CLINICS July 2017 by Clinics

Volume 72 Number 7 - July/2017

CLINICS Editor Edmund Chada Baracat

Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Anna Sara Shafferman Levin Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Antonio Egidio Nardi Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Berenice Bilharinho Mendonca Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Carlos Serrano Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Claudio Roberto Cernea Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Emilia Inoue Sato Universidade Federal de São Paulo São Paulo, SP, Brazil Flair José Carrilho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Fulvio Alexandre Scorza Universidade Federal de São Paulo São Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Heloisa de Andrade Carvalho Hospital das Clı´nicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Jesus Paula Carvalho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil José Guilherme Cecatti Universidade Estadual de Campinas Campinas, SP, Brazil José Maria Soares Júnior Hospital das Clı´nicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Luiz Eugenio Garcez-Leme Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Luíz Fernando Onuchic Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de São Paulo São Paulo, SP, Brazil Marcos Intaglietta University of California, San Diego San Diego, CA, USA Maria José Carvalho Carmona Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Mauricio Etchebehere Universidade Estadual de Campinas Campinas, SP, Brazil Michele Correale University of Foggia Foggia, Italy Naomi Kondo Nakagawa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Paulo Pêgo-Fernandes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Duke University Medical Center Durham, NC, USA Ricardo Bassil Lasmar Universidade Federal Fluminense Niterói, RJ, Brazil Ricardo Nitrini Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rosa Maria Rodrigues Pereira Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rossana Francisco Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de São Paulo São Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de São Paulo São Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sandro Esteves ANDROFERT - Andrology & Human Reproduction Clinic Campinas, SP, Brazil Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Sigmar de Mello Rode Universidade Estadual Paulista Júlio de Mesquita Filho São José dos Campos, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Valeria Aoki Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil Adauto Castelo Universidade Federal de São Paulo São Paulo, SP, Brazil Ademar Lopes Fundação Antônio Prudente, Hospital do Câncer São Paulo, SP, Brazil Alberto Azoubel Antunes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Alexandre Roberto Precioso Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Andrea Schmitt University of Goettingen Goettingen, Germany

Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Artur Brum-Fernandes Université de Sherbrooke Québec, Canadá Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil Daniel Romero Muñoz Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Eduardo Ferreira Borba Neto Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany

Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Ernest Eugene Moore University of Colorado Denver Denver, CO, USA Euclides Ayres Castilho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Fábio Biscegli Jatene Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Francisco Laurindo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil

Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Ke-Seng Zhao Southern Medical University Guangzhou, China Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK Marcelo Zugaib Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Mauro Perretti William Harvey Research Institute London, UK

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

Pedro Puech-Leão Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center Houston, Texas, USA Rafael Andrade-Alegre Santo Tomás Hospital Republic of Panamai, Panamá Ricardo Antonio Reﬁnetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy

Milton de Arruda Martins Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA Naomi Kondo Nakagawa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Navantino Alves Faculdade de Ciências Médicas de Minas Gerais Belo Horizonte, MG, Brazil

Samir Rasslan Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Board of Governors Alberto José da Silva Duarte Aluisio Augusto Cotrim Segurado Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonça Carlos Alberto Buchpiquel Carlos Roberto Ribeiro de Carvalho Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Daniel Romero Muñoz Edivaldo Massazo Utiyama Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfá Euripedes Constantino Miguel Fábio Biscegli Jatene Flair José Carrilho Geraldo Busatto Gerson Chadi Gilberto Luis Camanho Giovanni Guido Cerri Irene de Lourdes Noronha Irineu Tadeu Velasco

Ivan Cecconello Jorge Elias Kalil José Antonio Franchini Ramires José Antonio Sanches José Eduardo Krieger José Otávio Costa Auler José Ricardo de Carvalho Mesquita Ayres Linamara Rizzo Battistella Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Miguel Srougi Milton de Arruda Martins Mirian Nacagami Sotto Nelson de Luccia Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hilário Nascimento Saldiva Paulo Manuel Pêgo Fernandes Paulo Marcelo Gehm Hoff Paulo Rossi Menezes

Editorial Director

Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Pedro Puech-Leão Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini Roberto Kalil Roberto Zatz Roger Chammas Rolf Gemperli Rosa Maria Rodrigues Perreira Sandra Joseﬁna Ferraz Ellero Grisi Selma Lancman Tarcísio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Vanderson Geraldo Rocha Venâncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistants

Nair Gomes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

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ISSN-1807-5932

CLINICS CONTENTS Clinics 2017 72(7)395–453

CLINICAL SCIENCE

Hyoscine butylbromide for colorectal polyp detection: prospective, randomized, placebo-controlled trial Carlos Eduardo Oliveira dos Santos, Hamilton Moreira, Julio Carlos Pereira-Lima, Carmen Australia Paredes Marcondes Ribas, Fernanda de Quadros Onófrio, Alexandre Eduardo Augusti Czecko, Rafael Koerich Ramos, Caroline Aragão de Carvalho . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

Validation of QF-PCR for prenatal diagnoses in a Brazilian population Renata Wendel de Moraes, Mario Henrique Burlacchini de Carvalho, Antonio Gomes de Amorim-Filho, Rossana Pulcineli Vieira Francisco, Renata Moscolini Romão, José Eduardo Levi, Marcelo Zugaib. . . . . . . . . . . . . . 400

Clinical Features of Refractory Ascites in Outpatients Wanda Regina Caly, Rodrigo Martins Abreu, Bernardo Bitelman, Flair José Carrilho, Suzane Kioko Ono . . . . . . . . 405

Intermittent claudication and severe renal artery stenosis are independently associated in hypertensive patients referred for renal arteriography Thiago Andrade Macedo, Luciano Ferreira Drager, Rodrigo Pinto Pedrosa, Henrique Cotchi Simbo Muela, Valeria Costa-Hong, Luiz Junia Kajita, Luiz Aparecido Bortolotto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

Hypovitaminosis D in patients undergoing kidney transplant: the importance of sunlight exposure Cristiane F. Vilarta, Marianna D. Unger, Luciene M. dos Reis, Wagner V. Dominguez, Elias David-Neto, Rosa M. Moysés, Silvia Titan, Melani R. Custodio, Mariel J. Hernandez, Vanda Jorgetti . . . . . . . . . . . . . . . . . . . . . 415

Laryngeal tube suction for airway management during in-hospital emergencies Haitham Mutlak, Christian Friedrich Weber, Dirk Meininger, Colleen Cuca, Kai Zacharowski, Christian Byhahn, Richard Schalk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422

Complete axillary dissection without drainage for the surgical treatment of breast cancer: a randomized clinical trial Ruffo Freitas-Junior, Luís Fernando Jubé Ribeiro, Marise Amaral Rebouc¸as Moreira, Geraldo Silva Queiroz, Maurício Duarte Esperidião, Marco Aurélio Costa Silva, Rubens José Pereira, Rossana Araújo Catão Zampronha, Rosemar Macedo Sousa Rahal, Leonardo Ribeiro Soares, Danielle Laperche dos Santos, Maria Virginia Thomazini, Cassiana Ferreira Silva de Faria, Régis Resende Paulinelli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426

Abnormal diastolic function underlies the different beneﬁcial effects of cardiac resynchronization therapy on ischemic and non-ischemic cardiomyopathy Qi Wang, Kang-Yu Chen, Fei Yu, Hao Su, Chun-Sheng An, Yang Hu, Dong-Mei Yang, Jian Xu, Ji Yan. . . . . . . . . 432

REVIEW

The diagnostic value of narrow-band imaging for early and invasive lung cancer: a meta-analysis Juanjuan Zhu, Wei Li, Jihong Zhou, Yuqing Chen, Chenling Zhao, Ting Zhang, Wenjia Peng, Xiaojing Wang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

Osteointegration of porous absorbable bone substitutes: A systematic review of the literature Maria JĂşlia Escanhoela Paulo, Mariana Avelino dos Santos, Bruno Cimatti, Nelson FabrĂcio Gava, Marcelo Riberto, Edgard Eduard Engel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449

CLINICAL SCIENCE

Hyoscine butylbromide for colorectal polyp detection: prospective, randomized, placebo-controlled trial Carlos Eduardo Oliveira dos Santos,I,* Hamilton Moreira,II Julio Carlos Pereira-Lima,III Carmen Australia Paredes Marcondes Ribas,II Fernanda de Quadros Ono´frio,III Alexandre Eduardo Augusti Czecko,II Rafael Koerich Ramos,II Caroline Araga˜o de CarvalhoII I Departamento de Endoscopia e Gastroenterologia, Santa Casa de Caridade, Bage, RS, BR. II Programa de Pos graduacao em Principios de Cirurgia, Faculdade Evangelica do Parana, Curitiba, PR, BR. III Departamento de Gastroenterologia e Endoscopia, Hospital Santa Casa, Porto Alegre, RS, BR.

OBJECTIVES: The removal of pre-malignant colorectal lesions prevents cancer. Hyoscine has been proposed as a means of improving diagnosis by reducing colonic movements. The aim of this study was to analyze whether this anti-spasmodic enhances the detection of pre-malignant colorectal lesions. METHODS: In a randomized, double-blinded fashion patients received hyoscine or a saline solution in all consecutive colonoscopies in which the cecum was reached. Lesions were analysed with respect to number, size, location, histology and capillary pattern. RESULTS: A total of 440 colonoscopies were randomized. The overall polyp detection rate (PDR) and the adenoma detection rate (ADR) were 65.2% and 49.3%, respectively. In the hyoscine group, non-polypoid lesions were detected significantly more often (p=0.01). In the placebo group 281 lesions were diagnosed (202 adenomas) and in the hyoscine group 282 lesions were detected (189 adenomas) (p=0.23). The PDR and ADR were similar between the placebo and hyoscine groups (64% vs 66% and 50% vs 47%, respectively). No differences were observed between the two groups in the advanced-ADR or advanced neoplasia detection rate, as well the mean numbers of polyps, adenomas, advanced adenomas and advanced neoplasias detected per patient. The administration of hyoscine also did not improve the diagnostic accuracy of digital chromoendoscopy. The presence of adenomatous polyps in the right colon was detected significantly more frequently in the hyoscine group (OR 5.41 95% CI 2.7 - 11; po0.01 vs OR 2.3 95% CI 1.1 - 4.6; p=0.02). CONCLUSION: The use of hyoscine before beginning the withdrawal of the colonoscope does not seem to enhance the PDR and the ADR. KEYWORDS: Colonoscopy; Adenoma; Colonic Polyps; Scopolamine Hydrobromide. Santos CE, Moreira H, Pereira-Lima JC, Ribas CA, Ono´frio FQ, Czecko AE, et al. Hyoscine butylbromide for colorectal polyp detection: prospective, randomized, placebo-controlled trial. Clinics. 2017;72(7):395-399 Received for publication on September 30, 2016; First review completed on November 29, 2016; Accepted for publication on January 13, 2017 *Corresponding author. E-mail: ddendo@uol.com.br

’ INTRODUCTION

interval cancer, especially in the right colon (5). Interval cancer in the right colon could be partially explained by the nondiagnosis of non-polypoid lesions, since these neoplasms are more common in the right colon and are more aggressive (6). Many factors could contribute to the failure to diagnose these lesions, including inadequate bowel preparation (7,8), colonoscopy technique (9), colonoscope withdrawal time (10) and polyp location (11,12). New techniques have been developed to increase the ADR, such as high-definition colonoscopes (13), chromoendoscopy (14), G-EYE colonoscope (15), the Third Eye colonoscope (16), full spectrum endoscopy (17), waterimmersion colonoscopy (18), retroflexion in the right colon (19), cap-assisted colonoscopy (20), endocuff-assisted colonoscopy (21), and endorings (22). It has been suggested that colonic peristalsis may hinder the analysis of the mucosal surface, and consequently the discovery of colonic lesions. Hyoscine is a spasmolitic agent of the gastrointestinal tract. The peripheral anticholinergic action of hyoscine results from the blockade of the intramural ganglia of hollow viscera, as

Colorectal cancer (CRC) represents one of the leading causes of cancer-related deaths worldwide. The diagnosis of premalignant lesions, and the subsequent removal of such lesions is a well-recognized strategy in the secondary prevention of CRC (1). Colonoscopy is considered the gold standard in achieving this goal, with the adenoma detection rate (ADR) (defined as the index of procedures in which at least one adenoma is diagnosed) considered a known indicator of the quality of the method (2). Nevertheless, a considerable number of adenomas are missed (3,4), which are related to

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2017(07)01

395

Hyoscine butylbromide for colorectal polyp Santos CE et al.

CLINICS 2017;72(7):395-399

mannitol solution on the day of lower endoscopy. All colonoscopies were performed under conscious sedation with midazolamand fentanyl. One millilitre of hyoscine (20 mg) or placebo was infused intravenously as soon as the cecum was reached during colonoscopy (Fujinon 590ZW5 with high resolution and magnification, Fujifilm Corp., Saitama, Japan) with the EPX4400 processor. We waited for at least one minute before starting withdrawal, of the coloscope because the antispasmodic effect begins within one minute and lasts for 10-15 minutes (27). All procedures were performed by one of the authors (CEOS) who had already performed more than 12,000 colonoscopies with magnification and real or digital chromoendoscopy. The colonoscope withdrawal time was defined as the time spent between cecum examination after the intravenous administration of the drug or placebo and the removal of the colonoscope through the anus. In all cases, this procedure lasted more than 6 minutes. Patients were monitored continuously during the examination by means of a pulse oximeter. The alarm remained off during the colonoscopies and the monitor was seen only by the nurse and not by the endoscopist. To maintain patient safety, the nurse was instructed to communicate with the endoscopist in case of significant tachycardia, which was considered relevant when the heart beat rate was greater than 140/minute for more than 30 seconds (28).

well as an anti-muscarinic activity. The antiperistaltic effect of this compound on the small intestine was demonstrated by Gutzeitet et al. (23), with an average onset of action at 85 seconds after intravenous infusion and an average duration of 21 minutes. The administration of hyoscine during colonoscopy could be an alternative in controlling colonic contractility and could in this way facilitate the detection and characterization of colonic lesions. However, anti-spasmodic agents such as glucagon (24) and atropine (25) have not shown benefits in this setting. Hyoscine is inexpensive, widely available and safe. Furthermore, there have been no studies to date evaluating the use of spasmolytic agents in the differential diagnosis of colorectal lesions by digital chromoendoscopy. It was hypothesized that the analysis of the capillary pattern of colorectal lesions would be facilitated under the effect of hyoscine, which decreases the peristaltic movements of the colon. The goal of this prospective, randomized, placebocontrolled double-blinded trial was to analyse whether the use of hyoscine enhances the detection of polyps and adenomas. As a secondary objective, this study aimed to evaluate whether this drug has any impact on the differential diagnosis of colorectal lesions during colonoscopy with magnification and digital chromoscopy.

’ METHODS The participating institutions were as follows: Santa Casa de Caridade de Bagé, RS; Evangelic Faculty of Parana, Curitiba, PR; and Santa Casa Hospital, Porto Alegre, RS. The study was approved by the Ethics Committee of Santa Casa de Caridade, Bagé, RS and was conducted according to the Declaration of Helsinki. Informed written consent (26) was obtained from all participants. Patients who were referred to our endoscopy unit for CRC screening, surveillance or a clinical suspicion of CRC were randomly assigned to receive 20mg of hyoscine (Buscopan, Boehringer Ingelheim of Brazil Quim. e Farm. Ltda, Itapecerica da Serra, Brazil) or placebo (saline solution) as soon as the colonoscope reached the cecum. The randomization was generated by the www.researchrandomizer.org site. Sealed envelopes were opened by the nurse taking care of the patient’s sedation. The endoscopist was blinded to the randomization. The exclusion criteria were as follows: inadequate bowel preparation, incomplete colonoscopy, inflammatory bowel disease, advanced cancer, prior colorectal surgery, reported or known allergy to hyoscine, and patients referred to polypectomy or endoscopic mucosal resection due to previously diagnosed lesions. Between March and July 2015, 517 consecutively performed colonoscopies were analysed for inclusion in the study. A total of 77 cases were excluded: 20 had previously diagnosed colon lesions and were referred for colonoscopic removal; 13 had previously undergone colon resection surgery; 11 had inadequate bowel preparation; 7 had advanced carcinomas; 7 were aged less than 30 years; 7 being allergic to hyoscine and in 2 cases the cecum was not reached. Therefore, 440 cases were randomized, with 220 cases in each arm. The patients were analysed with respect to age, gender, colonoscope withdrawal time, endoscopic findings and histopathology.

The lesion size was estimated with an open biopsy forceps (7mm). The lesions morphology was determined according to the Paris classification (29), in which non-polypoid lesions are considered those less than 2.5mm in height, which was estimated with a closed biopsy forceps touching the lateral margins of the lesion, (types 0-IIa, 0-IIa+IIc, 0-IIc+IIa, 0-IIc, and 0-IIb and laterally spreading tumors - LSTs). Polypoid lesions are those greater than 2.5mm in height (types 0-Is, 0-Isp, and 0-Ip). All lesions included in the study had the endoscopic appearance of lesions known as superficial lesions, limited to the mucosa or submucosa, according to Kudo (30). The right colon was considered the cecum, and the ascending and transverse colon, while the left colon was the rectum, the sigmoid and the descending colon. All lesions were evaluated using digital chromoendoscopy (Flexible Spectral Imaging Color Enhancement – FICE) to analyse capillary patterns for the real time differential diagnosis between neoplastic and non-neoplastic lesions. According to the Teixeira Classification (31), lesions with I-II patterns were considered non-neoplastic, and lesions with III-V patterns were considered neoplastic. Non-neoplastic lesions were hyperplastic and inflammatory polyps, and neoplastic lesions were tubular, tubulovillous, villous, sessile serrated, and traditional serrated adenomas and early carcinomas. Those lesions greater than 1 cm, with villous histology or high-grade dysplasia were considered advanced adenomas. Advanced neoplasias were the advanced adenomas and early carcinomas. All the lesions were removed endoscopically and were analysed by the same pathologist who was blinded to the endoscopic diagnosis.

Procedures

Statistical Analysis

Bowel preparation was undergone with a clear liquids diet on the day prior to the procedure and 1000 ml of a 10%

The data were inserted into the Stata software version 11.2. Categorical variables were described using absolute and

Lesions characteristics

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Table 1 - Patient and lesion characteristics.

relative frequencies. Numerical variables were described as the mean and standard deviation (SD) when the distribution of the data was normal or as the median and interquartile interval when their distribution was not normal. Bivariate analyses of the categorical variables comparing the hyoscine and placebo groups were performed using Fisher’s exact test. Bivariate analyses of numerical variables comparing the groups with and without hyoscine were performed using a t-test (when comparing means) or the MannWhitney U test (when medians were compared). A logistic regression was used to analyse the adenoma detection rate (ADR) with the odds ratio (OR) and confidence interval (95% CI). For the sample size calculation, the analysis of the detection rate of adenomas was considered as an outcome, stratified according to the treatment group (hyoscine) and control group (placebo). The parameters considered were as follows: power of 80%, alpha-error of 5%, prevalence of 50% outcome, 10-75% exposure frequency and relative risk of at least 1.7 or an odds ratios of 2.6 between exposed versus unexposed, resulting in a patient number of 216. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the capillary pattern analysis were reported. The significance level adopted was 5% for the bicaudal tests.

Variable

Sex Female Male Age (years) o50 X50 Lesions per patient 0 1 2 3 X4 Size (mm) o10 X10 Morphology Non-polypoid Polypoid Capillary pattern Non-neoplastic Neoplastic Pathology Non-neoplastic Neoplastic Adenomas No Yes Advanced adenoma No Yes Advanced neoplasia No Yes Advanced histology No Yes Location Right Colon Left Colon

’ RESULTS From a total of 517 consecutive patients, 77 (14.9%) did not meet the inclusion criteria. Therefore, 220 patients were randomized to the hyoscine group, and the other 220 were included in the placebo group. A total of 563 lesions were diagnosed in 287 colonoscopies (65.2%), with 391 adenomas in 217 patients (49.3%). These figures are described in Table 1. Sex, age, mean lesion size (4.9 mm +/- 4.2 vs 5.3 mm +/- 6.2, p=0.46), and mean colonoscope withdrawal time (9.8 min +/- 5.7 vs 9.8 min +/- 8.3, p=0.99) were similar between the two groups. However, the median and the interquartile interval were higher in the placebo group with respect to the colonoscope withdrawal time [7.7 min (7.1-9.8) vs 7.5 min (6.8-8.6), p=0.05]. In the hyoscine group, significantly more non-polypoid lesions were detected than in the placebo group (79.8% vs 69.4%); nevertheless, polypoid lesions composed 20.2% of the lesions in the hyoscine group and 30.6% in the placebo group (p=0.01). The proportion of adenomas and non-polypoid adenomas found in the right colon was similar between the two groups. Analysing the lesions via digital chromoendoscopy in the placebo group, the accuracy was 94.3%, the sensitivity 94.1%, the specificity 94.7%, the PPV 98% and the NPV 85.7%, while these values were 95%, 93.7%, 97.8%, 98.9% and 88.1%, respectively, in the hyoscine group (Table 2, all these data were not significantly different). The detection of advanced adenomas (12.7% vs 11.8%) and advanced neoplasias (13.6% vs 12.3%), the PDR (64.6% vs 65.9% ) and the ADR (50.5% vs 46.8%) were also similar between the placebo and the hyoscine groups, respectively (Table 1), as well as the mean number of polyps (1.3 vs 1.3), adenomas (0.92 vs 0.86), advanced adenomas (0.16 vs 0.18) and advanced neoplasias (0.16 vs 0.18) detected per patient (p=0.97). The logistic regression analysis demonstrated that 1 additional minute of colonoscope withdrawal time and the presence of one more lesion were associated with the detection of one more adenoma in the placebo group, and

Placebo group (%)

Hyoscine group (%)

N=220 patients

151 (68.6) 69 (31.4)

147 (66.8) 73 (33.2)

47 (21.4) 173 (78.6)

54 (24.6) 166 (75.4)

78 (35.4) 68 (30.9) 38 (17.3) 24 (10.9) 12 (5.5) N= 281 lesions 262 (93.2) 19 (6.8)

75 (34.0) 71 (32.3) 42 (19.1) 16 (7.3) 16 (7.3) N=282 lesions 262 (92.9) 20 (7.1)

195 (69.4) 86 (30.6)

225 (79.8) 57 (20.2)

84 (29.9) 197 (70.1)

101 (35.8) 181 (64.2)

76 (27.1) 205 (72.9)

91 (32.3) 191 (67.7)

79 (28.1) 202 (71.9)

93 (33.0) 189 (67.0)

247 (87.9) 34 (12.1)

243 (86.2) 39 (13.8)

245 (87.2) 36 (12.8)

242 (85.2) 40 (14.8)

265 (94.3) 16 (5.7)

270 (95.7) 12 (4.3)

152 (54.1) 129 (45.9)

136 (48.2) 146 (51.8)

p value*

0.76

0.50

0.65

1.00

0.01

0.15

0.20

0.23

0.62

0.71

0.45

0.18

Table 2 - Diagnostic criteria of the lesions using digital chromoendoscopy. Placebo group (n=281) Sensitivity (%) Specificity (%) PPV (%) NPV (%) Accuracy (%) Kappa

94.1 94.7 98.0 85.7

(90.0 (87.1 (94.9 (76.4 94.3 0.86 (0.79 -

95.9) 98.5) 99.4) 92.4) 0.93)

Hyoscine group (n=282) 93.7 97.8 98.9 88.1

(89.3 (92.3 (96.1 (80.2 95 0.89 (0.83

95.7) 99.7) 99.9) 93.7)

- 0.95)

the presence of a lesion in the right colon was associated with the detection of more lesions in the hyoscine group. Polypoid lesions were a risk factor for adenoma only in the placebo group. The complete logistic regression analysis is depicted in Table 3. No serious side effects were observed in the study groups. Three patients presented with significant tachycardia lasting less than 30 seconds in the hyoscine group.

’ DISCUSSION We hypothesized that the spasmolytic action of hyoscine would favour a greater detection of polyps and adenomas,

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Table 3 - Logistic regression analysis of the adenoma detection

The analysis of the capillary pattern by magnification colonoscopy with digital chromoscopy has yielded excellent results in the differential diagnosis of neoplastic and nonneoplastic colorectal lesions, with good to excellent intraand interobserver agreement (42-45). We also hypothesized that the abolishment of colonic contractility would allow a better evaluation of these lesions by digital chromoendoscopy with magnification. However, no differences regarding the diagnostic sensitivity, specificity, PPV, NPV or accuracy were observed when comparing patients who received placebo or hyoscine. Perhaps, the use of hyoscine could improve the discrimination between neoplastic and nonneoplastic colorectal lesions, and improve the PDR and ADR among beginners, but not after the acquisition of expertise. Indeed, one of the limitations of this study is that all the examinations were performed by a single endoscopist, who is very familiar with the Japanese Classification of Colorectal Carcinoma (46), which may explain the large number of lesions morphologically classified as NPLs. In summary, we found a higher number of non-polypoid lesions in the hyoscine group and more polypoid lesions in the placebo group, but with no difference with respect to the colonic segment (left or right). This double-blind, prospective, placebo-controlled trial did not show any evidence supporting the routine use of hyoscine during colonoscopy to improve the PDR and ADR or to augment the diagnosis of advanced adenomas or advanced neoplasias. We could also not demonstrate an impact of the drug in differentiating neoplastic from non-neoplastic lesions by means of digital chromoendoscopy with magnification.

rate. Placebo group Variable Male sex X50 years Leions (number) Polypoid lesions Right colon Time (min)

OR IC95% 1.4 2.8 24 8.3 1.8 3.6

(0.8-2.4) (1.4-5.5) (19-60) (1.9-35) (0.9-3.7) (2.4-5.4)

p value 0.3 o0.01 o0.01 o0.01 0.1 o0.01

Hyoscine group OR IC95% 1.2 2.4 7 1.7 5.8 2.0

(0.7-2.2) (1.3-4.6) (4.2-12) (0.7-3.7) (2.8-12) (1.5-2.5)

p value 0.5 0.01 o0.01 0.2 o0.01 o0.01

thus increasing the ADR. Other studies have suggested that the administration of a spasmolytic drug could also facilitate the insertion of the colonoscope to the cecum (32,33) or could improve the visualization of the colonic surface due to the reduction of spasms and the flattening of the colonic haustrations (34). However, these results were not reproduced by other authors (35,36). Employing a grading scale for colonic spasms, Lee et al. (37) observed a significant decrease in the number of spasms between colonoscope insertion and withdrawal in patients receiving hyoscine in comparison to those receiving placebo. However, this group did not show any difference in the number of polyps per patient or the ADR. Nonetheless, Lee et al. found a trend for a higher polyp detection rate in the hyoscine group when comparing a subgroup of placebo patients with severe spasms. Our study did not show an advantage to injecting hyoscine when the colonoscope reached the cecum in terms of the PDR or ADR, which is in line with two recently published metanalyses (38,39). There was also no diagnostic advantage either in the detection of advanced adenomas, or advanced neoplasias. In a study of 601 patients, Corte et al. (40) identified a higher polyp rate per patient in a hyoscine group than in a placebo group (0.91 vs 0.7 p=0.04), but the PDR and ADR were similar for both arms (43.6 vs 36.6% and 27.1% vs 21.8%, respectively). In a study by de Brouwer et al. (41), there was no difference in the PDR, ADR or mean number of polyps detected per patient, in an Italian double-blind, randomized trial comparing hyoscine and placebo (28). The present study also did not demonstrate any difference in the PDR, ADR, or mean number of detected polyps or adenomas per patient when comparing hyoscine with placebo. In contrast to the findings of Rondonotti et al. (28), who encountered significantly fewer non-polypoid colorectal lesions in the hyoscine group, we found significantly more nonpolypoid colorectal lesions in the hyoscine arm. Our findings refute the hypothesis that spasmolytic agents would hinder the identification of flat lesions by stretching of the colon. However, de Brouwer et al. (41) identified no difference in the morphology of the diagnosed colorectal lesions, when comparing hyoscine and placebo. We observed a longer withdrawal time in the placebo group, which in our study, was associated with the ADR according to the logistic regression analysis. In contrast, in the hyoscine group, the main factor associated with the ADR was the detection of a lesion in the right colon. These figures are comparable to those reported by Corte et al. (40). Perhaps this longer withdrawal time in the placebo group can be explained by waiting for the colonic spasms to end during the examination.

’ AUTHOR CONTRIBUTIONS Santos CE, Pereira-Lima JC, Onófrio FQ were involved in subjects recruitment, data collection, interpretation of the results, provided assistance in statistical analyses and manuscript drafting. Moreira H, Ribas CA reviewed the manuscript, conceived and designed the study. Czeczko AE, Ramos RK, de Carvalho CA were involved in data collection, literature review and contributed to the results.

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CLINICAL SCIENCE

Validation of QF-PCR for prenatal diagnoses in a Brazilian population Renata Wendel de Moraes,I Mario Henrique Burlacchini de Carvalho,I,* Antonio Gomes de Amorim-Filho,I Rossana Pulcineli Vieira Francisco,I Renata Moscolini Roma˜o,I Jose´ Eduardo Levi,II Marcelo ZugaibI I Departamento de Ginecologia e Obstetricia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. Hemocentro de Sa˜o Paulo, SP, BR.

Fundac¸a˜o Pro´-Sangue,

OBJECTIVES: Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid and reliable method for screening aneuploidies, but in Brazil, it is not used in public services. We investigated the accuracy of QF-PCR for the prenatal recognition of common aneuploidies and compared these results with cytogenetic results in our laboratory. METHOD: A ChromoQuant QF-PCR kit containing 24 primer pairs targeting loci on chromosomes 21, 13, 18, X and Y was employed to identify aneuploidies of the referred chromosomes. RESULTS: A total of 162 amniotic fluid samples analyzed using multiplex QF-PCR were compared with karyotyping analysis. The QF-PCR results were consistent with the results of cytogenetic analysis in 95.4% of all samples. CONCLUSION: QF-PCR was demonstrated to be efficient and reliable for prenatal aneuploidy screening. This study suggests that QF-PCR can be used as a rapid diagnostic method. However, rearrangements and some mosaic samples cannot be detected with this test; thus, those exceptions must undergo cytogenetic analysis. KEYWORDS: Prenatal Diagnosis; Aneuploidy; QF-PCR; Brazilian Population. Moraes RW, de Carvalho MH, de Amorim-Filho AG, Francisco RP, Roma˜o RM, Levi JE, et al. Validation of QF-PCR for prenatal diagnoses in a Brazilian population. Clinics. 2017;72(7):400-404 Received for publication on November 24, 2016; First review completed on January 10, 2017; Accepted for publication on February 14, 2017 *Corresponding author. E-mail: marioburlacchini@uol.com.br

’ INTRODUCTION

culture has increased. These tests can detect aneuploidies using molecular techniques, such as quantitative fluorescent polymerase chain reaction (QF-PCR) (6). QF-PCR involves the amplification of small repetitive DNA sequences – short tandem repeats (STRs) – using fluorescent primers, followed by quantitative analysis of the products to evaluate the numbers of copies of specific chromosomes (7). Although a microsatellite marker is heterozygous, the ratio of its allele peak areas represents a disomic (1:1) or trisomic (2:1, 1:2, or 1:1:1) chromosome complement. A marker is uninformative if only a single peak is observed (8). In 2000, QF-PCR was first introduced to the UK National Health Service as a validated and efficient diagnostic test (9). In Stockholm, since 2005, women have been able to choose between QF-PCR alone or full karyotype for prenatal diagnosis (10), and in the USA, since 2006, this method has been validated and available (11). In Brazil, this technique is not widely available: only a few private services offer either QF-PCR or fluorescence in situ hybridization (FISH) in association with cytogenetic culture; furthermore, none of them are offered as a routine public service. The lack of studies involving prenatal diagnosis using QF-PCR methods in Brazil contributed to this study. The aims of this study were to investigate the diagnostic test performance of QF-PCR for the detection of trisomy 13, 18, and 21 and sex chromosome aneuploidies in high-risk pregnancies, to compare these results with karyotypes and to introduce this method as routine practice in our university hospital.

The most frequent chromosome abnormality identified in humans is aneuploidy, which occurs in 5% of all pregnancies and is the leading cause of pregnancy loss (1). Numerical and structural chromosome abnormalities are detected in approximately one in 200 newborns, and these abnormalities are the most common causes of developmental disabilities and congenital malformations in humans (2). In most countries, cytogenetic analysis has become an important component of prenatal diagnosis. Since the origin of cytogenetics, karyotyping has been used as a gold standard test for aneuploidy diagnosis (3,4). The accuracy of this test is approximately 99%; however, this technique is laborious and involves a long reporting time because of the necessity for cell culture (2-5). For most clinical genetics laboratories, the results can take 10 to 15 days, which is a long waiting period for anxious parents. To reduce parent stress and the time for diagnosis, the demand for rapid diagnostic methods that do not require cell

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’ MATERIALS AND METHODS

Genescan analysis and reporting

The amplified samples were pooled together (1.0 mL) with 12 mL of Hi-Di formamide (Applied Biosystems, USA) and 0.3 mL of ROX500 (Applied Biosystems, USA). This mixture was denatured at 95 oC for 3 min, transferred to ice and left until the sample was loaded on an ABI 3130 Genetic Analyzer. The samples were run on a POP7 polymer and a 36-cm capillary, and the obtained results were analyzed with Gene Mapper V4.0 (Softgenetics, USA). For reporting, the peak height ratio or area ratio was calculated. We considered a minimum of two markers to be informative for reporting, following the manufacturer’s protocol. The allele ratio for the normal range was from 0.8 to 1.4. If the ratio ranged from 1.8 to 2.4 or 0.45 to 0.65, it was considered trisomy positive for three individual peaks (1:1:1) with respect to each marker considered trisomy positive.

Study samples The data for this study were derived from the analysis of stored amniotic fluid obtained during prospective amniocentesis for prenatal diagnosis. A total of 162 samples of amniotic fluid were collected from pregnant women who were referred to the Obstetric Clinic at the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), São Paulo, Brazil, from August 2009 to May 2013. This study was approved by the University Ethics Committee (CAPPESq0789/08) in accordance with the ethical standards of the responsible committee on human experimentation. All participants in the study provided written informed consent. QF-PCR tests were performed by the same person (first author), and samples were analyzed with the karyotype results blinded. All women received genetic counseling, and routine informed consent was obtained in all cases included in the study. The clinical indications for karyotyping investigation included increased nuchal translucency (NT) thickness, previous child with chromosome abnormalities and structural fetal malformations. Most prenatal samples were collected between 12 and 34 weeks of gestation. At least 20 mL of amniotic fluid was collected from each pregnant woman, and 1 mL was allocated for our research and stored at – 80 oC for posterior analysis. Conventional cytogenetic analyses were performed on all the prenatal samples, which were cultured according to standard procedures in the HC-FMUSP, and results were issued between 14 and 21 days later.

Cytogenetic analysis Amniocytes were cultured and G banding was performed for all cases. These samples were analyzed at the cytogenetic laboratory at the Hospital das Clínicas da Universidade de São Paulo. Routine evaluation of each case involved the analysis of 20 random metaphase spreads from two independent cultures. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN2013).

Statistical analysis To determine appropriate sample size, a power calculation using a sensitivity of 83.73% was performed as described by Rostami et al. (12) in a group of pregnant women selected for karyotype studies according to increased maternal age and positive screen test; the prevalence of an abnormal karyotype was 4%. Considering that 55% of the samples in the present study presented abnormal karyotypes with a maximum estimated error of 10%, the 95% confidence interval would be achieved in a sample size of 95 cases.

DNA extraction DNA was extracted from fetal cells, which were obtained from amniotic fluid (1.0 mL) using a QIAamp DNA Blood Mini kit (Qiagen, Germany; CITOGEM Biotecnologia Ltda). Nucleic acids were eluted in a final volume of 65 mL of elution buffer. After extraction, the quality and quantity of DNA were evaluated by spectrophotometry on a NanoDrop 2000 (Thermo Fisher Scientific, USA).

’ RESULTS Patient demographics

Markers used

The samples obtained from the Brazilian population included 105 white volunteers, 45 brown volunteers, 9 black volunteers and 2 indigenous volunteers; 1 was unknown. Classifications of ethnicity were made according to the self-assessment of the volunteers. The mean maternal age was 29 years old (range, 14-49 years); 55 women (33.95%) were 35 years of age or older. The median gestational age of amniocentesis was 23 weeks (range: 11 weeks to 34 weeks). The results of 162 QF-PCR samples were compared to results obtained by culture conventional cytogenetic analysis, as presented in Table 1. There were two false-positives and one false-negative. The false-negative QF-PCR result was observed in one mosaicism for Turner syndrome; this sample was analyzed and reported as a normal female fetus. False-positives were found in 2 cases: one normal male with trisomy 18 and one normal female with polyploidy metaphases that showed abnormal results for chromosomes 21, 18, 13 and X. There were 151 samples corresponding with cytogenetic results (98.05%), excluding cases with chromosomal rearrangement. Table 2 shows the sensitivity, specificity, and positive and negative predictive values for all cases excluding those with rearrangements and mosaicism. The number and percentage of aneuploidies detected in

The assay uses multiplex PCR targeting STR markers to assess the copy numbers of chromosomes 13, 18, 21, X and Y. A total of 24 markers were selected for this study: 5 markers for chromosome 13 (D13S797, D13S742, D13S634, D13S628, and D13S305), 6 for chromosome 18 (D18S391, D18S976, D18S819, D18S390, D18S386, and D18S535), 6 for chromosome 21 (D21S1409, D21S11, D21S1411, D21S1246, D21S1444, and D21S1435), 3 for chromosome X (DXS6854, DXS6803, and XHPRT), 1 STR on the Y chromosome (SRY) and 2 for regions on the X and Y chromosomes (DXYS218 and X22). The amelogenin gene (AMXY) and SRY were used to allow for the assessment of fetal sex. QF-PCR is a quantitative method: the areas and the heights of the peaks are comparable.

Multiplex QF-PCR PCR amplification was performed in two reactions using 10 mL of the extracted DNA and 15 mL of ChromoQuant QF-PCR v.3 (Cybergene AB, Sweden) mix. After initial denaturation at 95 oC for 15 min, 26 cycles of amplification were performed (denaturation at 94 oC for 30 s, annealing at 57 oC for 1 min and extension at 71 oC for 2 min) followed by final extension at 71 oC for 5 min and 60 oC for 1 hour. The reaction was performed in a thermal cycler (Eppendorf, Germany).

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The QF-PCR results alone were in accordance with 98.05% of all karyotypes excluding cases with chromosomal rearrangements. These results are compatible with other recent reports in the literature. Lildballe et al. (17) analyzed 2,550 samples from chorionic villus sampling (CVS) and amniotic fluid from high-risk pregnancies and reported positive and negative predictive values greater than 99.8%. In this work, different predictive values were reported for each chromosomal abnormality, and even for mosaic trisomies, the detection

fetuses were compared with different ages of the mothers, as shown in Table 3. Among the discrepant results, 8 were due to chromosomal rearrangements, including one addition (46, XX, +add (7) (q33)), two translocations (46, XX, +13 rob (13, 14) (q10q10), 46, Y, t (X,14) (q22q31)), three inversions (46, XX, +i (1) (q10), 46, XY inv (9), 46, XY, inv (9) (p12q13)), one deletion (46, XX, del (11) (q22)), and one duplication (46, XY, dup (3) (p21.2p25)). In five cases of mosaicism, QF-PCR identified four abnormal trisomy karyotypes of the corresponding chromosomes (Table 4).

Table 4 - Discrepant cases of QF-PCR. Rearrangements

Analysis of the heterozygosities of STR markers

46, 46, 46, 46, 46, 46, 46, 46,

The heterozygosities of selected markers for QF-PCR are shown in Table 5. The markers D21S1435, D21S1409, and D21S1246 more commonly showed in a triallelic pattern, whereas the markers D13S742, D21S11, and D18S386 showed a diallelic pattern. For the sex chromosomes, the marker X22 had the highest frequency of heterozygosity.

’ DISCUSSION

45, 46, 46, 47, 47,

Table 1 - Comparison of QF-PCR and conventional cytogenesis results in AF samples. Cytogenetic, N (%)

QF-PCR, N (%)

72 (44) 28 (17) 21 (13) 9 (6) 17 (10) 2 (1) 5 (3) 8 (5) 90 (56) 100

70 (43) 28 (17) 21 (13) 9 (6) 17 (10) 2 (1) 4 (2) 0 (0) 81 (50) 93

46, XX; 46, XY 47, XX +21; 47, XY +21 47, XX +18; 47, XY +18 47, XX +13; 47, XY +13 Turner syndrome (45, X) Triploidy (69, XXX; 69, XXY) Mosaics Rearrangements Total abnormalities Test accuracy (%)

46, 46, 46, 46, 46, 46, 46, 46,

AF samples without rearrangements cases (n=154) (%)

Sensitivity Specificity Positive predictive value Negative predictive value Accuracy

98.78 97.22 97.59 98.59 98.05

XX XX XY XX XY XY XX XY

QF-PCR

X0 / 46, XX, +mar XY / 47, XY, +18 XY / 47, XY, +18 XX, +13 / 48, XX, +13 mar XX, +mar / 47, XX, +13

46, XX 47, XY, +18 47, XY, +18 47, XX, +13 47, XX, +13

Table 5 - QF-PCR results for each STR marker of chromosomes 13, 18, 21, X and Y. STR

AMEL DXYS218 DXS6803 DXS6854 XHPRT X22 D21S11 D21S1246 D21S1409 D21S1411 D21S1435 D21S1444 D18S386 D18S390 D18S391 D18S535 D18S819 D18S976 D13S305 D13S628 D13S634 D13S742 D13S797

Table 2 - Statistical analysis of AF samples tested using QF-PCR in high-risk pregnancies with an aneuploid fetus. Statistical analysis

XX, +add (7) (q33) XX, +13 rob (13, 14) (q10q10) Y, t (X,14) (q22q31) XX, +i (1) (q10) XY inv (9) XY, inv (9) (p12q13) XX, del (11) (q22) XY, dup (3)(p21.2p25)

Mosaics

In recent years, QF-PCR for the detection of common chromosomal trisomies has been introduced as a validated method at a number of cytogenetic centers (13-16). Our study presents results based on the application of QF-PCR for the rapid detection of aneuploidies in chromosomes 13, 18, 21, X and Y on 162 amniotic fluid samples in the Brazilian population.

Karyotype

QF-PCR

AF samples without mosaicism cases (n=157) (%)

90.59 97.22 97.44 89.74 93.63

Chromosome location

Mono allelic, N (%)

Diallelic, N (%)

Triallelic, N (%)

Xp22.31 – Xp22.1 Yp11.2 Xp22.32 / Yp11.3 Xq21.31 Xq26.1 Xq26.1 Xq28Yq 21q21.1 21q22.2 21q21.2 21q22.3 21q21.1 21q22.13 18q22.1 18q22.3-18q23 18p11.31 18q12.3 18q11.2 18p11.31 13q13.3 13q31.1 13q21.33 13q12.13 13q33.2

93 (58)

66 (41)

2 (1)

67 100 107 97 38 26 40 48 25 36 33 22 72 39 31 39 41 57 45 50 14 36

(41) (62) (66) (60) (66) (44) (25) (30) (42) (23) (49) (37) (44) (58) (53) (60) (57) (38) (71) (32) (35) (60)

91 58 52 63 10 11 97 86 11 96 10 11 80 10 10 10 10 80 10 96 13 11

(56) (36) (32) (39) (17) (19) (60) (53) (19) (60) (15) (19) (49) (15) (17) (15) (14) (54) (16) (61) (33) (18)

4 3 3 2 10 22 25 27 23 28 25 26 10 18 18 16 21 12 8 12 13 13

(2) (2) (2) (1) (17) (37) (15) (17) (39) (18) (37) (44) (6) (27) (31) (25) (29) (8) (13) (8) (33) (22)

Table 3 - Number and percentage of aneuploidies in fetuses detected using QF-PCR as stratified by the mother’s age. Mother’s age o35 X35 Total

Total Aneuploidies N (%)

47, XX/XY +21

47, XX/XY +18

47, XX/XY +13

45, X

69, XXX, 69, XXY

48 (63) 29 (37) 77 (100)

13 (17) 15 (19) 28 (36)

14 (18) 7 (9) 21 (27)

7 (9) 2 (3) 9 (12)

12 (16) 5 (6) 17 (22)

2 (3) 0 (0) 2 (3)

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97% of the predicted chromosomal anomalies when a fetal karyotype is requested. As for any other prenatal test, employing this assessment requires investment in infrastructure, equipment and training for the lab staff. This test can decrease the demand for conventional cytogenetic analysis and can reduce parental anxiety as well as expand the reach of this test throughout the population.

rate was higher than 99.8%. Rostami et al. (12) reported 4,058 samples analyzed for QR-PCR with a detection rate of 98.59%. Tekcan et al. (18) compared 100 amniotic fluid samples with karyotype results and obtained 99% concordance on 100 samples, including 4 abnormalities. These three authors reported higher detection rates than in our study; however, the percentages of total chromosomal abnormalities in these studies were 7.6% and 4.1%, respectively, whereas in the current study, approximately half of the population was abnormal. This difference in the prevalence of aneuploidy could be explained by the indication of the karyotype that was primarily due to ̌ fetal malformation in our study. ̌L /aczman´ńska et al. (19) analyzed 100 samples of amniotic fluid and obtained compatibility in 95 cases (95%), which agrees with our study due to the higher number of chromosomal abnormalities, which were found in 28 pregnancies (29.5%). Our detection rate of 98.8% included five cases of mosaicisms; among which four of these samples returned abnormal QF-PCR results, which was in agreement with the chromosome involved in the abnormality. If we consider that an abnormal result should be further confirmed by cytogenetic analysis, these four cases could be considered positive screen tests for aneuploidy in QF-PCR. Additionally, one Turner mosaic sample was revealed to be normal in QF-PCR testing. This result is in accordance with the literature and can be explained by lower rates of mosaic cells (less than 15%) (19-22). Of the two false-positive results, one revealed a triploid female upon QF-PCR with a normal polyploidy metaphases karyotype upon cytogenetic analysis. These polyploidy metaphases might be the reason for the false-positive result, though its significance is unknown. A second falsepositive was a normal male, which was revealed as trisomy 18. The sample was bloodstained, so this false result might be explained by maternal contamination. The markers D21S1435, D13S742, D13S797, and D18S1386 were the most frequently informative in all cases of trisomies 21, 13 and 18, respectively, in our population, displaying a higher frequency of heterozygosity. QF-PCR is a rapid, robust and accurate diagnostic method for detecting common aneuploidies in high-risk pregnancies, with results available in two days because fetal cells do not have to be cultured. The Implementation of QF-PCR at a public referral center or laboratory could improve patient care and reduce overall health costs. A strategy could be proposed in which, in combination with traditional 1st trimester screening tests, pregnant women could be referred for karyotyping via QF-PCR due to advanced maternal age, anxiety, NT o3 mm or a high-risk for trisomies; this would eliminate the need for karyotyping by culture. Karyotyping still should be applied in pregnancies with normal QF-PCR results and the presence of either structural abnormalities detected by ultrasound or two or more soft markers for Down syndrome as well as a family history of chromosome rearrangement. This approach has been implemented in London and has led to a 99.9% detection rate of any chromosomal abnormality, which has reduced the need for a full karyotype analysis to only 25% of pregnancies (23). For the public health system in Brazil, where most pregnant women do not have access to fetal karyotyping, an initial investigation with a rapid and feasible assay such as QF-PCR would provide the opportunity to offer tests at a central laboratory and include more patients. Although QF-PCR cannot detect all chromosomal abnormalities, this assay can cover more than

’ ACKNOWLEDGMENTS This research was supported by FAPESP (2012/51806-2) to Mário Henrique Burlacchini de Carvalho and a Master’s Scholarship supported by CAPES to Renata Wendel de Moraes. We are also grateful to all families who took part in this study and kindly donated their amniotic ﬂuid samples.

’ AUTHOR CONTRIBUTIONS Moraes RW was responsible for the data collection, analysis and interpretation and manuscript drafting. de Carvalho MH was responsible for the study conception and design, manuscript drafting, critical review of the manuscript for intellectual content, approval of the final version of the manuscript, supervision of all aspects of the project. De Amorim-Filho AG was responsible for the data collection, analysis and interpretation, critical review of the manuscript for intellectual content. Francisco RP was responsible for the critical review of the manuscript for intellectual content, approval of the final version of the manuscript for submission. Romão RM was responsible for the data collection. Levi JE was responsible for the study conception and design, manuscript drafting, critical review of the manuscript for intellectual content, approval of the final version of the manuscript for submission, supervision of all aspects of the project. Zugaib M was responsible for the approval of the final version of the manuscript for submission.

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11. Brown L, Abigania M, Warburton D, Brown S. Validation of QF-PCR for prenatal aneuploidy screening in the United States. Prenat Diagn. 2006; 26(11):1068-74, http://dx.doi.org/10.1002/pd.1558. 12. Rostami P, Valizadegan S, Ghalandary M, Mehrjouy MM, Esmail-Nia G, Khalili S, et al. Prenatal screening for aneuploidies using QF-PCR and karyotyping: A comprehensive study in Iranian population. Arch Iran Med. 2015;18(5):296-303. 13. Cho EH, Park BY, Kang YS, Lee EH. Validation of QF-PCR in a Korean population. Prenat Diagn. 2009;29(3):213-6, http://dx.doi.org/10.1002/ pd.2190. 14. Papoulidis I, Siomou E, Sotiriadis A, Efstathiou G, Psara A, Sevastopoulou E, et al. Dual testing with QF-PCR and karyotype analysis for prenatal diagnosis of chromosomal abnormalities. Evaluation of 13,500 cases with consideration of using QF-PCR as a stand-alone test according to referral indications. Prenat Diagn. 2012;32(7):680-5, http://dx.doi.org/10.1002/ pd.3888. 15. Xu AQ, Xia M, Liu JT, Yao FX, Zhang WM, Hao N, et al. Validation of quantitative fluorescent-PCR for rapid prenatal diagnosis of common aneuploidies in the Chinese population. Genet Mol Res. 2013;12(4):6379-88, http://dx.doi.org/10.4238/2013.December.9.1. 16. Guzel A, Yilmaz M, Demirhan O, Pazarbasi A, Kocaturk-Sel S, Erkoc M, et al. Rapid detection of fetal aneuploidies by quantitative fluorescentpolymerase chain reaction for prenatal diagnosis in the Turkish population. Balkan J Med Genet. 2012;15(1):11-7. 17. Lildballe DL, Vogel I, Petersen OB, Vestergaard EM. Diagnostic performance of quantitative fluorescence PCR analysis in high-risk

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pregnancies after combined first-trimester screening. Dan Med J. 2014; 61(11):A4964. Tekcan A, Tural S, Elbistan M, Kara N, Guven D, Kocak I. The combined QF-PCR and cytogenetic approach in prenatal diagnosis. Mol Biol Rep. 2014;41(11):7431-6, http://dx.doi.org/10.1007/s11033̌014-3630-7. ̌/ L aczman´ńska I, Gil J, Stembalska A, Makowska I, Koz"owska J, Skiba P, et al. [Rapid diagnosis of the most common fetal aneuploidies with the QF-PCR method--a study of 100 cases]. Ginekol Pol. 2015;86(9):694-9, http://dx.doi.org/10.17772/gp/59024. Cirigliano V, Voglino G, Marongiu A, Cañadas P, Ordoñez E, Lloveras E, et al. Rapid prenatal diagnosis by QF-PCR: evaluation of 30,000 consecutive clinical samples and future applications. Ann N Y Acad Sci. 2006;1075:288-98, http://dx.doi.org/10.1196/annals.1368.039. Cirigliano V, Voglino G, Ordoñez E, Marongiu A, Paz Cañadas M, Ejarque M, et al. Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR, results of 9 years of clinical experience. Prenat Diagn. 2009; 29(1):40-9, http://dx.doi.org/10.1002/pd.2192. Mann K, Hills A, Donaghue C, Thomas H, Ogilvie CM. Quantitative fluorescence PCR analysis of 440,000 prenatal samples for the rapid diagnosis of trisomies 13, 18 and 21 and monosomy X. Prenat Diagn. 2012;32(12):1197-204, http://dx.doi.org/10.1002/pd.3986. Hills A, Donaghue C, Waters J, Waters K, Sullivan C, Kulkarni A, et al. QF-PCR as a stand-alone test for prenatal samples: the first 2 years’ experience in the London region. Prenat Diagn. 2010;30(6):509-17, http://dx.doi.org/10.1002/pd.2503.

CLINICAL SCIENCE

Clinical Features of Refractory Ascites in Outpatients Wanda Regina Caly, Rodrigo Martins Abreu, Bernardo Bitelman, Flair Jose´ Carrilho, Suzane Kioko Ono * Departamento de Gastroenterologia, Divisao de Gastroenterologia e Hepatologia Clinica, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVES: To present the clinical features and outcomes of outpatients who suffer from refractory ascites. METHODS: This prospective observational study consecutively enrolled patients with cirrhotic ascites who submitted to a clinical evaluation, a sodium restriction diet, biochemical blood tests, 24 hour urine tests and an ascitic fluid analysis. All patients received a multidisciplinary evaluation and diuretic treatment. Patients who did not respond to the diuretic treatment were controlled by therapeutic serial paracentesis, and a transjugular intrahepatic portosystemic shunt was indicated for patients who required therapeutic serial paracentesis up to twice a month. RESULTS: The most common etiology of cirrhosis in both groups was alcoholism [49 refractory (R) and 11 nonrefractory ascites (NR)]. The majority of patients in the refractory group had Child-Pugh class B cirrhosis (p=0.034). The nutritional assessment showed protein-energy malnutrition in 81.6% of the patients in the R group and 35.5% of the patients in the NR group, while hepatic encephalopathy, hernia, spontaneous bacterial peritonitis, upper digestive hemorrhage and type 2 hepatorenal syndrome were present in 51%, 44.9%, 38.8%, 38.8% and 26.5% of the patients in the R group and 9.1%, 18.2%, 0%, 0% and 0% of the patients in the NR group, respectively (p=0.016, p=0.173, p=0.012, p=0.012, and p=0.100, respectively). Mortality occurred in 28.6% of the patients in the R group and in 9.1% of the patients in the NR group (p=0.262). CONCLUSION: Patients with refractory ascites were malnourished, suffered from hernias, had a high prevalence of complications and had a high postoperative death frequency, which was mostly due to infectious processes. KEYWORDS: Ascites; Hepatic Encephalopathy; Liver; Liver Cirrhosis; Outpatients. Caly WR, Abreu RM, Bitelman B, Carrilho FJ, Ono SK. Clinical Features of Refractory Ascites in Outpatients. Clinics. 2017;72(7):405-410 Received for publication on October 18, 2016; First review completed on January 12, 2017; Accepted for publication on February 16, 2017 *Corresponding author. E-mail: skon@usp.br

’ INTRODUCTION

activation of neurohumoral systems, the renin-angiotensinaldosterone system, and the sympathetic nervous system (SNS); subsequently, ascites triggers the non-osmotic activation of antidiuretic hormone, which is a compensatory mechanism of organic homeostasis. These changes result in an increased retention of salt and water. During the later stages of cirrhosis, in addition to the maximal activation of neurohumoral systems, some patients will have an imbalance of the intrarenal regulatory hormones, leading to hepatorenal syndrome (6). Depending on the duration of the underlying disease, this cascade of pathophysiological changes may lead to the development of ascites that can be easy or difficult to mobilize; thus, treatment is not restricted to the typical forms of therapy. In accordance with the most accepted pathophysiology, the typical treatment of ascites includes the restriction of salt in the diet and, in most cases, a combination of diuretics to obtain a satisfactory response, i.e., a negative balance of salt and water and the disappearance of the ascites and/or edema (7). However, approximately 10% of patients with ascites will have no resolution even when they adhere to a low sodium diet (a maximum intake of 2.0 g salt/day, which is associated with the use of diuretics). This group includes patients who have an inability to mobilize ascites despite a confirmed adherence to the dietary sodium restriction (88 mEq salt/day)

Ascites is one of the most frequent complications of liver cirrhosis and occurs in approximately 50% of patients with compensated liver disease during an observation period of approximately 10 years (1). The development of ascites is associated with a worse disease prognosis, and the estimated average life expectancy is approximately two years for 50% of cirrhotic patients with ascites and only approximately six months for those with a urinary sodium excretion that is less than 10 mEq/L (2-4). This condition is observed particularly in cirrhotic patients with refractory ascites, which is thus indicative of the need for definitive treatment with a liver transplantation. According to the most-widely accepted pathophysiological hypothesis (5), ascites occurs only in the presence of portal hypertension, which leads to peripheral arterial vasodilatation and, consequently, effective arterial hypovolemia. Ascites triggers the

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performed in other studies (11,12). The TSF was measured in mm using an appropriate caliper that was placed parallel to the major axis of the non-dominant arm between the acromion and the olecranon. The arm circumference (AC) was measured in cm using a conventional measuring tape without a clearance space that was placed at the midpoint between the acromion and the olecranon on the nondominant arm during normal posture and relaxation. The MAMC was measured in cm and was calculated based on the AC. These parameters, including the estimation of the percentage of depletion, were compared with the normal range. The patients were then grouped into mild, moderate and severe malnutrition groups based on these comparisons (13). A nutritional assessment was performed, and dietary guidance regarding sodium restriction and individualized supplementation was provided as necessary. The included patients then received the treatment protocol and were evaluated weekly for the volume of the ascites, body weight, renal function parameters, and electrolyte dosages. If necessary, changes to their prescriptions or other changes to the massive paracentesis with the intravenous albumin replacement (8 g/liter of ascites removed) were performed (13,14). In all outpatient care visits, the ChildPugh, MELD and MELD-Na scores were updated. All patients were under hepatocellular carcinoma (HCC) surveillance with abdominal ultrasound and alpha-fetoprotein. The ascites treatment protocol initiated therapy in combination with a diuretic schedule, which was supplemented with spironolactone (100 mg once daily) and furosemide (40 mg once daily) in patients who showed an inefficiency in weight loss after one week on the low sodium diet. Before the start of the diuretic therapy, an evaluation of the renal function parameters, urea, creatinine, and electrolytes, such as sodium and potassium was performed, and the dosage of sodium and potassium in the 24 hour urine test was assessed (15). Patients returned for weekly reevaluation to verify the effectiveness of the applied management and/or the appearance of complications due to the use of diuretics. In any of these events, the diuretic therapy had an optimized dosage until it reached the maximum allowed or until the administration was interrupted in the case of the onset of complications, indicating the failure of this treatment modality according to the criteria described for refractory ascites (7,16). Diuretic-resistant ascites was defined according to the International Ascites Club (7,8). In summary, at least one of the following criteria is fulfilled in the absence of therapy with a nonsteroidal anti-inflammatory drug: An inability to mobilize the ascites (manifested as minimal to no weight loss) despite the confirmed adherence to the dietary sodium restriction (88 mEq [2000 mg] per day) and the administration of the maximum tolerable doses of oral diuretics (400 mg of spironolactone and 160 mg of furosemide once daily); Rapid re-accumulation of fluid after therapeutic paracentesis despite adherence to a sodium-restricted diet; and The development of diuretic-related complications, such as progressive azotemia, HE, or progressive electrolyte imbalances. Patients who were withdrawn from the conventional treatment of ascites protocol were followed-up every two weeks at most and were controlled by TSP with an intravenous albumin replacement. The insertion of TIPS was indicated for those patients who were controlled by TSP, required this procedure more than twice a month and did not

and the administration of the maximum doses of oral diuretics, such as 400 mg/day of spironolactone and 160 mg/day of furosemide (refractory resistant); these patients may experience a rapid re-accumulation of fluid after the therapeutic paracentesis despite their adherence to a sodium-restricted diet or develop diuretic-related complications, such as progressive azotemia, hepatic encephalopathy (HE), or progressive electrolyte imbalances, which require the discontinuation of the therapy (refractory intolerant) (8). Thus, these patients are considered refractory to the conventional treatment of ascites and should be subjected to optional treatments, such as therapeutic serial paracentesis (TSP) with albumin replacement and intravenous insertion of a transjugular intrahepatic portosystemic shunt (TIPS); LeVeen or Denver valve insertions may be suitable for patients who are not eligible for TSP or TIPS. Moreover, a liver transplantation should be considered in the absence of contraindications (9,10). Although this group of patients presents a poor clinical situation, in most cases, they do not reach a sufficient score on the Model for End-Stage Liver Disease (MELD) scoring system to justify a liver transplantation, which increases the risk of morbidity and mortality while awaiting definitive treatment (2,10). Considering this scientific perspective, the objectives of this study were to present the clinical features and outcomes of outpatients who suffer from refractory ascites.

â&#x20AC;&#x2122; METHODS This observational study demonstrated our initial experience with such management at our institution and involved patients with cirrhotic ascites who were enrolled consecutively and prospectively and referred to the Clinic for Refractory Ascites of the Division of Gastroenterology and Hepatology â&#x20AC;&#x201C; Hospital das ClĂnicas, University of SĂŁo Paulo School of Medicine, SĂŁo Paulo, Brazil, between March 2009 and August 2010. Patients were excluded if they were cirrhotic with ascites considered to have an easy resolution, ascites of a different etiology, and ascites with concomitant neoplasm or had current alcoholic hepatitis. Non-compliance with the dietary restrictions, drug therapy and ascites treatment guidelines was considered a cause of the false refractoriness. Patients were included only after undergoing a comprehensive clinical evaluation to confirm the refractory ascites, which was based on their medical history, a physical examination and a plasma/ascitic fluid analysis (such as biochemistry, leucometry and culture). The patients participated in a one-week orientation, during which a nutritionist focused on the diuretic treatment withdrawal and the sodium restriction diet; the orientation was followed by 24 hour urine tests to quantify sodium and total proteins. Additional examinations were usually performed, such as a chest X-ray, an electrocardiogram, an echocardiogram, an abdominal Doppler ultrasound, an upper gastrointestinal endoscopy, and arterial blood gases. The total count number and differential leukocyte/mm3 ascites analyses were performed in all patients who were subjected to the various paracenteses. All patients were referred to a clinical multidisciplinary evaluation with a cardiologist, nutritionist and psychologist. The nutritional assessment was performed with anthropometric parameters using the triceps skinfold (TSF) and midarm muscle circumference (MAMC) according to procedures

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Table 2 - Nutritional Status.

present contraindications; even in patients with no urinary sodium excretion, performing paracentesis every two weeks controls the ascites (17). Currently, this form of therapy is considered a secondary option in the treatment of refractory ascites (7,9,18,19). The patients with esophageal varices were treated according to the Consensus of Variceal Bleeding of the Brazilian Society of Hepatology (20) with an elastic bandage on the medium and/or thick caliber veins with previous bleeding or hematocystic spots, whose presence is associated with beta-blocker drug treatments. The average blood pressure of each patient, spontaneous bacterial peritonitis (SBP) background and previous or current renal failure were considered. In selected cases, the treatment was exclusively an endoscopic treatment of the varicose veins. The follow-up was performed from the date on which the refractory ascites was diagnosed in each patient to the closure of the study date (August 2010). The evaluation period ranged from 3 to 17 months.

Nutritional Evaluation Normal weight Mild malnutrition Moderate malnutrition Severe malnutrition Overweight Obese

HRS type 2 HE Hernias SBP Hydrothorax UDH Transplantation Mortality rate

’ RESULTS During the study period, 60 patients were enrolled as follows: 49 refractory ascites patients (R) and 11 non-refractory ascites patients (NR). The most common etiology of the cirrhosis in both the refractory and non-refractory groups was alcoholism. The average age was 57.5±8.8 years in the R group and 50.3± 8.1 years in the NR group (p=0.010), and males were predominant in both groups (72.9% in the R group and 66.7% in the NR group; p=0.712) (Table 1). The nutritional assessment showed a protein-energy malnutrition in 81.6% of the patients in the R group and 35.5% of the patients in the NR group (Table 2). HE, hernia, SBP and hepatorenal syndrome (HRS) type 2 were present in 51%, 44.9%, 38.8% and 26.5%, respectively, of the patients in the refractory ascites group and 9.1%, 18.2%, 0% and 0%, respectively, of the patients in the non-refractory ascites group (p=0.016, p=0.173, p=0.012 and p=0.100, respectively). Table 1 - Clinical Results.

Age Male gender (%) Child-Pugh B (%) MELD ± SD MELD-Na ± SD

57.5±8.8 73.5 71.4 13.4±4.2 16.1±5.1

49.9±7.9 63.6 36.4 11.2±4.0 12.8±5.1

0.010 0.712 0.034 0.126 0.054

(36.4%) (18.2%) (18.2%) (9.1%) (9.1%) (9.1%)

8 9 24 12 3 4

(13.3%) (15.0%) (40%) (20%) (5.0%) (6.7%)

R (49) 13 25 22 19 11 19 9 14

(26.5%) (51.0%) (44.9%) (38.8%) (22.4%) (38.8%) (18.4%) (28.6%)

NR (11)

0 1 (9.1%) 2 (18.2%) 0 1 (9.1%) 0 0 1 (9.1%)

0.100 0.016 0.173 0.012 0.435 0.012 0.189 0.262

Of the 49 refractory patients, only 5 underwent the insertion of TIPS. TIPS were inserted in patients who lost control of the ascites by paracentesis or if this procedure had to be performed twice a month without any absolute contraindications for TIPS. Although some patients needed three or more paracenteses per month, several criteria contraindicated the placement of TIPS. Although we had patients with MELD scores o18 and Child-Pugh Class B, some patients had persistent encephalopathies and/or grade 42, HRS type 2 and/or other changes to the procedure that contraindicated or would be controversial to its effectiveness. In total, 12 (24.49%) patients in the refractory group and 1 patient in the non-refractory group required hospitalization during the evaluation period. The causes of admission varied, including decompensated ascites preventing the insertion of TIPS (5), severe gastrointestinal bleeding (1), infection (3), and HE (3). Five TIPS were inserted, but 4/5 (80%) patients presented HE and required diuretics to control the ascites; 3/5 (60%) patients who received TIPS required low doses of diuretics to control the ascites. Furthermore, despite the use of diuretics at tolerated doses, 1/5 (20%) patients returned to controlling the ascites by serial paracentesis because of TIPS obstruction 30 days after the insertion, and this patient presented with a worsening clinical condition and died on the 4th month after the TIPS insertion due to an acute myocardial infarction. Of the total number of enrolled patients, 6 patients (54.54%) in the non-refractory group and 20 patients (40.81%) in the refractory group received propranolol for more than 30 days as a primary or secondary bleeding prophylaxis from gastroesophageal varices. Mortality occurred in 28.6% of the patients in the R group and 9.1% of patients in the NR group (p=0.262). The results of the clinical complications diagnosed in both groups are shown in Table 3. Of the 15 total deaths, 5 patients were post liver transplantation, and 10 patients were on the waiting list for transplantation. According to the total deaths, 14 were in the refractory group; 9/14 patients were on the liver transplantation list; 3/9 deaths were caused by umbilical hernia rupture; and 2/9 deaths were caused by SBP, one of

For the qualitative variables, comparisons between the two groups were performed using the chi-square test and Fisher’s exact test. This study adopted the 5% level of significance, and analysis of variance (ANOVA) was used for all statistical calculations.

p-value

4 2 2 1 1 1

Total

Legend: HRS type 2: hepatorenal syndrome type 2; HE: hepatic encephalopathy; SBP: spontaneous bacterial peritonitis; UDH: upper digestive hemorrhage.

Statistical Analysis

NR (11)

(8.2%) (14.3%) (44.9%) (22.4%) (4.1%) (6.1%)

Clinical complications

The study protocol was approved by the Ethics Committee of the Hospital das Clínicas, University of São Paulo School of Medicine and conformed with the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008). Because this is an observational study, the ethics committee waived the requirement of informed consent from each patient included in the study.

R (49)

4 7 22 11 2 3

Table 3 - Results of Clinical Complications.

Ethics

Parameters

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complications and occurred in 38.8%, 38.8%, 51%, 26.5%, and 44.9% of the patients, respectively. According to the pathophysiology of ascites, the appearance of HRS type 2 is one of the expected complications in advanced stage cirrhotic patients with ascites, when the activation of the neurohumoral system, renin-angiotensin-aldosterone, SNS, and antidiuretic hormone (ADH) are extreme and can no longer return the balance of the hemodynamic parameters to normal levels of blood pressure. Thus, in some patients, there will be an increased activation of vasodilator hormones or a moderate production of vasoconstrictors in the kidney, which often reduces the effect of systemic and renal vasoconstriction but elicits a renal failure that is stable for months. This renal failure is likely to develop into a more serious impairment, depending on external aggravating factors that are related to the presence of an acute circulatory dysfunction as it occurs, for example, during the onset of SBP (8, 21). During the advanced stage of cirrhosis, patients may not respond to the conventional treatment with diuretics, and several complications arising from its use can be diagnosed. In addition, the patients frequently present asthenia, anorexia, malnutrition, hernias, HE, SBP and renal dysfunction. Thus, our results described findings that are related to severe cirrhotic patients with refractory ascites (22). Although there were more patients with HRS and hernias in the refractory ascites group, no significant difference was observed in the frequency of these complications, which is likely due to the small number of patients included in the study. However, according to our results and the results of others, even in this condition, this group of patients did not achieve a MELD score that prioritizes liver transplantation, which is of main importance since this may have been related to the high percentage of deaths in the patients waiting for liver transplantation (23, 24). The MELD-Na appears to be a valid method to more readily benefit these patients (25, 26, 32). We emphasize that refractory ascites patients are extremely and seriously ill (end-stage of liver disease). However, these patients do not need to reach a certain MELD score to be competitive with other cirrhotic patients on the liver transplantation list. In addition, only some patients obtain a score due to a special situation. Occasionally, the MELD score increases due to the absence of major coagulation disorders or a low alteration in the serum bilirubin level. Therefore, it is important to emphasize that the MELD-Na score could help hyponatremic patients with a worse prognosis, but it is not currently available. Thus, these patients progressively evolve with renal dysfunction and recurrent infections and may die before liver transplantation. It is highly important to frequently diagnose the presence of hernias in the patients with abdominal distensions due to ascites since hernias were found to be a significant cause of death in the group waiting for liver transplantation. Complications of spontaneous perforations led to discussions regarding whether these patients would benefit from elective surgery for the hernias, particularly for those who presented with thinner and/or eroded skin (27, 33, 34). Although five TIPS were inserted in the refractory patient group in accordance with the inclusion criteria for this type of therapy (9, 10, 18), we found that four of these patients developed HE, either grade 1 or 2, which was easily resolved and required diuretics at lower doses to control the ascites. We also emphasize that at the time of this study, the released

which progressed to sepsis, and the other progressed to HRS. In the other four cases (4/9), 3/9 deaths were not directly related to the presence of the ascites (rather, they were related to acute arterial thrombosis of the lower limbs, upper gastrointestinal bleeding and pulmonary embolism), and the other died due to cachexia in the course of the refractory ascites with contraindications to liver transplantation, TIPS and paracentesis. Five of the 14 deaths occurred post liver transplantation, and three of these occurred due to infectious complications. During this study, there were no cases with HCC since it was an exclusion criterion. SBP occurred in two cases (4.08%) of refractory patients.

â&#x20AC;&#x2122; DISCUSSION The treatment of ascites in liver cirrhosis is typically performed by different medical specialists. However, professionals other than gastroenterologists and hepatologists can also perform the treatment procedures, including gastroenterological surgeons, nephrologists and infectologists. Different medical doctors follow these patients depending on their degree of familiarity with ascites treatment, and they play a key role in the ultimate goal of therapy. However, many professionals do not follow any established treatment protocol, which may lead to the emergence of several complications, such as HE and renal dysfunction, which are the most frequent complications (7). Because many physicians note difficulties in managing massive ascites, we aimed to introduce a protocol that identifies outpatient cirrhotic patients with ascites that is considered difficult to manage (due to cirrhosis decompensation). The patients were followed-up with nutritional assessments to verify their adherence to the diet guidelines according to their individual needs because there is a direct relationship between malnutrition and increasing complications in cirrhotic patients with refractory ascites. Nutritional parameters, such as body mass index and percentage of ideal weight, have limitations in chronic liver disease mainly because of fluid retention, which is often exhibited by patients, including those without detectable ascites (21). According to our results, 80% of the patients had some degree of malnutrition in the initial evaluation, which was frequently related to moderate or advanced cirrhosis. This was also the profile of our patients, and the majority of them were classified as Child-Pugh B (22,23); our results are consistent with results of other studies (24). In several studies, protein and calorie malnutrition have been associated with an increased number of serious complications, including ascites, variceal bleeding, increased surgical morbidity and mortality, and reduced survival (25,26). This fact is very important considering the results obtained in a recent study (27), which showed that better nutritional support for patients with refractory ascites led to lower rates of morbidity and mortality compared with patients with refractory ascites who did not have nutritional supplementation (23). Thus, after excluding the other causes of ascites, which were not related to liver cirrhosis and/or associated with malignancies, the patients followed the treatment protocol for ascites and always paid attention to the exclusion of causes of false refractoriness (28-31). Interestingly, among the complications presented during the treatment in our refractory patients, upper digestive hemorrhage (UDH), SBP, HE, HRS type 2, and umbilical and/or inguinal-scrotal hernia were the most prevalent

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TIPS were not covered, increasing the possibility of dysfunction and clinical complications (35). Several reports in the literature show the benefits of TIPS in controlling ascites compared to serial voluminous paracentesis (36-38). TIPS was performed in refractory ascites patients who underwent more than two paracenteses each month for ascites control. This frequency shows a loss of ascites control by paracentesis and highlights a poor life quality and malnutrition in this group of patients (39). Our results are consistent with those reported in the literature; in five meta-analysis studies, the insertion of TIPS was shown to control ascites better than serial paracentesis therapies but leads to a higher incidence of HE (3, 40-44). This finding was highlighted in the EASL guidelines, which described that the reported management of ascites resulted more frequently in HE in the TIPS group compared to the paracentesis group (45). More recently, a retrospective study evaluating 70 covered TIPS and 80 serial paracenteses for refractory ascites treatment concluded that covered "stents" improved the survival to medium- and long-term without a significant increase in the short-term mortality of patients with refractory ascites in clinical treatment failure (46). None of our clinical cases were consistent with postparacentesis circulatory dysfunction, but this finding was not an aim of this study. Regarding the frequency of deaths in our refractory patients, complicated hernias were the most frequent causes of death before transplantation, and bacterial infection was an important cause of death post liver transplantation, which was likely due to the advanced stage of the underlying disease and the degree of malnutrition (23, 47-49). Among the refractory patients who died pre-transplantation, 4/9 (44.4%) were taking propranolol, and no cause of death was directly related to the refractory ascites; however, among the refractory deaths after transplantation, 2/5 (40%) had histories of propranolol use, and sepsis was the cause death. In the non-refractory group, one patient who received betablockers died due to SBP that progressed to sepsis. However, the sample presented was small. In conclusion, patients with refractory ascites were very malnourished, frequently suffered from hernias and had a high prevalence of renal dysfunction, SBP, upper gastrointestinal hemorrhage, and episodes of HE. Moreover, when the patients reached a score eligible for liver transplantation, they had a high frequency of postoperative death mostly due to infectious processes. Thus, the developmental profile of refractory patients based on our results shows the necessity of reviewing the current criteria to prioritize liver transplantation and minimize the high morbidity and mortality in this group of patients. However, further studies with larger numbers of patients are needed to confirm these data.

Bitelman B, Carrilho FJ and Ono SK provided clinical data regarding the patients.

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’ ACKNOWLEDGMENTS Alves de Queiroz Family Fund for Research.

’ AUTHOR CONTRIBUTIONS Caly WR and Ono SK conceived and designed the study. Caly WR, Abreu RM, Bitelman B, Carrilho FJ and Ono SK performed the study, analyzed the data, were responsible for the reagents/materials/analysis tools, provided clinical data regarding the patients and critical advice on writing the manucript. Abreu RM, Caly WR and Ono SK wrote the manuscript. Caly WR,

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CLINICAL SCIENCE

Intermittent claudication and severe renal artery stenosis are independently associated in hypertensive patients referred for renal arteriography Thiago Andrade Macedo,I,* Luciano Ferreira Drager,I Rodrigo Pinto Pedrosa,I Henrique Cotchi Simbo Muela,I Valeria Costa-Hong,I Luiz Junia Kajita,II Luiz Aparecido BortolottoI I Unidade de Hipertensao, Divisao de Cardiologia, Instituto do Coracao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Laboratorio de Hemodinamica, Divisao de Cardiologia, Instituto do Coracao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVE: The purpose of this study was to evaluate the association between the presence of clinical symptoms of peripheral artery disease and severe renal artery stenosis in patients referred for renal angiography. METHOD: We included 82 patients with clinical suspicion of renovascular hypertension and performed an imaging investigation (renal Doppler ultrasound and/or renal scintigraphy) for possible renal artery stenosis. All patients underwent renal arteriography and were examined for peripheral artery disease based on the presence of intermittent claudication and ankle-brachial index test results. Severe renal artery stenosis was defined as a lesion causing 70% obstruction. RESULTS: Severe renal artery stenosis was present in 32 of 82 (39%) patients. Patients with severe renal artery stenosis were older (63±12 vs 56±12 years, p=0.006), had more intermittent claudication (55 vs 45%, p=0.027), and had a greater prevalence of an ankle-brachial index o0.9 (44% vs 20%, p=0.021) than patients without severe renal artery stenosis. Multivariate logistic regression analysis showed that the presence of intermittent claudication was independently associated with renal artery stenosis X70% (OR: 3.33; 95% CI 1.03–10.82, p=0.04), unlike the ankle-brachial index, which showed no association (OR: 1.44; 95% CI 0.37–5.66, p=0.60). CONCLUSION: Intermittent claudication is independently associated with severe renal artery stenosis (X70%) in patients clinically suspected of having renovascular hypertension. KEYWORDS: Intermittent Claudication; Renal Artery Stenosis; Renal Angiography. Macedo TA, Drager LF, Pedrosa RP, Muela HC, Costa-Hong V, Kajita LJ, et al. Intermittent claudication and severe renal artery stenosis are independently associated in hypertensive patients referred for renal arteriography. Clinics. 2017;72(7):411-414 Received for publication on July 24, 2016; First review completed on August 24, 2016; Accepted for publication on March 9, 2017 *Corresponding author. E-mail: thiago.macedo@incor.usp.br

’ INTRODUCTION

with hypertension. During the clinical investigation, the presence of symptoms suggestive of PAD should be further investigated using the ankle-brachial index (ABI) test. This test, besides being an important diagnostic tool, provides significant information about the presence of subclinical atherosclerosis (2) and the increased risk of cardiovascular morbidity and mortality (3). Furthermore, in the presence of vascular risk factors such as hypertension, the detection of asymptomatic PAD may represent the need for a change from primary to secondary prevention, resulting in more rigorous treatment strategies. Evidence has shown that RAS is a predictor of coronary artery disease (CAD) and a marker of diffuse atherosclerosis (9-11). In a previous study, the authors identified predictors of serious CAD (X70%) in patients with clinical suspicion of severe RAS (X70%). Severe RAS is a strong predictor of serious CAD independent of angina, and dual investigation should be considered in patients referred for renal angiography (9). The purpose of our study was to determine whether IC could be a clinical predictor of severe RAS in

Atherosclerotic renal stenosis is a significant cause of secondary arterial hypertension and is strongly associated with cardiovascular events (1,2), independent of the degree of hypertension (3,4). Previous studies have shown the coexistence of renal artery stenosis (RAS) with atherosclerosis at other vascular sites (5-7) and high blood pressure is linked to an increased incidence of peripheral artery disease (PAD) (8). Most patients with PAD have no symptoms but do experience intermittent claudication (IC), the main symptom of PAD, which is frequently underdiagnosed (2) in patients

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artery, left posterior tibial artery, and right brachial artery. The ABI was calculated for each lower limb to determine the ratio between the highest ankle pressure (dorsalis pedis or posterior tibial) and highest blood pressure (right or left arm) (6). The lowest ABI value obtained was used for the analysis of the results (right or left ABI).

hypertensive patients referred for renal angiography because the relationship between severe RAS and the presence of clinical symptoms of PAD is not clear.

’ MATERIALS AND METHODS All subjects included in the study had established hypertension and suspicion of RAS based on clinical data. One or more of the following conditions were considered: resistant hypertension (sustained hypertension despite the use of 3 different classes of antihypertensive agents), previous hypertensive pulmonary edema, congestive heart failure, malignant hypertension or progressive renal failure. Subjects were referred to the Hypertension Unit of the Heart Institute (InCor) - Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo over a 2-year period. The procedures were conducted in accordance with institutional guidelines, and the institutional review committee approved the protocol (1125/07 – Ethics Committee for Analysis of Research Projects HC-FMUSP). All patients gave written informed consent. Patients were selected whose angiography would be useful for the diagnosis of significant RAS and according to the risk of RAS based on clinical data and additional diagnostic noninvasive imaging that suggested the presence of RAS as follows: decreased renal perfusion by renal scintigraphy, increased renal blood flow velocity (X180 cm/s) observed on Doppler ultrasound of the renal arteries, and/or magnetic resonance angiography of renal arteries suggesting stenosis. Patients with known PAD (prior ABI o0.9, peripheral lower extremity vascular procedures, or major lower extremity amputation) or RAS detected by prior renal angiography were excluded. Risk factors were measured at each clinical examination and included age, resting blood pressure, smoking status, presence of diabetes, and measurement of serum cholesterol levels. Blood tests were performed to measure total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and serum creatinine. The diagnosis of hypercholesterolemia was based on whether the patients had been prescribed cholesterol-lowering agents or had a fasting total cholesterol level 4200 mg/dL. Patients were considered to have diabetes mellitus if dietary or pharmacological interventions were required to maintain normal blood glucose levels (o126 mg/dL) or if they were using specific medications. Blood pressure (BP) was measured using an automatic digital sphygmomanometer (OMRON-705CP, Japan) after the patient had been in a resting position for 5 minutes. The classification of BP was based on the average of 2 readings taken by the examining physician and according to the recommendations of the Eighth Joint National Committee on High Blood Pressure (4). Evaluation for the presence of IC was based on a structured history and questions from the Edinburgh Claudication Questionnaire (5). Patients with a positive questionnaire (6 answers) were diagnosed as having IC. The clinical evaluation also included the ABI measured with the patient in the supine position after a 10-minute rest. A portable vascular Doppler device (DV 610, Medmega, SP, Brazil) and a mercury sphygmomanometer with an appropriate cuff size for the patient’s arm circumference were used for measurements. Systolic blood pressure measurements were performed in the following order: right brachial artery, right dorsalis pedis artery, right posterior tibial artery, left dorsalis pedis

Evaluation of renal arteries All patients underwent renal angiography after a detailed medical history and complete clinical examination were completed. For a statistical comparison of clinical and laboratory variables, the study population was divided into 2 groups, according to the presence (X70%) or absence (o70%) of severe renal obstruction verified by renal angiography. Serum creatinine levels were determined twice during the evaluation, 1 day before the procedure and 3 days afterwards. The glomerular filtration rate (GFR) was estimated with the Modification of Diet in Renal Disease (MDRD) formula (7). To prevent nephropathy induced by iodinated contrast, patients who had an estimated GFR (eGFR) below 60 mL/min/m2 received a normal saline infusion (1 mL/kg/h) before and 24 h after the procedure plus 600 mg of oral N-acetyl-cysteine twice daily and low-osmolar contrast for the procedure, as previously described (8).

Statistical analysis Data were analyzed using SPSS 20.0 statistical software. Descriptive analysis was used to define the study population. Parametric data are expressed as the mean ± standard deviation, and nonparametric data are expressed as the median followed by the interquartile range or as percentages, when appropriate. Student’s t test for independent samples and the Mann-Whitney U test were used to compare quantitative variables of groups with and without significant RAS. A Chisquared test was used to analyze qualitative variables, and the Fisher correction was used when necessary. Univariate and multiple logistic regression analyses were used to determine the factors associated with significant RAS in the entire population. Variables with a value of po0.1 in the univariate analysis were included in multivariate models. A p value o0.05 was considered significant.

’ RESULTS We evaluated 82 patients with clinical suspicion of RAS, consisting of 62% females, with a mean age of 59±13 years. The main characteristics of the total sample and groups categorized by the presence or absence of severe RAS are shown in Table 1. A high frequency of diabetes, IC, and smoking were observed. Severe RAS diagnosed by renal arteriography was present in 32 of 82 (39%) patients. The prevalence of atherosclerosis risk factors was compared between groups (RAS o70% and RAS X70%). Patients with severe RAS were older (63±12 vs 56±12, p=0.006) and had a greater prevalence of IC (55% vs 45%; p=0.027) and ABI o0.9 (44% vs 20%, p=0.021). No significant differences were observed between the 2 groups concerning diabetes, smoking status, angina, blood pressure, renal function, and dyslipidemia. The average contrast volume used in renal arteriography was 110 mL. Only 7 (8.5%) patients had a transient and mild/moderate increase in creatinine levels suggestive of contrast-induced nephropathy. Dialysis was not required, and no patient developed serious complications during renal arteriography. In the univariate analysis, the variables that

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Table 1 - Clinical characteristics of the 82 patients according to the presence of severe renal artery stenosis. Variables

Total (n=82)

RAS (o70%) (n=50)

RAS (X70%) (n=32)

p value

Age, years Female, % Mulatto, % Diabetes mellitus, % Smoking, % Angina, % IC, % SBP, mmHg DBP, mmHg Antihypertensives, n Serum creatinine, mg/dL eGFR o60 mL/min/m2, % ABI o0.9, % LDL, mg/dL HDL, mg/dL Triglycerides, mg/dL

59±13 62 40 44 32 26 35 149±29 78±12 4 (3-5) 1.4 (1.1-2.3) 63 29 110±35 40 (35-48) 134 (95-178)

56±12 62 34 42 30 28 45 146±31 77±13 5 (3.7-5) 1.38 (1.0-2.3) 58 20 107±32 41 (36-48) 132 (98-171)

63±12 66 50 47 34 22 55 153±25 80±11 4 (3-5) 1.51 (1.1-2.2) 72 44 113±40 36 (32-46) 153 (91-202)

0.006* 0.96 0.32 0.66 0.68 0.55 0.027* 0.29 0.18 0.18 0.49 0.20 0.021* 0.46 0.09 0.31

RAS: renal artery stenosis; IC: intermittent claudication; SBP: systolic blood pressure; DBP: diastolic blood pressure; eGFR: estimated glomerular filtration rate; ABI: Ankle-brachial index; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol.

Table 2 - Unadjusted and adjusted odds ratio variables in patients according to severe renal artery stenosis (X70%). Univariate Variables Age ABI IC

life and is strongly associated with impaired functional capacity (15). In addition, IC is related to a twofold to fourfold increased risk of mortality, predominantly from cardiovascular disease (16), and its presence is a predictor of higher rates of myocardial infarction, stroke, and disability in older adults (17). Similar to the elevated prevalence of the clinical presentation of IC, we found an elevated prevalence of an ABI o0.9 (29%). This parameter was observed more frequently in patients with severe RAS (44% vs 20%, p=0.021) and is equally associated with an unfavorable prognosis as described in previous studies (6,13,18). The clinical expression of severity in the group with RAS X70% can be verified by uncontrolled blood pressure (SBP median=149 mmHg) despite patients’ use of 4 different classes of antihypertensive medications (Table 1). The prevalence of diabetes mellitus in our study was 44%, which is higher than that reported in other studies of patients with PAD (between 20 and 29%) (19). The group with severe RAS was older and possibly had more diffuse and serious atherosclerosis, which could explain the higher prevalence of PAD (20). We did not find studies that investigated the prediction of RAS based on the presence of PAD defined by clinical parameters or the ABI. The main strength of the present investigation was the systematic evaluation of IC symptoms and PAD in consecutive patients diagnosed according to the gold standard method used for identifying RAS. We found a potential role for IC symptoms, which can be easily identified when the clinical history is obtained, for predicting severe RAS in highrisk patients. This finding may reinforce the indication of RAS investigation as important in patients with resistant hypertension. Some limitations should also be addressed. First, the sample size was small, which is primarily explained by our single-center study design, and we only investigated patients at high risk, suggesting the presence of RAS. Second, the selection of patients with hypertension at high risk for atherosclerotic RAS could overestimate the number of individuals affected by atherosclerosis in other vessels, including the peripheral arteries. However, both limitations are related to the standard indications for investigating RAS per se and could partially explain the high rate (35%) of IC in selected patients despite of prevalence of severe RAS in the total group.

Multivariate

OR (CI)

p value

OR (CI)

p value

1.05 (1.01-1.10) 3.11 (1.16-1.01) 2.85 (1.11-7.27)

0.009 0.009 0.029

1.26 (0.97-1.08) 1.44 (0.37-5.66) 3.33 (1.03-10.82)

0.70 0.60 0.04*

ABI: Ankle-brachial index; OR: odds ratio; CI: confidence interval; IC: intermittent claudication. * Adjusted for age, ABI, and IC.

were significantly correlated with the presence of severe RAS included age, ABI, and IC (Table 2). In the multivariate logistic regression analysis, the presence of IC was an independent variable associated with severe RAS (OR: 3.33, 95% CI: 1.03–10.82, p=0.04).

’ DISCUSSION The present study produced the following important findings: a) in patients with hypertension who were referred for renal angiography under clinical suspicion of renovascular hypertension, the frequencies of IC and an ABI o0.9 were 35% and 28%, respectively; and b) a validated clinical parameter (presence of IC defined by a standardized questionnaire) was independently associated with severe RAS in patients with hypertension. Atherosclerotic RAS is a clinical condition frequently observed in patients with multiple cardiovascular risk factors and concomitant atherosclerosis in other arterial beds (10,11). Because atherosclerosis is a systemic disease, it is conceivable that a significant proportion of patients with hypertension who have confirmed RAS also have PAD; however, current evidence is scant, even though PAD has a high prevalence (12) and is associated with increased cardiovascular morbidity and mortality (13). Although IC is the symptomatic expression of PAD, approximately 50% of patients with IC remain asymptomatic (14). Conversely, we found a high prevalence (35%) of IC among patients with suspicion of RAS, and IC was more prevalent in the group with RAS X70% than in the group without severe RAS (55% vs 45%, p=0.027). IC has a significant effect on quality of

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The presence of IC was independently associated with severe RAS in patients suspected of having renovascular hypertension based on clinical evidence.

’ AUTHOR CONTRIBUTIONS 7.

Macedo TA conceived and designed the work, wrote the draft and contributed to the correction of the manuscript, was responsible for the acquisition, analysis and interpretation of data for the work, managed the literature searches, conducted research procedures, supervised the work, agreed to be accountable for all aspects of the work and to answer questions related to the accuracy or integrity of any part of the work and approved the final version of the manuscript to be published. Drager LF conceived and designed the work, wrote the manuscript draft and contributed to the correction of the manuscript, supervised the work and approved the final version of the manuscript to be published. Muela HC, Pedrosa RP, Costa-Hong V and Kajita LJ were responsible for the acquisition, analysis and interpretation of data for the work, managed the literature searches, conducted research procedures and approved the final version of the manuscript to be published. Bortolotto LA conceived and designed the work, wrote the draft and contributed to the correction of the manuscript, was responsible for the acquisition, analysis and interpretation of data for the work, managed the literature searches, supervised the work, agreed to be accountable for all aspects of the work and to answer questions related to the accuracy or integrity of any part of the work, approved the final version of the manuscript to be published. All authors read and approved the final manuscript and have participated sufficiently in the work to take public responsibility for appropriate portions of the content.

10. 11.

12.

13.

’ REFERENCES 14.

1. Colyer WR Jr, Cooper CJ. Cardiovascular morbidity and mortality and renal artery stenosis. Prog Cardiovasc Dis. 2009;52(3):238-42, http://dx. doi.org/10.1016/j.pcad.2009.09.004. 2. Bendermacher BL, Teijink JA, Willigendael EM, Bartelink ML, Büller HR, Peters RJ, et al. Symptomatic peripheral arterial disease: the value of a validated questionnaire and a clinical decision rule. Br J Gen Pract. 2006;56(533):932-7. 3. Fowkes FG, Price JF, Stewart MC, Butcher I, Leng GC, Pell AC, et al. Aspirin for prevention of cardiovascular events in a general population screened for a low ankle brachial index: a randomized controlled trial. JAMA. 2010;303(9):841-8, http://dx.doi.org/10.1001/jama.2010.221. 4. James PA, Oparil S, Carter BL, Cushman WC, Dennison-Himmelfarb C, Handler J, et al. 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014;311(5): 507-20, http://dx.doi.org/10.1001/jama.2013.284427. 5. Leng GC, Fowkes FG. The Edinburgh Claudication Questionnaire: an improved version of the WHO/Rose Questionnaire for use in epidemiological surveys. J Clin Epidemiol. 1992;45(10):1101-9, http://dx.doi.org/ 10.1016/0895-4356(92)90150-L. 6. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, et al. ACC/AHA 2005 guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): executive summary a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA

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CLINICAL SCIENCE

Hypovitaminosis D in patients undergoing kidney transplant: the importance of sunlight exposure Cristiane F. Vilarta,I Marianna D. Unger,I Luciene M. dos Reis,I Wagner V. Dominguez,I Elias David-Neto,II Rosa M. Moyse´s,I,III Silvia Titan,I Melani R. Custodio,I Mariel J. Hernandez,IV Vanda JorgettiI,* I Divisao de Nefrologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Divisao de Urologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. III Mestrado em Medicina, Universidade Nove de Julho (UNINOVE), Sao Paulo, SP, BR. IV Servicio de Nefrologı´a y Trasplante Renal, Hospital Universitario de Caracas, Universidad Central de Venezuela, Caracas, Venezuela.

OBJECTIVES: Recent studies have shown a high prevalence of hypovitaminosis D, defined as a serum 25-hydroxyvitamin D level less than 30 ng/ml, in both healthy populations and patients with chronic kidney disease. Patients undergoing kidney transplant are at an increased risk of skin cancer and are advised to avoid sunlight exposure. Therefore, these patients might share two major risk factors for hypovitaminosis D: chronic kidney disease and low sunlight exposure. This paper describes the prevalence and clinical characteristics of hypovitaminosis D among patients undergoing kidney transplant. METHODS: We evaluated 25-hydroxyvitamin D serum levels in a representative sample of patients undergoing kidney transplant. We sought to determine the prevalence of hypovitaminosis D, compare these patients with a control group, and identify factors associated with hypovitaminosis D (e.g., sunlight exposure and dietary habits). RESULTS: Hypovitaminosis D was found in 79% of patients undergoing kidney transplant, and the major associated factor was low sunlight exposure. These patients had higher creatinine and intact parathyroid hormone serum levels, with 25-hydroxyvitamin D being inversely correlated with intact parathyroid hormone serum levels. Compared with the control group, patients undergoing kidney transplant presented a higher prevalence of 25-hydroxyvitamin D deficiency and lower serum calcium, phosphate and albumin but higher creatinine and intact parathyroid hormone levels. CONCLUSIONS: Our results confirmed the high prevalence of hypovitaminosis D in patients undergoing kidney transplant. Therapeutic strategies such as moderate sunlight exposure and vitamin D supplementation should be seriously considered for this population. KEYWORDS: Vitamin D; Kidney Transplantation; Parathyroid Hormone; Hypovitaminosis D; Chronic Kidney Disease. Vilarta CF, Unger MD, dos Reis LM, Dominguez WV, David-Neto E, Moyse´s RM, et al. Hypovitaminosis D in patients undergoing kidney transplant: the importance of sunlight exposure. Clinics. 2017;72(7):415-421 Received for publication on November 24, 2016; First review completed on January 24, 2017; Accepted for publication on March 14, 2017 *Corresponding author. E-mail: vandajor@usp.br

’ INTRODUCTION

vitamin D deficit and the development of cardiovascular and immunological disease, cancer, diabetes mellitus and arterial hypertension (2). Vitamin D deficiency in patients with chronic kidney disease (CKD) can lead to alterations in bone mineral metabolism, resulting in frequent fractures and increased cardiovascular mortality (4,5). In this context, increases in the serum levels of 25(OH)D at 10 ng/ml are associated with a 14% decreased mortality risk in this population (5). Kidney transplant (KT) resolves some of these disorders and leads to improvements in the prognosis and quality of life of patients with end-stage renal disease. However, studies have also shown that hypo D is a persistent event in during both early and late transplant periods, requiring supplementation in most cases (4-10). The major causes of hypo D in patients undergoing KT include: the increased catabolism of vitamin D secondary to the use of glucocorticoids (11), restricted sunlight exposure, and sunscreen use by medical indication because immunosuppression is associated with skin cancer in these patients (12).

Several studies have demonstrated a high prevalence of hypovitaminosis D (hypo D) in different regions of the world including Latin America, even though these places receive the necessary ultraviolet radiation (UVR) required to produce adequate levels of 25-hydroxyvitamin D [25(OH)D] (1-3). Recently, vitamin D and its metabolites have been extensively studied because of the role they play in functions such as the regulation of calcium (Ca) and phosphorus (P) levels, parathyroid hormone suppression and bone mineralization in adults (4). In addition, there are associations between

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group and because it has less UV radiation, enabling us to detect a greater number of patients with hypo D. The following parameters were analyzed in the selected patients: age, gender, race (white or non-white), donor type, time after KT, immunosuppressive therapy, and post-KT fractures (spontaneous or minimal trauma). In addition, the serum concentrations of creatinine, total Ca, P, albumin, ALP, GOT, GPT, GGT, PTHi and 25(OH)D were measured. The GFR was estimated using the MDRD simplified formula (14). The patients completed a questionnaire on dietary food frequency that evaluated the consumption frequency of major food sources of vitamin D (e.g., fatty fish and fish oil). Sunlight exposure habits were analyzed based on the frequency of exposure (3 or more times a week or less than 3 times a week) and a sunscreen protection factor (yes or no). Total serum Ca, serum P, albumin, ALP, GOT and GGT were determined using routine laboratory techniques. PTHi was measured using the Immulite analyzer (Diagnostic Products Corporation, Los Angeles, CA, USA; reference range, 10-87 pg/ml). 25(OH)D was measured using a chemiluminescent assay (Dia-Sorin Inc., Stillwater, MN, USA). Serum concentrations of 25(OH)D between 15 and 30 ng/mL were considered insufficient, and concentrations less than 15 ng/ml were considered deficient. All procedures in this study were performed after approval from the local institutional committee, Comissão de Ética para Análise de Projetos de Pesquisa (CAPPesq no 1035/07), and in accordance with the ethical standards of the 1964 Helsinki Declaration.

In addition to the above known effects, Obi et al. showed that vitamin D deficiency/insufficiency in patients undergoing KT is associated with an increased risk of renal interstitial fibrosis, renal tubular atrophy, and glomerular filtration rate (GFR) decline (13). Given the recent developments of its molecular properties, vitamin D plays a multifaceted role and has led us to determine the prevalence and clinical characteristics of hypo D in a cohort of patients with KT compared with a control group.

’ PATIENTS AND METHODS Patients undergoing KT were followed at the Transplant Unit of Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo. Of these patients, we selected those who were older than 18 years, showed stable GFR, and were more than 1 year removed from KT. A total of 752 patients was selected (355 females). In this cohort, we determined a representative sample size calculation. In this calculation, we considered a 32.5% prevalence of 25(OH)D deficiency and 5% alpha and a 1% beta risks for a test power ratio of 99%. The final KT population was composed of 149 patients; race, gender, age, donor type, serum creatinine, proteinuria, glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), gammaglutamyltransferase (GGT) levels, immunosuppressive therapy, body mass index (BMI) and vitamin supplementation were controlled. Concomitantly, we selected 398 individuals (333 females) from a sample of 603 volunteering outpatient employees of the Hospital Universitário da Universidade de São Paulo and graduate students from the Faculdade de Medicina da Universidade de São Paulo previously included in Unger et al. We excluded individuals with diabetes mellitus, arterial hypertension, serum creatinine levels higher than 1.2 mg/dL or those on medications/supplements such as Ca, bisphosphonates, vitamin D and corticosteroids (see Figure 1) (2). A blood sample was collected during late winter of 2006, and serum Ca, P, creatinine, albumin, intact parathyroid hormone (PTHi), total alkaline phosphatase (ALP), 25(OH)D and BMI were analyzed. KT recipients were submitted to a single blood collection during late winter. We chose late winter to coincide with the period in which samples were collected from the control

Statistical analysis

The data are expressed as either means ± SD or medians and ranges; categorical data are described as percentages. To determine the relationship between the 25(OH)D levels and other categorical variables, we used chi-square test and, when necessary, Fisher’s exact test. We used the Box and Cox transformation for the variables age, time after KT, albumin, creatinine, PTHi, GOT, GPT, GGT and ALP to ensure the normality assumption. Even with the Box and Cox transformation, the variables age, time after KT and GOT did not reach normality. Because these transformed variables presented homoscedasticity (according to Levene’s test), they were also considered normally distributed to compare the serum 25(OH)D levels.

Figure 1 - Participant data. KT: kidney transplant; GFR: glomerular filtration rate; 25(OH)D: 25 hydroxyvitamin D; Ca: calcium; P: phosphorus; PTHi: intact parathyroid hormone; ALP: alkaline phosphatase; GOT: glutamic-oxaloacetic transaminase; GPT: glutamicpyruvic transaminase; GGT: gamma-glutamyltransferase.

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patients with and without fractures regarding the rates of 25(OH)D deficiency or insufficiency. In addition to the data described above, Table 1 shows the demographic and biochemical parameters. The patients under going KT showed serum PTHi above the reference range. We did not find differences in the 25(OH)D levels (21.1±12.1 vs. 22.6±13.4) between patients with abnormal liver function (GGT levels450 U/L) and those with preserved liver function.

An ANOVA was used to determine the relationship between the clinical and biochemical variables and serum 25(OH)D (deficient, insufficient or normal). If results were significant, then we applied the Bonferroni post hoc test. The correlation between the 25(OH)D levels and other biochemical parameters was analyzed using Spearman’s correlation coefficients. The patients undergoing KT and the control group differed with regard to gender; therefore, we used an ANOVA adjusted for gender to compare these two groups with regard to BMI, P, Ca, albumin, creatinine, PTHi, ALP and 25(OH)D for each variable separately. The level of significance was set at 5% (p-valueo0.05).

Clinical and demographic characteristics according to the 25(OH)D categories among patients undergoing KT Table 2 describes the clinical and demographic characteristics according to the 25(OH)D categories (deficient, insufficient and normal). Donor type was associated with important differences, although they were not significant. Specifically, we found that 28% of patients who received a graft from a living related donor had normal 25(OH)D levels, whereas only 12% of patients receiving a graft from a standard cadaveric donor showed normal 25(OH)D levels (p=0.055). Regarding the frequency of sunlight exposure, we observed a significant difference between those who were exposed to the sun more than 3 times a week and those who had less exposure with regard to 25(OH)D levels; specifically, 55.8% of patients with higher sun exposure had a normal concentration of 25(OH)D versus 10.4% in the group with lower sun exposure (po0.0001). The use of sunscreen did not affect the serum concentrations of this vitamin. We did not find a significant relationship between 25(OH)D serum levels and

’ RESULTS Basic characteristics of the patients undergoing KT Of the 149 patients undergoing KT, 56.4% were female, and 68.4% were white. The median age was 44 (18 - 81) years, and the median time after KT of was 6 (1 - 36) years. Approximately 55% of these patients received a graft from a living donor. Regarding their 25(OH)D serum levels, 56 (37.5%) patients presented deficiency, 62 (41.6%) insufficiency, and only 31 (20.8%) a normal concentration. Thus, the prevalence of hypo D in the study sample was 79.2%. Post-KT insufficiency fractures (spontaneous or minimal trauma) were reported in 10% of the patients (N=15). The mean 25(OH)D serum level was similar between patients without and those with fractures (21.8±13.2 ng/mL vs. 21.9±8.3 ng/mL). Likewise, we found no differences between

Table 1 - Demographic and biochemical parameters of patients undergoing KT. Demographic parameters Gender Female Male Race White Non-white Time after KT(years) Age (years) Type of donor Living related donor Standard cadaveric donor Insufficiency fracture after KT BMI (kg/m2)

Biochemical parameters

84 (56) 65 (44) 102 47 6 44

(68) (31) (1 - 36) (18 - 81)

82 67 15 25.4

(55) (45) (10) ± 5.3

Reference range

21.8 ± 12.8

25(OH)D (ng/mL) 25(OH)D category Normal Deficiency Insufficiency PTHi (pg/mL) Ca (mg/dL) P (mg/dL) Creatinine (mg/dL) Albumin (g/dL) ALP (U/L) GOT (U/L) GPT (U/L) GGT (U/L)

31 56 62 80.1 8.9 3.0 1.5 4.4 65.8 23 10 28

430 ng/ml o15 ng/ml 15 - 30 ng/ml 10 - 87 pg/ml 8.7 - 10.3 mg/dL 2.5 - 4.5 mg/dL 0.5 - 1.0 mg/dL 3.5 - 5.0 g/dL 35 - 100 U/L 0 - 35 U/L 3 - 36 U/L 5 - 36 U/L

(21) (37) (42) ± 78.3 ± 0.6 ± 0.8 (0.6 - 4.5) (3.4 - 5.5) (29.7 - 364.5) (6 - 121) (3 - 81) (7 - 704)

Values are expressed as the mean±SD, median (range) or number of patients (%); KT: kidney transplant; BMI: body mass index; 25(OH)D: 25 hydroxyvitamin D; Ca: calcium; P: phosphorus; PTHi: intact parathyroid hormone; ALP: alkaline phosphatase; GOT: glutamic-oxaloacetic transaminase; GPT: glutamic-pyruvic transaminase; GGT gamma-glutamyltransferase

Table 2 - Demographic and clinical parameters based on 25(OH)D category among patients undergoing KT. Race

25(OH)D levels White Deficient Insufficient Normal Total

35 43 24 102

(34.3) (42.1) (23.5) (100)

Gender NonWhite

21 19 7 47

(44.7) (40.4) (14.8) (100)

Female

30 35 19 84

(35.7) (41.6) (22.6) (100)

BMI

Male

26 27 12 65

(40) (41.5) (18.5) (100)

Nonobese 29 34 16 79

(36.7) (43) (20.2) (100)

Type of donor* Obese

27 28 15 70

(38.6) (40) (21.4) (100)

Living donor 28 31 23 82

(34.1) (37.8) (28) (100)

Bone fracture after KT

Cadaveric donor 28 31 8 67

(41.8) (46.3) (12) (100)

52 53 29 134

(38.8) (39.5) (21.6) (100)

Yes

4 9 2 15

(26.6) (60) (13.3) (100)

Immunosuppressive therapy CNI use

44 43 20 107

(41.1) (40.2) (18.7) (100)

CNI non-use 12 19 11 42

(28.6) (45.2) (26.2) (100)

Values are expressed as number of patients (%); 25(OH)D: 25 hydroxyvitamin D; BMI: body mass index; KT: kidney transplant; CNI: calcineurin inhibitor. * Chi-square test: p=0.055.

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levels of creatinine and PTHi as well as lower levels of P than the control group. However, patients undergoing KT presented lower levels of this hormone, regardless of 25(OH)D category, when compared to the control group ( p=0.001); these data are shown in Table 6. We analyzed the correlations between the serum 25(OH)D levels and biochemical parameters but found only one negative correlation with regard to serum PTHi levels (r=-0.24; po0.03).

consumption of food sources of vitamin D. The data are shown in Table 3.

Biochemical parameters by 25(OH)D category among patients undergoing KT Table 4 compares the biochemical parameters across 25(OH)D categories. The results revealed that only serum creatinine and PTHi significantly differed with regard to 25(OH)D levels. Specifically, we found lower serum levels of creatinine and PTHi among patients with normal 25(OH)D.

’ DISCUSSION

Comparison of the demographic and biochemical parameters between the control group and patients undergoing KT

Despite the growing interest in vitamin D status and its supplementation forms in both the normal population and patients with CKD, a lack of data remains regarding vitamin D levels in KT recipients. Little research has compared the 25(OH)D serum levels between patients undergoing KT and the healthy population. Our findings coincide with those of Ebbert et al., who analyzed a pediatric population,

Table 5 shows that the control group and patients undergoing KT had different distributions based on gender. Regarding the biochemical data, we found significant differences in creatinine, albumin, Ca, P and PTHi between men and women. As expected, patients undergoing KT showed higher

Table 3 - Sunlight exposure habits and vitamin D food source consumption frequency according to 25(OH)D category in patients undergoing KT. Sunlight exposure*

25(OH)D levels

3 or more times a week Deficient Insufficient Normal Total

2 13 19 34

Use of sunscreen

Fewer than 3 times a week

(5.8) (38.2) (55.8) (100)

51 52 12 115

Yes

(44.3) (45.2) (10.4) (100)

16 14 13 43

(37.2) (32.5) (30.2) (100)

Vitamin D food source

40 47 18 105

(38) (44.7) (17.1) (100)

Frequent

Infrequent

5 6 7 18

48 57 24 129

(27.7) (33.3) (38.8) (100)

(37.2) (44.2) (18.6) (100)

Values are expressed as number of patients (%); KT: kidney transplant; 25(OH)D: 25 hydroxyvitamin D; * Chi-square test: po0.0001.

Table 4 - Biochemical parameters of patients undergoing KT according to 25(OH)D category.

Creatinine (mg/dL)

Albumin (g/dL)

Ca (mg/dL)

P (mg/dL)

PTHi (pg/dL)

ALP (U/L)

GOT (U/L)

GPT (U/L)

GGT (U/L)

Deficient

Insufficient

Normal

53 2.0 ± 0.9 1.7 (1.0 - 4.5) 52 4.4 ± 0.5 4.4 (3.4 - 5.3) 51 8.9 ± 0.7 9.0 (7.0 - 10.2) 55 3.0 ± 0.8 2.9 (1.2 - 5.4) 55 104.7 ± 104.6 68.6 (18.2 - 562.5) 53 82.8 ± 54.5 69.6 (29.7 - 364.5) 52 27.9 ± 22.8 21 (6 - 121) 53 14.2 ± 13.2 10.0 (4 - 81) 51 67.5 ± 116.9 31 (7 - 704)

60 1.7 ± 0.7 1.5 (0.6 - 4.1) 60 4.4 ± 0.3 4.4 (3.8 - 5.2) 59 8.9 ± 0.5 8.9 (7.8 - 9.9) 61 3.0 ± 0.8 2.9 (1.3 - 5.3) 61 72.5 ± 58.6 53.2 (9.5 - 277.0) 60 71.6 ± 30.3 64.8 (34.2 - 191.0) 60 27.8 ± 11.7 25 (9 - 64) 60 13.3 ± 8.5 10.5 (3 - 44) 58 44.1 ± 50.1 28 (10 - 334)

31 1.5 ± 0.5 1.3 (1.0 - 2.7) 31 4.4 ± 0.4 4.5 (3.4 - 5.5) 31 8.9 ± 0.5 8.8 (7.8 - 10.1) 31 2.7 ± 0.6 2.7 (1.2 - 3.8) 31 51.6 ± 37.0 44.1 (13.3 - 229.8) 31 65.8 ± 23.4 65.6 (34.5 - 166.4) 31 23.5 ± 12.2 19 (13 - 67) 31 13.2 ± 14.2 9.0 (4 - 77) 31 35.6 ± 37.2 22 (12 - 206)

Values are expressed as number of patients, mean±SD and median (range). KT: kidney transplant; 25(OH)D: 25 hydroxyvitamin D; Ca: calcium; P: phosphorus; PTHi: intact parathyroid hormone; ALP: alkaline phosphatase; GOT: glutamic-oxaloacetic transaminase; GPT: glutamic-pyruvic transaminase; GGT: gamma-glutamyltransferase. *Bonferroni’s post-hoc test: po0.05, Normal vs. Deficient or Insufficient.

418

p 0.013*

0.660

0.997

0.154

0.011*

0.441

0.445

0.387

0.324

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Table 5 - Comparison of demographic and biochemical parameters between the control group and patients undergoing KT.

Age 18 to 30 years 31 to 50 years 51 to 65 years More than 65 years Gender Female Male Race White Non-white BMI (kg/m2)

Creatinine (mg/dL)

Albumin (g/dL)

Ca (mg/dL)

P (mg/dL)

PTHi (pg/dL)

25(OH)D (ng/mL)

ALP (U/L)

Control group

Patients undergoing KT

N=395 68 (17) 210 (53) 97 (25) 20 (5) N=398 333 (84) 65 (16) N=385 269 (68) 116 (29) N=348 25.9 ± 4.9 25.2 (16.8 - 56.3) N=382 0.8 ± 0.1 0.8 (0.4 - 1.2) N=384 4.5 ± 0.3 4.5 (3.3 - 5.4) N=382 9.6 ± 0.5 9.6 (6.7 - 10.8) N=383 3.8 ± 0.7 3.8 (1.8 - 7.6) N=352 64.9 ± 24.8 60.5 (5.0 - 144.0) N=398 23.8 ± 10.4 21.6 (6.0 - 64.3) N=381 70.9 ± 26.1 69.0 (24.0 - 369.0)

N=149 29 (19) 71 (48) 46 (31) 3 (2) N=149 84 (56) 65 (44) N=149 102 (68) 47 (32) N=149 25.4 ± 5.3 24.9 (16.4 - 44.0) N=145 1.8 ± 0.7 1.5 (0.6 - 4.5) N=144 4.4 ± 0.4 4.4 (3.4 - 5.5) N=142 8.9 ± 0.6 8.9 (7.0 - 10.2) N=147 3.0 ± 0.8 2.9 (1.2 - 5.4) N=148 80.1 ± 78.3 53.9 (9.5 - 562.5) N=149 21.8 ± 12.8 20.7 (4.0 - 70.5) N=145 74.4 ± 40.1 65.8 (29.7 - 364.5)

p 0.078

o0.001

0.151

0.118

o0.001

0.056

0.306

Values are expressed as number of patients (%), mean±SD and median (range). KT: kidney transplant; BMI: body mass index; Ca: calcium; P: phosphorus; PTHi: intact parathyroid hormone; 25(OH)D: 25 hydroxyvitamin D; ALP: alkaline phosphatase.

Table 6 - Patients undergoing KT and the control group according to 25(OH)D category. 25(OH)D levels Deficient Insufficient Normal

Control group

Patients undergoing KT

90 (23) 209 (52) 99 (25)

56 (37) 62 (42) 31 (21)

0.001

with hypo D. Researchers who plan future studies on vitamin D and clinicians who monitor and manage vitamin D deficiencies in patients undergoing KT must consider this seasonal variation. Nutritional status did not affect serum 25(OH)D levels, and in contrast to other published data, we did not find a higher prevalence of hypo D in obese patients, even though the bioavailability of vitamin D can be influenced by its storage in adipocytes (16,17). Hypo D is one of the major causes of secondary hyperparathyroidism (SHPT). Unger et al. showed that healthy subjects develop SHPT in the context of vitamin D deficiency during winter but show improvements in summer (2). Therefore, this seasonal variation in 25(OH)D and PTH is likely a normal response to physiologically compensate for the drop in vitamin D levels during winter. Among patients undergoing KT, studies have been conducted during different periods of the year, making it difficult to know whether they follow the trend observed in the general population. The literature commonly describes parathyroid hormone stimulation due to 25(OH)D deficiency during the earliest periods after KT as a consequence of the high doses of corticosteroids and persistent elevation of phosphatonins (6,18,19). Some authors have also found this correlation during later periods of KT recovery (7,20). We observed the same trends in our patients, showing a negative correlation between PTHi and 25(OH)D serum levels.

Values are expressed as number of patients (%); KT: kidney transplant; 25(OH)D: 25 hydroxyvitamin D.

reporting the presence of hypo D in patients undergoing KT as well as control groups (15). However, our findings showed that patients undergoing KT had a higher prevalence of deficiency. The current study demonstrated that 79% of patients undergoing KT at the Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo presented with an insufficiency or deficiency of 25(OH)D during late winter. These results are similar to those obtained in other studies conducted throughout the world and across climatic seasons, revealing a total approximate prevalence of 76-97% (7-10). Stavroulopoulos et al. analyzed patients undergoing KT during the summer and winter and confirmed that levels of 25(OH)D are lower during winter (7). That study explains why the current evaluation was performed during late winter, i.e., to detect the highest possible number of patients

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(28%) in the group receiving a renal graft from a living donor, compared to only 12% in the group receiving a renal graft from a cadaveric donor. These results suggest that the immunological tolerance in these patients could be due to the vitamin D status and that the concentration of this hormone depends on the type of renal graft received. Specifically, better compatibility conditions ensure a briefer cold ischemia time, and a minor ischemia-reperfusion effect predicts higher deposits of 25(OH)D. In conclusion, vitamin D deficiency is prevalent among patients undergoing KT, even in geographic areas with high UV exposure. Additional studies are needed to clarify the role that donor type plays in vitamin D deficiency. Furthermore, studies are needed to define the efficacy and safety of UVR exposure as a therapeutic strategy for these patients. However, individual sunlight exposure in moderation combined with vitamin D supplementation are the easiest and safest forms to guarantee adequate levels of 25(OH)D, thereby leading to the beneficial effects of this hormone.

In addition, regarding the biochemical associations described above in the general population, vitamin D insufficiency contributes to the development of osteoporosis. Moreover, adequate supplementation can reduce the fracture risk in approximately 20% of this population (21). However, the effect of this hormone on the prevalence of fracture during later periods of KT is unclear, which suggests that low mineral bone density and hypo D are multifactorial conditions (7). In this regard, we found no difference between patients with and without fracture. The reasons for hypovitaminosis after KT include low sunlight exposure and the accelerated catabolism of vitamin D secondary to glucocorticoids use (22). Exposure to UV-A and UV-B radiation via sunlight can induce skin cancer through induced mutations in the DNA structure. This radiation is clearly the most important physical environmental carcinogen for the development of skin cancer, and nonmelanoma skin cancer (NMSC) is the most common malignancy among patients undergoing KT (23,24). The risk of developing this complication is associated with the duration and intensity of immunosuppression and cumulative sun exposure (18). However, some guidelines suggest that sunlight exposure for short periods of time is beneficial and does not increase the risk of skin cancer (25,26). In this sense, sunlight exposure at least 3 times a week predicted higher 25(OH)D serum levels in our patients, despite the use of sunscreen. However, Penny et al. found an association between low 25(OH)D levels and a high incidence of NMSC, limiting the use of this therapeutic strategy to low-risk groups defined by skin type, age, gender and immunosuppressive regimen (18,27). Regarding the effect of immunosuppression on vitamin D status after KT, steroids accelerate catabolism of vitamin D. Despite not knowing the steroid doses in our patients, the development time of transplantation suggests that they received a minimal and stable glucocorticoid dose, which most likely would not explain their vitamin D deficiency. Regarding other immunosuppressive drugs (e.g., calcineurin inhibitors), we similarly did not find an association, contrary to results reported by Filipov et al. and Eyal et al., who showed that high tacrolimus doses are associated with hypo D through hepatic CYP3A4 suppression (28,29). The potent immunomodulatory activity of 25(OH)D through the suppression of T cell receptor proliferation and its direct effect on B cells through the inhibition of immunoglobulin production are additional physiological effects to consider among patients undergoing KT (30). Sezer et al. demonstrated that patients undergoing KT showed an association between 25(OH)D deficit and renal graft failure after one year (31), whereas other authors have shown a direct relationship between 25(OH)D deficit and renal graft survival (11,32). In this respect, our patients presented with an association between low serum levels of 25(OH)D and higher creatinine serum levels. In addition to the clinical aspects described above, Bienaimé et al. found a correlation between vitamin D status and graft histological findings; specifically, 25(OH)D serum levels 3 months after transplantation independently predictor the progression of interstitial fibrosis and tubular atrophy (IF/TA) at 12 months (32). Other studies have shown an association between hypo D and the reception of a renal graft from a cadaver donor (32). In this regard, although our study was retrospective and did not evaluate the characteristics of the renal graft, we found a higher percentage of patients with normal 25(OH)D levels

’ ACKNOWLEDGMENTS We acknowledge the substantial contributions of the Fundac¸ão de Amparo à Pesquisa de Estado de São Paulo (FAPESP) (2008/50782-7) in providing support for the realization of this work.

’ AUTHOR CONTRIBUTIONS Vilarta CF participated in study design, data collection, analysis and interpretation, and manuscript writing. Unger MD, dos Reis LM, Dominguez WV, David-Neto E, Moyses RM and Custodio MR participated in data interpretation and manuscript writing. Titan S and Hernandez MJ participated in statistical analysis, data interpretation and revision of the edited manuscript. Jorgetti V participated in study design, data analysis and interpretation, and manuscript writing.

’ REFERENCES 1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357(3):266-81, http://dx.doi.org/10.1056/NEJMra070553. 2. Unger MD, Cuppari L, Titan SM, Magalhães MC, Sassaki AL, dos Reis LM, et al. Vitamin D status in a sunny country: where has the sun gone? Clin Nutr. 2010;29(6):784-8, http://dx.doi.org/10.1016/j.clnu.2010.06.009. 3. Binkley N, Novotny R, Krueger D, Kawahara T, Daida YG, Lensmeyer G, et al. Low vitamin D status despite abundant sun exposure. J Clin Endocrinol Metab. 2007;92(6):2130-5, http://dx.doi.org/10.1210/jc.20062250. 4. Çankaya E, Bilen Y, Keles¸ M, Uyanık A, Akbas¸ M¸ Güngör A, et al. Comparison of serum vitamin D Levels among patients with chronic kidney disease, patients in dialysis, and renal transplant patients. Transplant Proc. 2015;47(5):1405-7, http://dx.doi.org/10.1016/j.transproceed. 2015.04.036. 5. Pilz S, Iodice S, Zittermann A, Grant WB, Gandini S. Vitamin D status and mortality risk in CKD: a meta-analysis of prospective studies. Am J Kidney Dis. 2011;58(3):374-82, http://dx.doi.org/10.1053/j.ajkd.2011. 03.020. 6. Sadlier DM, Magee CC. Prevalence of 25(OH) vitamin D (calcidiol) deficiency at time of renal transplantation: a prospective study. Clin Transplant. 2007;21(6):683–8, http://dx.doi.org/10.1111/j.1399-0012.2007.00696.x. 7. Stavroulopoulos A, Cassidy MJ, Porter CJ, Hosking DJ, Roe SD. Vitamin D status in renal transplant recipients. Am J Transplant. 2007;7(11):254652, http://dx.doi.org/10.1111/j.1600-6143.2007.01978.x. 8. Lomonte C, Antonelli M, Vernaglione L, Cazzato F, Casucci F, Chimienti D. Are low plasma levels of 25-(OH)vitamin D a major risk factor for hyperparathyroidism independent of calcitriol in renal transplant patients? J Nephrol. 2005;18(1):96-101. 9. Boudville NC, Hodsman AB. Renal function and 25-hydroxyvitamin D concentrations predict parathyroid hormone levels in renal transplant patients. Nephrol Dial Transplant. 2006;21(9):2621-4, http://dx.doi.org/ 10.1093/ndt/gfl201. 10. Lim WH, Coates PS, Russ GR, Coates PT. Hyperparathyroidism and vitamin D deficiency predispose to bone loss in renal transplant recipients. Transplantation. 2009;88(5):678-83, http://dx.doi.org/10.1097/ TP.0b013e3181b27c4d.

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22. Hesketh CC, Knoll GA, Molnar AO, Tsampalieros A, Zimmerman DL. Vitamin D and kidney transplant outcomes: a protocol for a systematic review and meta-analysis. Syst Rev. 2014;3:64, http://dx.doi.org/10.1186/ 2046-4053-3-64. 23. Attard NR, Karran P. UVA photosensitization of thiopurines and skin cancer in organ transplant recipients. Photochem Photobiol Sci. 2012; 11(1):62–8, http://dx.doi.org/10.1039/C1PP05194F. 24. Moloney FJ, Comber H, O’Lorcain P, O’Kelly P, Conlon PJ, Murphy GM. A population-based study of skin cancer incidence and prevalence in renal transplant recipients. Br J Dermatol. 2006;154(3):498-504, http:// dx.doi.org/10.1111/j.1365-2133.2005.07021.x. 25. Reichrath J. The challenge resulting from positive and negative effects of sunlight: how much solar UV exposure is appropriate to balance between risks of vitamin D deficiency and skin cancer? Prog Biophys Mol Biol. 2006;92(1):9-16, http://dx.doi.org/10.1016/j.pbiomolbio.2006.02.010. 26. Moan J, Porojnicu AC, Dahlback A, Setlow BR. Addressing the health benefits and risks, involving vitamin D or skin cancer, of increased sun exposure. Proc Natl Acad Sci U S A. 2008;105(2):668-73, http://dx.doi. org/10.1073/pnas.0710615105. 27. Ferreira FR, Ogawa MM, Nascimento LF, Tomimori J. Epidemiological profile of nonmelanoma skin cancer in renal transplant recipients: experience of a referral center. An Bras Dermatol. 2014;89(5):745-50, http://dx.doi.org/10.1590/abd1806-4841.20142590. 28. Filipov JJ, Zlatkov BK, Dimitrov EP, Svinarov D. Relationship between vitamin D status and immunosuppressive therapy in kidney transplant recipients. Biotechnol Biotechnol Equip. 2015;29(2):331-5, http://dx.doi. org/10.1080/13102818.2014.995415. 29. Eyal O, Aharon M, Safadi R, Elhalel MD. Serum vitamin D Levels in kidney transplant recipients: the importance of an immunosuppression regimen and Sun exposure. Isr Med Assoc J. 2013;15(10):628-33. 30. Kamen DL, Tangpricha V. Vitamin D and molecular actions on the immune system: modulation of innate and autoimmunity. J Mol Med. 2010;88(5):441–50, http://dx.doi.org/10.1007/s00109-010-0590-9. 31. Sezer S, Yavuz D, Canoz MB, Ozdemir FN, Haberal M. Vitamin D status, bone mineral density, and inflammation in kidney transplantation patients. Transplant Proc. 2009;41(7):2823-5, http://dx.doi.org/10.1016/ j.transproceed.2009.06.141. 32. Bienaimé F, Girard D, Anglicheau D, Canaud G, Souberbielle JC, Kreis H, et al. Vitamin D status and outcomes after renal transplantation. J Am Soc Nephrol. 2013;24(5):831–41, http://dx.doi.org/10.1681/ASN.2012060614.

11. Eyal O, Aharon M, Safadi R, Elhalel MD. Serum vitamin D levels in kidney transplant recipients: the importance of an immunosuppression regimen and sun exposure. Isr Med Assoc J. 2013;15(10):628-33. 12. Euvrard S, Kanitakis J, Claudy A. Skin cancers after organ transplantation. N Engl J Med. 2003;348(17):1681-91, http://dx.doi.org/10.1056/ NEJMra022137. 13. Obi Y, Hamano T, Ichimaru N, Tomida K, Matsui I, Fujii N, et al. Vitamin D deficiency predicts decline in kidney allograft function: a prospective cohort study. J Clin Endocrinol Metab. 2014;99(2):527–35, http://dx.doi. org/10.1210/jc.2013-2421. 14. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999;130(6):461–70, http://dx.doi.org/ 10.7326/0003-4819-130-6-199903160-00002. 15. Ebbert K, Chow J, Krempien J, Matsuda-Abedini M, Dionne J. Vitamin D insufficiency and deficiency in pediatric renal transplant recipients. Pediatr Transplant. 2015;19(5):492–8, http://dx.doi.org/10.1111/petr.12527. 16. Cofán F, Vela E, Clèries M, Catalan Renal Registry. Obesity in renal transplantation: analysis of 2691 patients. Transplant Proc. 2005;37(9): 3695-7, http://dx.doi.org/10.1016/j.transproceed.2005.10.069. 17. Gore JL, Pham PT, Danovitch GM, Wilkinson AH, Rosenthal JT, Lipshutz GS, et al. Obesity and outcome following renal transplantation. Am J Transplant. 2006;6(2):357-63, http://dx.doi.org/10.1111/j.1600-6143.2005. 01198.x. 18. Penny H, Frame S, Dickinson F, Garrett G, Young AR, Sarkany R, et al. Determinants of vitamin D status in long-term renal transplant patients. Clin Transplant. 2012;26(6):E617–23, http://dx.doi.org/10.1111/ctr.12039. 19. Palmer SC, Mc Gregor DO, Strippoli GF. Interventions for preventing bone disease in kidney transplant recipients. Cochrane Database Syst Rev. 2007;18(3):CD005015, http://dx.doi.org/10.1002/14651858.CD005015.pub2. 20. Giannini S, Sella S, Silva Netto F, Cattelan C, Dalle Carbonare L, Lazzarin R, et al. Persistent secondary hyperparathyroidism and vertebral fractures in kidney transplantantion: role of calcium-sensing receptor polymorphisms and vitamin D deficiency. J Bone Min Res. 2010;25(4):841-8, http:// dx.doi.org/10.1359/jbmr.091025. 21. Bischoff-Ferrari HA, Willett WC, Wong JB, Stuck AE, Staehelin HB, Orav EJ, et al. Prevention of nonvertebral fractures with oral vitamin D and dose dependency: a meta-analysis of randomized controlled trials. Arch Intern Med. 2009;169(6):551-61, http://dx.doi.org/10.1001/archinternmed.2008.600.

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CLINICAL SCIENCE

Laryngeal tube suction for airway management during in-hospital emergencies Haitham Mutlak,I,* Christian Friedrich Weber,I Dirk Meininger,I,III Colleen Cuca,I Kai Zacharowski,I Christian Byhahn,II Richard SchalkI I

Department of Anesthesiology, Intensive Care Medicine and Pain Therapy, Goethe-University Hospital, Theodor-Stern Kai 7-10, 60590 Frankfurt, Germany. II Department of Anesthesiology and Intensive Care Medicine, Medical Campus, University of Oldenburg, Evangelisches Krankenhaus, Steinweg 13-17, 26122 Oldenburg, Germany. III Department of Anesthesiology, Main-Kinzig-Kliniken, Herzbachweg 14, 63571 Gelnhausen, Germany.

OBJECTIVE: The role of supraglottic airway devices in emergency airway management is highlighted in international airway management guidelines. We evaluated the application of the new generation laryngeal tube suction (LTS-II/LTS-D) in the management of in-hospital unexpected difficult airway and cardiopulmonary resuscitation. METHODS: During a seven-year period, patients treated with a laryngeal tube who received routine anesthesia and had an unexpected difficult airway (Cormack Lehane Grade 3-4), who underwent cardiopulmonary resuscitation, or who underwent cardiopulmonary resuscitation outside the operating room and had a difficult airway were evaluated. Successful placement of the LTS II/LTS-D, sufficient ventilation, time to placement, number of placement attempts, stomach content, peripheral oxygen saturation/end-tidal carbon dioxide development (SpO2/etCO2) over 5 minutes, subjective overall assessment and complications were recorded. RESULTS: In total, 106 adult patients were treated using an LTS-II/LTS-D. The main indication for placement was a difficult airway (75%, n=80), followed by cardiopulmonary resuscitation (25%, n=26) or an overlap between both (18%, n=19). In 94% of patients (n=100), users placed the laryngeal tube during the first attempt. In 93% of patients (n=98), the tube was placed within 30 seconds. A significant increase in SpO2 from 97% (0-100) to 99% (5-100) was observed in the whole population and in cardiopulmonary resuscitation patients. The average initial etCO2 of 39.5 mmHg (0-100 mmHg) decreased significantly to an average of 38.4 mmHg (10-62 mmHg) after 5 minutes. A comparison of cardiopulmonary resuscitation patients with non-cardiopulmonary resuscitation patients regarding gastric contents showed no significant difference. CONCLUSIONS: LTS-D/LTS-II use for in-hospital unexpected difficult airway management provides a secure method for primary airway management until other options such as video laryngoscopy or fiber optic intubation become available. KEYWORDS: Difficult Airway Management; Laryngeal Tube; Supraglottic Airway Devices; In-Hospital Emergencies. Mutlak H, Weber CF, Meininger D, Cuca C, Zacharowski K, Byhahn C, et al. Laryngeal tube suction for airway management during in-hospital emergencies. Clinics. 2017;72(7):422-425 Received for publication on March 7, 2017; First review completed on April 4, 2017; Accepted for publication on May 15, 2017 *Corresponding author. E-mail: haitham.mutlak@kgu.de

â&#x20AC;&#x2122; INTRODUCTION

operating room (OR). Anesthesia outside the OR is a highrisk procedure (3). Airway management outside the OR is mainly influenced by environmental, equipment, and assistance issues and is associated with a 10-fold higher risk of a failed intubation (3-5). In these situations, sufficient oxygenation and ventilation receive the highest priority, followed by avoiding aspiration to prevent serious complications. If intubation fails, further algorithms should be available. In cases of failure to intubate conventionally, the placement of alternative devices, e.g., supraglottic airway devices (SADs), is recommended in many guidelines for difficult airway (6, 7). SADs play an important role in the management of patients with difficult airways (8-11). They enable ventilation, even in patients with difficult facemask ventilation, and can furthermore be used as a conduit for endotracheal intubation (10). In the preclinical setting, SADs, such as the laryngeal tube (e.g., the laryngeal tube suction II (LTS II) or LTS-D), have been

Patients with an unexpected or anticipated difficult airway requiring emergency airway management remain a challenge, even for experienced anesthesiologists and emergency physicians. This condition is responsible for major anesthetic complications, which result in significant morbidity and mortality (1). The most important problems reported among anesthesiologists are difficult or delayed intubation, aspiration of gastric contents and failed intubation (2). A substantial number of in-hospital difficult airway situations occur outside the

Copyright & 2017 CLINICS â&#x20AC;&#x201C; This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. DOI: 10.6061/clinics/2017(07)06

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’ RESULTS

evaluated and serve as a secure alternative method for maintaining oxygenation and preventing aspiration (12). Intratracheal intubation may then be performed in a standardized environment with adequate equipment. This approach is sometimes defined as ‘‘dual’’ airway management (13). The aim of the investigation was to evaluate the use of a laryngeal tube for in-hospital emergency airway management.

Within the seven-year study period, data were available for 106 adult patients (78 males, 28 females) who had their airway managed with the LTS-II/LTS-D in the context of an unanticipated difficult airway and/or in-hospital resuscitation. The mean age was 55 years (standard deviation ±16 years), the mean height was 174 cm (standard deviation ±9.1 cm) and the body mass index was 30 (standard deviation ±8.4 kg/m2). The most frequent indication for the use of an LTS II/ LTS-D was a difficult airway situation (75%, n=80), followed by use during CPR (25%, n=26) or an overlap between both (18%, n=19). Predictors for a difficult airway (receding chin, maxillary hypoplasia, micrognathia, retrognathia, reduced mouth opening, short neck, reduced cervical mobility, and obesity) were present in 75 patients (70%). While performing direct laryngoscopy on these patients, a corresponding Cormack and Lehane score of CL3 was observed in 69% (n=52), and a score of CL4 was present in 15% (n=11). In 94% (n=100) of the cases, users were able to place the laryngeal tube during the first attempt; with two attempts, the success rate increased to 99% (n=105). In 93% (n=99) of the cases, the tube could be placed within 30 seconds; 5% (n=5) of the cases required between 30 and 60 seconds. Only one placement (n=1) required more than 60 seconds. In one case, the laryngeal tube could not be placed. In three cases (3%), the laryngeal tube failed to provide sufficient ventilation after the first placement. In eight cases (7.5%), leakage was detected. In CPR and non-CPR patients, sufficient tidal volume within the first five minutes was measured without significant differences between the two groups. The majority of the participating anesthetists (87%; n=92) had performed laryngeal tube placement 410 times before, 8% (n=9) had used a laryngeal tube 5-10 times previously, and only 5% (n=5) had placed o5 laryngeal tubes prior to our investigation. During the first five minutes, a significant increase in SpO2 from 70% (0-100%) to 96% (20-100%) was observed in CPR patients (Figure 1A). An increase in CO2 in CPR patients from 19 mmHg (0-70 mmHg) to 36 mmHg (10-62 mmHg) and then a significant decrease in CO2 values after five minutes was observed. Patients without CPR exhibited a decrease in CO2 values from 40 mmHg (10-100 mmHg) (Figure 1A) to 38 mmHg (30-52 mmHg) (Figure 1B). An average of 234.2 ml (10-2000 ml) of stomach content was drained through the placed gastric tube, either passively or through active suction, in 28.8% (n=31) of all treated patients. A comparison of CPR patients with non-CPR patients regarding gastric contents showed no significant difference. None of the patients aspirated (if aspiration before LTS placement was suspected, bronchoscopy was performed). For 60% of all placed gastric tubes (n=91), no stomach content was drained. No complications were recorded. The laryngeal tube was evaluated by the clinical staff after insertion with a score range from 1 (very bad) to 10 (very good). Placement was evaluated as excellent at 9.7, prevention of leakage was evaluated as 9.6 and ventilation was evaluated as 9.7.

’ METHODS After approval by the Local Ethical Review Board (455/13), data from 106 adult patients treated using routine anesthesia with an unexpected difficult airway or during resuscitation outside the OR between December 2006 and February 2013 were collected. As a part of a quality assurance program, an observational clinical trial was performed over a period of seven years by anesthetists using a standardized questionnaire. The use of the LTS II/LTS-D in difficult airway situations and in-hospital cardiopulmonary resuscitation (CPR) was examined. The inclusion criteria consisted of a difficult airway (Cormack Lehane Grade 3-4), CPR, and CPR with a difficult airway. Patients o18 years of age were excluded. After an initial failed intubation, the LTS-II/LTS-D was used as a second-line rescue device by the resuscitation team or the responsible anesthetist in the OR. The time of placement of the LTS-II/LTS-D was measured by the anesthetist or nurse specialist as the time the facemask was removed until the first successful ventilatory hub. The LTS-II/LTS-D was inserted by anesthetists and experienced nurse specialists. The number of intubation attempts before using the LTS-II/LTS-D was at the discretion of the responsible resuscitation team member or anesthetist. Placement of the LTS-II/LTS-D was classified as ‘‘failed’’ if the time for insertion was longer than 120 seconds. The cuff pressure in non-fasting patients was adjusted to 60 cmH2O to prevent aspiration; in all other patients, the cuff pressure was gradually decreased until reaching the ‘‘best cuff pressure’’ without leakage or a change in tongue color was achieved. Apart from the LTS-II/LTS-D, a videolaryngoscopy system (Karl Storz GmbH&Co.KG, Tuttlingen, Germany) with a Macintosh blade served as a rescue device for securing the airway. The main goal of the study was the successful placement of the LTS II/LTS-D with sufficient ventilation in terms of tidal volume after placement. The secondary variables included demographic data, time to placement, classified as o30 seconds, 30-60 seconds or 460 seconds, number of placement attempts (1, 2, 3 or 43), gastric content, SpO2/ etCO2 development over 5 minutes, subjective overall assessment, prior experience using a laryngeal tube and complications related to the placement of the LTS-II/LTS-D, such as leakage, tongue swelling or bleeding. Analysis of the data confirmed that a Gaussian distribution was only observed for demographic data such as age and height. Demographic data were summarized as means and standard deviations or numbers and percentages. SpO2/ etCO2-development and gastric content were summarized as medians and ranges. The statistical analysis was performed using software packages (Microsoft Excel 2013, Microsoft Deutschland GmbH, Unterschlei heim, Germany and GraphPad Prism 6, GraphPad Software Inc., San Diego, CA). Data were analyzed using the non-parametric Mann-Whitney test. Statistical significance was assumed at a probability of a type I error of less than 5% (po0.05).

’ DISCUSSION Our data indicate that laryngeal tubes such as the LTS-II/D are a valuable tool in ‘‘in-hospital’’ emergency airway management.

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Figure 1 - (A) SpO2 development and (B) CO2 development within the first five minutes in patients with CPR and without CPR.

users in our investigation and the fact that in our institution, the laryngeal tube is the SAD of choice in cases of an unexpected difficult airway in preclinical and clinical settings and is thus implemented in the standard operating procedures for airway management. No complications associated with the insertion of the LTS were recorded. Our own study group recently published data regarding LTS-associated complications in the preclinical setting (17). Complications consisted of significant tongue swelling as a result of cuff overinflation and gastric overinflation when the laryngeal tube was used without a gastric tube. Our lack of complications may have occurred due to strict cuff pressure monitoring after inflation and also because after securing the airway via the LTS, intubation was performed either via flexible bronchoscopy or (after 2008) video laryngoscope using a technique described earlier (13) under safe environmental circumstances. The fact that video laryngoscopy was implemented in our inner-clinical difficult airway management algorithm after 2009 may explain the small sample size. The use of a second-generation LTS with a gastric drainage (LTS-II 16 Ch. and LTS-D 18 Ch.) channel may be the reason for the absence of gastric distension. In the OR, when an unexpected difficult airway occurred and SAD utilization

Placement is safe and can be easily performed by anesthetists and nurse specialists, even outside the OR, within a short time. In over 94% of cases, placement was possible during the first attempt and in less than 30 seconds. This finding is inconsistent with recently published data in the preclinical setting. Sunde et al. retrospectively analyzed laryngeal tube insertion in out-of-hospital cardiac arrest and reported 74% of first-attempt placements and overall laryngeal tube insertion of 85%. Nevertheless, only a minority of cases (13%) required more than 30 seconds for placement (14). This difference may be related to the experienced users in our study, the majority of whom had performed more than 10 placements before and used an additional chin lift during insertion of the laryngeal tube. Russo (15) compared three different types of SAD (i-gel, laryngeal mask airway (LMA) Supreme and LTS-D) during elective anesthesia. Although a minimum experience of 15 placements for each device was required, the first attempt success rate was only 53%, and the overall success rate was 70% for the LTS-D; these rates were significantly lower than those found using the i-gel or LMA Supreme. Langenstein and Moeller reported 92% vs 93% success rates for insertion and ventilation using the classic LMA (cLMA) and intubating LMA (ILMA) in patients with a difficult airway (16). Again, this difference may be related to the experienced

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â&#x20AC;&#x2122; REFERENCES

was feasible, the surgical procedure was performed with the LTS under intermittent cuff pressure control (18). Furthermore, no aspiration after LTS insertion was recorded, but this possibility cannot be completely ruled out because intubation failure could be consistent with aspiration before placement of the LTS. The reason for the failed placement in one case remained unclear. Reasons for insufficient ventilation included one case of insufficient depth of anesthesia with bronchospasm and two cases of a reverted tip of the SGA. We observed gastric distension in four patients with multiple intubation attempts and therefore prolonged mask ventilation, but the data were insufficient to correlate these findings. By deepening anesthesia or appropriately placing a gastric tube, ventilation was possible without complications. Predictors of a difficult airway (receding chin, maxillary hypoplasia, micrognathia, retrognathia, reduced mouth opening, short neck, reduced cervical mobility, and obesity) were present in 70% of cases. This may have occurred because CPR patients were included without a preoperative anesthesiological examination. However, even in the absence of data regarding Mallampati scores or thyromental distance (TMD), our results led to the assumption that these tests have a poor predictive value for a difficult airway in the clinical setting; this finding has been previously described in the literature (19, 20). We showed that oxygenation and removal of CO2 was improved in our population. In CPR patients, the increases in SpO2 and CO2 are likely related to sufficient CPR by the resuscitation team. In non-CPR patients, the decrease in CO2 was significant and may be related to sufficient ventilation via the laryngeal tube. Nevertheless, these significant results are not clinically relevant in our opinion. They only indicate sufficient ventilation. In fact, our data emphasize that the laryngeal tube is a valuable tool for unexpected difficult airway management that can also provide sufficient ventilation outside the OR. In summary, we highlighted the importance of SGAs in unexpected difficult airway management and in innerclinical emergency situations, as noted in different difficult airway guidelines (7). We showed that the use of the LTS-D/ LTS-II in inner-clinical unexpected difficult airway management provides a secure method to initially secure the airway until other options for securing the airway, such as video laryngoscopy or fiber optic intubation, are available.

1. Cook TM, Woodall N, Frerk C, Fourth National Audit Project. Major complications of airway management in the UK: results of the Fourth National Audit Project of the Royal College of Anaesthetists and the Difficult Airway Society. Part 1: anaesthesia. Br J Anaesth. 2011;106(5): 617-31, http://dx.doi.org/10.1093/bja/aer058. 2. Cook TM, MacDougall-Davis SR. Complications and failure of airway management. Br J Anaesth. 2012;109 Suppl 1:i68-i85, http://dx.doi.org/ 10.1093/bja/aes393. 3. Mort TC. Emergency tracheal intubation: complications associated with repeated laryngoscopic attempts. Anesth Analg. 2004;99(2):607-13, http://dx.doi.org/10.1213/01.ANE.0000122825.04923.15. 4. Nolan JP, Kelly FE. Airway challenges in critical care. Anaesthesia. 2011;66 Suppl 2:81-92, http://dx.doi.org/10.1111/j.1365-2044.2011. 06937.x. 5. Schwartz DE, Matthay MA, Cohen NH. Death and other complications of emergency airway management in critically ill adults. A prospective investigation of 297 tracheal intubations. Anesthesiology. 1995;82(2):367-76, http://dx.doi.org/10.1097/00000542-199502000-00007. 6. Nolan JP, Hazinski MF, Billi JE, Boettiger BW, Bossaert L, de Caen AR, et al. Part 1: Executive summary: 2010 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations. Resuscitation. 2010;81 Suppl 1:e1-25, http://dx.doi.org/10.1016/j.resuscitation.2010.08.002. 7. Piepho T, Cavus E, Noppens R, Byhahn C, Dorges V, Zwissler B, et al. S1 guidelines on airway management : Guideline of the German Society of Anesthesiology and Intensive Care Medicine. Anaesthesist. 2015;64 Suppl 1: 27-40, http://dx.doi.org/10.1007/s00101-015-0109-4. 8. Thierbach A, Piepho T, Gobler S, Rutzler K, Frass M, Kaye AD, et al. Comparative study of three different supraglottic airway devices in simulated difficult airway situations. Minerva Anestesiol. 2015;81(12):1311-7. 9. Thierbach A, Piepho T, Wolcke B, Kuster S, Dick W. [Prehospital emergency airway management procedures. Success rates and complications]. Anaesthesist. 2004;53(6):543-50, http://dx.doi.org/10.1007/s00101-004-0679-z. 10. Timmermann A. Supraglottic airways in difficult airway management: successes, failures, use and misuse. Anaesthesia. 2011;66 Suppl 2:45-56, http://dx.doi.org/10.1111/j.1365-2044.2011.06934.x. 11. Wetsch WA, Schneider A, Schier R, Spelten O, Hellmich M, Hinkelbein J. In a difficult access scenario, supraglottic airway devices improve success and time to ventilation. Eur J Emerg Med. 2015;22(5):374-6, http://dx.doi. org/10.1097/MEJ.0000000000000230. 12. Schalk R, Auhuber T, Haller O, Latasch L, Wetzel S, Weber CF, et al. [Implementation of the laryngeal tube for prehospital airway management: training of 1,069 emergency physicians and paramedics]. Anaesthesist. 2012;61(1):35-40, http://dx.doi.org/10.1007/s00101-011-1966-0. 13. Schalk R, Weber CF, Byhahn C, Reyher C, Stay D, Zacharowski K, et al. [Reintubation using the C-MAC videolaryngoscope. Implementation in patients with difficult airways initially managed with in situ laryngeal tubes]. Anaesthesist. 2012;61(9):777-82, http://dx.doi.org/10.1007/s00101-012-2072-7. 14. Sunde GA, Brattebo G, Odegarden T, Kjernlie DF, Rodne E, Heltne JK. Laryngeal tube use in out-of-hospital cardiac arrest by paramedics in Norway. Scand J Trauma Resusc Emerg Med. 2012;20:84, http://dx.doi. org/10.1186/1757-7241-20-84. 15. Russo SG, Cremer S, Galli T, Eich C, Brauer A, Crozier TA, et al. Randomized comparison of the i-gel, the LMA Supreme, and the Laryngeal Tube Suction-D using clinical and fibreoptic assessments in elective patients. BMC Anesthesiol. 2012;12:18, http://dx.doi.org/10.1186/14712253-12-18. 16. Langenstein H, Moller F. [The importance of the laryngeal mask in the difficult intubation and early experience with the intubating laryngeal mask airway--ILMA--Fastrach]. Anasthesiol Intensivmed Notfallmed Schmerzther. 1998;33(12):771-80, http://dx.doi.org/10.1055/s-2007-994852. 17. Schalk R, Seeger FH, Mutlak H, Schweigkofler U, Zacharowski K, Peter N, et al. Complications associated with the prehospital use of laryngeal tubes--a systematic analysis of risk factors and strategies for prevention. Resuscitation. 2014;85(11):1629-32, http://dx.doi.org/10.1016/j.resuscitation. 2014.07.014. 18. Schalk R. [Preclinical duty of care during cuff pressure management]. Med Klin Intensivmed Notfmed. 2016;111(8):737-42, http://dx.doi.org/ 10.1007/s00063-015-0115-y. 19. Krobbuaban B, Diregpoke S, Kumkeaw S, Tanomsat M. The predictive value of the height ratio and thyromental distance: four predictive tests for difficult laryngoscopy. Anesth Analg. 2005;101(5):1542-5, http://dx. doi.org/10.1213/01.ANE.0000181000.43971.1E. 20. Lee A, Fan LT, Gin T, Karmakar MK, Ngan Kee WD. A systematic review (meta-analysis) of the accuracy of the Mallampati tests to predict the difficult airway. Anesth Analg. 2006;102(6):1867-78, http://dx.doi.org/ 10.1213/01.ane.0000217211.12232.55.

Conflicts of Interest R.S. and C.B. receive material support for research from VBM Medizintechnik GmbH and Karl Storz GmbH & Co KG, the manufacturers of the laryngeal tube and the C-MAC video laryngoscope and Bonfils intubation fiberscope, respectively. C.B. is a member of the Karl Storz advisory board. None of the other authors has any conflict of interest with products and/or companies mentioned in the manuscript.

â&#x20AC;&#x2122; AUTHOR CONTRIBUTIONS Mutlak H, Byhahn C, Meininger D, Zacharowski K, Weber CF and Schalk R contributed to the writing and preparation of the manuscript. Mutlak H, Meininger D, Schalk R and Byhahn C conceived the study and performed the statistical analysis. Cuca C edited the manuscript as a native speaker.

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CLINICAL SCIENCE

Complete axillary dissection without drainage for the surgical treatment of breast cancer: a randomized clinical trial Ruffo Freitas-Junior,I,II,* Luı´s Fernando Jube´ Ribeiro,I Marise Amaral Rebouças Moreira,III Geraldo Silva Queiroz,I Maurıćio Duarte Esperidiaõ,I Marco Aure´lio Costa Silva,I Rubens Jose´ Pereira,I Rossana Arau´jo Cataõ Zampronha,I Rosemar Macedo Sousa Rahal,II Leonardo Ribeiro Soares,I Danielle Laperche dos Santos,I Maria Virginia Thomazini,II Cassiana Ferreira Silva de Faria,II Re´gis Resende PaulinelliI,II I Gynecology and Breast Unit, Hospital Arau´jo Jorge, Goia´s Anticancer Association, Goiaˆnia, GO, BR. II Breast Program, Department of Gynecology and Obstetrics, School of Medicine, Federal University of Goia´s, Goiaˆnia, GO, BR. III Department of Pathology, School of Medicine, Federal University of Goia´s, Goiaˆnia, GO, BR.

OBJECTIVE: This randomized clinical trial evaluated the possibility of not draining the axilla following axillary dissection. METHODS: The study included 240 breast cancer patients who underwent axillary dissection as part of conservative treatment. The patients were divided into two groups depending on whether or not they were subjected to axillary drainage. ClinicalTrials.gov: NCT01267552. RESULTS: The median volume of fluid aspirated was significantly lower in the axillary drainage group (0.00 ml; 0.00 – 270.00) compared to the no drain group (522.50 ml; 130.00 - 1148.75). The median number of aspirations performed during conservative breast cancer treatment was significantly lower in the drainage group (0.5; 0.0 - 4.0) compared to the no drain group (5.0; 3.0 - 7.0). The total volume of serous fluid produced (the volume of fluid obtained from drainage added to the volume of aspirated fluid) was similar in the two groups. Regarding complications, two cases (2.4%) of wound dehiscence occurred in the drainage group compared to 13 cases (13.5%) in the group in which drainage was not performed, with this difference being statistically significant. Rates of infection, necrosis and hematoma were similar in both groups. CONCLUSION: Safety rates were similar in both study groups; hence, axillary dissection can feasibly be performed without drainage. However, more needle aspirations could be required, and there could be more cases of wound dehiscence in patients who do not undergo auxiliary drainage. KEYWORDS: Breast Cancer; Breast-conserving Surgery; Lymph Node Excision; Drainage; Postoperative Complications. Freitas-Junior R, Ribeiro LF, Moreira MA, Queiroz GS, Esperidia˜o MD, Silva MA, et al. Complete axillary dissection without drainage for the surgical treatment of breast cancer: a randomized clinical trial. Clinics. 2017;72(7):426-431 Received for publication on February 16, 2017; First review completed on May 16, 2017; Accepted for publication on May 16, 2017 *Corresponding author. E-mail: ruffojr@terra.com.br

’ INTRODUCTION

(1-4). In addition, some patients complain of considerable discomfort and difficulty with shoulder movement (5). The systematic use of continuous suction drainage may reduce postoperative morbidity (3). Several other approaches have been tested in an attempt to decrease the accumulation of serous fluid or seroma formation, including reducing the duration of drainage (6), the use of sclerosants (7) or fibrin glue (8) at the surgical site, and the obliteration of dead space in axillary dissection (3,9,10). Axillary drainage has become a subject of debate in recent years since it tends to prolong hospitalization times (11) and creates discomfort, exerting a negative psychological impact on patients who have undergone this treatment (5,12,13). Drainage is also associated with limitations to activities of daily living that may include difficulty in dressing and

The complications directly associated with the surgical treatment of breast cancer involving lymphadenectomy have been well established. They include seroma (the most common complication), hematoma, infection of the surgical wound, skin necrosis, winged scapula and lymphedema of the arm

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of small breasts, the maximum diameter of the tumor that would permit conservative surgery to be performed was 2 cm. Although the simultaneous presence of multicentric foci or contralateral tumors did not a priori constitute a contraindication to conservative surgery, these were assessed on a case-by-case basis by the surgeon to determine whether this procedure was appropriate.

problems with sleeping (13). Consequently, some studies have been conducted to evaluate the possibility of not draining the surgical site following axillary dissection (9,11,12). However, controversy persists regarding the need to use continuous drainage (9), with the debate being mainly motivated by doubts that not performing drainage could increase the rate of complications, particularly in patients undergoing complete axillary dissection (levels I, II and III). The primary objective of the present study was to investigate the safety of not draining the axilla. The evaluation was conducted by comparing the total number of breast cancer patients who did not develop complications following conservative surgery with complete axillary dissection according to whether or not axillary drainage was performed. The number of needle aspirations, the volume of fluid drained or aspirated and the total volume of fluid produced were compared between the two groups as a secondary objective.

Surgical technique Conservative breast cancer treatment consisted of excising the tumor, taking care to leave wide margins, and then performing complete axillary dissection, including levels I, II and III. After performing the cutaneous incision, the lymph nodes and all the adipose tissue located in the lower anterior portion in relation to the axillary vein were removed from its entry into the thoracic wall up to the anterior edge of the latissimus dorsi muscle. The pectoralis minor muscle was preserved at the surgeon’s discretion. The thoracodorsal nerve and the long thoracic nerve were preserved.

’ PATIENTS AND METHODS The internal review board of the Federal University of Goiás teaching hospital approved the protocol of the present study under reference number 017/2000. The procedures were in accordance with the ethical standards of the responsible committee on human research and with the Helsinki Declaration of 1975, as revised in 1983. ClinicalTrials.gov: NCT01267552. Following approval, 240 patients with breast cancer were included in the study and submitted to conservative surgery with axillary dissection. Only patients who voluntarily agreed to participate in the study and signed the informed consent form were enrolled. Sample size was calculated based on the number of complications detected in a pilot study conducted previously in this same institute. In that pilot study, the safety rate (i.e., absence of complications) for patients who were not submitted to axillary drainage was 72%. Taking into consideration a possible difference of 12% between the groups, with a=0.05 and b=0.20, a total of 202 patients were required for the study. The possibility of losses was estimated at 19%; therefore, the total number of patients to be enrolled to the study was increased to 240, a number intended to be distributed equally between the two groups.

Axillary drainage In the group of women randomized to drainage, a continuous suction drain was installed in the surgical site before the wound was closed. The drain was exteriorized through a counter-opening and attached using non-absorbable suture thread. Inside the surgical wound, the drain was placed in such a way to drain the region overlying the pectoralis major muscle, the axillary hollow and the edge of the latissimus dorsi muscle. The external extremity of the drain was connected to a continuous suction system.

Follow-up of the women in the drainage group In this group of patients, the fluid was removed from the reservoir bulb twice daily, and its volume was recorded on a chart specifically designed for this purpose. In the ward, dressings were applied according to the standard procedures in the institute until the patient was discharged on the second postoperative day, at which time she was referred to the outpatient clinic for follow-up. Between the fifth and seventh postoperative days, the drain was removed. Whenever necessary, needle aspiration was performed, and the volume of fluid aspirated was recorded. The patients were then evaluated weekly, and any complications occurring up to the 30th postoperative day were recorded.

Inclusion and exclusion criteria Female patients with clinical stage I or II breast cancer were included in the study. The recommendation for all these patients was conservative surgery, including a wide resection and complete axillary dissection (levels I, II and III). The procedures were to be performed either at the Araújo Jorge Hospital (ACCG) or at the university teaching hospital (UFG). Patients were only considered for inclusion if they fulfilled the inclusion criteria for both groups and if they agreed to participate in the study. Patients with diabetes were excluded.

No drain group In the group of women in whom drainage was not performed, needle aspiration was carried out whenever necessary up to the time of their discharge from hospital. The patients were advised to return once a week for assessment and for seroma aspiration as required. As in the drainage group, the volume of fluid aspirated was recorded, as were any other complications.

Randomization Randomization was performed by means of a computergenerated list of consecutive random numbers. The information regarding the group to which each patient had been allocated was given to the surgeon minutes prior to the start of the surgical procedure.

Definition of the variables Age: The patient’s age at the time of breast cancer diagnosis. Tumor size: Measured across the largest diameter at the time of diagnosis. Clinical stage: The clinical staging of the disease was defined in accordance with the International Union Against Cancer TNM classification.

Breast conservation criteria Conservative surgery was an option when the patients’ tumors were no greater than 4 cm in diameter. In the case

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number of histologically compromised lymph nodes ranged from 0 to 34, with a median of 0 (IQR: 0 - 2). The most frequent histological type was invasive ductal carcinoma, which accounted for 215 cases: 104 in the drainage group and 111 in the no drain group. There were 8 cases of lobular carcinoma in women in the drainage group and 3 in the no drain group. Other histological types were found in the remaining 14 patients, with 7 in each group. No statistically significant differences were found between the two groups with respect to the distribution of histological types (w2=3.08; p=0.68). Regarding the degree of tumor differentiation, grade II was the most common, accounting for 143 cases (59.6%), followed by grade III with 40 cases (16.7%), undetermined grade with 33 cases (13.7%) and grade I with 24 cases (10%). Analysis of the distribution of the histological grades also showed no statistically significant differences between the groups (w2=1.03; p=0.79). The two groups were similar with respect to the other control variables including age, tumor size, number of lymph nodes resected, and the number of compromised lymph nodes (Table 1). The median volume of fluid drained was 0 ml (range 0 2,040 ml). The median volume of fluid aspirated was 240 ml (range 0 - 11,998 ml). The median number of aspirations was 3.00 (range 0 - 48). The median overall volume of all the fluid drained or aspirated was 649.00 ml (range 0 - 11,998 ml). Table 2 compares the two study groups. The analysis of complications included the data from 179/ 240 patients, with the remaining 61 patients being lost to follow-up. Infections occurred in 25 cases (14%): 20 cases of hyperemia (11.2%) and 5 cases of abscess (2.8%). Wound dehiscence occurred in 15 cases (8.4%), necrosis in 4 cases (2.2%) and hematoma in 15 cases (8.4%). The two groups were significantly different in relation to wound dehiscence, which was more common in the no drain group. No other statistically significant differences were found between the two groups for any of the other complications registered (infection, necrosis or hematoma). The safety rate was similar for both groups, as shown in Table 3. Analysis of the individual subgroups showed that none of the control variables had any effect on safety (Figure 1).

Number of lymph nodes resected: The number of lymph nodes histologically identified in the surgical specimen obtained at axillary dissection. Axillary lymph node status: Defined in accordance with the number of lymph nodes histologically affected by neoplastic cells. Histological type: Defined in accordance with the World Health Organization (WHO) classification. Seroma formation: Seroma was defined as any accumulation of serous fluid clinically detected beneath the flaps or in the axilla following surgery. Volume of fluid drained: The total volume of fluid aspirated by the suction drain only for the patients in the drainage group, expressed in milliliters. Volume of fluid aspirated: The total volume of serous fluid aspirated by needle aspiration, expressed in milliliters. Number of needle aspirations: The number of times needle aspiration was required to drain the seroma. Total volume drained: The sum of the volume of fluid drained and aspirated, expressed in milliliters. Infection of the wound: The formation of cellulitis and/or accumulation of purulent secretion in the surgical area were considered indications of an infection in the surgical wound. Wound dehiscence: Wound dehiscence was defined as the opening of the skin including exposure of the underlying tissue prior to wound healing. Necrosis: Necrosis was defined as the presence of blackening areas on the skin flaps caused by deficient vascularization in the region. Safety rate: The total number of patients with no complications divided by the total number of patients in each group.

Statistical analysis

Means (± standard deviation) and a Student’s t-test were used for the variables with a normal distribution. In the case of numerical values with a non-normal distribution, the medians, interquartile ranges (IQR) and a Mann-Whitney test were used. For categorical variables, a chi-squared test (w2) or Fisher’s exact test was used, as appropriate. In the case of complications, the relative risks of each complication were analyzed together with their respective 95% confidence intervals. The significance level was set at 5%. The statistical software program SPSS (Chicago, IL, USA) was used throughout the statistical analysis.

’ DISCUSSION Investigation of sentinel lymph nodes has been the routine technical approach for dealing with the axillae of patients with early stage breast cancer (14,15). Nevertheless, axillary dissection with drainage continues to be performed during surgery in a large number of cases because of compromised lymph nodes or in cases in which identification of the sentinel lymph node proves impossible. Such procedures involve adverse events, with seroma formation constituting

’ RESULTS Of the 240 patients randomized, 119 were allocated to the axillary drainage group and 121 to the no drain group. The mean age of the patients was 49.64±11.93 years. The median tumor size was 30 mm (range 20 - 40 mm). The median number of lymph nodes resected was 17 (range 14 - 21). The

Table 1 - Demographic analysis comparing the group in which axillary drainage was performed with the group in which this procedure was not performed.* Drainage n=119 Mean age (± standard deviation) Median tumor size (25th – 75th percentile) Median histological grade (25th – 75th percentile) Median number of lymph nodes resected (25th – 75th percentile) Median number of positive lymph nodes (25th – 75th percentile)

49.38 30.00 2.00 17.00 0.00

* Mann-Whitney test/Student’s t-test.

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(± 10.95) (20.00 – 40. 00) (2.00 – 3.00) (13.00 – 21.00) (0.00 – 2.00)

No drainage n=121 49.91 28.00 2.00 17.00 0.00

(±12.86) (20.00 – 40.00) (2.00 – 3.00) (14.00 – 21.00) (0.00 – 2.00)

p-value 0.87 0.89 0.65 0.46 0.49

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Table 2 - Comparison of the volume of drained and aspirated serous fluid in the drainage and no drain groups.* Drainage (25th Volume drained (ml) Volume aspirated (ml) Number of aspirations Total drained (ml)

500.00 0.00 0.50 720.00

Median – 75th percentile) (233.50 – 864.00) (0.00 – 270.00) (0.00 – 4.00) (395.00 – 1145.00)

No drain Mean (± SD) 581.80 176.60 2.10 779.30

(25th

(442.60) (298.20) (3.10) (451.90)

0.00 522.50 5.00 540.00

Median – 75th percentile)

Mean (± SD)

(0.00 – 0.00) (130.00 – 1148.80) (3.00 – 7.00) (215.00 – 1152.50)

21.50 858.40 5.90 890.60

(107.90) (1434.50) (6.20) (1434.30)

p-value o0.01 o0.01 o0.01 0.19

* Mann-Whitney test/Student’s t-test.

Table 3 - Analysis of the complications observed according to group (axillary drainage or no drain). Drainage n (%) Infection Dehiscence Necrosis Hematoma Safety rate

10 2 1 6 67

(12.20%) (2.40%) (1.20%) (7.20%) (81.70%)

No drain n (%) 15 13 3 9 68

(15.50%) (13.50%) (3.10%) (9.40%) (70.10%)

RR (95%CI) 1.17 3.70 1.87 1.17 0.52

(0.70-1.94) (1.01-13.59) (0.34-10.31) (0.62-2.23) (0.25-1.06)

p-value 0.53 o0.01 0.39 0.61 0.07

RR = relative risk; CI = confidence interval. Safety rate: total number of patients without complications divided by the number of patients in each group.

Figure 1 - Forest plot of the effect of each individual group on the safety rate. The safety rate was defined as the total number of patients without complications divided by the total number of patients in each group.

of extensive surgical dissection and disruption of tissue planes, creating a dead space (18). To the best of our knowledge, the results of the other randomized studies comparing drainage versus no drainage have been inconsistent, and furthermore, the previous studies also differ in some points from the results of the present study. Classe et al. reported the results of breast-conserving surgeries (19), while Purushotham et al. reported on mastectomies and breast-conserving surgeries, with both groups performing axillary dissection only at levels I and II (20),

the principal complication, followed by infection, necrosis, dehiscence and hematoma (1,16). Systematic use of suction drainage has been performed in an attempt to reduce the rates of seroma formation. Among breast cancer patients undergoing surgery, this rate can be as high as 90% (16,17). However, over recent years, the possibility of not draining the axilla has been studied (11,12) since seroma formation rates tend to remain very high even with the use of drains (18). This complication occurs secondary to the disruption of lymph channels, an inevitable consequence

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drainage group. If we consider only the data from the drainage group, then the rate encountered was similar to rates reported from other previously published studies (1,6,23). In the present study, the rate of necrosis in the skin flaps was 2.2%, which is similar to the rate of 2.35% reported by Chintemani et al. (24). On the other hand, the rate of hematoma of 8.4% found in the present study was higher than rates reported by other groups, which ranged from 2.1% to 3.9% (1,3); however, the presence or absence of a drain did not affect these rates. In this study, the total number of patients with no complications (the safety rate) was similar in both groups; however, there was a tendency toward greater numbers of complications in the no drain group, influenced by the greater number of cases of dehiscence. These data are in agreement with the findings by Tylor et al., who also failed to find any difference between the two groups insofar as the overall number of complications was concerned (11). In the exploratory analysis, the individual subgroups exerted no effect on the safety rate. The results presented here allow the possible alternatives to be discussed with patients, i.e., the inconvenience of drainage versus a greater number of needle aspirations, with the greater potential risk of wound dehiscence. Patients are thus able to participate with the physicians in the decision-making process regarding their treatment. The safety rate (i.e., the total number of patients with no complications) was similar in both study groups, irrespective of whether or not axillary drainage was used. Therefore, axillary lymph node dissection can be performed safely without axillary drainage; however, there is a greater possibility of wound dehiscence, and a greater number of needle aspirations are required for patients who do not undergo auxiliary drainage.

whereas in the present study, axillary dissection was performed at all three levels. On the other hand, although axillary dissection was performed at all three levels in the study conducted by Soon et al., those investigators also included patients submitted to mastectomy (16). In previously published randomized clinical trials on breast cancer surgery in which the use of drainage was compared with no drainage, and axillary dissection was performed both in cases of mastectomy and in breast-conserving surgery, no statistically significant differences were found between the groups insofar as seroma formation was concerned (16,20,21). In the present study, the rate of seroma formation was 44%, and unlike previous studies (11), seroma formation was around four times greater in the no drain group, a difference that was statistically significant. The mean volume of serous fluid drained was much greater in the group of women in whom drainage was performed, and the mean volume of fluid aspirated was much greater in the no drain group. However, when the total volume of fluid was analyzed, i.e., the volume of fluid from drainage added to the volume of aspirated fluid, there was no statistically significant difference between the groups. The mean number of needle aspirations was significantly greater in the no drain group and may be considered a sign of the severity of a seroma. The number of aspirations performed in the present study ranged from 0 to 7, a range that is similar to that reported from other series, such as the study conducted by Gonzalez et al., who performed between 1 and 6 aspirations after removal of the drain in patients submitted to breast cancer surgery with axillary dissection (22). These aspirations also generate additional costs and can reduce these patient’s postsurgical quality of life. According to these results, drainage does not prevent seroma formation. The fluid that is not drained will have to be aspirated by needle aspiration. The present findings are in agreement with results published by other investigators, such as Soon et al., who reported total fluid volumes of 538.8 ml in the drained group versus 856.7 ml in the no drain group (16). With regard to the other complications, the infection rate in the present study was 14%, 11.2% of which consisted of cellulitis that receded with the use of antibiotics. The remaining 2.8% consisted of abscesses. There was no statistically significant difference between the two groups. Hall and Hall (23) reported a rate of cellulitis of 11% and a rate of abscesses of 4.6% following breast surgery, a result that was similar to that found in the present study. Therefore, care should be taken to ensure that antisepsis of the surgical area is strictly observed. Comparison of the two study groups showed infection rates of 12.2% and 15.5% for the drainage and no drain groups, respectively. The infection rates detected in previous randomized clinical trials differed greatly, with reports of 1.9% (21) and 25% (16). As in the present study, none of the other authors observed any significant differences in the rate of infection between the drainage and no drain groups of patients (3,16,21). Nevertheless, in 2005, Soon et al. reported a tendency for infection in the group of women subjected to drainage, although patients submitted to mastectomy were also included in that study (16). The rate of wound dehiscence in the present study was 8.4%, which was higher than rates reported from other series (3). This complication was strongly influenced by the no drain group, in which the rate was 13.5% versus 2.4% in the

’ ACKNOWLEDGMENTS This study was supported by the Fundac¸ão de Amparo à Pesquisa do Estado de Goiás (FAPEG) under number 01/2007.

’ AUTHOR CONTRIBUTIONS Ribeiro LF and Freitas-Junior R wrote and revised the manuscript. FreitasJunior R, Ribeiro LF, Moreira MA, Queiroz GS, Esperidião MD, Silva MA, Pereira RJ, Zampronha RA, Rahal RM, Soares LR, Santos DL, Thomazini MV, Faria CF and Paulinelli RR were responsible for the clinical trial and for revising the manuscript.

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15. Truin W, Roumen RM, Siesling S, van der Heiden-van der Loo M, Lobbezoo DJ, Tjan-Heijnen VC, et al. Sentinel lymph node biopsy and isolated tumor cells in invasive lobular versus ductal breast cancer. Clin Breast Cancer. 2016;16(4):e75-82, http://dx.doi.org/10.1016/j.clbc.2016.03.007. 16. Soon PS, Clark J, Magarey CJ. Seroma formation after axillary lymphadenectomy with and without the use of drains. Breast. 2005;14(2):103-7, http://dx.doi.org/10.1016/j.breast.2004.09.011. 17. Nadkarni MS, Rangole AK, Sharma RK, Hawaldar RV, Parmar VV, Badwe RA. Influence of surgical technique on axillary seroma formation: a randomized study. ANZ J Surg. 2007;77(5):385-9, http://dx.doi.org/10.1111/ j.1445-2197.2007.04067.x. 18. Ouldamer L, Caille A, Giraudeau B, Body G. Quilting suture of mastectomy dead space compared with conventional closure with drain. Ann Surg Oncol. 2015;22(13):4233-40, http://dx.doi.org/10.1245/s10434-015-4511-6. 19. Classe JM, Berchery D, Campion L, Pioud R, Dravet F, Robard S. Randomized clinical trial comparing axillary padding with closed suction drainage for the axillary wound after lymphadenectomy for breast cancer. Br J Surg. 2006;93(7):820-4, http://dx.doi.org/10.1002/bjs.5433. 20. Purushotham AD, McLatchie E, Young D, George WD, Stallard S, Doughty J, et al. Randomized clinical trial of no wound drains and early discharge in the treatment of women with breast cancer. Br J Surg. 2002; 89(3):286-92, http://dx.doi.org/10.1046/j.0007-1323.2001.02031.x. 21. Ruggiero R, Procaccini E, Piazza P, Docimo G, Iovino F, Antoniol G, et al. Effectiveness of fibrin glue in conjuntion with collagen patches to reduce seroma formation after axillary lynphadenectomy for breast cancer. Am J Surg. 2008;196(2):170-4, http://dx.doi.org/10.1016/j.amjsurg.2007.09.042. 22. Gonzales EA, Saltzstein EC, Riedner CS, Nelson BK. Seroma formation following breast cancer surgery. Breast J. 2003;9(5):385-8, http://dx.doi. org/10.1046/j.1524-4741.2003.09504.x. 23. Hall JC, Hall JL. The measurement of wound infection after breast surgery. Breast J. 2004;10(5):412-5, http://dx.doi.org/10.1111/j.1075-122X.2004.21401.x. 24. Chintamani, Singhal V, Singh J, Bansal A, Saxena S. Half versus full vacuum suction drainage after modified radical mastectomy for breast cancer – a prospective randomized clinical trial [ISRCTN24484328]. BMC Cancer. 2005;5:11, http://dx.doi.org/10.1186/1471-2407-5-11.

6. Kottayasamy Seenivasagam R, Gupta V, Singh G. Prevention of seroma formation after axillary dissection – a comparative randomized clinical trial of three methods. Breast J. 2013;19(5):478-84. 7. Catsman CJ, Beek MA, Rijken AM. Talc seromadesis in patients with chronic seroma formation after breast surgery. Springerplus. 2016;5:3, http:// dx.doi.org/10.1186/s40064-015-1648-5. 8. Sajid MS, Hutson KH, Rapisarda IF, Bonomi R. Fibrin glue instillation under skin flaps to prevent seroma-related morbidity following breast and axillary surgery. Cochrane Database Syst Rev. 2013;(5):CD009557, http:// dx.doi.org/10.1002/14651858.CD009557 9. Srivastava V, Basu S, Shukla VK. Seroma formation after breast cancer surgery: what we have learned in the last two decades. J Breast Cancer. 2012;15(4):373-80, http://dx.doi.org/10.4048/jbc.2012.15.4.373. 10. van Bastelaar J, Beckers A, Snoeijs M, Beets G, Vissers Y. Flap fixation reduces seroma in patients undergoing mastectomy: a significant implication for clinical practice. World J Surg Oncol. 2016;14:66, http://dx.doi. org/10.1186/s12957-016-0830-8. 11. Taylor JC, Rai S, Hoar F, Brown H, Vishwanath L. Breast cancer surgery without suction drainage: the impact of adopting a ‘no drains’ policy on symptomatic seroma formation rates. Eur J Surg Oncol. 2013;39(4):334-8, http://dx.doi.org/10.1016/j.ejso.2012.12.022. 12. Ebner F, deGregorio N, Vorwerk E, Janni W, Wöckel A, Varga D. Should a drain be placed in early breast cancer surgery? Breast Care. 2014;9(2): 116-22, http://dx.doi.org/10.1159/000360928. 13. Freitas-Junior R, Cavalcante AF, Soares LR, Pádua AP, Sousa PT, Ribeiro LF, et al. Estudo comportamental sobre a drenagem axilar no câncer de mama. Rev Bras Mastologia. 2014;24(2):42-6, http://dx.doi.org/10.5327/ Z201400020003RBM. 14. Giuliano AE, Ballman K, McCall L, Beitsch P, Whitworth PW, Blumencranz P, et al. Locoregional recurrence after sentinel lymph node dissection with or without axillary dissection in patients with sentinel lymph node metastases: long-term follow-up from the American College of Surgeons Oncology Group (Alliance) ACOSOG Z0011 Randomized Trial. Ann Surg. 2016;264(3):413-20, http://dx.doi.org/10.1097/SLA.00000000 00001863.

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CLINICAL SCIENCE

Abnormal diastolic function underlies the different beneficial effects of cardiac resynchronization therapy on ischemic and non-ischemic cardiomyopathy Qi Wang,I,# Kang-Yu Chen,I,# Fei Yu,I Hao Su,I Chun-Sheng An,I Yang Hu,I Dong-Mei Yang,I Jian Xu,I Ji YanI,II,* I

Department of Cardiology, Provincial Hospital Affiliated to Anhui Medical University, Hefei, Anhui, China. II Cardiovascular Institute of Anhui, Hefei, China.

OBJECTIVES: To investigate the association between diastolic function and the different beneficial effects of cardiac resynchronization therapy in patients with heart failure due to different causes. METHODS: The 104 enrolled patients were divided into an ischemic cardiomyopathy group (n=27) and a nonischemic cardiomyopathy group (n=77) according to the cause of heart failure. Before implantation, left ventricular diastolic function was evaluated in all patients using echocardiography. After six months of followup, the beneficial effects of cardiac resynchronization therapy were evaluated using a combination of clinical symptoms and echocardiography parameters. RESULTS: The ischemic cardiomyopathy group included significantly more patients with restrictive filling than the non-ischemic cardiomyopathy group. The response rate after the implantation procedure was significantly higher in the non-ischemic cardiomyopathy group than in the ischemic cardiomyopathy group. Degrees of improvement in echocardiography parameters were significantly greater in the non-ischemic cardiomyopathy group than in the ischemic cardiomyopathy group. Multivariate regression analysis showed that a restrictive filling pattern was an independent factor that influenced responses to cardiac resynchronization therapy. CONCLUSIONS: This study again confirmed that the etiology of heart failure affects the beneficial effects of cardiac resynchronization therapy and a lower degree of improvement in ventricular systolic function and remodelling was observed in ischemic cardiomyopathy patients than in non-ischemic cardiomyopathy patients. In addition, systolic heart failure patients with severe diastolic dysfunction had poor responses to cardiac resynchronization therapy. Ischemic cardiomyopathy patients exhibited more severe diastolic dysfunction than non-ischemic cardiomyopathy patients, which may be a reason for the reduced beneficial effect of cardiac resynchronization therapy. KEYWORDS: Heart Failure; Cardiac Resynchronization Therapy; Diastole. Wang Q, Chen KY, Yu F, Su H, An CS, Hu Y, et al. Abnormal diastolic function underlies the different beneficial effects of cardiac resynchronization therapy on ischemic and non-ischemic cardiomyopathy. Clinics. 2017;72(7):432-437 Received for publication on January 24, 2017; First review completed on March 20, 2017; Accepted for publication on May 17, 2017 *Corresponding author. E-mail: yanji111111@yeah.net #

These authors contributed equally.

â&#x20AC;&#x2122; INTRODUCTION

etiology of heart failure. The beneficial effects of CRT are smaller in patients with ischemic cardiomyopathy (ICM) than in patients with non-ischemic cardiomyopathy (NICM); therefore, ICM is a predictive factor for CRT non-response (3,4). Given this difference in the beneficial effects of CRT for different patients, prior studies have examined quantity of viable myocardium, myocardial scar burden, degree of scar transmurality, scar location, and left ventricular (LV) pacing to assess the potential relevance of these factors (5-7). However, the specific mechanism of these different effects remains unclear. Diastolic function is an important component of overall cardiac function (8). The process and degree of diastolic dysfunction differ in different etiologies. Based on previous findings, we hypothesized that ICM patients who meet CRT indications exhibit more serious diastolic dysfunction than NICM patients, which may be a reason for the differing beneficial effects of CRT.

Cardiac resynchronization therapy (CRT) can improve symptoms, increase quality of life, reduce the risk of hospital readmission, and decrease the mortality of chronic heart failure patients (1). However, approximately 30% of patients present non-response to CRT based on current standard guidelines (2). Substantial evidence-based medical data indicate that the beneficial effect of CRT depends on the

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’ METHODS

and interventricular septum, to record mitral annular early diastolic peak velocity (Em lateral and Em septal), and to calculate the mean Em value and E/Em ratio. According to the mitral valve flow spectrum and the tissue Doppler spectrum of the mitral annulus, patients were divided into restrictive filling pattern (RFP) (E/AX2, DTo160 ms, and E/EmX13) and non-RFP (E/Ao2, DTX160 ms, and E/ Emo13) groups (11).

Study subjects Consecutive patients who were scheduled to receive CRT pacemaker/defibrillator (CRT-P/D) treatment for chronic heart failure at Provincial Hospital Affiliated with Anhui Medical University from April 2013 to January 2015 were selected. Patients who met the following criteria were enrolled in the study (9): (1) grade III-IV on the New York Heart Association (NYHA) functional classification after treatment with standard drugs for heart failure; (2) sinus rhythm, left bundle branch block (LBBB), and QRS duration X120 ms; and (3) left ventricular ejection fraction (LVEF) p0.35. The exclusion criteria consisted of (1) atrial fibrillation; (2) acute myocardial infarction within three months before implantation; and (3) primary organic valvular heart disease. A total of 106 patients were enrolled. Because two patients suffered LV lead implantation failure, 104 patients were included in the statistical analysis. The NICM group consisted of 77 patients, and the ICM group comprised 27 patients. ICM was defined as X70% stenosis of at least one major coronary artery and its large branch on coronary angiography or a history of myocardial infarction.

Placement and optimization of CRT CRT was placed using the transvenous approach. Subclavian vein puncture was performed. After coronary sinus intubation, retrograde angiography was performed to fully display all branches of the coronary veins. The LV electrode lead was placed in the lateral or posterolateral cardiac vein to ensure pacing capture, with high voltage (10 V) used to demonstrate a lack of phrenic nerve stimulation. The right ventricular electrode lead was placed in the right ventricular apex or septum. The right atrial electrode lead was conventionally placed in the right atrial appendage. After the pacing tests were completed, the electrode leads were connected to a pulse generator and embedded in a subcutaneous or a deep to pectoralis major pouch in the chest wall. The incision was sutured in layers. Within one week of implantation, atrioventricular (AV) delay and interventricular (VV) delay optimization were performed under echocardiographic guidance. AV delay optimization was conducted using the iterative method. In particular, the optimal AV delay was determined based on the maximum left ventricular filling times at 6 selected AV delays: 180, 160, 140, 120, 100, and 80 ms. VV delay optimization was performed after AV delay optimization; the optimal VV delay was determined using the maximum left ventricular outflow tract velocity time integral at peak velocity (LVOT-VTImax) (12). Six months after implantation, a X15% reduction in LVESV compared with the preoperative value was defined as a CRT response.

Clinical evaluation The NYHA functional classification of patients was determined before implantation, and the disease course of heart failure, medications, and serum creatinine values were recorded. All patients underwent 12-lead electrocardiography (ECG) to determine heart rhythm, QRS duration, and morphology as well as echocardiography to evaluate cardiac structure and systolic and diastolic function. Six months after implantation, the NYHA functional classification was reevaluated, and ECG and echocardiography were performed to assess the changes.

Echocardiography Each patient underwent conventional echocardiography before implantation and at six months after implantation. A Philips iE33 ultrasound system with an S5-1 probe was used (1.0-5.0 MHz). Left atrial as well as LV end-systolic and end-diastolic diameters (LAD, LVESD and LVEDD, respectively) were obtained in M mode. LV volumes and the ejection fraction (LVESV, LVEDV and LVEF) were assessed using the biplane Simpson’s rule. The LV outflow tract-velocity time integral (LVOT-VTI) was measured within 0.5-1 cm below the aortic valve, and the systolic pulmonary artery pressure (SPAP) was estimated based on the tricuspid regurgitation velocity. LV dyssynchrony was assessed using the standard deviation of the time to peak systolic velocity in 12 LV segments (Ts-SD) derived from tissue Doppler images (10). The degree of mitral regurgitation (MR) was evaluated according to the ratio between the maximal mitral regurgitant jet area and the left atrial area as follows: mild (Grade 1) o20%, moderate (Grade 2) 20-40%, and severe (Grade 3) 440%. Diastolic function was assessed by transmitral Doppler and tissue Doppler. Pulse Doppler was used to acquire the diastolic mitral valve flow spectrum and record peak early diastolic velocity (E), peak late diastolic velocity (A), and diastolic early filling deceleration time (DT) and to calculate the E/A ratio. Tissue Doppler was used to acquire mitral annular velocity curves corresponding to the lateral wall

Statistical analysis The statistical analysis was performed using SPSS 19.0 software. Continuous variables are presented as the mean± standard deviation. Categorical variables are presented as frequencies and percentages. Comparisons of data within groups were performed with paired Student’s t-tests (continuous variables) and Wilcoxon signed-rank tests (NYHA classification, MR grade). Comparisons of data between groups were performed with unpaired Student’s t-tests (continuous variables) and Mann-Whitney U tests (NYHA classification, MR grade). Comparisons of gender and CRT response rates between groups were performed with x2 tests. Influencing factors on CRT responses were analyzed using univariate and multivariate logistic regression models. A value of po0.05 indicated a statistically significant difference.

’ RESULTS 1. Baseline information (Table 1). In this study, 104 patients were enrolled, including 77 patients in the NICM group and 27 patients in the ICM group. Before implantation, age, gender, NYHA functional classification, PR interval, QRS duration, serum creatinine level, echocardiography parameters (LAD, LVEDV, LVESV, LVEF, SPAP, MR, LVOT-VTI, and Ts-SD), and drug treatment conditions were not significantly

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Table 1 - Basic Patient Data. Parameter Age, y Male, n(%) Disease course, y NYHA class, n(%) III/IV Heart rate (beats/min) PR interval, ms AV delay, ms QRS duration, ms SCr, mmol/l LAD, mm LVEDD, mm LVESD, mm LVEDV, ml LVESV, ml LVEF, % FS, % MR, n(%) grade 1 grade 2 grade 3 SPAP, mmHg E, cm/s A, cm/s E/A DT, ms Em septal, cm/s Em lateral, cm/s E/Em LVOT-VTI, cm Ts-SD, ms RFP, n(%) Diuretics, n(%) ACE-I or ARB, n(%) b-Blocker, n(%) Amiodarone, n(%) Spironolactone, n(%)

Total n=104

NICM n=77

ICM n=27

p-value (NICM vs. ICM)

59.8±10.6 78 (75.0) 5.2±2.9 81 (77.9)/23 (22.1) 74.1±12.2 197.8±37.3 122.1±17.8 154.3±24.8 88.8±29.5 47.2±5.7 74.8±8.0 64.8±7.8 300.7±73.3 217.6±61.4 28.2±4.9 13.4±2.8

59.3±11.2 57 (74.0) 5.3±3.0 60 (77.9)/17 (22.1) 73.1±12.3 199.4±38.2 123.1±17.2 155.6±24.7 88.3±28.5 46.9±6.0 74.7±7.6 64.4±7.4 298.9±70.2 214.7±58.3 28.4±4.7 13.7±2.5

61.3±8.8 21 (77.8) 5.0±2.7 21 (77.8)/6 (22.2) 77.0±11.9 193.3±35.1 119.3±19.6 150.7±25.0 90.1±32.7 48.1±4.4 75.2±9.1 65.7±9.0 305.8±82.8 225.8±69.9 27.4±5.4 12.8±3.4

0.392 0.698 0.600 0.988 0.158 0.474 0.336 0.384 0.794 0.279 0.771 0.454 0.676 0.421 0.332 0.241 0.256

22 (21.2) 68 (65.4) 14 (13.5) 39.1±11.9 75.9±25.6 64.6±17.1 1.3±0.7 187.3±43.8 5.2±1.3 7.8±1.7 12.3±5.6 9.9±1.7 70.3±31.5 33 (31.7) 104 (100) 86 (82.7) 73 (70.2) 51 (49.0) 100 (96.2)

17 (22.1) 52 (67.5) 8 (10.4) 38.4±12.0 71.7±25.8 67.1±17.0 1.2±0.7 193.9±42.1 5.4±1.4 7.9±1.6 11.2±5.1 10.0±1.7 72.6±30.6 19 (24.7) 77 (100) 66 (85.7) 54 (70.1) 34 (44.2) 75 (97.4)

5 (18.5) 16 (59.3) 6 (22.2) 41.2±11.4 87.8±21.3 57.5±15.8 1.7±0.7 168.3±43.9 4.7±1.0 7.5±2.0 15.4±6.1 9.6±1.5 63.8±33.7 14 (51.9) 27 (100) 20 (74.1) 19 (70.4) 17 (63.0) 25 (92.6)

0.290 0.005 0.012 0.003 0.008 0.010 0.253 0.001 0.242 0.215 0.009 1.000 0.280 0.981 0.093 0.591

SCr, serum creatinine; LVEDV, left ventricular end diastolic volume; LVESV, left ventricular end systolic volume; LVEF, left ventricular ejection fraction; MR, mitral regurgitation; SPAP, systolic pulmonary arterial pressure; LVOT-VTI, left ventricular outflow tract-velocity time integral; ACE-I, angiotensinconverting enzyme inhibitors; ARB, angiotensin receptor blockers.

Table 2 - Clinical and echocardiographic characteristics of the overall population at baseline and follow-up.

different between these two groups. Compared with the NICM group, the ICM group exhibited higher values of E/A and E/ Em and shorter DT values (po0.05 for all comparisons). The RFP was significantly higher in patients in the ICM group than in patients in the NICM group (51.9% vs. 24.7%, x2=6.816, p=0.009). One week after implantation, the AV delay optimization was not significantly different between the ICM group (119.3±19.6 ms) and the NICM group (123.1±17.2 ms). 2. CRT response rate. Six months after the implantation procedure, 66 out of the 104 patients (63.5%) exhibited responses to CRT. The CRT response rate was significantly higher in the NICM group than in the ICM group (70.1% vs. 44.4%, x2=5.688, p=0.017). 3. Clinical beneficial effects of CRT. NYHA functional classification, LAD, LVEDV, LVESV, LVEF, FS, and LVOT-VTI were improved six months after the implantation procedure relative to before implantation (po0.001 for all comparisons). The degree of MR was lower than before implantation (po0.05). The SPAP did not change significantly. A comparison of parameters before and after CRT is presented in Table 2. In the NICM group, the postoperative NYHA functional classification, LAD, LVEDV, LVESV, LVEF, FS, and LVOT-VTI were all significantly improved compared with the values recorded before implantation (all po0.001); the degrees of SPAP and MR were both lower than those recorded before

Parameter NYHA class, I/II/III/IV LAD, mm LVEDV, ml LVESV, ml LVEF, % FS, % SPAP, mmHg MR grade, 1/2/3 LVOT-VTI, cm

Baseline

Follow-up

p-value

0/0/81/23 47.2±5.7 300.7±73.3 217.6±61.4 28.2±4.9 13.4±2.8 39.1±11.9 22/68/14 9.9±1.7

15/58/23/8 45.9±6.5 279.8±84.9 183.0±72.9 36.0±8.9 17.6±4.7 37.8±12.8 42/43/19 12.9±3.8

o0.001 o0.001 o0.001 o0.001 o0.001 o0.001 NS 0.043 o0.001

implantation (both po0.05). In the ICM group, the postoperative NYHA functional classification, LVESV, LVEF, FS, and LVOT-VTI were all significantly improved (all po0.05), whereas the LAD, LVEDV, SPAP, and MR did not exhibit significant changes. Six months after implantation, the differences in echocardiography parameters (LVESV, LVEF, FS, SPAP, and LVOT-VTI) between the two groups were significant (all po0.05). A comparison of parameters between the NICM and ICM groups is shown in Table 3. 4. Factors that influence the CRT response (Table 4). The univariate logistic regression analysis results suggested that

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Table 3 - Clinical and echocardiographic characteristics of patients with ICM and NICM. Parameter

NYHA class, I/II/III/IV Baseline Follow-up LAD, mm Baseline Follow-up LVEDV, ml Baseline Follow-up LVESV, ml Baseline Follow-up LVEF, % Baseline Follow-up FS, % Baseline Follow-up SPAP, mmHg Baseline Follow-up MR grade, 1/2/3 Baseline Follow-up LVOT-VTI, cm Baseline Follow-up

p-value

NICM

ICM

n=77

n=27

0/0/60/17 12/45/16/4*

0/0/21/6 3/13/7/4#

NS NS

46.9±6.0 45.5±6.6*

48.1±4.4 47.0±6.2

NS NS

298.9±70.2 272.6±79.9*

305.8±82.8 300.4±96.5

NS NS

214.7±58.3 174.5±67.2*

225.8±69.9 207.2±84.0#

NS 0.045

28.4±4.7 37.3±8.4*

27.4±5.4 32.4±9.3#

NS 0.013

13.7±2.5 18.3±4.3*

12.8±3.4 15.6±5.2#

NS 0.008

38.4±12.0 36.0±12.1#

41.2±11.4 42.9±13.7

NS 0.015

17/52/8 33/34/10#

5/16/6 9/9/9

NS NS

10.0±1.7 13.5±3.5*

9.6±1.5 11.1±3.9#

NS 0.003

* po0.001; #po0.05; follow-up vs. baseline.

Table 4 - Univariate and multivariate logistic regression analyses: estimates of correlations between baseline clinical and echocardiographic characteristics and response to CRT. Parameter

Univariate

Multivariate

HR (95% CI) Age, y Male sex Ischemic etiology Disease course, y NYHA class IV SCr, mmol/l QRS duration, ms LAD, mm LVEDV baseline, ml LVEF baseline, % SPAP baseline, mmHg MR baseline grade 3 Ts-SD, ms RFP

0.986 0.261 0.341 0.815 0.487 0.979 1.038 0.821 0.997 1.019 0.919 0.221 1.006 0.123

(0.949-1.025) (0.082-0.831) (0.138-0.840) (0.701-0.948) (0.189-1.252) (0.963-0.994) (1.016-1.060) (0.749-0.901) (0.992-1.003) (0.938-1.108) (0.879-0.961) (0.052-0.944) (0.993-1.020) (0.048-0.311)

p-value

HR (95% CI)

p-value

0.487 0.023 0.019 0.008 0.135 0.007 0.001 o0.001 0.297 0.653 o0.001 0.042 0.361 o0.001

1.045 (1.013-1.077) 0.846 (0.730-0.980)

0.006 0.026

0.100 (0.025-0.397)

0.001

HR, indicates hazard ratio; CI, confidence interval.

non-ischemic cardiomyopathy patients; (2) compared with non-ischemic cardiomyopathy patients, ischemic cardiomyopathy patients exhibited more severe diastolic dysfunction; and (3) severe diastolic dysfunction (restrictive filling) was an independent factor influencing the CRT response. Many randomized controlled trials and observational studies (3,4,13-15) have shown that CRT results in less improvement in LV systolic function and remodeling in ICM patients than in NICM patients. This study showed that the ICM group had lower CRT response rates six months after implantation; improvements in LVEF and LVOT-VTI, which reflect LV function, and improvements in LVESV, which reflect remodeling, were smaller in the ICM group than in

male gender, an etiology of ischemia, a disease course of heart failure, serum creatinine level, QRS duration, LAD, SPAP, severe MR, and RFP influenced the CRT response. When these factors were included in the multivariate logistic regression analysis, the results showed that QRS duration, LAD, and RFP were independent factors that influenced the CRT response.

’ DISCUSSION This study showed that (1) the improvement of left ventricular systolic function and remodeling was reduced in ischemic cardiomyopathy patients compared with

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Limitations

the NICM group. These results are not surprising. Several groups reported similar findings as long as a decade ago (16-18). Hummel et al. proposed that their similar findings were associated with the amount of viable myocardium (19). Because the ICM group had a smaller amount of viable myocardium, the improvement in LV function due to CRT could not be maintained. Other studies have shown that myocardial scars are associated with CRT nonresponse (5,6,20), which has been described in relation to the myocardial scar burden, degree of transmurality, and locations of the scars and LV lead. Chalil et al. further proposed that a scar size X33%, transmurality X51%, and pacing over a posterolateral scar in ICM patients were associated with suboptimal CRT responses (7). However, the specific mechanism that causes different CRT curative effects in heart failure patients with different etiologies remains unclear. Waggoner et al. reported that even when ICM patients exhibited improved LV systolic function, their diastolic function was not improved compared with NICM patients, and this result was attributed to the presence of relatively restrictive LV filling before CRT in ICM patients and a lack of improvement of end-diastolic volume and diastolic synchrony after CRT (21). The present study also showed that the ICM group presented with elevated LV filling pressures (i.e., elevated mitral E/A and E/Em) before CRT and that LVEDV was not improved six months after CRT. Although diastolic function is an important component of overall cardiac function, it has long received much less attention than systolic function. Abnormal diastolic function is important for the development of heart failure symptoms and signs. Even during systolic heart failure, increased LV filling pressure is closely associated with exercise limitation and is independent of the degree of systolic dysfunction (8). Studies have increasingly indicated that severe diastolic dysfunction (restrictive filling) suggests a worse hemodynamic and clinical status of heart failure as well as increased mortality and heart transplantation rates (22), which is also true for patients undergoing drug treatment (23,24), surgical ventricular restoration (25,26), and CRT (27,28). Using multivariate regression analysis, this study confirmed that severe diastolic dysfunction was an independent factor that influenced the CRT response. Diastolic dysfunction is a typical presentation of ICM. Abnormal diastolic function occurs at the early stage of myocardial ischemia, even earlier than systolic dysfunction (29,30). During myocardial ischemia, ATP production, ATP-dependent Ca2+ pump activity, Ca2+ uptake into the sarcoplasmic reticulum, and Ca2+ outflow decrease, and myosin-actin complex dissociation disorder occurs, resulting in a delayed and incomplete active diastole (31). Long-term recurrent attacks of ischemia and infarction, myocardial necrosis, advanced interstitial fibrosis, scar formation, increases in ventricular stiffness, and reduction in compliance will cause passive diastolic dysfunction, sustained increase in ventricular filling pressure, and eventually, development of restrictive filling (32). This phenomenon is different from dilated cardiomyopathy, which mainly causes ventricular enlargement and systolic dysfunction. In this study, the ICM group exhibited more severe diastolic dysfunction than the NICM group. Therefore, we propose that in addition to a lack of viable myocardium and the presence of myocardial scars, the secondary severe diastolic dysfunction in ICM patients might also cause suboptimal improvement in LV function and structure after CRT.

This study is subject to the following limitations. (1) This was a single-center observational study with no randomized control group. (2) The composition of the underlying etiologies was not balanced; fewer cases of ICM were included. (3) Invasive measurements (such as left ventricle end diastolic pressure (LVEDP)) were not performed for the evaluation of diastolic function. Therefore, it is necessary to increase the sample size and validate invasive measurements to further clarify the reason for the different curative effects of CRT for different disease etiologies. In summary, this study again confirmed that the etiology of heart failure affected the curative effects of CRT and that a lower degree of improvement of ventricular systolic function and remodeling was observed in ICM patients than in NICM patients. In addition, systolic heart failure patients with severe diastolic dysfunction showed poor CRT responses. ICM patients exhibited more severe diastolic dysfunction than NICM patients, which may be a reason for the reduced beneficial effect of CRT. The findings of our study are similar to those published as long as a decade ago, but the intervening period has seen little attention to this important aspect of the CRT response. We feel that it may now be reasonable to refocus on this aspect of CRT and that a study from an Asian country may reignite interest.

’ ACKNOWLEDGMENTS The authors thank Kai Hu and Zhi-Quan Liu for statistical guidance and Zhi-Wei Du and Cui-Ping Xie for valuable discussions.

’ AUTHOR CONTRIBUTIONS Wang Q, Chen KY and Yan J conceived and designed the study. Wang Q and Chen KY drafted the manuscript. Wang Q, Hu Y and Yang DM were responsible for the acquisition of data. Chen KY, Yu F, An CS and Su H were responsible for the analysis and interpretation of data. Yan J was responsible for the administrative support. Xu J, Su H and Yang DM were responsible for the critical revision of the manuscript for important intellectual content. All authors have read and approved the ﬁnal version of this manuscript.

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REVIEW

The diagnostic value of narrow-band imaging for early and invasive lung cancer: a meta-analysis Juanjuan Zhu,I,III Wei Li,I,III,* Jihong Zhou,V Yuqing Chen,I,III Chenling Zhao,I,III Ting Zhang,IV Wenjia Peng,II Xiaojing WangI,III I Department of Respiratory Disease, the First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, China. II Department of Epidemiology and Health Statistics, Bengbu Medical College, Bengbu 233030, China. III Provincial Key Laboratory of Respiratory Disease in Anhui, Bengbu 233004, China IV Department of Respiratory Disease, No.2 People’s Hospital of Fuyang City, Fuyang 236015, China. V Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu 233004, China.

This study aimed to compare the ability of narrow-band imaging to detect early and invasive lung cancer with that of conventional pathological analysis and white-light bronchoscopy. We searched the PubMed, EMBASE, Sinomed, and China National Knowledge Infrastructure databases for relevant studies. Meta-disc software was used to perform data analysis, meta-regression analysis, sensitivity analysis, and heterogeneity testing, and STATA software was used to determine if publication bias was present, as well as to calculate the relative risks for the sensitivity and specificity of narrow-band imaging vs those of white-light bronchoscopy for the detection of early and invasive lung cancer. A random-effects model was used to assess the diagnostic efficacy of the above modalities in cases in which a high degree of between-study heterogeneity was noted with respect to their diagnostic efficacies. The database search identified six studies including 578 patients. The pooled sensitivity and specificity of narrow-band imaging were 86% (95% confidence interval: 83–88%) and 81% (95% confidence interval: 77–84%), respectively, and the pooled sensitivity and specificity of white-light bronchoscopy were 70% (95% confidence interval: 66–74%) and 66% (95% confidence interval: 62–70%), respectively. The pooled relative risks for the sensitivity and specificity of narrow-band imaging vs the sensitivity and specificity of white-light bronchoscopy for the detection of early and invasive lung cancer were 1.33 (95% confidence interval: 1.07–1.67) and 1.09 (95% confidence interval: 0.84–1.42), respectively, and sensitivity analysis showed that narrow-band imaging exhibited good diagnostic efficacy with respect to detecting early and invasive lung cancer and that the results of the study were stable. Narrow-band imaging was superior to white light bronchoscopy with respect to detecting early and invasive lung cancer; however, the specificities of the two modalities did not differ significantly. KEYWORDS: Invasive Lung Cancer; Early Lung Cancer; Narrow-Band Imaging; Meta-Analysis. Zhu J, Li W, Zhou J, Chen Y, Zhao C, Zhang T, et al. The diagnostic value of narrow-band imaging for early and invasive lung cancer: a meta-analysis. Clinics. 2017;72(7):438-448 Received for publication on November 24, 2016; First review completed on January 19, 2017; Accepted for publication on February 14, 2017 *Corresponding author. E-mail: weilidc@163.com

’ INTRODUCTION

both in China and in other countries around the world and has imposed significant medical and economic burdens on society (1,2). Surgery offers a relatively good possibility of a cure in patients with early-stage lung cancer, and the 5-year survival rate for patients with stage Ia disease is 73%. However, the 5-year survival rates range from 9% to 46% in patients with more advanced disease (stages II–IV) (3). Currently, only 16% of lung cancer are diagnosed when the disease is localized, and even fewer lung cancer are diagnosed at stage 0, resulting in an overall 5-year survival rate of only 15%. Therefore, early diagnosis and treatment are important, as these factors are associated with an improved 5-year survival rate and a better prognosis in patients with lung cancer (4). Endoscopic examination remains the most direct and effective method for detecting lung cancer. Narrow-band imaging (NBI) is a new endoscopic technique designed to detect pathologically altered mucosal and submucosal microvascular patterns that uses the following two narrow

Trend analyses of high-quality data from 22 registries, including several population-based registries currently available in the National Central Cancer Registry of China, were conducted in 2000–2011 and published in 2016. These analyses suggested that an estimated 4,292,000 new cancer cases and 2,814,000 cancer deaths would occur in China in 2015, and they identified lung cancer as the most common incident cancer and the leading cause of cancer-related death in the country. Lung cancer is considered an important health threat

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using histopathological analysis as the reference standard (i.e., the gold standard); (d) studies reporting sufficient data for the construction of 2 2 contingency tables for the true positive (TP), false positive (FP), false negative (FN), and true negative (TN) rates of early or invasive lung cancer, as well as studies reporting ‘‘per-lesion’’ or ‘‘per-patient’’ statistics; and (e) studies including patients who underwent clinical or radiological examinations because they were suspected of having or known to have a lung malignancy or were at high risk for lung cancer. When the same data sets or subsets were presented in more than one article, only the article that provided the greatest detail, was published most recently, or had the highest level of quality was chosen. When abstracts and studies did not report sufficient data, we did not attempt to contact their authors to request additional information. The following studies were excluded from the analysis: (1) studies reporting data pertaining to histologically unconfirmed lesions; (2) studies reporting incomplete data; (3) studies with overlapping data; and (4) letters, editorials, expert opinions, reviews without original data, conference abstracts, meta-analyses, case reports, studies with fewer than 10 cases, and articles for which only the abstract could be obtained.

light bandwidths: blue light (390–445 nm), which is absorbed by superficial capillaries; and green light (530–550 nm), which is absorbed by blood vessels inside capillary membranes. These narrow bandwidths reduce light scattering (i.e., the deeper the light penetrates into the mucosa, the stronger the scattering) and enable enhanced blood vessel visualization. This approach consequently improves contrast on the mucosal surface, reduces examination times, and eliminates unnecessary biopsies (5). Following the successful introduction of NBI in the field of gastroenterology and the confirmation of its diagnostic value for gastrointestinal malignancies, clinicians incorporated this modality into interventional pulmonology. NBI has since been used to diagnose lung diseases, especially early and invasive lung cancer. Several studies have compared the ability of NBI to detect early and invasive lung cancer (defined as moderate and severe dysplasia or carcinoma in situ [CIS]) and invasive lung cancer with that of pathological analysis (6-14). The simplicity, reproducibility, and straightforward nature of NBI evaluations of the tracheo-bronchial tree ensure that NBI videobronchoscopy will play a significant role in lung cancer detection and staging in the future. However, previous publications have shown that the sensitivity and specificity of NBI for these lesions vary widely (0.53–1.00 and 0.43–0.90, respectively). However, most of these studies confirmed that NBI was superior to white-light bronchoscopy (WLB) with respect to detecting both early and invasive lung cancer (7-10,13,14). Therefore, the present study aimed to conduct a meta-analysis to evaluate the diagnostic efficacies of NBI and WLB and to compare the ability of NBI to diagnose early and invasive lung cancer with that of WLB.

Qualitative assessment Study quality was evaluated using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool (15), a validated tool that assesses diagnostic accuracy studies using 14 domains pertaining to the design and presentation of study data. The possible responses to each question are ‘‘yes’’, ‘‘no’’, or ‘‘unclear’’ and are scored as 1, -1, and 0, respectively. The assessment was performed by two independent authors (WL and JJZ), and disagreements were resolved by discussion.

’ MATERIALS AND METHODS Search strategy We searched the PubMed, EMBASE, Sinomed, and China National Knowledge Infrastructure databases for relevant studies published up to September 14, 2015. We focused on English- and Chinese-language publications reporting the diagnostic efficacy of NBI for early and invasive lung cancer. The following search terms were used to ensure the list of studies retrieved via the search was comprehensive: (‘‘Narrow Band Imaging’’ OR ‘‘Narrowband Imaging’’ OR ‘‘Band Imaging, Narrow’’ OR ‘‘Band Imagings, Narrow’’ OR ‘‘Imaging, Narrow Band’’ OR ‘‘Imagings, Narrow Band’’ OR ‘‘Narrow Band Imagings’’ OR ‘‘Imaging, Narrowband’’ OR ‘‘Imagings, Narrowband’’ OR ‘‘Narrowband Imagings’’ OR ‘‘NBI’’ OR ‘‘Narrow-band Imaging’’) AND (‘‘Lungs’’ OR ‘‘Lung’’ OR ‘‘Pulmonary’’) AND (‘‘Humans’’ OR ‘‘Human’’). First, all the records from the database search were imported into Endnote to eliminate duplicate studies. Then, two evaluators (WL and JJZ) independently read the titles and abstracts of the articles identified through the above searches to exclude studies that did not meet the analysis inclusion criteria. Any disagreements were resolved through discussion. Finally, the full texts of the remaining studies were carefully reviewed so that informed final decisions regarding their inclusion in the study could be made. The reference lists of the included records were also screened for relevant articles (Figure 1).

Data extraction Data pertaining to the numbers of TP, FP, FN, and TN diagnoses were extracted, and histological pathological findings served as the gold standard with which the above modalities were compared. We constructed 2 2 tables containing the number of cases of early or invasive lung cancer. These data were extracted on both a ‘‘per-patient’’ and a ‘‘per-lesion’’ basis when possible. We also extracted data regarding the first author’s name, year of publication, nation of research origin, mean patient age, ratio of men to women, number of lesions, number of patients, pathological results, number of endoscopists and endoscopes, type of NBI system, study design, and other relevant items (Table 1).

Data synthesis and analysis Meta-disc (version 1.4) software was used to test the threshold effect among the included studies by calculating the Spearman correlation coefficient, to combine the indices used to describe the diagnostic efficacy of NBI, and to conduct heterogeneity tests. Study heterogeneity was assessed using the w2 test and I2 statistic. I2 values of 25%, 50%, and 75% were indicative of low, moderate, and high heterogeneity, respectively. If the I2 value exceeded 50%, or the likelihood ratio w2 test yielded a p-value o0.05, the data were considered to exhibit significant statistical heterogeneity. Therefore, we pooled the data pertaining to the sensitivity and specificity of the above diagnostic tests using a randomeffects model to generate a more conservative estimate.

Literature inclusion and exclusion criteria The following studies were included in the analysis: (a) studies published in English or Chinese; (b) studies using NBI to diagnose early and invasive lung cancer; (c) studies

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Figure 1 - Study flow diagram.

cancer. For these analyses, a p-value o0.05 was considered significant. An RR 41 indicated that NBI was more sensitive than WLB for the detection of early invasive lung cancer. Publication bias was analysed using a weighted linear regression model and a Deeks funnel plot (STATA software). An asymmetric funnel plot was suggestive of possible publication bias. The significance of the intercept was determined using a t test, and a p-value o0.05 was indicative of statistically significant publication bias. All p-values were two-sided, and all confidence intervals (CIs) had a two-sided probability coverage of 95%.

The area under the summary receiver operating characteristic curve (AUC) was used to analyse the lung cancer diagnostic efficacy of NBI. If significant heterogeneity was noted, meta-regression was used to identify the sources. Subgroup analysis was conducted to examine the diagnostic value of various statistical indices, and sensitivity analysis was used to assess the stability of the study results. STATA software (version 11.0; STATACorp, College Station, TX, USA) was used to calculate the relative risks (RRs) for the sensitivity and specificity of NBI vs the sensitivity and specificity of WLB for the diagnosis of early or invasive lung

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Table 1 - Characteristics of the studies included in this meta-analysis Study/year

Gao et al./2014 (12) Li et al./2014 (9) Chen et al./2014 (11) Herth et al./2009 (7) Bojan et al./2009 (10) Vincent et al./2007 (8)

Nation

No. of subjects (n)

No. of biopsies (n)

Average age (yr)

M/F

Control

Statistical index

Blind

QUADAS score

China China China Germany Serbia America

44 196 153 57 106 22

Unclear 1072 Unclear 98 636 64

58.3±13.8 58±10 57±11 Unclear 55 59

34/10 152/44 106/47 Unclear 85/21 8/14

No No No No Yes Yes

Per-patient Per-patient Per-patient Per-patient Per-lesion Per-lesion

Unclear Yes Yes (partially blind) Yes Yes Yes (partially blind)

11 12 12 11 13 11

Table 1 - Characteristics of the studies included in this meta-analysis (continued) Study/year

Gao et al./2014 (12) Li et al./2014 (9)

Type of investigation

NBI System

No. of endoscopists

Lesion characterization

Study design

Pathological results

NBI

EVIS LUCERA (CV-260SL) EVIS LUCERA (CV-260SL) EVIS LUCERA (CV-260SL) EVIS EXERA

Shibuya’s descriptors Shibuya’s descriptors Shibuya’s descriptors Shibuya’s descriptors Shibuya’s descriptors Shibuya’s descriptors

Retrospective

Cancer

Prospective

Moderate to severe dysplasia and Cancer Cancer

NBI, WLB

Chen et al./2014 (11)

NBI, AFI+NBI, AFI

Herth et al./2009 (7) Bojan et al./2009 (10)

NBI, AFI, WLB and combination NBI, WLB

Vincent et al./2007 (8)

NBI, WLB

EVIS LUCERA (CV-260SL) EVIS EXERA II

Unclear Unclear Unclear (more than one) Unclear 2

Prospective Prospective Prospective

Moderate to severe dysplasia and carcinoma in situ Cancer

Prospective

Moderate to severe dysplasia and cancer

Table 1 - Characteristics of studies included in this meta-analysis (continued) Study/year Gao et al./2014 (12) Li et al./2014 (9) Chen et al./2014 (11) Herth et al./2009 (7) Bojan et al./2009 (10) Vincent et al./2007 (8)

No. of subjects (n)

44 196 153 57 106 22

29 157 87 9 267 13

4 3 4 4 51 29

2 21 50 8 14 0

9 15 12 36 304 22

’ RESULTS

indicating that a high level of between-study heterogeneity was present with respect these parameters because of the nonthreshold effect. When a random-effects model was used to analyse the sensitivity and specificity of NBI for the detection of early or invasive lung cancer, the pooled effect values for the sensitivity, specificity, and AUC were 86% (95% CI: 83– 88%, I2=94.1%), 81% (95% CI: 77–84%, I2=88.9%), and 0.89 (Figure 2).

Study identification and study quality Of the 207 reports retrieved during the database search, 159 remained after the duplicate studies were eliminated, and 28 remained after the titles and abstracts were screened. Twenty-two studies were excluded after a full-text review for the following reasons: (a) their biopsies were performed at sites of abnormal fluorescence, (b) they reported insufficient data with respect to their sensitivity and specificity calculations, (c) they were review articles, (d) their data were presented in more than one article, and (e) their full text was not available (Figure 1).

The efficacies of NBI and WLB Four of the included articles (7-10) compared the diagnostic efficacies of NBI and WLB, and three of these articles included data pertaining to patients with of moderate and severe dysplasia. These four studies included a total of 381 patients and 1870 lesions. The pooled sensitivity and specificity of NBI for the detection of early and invasive lung cancer were 91% (95% CI: 88–94%, I2=89%) and 81% (95% CI: 77–85%, I2=93.1%), respectively, with an AUC of 0.92 (Figure 3). The pooled sensitivity and specificity of WLB for the detection of early and invasive lung cancer were 70% (95% CI: 66–74%, I2=90.3%) and 66% (95% CI: 0.62–0.70, I2=72.8%), respectively, with an AUC of 0.70 (Figure 4). The pooled RR for the sensitivity of NBI vs the sensitivity of WLB for the detection of early and invasive cancer was 1.33 (95% CI: 1.07–1.67), and the corresponding pooled RR for the specificity of NBI vs the specificity of WLB for the detection of early or invasive

Quality assessment The results of the quality assessment of the eligible studies are shown in Table 1. The studies included in the analysis were generally of an ideal design.

NBI accuracy Five prospective studies and one retrospective study including a total of 578 patients were included in the analysis. The Spearman correlation coefficient for these studies was 0.600, p=0.208, indicating that the lack of a definite threshold effect produced heterogeneity. The I2 values for sensitivity and specificity were 94.1% and 88.9%, respectively,

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Figure 2 - Six-study analysis of the ability of NBI to diagnose early lung cancer and invasive lung cancer. A. Pooled sensitivity of NBI for the diagnosis of early lung cancer and invasive lung cancer; B. Pooled specificity of NBI for the diagnosis of early lung cancer and invasive lung cancer; C. The summary receiver operating characteristic (SROC) curve for the diagnosis of early lung cancer and invasive lung cancer by NBI.

Figure 3 - Four-study analysis of the ability of NBI to diagnose early lung cancer and invasive lung cancer. A. Pooled sensitivity of NBI for the diagnosis of early lung cancer and invasive lung cancer; B. Pooled specificity of NBI for the diagnosis of early lung cancer and invasive lung cancer; C. The SROC curve for the diagnosis of early lung cancer and invasive lung cancer by NBI.

ability of NBI to accurately diagnose early lung cancer only (7,8). The sensitivity and specificity of NBI for the diagnosis of early lung cancer were 62% (95% CI: 38–82%, I2=77.3%) and 58% (95% CI: 48–68%, I2=96.8%), respectively, whereas the corresponding values for WLB were 14% (95% CI: 3–36%, I2=27.6%) and 69% (95% CI: 59–78%, I2=91.4%), respectively. The RR for the sensitivity of NBI vs the sensitivity of WLB for

cancer was 1.09 (95% CI: 0.84–1.42) (Figure 5). Thus, NBI was significantly more sensitive than WLB with respect to detecting early and invasive lung cancer; however, NBI and WLB did not differ significantly with respect to specificity. The value of NBI vs that of WLB for the diagnosis of early lung cancer. We also searched for data regarding the

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Figure 4 - Four-study analysis of the ability of WLB to diagnose early lung cancer and invasive lung cancer. A. Pooled sensitivity of WLB for the diagnosis of early lung cancer and invasive lung cancer; B. Pooled specificity of WLB for the diagnosis of early lung cancer and invasive lung cancer; C. The SROC curve for the diagnosis of early lung cancer and invasive lung cancer by WLB.

and invasive lung cancer were available; thus, additional studies will be needed to confirm the abovementioned findings.

the diagnosis of early lung cancer was 3.86 (95% CI: 1.38– 10.75), and the RR for the specificity of NBI vs the specificity of WLB for the diagnosis of early lung cancer was 0.83 (95% CI: 0.45–1.53). Therefore, we concluded that NBI is superior to WLB with respect to detecting early lung cancer. The sensitivity and specificity of NBI were lower in the above studies than in the cumulative results.

Sensitivity and meta-regression analysis As shown in Table 2, we noted high heterogeneity in the pooled effect sizes from the included studies. Therefore, we performed sensitivity analysis of the ability of NBI to diagnose early and invasive lung cancer using characteristics from different types of studies, including prospective studies and studies including X50 patients and using per-patient statistics and different video endoscopy systems or patient populations, as well as other studies (Table 2). In addition, we sequentially excluded studies whose results deviated significantly from the pooled effect size (7,8). The remaining five and four studies were subjected to sensitivity analyses. The AUCs of NBI for the diagnosis of early and invasive cancer ranged from 0.83 to 0.93. These results indicated that the diagnostic efficacy of NBI for early and invasive cancer remained high and that the results of the study were stable. Several of the previously mentioned factors were also selected for the meta-regression analysis (Table 3). Although all the factors could be extracted from the six studies, the sources of between-study heterogeneity were not identified. Notably, the heterogeneity may have been caused by factors that could not be assessed in the present analysis, namely, patient age and sex ratios, because a portion of their data were lost or were not reported.

The value of NBI vs that of autofluorescence imaging and NBI + autofluorescence imaging. In this study, we also searched for data comparing the ability of NBI to accurately diagnose early and invasive lung cancer with that of autofluorescence imaging (AFI) and NBI + AFI (7,11). Only two studies including 210 patients were included in this analysis, and we applied a fixed-effects model to pool the effect sizes. The diagnostic sensitivity and specificity of NBI for the diagnosis of early and invasive lung cancer were 62% (95% CI: 54–70%, I2=0.0%) and 86% (95% CI: 0.74–0.94, I2=48.2%), respectively, whereas the corresponding values of AFI were 91% (95% CI: 85–95%, I2=90.7%) and 38% (95% CI: 25–51%, I2=0.0%), respectively. The RRs for the sensitivity and specificity of NBI for the diagnosis of early and invasive lung cancer were 0.69 (95% CI: 0.60– 0.78) and 2.29 (95% CI: 1.61–3.25), respectively. The sensitivity and specificity of AFI + NBI for the detection of early and invasive lung cancer were 93% (95% CI: 88–96%, I2=89.3%) and 54% (95% CI: 40–67%, I2=91.3%), respectively, and the corresponding RRs were 0.67 (95% CI: 0.59–0.76) and 1.38 (95% CI: 0.49–3.90), respectively. Therefore, we concluded that the sensitivity of NBI for the diagnosis of early and invasive lung cancer was lower than that of AFI + NBI; however the specificity of NBI for the diagnosis of early and invasive lung cancer did not differ significantly from that of AFI, suggesting that NBI and AFI are complementary tests. Unfortunately, only two relevant studies regarding the ability of these tests to diagnosis early

Assessment of publication bias In the publication bias analysis (STATA), the Deeks funnel plot suggested that publication bias may have been present among the included studies, and the corresponding p-value of 0.02 indicated that statistically significant publication bias was present among the included studies. As shown in the

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Figure 5 - A. The pooled RR for the sensitivity of NBI vs the sensitivity of WLB for the detection of early lung cancer and invasive lung cancer; B. The pooled RR for the specificity of NBI vs the specificity of WLB for the detection of early lung cancer and invasive lung cancer.

Table 2 - Outcomes of the sensitivity analysis of selected studies assessing the stability of the accuracy of NBI for diagnosing early and invasive lung cancer Study characteristics Inclusion populationa (8-12) Prospective studies (8-11) Studies with X50 patients (7,9-11) EVIS LUCERA videoendoscopy system (9-12) Final outcomeb (10-12) Per-patient analysis (7, 9, 11, 12) Blind (7-11) No control (7,9,11,12) Five studiesc (7,9-12) Four studiesc (9-12)

No. of studies

No. of patients

5 5 4 4 3 4 5 4 5 4

521 534 512 499 303 450 534 450 556 499

Pooled estimatesd Sensitivity Specificity 0.86 0.85 0.85 0.86 0.86 0.78 0.85 0.78 0.85 0.86

(0.84-0.89) (0.82-0.88) (0.82-0.88) (0.83-0.89) (0.81-0.90) (0.73-0.82) (0.82-0.88) (0.73-0.82) (0.82-0.88) (0.83-0.89)

0.80 0.81 0.86 0.85 0.67 0.83 0.81 0.83 0.85 0.85

(0.76-0.84) (0.77-0.84) (0.82-0.89) (0.81-0.88) (0.57-0.76) (0.73-0.90) (0.77-0.84) (0.73-0.90) (0.81-0.88) (0.81-0.88)

AUC

0.89 0.89 0.91 0.87 0.89 0.87 0.89 0.87 0.90 0.87

a: Patients with known or suspected lung malignant lesions. b: Pathological positive index was cancer. c: Studies whose results deviated significantly from the pooled effect size were excluded, and sensitivity analyses of the remaining five and four studies were performed. d: The 95% confidence interval is given in parentheses.

Complications

Deeks funnel plot (Figure 6), the angle of the regression line and the diagnostic odds ratio axis diverged from 90o, suggesting that publication bias was present among the included studies.

Two studies reported adverse effects. In one study, all the adverse effects were attributed to bronchoscopy rather than to the NBI devices. The other study reported slight bleeding

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Table 3 - Meta-regression analysis of the potential sources of heterogeneity Variables

Coefficient

Study design (prospective vs retrospective) Number of patients (X50 vs o50) Histology outcome (invasive cancer vs early lung cancer) Videoendoscopy system (Lucera vs EXERA) Statistical index (per-patient vs per-lesion) Included population (known or suspected lung malignancy vs high risk of lung malignancy)

0.311 1.768 -0.139 -0.604 1.400 -0.083

Relative DOR (95% CI) 1.36 5.86 0.87 0.55 4.06 0.92

(0.01-285.42) (0.05-750.93) (0.02-33.32) (0.01-32.01) (0.14-118.82) (0.00-801.28)

p-value 0.8650 0.3303 0.9111 0.6691 0.2787 0.9712

however, it is also an accepted endoscopic diagnostic method with defined standards for the diagnosis of early and invasive lung cancer. The most widely used set of current diagnostic standards was proposed by Shibuya, whose ‘‘descriptors’’ include dotted, tortuous, abruptly terminating blood vessels (27). NBI can distinguish cancer from benign lesions with a high level of accuracy (6,25,26). Several articles have reported the superiority of NBI over AFI and WLB because the former detects cancerous lesions with higher specificity than the latter; however its sensitivity is not significantly compromised by its higher specificity (7-11,13,14). In a meta-analysis of 14 studies (15 data sets), Chen et al. (23) compared the ability of autofluorescence bronchoscopy (AFB) to detect lung cancer and paraneoplastic lesions with that of WLB. The pooled sensitivity and specificity for AFB were 90% (95% CI: 84–93%) and 56% (95% CI: 45–66%), respectively, and the pooled sensitivity and specificity for WLB were 66% (95% CI: 58–73%) and 69% (95% CI: 57–79%), respectively. Wang et al. (21) also compared the ability of AFB to detect lung cancer and precancerous lesions with that of WLB in a metaanalysis of six studies. The pooled sensitivities of AFI and WLB were 89% (95% CI: 81–94%) and 67% (95% CI: 46–83%), respectively, and the pooled specificities of AFI and WLB were 64% (95% CI: 37–84%) and 84% (95% CI: 74–91%), respectively. Thus, AFI was more sensitive and less specific than WLB for the detection for early and invasive lung cancer. However, in the present meta-analysis, NBI had a sensitivity of 86% (95% CI: 83–88%, I2=94.1%) and a specificity 81% (95% CI: 77–84%, I2=88.9%) for the detection of early and invasive lung cancer. One systematic review (28) from 2001 that examined the utility of positron emission tomography (PET) with respect to the diagnosis of pulmonary nodules and mass lesions included 40 studies that met its specific inclusion criteria. PET was used to examine 1474 focal pulmonary lesions of any size and was found to have a maximum joint sensitivity and specificity (upper-left point on the receiver operating characteristic curve at which sensitivity and specificity were equal) of 91.2% (95% CI: 89.1–92.9%). The diagnostic sensitivity and specificity of PET are superior to those of NBI; however, PET is expensive, cannot be used to obtain histological specimens, and exposes the patient to radiation. The prognoses of patients with lung cancer depend heavily on the stage at which patients are diagnosed with the disease. According to the stepwise progression theory of carcinogenesis, early detection of preinvasive lesions, such as moderate or severe dysplasia or CIS, and subsequent prompt surgical resection or endobronchial treatment will provide the patient with the best chance of survival and lung function conservation. Consistent with this idea, the reported 5-year survival rate for patients treated for preinvasive (stage 0) lung cancer exceeds 90% (29). However, in the current study, only two articles (7,8) evaluated the ability of NBI and WLB

Figure 6 - Deeks funnel plot for the evaluation of publication bias.

that was attributed to NBI but described no other NBIrelated complications.

’ DISCUSSION Diagnosing lung cancer early is essential for effectively treating the disease and optimizing survival. Bronchoscopy is ideal for detecting lung cancer arising from the central airway; however, detecting early and invasive lung cancer and precancerous lesions using WLB remains difficult. According to a previous report, only 29% of CIS and 69% of microinvasive cancer cases are identified by WLB, regardless of the experience level of the bronchoscopist (16). The more advanced AFI bronchoscopy exploits the autofluorescent nature of the bronchial mucosa to detect tiny and subtle superficial lesions. AFI has been shown to increase the yield of diagnostic testing for early lung cancer (17-22); however, its role in detecting lung neoplasia is limited by its specificity, which is actually lower than that of WLB and leads to more false positives (7,21,23,24). NBI is an optical technique based on the use of narrower blue and green light filters to enhance visualization of the microvascular architecture and microsurface structures. This technique, which offers chromoendoscopy without requiring additional dye sprays, clearly highlights the micromorphology and microvascular structures of mucosal lesions and facilitates the identification of cancerous lesions (5,25-27). NBI is simple and can be performed by general endoscopists;

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than those of AFI, respectively, suggesting that NBI and AFI are complementary tests. In that study, NBI + AFB and AFB alone differed significantly with respect to their specificities (po0.01) but not their sensitivities (p40.05) for the diagnosis of early and invasive lung cancer, whereas NBI + AFB and NBI alone differed significantly with regards to both their sensitivities and their specificities for the diagnosis of early and invasive lung cancer (po0.01 and p=0.03, respectively). Chen concluded that NBI + AFB could diagnose lung cancer with greater specificity than AFB alone, a notable finding that contradicted those reported by Herth. This discrepancy may be attributable to the following factors: 1. The study by Chen addressed obvious bronchial and pulmonary masses and used the above diagnostic techniques only to observe mucosal abnormalities. Moreover, the patients eligible for inclusion in the study were not at high risk for lung cancer. Therefore, that study differed from the study performed by Herth, as it lacked data regarding certain study parameters, as well as data regarding precancerous lesions. 2. The positive diagnosis rate reported by Chen pertained to patients rather than lesions. The discrepancies between these two studies have necessitated additional high quality research to determine whether NBI + AFB is superior to either modality alone with respect to diagnosing cancer. Despite these differences, the study by Chen demonstrated the distinct advantages of the above modalities. AFB is used to observe surface mucosal changes, and NBI is used to observe surface mucosal vascular lesions. When used in combination, each modality can compensate for the shortcomings of the other. However, AFI and NBI are not currently integrated into single bronchoscope units. In the future, the combined use of these technologies may reduce inspection times and patient discomfort. In our metaanalysis, which included 578 patients, only one article reported slight bleeding. Therefore, we believe that NBI, which has high diagnostic value and a low complication rate, may be an accurate, safe, and cost-effective tool for diagnosing early lung and invasive lung cancer.

to accurately diagnose early lung cancer. The sensitivity and specificity of NBI for the diagnoses of early lung cancer were 62% and 58%, respectively, and the corresponding values for WLB were 14% and 69%, respectively. The corresponding RRs for the sensitivity and specificity were 3.86 (95% CI: 1.38–10.75) and 0.83 (95% CI: 0.45–1.53), respectively. Thus, NBI appears to be superior to WLB with respect to detecting early lung cancer. Unfortunately, only two relevant studies regarding this issue were identified, and additional research is needed to confirm the findings described herein. NBI, a novel observational method, has facilitated pathological diagnoses of lung cancer by allowing mass observation, guiding targeted biopsies, and enabling predictions of pathological type (30); has improved assessments of extension; and has influenced therapeutic strategies (9,10,14). The results of our meta-analysis suggest that NBI is highly accurate with respect to diagnosing lung neoplasms. An article (7) included in the analysis had accepted patients who were at high risk for lung cancer (i.e., patients without a known diagnosis or clinical findings suggestive of central airway malignancies). In that study, a histologic tumour grade of moderate to severe dysplasia or CIS (via biopsy) was considered a positive diagnosis of intraepithelial neoplasia. However, that study reported a lower NBI sensitivity for the detection of lung cancer than other recent articles (13,18). This discrepancy can be explained by the ability of endoscopy to distinguish between ‘‘abnormal’’ and ‘‘suspicious’’ lesions and the researchers’ subsequent decision to consider only suspicious lesions to be positive diagnostic findings on bronchoscopy. Not all previous studies made this distinction, and if that study had considered both abnormal and suspicious lesions to be ‘‘positive bronchoscopic findings’’, NBI would have identified 100% of the high-grade dysplastic lesions and CIS affecting the patients enrolled therein. Similarly, Vincent et al. (8) reported that NBI identified dysplasias or malignancies that were not detected by WLB in 23% of patients. However, NBI did not detect these lesions with greater accuracy in areas with abnormal WLB findings. Further studies are needed to determine the efficacy of NBI with respect to the detection of premalignant airway lesions in at-risk populations. In an earlier article, Herth reported that AFI, NBI, and NBI + AFI exhibited sensitivities of 65%, 53%, and 71% and specificities of 40%, 90%, and 40%, respectively, for the diagnosis of early lung cancer (7). Although NBI was significantly more specific than AFI with respect to diagnosing cancer (po0.001), these modalities did not differ significantly with regards to sensitivity (p=0.49). Furthermore, although the relative sensitivity for the combination of AFI and NBI vs WLB was 4.0, the combined test did not exhibit improved specificity compared with either AFI alone or NBI alone (p=0.56). The specificity of AFI + NBI was equal to that of AFI and significantly lower than that of NBI; thus, this combination led to a nonsignificant increase in lung cancer diagnostic yield. In summary, this study demonstrated that NBI can be used alone as a first-choice lung cancer diagnostic modality rather than in combination with other diagnostic modalities. According to Chen (11), the diagnostic sensitivities of NBI, AFI, and NBI + AFI for the detection of central lung cancer were 63.5%, 94.2%, and 95.6%, respectively, and the specificities of these tests for the detection of central lung cancer were 75.0%, 31.3%, and 87.5%, respectively. The specificity and sensitivity of NBI were significantly higher and lower

Limitations Our analysis had some limitations. First, we included only six studies in the analysis. Second, we were unable to identify the sources of heterogeneity among the studies included herein. Although one could argue that heterogeneity is inevitable in a meta-analysis regardless of whether its presence is detectable via statistical analysis, its existence limits the clinical application of the results of the analysis. The heterogeneity noted herein may be attributed to differences in the time spans of the included studies, the use of different types of bronchoscopes, differences in the experience levels of the endoscopists and pathologists who performed the bronchoscopies and biopsies, diagnostic research on small sample sizes, differences in sex ratios and age distributions, and differences in pathological results. Furthermore, in some of the studies included in the analysis, tissue samples biopsied from visually normal areas, as demonstrated by both NBI and WLB, were used as controls; however, other studies included in the analysis did not perform control biopsies. Third, in our subgroup analysis, we compared the diagnostic accuracies of NBI, AFI and AFI + NBI; however, we identified only two studies that performed these comparisons. Therefore, our conclusions regarding the relative efficacies of those modalities require further

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verification in future studies. Fourth, all the included studies used an earlier bronchoscope model. The newest NBI bronchoscope is the OLYMPUS BF-H290, which produces clearer images than earlier models but is expensive. To date, no published studies have used this bronchoscope. Finally, publication bias was inevitable because we did not search for unpublished data, and data regarding TP, FP, TN, and FN diagnoses could not be extracted from the sensitivity and specificity calculations of two relevant studies (13,14). In addition, we included only English- and Chinese-language studies from four major databases and therefore may have overlooked some important articles published in other countries and languages. Furthermore, the reports included herein seldom described whether their patients were consecutively or randomly selected, an omission that may also have been a source of bias. NBI is an accurate, safe, and cost-effective tool for diagnosing lung cancer. The current evidence indicates that NBI is more valuable than WLB with respect to diagnosing both early and invasive lung cancer and that it is both feasible and convenient to switch between these two modalities as technologies advance. Our findings indicate that NBI is superior to WLB and that its use may lead to improvements in diagnostic yields, as well as improvements in sensitivity for the diagnosis of cancer that do not compromise specificity. However, additional high-quality prospective studies regarding the ability of NBI to diagnose early and invasive lung cancer remain necessary.

10.

11.

12. 13.

14.

15.

’ ACKNOWLEDGMENTS

16.

This work was funded by the Key Project of Natural Science Research Project in Higher Education of Anhui(KJ2016A487),National Natural Science Foundation of Anhui Province (grant no.1408085MH144), Key Laboratory Project on Department of Science and Technology of Anhui province (grant no.1206c0805026) and Key Programs for Science and Technology Development of Anhui province (12010402127).

17. 18.

’ AUTHOR CONTRIBUTIONS Li W designed the study, acquired some of the data, edited the manuscript and critically reviewed the ﬁnal version of the manuscript. Zhu J acquired some of the data, conducted the statistical analysis, and drafted the manuscript. Chen Y, Zhao C, Zhang T, Peng W, Wang X performed the study and performed the data collection and analysis. All authors read and approved the ﬁnal version of the manuscript.

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26. Miyazu YM, Ishida A, Nakamura M, Miyazawa T. Possible clinical applications of high magnification bronchoscpy combined with narrow band imaging. Chest. 2006;130:146S, http://dx.doi.org/10.1378/chest. 130.4_MeetingAbstracts.146S-b. 27. Shibuya K, Nakajima T, Fujiwara T, Chiyo M, Hoshino H, Moriya Y, et al. Narrow band imaging with high-resolution bronchovideoscopy: a new approach for visualizing angiogenesis in squamous cell carcinoma of the lung. Lung Cancer. 2010;69(2):194-202, http://dx.doi.org/10.1016/ j.lungcan.2010.04.023. 28. Gould MK, Maclean CC, Kuschner WG, Rydzak CE, Owens DK. Accuracy of positron emission tomography for diagnosis of pulmonary

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REVIEW

Osteointegration of porous absorbable bone substitutes: A systematic review of the literature Maria Ju´lia Escanhoela Paulo, Mariana Avelino dos Santos, Bruno Cimatti, Nelson Fabrı´cio Gava, Marcelo Riberto, Edgard Eduard Engel * Departamento de Biomecanica, Medicina e Reabilitacao do Aparelho Locomotor, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, BR.

Biomaterials’ structural characteristics and the addition of osteoinductors influence the osteointegration capacity of bone substitutes. This study aims to identify the characteristics of porous and resorbable bone substitutes that influence new bone formation. An Internet search for studies reporting new bone formation rates in bone defects filled with porous and resorbable substitutes was performed in duplicate using the PubMed, Web of Science, Scielo, and University of Sa˜o Paulo Digital Library databases. Metaphyseal or calvarial bone defects 4 to 10 mm in diameter from various animal models were selected. New bone formation rates were collected from the histomorphometry or micro-CT data. The following variables were analyzed: animal model, bone region, defect diameter, follow-up time after implantation, basic substitute material, osteoinductor addition, pore size and porosity. Of 3,266 initially identified articles, 15 articles describing 32 experimental groups met the inclusion criteria. There were no differences between the groups in the experimental model characteristics, except for the follow-up time, which showed a very weak to moderate correlation with the rate of new bone formation. In terms of the biomaterial and structural characteristics, only porosity showed a significant influence on the rate of new bone formation. Higher porosity is related to higher new bone formation rates. The influence of other characteristics could not be identified, possibly due to the large variety of experimental models and methodologies used to estimate new bone formation rates. We suggest the inclusion of standard control groups in future experimental studies to compare biomaterials. KEYWORDS: Biomaterials; Osteointegration; Systematic Review; Bone Substitute. Paulo MJ, dos Santos MA, Cimatti B, Gava NF, Riberto M, Engel EE. Osteointegration of porous absorbable bone substitutes: A systematic review of the literature. Clinics. 2017;72(7):449-453 Received for publication on December 28, 2016; First review completed on March 21, 2017; Accepted for publication on May 5, 2017 *Corresponding author. E-mail: engel@fmrp.usp.br

’ INTRODUCTION

inside the biomaterial, accelerates absorption of absorbable biomaterials and decreases the peak temperature of cements during setting (4,5). Most authors believe that pore size, porosity and interconnection of pores enhances new bone ingrowth. The ideal magnitude of these characteristics, however, has not yet been established (5-7). Progressive reabsorption and replacement of the biomaterial by normal bone is also considered an advantageous property because inert substitutes affect bone remodeling and can compromise its structure and mechanical resistance (8). The addition of growth factors and other osteoinductive factors seems to increase osteointegration, but conflicting data also exist (9). Comparing the many combinations of materials is a demanding task, and the use of many different analysis methods makes comparisons even more challenging. Computed microtomography (micro-CT) and histomorphometry (HMM) have frequently been used to quantify new bone formation (NBF) in bone defects created in animal models and inside the porous biomaterial (10). The aim of this study was to identify the chemical and structural characteristics that influence the capacity of new bone formation of porous and absorbable bone substitutes implanted in animal models using a systematic review.

The use of autografts for the treatment of bone defects has well-known restrictions, including limited availability and donor site morbidity (1). This fact has sparked an intense search for bone substitutes of different compositions and structural conformations (2). Some are already available on the market, whereas many still await evidence attesting their capacity for osteointegration and consequent commercial viability. A large number of new materials and material combinations have been developed. Structural characteristics have also been improved. The presence of pores significantly increases the osteointegration capacity, whereas solid biomaterials tend to form a fibrosis layer on the surface (3). Furthermore, the presence of pores allows fluid circulation

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Methodology

3. Implantation of porous and resorbable bone substitutes in the form of premolded blocks or cement with a clear description of the composition, porosity and pore size; 4. NBF indicated as a rate according to HMM or micro-CT data.

A systematic review was performed to evaluate how specific characteristics of porous and absorbable bone substitutes used to fill bone defects in experimental in vivo studies affects the capacity of new bone formation using micro-CT or HMM quantifications.

Exclusion criteria: 1. Clinical trials, implantation in humans or in vitro studies; 2. Insufficient description of the substitute characteristics, methodology or results; 3. Non-porous or non-absorbable substitutes; 4. Presentation in granular form. This form was excluded due to interference with the porosity and pore size of the substitute.

Search strategy An electronic search was independently performed by two researchers (MJEP and MAS) between July 2014 and February 2015 without restrictions on the publication date. The following databases were used: PubMed, Web of Science, Scielo, and Theses and Dissertations of the University of São Paulo Digital Library. Only articles written in English or Portuguese were selected. The following search term combinations were used: (bone substitute AND porous) OR (bone substitute AND cancellous) OR (bone substitute AND spongy) OR (bone substitute AND pore) OR (cement AND porous) OR (cement AND cancellous) OR (cement AND spongy) OR (cement AND pore) OR (bone cement AND porous) OR (bone cement AND cancellous) OR (bone cement AND spongy) OR (bone cement AND pore).

Variables The end point variable was the rate of new bone formation (NBF), which was based on the histomorphometry (HMM) or micro-CT analysis data. The remaining dependent variables were as follows: animal model, bone region of the defect, diameter of the defect (in mm), follow-up time after implantation (in weeks), substitute basic material (calcium phosphate, hydroxyapatite, bioglass, etc.), osteoinductor addition (fibroblast growth factor (FGF), BMP or bone marrow mesenchymal stem cells (BMSCs)), maximum pore size and porosity.

Article selection strategy Two researchers (MJEP and MAS) independently performed the article selection based on the eligibility criteria by reading the titles, summaries or full text, according to the search strategy (Figure 1). Consensus meetings, with the participation of a third researcher (EEE), were utilized to resolve conflict situations. Some articles were included from the reference lists of the selected articles. The experimental groups of each article that met eligibility criteria were analyzed independently; therefore, each article could have more than one group.

Presentation of results The NBF rates are described as the means, maximum and minimum values. The categorical variables and maximum defect size were grouped and compared using the KruskalWallis test. The defect size, follow-up time and porosity were correlated to NBR using the Spearman’s correlation coefficient. PASW software version 17 (IBM SPSS, Armonk, USA) was used for the data analysis and the level of significance was set as 5%.

Eligibility criteria Inclusion criteria:

’ RESULTS

1. Experimental, in vivo studies in animal models; 2. Orificial defects, 4.0 mm to 10.0 mm in diameter, produced by curettage or drilling of holes;

The initial search identified 3,266 studies. Figure 1 illustrates the selection flow. A total of 3,143 articles were excluded because the biomaterial characteristics, experimental

Figure 1 - Search strategy.

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3,266 studies were published describing porous and absorbable bone substitutes filling bone defects in experimental animals. A large number of materials have been developed and tested, separately or combined, to increase the capacity of new bone formation. This continuous and effervescent development hinders the classification of these materials (9). In this review, we aimed to classify these materials according to the basic material used in the production of the bone substitute; however, there are overlaps between groups and many materials that are classified into the same group are not always similar. Most of the substitutes contained HA or other types of CPs or a combination of them as the basic material. The accumulated experience on modulating the resorption rate and mechanical resistance of these compounds make this an attractive combination (10,18). The number of other materials was too small to allow a statistical analysis. The influence of the other chemical and structural characteristics of the biomaterials had to be very large to be detected in such a heterogeneous sample. For this reason, it was not possible to identify differences in the capacity of new bone formation by comparing the different groups of experimental animals, the sizes and locations of bone defects, the association with osteoinducers and the pore sizes. The follow-up time presented a weak to moderate correlation to NBF, depending on the analysis method (HMM or microCT). However, the porosity presented significant differences, suggesting that this parameter has a strong impact on the NBF rate.

model, NBF measurement or article language were not eligible. Another 108 articles were excluded due to insufficient data or incomplete description of the biomaterial, methodology or defect characteristics. From the remaining 15 articles, 32 experimental groups with different implanted bone substitutes were considered for the analysis (Table 1). HMM was used more frequently than micro-CT for NBF quantification (20 groups, 62.5%). In both analysis methods, no significant differences were found when the NBF means were grouped according to the animal model and bone region (Table 2). The basic materials of the bone substitutes could not be compared because many of the material groups became too small due to uneven distribution of the experimental groups. The addition of osteoinductors to the bone substitute did not result in a significant increase in NBF. In addition, the influence of the pore size on NBF could not be detected. There was no correlation between the defect size and NBF in the HMM analysis (r=â&#x20AC;&#x201C;0.181, p=0.446) and micro-CT analysis (r=0.103, p=0.751). NBR showed a moderate correlation to the follow-up time in the HMM analysis (r=0.441, p=0.052) but a weak correlation in the micro-CT analysis (r=0.122, p=0.705). NBF was significantly influenced by porosity (Figure 2).

â&#x20AC;&#x2122; DISCUSSSION The search for bone substitutes is a contemporary and relevant subject (9). The large number of studies that present new biomaterials or the further development of known biomaterials confirms this statement. Prior to February 2015, Table 1 - Characteristics of the experimental groups. Author

Animal

Region

Defect size (mm)

Follow-up (weeks)

Basic material

Osteoinductor

Maximum pore size (mm)

Porosity (%)

Evaluation method

NBF (%)

Del Real et al. (11) Del Real et al., 2002 Hing et al. (12) Hasegawa et al. (13) Hasegawa et al. (13) von Doernberg et al. (14) von Doernberg et al. (14) von Doernberg et al. (14) von Doernberg et al. (14) Kroese-Deutman et al. (15) Kroese-Deutman et al. (15) Xu et al. (16) Xu et al. (16) Tang et al. (3) Keiichi et al. (17) Keiichi et al. (17) Keiichi et al. (17) Calvo-Guirado et al. (18) Okanoue et al. (19) Okanoue et al. (19) Su et al. (20) Su et al. (20) Zhao et al. (21) Zhao et al. (21) Zhao et al. (21) Klijn et al. (22) Klijn et al. (22) Klijn et al. (22) Liu et al. (23) Liu et al. (23) Tayton et al. (24) Tayton et al. (24)

Goat Goat Rabbit Rabbit Rabbit Goat Goat Goat Goat Rabbit Rabbit Rabbit Rabbit Rabbit Rat Rat Rat Rabbit Rabbit Rabbit Rabbit Rabbit Rat Rat Rat Rat Rat Rat Rat Rat Goat Goat

Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Calvaria Calvaria Calvaria Calvaria Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Metaphysis Calvaria Calvaria Calvaria Calvaria Calvaria Calvaria Calvaria Calvaria Metaphysis Metaphysis

6.3 6.3 4.5 6.0 6.0 8.0 8.0 8.0 8.0 6.0 9.0 10.0 10.0 5.5 4.3 4.3 4.3 6.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 6.0 6.0 6.0 4.6 4.6 8.0 8.0

10 10 26 26 26 24 24 24 24 12 12 16 16 16 12 12 12 8 12 12 12 12 8 8 8 12 12 12 24 24 13 13

CP CP HA HA HA CP CP CP CP CP CP CS CP HA HA HA HA HA+CP HA CP Bioglass Bioglass HA HA HA CP CP CP Bioglass Bioglass HA HA

FGF FGF BMP-2 BMP-2 BMSCs -

100 100 350 480 200 150 260 510 1220 150 150 400 400 394 500 500 500 450 300 400 500 500 450 450 450 500 500 500 150 500 192 192

47.0 59.0 60.0 70.0 70.0 75.0 75.0 75.0 75.0 71.0 74.0 74.9 72.3 36.0 52.0 52.0 52.0 95.0 85.0 75.0 76.0 76.0 75.0 75.0 75.0 53.2 44.5 42.0 50.0 80.0 63.4 63.4

HMM HMM HMM HMM HMM HMM HMM HMM HMM HMM HMM HMM HMM HMM Micro-CT Micro-CT Micro-CT HMM Micro-CT Micro-CT Micro-CT Micro-CT Micro-CT Micro-CT Micro-CT HMM HMM HMM HMM HMM Micro-CT Micro-CT

0.0 20.0 28.0 27.0 23.0 22.0 37.0 25.0 20.0 17.0 18.0 28.4 18.8 32.2 20.4 28.7 28.5 60.0 43.0 15.6 80.0 48.0 43.0 27.0 10.0 10.8 7.0 6.8 24.0 36.0 38.1 24.8

NBF, new bone formation; HA, hydroxyapatite; CP, calcium phosphate; CS, calcium silicate; FGF, fibroblast growth factor; BMP-2, bone morphogenetic protein-2; BMSC, bone marrow mesenchymal stem cells; HMM, histomorphometry.

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Table 2 - Median, minimum and maximum values for NBF rates and the experimental group counts according to the experimental model and biomaterial characteristics. HMM

Animal Rat Goat Rabbit Region Calvaria Metaphisis Basic material CP HA CS Glass HA+CP Osteoinductor None FGF BMP-2 BMSCs Maximum pore size o 300 mm 300 - 500 mm 4 500 mm Porosity o 5% 5% - 75% 4 75% Total

Micro-CT

NBF

Count

p-value

NBF

Count

p-value

16.92 (6.80-36.00) 20.67 (0.00-37.00) 28.04 (17.00-60.00)

5 6 9

0.377

26.27 (10.00-43.00) 31.45 (24.80-38.10) 46.65 (15.60-80.00)

6 2 4

0.340

18.53 (6.80-36.00) 26.75 (0.00-60.00)

9 11

0.102

26.67 (10.00-43.00) 36.34 (15.60-80.00)

3 9

0.459

16.87 27.55 28.36 30.00 60.00

12 4 1 2 1

15.60 (15.60-15.60) 29.28 (10.00-43.00) 80.00 (48.00-80.00) -

1 9 0 2 0

23.05 (0.00-60.00) -

20 0 0 0

34.54 28.60 35.00 38.10

(10.00-80.00) (28.50-28.70) (27.00-43.00) (38.10-38.10)

7 2 2 1

0.626

20.13 (0.00-37.00) 25.50 (6.80-60.00) 22.50 (20.00-25.00)

8 10 2

0.588

31.45 (24.80-38.10) 34.42 (10.00-80.00) -

2 10 -

0.829

11.50 21.50 33.33 23.05

4 10 6 20

0.035

28.10 (20.40-38.10) 38.09 (10.00-80.00) 33.93 (10.00-80.00)

5 7 12

(0.00-37.00) (23.00-32.20) (28.36-28.36) (24.00-36.00) (60.00-60.00)

(0.00-32.20) (10.80-28.36) (20.00-60.00) (0.00-60.00)

0.016

Figure 2 - Boxplot of the NBR rates of the different porosity groups according to the analysis method (HMM or micro-CT). NBF, new bone formation; HMM, histomorphometry. p-values from Kruskalâ&#x20AC;&#x201C;Wallis test.

defects (27). The wide variety and geometrical shapes of bone materials indicates that many questions remain to be answered. More research is necessary to better understand how NBF can be increased. This review demonstrates that the lack of standardization of NBF analyses has hampered the comparison of the various types of porous and absorbable bone substitutes. Even when limiting the evaluation to two types of quantitative analyses for NBF, it was not possible to obtain accurate results. A better description of the analyzed region of interest (ROI) by each method could offer more precise data interpretation, and consequently, a more consistent comparison with other

Despite the large number, few studies could be included due to the significant variation in the presentation of the bone substitutes and experimental models. In addition, the NBF assessment method varied greatly, making comparison unsuitable. In this review, we used the two most cited methods, which, according to some articles, do not present comparable values but are related (10,25,26). Therefore, the HMM and micro-CT results were analyzed separately. Although a substantial effort has been made by the scientific community in recent years to improve bone substitutes, some skepticism still exists regarding the effectiveness of the currently available biomaterials to cure critical size bone

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similar studies. Additionally, the inclusion of standard control groups with autografts or a commonly used substitute would allow the calibration of the results and the comparison of many different study groups. We conclude that porosity has a high impact on NBF rates and that the current lack of standardized analysis methods and the broad variety of experimental models makes identifying the chemical and structural characteristics that provide a greater capacity for NBF almost impossible. We suggest that standard control groups should always be used to allow for a better comparison of the results.

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’ AUTHOR CONTRIBUTIONS Paulo MJ and dos Santos MA conducted the electronic search and data collection. Cimatti B and Gava NF reviewed the electronic search and data collection. Riberto M revised the manuscript and conducted the data analysis. Engel EE wrote the manuscript, reviewed the data analysis and supervised the study.

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