, 235 of whom tested negative and underwent a second testing for new candidate genes, including SVEP1, as reported here. We found three samples with rare heterozygous missense single-nucleotide variants in the SVEP1 gene. In one family, healthy members were found to carry the same variants and reported some subclinical edema. Based on our findings and a review of the literature, we propose SVEP1 as a candidate gene that should be sequenced in patients with lymphatic malformations, with or without lymphedema, in order to investigate and add evidence on its possible involvement in the development of lymphedema.

Rare PECAM1 variants in three families with lymphedema.

PECAM1 is a member of the immunoglobulin superfamily and is expressed in monocytes, neutrophils, macrophages and other types of immune cells as well as in endothelial cells. PECAM1 function is crucial for the development and maturation of B lymphocytes. The aim of this study was to link rare PECAM1 variants found in lymphedema patients with the development of lymphatic system malformations. Using NGS, we previously tested 246 Italian lymphedema patients for variants in 29 lymphedema-associated genes and obtained 235 negative results. We then tested these patients for variants in the PECAM1 gene. We found three probands with rare variants in PECAM1. All variants were heterozygous missense variants. In Family 1, the unaffected mother and brother of the proband were found to carry the same variant as the proband. Lymphoscintigraphy was performed to determine possible lymphatic malformations and showed that in both cases a bilateral slight reduction in the speed and lymphatic clearance of the lower limbs. PECAM1 function is important for lymphatic vasculature formation. We found variants in PECAM1 that may be associated with susceptibility to lymphedema.

Review of the function of SEMA3A in lymphatic vessel maturation and its potential as a candidate gene for lymphedema: Analysis of three families with rare causative variants.

SEMA3A is a semaphorin involved in cell signaling with PlexinA1 and Neuropilin-1 (NRP1) receptors and it is responsible for recruiting dendritic cells into lymphatics. Mutations in the SEMA3A gene result in abnormalities in lymphatic vessel development and maturation. We investigated the association of SEMA3A variants detected in lymphedema patients with lymphatic maturation and lymphatic system malfunction. First, we used NGS technology to sequence the SEMA3A gene in 235 lymphedema patients who carry wild type alleles for known lymphedema genes. We detected three different missense variants in three families. Bioinformatic results showed that some protein interactions could be altered by these variants. Other unaffected family members of the probands also reported different episodes of subclinical edema. We then evaluated the importance of the SEMA3A gene in the formation and maturation of lymphatic vessels. Our results determined that SEMA3A variants segregate in families with lymphatic system malformations and recommend the inclusion of SEMA3A in the gene panel for testing of patients with lymphedema.

CYP26B1 and its implications in lymphangiogenesis: Literature review and study of rare variants in two families.

CYP26B1 is a member of the cytochrome P450 family and is responsible for the break-down of retinoic acid for which appropriate levels are important for normal development of the cardiovascular and lymphatic systems. In a cohort of 235 patients with lymphatic malformations, we performed genetic testing for the CYP26B1 gene. These probands had previously tested negative for known lymphedema genes. We identified two heterozygous missense CY-P26B1 variants in two patients. Our bioinformatic study suggested that alterations caused by these variants have no major effect on the overall stability of CYP26B1 protein structure. Balanced levels of retinoic acid maintained by CYP26B1 are crucial for the lymphatic system. We identified that CYP26B1 could be involved in predisposition for lymphedema. We propose that CYP26B1 be further explored as a new candidate gene for genetic testing of lymphedema patients.

Molecular epidemiology of Mycobacterium tuberculosis complex strains isolated from livestock and wild animals in Italy suggests the need for a different eradication strategy for bovine tuberculosis.

Bovine tuberculosis (bTB) is an important zoonosis, which has been re-emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis-like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU-VNTR profiles. Spoligotype SB0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU-VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection.

A review of the methods for storing floral nectars in the field.

The knowledge of floral nectar sugar characteristics, such as concentration, ratio and mass, is essential to understand the complex nature of nectar production and pollination systems. Although nectar is commonly stored in ecology, storage reliability and effectiveness have rarely been quantified. Inappropriate nectar storage between sampling and analysis can alter nectar chemistry as a result of enzyme or microbial action. Our review of the literature indicates that measures to preserve nectar sugars before analysis include refrigeration, freezing, the addition of an antimicrobial agent, spotting and drying on filter paper, the addition of a desiccant or a combination of these storage treatments. Nectar stored on filter paper is removed by washing with a solvent before analysis. Elution methods are often complex, not standardised and poorly reported in published work. Existing storage methods have generally been used without an evaluation of their impact on results, but evidence suggests a potentially large impact on result accuracy. Future studies should report storage treatments and elution methods to legitimise comparison among independent studies and provide unbiased evaluation of the results. In view of the wide range of storage methods used and lack of verification of their appropriateness, is imperative that standardised and effective methods be developed to ensure that results are reliable. We recommend the prompt analysis of nectar, detailed description of methods, including size of filter paper and method of elution, and use of sterile techniques.

Painful bone metastasis in elderly treated with radiation therapy: Single- or multiple-fraction regimen? A multicentre retrospective observational analysis.

BACKGROUND: Bone metastases are a frequent complication of advanced oncologic disease. Pain associated to bone metastasis is a major cause of morbidity in cancer patients, especially in elderly. AIMS: The aim of this multicentric retrospective observational study is to evaluate the efficacy of different schedules of radiation therapy in elderly patients in terms of pain relief. METHODS: 206 patients over the age of 60 were enrolled in 1 year time for a multicentre retrospective observational study. Patients were treated with palliative purposes for painful bone metastases. RESULTS: Pain intensity difference (PID) was found in 72% of patients. Reported PID was statistically significant for p < 0.01. Pain intensity measured by a point numeric rating scale was statistically significant reduced for p < 0.05 by one-fraction regimen compared to other two regimens. DISCUSSION: In recent years, numerous studies have evaluated the most appropriate regimen of fractionation in individual cases, despite this, a consensus about the best schedule is still debated. CONCLUSIONS: On our analysis, single-fractionation scheme (8 Gy) confirmed to be statistical significant effective in providing pain reduction due to bone metastases. Radiation therapy provides significant pain relief of symptomatic bone metastases, but appropriate radiotherapy scheduled is needed in order to get significant response to treatment. Multidisciplinary approach is warranted to value the balance between the therapeutic objectives and the patient quality of life.

Colorectal surgery in elderly patients: our experience with DaVinci Xi® System.

BACKGROUND: Robotic technology for colorectal surgery was introduced by Weber in 2002 to improve the benefits of the minimally invasive surgery already offered by the laparoscopic approach. AIMS: To evaluate the feasibility and the efficacy of the application of robotic surgery in elderly patients affected by colorectal diseases. METHODS: We reported the outcomes obtained during our first 50 colorectal robotic surgical performances with DaVinci Xi® System, and we compared the results assessed for patients younger or older than 70 years. RESULTS: We examined 28 patients younger and 22 older than 70 years who underwent colorectal robotic surgery in our institution from September 2014 to June 2016. We performed 15 right colectomies, 20 left colectomies, 15 rectal resections. Mean ASA score was significantly higher in the Elderly Group. No statistically significant differences have been revealed in terms of post-operative morbidity, hospital stay, first diet intake, first flatus canalization and oncological outcome. DISCUSSION: According to the prolonged operative time, robotic technology was initially reserved to young patients with good performance status in order to avoid systemic failures in elderly patients suffering from pre-existent comorbidities. Otherwise, once robotic approach safety and benefits in terms of better systemic outcomes were demonstrated, it started to be performed in elderly patients with satisfactory outcomes. CONCLUSION: Our experience revealed that robotic surgical approach is safe, feasible and offers many systemic benefits in elderly patients also with high ASA score. Age alone has not to be considered as exclusion criteria for robotic approach.

Stimulation of Bovine Whole-Blood Samples Cultured in Media Supplemented with Recombinant Interleukin-7 (IL-7) and IL-12 Extends the Life Span of the Gamma Interferon Assay To Detect Mycobacterium bovis-Infected Cattle.

The gamma interferon (IFN-γ) assay is widely used to measure cell-mediated immune (CMI) response for the early detection of tuberculosis infection. Processing whole-blood samples for CMI-based diagnostics is time sensitive and usually must occur within 8 h of collection to ensure optimal assay performance. In this study, we developed and tested a modified protocol, in which whole-blood samples from Mycobacterium bovis-infected cattle were diluted 1:1 in RPMI medium containing 0.3% fetal bovine serum (FBS) added or not to recombinant mouse interleukin-7 (rmIL-7) or rmIL-12, alone or in combination, and stored at 4°C. At 3 and 6 days postcollection, the diluted blood samples were adjusted to 10% FBS, dispensed into culture trays, stimulated with a bovine purified protein derivative from M. bovis, and incubated at 37°C in 5% CO2 in air. Plasma was removed and assayed for an IFN-γ response using bovine IFN-γ enzyme-linked immunosorbent assay (ELISA) (Bovigam). The results were then compared with those obtained from the conventional procedure. The IFN-γ responses of the samples stored up to 6 days postcollection in the supplemented RPMI medium were similar to those observed in the samples processed within 8 h after sampling, indicating that lymphocyte vitality and response were preserved. The addition of rmIL-7 and rmIL-12, alone or in combination, to culture medium can enhance lymphocyte survival and thus extends the time limit within which the IFN-γ assay can be applied as a diagnostic tool in bovine tuberculosis surveillance and eradication.

Surgical management of cervico-mediastinal goiters: Our experience and review of the literature.

AIM: We analyze and discuss the clinical presentation, the diagnostic procedures and the surgical technique in relation to post-operative complications and results in cervico-mediastinal thyroid masses admitted in Thoracic Surgery Unit of AOU Second University of Naples from 1991 to 2006 and in Thoracic Surgery Unit of AOU "S. Giovanni di Dio & Ruggi D'Aragona" of Salerno over a period of 3 years (2011-2014). METHODS: We reviewed 97 patients who underwent surgical treatment for cervico-mediastinal goiters. 47 patients (49.2%) had cervico-mediastinal goiter, 40 patients (40%) had mediastino-cervical goiter and 10 patients (10.8%) had mediastinal goiter. 73 cases were prevascular goiters and 24 were retrovascular goiters. We performed total thyroidectomy in 40 patients, subtotal thyroidectomy in 46 patients and in 11 cases the resection of residual goiter. In 75 patients we used only a cervical approach, in 21 patients the cervical incision was combined with median sternotomy and in 1 patient with transverse sternotomy. RESULTS: Three patients (3.1%) died in the postoperative period (2 cardio-respiratory failure and 1 pulmonary embolism). The histologic study revelead 8 (7.7%) carcinomas. Postoperative complications were: dyspnea in 9 cases (10.7%), transient vocal cord paralysis in 6 patients (9.2%), temporary hypoparathyroidism in 9 patients (9.2%) and kidney failure in 1 case (0.9%). CONCLUSIONS: The presence of a cervico-mediastinal thyroid mass with or without respiratory distress requires a surgical excision as the only treatment option. Thyroid masses extending to the mediastinum can be excised successfully by cervical incision. Bipolar approach (cervical incision and sternotomy) has an excellent outcome, achieving a safe resection, especially in large thyroid masses extending to the mediastinum with close relations to mediastinal structures and in some limited cases (carcinoma, thyroiditis, retrovascular goiter, ectopic goiter). Postoperative mortality and morbidity is very low, independent of surgical techniques. Other surgical approaches for excision of a Posterior Mediastinal Thyroid Goiter reported in literature are: VATS techniques to remove an ectopic intrathoracic goiter, robot-assisted technique for the removal of a substernal thyroid goiter, with extension into the posterior mediastinum.

Surgical complications of carotid body tumors surgery: a review.

Carotid body tumor (CBT) is a rare neoplasm, although it represents about 65% of head and neck paragangliomas. Surgical excision is considered the appropriate therapy for CBTs. The aim of this study was to evaluate surgical outcomes on a large scale. We reviewed 19 studies between 2004 to 2014 with a total of 625 procedures. We observed a higher number of cases in women (62%). Only 3 (0,48%) deaths were reported as surgical complication. Total cranial nerve injuries were 302 (48,32%) of which 194 (31,04%) were transient and 108 (17,28%) were permanent. We found a total of 174 (27,84%) arterial injuries, most of which are external carotid artery (ECA) injuries. Cerebrovascular accident due to surgery were 15 (2,4%). We concluded that surgical resection remains the treatment of choice for these disease despite the related morbidity.

The use of intraoperative ultrasound for diagnosis and stadiation in pancreatic head neoformations.

UNLABELLED: The intraoperative staging of the pancreatic cancer is important to make a proper treatment. For this reason the intraoperative echography is playing an important role in the right treatment choice. The intraoperative echography, that can be performed with an open or laparoscopic probe, is used to confirm the preoperative diagnosis and assess the pancreatic cancer resecability. The intraoperative echography (IOUS) or laparoscopic intraoperative echography (LIOUS) are useful to identify the patients with a non resecable cancer and perform a faster neoadjuvant treatment. The LIOUS can also avoid an useless laparotomy. The aim of this study is to assess, both in our experience and in the cited literature, the concordance rate between the pancreatic cancer preoperative staging, performed with TC and MRI (when it is available), and intraoperative staging, performed with intraoperative laparotomic or laparoscopic echography. MATERIAL AND METHODS: We have analyzed the treatment management of 34 patients, who were candidate to major surgery for suspected pancreatic head cancer and who underwent to intraoperative LIOUS or IOUS staging from 2001 to 2012. RESULTS: LIOUS and IOUS have allowed to detect cases in which preoperative diagnosis, proved by CT and MRI, was not agreeing with intraoperative diagnosis (22 patients on 34, 64% discordance rate), avoiding the execution of a demolitive and uneseful surgery in order to guarantee the surveillance and life's quality of patients. CONCLUSION: We suggest to perform in every patients undergone to pancreatic surgery an intraoperative ultrasound exam, to detect unresecable and unpredicted lesions.

Long-term survival after curative resection for pancreatic ductal adenocarcinoma--Surgical treatment.

INTRODUCTION: Recent reports have demonstrated improvement in the 5-years serviva with resecate ductal adenocarcinoma. The aim of study is to determinate the factor influencing long-term survival after DCP. MATERIALS AND METHODS: We have critically reviewed a group of 85 patients how were admitted to our department with diagnosis of ductal adenocarcinoma of the head of pancreas between January 1974 and January 1998. RESULTS: Patients were stratified according to stage using TNM classification; in stage I 31 patients with 5 T1aN0M0 disease, 11 patients with T1bN0M0 and 15 patients T2N0M0 disease. By contrast, in stage III 54 patients with 48 patients T2N1M0 and 6 patients with T3N1M0. Tumour size was recorded for 72 patients with a median of 3.8 cm. The R1 dissection was performed in 67 patients. The R2 resection was performed in 18 patients. In our series we verified and analysed the histopathologic features of 5 patients with 15-years survival (5.8%). The features regard age, male or female, tumours size, stage and positive lymph node resection. DISCUSSION: We found a specifically subset of patients where the combination of prognostic factors, in particular, negative surgical margins R0, tumour size ≤ 30 mm and the absence of lymph node metastasis independently reduced the mortality indicating that earlier tumour detection and histologically curative resection are important factors contributing to long term survival and healing of ductal adenocarcinoma of the head of the pancreas.

Acinic cell carcinoma of the breast: review of the literature.

INTRODUCTION: The breast and salivary gland tissue share embryologic and thus pathological similarities. Acinic cell carcinoma (ACC) is a typical tumor in salivary glands, but rarely arises in breast too. We reviewed 38 cases of mammary ACC reported in literature and our case, the first ACC born within a fibroadenoma. MATERIALS AND METHODS: Data were collected by a research for the key words acinic cell carcinoma breast on Pubmed in March 2014, including a case treated in our department. All reviewed cases were compared for clinical approach and histological pattern. RESULTS: To date 23 articles presenting cases of ACC of the breast are reported in literature. We included in our review 38 cases previously described and one new case. The histological pattern was predominantly solid with a microglandular structure. All the tumor cells were cytologically characterized by monotonous round cells with a finely granular, weakly eosinophilic, or clearly vacuolated cytoplasm. The most of the cells were intensely stained with anti-lysozime, anti-amylase, anti-α1-chimotripsin, anti-EMA and anti-S100 protein antisera. Immunohistochemistry was also performed to point out: estrogen receptor (ER), progesterone receptor (PR), androgen receptors (AR), human epidermal growth factor receptor 2 overexpression (HER2/neu), E-cadherin (E-cad), cytokeratin-7 (CK7), gross cystic disease fluid protein 15 (GCDFP15), smooth muscle actin (SMA). CONCLUSION: ACC of the breast is a rare tumor, showing similarities with the salivary gland counterpart, above all in terms of good prognosis, and differences from the ordinary invasive breast carcinoma. Further investigations are needed to elucidate the true histogenesis and the correct treatment.

Surgical margins of resection for breast cancer: current evidence.

Breast cancer is the most common form of cancer and second main cause of death in women in western countries. Breast-conserving therapy, consisting of lumpectomy and radiation therapy, has become the standard local treatment for T1-T2 breast tumors. There is general agreement that successful breast conservation requires complete tumor excision with a "tumor-free" or "negative" margin of resection, but the definition of a negative margin is controversial. A commonly accepted definition of adequate margins requires a 2-mm distance between ink and tumor but opinions range from the original National Surgical Adjuvant Breast and Bowel Project definition of "no ink on tumor", to a recommended width of 10 mm or more. The ability to perform real-time molecular imaging analysis of margins during surgery would clearly be a significant advance; several groups have engaged in this effort, with encouraging reports of preliminary data. Further development of such techniques promises to lead to a point at which accurate intraoperative margin evaluation may be possible and may even be combined with therapeutic interventions, using techniques such as photodynamic therapy.

Surgery for a gastric Dieulafoy's lesion reveals an occult bleeding jejunal diverticulum. A case report.

BACKGROUND: Jejunal diverticulosis is an uncommon disease and usually asymptomatic. It can be complicated not only by diverticulitis, but by hemorrhage, perforation, intussusception, volvulus, malabsorption and even small bowel obstruction due to enteroliths formed and expelled from these diverticula. METHODS: We describe a case of an occult bleeding jejunal diverticulum, casually discovered in a patient that was taken to surgery for a Dieulafoy's lesion after unsuccessful endoscopic treatment. We performed a gastric resection together with an ileocecal resection.Macroscopic and microscopic examinations confirmed the gastric Dieulafoy's lesion and demonstrated the presence of another source of occult bleeding in asymptomatic jejunal diverticulum. DISCUSSION: The current case emphasizes that some gastrointestinal bleeding lesions, although rare, can be multiple and result in potentially life-threatening bleeding. The clinician must be mindful to the possibility of multisite lesions and to the correlation between results of the investigations and clinical condition of the bleeding patient.

Feasibility of capsule endoscopy in elderly patients with obscure gastrointestinal bleeding. An up-to-date report.

BACKGROUND: Anemia is the most common hematologic abnormality in older populations. Furthermore, iron deficiency anemia is common and merits investigation and treatment, as it usually results from chronic occult bleeding from the gastrointestinal tract. In view of a wide use of capsule endoscopy as a diagnostic procedure for occult gastrointestinal bleeding and of the growth of aging population, we performed a literature review about the feasibility of capsule endoscopy in the elderly. METHODS: We conducted a literature search in the PubMed database in July 2012, and all English-language publications on capsule endoscopy in elderly patients since 2005 were retrieved. The potential original articles mainly focused on obscure gastrointestinal bleeding were all identified and full texts were obtained and reviewed for further hand data retrieving. RESULTS: We retrieved only six papers based on different primary end-points. Four were retrospective non randomized studies and two were prospective non randomized studies. In the end 65, 70, 80 and 85 years were used as an age cut-off. All studies evaluate the diagnostic yield of capsule endoscopy in iron deficiency anemia. Only three studies assess the feasibility of capsule examination of the elderly. CONCLUSIONS: Iron deficiency anemia in the elderly with or without obscure gastrointestinal bleeding is the major indication for capsule endoscopy after a negative esophago-gastro-duodenoscopy and colonoscopy. It is safe and effective to identify a small bowel pathology without a great discomfort for the elderly. Inability to swallow the capsule, battery failure before capsule reaches the cecum, and capsule retention are some of the important problems associated with capsule endoscopy in elderly as well as in younger patients.

Metronidazole single versus multiple daily dosing in serious intraabdominal/pelvic and diabetic foot infections.

The purpose of the study was to compare the clinical efficacy of once-daily versus multiple dose regimens of metronidazole in inpatients with serious/systemic Bacteroides fragilis infections, i.e., intraabdominal/pelvic and diabetic foot infections. A retrospective chart review was performed on 145 adult inpatients who received combination therapy with metronidazole for B. fragilis abdominal/pelvic infection or diabetic (deep) foot infections/osteomyelitis. Exclusion criteria included metronidazole given for indications other than those mentioned, patients who received only one dose of metronidazole, and patients who received oral metronidazole only. The 145 patients were in two groups: 66 patients in the metronidazole 1 g (i.v.) q24h (Group A) and 79 patients who received metronidazole 500 mg (i.v./p.o.) q6-8h dosing (Group B). Patient demographics included age, gender, indications of metronidazole, concomitant, antibiotics, and co-morbidities. Data collection also included length of stay (LOS), antibiotic days, and clinical outcomes. The 145 patients in our study had a mean age of 66 years, 61% were female and 39% male. Most patients were being treated for definitive intraabdominal/pelvic infections (82%), or probable intraabdominal/pelvic infections (22%). Only 6% had deep diabetic foot infections of osteomyelitis (percentages exceed 100% since a patient can have more than one indication) and were included since B. fragilis is also and important pathogen in diabetic osteomyelitis. Group A patients had more concomitant antibiotics and co-morbidities (p < 0.0001 and p < 0.05 respectively, chi-square test for trend) than Group B patients. There were no statistically significant differences between groups A and B for LOS and antibiotic days (p = 0.42 and p = 0.92 respectively, by rank-sum test), but after adjusting for concomitant antibiotics and co-morbidities Group A patients had clinically shorter LOS and fewer antibiotic days. Unadjusted mortality and failure rates were non-significantly higher in group A (relative ratios of 12.1%/6.3% = 6.3% = 1.91 and 18.2%/ 10.1% = 1.80 respectively), but after adjusting for concomitant antibiotics and co-morbidities with stratification analysis, groups A and B were virtually the same (risk differences of